U.S. patent application number 10/724598 was filed with the patent office on 2006-01-26 for methods of increasing the production of cobalamins using cob gene expression.
This patent application is currently assigned to Aventis Pharma S.A.. Invention is credited to Francis Blanche, Beatrice Cameron, Joel Crouzet, Laurent Debussche, Sophie Levy Schil, Denis Thibaut.
Application Number | 20060019352 10/724598 |
Document ID | / |
Family ID | 9393280 |
Filed Date | 2006-01-26 |
United States Patent
Application |
20060019352 |
Kind Code |
A1 |
Blanche; Francis ; et
al. |
January 26, 2006 |
Methods of increasing the production of cobalamins using cob gene
expression
Abstract
Novel polypeptides involved in the biosynthesis of cobalamines
and/or cobamides, in particular coenzyme B.sub.12, genetic material
responsible for expressing these polypeptides, and a method for
preparing them, are described. A method for amplifying the
production of cobalamines, and particularly coenzyme B.sub.12,
using recombinant DNA techniques, are also described.
Inventors: |
Blanche; Francis; (Paris,
FR) ; Cameron; Beatrice; (Paris, FR) ;
Crouzet; Joel; (Paris, FR) ; Debussche; Laurent;
(Paris, FR) ; Levy Schil; Sophie; (Paris, FR)
; Thibaut; Denis; (Paris, FR) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
Aventis Pharma S.A.
|
Family ID: |
9393280 |
Appl. No.: |
10/724598 |
Filed: |
December 1, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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08426630 |
Apr 21, 1995 |
6656709 |
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10724598 |
Dec 1, 2003 |
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07916151 |
Sep 14, 1992 |
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08426630 |
Apr 21, 1995 |
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Current U.S.
Class: |
435/86 ;
435/252.2; 435/252.3 |
Current CPC
Class: |
C12N 9/00 20130101; C12N
15/52 20130101; C12P 19/42 20130101 |
Class at
Publication: |
435/086 ;
435/252.3; 435/252.2 |
International
Class: |
C12P 19/42 20060101
C12P019/42; C12N 1/20 20060101 C12N001/20 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 31, 1990 |
FR |
90 01137 |
Jan 30, 1991 |
WO |
PCT/FR91/00054 |
Claims
1-57. (canceled)
58. A method for increasing the production of cobalamins,
cobamides, cobalamin precursors, or cobamide precursors, wherein
said method comprises: a) introducing a plasmid comprising a DNA
sequence selected from the group consisting of the cobA, cobB,
cobC, cobD, cobE, cobF, cobG, cobH, cobI, cobJ, cobK, cobL, cobM,
cobN, cobO, cobP, cobQ, cobS, cobT, cobU, cobV, cobW, and cobX
genes of P. denitrificans and homologs of said genes resulting from
the degeneracy of the genetic code into a microorganism capable of
producing cobalamins or cobamides; b) culturing said microorganism
under conditions suitable for the synthesis of cobalamins,
cobamides, cobalamin precursors, or cobamide precursors, wherein
said culture conditions are also suitable for expression of said
DNA; and c) recovering the cobalamins, cobamides, cobalamin
precursors, or cobamide precursors produced.
59. The method of claim 58, wherein said recovery step comprises:
a) solubilization; b) conversion to a cyanoform; and c)
purification.
60. The method of claim 58, wherein said cobalamin is coenzyme
B.sub.12.
61. The method of claim 58, wherein said cobalamin precursor or
cobamide precursor is selected from the group consisting of
decobaltocorrinoids and corrinoids.
62. A method for increasing the production of cobalamins,
cobamides, cobalamin precursors, or cobamide precursors, wherein
said method comprises: a) introducing a DNA selected from the group
consisting of the cobA, cobB, cobC, cobD, cobE, cobF, cobG, cobH,
cobI, cobJ, cobK, cobL, cobM, cobN, cobO, cobP, cobQ, cobS, cobT,
cobU, cobV, cobW, and cobX genes of P. denitrificans and homologs
of said genes resulting from the degeneracy of the genetic code
into a microorganism capable of producing cobalamins or cobamides;
b) culturing said microorganism under conditions suitable for the
synthesis of cobalamins, cobamides, cobalamin precursors, or
cobamide precursors, wherein said culture conditions are also
suitable for expression of said DNA; and c) recovering the
cobalamins, cobamides, cobalamin precursors, or cobamide precursors
produced.
63. The method of claim 62, wherein said recovery step comprises:
a) solubilization; b) conversion to a cyanoform; and c)
purification.
64. The method of claim 62, wherein said cobalamin is coenzyme
B.sub.12.
65. The method of claim 62, wherein said cobalamin precursor or
cobamide precursor is selected from the group consisting of
decobaltocorrinoids and corrinoids.
66. The method of claim 58 or 62 wherein said microorganism is P.
denitrificans strain SC510 RifR.
67. A method for increasing the industrial production of
cobalamins, cobamides, cobalamin precursors, or cobamide
precursors, wherein said method comprises: a) introducing at least
one plasmid comprising a DNA sequence selected from the group
consisting of cobA, cobB, cobC, cobD, cobE, cobF, cobG, cobH, cobI,
cobJ, cobK, cobL, cobM, cobN, cobO, cobP, cobQ, cobS, cobT, cobU,
cobV, cobW, and cobX genes of P. denitrificans and homologs of said
genes resulting from the degeneracy of the genetic code into a
microorganism producing cobalamins or cobamides; b) culturing said
microorganism under conditions suitable for the synthesis of
cobalamins, cobamides, cobalamin precursors, or cobamide
precursors, wherein industrial production comprises culture
conditions suitable for expression of said DNA and suitable for
production of at least 100 grams of cells; and c) recovering the
cobalamins, cobamides, cobalamin precursors, or cobamide precursors
produced, wherein the industrial production of cobalamins,
cobamides, cobalamin precursors, or cobamide precursors by said
microorganism is increased by the introduction of said plasmid.
68. The method of claim 67, wherein said host cell is selected from
Pseudomonas denitrificans, Rhizobium meliloti, and Agrobacterium
tumefaciens.
69. The method of claim 68, wherein said microorganism is P.
denitrificans strain SC510 RifR.
70. The method of any one of claims 67-69, wherein said cobalamin
is coenzyme B.sub.12.
71. The method of claim 67, wherein said cobalamin precursor or
cobamide precursor is selected from the group consisting of
decobaltocorrinoids and corrinoids.
72. The method of claim 67, wherein said at least one plasmid
comprises the cobF, cobG, cobH, cobI, cobJ, cobK, cobL, and cobM
genes of P. denitrificans or homologs of said genes resulting from
the degeneracy of the genetic code.
73. The method of claim 72, wherein said host cell is selected from
Pseudomonas denitrificans, Rhizobium meliloti, and Agrobacterium
tumefaciens.
74. The method of claim 73, wherein said microorganism is P.
denitrificans strain SC510 RifR.
75. The method of any one of claims 72-74, wherein said cobalamin
is coenzyme B.sub.12.
76. The method of claim 72, wherein said cobalamin precursor or
cobamide precursor is selected from the group consisting of
decobaltocorrinoids and corrinoids.
77. The method of claim 72, wherein said at least one plasmid
further comprises the cobA and cobE genes of P. denitrificans or
homologs of said genes resulting from the degeneracy of the genetic
code.
78. The method of claim 77, wherein said host cell is selected from
Pseudomonas denitrificans, Rhizobium meliloti, and Agrobacterium
tumefaciens.
79. The method of claim 78, wherein said microorganism is P.
denitrificans strain SC510 RifR.
80. The method of any one of claims 77-79, wherein said cobalamin
is coenzyme B.sub.12.
81. The method of claim 77, wherein said cobalamin precursor or
cobamide precursor is selected from the group consisting of
decobaltocorrinoids and corrinoids.
82. The method of claim 77, wherein said at least one plasmid
further comprises the cobA and cobE genes of P. denitrificans or
homologs of said genes resulting from the degeneracy of the genetic
code.
83. The method of claim 82, wherein said host cell is selected from
Pseudomonas denitrificans, Rhizobium meliloti, and Agrobacterium
tumefaciens.
84. The method of claim 83, wherein said microorganism is P.
denitrificans strain SC510 RifR.
85. The method of any one of claims 82-84, wherein said cobalamin
is coenzyme B.sub.12.
86. The method of claim 82, wherein said cobalamin precursor or
cobamide precursor is selected from the group consisting of
decobaltocorrinoids and corrinoids.
87. A method for increasing the industrial production of
cobalamins, cobamides, cobalamin precursors, or cobamide
precursors, wherein said method comprises: a) introducing at least
one plasmid comprising a DNA sequence selected from the group
consisting of cobA, cobB, cobC, cobD, cobE, cobF, cobG, cobH, cobI,
cobJ, cobK, cobL, cobM, cobN, cobO, cobP, cobQ, cobS, cobT, cobU,
cobV, cobW, and cobX genes of P. denitrificans and homologs of said
genes resulting from the degeneracy of the genetic code into a
microorganism capable of producing cobalamins or cobamides; b)
culturing said microorganism under conditions suitable for the
synthesis of cobalamins, cobamides, cobalamin precursors, or
cobamide precursors, wherein industrial production comprises
culture conditions suitable for expression of said DNA and suitable
for production of at least 100 grams of cells; and c) recovering
the cobalamins, cobamides, cobalamin precursors, or cobamide
precursors produced, wherein the industrial production of
cobalamins, cobamides, cobalamin precursors, or cobamide precursors
by said microorganism is increased by the introduction of said
plasmid.
88. The method of claim 87, wherein said host cell is selected from
Pseudomonas denitrificans, Rhizobium meliloti, and Agrobacterium
tumefaciens.
89. The method of claim 88, wherein said microorganism is P.
denitrificans strain SC510 RifR.
90. The method of any one of claims 87-89, wherein said cobalamin
is coenzyme B.sub.12.
91. The method of claim 87, wherein said cobalamin precursor or
cobamide precursor is selected from the group consisting of
decobaltocorrinoids and corrinoids.
92. The method of claim 87, wherein said at least one plasmid
comprises the cobF, cobG, cobH, cobI, cobJ, cobK, cobL, and cobM
genes of P. denitrificans or homologs of said genes resulting from
the degeneracy of the genetic code.
93. The method of claim 92, wherein said host cell is selected from
Pseudomonas denitrificans, Rhizobium meliloti, and Agrobacterium
tumefaciens.
94. The method of claim 93, wherein said microorganism is P.
denitrificans strain SC510 RifR.
95. The method of any one of claims 92-94, wherein said cobalamin
is coenzyme B.sub.12.
96. The method of claim 92, wherein said cobalamin precursor or
cobamide precursor is selected from the group consisting of
decobaltocornrinoids and corrinoids.
97. The method of claim 92, wherein said at least one plasmid
further comprises the cobA and cobE genes of P. denitrificans or
homologs of said genes resulting from the degeneracy of the genetic
code.
98. The method of claim 97, wherein said host cell is selected from
Pseudomonas denitrificans, Rhizobium meliloti, and Agrobacterium
tumefaciens.
99. The method of claim 98, wherein said microorganism is P.
denitrificans strain SC510 RifR.
100. The method of any one of claims 97-99, wherein said cobalamin
is coenzyme B.sub.12.
101. The method of claim 97, wherein said cobalamin precursor or
cobamide precursor is selected from the group consisting of
decobaltocorrinoids and corrinoids.
102. The method of claim 87, wherein said at least one plasmid
further comprises the cobA and cobE genes of P. denitrificans or
homologs of said genes resulting from the degeneracy of the genetic
code.
103. The method of claim 102, wherein said host cell is selected
from Pseudomonas denitrificans, Rhizobium meliloti, and
Agrobacterium tumefaciens.
104. The method of claim 103, wherein said microorganism is P.
denitrificans strain SC510 RifR.
105. The method of any one of claims 102-104, wherein said
cobalamin is coenzyme B.sub.12.
106. The method of claim 102, wherein said cobalamin precursor or
cobamide precursor is selected from the group consisting of
decobaltocorrinoids and corrinoids.
107. The method of any one of claims 67-69, 71-74, 76-79, 81-84,
87-89, 91-94, 96-99, 101-104, and 106, wherein said recovery step
comprises: a) solubilization; b) conversion to a cyanoform; and c)
purification.
108. The method of claim 70, wherein said recovery step comprises:
a) solubilization; b) conversion to a cyanoform; and c)
purification.
109. The method of claim 75, wherein said recovery step comprises:
a) solubilization; b) conversion to a cyanoform; and c)
purification.
110. The method of claim 80, wherein said recovery step comprises:
a) solubilization; b) conversion to a cyanoform; and c)
purification.
111. The method of claim 85, wherein said recovery step comprises:
a) solubilization; b) conversion to a cyanoform; and c)
purification.
112. The method of claim 90, wherein said recovery step comprises:
a) solubilization; b) conversion to a cyanoform; and c)
purification.
113. The method of claim 95, wherein said recovery step comprises:
a) solubilization; b) conversion to a cyanoform; and c)
purification.
114. The method of claim 100, wherein said recovery step comprises:
a) solubilization; b) conversion to a cyanoform; and c)
purification.
115. The method of claim 105, wherein said recovery step comprises:
a) solubilization; b) conversion to a cyanoform; and c)
purification.
Description
[0001] The present invention relates to new polypeptides involved
in the biosynthesis of cobalamins and/or cobamides, and especially
of coenzyme B.sub.12. It also relates to the genetic material
responsible for the expression of these polypeptides, as well as to
a method by means of which they may be prepared. It relates,
lastly, to a method for amplification of the production of
cobalamins, and more especially of coenzyme B.sub.12, by
recombinant DNA techniques.
[0002] Vitamin B.sub.12 belongs to the B group of vitamins. It is a
water-soluble vitamin which has been identified as the factor
enabling patients suffering from pernicious anaemia to be treated.
It is generally prescribed to stimulate haematopoiesis in fatigue
subjects, but it is also used in many other cases comprising liver
disorders and nervous deficiencies or as an appetite stimulant or
an active principle with tonic activity, as well as in dermatology
(Berck, 1982, Fraser et al., 1983). In the industrial rearing of
non-ruminant animals, the feed being essentially based on proteins
of vegetable origin, it is necessary to incorporate vitamin
B.sub.12 in the feed rations in amounts of 10 to 15 mg per tonne of
feed (Barrere et al., 1981).
[0003] Vitamin B.sub.12 belongs to a class of molecules known as
cobalamins, the structure of which is presented in FIG. 1.
Cobamides differ from cobalamins in the base of the lower
nucleotide, which is no longer 5,6-dimethylbenzimidazole but
another base, e.g. 5-hydroxybenzimidazole for vitamin
B.sub.12-factor III synthesised, inter alia, by Clostridium
thermoaceticum and Methanosarcina barkeri (Iron et al., 1984).
These structural similarities explain the fact that the metabolic
pathways of biosynthesis of cobalamins and cobamides are, for the
most part, shared.
[0004] Cobalamins are synthesised almost exclusively by bacteria,
according to a complex and still poorly understood process which
may be divided into four steps (FIG. 2):
[0005] i) synthesis of uroporphyrinogen III (or uro'gen III),
then
[0006] ii) conversion of uro'gen III to cobyrinic acid, followed
by
[0007] iii) conversion of the latter to cobinamide, and
[0008] iv) construction of the lower nucleotide loop with
incorporation of the particular base (5,6-dimethylbenzimidazole in
the case of cobalamins).
[0009] For coenzyme B.sub.12, it is probable that the addition of
the 5'-deoxyadenosyl group occurs shortly after the corrin
ring-system is synthesised (Huennekens et al., 1982).
[0010] In the case of cobamides, only the step of synthesis and
incorporation of the lower base is different.
[0011] The first part of the biosynthesis of cobalamins is very
well known, since it is common to that of haemes as well as to that
of chlorophylls (Battersby et al., 1980). It involves,
successively, .delta.-aminolevulinate synthase (EC 2.3.137),
.delta.-aminolevulinate dehydrase (EC 4.2.1.24), porphobilinogen
deaminase (EC 4.3.1.8) and uro'gen III cosynthase (EC 4.2.1.75),
which convert succinyl-CoA and glycine to uro'gen III. However, the
first step takes place in some organisms [e.g. E. coli (Avissar et
al., 1989) and in methanogenic bacteria (Kannangara et al., 1989),
for example) by the conversion by means of a multi-enzyme complex
of glutamic acid to .delta.-aminolevulinic acid.
[0012] Between uro'gen III and cobyrinic acid, only three
intermediate derivatives have been purified to date; they are the
factors FI, FII and FIII, which are oxidation products,
respectively, of the three intermediates precorrin-1, precorrin-2
and precorrin-3, which correspond to the mono-, di- and
trimethylated derivatives of uro'gen III (FIG. 3); these
intermediates are obtained by successive transfers of methyl groups
from SAM (S-adenosyl-L-methionine) to uro'gen III at positions C-2,
C-7 and C-20, respectively. The other reactions which take place to
give cobyrinic acid are, apart from five further transfers of
methyl groups from SAM at C-17, C-12, C-1, C-15 and C-5,
elimination of the carbon at C-20, decarboxylation at C-12 and
insertion of a cobalt atom (FIG. 4). These biosynthetic steps have
been deduced from experiments performed in vitro on acellular
extracts of Propionibacterium shermanii or of Clostridium
tetanomorphum. In these extracts, cobyrinic acid is obtained by
conversion of uro'gen III after incubation under suitable anaerobic
conditions (Batterby et al., 1982). No intermediate between
precorrin-3 and cobyrinic acid capable of being converted to
corrinoids by subsequent incubation with extracts of
cobalamin-producing bacteria has been isolated to date. The
difficulty of isolating and identifying these intermediates is
linked to
[0013] i) their great instability,
[0014] ii) their sensitivity to oxygen, and
[0015] iii) their low level of accumulation in vivo.
In this part of the pathway, only one enzyme of Pseudomonas
denitrificans has been purified and studied; it is SAM:uro'gen III
methyltransferase (Blanche et al., 1989), referred to as SUMT.
[0016] Between cobyrinic acid and cobinamide, the following
reactions are performed:
[0017] i) addition of the 5'-deoxyadenosyl group (if coenzyme
B.sub.12 is the compound to be synthesised),
[0018] ii) amidation of six of the seven carboxyl functions by
addition of amine groups, and
[0019] iii) amidation of the last carboxyl function (propionic acid
chain of pyrrole ring D) by addition of (R)-1-amino-2-propanol
(FIG. 2).
[0020] Whether there was really an order in the amidations was not
elucidated (Herbert et al., 1970). Lastly, no assay of activity in
this part of the pathway has been described, except as regards the
addition of the 5'-deoxyadenosyl group (Huennekens et al.,
1982).
[0021] The final step of the biosynthesis of a cobalamin, e.g.
coenzyme B.sub.12, comprises four successive phases described in
FIG. 5 (Huennekens et al., 1982), namely:
[0022] i) phosphorylation of the hydroxyl group of the
aminopropanol residue of cobinamide to cobinamide phosphate,
then
[0023] ii) addition of a guanosine diphosphate by reaction with
guanosine 5'-triphosphate; the compound obtained is GDP-cobinamide
(Friedmann, 1975), which
[0024] iii) reacts with 5,6-dimethylbenzimidazole, itself
synthesised from riboflavin, to give adenosylcobalamin 5'-phosphate
(Friedmann et al., 1968), which
[0025] iv) on dephosphorylation leads to coenzyme B.sub.12
(Schneider and Friedmann, 1972).
[0026] Among bacteria capable of producing cobalamins, the
following may be mentioned in particular:
[0027] Agrobacterium tumefaciens
[0028] Agrobacterium radiobacter
[0029] Bacillus megaterium
[0030] Clostridium sticklandii
[0031] Clostridium tetanomorphum
[0032] Clostridium thermoaceticum
[0033] Corynebacterium XG
[0034] Eubacterium limosum
[0035] Methanobacterium arbophilicum
[0036] Methanobacterium ivanovii
[0037] Methanobacterium ruminantium
[0038] Methanobacterium thermoautotrophicum
[0039] Methanosarcina barkeri
[0040] Pronionobacterium shermanii
[0041] Protaminobacter ruber
[0042] Pseudomonas denitrificans
[0043] Pseudomonas putida
[0044] Rhizobium meliloti
[0045] Rhodopseudomonas sphaeroides
[0046] Salmonella typhimurium
[0047] Spirulina platensis
[0048] Streptomvces antibioticus
[0049] Streptomyces aureofaciens
[0050] Streptomyces ariseus
[0051] Streptomvces olivaceus
[0052] At the industrial level, as a result of the great complexity
of the biosynthetic mechanisms, the production of cobalamins, and
especially of vitamin B.sub.12, is exclusively microbiological. It
is carried out by large-volume cultures of the bacteria Pseudomonas
denitrificans, Propionibacterium shermanii and Propionibacterium
freudenreichii (Florent, 1986). The strains used for the industrial
production are derived from wild-type strains; they may have
undergone a large number of cycles of random mutation and then of
selection of improved clones for the production of cobalamins
(Florent, 1986). The mutations are obtained by mutagenesis with
mutagenic agents or by physical treatments such as treatments with
ultraviolet rays (Barrere et al., 1981). By this empirical method,
random mutations are obtained and improve the production of
cobalamins. For example, it is described that, from the original
strain of Pseudomonas denitrificans initially isolated by Miller
and Rosenblum (1960, U.S. Pat. No. 2,938,822), the production of
this microorganism was gradually increased in the space of ten
years, by the techniques mentioned above, from 0.6 mg/l to 60 mg/l
(Florent, 1986). For bacteria of the genus Propionibacterium
[Pronionibacterium shermanii (ATCC 13673) and freudenreichii (ATCC
6207)), the same production values appear to be described in the
literature; e.g. a production of 65 mg/l has been described
(European Patent 87,920). However, no screen has yet been described
enabling either mutants overproductive of cobalamins or mutants
markedly improved in their production of cobalamins to be readily
selected or identified.
[0053] At the genetic level, little work has been performed to
date. The cloning of cob genes (coding for enzymes involved in the
biosynthetic process) has been described in Bacillus megaterium
(Brey et al., 1986). Eleven complementation groups have been
identified by complementation of cob mutants of Bacillus megaterium
with plasmids carrying different fragments of Bacillus megaterium
DNA. These genes are grouped on the same locus, carried by a 12-kb
fragment.
[0054] Studies have also been carried out on the cob genes of
Salmonella typhimurium. Although the cloning of these has not been
described, it has been shown that almost all the genes for
cobalamin biosynthesis are grouped together between minutes 40 and
42 of the chromosome (Jeter and Roth, 1987). Only the cysG locus,
which must permit the conversion of uro'gen III to precorrin-2,
does not form part of this group of genes. However, the activity
encoded by this locus and also its biochemical properties have not
been described.
[0055] In addition, some phenotypes have been associated with cob
mutations. In Salmonella typhimurium and in Bacillus megaterium,
cob mutants no longer show growth on minimum medium with
ethanolamine as a carbon source or as a nitrogen source (Roof and
Roth, 1988). This is due to the fact that an enzyme of ethanolamine
catabolism, ethanolamine ammonia-lyase (EC 4.3.1.7), has coenzyme
B.sub.12 as a cofactor; the cob mutants no longer synthesise
coenzyme B.sub.12, and they can no longer grow with ethanolamine as
a carbon source and/or as a nitrogen source. metE mutants of
Salmonella typhimurium retain only a methylcobalamin-dependent
homocysteine methyltransferase (EC 2.1.1.13). cob mutants of
Salmonella typhimurium metE are auxotrophic for methionine (Jeter
et al., 1984).
[0056] In Pseudomonas denitrificans and Agrobacterium tumefaciens,
phenotypes associated with a total deficiency of cobalamin
synthesis have not been described to date.
[0057] Finally, work on Pseudomosas denitrificans (Cameron et al.,
1989) has led to the cloning of DNA fragments carrying cob genes of
this bacterium. These are distributed in four complementation
groups carried by at least 30 kb of DNA. At least fourteen
complementation groups have been identified by heterologous
complementation of cob mutants of Agrobacterium tumefaciens and of
Pseudomonas outida with DNA fragments of Pseudomonas denitrificans
carrying cob genes.
[0058] However, hitherto, none of these genes has been purified,
and no nucleotide sequence has been described. Similarly, no
protein identification nor any catalytic function attributed to the
product of these genes has been described. Furthermore, no
improvement in production of cobalamins by recombinant DNA
techniques could be obtained. The amplification of cob genes of
Bacillus megaterium does not bring about, in the strain from which
they have been cloned, an improvement in production of cobalamins
(Brey et al., 1986). In Salmonella tyrhimurium, physiological
studies have been carried out in order to determine conditions
under which a strong transcription of the cob genes studied was
observed (Escalante and Roth, 1987). Under these conditions, there
is no improvement in the production of cobalamins, although genes
of the biosynthetic pathway are more expressed than under standard
culture conditions.
[0059] The present invention results from the precise
identification of DNA sequences coding for polypeptides involved in
the biosynthesis of cobalamins and/or cobamides. A subject of the
invention hence relates to the DNA sequences coding for the
polypeptides involved in the biosynthesis of cobalamines and/or
cobamides. More especially, the subject of the invention is the
cobA, cobB, cobC, cobD, cobE, cobF, cobG, cobH, cobI, cobJ, cobK,
cobL, cobM, cobN, cobO, cobP, cobQ, cobS, cobT, cobU, cobV, cobW,
cobX and corA genes, any DNA sequence homologous with these genes
resulting from the degeneracy of the genetic code, and also DNA
sequences, of any origin (natural, synthetic, recombinant), which
hybridise and/or which display significant homologies with these
sequences or with fragments of the latter, and which code for
polypeptides involved in the biosynthesis of cobalamins and/or
cobamides. The subject of the invention is also the genes
containing these DNA sequences.
[0060] The DNA sequences according to the present invention were
isolated from an industrial strain, Pseudomonas denitrificans
SC510, derived from strain MB580 (U.S. Pat. No. 3,018,225), by
complementation of cob mutants of A. tumefaciens and P. putida; and
of Methanobacterium ivanovii. The clones obtained could be analysed
precisely, in particular by mapping using insertions of a
derivative of transposon Tn5. These genetic studies have enabled
the cob or cor genes to be localised on the restriction map and
their sequencing to be carried out. An analysis of the open reading
frames then enabled the coding regions of these DNA fragments to be
demonstrated.
[0061] The subject of the present invention is also the use of
these nucleotide sequences for cloning the cob genes of other
bacteria. In effect, it is known that, for proteins catalysing the
same activities, sequences are conserved, the divergence being the
evolutionary divergence (Wein-Hsiung et al., 1985). It is shown in
the present invention that there is a significant homology between
the nucleotide sequences of different microorganisms coding for
polypeptides involved in the biosynthesis of cobalamins and/or
cobamides. The differences which are seen result from the
evolutionary degeneracy, and from the degeneracy of the genetic
code which is linked to the percentage of GC in the genome of the
microorganism studied (Wein-Hsiung et al., 1985).
[0062] According to the present invention, a probe may be made with
one or more DNA sequences of Pseudomonas denitrificans in
particular, or with fragments of these, or with similar sequences
displaying a specific degree of degeneracy in respect of the use of
the codons and the percentage of GC in the DNA of the bacterium
which it is desired to study. Under these conditions, it is
possible to detect a specific hybridisation signal between the
probe and fragments of genomic DNA of the bacterium studied; this
specific hybridisation signal corresponds to the hybridisation of
the probe with the isofunctional cob genes of the bacterium. The
cob genes as well as their products may then be isolated, purified
and characterised. The invention thus provides a means enabling
access to be gained, by hybridisation, to the nucleotide sequences
and the polypeptides involved in the biosynthesis of cobalamins
and/or cobamides of any microorganism.
[0063] The subject of the present invention is also a recombinant
DNA containing at least one DNA sequence coding for a polypeptide
involved in the biosynthesis of cobalamins and/or cobamides, and in
particular a recombinant DNA in which the said sequence or
sequences are placed under the control of expression signals.
[0064] In this connection, promoter regions may, in particular, be
positioned at the 5' end of the DNA sequence. Such regions may be
homologous or heterologous to the DNA sequence. In particular,
strong bacterial promoters such as the promoter of the tryptophan
operon Ptrp or of the lactose operon Plac of E. coli, the leftward
or rightward promoter of bacteriophage lambda, the strong promoters
of phages of bacteria such as Corynebacteria, the functional
promoters in Gram-negative bacteria such as the Ptac promoter of E.
coli, the PxylS promoter of the xylene catabolism genes of the TOL
plasmid and the amylase promoter of Bacillus subtilis Pamy may be
used. Promoters derived from glycolytic genes of yeasts may also be
mentioned, such as the promoters of the genes coding for
phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase,
lactase or enolase, which may be used when the recombinant DNA is
to be introduced into a eukaryotic host. A ribosome binding site
will also be positioned at the 5' end of the DNA sequence, and it
may be homologous or heterologous, such as the ribosome binding
site of the cII gene of bacteriophage lambda.
[0065] Signals necessary to transcription termination may be placed
at the 3' end of the DNA sequence.
[0066] The recombinant DNA according to the present invention may
then be introduced directly into a host cell compatible with the
chosen expression signals, or be cloned into a plasmid vector to
enable the DNA sequence in question to be introduced in a stable
manner into the host cell.
[0067] Another subject of the invention relates to the plasmids
thereby obtained, containing a DNA sequence coding for a
polypeptide involved in the biosynthesis of cobalamins and/or
cobamides. More specifically, these plasmids also contain a
functional replication system and a selectable marker.
[0068] The subject of the invention is also the host cells into
which one or more DNA sequences as defined above, or a plasmid as
defined hereinbefore, has/have been introduced.
[0069] Another subject of the invention relates to a method for
production of polypeptides involved in the biosynthesis of
cobalamins and/or cobamides. According to this method, a host cell
is transformed with a DNA sequence as described above, this
transformed cell is cultured under conditions for expression of the
said sequence and the polypeptides produced are then recovered.
[0070] The host cells which may be used for this purpose are either
prokaryotes or eukaryotes, animal cells or plant cells. Preferably,
they will be chosen from bacteria, and especially bacteria of the
genus E. coli, P. denitrificans, A. tumefaciens or R. meliloti.
[0071] Another use of the DNA sequences according to the present
invention lies in a method for amplification of the production of
cobalamins and/or cobamides, by recombinant DNA techniques. In
effect, if the limitation of the metabolic flux of the biosynthesis
of cobalamins and/or cobamides is due to a limitation in the
activity of an enzyme in the biosynthetic pathway, an increase in
this activity by increasing the expression of this same enzyme
using recombinant DNA techniques (gene amplification, substitution
of the transcription/translation signals with more effective
signals, etc.) will lead to an increase in the biosynthesis of
cobalamins and/or cobamides. It is also possible that the
limitation of the production of cobalamins and/or cobamides results
from a biochemical regulation. In this case, the cob gene or genes
corresponding to the regulated enzyme may be specifically
mutagenised in vitro in order to obtain mutated genes whose
products will have lost the regulation mechanisms impeding an
improvement in the production.
[0072] The method according to the present invention consists in
transforming a microorganism productive of cobalamins and/or
cobamides, or only potentially productive of these compounds (i.e.
deficient in one or more steps of the biosynthesis), with a DNA
sequence as defined above, then in culturing this microorganism
under conditions for expression of the said sequence and for
synthesis of cobalamins and/or cobamides, and lastly in recovering
the cobalamins and/or cobamides produced. Such a method is
applicable, in particular, to all the productive microorganisms
mentioned on pages 5 and 6, and more specifically to microorganisms
of the genus P. denitrificans, Rhizobium meliloti, or Agrobacterium
tumefaciens. In a preferred embodiment, the microorganism is P.
denitrificans, and especially strain SC510. As regards potentially
productive microorganisms, the DNA sequences used will be those
corresponding to the steps of the biosynthesis which the
microorganism cannot carry out.
[0073] Using the present invention, and by the various stragegies
described above, an improvement in the production of cobalamins
and/or cobamides may be obtained for any microorganism productive
or potentially productive of cobalamins and/or cobamides. It will
suffice to culture this recombinant microorganism under suitable
conditions for the production of cobalamins and for the expression
of the DNA sequences introduced. This culturing may be carried out
batchwise or alternatively in continuous fashion, and the
purification of the cobalamins may be carried out by the methods
already used industrially (Florent, 1986). These methods comprise,
inter alia:
[0074] i) solubilisation of the cobalamins and their conversion to
their cyano form (e.g. by heat treatment of the fermentation must,
with potassium cyanide in the presence of sodium nitrite), then
[0075] ii) purification of the cyanocobalamins in various steps
which can be, e.g. [0076] a) adsorption on different substrates
such as Amberlite IRC-50, Dowex 1.times.2 or Amberlite XAD-2,
followed by an elution with a water/alcohol or water/phenol
mixture, then [0077] b) extraction in an organic solvent, and
lastly [0078] c) precipitation or crystallisation from the organic
phase, either by the addition of reagents or dilution in a suitable
solvent, or by evaporation.
[0079] The present invention shows, furthermore, that it is
possible by recombinant DNA techniques to improve the cobalamin
production of a bacterium productive of cobalamins by cumulating
improvements. This amounts to obtaining a first improvement as
described above, and then in improving this improvement, still
using recombinant DNA techniques, i.e., e.g. by amplifying genes
for cobalamin biosynthesis.
[0080] Another subject of the present invention relates to the
polypeptides involved in the biosynthesis of cobalamins and/or
cobamides. In particular, the subject of the present invention is
all polypeptides, or derivatives or fragments of these
polypeptides, which are encoded by the DNA sequences described
above, and which are involved in the pathway of biosynthesis of
cobalamins and/or cobamides. The amino acid sequence of these
polypeptides is described, as well as some of their physicochemical
properties. An enzymatic activity or specific properties have also
been associated with each of them.
[0081] In this connection, the subject of the invention is the
polypeptides participating in the conversion of precorrin-3 to
cobyrinic acid a,c-diamide, and more especially in the transfer of
a methyl group from SAM to positions C-1, C-5, C-11, C-15 and
C-17.
[0082] The subject of the invention is also the polypeptides:
[0083] participating in the conversion of cobyric acid to
cobinamide, or
[0084] possessing an S-adenosyl-L-methionine:precorrin-2
methyltransferase (SP2MT) activity, or
[0085] possessing a cobyrinic and/or hydrogenobyrinic acid
a,c-diamide synthase activity, or
[0086] possessing a precorrin-8x mutase activity, or
[0087] possessing a nicotinate-nucleotide: dimethylbenzimidazole
phosphoribosyltransferase activity, or
[0088] possessing a cobalamin-5'-phosphate synthase activity,
or
[0089] possessing a cobyric acid synthase activity, or
[0090] possessing a cob(I)alamin adenosyl-transferase activity,
or
[0091] possessing a precorrin-6.times. reductase activity, or
[0092] participating in the conversion of hydrogenobyrinic acid
a,c-diamide to cobyrinic acid a,c-diamide.
[0093] Advantageously, the subject of the invention is a
polypeptide chosen from the COBA, COBB, COBC, COBD, COBE, COBF,
COBG, COBH, COBI, COBJ, COBK, COBL, COBM, COBN, COBO, COBP, COBQ,
COBS, COBT, COBU, COBV, COBW, COBX and CORA proteins presented in
FIGS. 15, 16, 40, 41 and 47.
[0094] Furthermore, the use of the hybridisation probes described
above makes it possible, from genes isolated in other
microorganisms, to characterise and isolate the isofunctional
polypeptides of other microorganisms. In this manner, the present
invention shows that the sequence of a COB protein of Pseudomonas
denitrificans is significantly homologous with the protein
sequences of other microorganisms displaying the same type of
activity. Between these COB proteins catalysing the same reaction
in different microorganisms, only the evolutionary distances have
introduced variations (Wein-Hsiung et al., 1985). The subject of
the present invention is also these isofunctional polypeptides.
[0095] The assignment of a particular enzymatic activity is the
result of an analysis which may be performed according to various
strategies. In particular, in vitro affinity studies with respect
to SAM (S-adenosyl-L-methionine) make it possible to assign a
methyl transferase activity to a protein capable of binding SAM,
and hence to assign its involvement in one of the steps of transfer
of methyl groups which occur between uro'gen III and cobyrinic
acid. Another means of assessing the activity of these polypeptides
consists in assaying the intermediates in the pathway of
biosynthesis of cobalamins which are accumulated in mutants
incapable of expressing these polypeptides (identified by
complementation experiments). These analyses enable it to be
deduced that the polypeptide in question has the accumulated
intermediate as its substrate, thereby enabling its activity in the
biosynthetic pathway to be situated and defined. The present
invention also describes a method for assaying the enzymatic
activities of the biosynthetic pathway, applicable to any strain
productive of cobalamins and/or cobamides. These assays enable the
enzymatic activity assayed to be purified from any strain
productive of these compounds. From this purified activity, the
NH.sub.2-terminal sequence of the COB protein in question, or
alternatively that of the subunits of this protein, may be
determined, thereby enabling the structural gene or genes which
code for the activity in question to be identified. For Pseudomonas
denitrificans, the structural genes which code for activities of
the biosynthetic pathway are identified by finding, for each
NH.sub.2-terminal sequence, the COB protein having the same
NH.sub.2-terminal sequence.
[0096] The present invention also describes a method enabling
intermediates in the pathway of biosynthesis of cobalamins or of
other corrinoids to be identified and assayed in strains productive
of cobalamins. These intermediates may be assayed both in culture
musts and in the cells themselves. The intermediates which may be
assayed are all the corrinoids which occur in the biosynthetic
pathway after cobyrinic acid, namely, apart from cobyrinic acid,
cobyrinic acid monoamide, cobyrinic acid diamide, cobyrinic acid
triamide, cobyrinic acid tetraamide, cobyrinic acid pentaamide,
cobyric acid, cobinamide, cobinamide phosphate, GDP-cobinamide,
coenzyme B.sub.12 phosphate and coenzyme B.sub.12. The
non-adenosylated forms of these products may also be assayed by
this technique.
[0097] Other subjects and advantages of the present invention will
become apparent on reading the examples and the drawings which
follow, which are to be considered as illustrative and not
limiting.
Definition of the Terms Employed and Abbreviations.
[0098] ATP: adenosine 5'-triphosphate [0099] bp: base pairs [0100]
BSA: bovine serum albumin [0101] CADAS: cobyrinic acid a,c-diamide
synthase [0102] cluster: group of genes [0103] Cob: corresponds to
the phenotype with a reduced level (at least 10-fold lower than the
control) of production of cobalamins [0104] cob gene: gene involved
in the biosynthesis of cobalamins and/or cobamides from uro'gen III
[0105] COB protein: protein participating either as a catalyst in
the pathway of biosynthesis of cobalamins, or as a regulatory
protein in the network of regulation of the cob genes, or both.
[0106] cor gene: gene involved in the biosynthesis of corrinoids
from uro'gen III [0107] COR protein: protein participating either
as a catalyst in the pathway of biosynthesis of corrinoids, or as a
regulatory protein in the network of regulation of the cor genes,
or both [0108] Corrinoids: cobyrinic acid derivatives possessing
the corrin ring-system [0109] dGTP: 2'-deoxyguanosine
5'-triphosphate [0110] DMBI: dimethylbenzimidazole [0111] dNTP:
2'-deoxyribonucleoside 5'-triphosphates [0112] DTT: dithiothreitol
[0113] HPLC: high performance liquid chromatography [0114] kb:
kilobases [0115] NN: DMBI PRT: nicotinate-nucleotide:
dimethylbenzimidazole phosphoribosyltransferase [0116] ORF: open
reading frame [0117] recombinant DNA: set of techniques making it
possible either to combine within the same microorganism DNA
sequences which are not naturally so combined, or to mutagenise
specifically a DNA fragment [0118] SAM: S-adenosyl-L-methionine
[0119] SDS: sodium dodecyl sulphate [0120] SP.sub.2MT:
SAM-L-methionine:precorrin-2 methyltransferase [0121] Stop codon:
translation termination codon [0122] SUMT: SAM:uro'gen III
methyltransferase [0123] Uro'gen III: uroporphyrinogen III
LEGENDS TO THE FIGURES
[0124] FIG. 1: Structure of coenzyme B.sub.12; the 5'-deoxyadenosyl
group is replaced by a CH.sub.3 group for methylcobalamin, by a
cyano group for cyanocobalamin, by a hydroxyl group for
hydroxocobalamin.
[0125] FIG. 2: Biosynthesis of cobalamins and various steps of this
biosynthesis. X: axial ligands of the cobalt; the ligand at a may
be different from the ligand at b. R: ligand at a of the cobalt
which defines the cobalamin type (see FIG. 1).
[0126] FIG. 3: Structures of uro'gen III, precorrin-1, precorrin-2
and precorrin-3.
[0127] FIG. 4: Structural formulae of uro'gen III and cobyrinic
acid. Between uro'gen III and cobyrinic acid, there occur 8
SAM-dependent methyl transfers successively at C-2, C-7, C-20,
C-17, C-12, C-1, C-15 and C-5, a decarboxylation at C-12,
elimination of the carbon at C-20 and insertion of the cobalt atom.
X: axial ligands of the cobalt; the ligand at a may be different
from the ligand at b.
[0128] FIG. 5: Final steps of the biosynthesis of cobalamins. In
order to clarify the diagram, details of the corrin ring-system
have been omitted. The five enzymatic steps are represented: 1,
cobinamide kinase; 2, cobinamidephosphate guanylyltransferase; 3,
cobalamin-5'-phosphate synthase; 4, cobalamin-5'-phosphate
phosphohydrolase; 5, nicotinatenucleotide:DMBI
phosphoribosyltransferase.
[0129] FIG. 6: Restriction maps of the 5.4-kb
ClaI-HindIII-HindIII-HindIII, 8.7-kb EcoRI, 4748-bp
SalI-SalI-SalI-SalI-SalI-BalI and 3855-bp SstI-SstI-BamHI
fragments. Only the 20 restriction enzymes which cut the DNA least
frequently are shown. The cleavage sites of each enzyme are
indicated by a vertical line.
[0130] FIG. 7: Nucleotide sequence of both strands of the 5378-bp
ClaI-HindIII-HindIII-HindIII fragment of Pseudomonas denitrificans.
The strand situated at the top is to be read from 5' to 3' in the
left-to-right direction which corresponds to the left-to-right
orientation of the sequenced fragment presented in FIG. 6. The ClaI
site occurs at position 23 (beginning of the cleavage site) since,
in this sequence, there occur PstI, SalI and XbaI restriction sites
which have appeared during clonings in multisites with a view to
sequencing. The sequence of the ClaI-HindIII-HindIII-HindIII
fragment hence begins at position 23.
[0131] FIG. 8: Nucleotide sequence of both strands of the 8753-bp
EcoRI fragment of Pseudomonas denitrificans. The strand situated at
the top is to be read from 5' to 3' in the left-to-right direction
which corresponds to the left-to-right orientation of the fragment
of the restriction map presented in FIG. 6.
[0132] FIG. 9: Analysis of the probabilities of the coding frames
on the basis of codon preference using the programme of Staden and
MacLachlan (1982) on the 6 reading frames of the 5378-bp
ClaI-HindIII-HindIII-HindIII fragment. For the frames belonging to
the same coding strand, the most probable frame corresponds to that
in which a dotted line, not interrupted by stop codons, is placed
under the probability line for this frame.
[0133] 1. Sequence extending from nucleotide 1 to nucleotide 1200.
By means of this analysis, open reading frame 1 is identified. It
begins at the ATG at position 549 and ends at the TGA at position
1011.
[0134] 2. Sequence extending from nucleotide 1000 to nucleotide
2200. By means of this analysis, open reading frame 2 is
identified. It begins at the ATG at position 1141 and ends at the
TGA at position 1981.
[0135] 3. Sequence extending from nucleotide 1800 to nucleotide
3400. By means of this analysis, open reading frame 3 is
identified. It begins at the ATG at position 1980 and ends at the
TGA at position 3282.
[0136] 4. Sequence extending from nucleotide 3000 to nucleotide
4500. By means of this analysis, open reading frame 4 is
identified. It begins at the ATG at position 3281 and ends at the
TGA at position 4280.
[0137] 5. Sequence extending from nucleotide 3800 to nucleotide
5378. By means of this analysis, open reading frame 5 is
identified. It begins at the GTG at position 4284 and ends at the
TGA at position 5253.
[0138] FIG. 10: Analysis of the probabilities of the coding frames
on the basis of codon preference using the programme of Staden and
MacLachlan (1982) on the 6 reading frames of the 8753-bp EcoRI
fragment. For the frames belonging to the same coding strand, the
most probable frame corresponds to that in which a dotted line, not
interrupted by stop codons, is placed under the probability line
for this frame.
[0139] 1. Sequence extending from nucleotide 650 to nucleotide
1650. By means of this analysis, open reading frame 6 is
identified. It begins at the ATG at position 736 and ends at the
TGA at position 1519.
[0140] 2. Sequence extending from nucleotide 1400 to nucleotide
3100. By means of this analysis, open reading frame 7 is
identified. It begins at the ATG at position 1620 and ends at the
TAG at position 2997.
[0141] 3. Sequence extending from nucleotide 2700 to nucleotide
3700. By means of this analysis, open reading frame 8 is
identified. It begins at the ATG at position 3002 and ends at the
TGA at position 3632.
[0142] 4. Sequence extending from nucleotide 3500 to nucleotide
4100. By means of this analysis, open reading frame 9 is
identified. It begins at the GTG at position 3631 and ends at the
TGA at position 4366.
[0143] 5. Sequence extending from nucleotide 4150 to nucleotide
5150. By means of this analysis, open reading frame 10 is
identified. It begins at the ATG at position 4365 and ends at the
TGA at position 5127.
[0144] 6. Sequence extending from nucleotide 5000 to nucleotide
6000. By means of this analysis, open reading frame 11 is
identified. It begins at the ATG at position 5893 and ends at the
TAG at position 5110.
[0145] 7. Sequence extending from nucleotide 5700 to nucleotide
7200. By means of this analysis, frame 12 is identified. It begins
at the ATG at position 5862 and ends at the TAA at position
7101.
[0146] 8. Sequence extending from nucleotide 7000 to nucleotide
8000. By means of this analysis, open reading frame 13 is
identified. It begins at the ATG at position 7172 and ends at the
TTG at position 7931.
[0147] FIG. 11: Construction of plasmids pXL556, pXL545 and
pXL723.
[0148] A 2.4-kb ClaI-EcoRV fragment containing the cobA and cobE
genes is excised from the 5.4-kb fragment and then purified. An
EcoRI linker is added at the EcoRV site and the fragment is then
inserted into pXL59 between the ClaI-EcoRI sites. The plasmid
thereby constructed is designated pXL556.
[0149] The construction is comparable for pXL545: a 1.9-kb
ClaI-HindIII-HindIII fragment is excised from the 5.4-kb fragment
and then purified. This fragment contains only the CobE gene. An
EcoRI linker is added at the HindIII site and the fragment is then
inserted into pXL59 between the ClaI-EcoRI sites. pXL723 is
constructed as follows: a 2.3-kb EcoRI-HindIII fragment is excised
from the 5.4-kb fragment and purified, and the ends are then filled
in with the large fragment of E. coli DNA polymerase I. This
fragment is cloned into pRK290 (Ditta et al., 1981) digested with
EcoRI and then treated with the large fragment of E. coli DNA
polymerase I in order to fill in the ends.
[0150] The restriction sites which are shown in brackets correspond
to sites which have disappeared after treatment with the large
fragment of E. coli DNA polymerase I.
[0151] 1, PstI-SstI fragment of RSF1010 (De Graff et al., 1978); 2,
PstI-BamHI fragment of pACYC177 (Bagdasarian et al., 1981); 3,
BamHI-SstI fragment containing the lactose operon of E. coli
without its promoter, the operator, the translation initiation site
and the first 8 non-essential codons of lacZ (Casadaban et al.,
1983); 4, Sau3AI fragment of Pseudomonas putida KT2440 (Bagdasarian
et al., 1981); ori, origin of replication; nic, relaxation site;
mob, locus essential for mobilisation; Km', kanamycin resistance
gene (Bagdasarian et al., 1981); B. BamHI; C, ClaI; E, EcoRI; H,
HindIII; P, PstI; S. SstI; Sa, SalI; X, XhoI; Xb, XbaI.
[0152] FIG. 12: Studies of the insertions of transposons Tn5Sp' and
Tn5 into the 5378-bp fragment. The insertions of transposon Tn5
into plasmid pXL723 are shown as in FIG. 14; those of transposon
Tn5Sp', into the chromosome of strain G2 Rif', are boxed; the
insertions into the chromosome of SC510 Rif' of cassettes carrying
the kanamycin resistance gene (1630 and 1631) are shown with an
arrow, according to the orientation of transcription of the
kanamycin resistance gene, under the insertion number. The open
reading frames deduced from the sequence are given in this figure
(from cobA to cobE); + or - signs are shown under each insertion of
transposon or of resistance cassette to indicate that the insertion
is inactivating (-) or otherwise (+), i.e. for the complementation
of different mutants (the case with the insertions of transposons
Tn5), or that the insertion abolishes the cobalamin production of
the strain in which it takes place. There is an absence of
complementation when the recombinant mutant synthesises less than
threefold less cobalamins than the level of synthesis of the strain
from which the mutant is derived. The inserts of plasmids pXL545,
pXL1500, pXL1397 and pXL302 are shown with the restriction sites
occurring at their ends. These inserts are cloned into broad host
range plasmids, pXL435 and pXL59 (Cameron et al., 1989):
[0153] plasmid pXL545 corresponds to plasmid pXL545 described in
FIG. 11 with, in addition, the 2-kb BamHI fragment of pHP45
(Prentki and Krisch) containing a spectinomycin resistance gene
cloned at the BamHI site of pXL545;
[0154] plasmid pXL1500 corresponds to the 4.2-kb BqlII-SstI
fragment presented in this figure, cloned at the BamHI and SstI
sites of pKT230 (Bagdasarian et al., 1981); presented in FIG.
30;
[0155] plasmid pXL1397 corresponds to the 2.4-kb HindIII-SstI
fragment indicated in the figure, inserted between the HindIII and
SstI sites of the multisite of pXL435 (Cameron et al., 1989)
described in FIG. 30; plasmid pXL302 corresponds to the 2.3-kb
EcoRI-HindIII fragment as described in the figure, inserted between
the EcoRI and HindIII sites of pXL59 (Cameron et al., 1989)
described in FIG. 30, the HindIII site used being the site
occurring in the cloning multisite of pXL59;
[0156] pXL723 is described in FIG. 11, like pXL545.
[0157] +or - signs are shown above each of these inserts to
indicate whether there is complementation by the plasmid in
question of the chromosomal insertions shown underneath. C, ClaI;
E, EcoRI; H, HindIII; RV, EcoRV; Sau, Sau3AI; S, SstI.
[0158] FIG. 13: Construction of plasmids pXL253 and pXL367. The
8.7-kb EcoRI fragment is excised and then purified from plasmid
pXL151. It is cloned at the EcoRI site of pKT230 to give pXL253.
This same fragment is inserted at the EcoRI site of pRK290 (Ditta
et al., 1981) to give pXL3E7. 1, PstI-SstI fragment of RSF1010 (De
Graff et al., 1978); 2, PstI-BamHI fragment of pACYC177
(Bagdasarian et al., 1981); ori, origin of replication; nic,
relaxation site; mob, locus essential for mobilisation (Bagdasarian
et al., 1981); B, BamHI; C, ClaI; E, EcoRI; H. HindIII; P, PstI; S,
SstI; Sa, SalI; X, XhoI; Xb, XbaI; tet', tetracycline resistance
gene; Km', kanamycin resistance gene.
[0159] FIG. 14: Studies of the insertions of transposons Tn3lacZ
and Tn5 into the 8.7-kb EcoRI fragment cloned into pRK290 (Ditta et
al., 1980). The insertions of transposons Tn3lacZ are underlined,
in contrast to those of transposons Tn5. The open reading frames
deduced from the sequence (cobF to cobM) are given in this figure,
and the eight groups of inactivating insertions (numbered from 1 to
8) are presented; +or - signs are shown under each transposon
insertion to indicate that the insertion is inactivating (-) or
otherwise (+) for the complementation of different mutants. There
is an absence of complementation when the recombinant mutant
synthesises less than threefold less cobalamins than the level of
synthesis of the strain from which the mutant is derived. These
groups of inactivating insertions correspond to the following
mutants: 1, G615; 2, G614 and G616; 3, G613 and G614; 4, G620; 5,
G638; 6, G610 and G609; 7, G612; 8, G611. These mutants are Cob
mutants of Agrobacterium tumefaciens already described (Cameron et
al., 1989). A restriction map of the 8.7-kb fragment is given at
the bottom of the figure.
[0160] FIG. 15: The coding sequences of each of the genes of the
5.4-kb fragment, cobA to cobE, respectively, are indicated. The
sequences of the proteins COBA to COBE encoded by these sequences
appear under their respective coding sequence, cobA to cobE. The
amino acid composition of each protein, in number and in
percentage, respectively, of COBA to COBE, is presented, as well as
the molecular weight, the index of polarity, the isoelectric point
and the optical density at 260 nm and 280 nm of a solution
containing 1 mg/ml of purified protein. The hydrophilicity profile
of each COBA to COBE protein, respectively, is shown; it was
calculated on the basis of the programme of Hopp and Woods (1981).
Positive values correspond to regions of the protein which are
hydrophilic. The position of the amino acids is indicated as
abscissa, while the value of the index of hydrophilicity is shown
as ordinate; when this value is positive, this indicates that the
region of the protein is hydrophilic.
[0161] FIG. 16: The coding sequences of each of the genes of the
8.7-kb fragment, cobF to cobM respectively, are indicated. The
sequences of the COBF to COBM proteins encoded by these sequences
appear under their sequence. The legend is identical to that for
FIG. 15. NB. We have shown the COBF protein as beginning at the ATG
located at position. 736; it is possible that the ATG located at
position 751 is the true initiation codon of this protein.
[0162] FIG. 17: Reaction catalysed by cobyrinic acid a,c-diamide
synthase. CADAS catalyses the amidation of the carboxylic acid
functions of the peripheral acetate chains a and c of cobyrinic
acid to give cobyrinic acid diamide; the donor of the amine group
used in the enzymatic test is L-glutamine; it gives L-glutamic acid
on deamination. X corresponds to the axial ligands of the cobalt,
which may be different from one another.
[0163] FIG. 18: Reaction catalysed by SP.sub.2MT. SP.sub.2MT
catalyses the transfer of a methyl from SAM to
dihydrosirohydrochlorin or precorrin-2 to give precorrin-3. The
methyl group is transferred to position C-20 of the porphyrin
ring-system.
[0164] FIG. 19: Structure of hydrogenobyrinic acid and of
hydrogenobyrinic acid a,c-diamide.
[0165] FIG. 20: Affinities of the COBA and COBF proteins for SAM.
The curves give in arbitrary units the radioactivity at emergence
from the TSK-125 column for each protein applied to this column.
The retention times are indicated in minutes and the radioactivity
peak corresponding to free SAM is observed at the time of 10 min 30
sec.
[0166] FIG. 21: Comparison of the sequences of COBA and COBI. Only
the regions 1, 2 and 3, of strong homology, are presented. =signs
are placed between identical residues and--signs between homologous
residues (H K R, LIVM, A G S T, Y F W, D E Q N B Z, P, C).
[0167] FIG. 22: Comparison of the primary sequences of the proteins
COBA of Pseudomonas denitrificans and CYSG of E. coli. The
alignment has been done according to the programme of Kanehisa,
1984. =signs are placed between identical residues and - signs
between homologous residues (HKR, L I V M, A G S T, Y F W, D E Q N
B Z, P, C). The regions 1, 2 and 3 correspond to zones of strong
homology between the proteins.
[0168] FIG. 23: Comparison of the sequences of CYSG of E. coli with
COB proteins of Pseudomonas denitrificans (COBA, COBF, COBI, COBJ,
COBL and COBM). The comparisons concern the regions 1, 2 and 3, of
strong homologies, which exist between CYSG, COBA and COBI. The
positions in the protein sequences of the regions displaying
homologies are presented in the figure. We have considered the
following groups of homologous residues: H K R, L I V M, A G S T, Y
F W, D E Q N B Z, P, C. If there are at least 3 homologous residues
at the same position, we have boxed these amino acids.
[0169] FIG. 24: Construction of plasmids pXL1148 and pXL1149.
pXL1148 is constructed as follows: the 1.9-kb BamHI-BamHI-SstI-SstI
fragment of the 8.7-kb fragment containing the cobH and cobI genes
is purified, and XbaI and EcoRI linkers are placed at the BamHI and
SstI ends respectively. This fragment is then inserted between the
XbaI and EcoRI sites of the broad host range plasmid pXL59 (Cameron
et al., 1989) to give plasmid pXL1148.
[0170] pXL1149 is constructed like pXL1148, apart from the fact
that the fragment initially purified is the 1.5-kb BamHI-BamHI-SstI
fragment instead of the fragment additionally containing the small
400-bp SstI fragment used for pXL1148. The fragment then undergoes
the same enzymatic treatments and the same cloning into pXL59.
[0171] 1, PstI-SstI fragment of RSF1010 (De Graff et al., 1978); 2,
PstI-BamHI fragment of pACYC177 (Bagdasarian et al., 1981); 3,
BamHI-SstI fragment containing the lactose operon of E. coli
without promoter, operator, translation initiation site and the
first 8 non-essential codons of lacZ (Casadaban et al., 1983); 4,
Sau3AI fragment of Pseudomonas putida KT2440 (Bagdasarian et al.,
1981); ori, origin of replication; nic, relaxation site; Km',
kanamycin resistance gene; mob, locus essential for mobilisation
(Bagdasarian et al., 1981); B, BamHI; C, ClaI; E, EcoRI; H,
HindIII; P, PstI; S, SstI; Sa, SalI; X, XhoI; Xb, XbaI.
[0172] FIG. 25: Total proteins of strains SC510 Rif', SC510 Rif'
pKT230, SC510 Rif' pXL1148, SC510 Rif' pXL1149 analysed in 10%
SDS-PAGE as described. The bacteria were cultured for 4 days in PS4
medium, and lysates of the total proteins were then made. Lane 1,
SC510 Rif'; lane 2, SC510 Rif' pXL1149; lane 3, SC510 Rif' pXL1148;
lane 4, SC510 Rif' pKT230. The molecular masses of the molecular
mass markers are indicated. The positions to which the COBI and
COBH proteins migrate are indicated.
[0173] FIG. 26: Construction of plasmids pXL1496 and pXL1546.
Plasmid pXL1496 enables the COBF protein to be overexpressed in E.
coli, and plasmid pXL1546 enables COBF to be overexpressed in
Pseudomonas denitrificans.
[0174] The 2.2-kb EcoRI-XhoI fragment is excised and purified from
the 8.7-kb fragment. It is cloned at the EcoRI site of phage
M13mp19 to give plasmid pXL1405. An NdeI site is then introduced by
directed mutagenesis, as described above, at position 733 of this
fragment; in this manner, an NdeI site occurs exactly on the
presumed initiation codon of the cobF gene. The new plasmid thereby
obtained is designated pXL1406. A 1.5-kb NdeI-SphI-sphI fragment,
containing the cobF gene starting from its presumed initiation
codon, is purified after partial digestion with the appropriate
enzymes and ligated with the appropriate fragments of plasmid
pXL694 (120-bp EcoRI-NdeI fragment containing expression signals of
E. coli--see text--and 3.1-kb EcoRI-SphI fragment containing the
ampicillin resistance gene, the replication functions of the
plasmid and also the terminators of the rrnB operon of E. coli, as
described in the text). The plasmid thereby constructed is
designated pXL1496. pXL1546 is constructed as follows: the 2-kb
EcoRI-BamHI-BamHI fragment of pXL1496 is purified by partial
digestion with the appropriate enzymes; this fragment contains the
expression signals of E. coli, followed by the cobF gene and then
the 5' portion of the cobG gene, this portion itself being followed
by terminators of the rrnB operon of E. coli, as described in the
text. This fragment is cloned into the multihost plasmid pKT230
(Bagdasarian et al., 1981) described in FIG. 30. B, BamHI; C, ClaI;
E, EcoRI; H, HindIII; P. PstI; S, SstI, Sa, SalI; X, XhoI; Xb,
XbaI; Km', kanamycin resistance gene; Amp, ampicillin resistance
gene.
[0175] FIG. 27: Total proteins of strains SC510 Rif', SC510 Rif'
pKT230, SC510 Rif' pXL1546 analysed in 10% SDS-PAGE as described.
The bacteria were cultured for 4 days in PS4 medium, and lysates of
the total proteins were then made. Lane 1, SC510 Rif'; lane 2,
SC510 Rif' pKT230; lane 3, SC510 Rif' pXL1546. The molecular masses
of the molecular mass markers are indicated. The position to which
the COBF protein migrates is indicated.
[0176] FIG. 28: Total proteins of the strains E. coli B and E. coli
B pXL1496 analysed in 10% SDS-PAGE as described. Lane 1, E. coli
pXL1496 cultured in the absence of tryptophan; lane 2, E. coli
pXL1496 cultured under the same conditions in the presence of
tryptophan; lane 3, E. coli cultured in the absence of tryptophan;
lane 4, E. coli cultured under the same conditions in the presence
of tryptophan. The molecular masses of the markers are indicated.
The position of migration of the COBF protein is indicated.
[0177] FIG. 29: Construction of plasmids pXL525 and pXL368. Plasmid
pXL368 is constructed as follows: the 2.4-kb EcoRV-ClaI fragment
(containing the cobA and cobE genes) is purified from plasmid
pXL556, thereby enabling this fragment to be obtained with a BamHI
site and an XbaI site at the ends; this fragment is cloned into
pXL203 at the BamHI and XbaI sites.
[0178] For the construction of pXL525, an XbaI linker is added at
the EcoRI site situated at the right-hand end of the 8.7-kb EcoRI
fragment; this 8.7-kb EcoRI-XbaI fragment is then cocloned with the
2.4-kb EcoRI-XbaI fragment originating from pXL556 and containing
cobA and cobE.
[0179] The restriction sites which are shown in brackets correspond
to sites which have disappeared after treatment with the large
fragment of E. coli DNA polymerase I. 1, PstI-SstI fragment of
RSF1010 (De Graff et al., 1978); 2, PstI-BamHI fragment of pACYC177
(Bagdasarian et al., 1981); ori, origin of replication; nic,
relaxation site; mob, locus essential for mobilisation; Km',
kanamycin resistance gene (Bagdasarian et al., 1981); B, BamHI; C,
ClaI; E, EcoRI; H, HindIII; P, PstI; S. SstI; Sa, SalI; X, XhoI;
Xb, XbaI; tet, tetracycline resistance gene; Amp' and Amp,
ampicillin resistance gene.
[0180] FIG. 30: Plasmids of the incompatibility group Q having a
broad host range in Gram-negative bacteria. These plasmids are
described in a previous publication (Cameron et al., 1989) and are
used in the present invention.
[0181] 1, PstI-SstI fragment of RSF1010 (De Graff et al., 1978); 2,
PstI-BamHI fragment of pACYC177 (Bagdasarian et al., 1981); 3,
BamHI-SstI fragment containing the lactose operon of E. coli
without promoter, operator, translation initiation site and the
first 8 non-essential codons of lacZ (Casadaban et al., 1983); 4,
Sau3AI fragment of Pseudomonas putida KT2440 (Bagdasarian et al.,
1981); ori, origin of replication; nic, relaxation site; Km',
kanamycin resistance gene; Sm', streptomycin resistance gene; mob,
locus essential for mobilisation (Bagdasarian et al., 1981); B,
BamHI; C, ClaI; E, EcoRI; H, HindIII; P, PstI; S, SstI; Sa, SalI;
X, XhoI; Xb, XbaI.
[0182] FIG. 31: Retention time of different corrinoid standards (1
mg/standard) on the separation system described in Example 7. The
column used is a Nucleosil C-18 column (Macherey-Nagel). Against
each absorbance peak, a number is shown corresponding to the
corrinoid described below. The retention time is shown as abscissa
and the absorbance at 371 nm appears as ordinate.
[0183] 1, cobyrinic acid; 2, cobyrinic acid a-amide; 3, cobyrinic
acid g-amide; 4, cobyrinic acid a,g-diamide; 5, cobyrinic acid
c-amide; 6, cobyrinic acid c,g-diamide; 7, cobyrinic acid
a,c-diamide; 8, cobyrinic acid triamide; 9, cobyrinic acid
tetraamide; 10, cobyrinic acid pentaamide; 11, cobyric acid; 12,
GDP-cobinamide; 13, cobinamide phosphate; 14, cobinamide; 15,
cyanocobalamin 5'-phosphate; 16, cyanocobalamin.
[0184] FIG. 32: Nucleotide sequence of both strands of the 4748-bp
SalI-SalI-SalI-SalI-SalI-BqlI fragment of Pseudomonas
denitrificans. The strand situated at the top is to be read from 5'
to 3' in the left-to-right direction which corresponds to the
left-to-right orientation of the fragment of the restriction map
presented in FIG. 6.
[0185] FIG. 33: Nucleotide sequence of both strands of the 3855-bp
SstI-SstI-BamHI fragment of Pseudomonas denitrificans. The strand
situated at the top is to be read from 5' to 3' in the
left-to-right direction which corresponds to the left-to-right
orientation of the fragment of the restriction map presented in
FIG. 6.
[0186] FIG. 34: Analysis of the probabilities of the coding frames
on the basis of codon preference using the programme of Staden and
MacLachlan (1982) on the six reading frames of the 4748-bp
SalI-SalI-SalI-SalI-SalI-BglI fragment. For the frames belonging to
the same coding strand, the most probable frame corresponds to that
in which a dotted line, not interrupted by stop codons, is placed
under the probability line for this frame. 4a. Analysis of the
sequence corresponding to nucleotides 200 to 800. This analysis
enables open reading frame 14 to be identified. It begins at the
ATG at position 660 and ends at the TGA at position 379. 4b.
Analysis of the sequence corresponding to nucleotides 800 to 1500.
This analysis enables open reading frame 15 to be identified. It
begins at the GTG at position 925 and ends at the TAA at position
1440. 4c. Analysis of the sequence corresponding to nucleotides
1450 to 2600. This analysis enables open reading frame 16 to be
identified. It begins at the ATG at position 1512 and ends at the
TGA at position 2510. 4d. Analysis of the sequence corresponding to
nucleotides 2500 to 4650. This analysis enables open reading frame
17 to be identified. It begins at the GTG at position 2616 and ends
at the TGA at position 4511.
[0187] FIG. 35: Analysis of the probabilities of the coding frames
on the basis of codon preference using the programme of Staden and
MacLachlan (1982) on the six reading frames of the 3855-bp
SstI-SstI-BamHI fragment. For the frames belonging to the same
coding strand, the most probable frame corresponds to that in which
a dotted line, not interrupted by stop codons, is placed under the
probability line for this frame. 5a. Analysis of the sequence
corresponding to nucleotides 1 to 905. This analysis enables open
reading frame 18 to be identified. It begins at the ATG at position
809 and ends at the TGA at position 108. 5b. Analysis of the
sequence corresponding to nucleotides 955 to 2105. This analysis
enables open reading frame 19 to be identified. It begins at the
ATG at position 1971 and ends at the TGA at position 1063. 5c.
Analysis of the sequence corresponding to nucleotides 2000 to 3300.
This analysis enables open reading frame 20 to be identified. It
begins at the ATG at position 2099 and ends at the TAG at position
3115. 5d. Analysis of the sequence corresponding to nucleotides
3250 to 3855. This analysis enables open reading frame 21 to be
identified. It begins at the ATG at position 3344 and ends at the
TGA at position 3757.
[0188] FIG. 36: Construction of plasmids pXL233, pXL843 and pXL1558
from pXL154.
[0189] The plasmids are constructed in the following manner. The
3.5-kb EcoRI fragment containing the truncated cobs gene and the
sequence upstream is excised from pXL154, then purified and cloned
to the EcoRI site of pKT230. The plasmid thereby constructed is
designated pXL233. The 3.5-kb EcoRI-XhoI-XhoI fragment containing
the cobT gene and the sequence downstream is excised and purified
from pXL154 by partial digestions. The 4.3-kb EcoRI-EcoRI-EcoRI
fragment containing the cobs gene and the sequence upstream is
excised and purified from pXL154 and then ligated to the above
3.5-kb fragment. The approximately 8-kb EcoRI-XhoI fragment thereby
attained is cloned into the EcoRI and SalI sites of pXL59 to
generate plasmid pXL843. Plasmid pXL1558 is constructed in the
following manner: the 12-kb HindIII-HindIII fragment is excised
from pXL154 and purified, and the ends are then filled in with the
large fragment of E. coli DNA polymerase I. This insert is cloned
in pRK29O (Ditta et al., 1981) digested with EcoRI and then treated
with the large fragment of E. coli DNA polymerase I in order to
make the ends blunt. Restriction sites which are shown in brackets
correspond to sites which have disappeared during cloning. 1,
PstI-SstI fragment of RSF1010 (Degraff et al., 1978); 2, PstI-BamHI
fragment of pACYC177 (Bagdasarian et al., 1981); B, BamHI; C, ClaI;
E, EcoRI; H, HindIII; P, PstI; S, SstI; Sa, SalI; X, XhoI; Xb,
XbaI;
[0190] Tet tetracycline resistance gene; Km', kanamycin resistance
gene; Sm', streptomycin resistance gene.
[0191] FIG. 37: Study of the insertions of the transposon Tn5Sp
into the 12-kb HindIII-HindIII insert of pXL154.
[0192] The insertions of the transposon are mapped on the 12-kb
HindIII-HindIII insert cloned into pXL1558. The chromosomal
insertions into strain SC510 Rif' are boxed, that which is not is
introduced into strain SBL27 Rif'. A plus or minus sign is shown
under each insertion to indicate the Cob phenotype of the strain
having this insertion. Absence of complementation (or
complementation) of strain G2035 by plasmids pXL1558::Tn5Sp is
indicated by minus (or plus) signs below each insertion. The
inserts of the plasmids described in FIG. 36 are shown. The plus
(or minus) signs over these plasmids, and aligned with the
transposon insertions, show diagrammatically the complementation
(or absence) of the transposon-mutated strain by the plasmid. The
open reading frames deduced from the sequence are also given in
this figure (ORF14 to 17, as well as the corresponding cob genes
(cobS and cobT)). E: EcoRI; H: HindIII; X: XhoI.
[0193] FIG. 38: Construction of plasmids pXL1286, pXL1303, pXL1324,
pXL1490B and pXL1557 from pXL519. The position of the sequenced
fragment appears in the upper part of the figure above the
restriction map of the cluster; it is a 3.9-kb SstI-SstI-SstI-BamHI
fragment. The plasmids are constructed in the following manner. The
2-kb BqlII-EcoRI fragment containing the cobU gene and the sequence
downstream is excised from pXL519, then purified and cloned at the
BamHI and EcoRI sites of pKT230 to generate plasmid pXL1286. The
2.7-kb SstI-EcoRI fragment containing the truncated cobV gene, cobU
gene and the sequence downstream is excised on pXL519, then
purified and cloned at the SstI and EcoRI sites of pKT230 to
generate plasmid pxL1324. The 1.6-kb SstI-SstI fragment containing
the truncated cobV gene and the sequence upstream is excised from
pXL519, then purified and cloned at the SstI site of pKT230 to
generate plasmid pXL1303. The 3.85-kb SstI-SstI-BamHI fragment is
purified after total digestion of pXL519 with BamHI and partial
digestion with SstI. This fragment is then cloned at the BamHI and
SstI sites of pKT230 to generate pXL1490B. Plasmid pXL1557 is
constructed in the following manner: the 9-kb HindIII-BamHI
fragment is excised from pXL519 and purified, and the ends are then
filled in with the large fragment of E. coli DNA polymerase I. This
insert is cloned into pRK290 (Ditta et al., 1981) digested with
EcoRI and then treated with the large fragment of E. coli DNA
polymerase I to make the ends blunt. The restriction sites which
are shown in brackets correspond to sites which have disappeared
during cloning. 1, PstI-SstI fragment of RSF1010 (Degraff et al.,
1978); 2, PstI-BamHI fragment of pACYC177 (Bagdasarian et al.,
1981); B, BamHI;Bg, BqlII; C, ClaI; E, EcoRI; H, HindIII; P, PstI;
S, SstI; Sa, SalI; X, XhoI; Xb, XbaI; Tet', tetracycline resistance
gene; Km' kanamycin resistance gene; Sm', streptomycin resistance
gene.
[0194] FIG. 39: Study of the insertions of the transposon Tn5Sp
into the 9-kb HindIII-BamHI insert of pXL519. The insertions of the
transposon are mapped on the 9-kb HindIII-BamHI insert cloned into
pXL1557. The chromosomal insertions into strain SC510 Rif' are
boxed, those which are not-are introduced into strain SBL27 Rif'. A
plus or minus sign is shown under each insertion to indicate the
Cob phenotype of the strain having this insertion. Absence of
complementation (or complementation) of strain G2040 by plasmids
pXL1557::Tn5Sp is indicated by minus (or plus) signs below each
insertion. The inserts of the plasmids described in FIG. 6 are
shown. The plus (or minus) signs over these plasmids and aligned
with the transposon insertions, show diagrammatically the
complementation (or absence) of the transposon-mutated strain by
the plasmid. The open reading frames deduced from the sequence are
also given in this figure (ORF18 to 21), as well as the
corresponding cob genes (cobU and cobV).
[0195] FIG. 40: Coding sequences of each of the genes of the 4.8-kb
fragment, cobx, cobS and cobT, respectively, are indicated. The
sequence of the COBX, COBS and COBT proteins encoded by these
sequences appears under the respective coding sequences cobX, cobS
and cobT. The legend is identical to that for FIG. 15.
[0196] FIG. 41: Coding sequences of each of the genes of the 3.9-kb
fragment, cobU and cobV, respectively, are indicated. The sequence
of the COBU and COBV proteins encoded by these sequences appears
under the respective coding sequences cobU and cobV. The legend is
identical to that of FIG. 15.
[0197] FIG. 42: A. Total proteins of the strains E. coli BL21 pLysS
pET3b and E. coli BL21 pLysS pXL1937 analysed in 10% SDS-PAGE. Lane
1, BL21 pLyspET3b; lane 2, E. coli BL21 pLysS pXL1937. B. Total
proteins of the strains E. coli BL21, E. coli BL21 pXL1874 and E.
coli BL21 pXL1875 analysed in 10% SDS-PAGE. Lane 1, E. coli BL21;
lane 2, E. coli BL21 pXL1874; lane 3, E. coli BL21 pXL1875.
[0198] The molecular masses of the markers are indicated. The band
corresponding to the overexpressed protein is indicated by an
arrow.
[0199] FIG. 43: Nucleotide sequence of both strands of the 13144-bp
SstI-SstI-SstI-SstI-BqlII-BqlII fragment of Pseudomonas
denitrificans. The strand situated at the top is to be read from 5'
to 3' in left-to-right direction which corresponds to the
left-to-right orientation of the fragment of the restriction map
presented in FIG. 46.
[0200] FIG. 44: Restriction map of the 13144-bp
SstI-SstI-SstI-SstI-BqlII-SstI-BqlII fragment of Pseudomonas
denitrificans. The position or positions of restriction sites
occurring are indicated in increasing order of the cut number on
the fragment sequenced; the positions correspond to the sequence
presented in FIG. 43.
[0201] FIG. 45: Analysis of the probabilities of the coding frames
on the basis of codon preference using the programme of Staden and
MacLachlan (1982) on the six reading frames of the 13144-bp
SstI-SstI-SstI-SstI-BqlII-SstI-BqlII fragment of Pseudomonas
denitrificans. For the frames belonging to the same coding strand,
the most probable frame corresponds to that in which a dotted line,
not interrupted by stop codons, is placed under the probability
line for this frame.
[0202] 1. Sequence corresponding to nucleotides 1 to 2266. This
analysis enables open reading frame 22 to be identified. It begins
at the ATG at position 429 and ends at the TAG at position
1884.
[0203] 2. Sequence corresponding to nucleotides 2266 to 4000. This
analysis enables open reading frame 23 to be identified. It begins
at the ATG at position 3364 and ends at the TGA at position
3886.
[0204] 3. Sequence corresponding to nucleotides 3800 to 5000. This
analysis enables open reading frame 24 to be identified. It begins
at the ATG at position 3892 and ends at the TAG at position
4954.
[0205] 4. Sequence corresponding to nucleotides 5000 to 9000. This
analysis enables open reading frame 25 to be identified. It begins
at the ATG at position 5060 and ends at the TAG at position
8885.
[0206] 5. Sequence corresponding to nucleotides 9000 to 9700. This
analysis enables open reading frame 26 to be identified. It begins
at the ATG at position 9034 and ends at the TGA position 9676.
[0207] 6. Sequence corresponding to nucleotides 9600 to 13144. This
analysis enables open reading frames 27, 28, 29 and 30 to be
identified. They begin, respectively, at the ATGs at positions
9678, 10895, 11656 and 13059, and end at the stop codons at
positions 10101, 10304, 12181 and 12366. Open reading frames 28 and
30 occur on the strand complementary to the coding strand
corresponding to all the other open reading frames.
[0208] FIG. 46: 13.4-kb EcoRI-BqlII-EcoRI-BglII fragment, positions
of the insertions of transposons Tn5Sp into the 9.1-kb EcoRI
fragment, positions of the insertions of transposons Tn5 into the
insert of plasmid pXL189 as well as the inserts of the various
plasmids used during the experiments on complementation of strains
SC510 Rif'::Tn5Sp. The complementations of the mutants SC510
Rif'::Tn5Sp by the plasmids are indicated (+)--between 5% and 100%
of the level of the parent strain SC510 Rif'-, (.)--partial
complementation, between 0.5 and 5% of the level of SC510 Rif'-, or
(-)--absence of complementation, i.e. less than one thousand times
less than SC510 Rif'-, positioned immediately above the lines
showing diagrammatically the insert of the plasmids and aligned
with the insertion sites of the corresponding mutants. Below the
mapping of the insertions of transposons Tn5 into the insert of
plasmid pXL189, the complementation (+) or absence of
complementation (-) of these mutant plasmids for the Agrobacterium
tumefaciens mutants G632 and G633 is shown. On the right-hand part
of the figure, there is a table showing the complementation of the
mutants G622, G623 and G630 (Cameron et al., 1989) by different
plasmids; (+)--total complementation, 100% of the level of the
parent strain C58C9 Rif''', (.)--partial complementation, between
10 and 50% of the level of C58C9 Rif'-, or (-)--absence of
complementation.
[0209] The different plasmids whose insert is shown are constructed
as follows (the fragments are excised either from pXL156 or from
pXL157):
[0210] pXL618 corresponds to the 2.5-kb EcoRI-BamHI fragment cloned
at the same sites of pKT230 (Bagdasarian et al., 1981);
[0211] pXL593 corresponds to the 3.1-kb BamHI fragment cloned at
the BamHI site of pKT230 (Bagdasarian et al., 1981);
[0212] pXL623 corresponds to the 1.9-kb BamHI-XhoI fragment cloned
at the BamHI-SalI sites of pXL59 (Cameron et al., 1989);
[0213] pXL1909 corresponds to the 8.4-kb BamHI-BamHI-BamHI fragment
cloned at the BamHI of pKT230 (Bagdasarian et al., 1981);
[0214] pXL221 corresponds to the 1.6-kb EcoRI-ClaI fragment cloned
at the same sites of pXL59 (the ClaI site into which this fragment
is cloned is the ClaI site of the multisite of pXL59) (Cameron et
al., 1989);
[0215] pXL1908 and 1938 correspond to the same insert, 6.5-kb
XhoI-BamHI-BamHI fragment, to which XbaI linkers have been added;
this insert is cloned in both orientations at the XbaI site of
pXL435 (Cameron et al., 1989); an arrow positioned on the figure
indicates the position of the kanamycin resistance gene with
respect to the ends of the insert of the two plasmids;
[0216] pXL208 corresponds to the 5.2-kb BamHI fragment cloned at
the BamHI site of pKT230 (Bagdasarian et al., 1981);
[0217] pXL297 corresponds to the 9.1-kb EcoRI fragment cloned at
the EcoRI site of pKT230 (Bagdasarian et al., 1981).
[0218] The open reading frames (ORF) defined by the sequencing of
the fragment (ORF 22 to 30) are shown, as well as the corresponding
cob genes; an arrow indicates the polarity of the
transcription.
[0219] E, EcoRI; B, BamHI; Bg, BqlII; Cl, ClaI; Sau, Sau3AI; X,
XhoI;
[0220] FIG. 47: Coding sequences of each of the genes of the
13.4-kb fragment, cobQ, cobP and cobW, cobN and cobO, respectively,
are indicated. The sequences of the COBQ, COBP, COBW, COBN and COBO
proteins encoded by these sequences appear under their respective
coding sequence cobQ, cobP, cobW, cobN and cobO. The legend is
identical to that for FIG. 15.
[0221] FIG. 48: A-NH.sub.2-terminal sequence of SUMT of M. ivanovii
and sequence of the oligonucleotides 923, 946, 947; -, means that,
at this position, the residue could not be determined; for the
antisense oligonucleotide, the amino acids indicated below the
sequence correspond to the anticodons shown. B--Presentation of the
enzymatic amplification of a fragment internal to the structural
gene of SUMT of M. ivanovii with the oligonnucleotides 946 and
947.
[0222] FIG. 49: Construction of the recombinant replicative form
pG10. The 615-bp fragment obtained by amplification is digested
with HindIII and EcoRI and then purified as described. This
fragment is then ligated with the replicative form of phage M13mp19
digested with the same enzymes. The recombinant clone is found as
described in the text.
[0223] FIG. 50: Autoradiograph of a genomic DNA blot of M. ivanovii
digested with various enzymes, separated by agarose gel
electrophoresis and then transferred onto a nylon membrane as
described previously. The membrane is hybridised with the pG10
probe as described previously. 1, HindIII-BqlII; 2, KpnI-BclII; 3,
EcoRI-BqlII; 4, BqlII-PstI. The sizes of the different fragments
which hybridise with the probe are shown in kb.
[0224] FIG. 51: Nucleotide sequence of both strands of the 955-pb
fragment of M. ivanovii. The strand situated at the top is to be
read from 5' to 3' in the left-to-right direction.
[0225] FIG. 52: Coding sequence of the corA gene of M. ivanovii
obtained from the 955-bp sequence. The primary sequence of the CORA
protein is also shown. The amino acids are shown above their codon
and the stop codon is designated by a star. The main physical
properties of the CORA protein of M. ivanovii, namely the amino
acid composition, in number and in percentage, the molecular
weight, the index of polarity, the isoelectric point and the
optical density at 280 nm of a solution containing 1 mg/l of
purified protein. The hydrophobicity profile of the CORA protein of
M. ivanovii; this profile was obtained on the basis of the
programme of Hopp and Woods (1981). Positive values correspond to
regions of the protein which are hydrophilic. The position of the
amino acids is indicated as abscissa, and the value of the index of
hydrophilicity as ordinate; when this value is positive, this
indicates that the protein is hydrophilic in this region.
[0226] FIG. 53: Comparison of the primary sequences of the proteins
COBA of P. denitrificans and CORA of M. ivanovii. The proteins have
been aligned by means of the programme of Kanehisa (1984). =,
identical amino acids; -, homologous amino acids on the basis of
the criteria defined above (see FIGS. 22 and 23).
[0227] FIG. 54: Construction of plasmids pXL1832 and pXL1841. The
legends described, placed on the figure, enable the constructions
to be followed.
GENERAL TECHNIQUES OF CLONING, MOLECULAR BIOLOGY AND
BIOCHEMISTRY
[0228] The classical methods of molecular biology, such as
centrifugation of plasmid DNA in a caesium chloride/ethidium
bromide gradient, digestions with restriction enzymes, gel
electrophoresis, electroelution of DNA fragments from agarose gels,
transformation in E. coli, and the like, are described in the
literature (Maniatis et al., 1982, Ausubel et al., 1987).
[0229] Restriction enzymes were supplied by New-England Biolabs
(Biolabs), Bethesda Research Laboratories (BRL) or Amersham Ltd
(Amersham). Linker oligonucleotides were supplied by Biolabs.
[0230] For the ligations, the DNA fragments are separated according
to their size on 0.7% agarose or 8% acrylamide gels, purified by
electroelution, extracted with phenol, precipitated with ethanol
and then incubated in 50 mM Tris-HCl buffer pH 7.4, 10 mM MgCl, 10
mM DTT, 2 mM ATP, in the presence of phage T4 DNA ligase
(Biolabs).
[0231] If necessary, DNA fragments having protuberant 5' ends are
dephosphorylated by a treatment with calf intestinal alkaline
phosphatase (CIP, Pharmacia) at 37.degree. C. for 30 min in the
following buffer: 100 mM glycine, 1 mM MgCl.sub.2, 1 mM ZnCl.sub.2,
pH 10.5. The same technique is used for dephosphorylation of
protuberant or blunt 3' ends, but the treatment is for 15 min at
37.degree. C. and then 15 min at 56.degree. C. The enzyme is
inactivated by heating the reaction mixture to 68.degree. C. for 15
min in the presence of 1% SDS and 100 mM NaCl, followed by a
phenol/chloroform extraction and an ethanol precipitation.
[0232] Filling-in of protuberant 5' ends is performed with the
Klenow fragment of E. coli DNA polymerase I (Biolabs). The reaction
is performed at room temperature for 30 min in 50 mM Tris-HCl
buffer pH 7.2, 0.4 mM dNTPs, 10 mM MgSO.sub.4, 0.1 mM DTT, 50 mg/ml
BSA. Filling-in of protuberant 3' ends is performed in the presence
of phage T4 DNA polymerase (Biolabs) according to the
manufacturer's recommendations. Digestion of the protuberant ends
is performed by limited treatment with S1 nuclease (BRL) according
to the manufacturer's recommendations. Linker oligonucleotides are
added onto ends of DNA fragments as already described (Maniatis,
1982).
[0233] In vitro mutageneis with oligodeoxynucleotides is performed
according to the method developed by Taylor et al., 1985, using the
kit distributed by Amersham.
[0234] The ligated DNAs are used for transforming the strain
rendered competent: E. coli MC 1060 [D(lacIOPZYA)X74, galU, galK,
strA', hsdR] for plasmids or E. coli TG1[D(lac proA,B), supE, thi,
hsdD5/ F' traD36, proA.sup.+, B.sup.+, lacI.sup.q, lacZDM15) for
replicative forms of phages derived from bacteriophage M13.
[0235] Plasmid DNAs are purified according to the technique of
Birnboim and Doly, 1979. Minipreparations of plasmid DNA are made
according to the protocol of Klein et al., 1980. Preparations of
chromosomal DNA of Gram-negative bacteria are produced as already
described (Cameron et al., 1989).
[0236] Radioactive probes are prepared by nick translation
according to the method already detailed (Rigby et al., 1977).
Hybridisations between DNA sequences as well as the immobilisation
of nucleic acids on nitrocellulose membranes are performed as
already described (Cameron et al., 1989). In clonings for which
there is a small probability of finding the desired recombinant
clone, the latter are found after hybridisation on filters as
already described (Maniatis et al., 1982).
[0237] The nucleotide sequence of DNA fragments is determined by
the chain-termination method (Sanger et al., 1977). In the reaction
mixture, dGTP is replaced by 7-deaza-dGTP, in order to avoid
compression of bands during acrylamide gel electrophoresis caused
by the high percentage of GC in the DNA.
[0238] The culture media used for the bacteriological part have
already appeared (Maniatis et al., 1982). Culturing in PS4 medium
is carried out as already described (Cameron et al., 1989);
Pseudomonas denitrificans strains SC510 Rif' and G2 Rif' are
cultured in PS4 medium as follows: 250-ml Erlenmeyers containing
PS4 medium (25 ml), with, if necessary, the selective antibiotic
for the plasmid carried by each strain, are inoculated-with a 1/100
dilution of saturated preculture in L medium (Miller 1972), with,
if necessary, the selective antibiotic for the plasmid carried by
each strain; these cultures are incubated for 6 days at 30.degree.
C. and the musts are then analysed for their cobalamin content or
alternatively the enzymatic activity of some enzymes of the
pathway. Strains of Agrobacterium tumefaciens, Pseudomonas putida
and Rhizobium meliloti are cultured at 30.degree. C.; except where
otherwise stated, they are cultured in L medium.
[0239] Bacterial conjugations are carried out as already described
(Cameron et al., 1989).
[0240] Extracts of total proteins are produced as already described
(Ausubel et al., 1987).
[0241] Analytical electrophoresis (SDS-PAGE) of proteins in
acrylamide gel under denaturing conditions is performed as already
described (Ausubel et al., 1987). The PhastSystem apparatus
(Pharmacia) using Laemli's discontinuous-buffer system (Laemli,
1970) is also used; different gels are used in accordance with the
molecular weights of the proteins to be analysed as well as their
purity:
[0242] PhastGel gradient 8-25
[0243] PhastGel Homogeneous 12.5
[0244] Staining is performed either with Coomassie blue with the
aid of PhastGel Blue R (Pharmacia), or with silver nitrate using
the PhastGel silver Kit (Pharmacia) in accordance with the
manufacturer's instructions.
[0245] NH.sub.2-terminal sequences of the proteins are determined
by the Edman degradation technique, using an automated sequencer
(Applied Biosystems model 407A) coupled to an HPLC apparatus for
identification of the phenylthiohydantoin derivatives.
EXAMPLE 1
Isolation of DNA fragments of P. denitrificans Containing Cob
Genes
[0246] This example describes the isolation of DNA fragments of
Pseudomonas denitrificans carrying Cob genes. These fragments were
demonstrated by complementation experiments on Cob mutants of A.
tumefaciens and P. putida (Cameron et al., 1989).
[0247] These Cob mutants were obtained by mutagenesis with
N-methyl-N'-nitro-N-nitrosoguanidine according to the technique of
Miller (Miller et al., 1972), or by insertions of transposon Tn5.
In this manner, strains incapable of synthesising cobalamins were
demonstrated, and especially the Cob mutant G572 of P. putida and
the Cob mutants G159, G161, G164, G169, G171, G258, G609, G610,
G611, G612, G613, G614, G615, G616, G620, G622, G623, G630, G632,
G633, G634, G638, G642, G643, G2034, G2035, G2037, G2038, G2039,
G2040, G2041, G2042 and G2043 of A. tumefaciens.
[0248] At the same time, a library of genomic DNA of P.
denitrificans is produced in a mobilisable broad host range vector
pXL59, by digestion of 5 .mu.g of DNA in the presence of
restriction enzymes (Cameron et al., 1989).
[0249] By complementation, several plasmids could be isolated,
enabling the Cob mutants of P. putida and of A. tumefaciens to be
complemented. Among these, plasmids pXL151, pXL154, pXL156, pXL157
and pXL519 will be noted more especially.
[0250] These plasmids were isolated and DNA fragments could be
excised, purified and analysed by restriction. These fragments are
presented in FIGS. 6 and 44: a 5.4-kb ClaI-HindIII-HindIII-HindIII
fragment, an 8.7-kb EcoRI-EcoRI fragment, a 4.8-kb
SalI-SalI-SalI-SalI-SalI-BglI fragment, a 3.9-kb SstI-SstI-BamHI
fragment and a 13.4-kb EcoRI-BglII-EcoRI-BglII fragment.
EXAMPLE 2
Sequencing of the DNA Fragments Isolated
[0251] This example illustrates the sequencing of DNA fragments
carrying cob genes of Pseudomonas denitrificans SC510.
[0252] 2.1. Sequencing of a 5.4-kb ClaI-HindIII-HindIII-HindIII
Fragment.
[0253] This fragment is contained in plasmid pXL157 described in
Example 1. After excision, the subfragments of the 5.4-kb fragment
were cloned into phages M13mp18 or M13mp19 (Norrander et al., 1983)
or M13tg130 or M13tg131 (Kieny et al., 1983) in both orientations.
Deletions were then produced in vitro by the method of Henikoff
(1987). These deletions were then sequenced with the "universal
primer" as a synthetic primer of chain-termination reactions. The
overlap between these different deletions enabled the total
sequence, over both strands, of the 5.4-kb fragment to be
established (FIG. 7). This fragment comprises 5378 bp. In the
sequence described in FIG. 7, there are seen, before the ClaI site,
three restriction sites (PstI, SalI and XbaI) which have appeared
during the cloning of the fragment in question with a view to
sequencing in cloning multisites. When subsequent reference is
made, in the present invention, to the sequence of this
ClaI-HindIII-HindIII-HindIII fragment, this will be to the sequence
presented in FIG. 7 in which the first 22 bases do not correspond
to the DNA of Pseudomonas denitrificans (thus, all the positions of
restriction site or of beginning of open reading frame refer to the
sequence presented in FIG. 7).
[0254] 2.2. Nucleotide Sequence of an 8.7-kb EcoRI-EcoRI
Fragment.
[0255] This fragment is carried by pXL151 described in Example 1.
The EcoRI site as well as the adjacent 70 bp located to the right
of this fragment originate from pXL59, which is the vector used for
constructing pXL151 by cloning an Sau3AI fragment of Pseudomonas
denitrificans SC510. After excision, subfragments of the 8.7-kb
fragment were cloned into phages M13mp18 or M13mp19 (Norrander et
al., 1983) or M13tg130 or M13tg131 in both orientations (Kieny et
al., 1983). Deletions were then produced in vitro by the method of
Henikoff (1987). These deletions were then sequenced with the
"universal primer" as a synthetic primer of chain-termination
reactions. The overlap between these different deletions enabled
the total sequence, over both strands, of the 8.7-kb fragment to be
established (FIG. 8). This fragment comprises 8753 bp.
[0256] 2.3. Sequencing of a 4.8-kb SalI-SalI-SalI-SalI-SalI-BglI
Fragment.
[0257] This fragment is contained in plasmid pXL154 described in
Example 1. The protocol is identical to that used in Example 2.2.
The total sequence on both strands of the 4.8-kb fragment is
presented in FIG. 32. This fragment contains 4749 bp.
[0258] 2.4. Nucleotide Sequence of a 3.9-kb SstI-SstI-BamHI
Fragment.
[0259] This fragment is included in plasmid pXL519 described in
Example 1. The protocol is identical to that used in Example 2.2.
The total sequence on both strands of the 3.9-kb fragment is
presented in FIG. 33. This fragment contains 3855 bp.
[0260] 2.5. Nucleotide Sequence of a 13.4-kb
EcoRI-BglII-EcoRI-BglII Fragment.
[0261] This fragment is contained in plasmids pXL156 and pXL157
described in Example 1. The protocol used is identical to that of
Example 2.2. The sequence on both strands of the 13.15-kb fragment
is presented in FIG. 43. It corresponds to the total sequence of
the 13.4-kb fragment except for 250 bp, corresponding to an
EcoRI-SstI fragment, occurring at the left-hand end of the
fragment.
[0262] From these nucleotide sequences, restriction maps were
obtained for the enzymes which cut least frequently (FIGS. 6 and
44). The percentage of GC bases in Pseudomonas denitrificans SC150
DNA is relatively high (65.5%) and manifests itself in compression
on the sequencing gels. To avoid these problems, two approaches are
adopted:
[0263] i) the use of 7-deaza-dGTP instead of dGTP in the sequencing
reactions to decrease the secondary structures which form during
electrophoresis in the sequencing gel, and
[0264] ii) the sequencing of both strands.
EXAMPLE 3
Analysis of these Nucleotide Sequences Determination of the Open
Reading Frames
[0265] The nucleotide sequences of the 5.4-kb
ClaI-HindIII-HindIII-HindIII (FIG. 7), 8.7-kb EcoRI-EcoRI (FIG. 8),
4.8-kb SalI-SalI-SalI-SalI-SalI-BglI (FIG. 32), 3.9-kb
SstI-SstI-BamHI (FIG. 33) and 13.4-kb EcoRI-BglII-EcoRI-BglII (FIG.
43) fragments enable open reading frames to be defined. Since the
DNA in question contains a high percentage of GC, the open reading
frames are numerous in view of the low frequency of translation
stop codons. A study of the probability of the coding frames on the
basis of codon preference using the method of Staden and MacLachlan
(1982) is carried out. It characterises the open reading frames
which have the greatest probability of being coding relative to the
other frames of the same DNA strand, this probability being
dependent on the codon preference of genes already sequenced
originating from bacteria of the genus Pseudomonas. In this
manner:
[0266] 3.1. Five open reading frames are characterised for the
5.4-kb ClaI-HindIII-HindIII-HindIII fragment. They are designated
frames 1 to 5, and their positions in the sequence of the 5.4-kb
fragment are as follows (in the 5'-3' sequence from the ClaI site
to the HindIII sites): Table: Probable open reading frames of the
5.4-kb ClaI-HindIII-HindIII-HindIII fragment. The positions in the
sequence correspond to the positions in the sequence described in
FIG. 7; the coding strand is the 5'-3' strand corresponding to the
upper strand in this figure. TABLE-US-00001 Translation Molecular
weight Frame initiation Stop in kD of the number codon codon
encoded protein 1 549 1011 15.5 2 1141 1980 29.2 3 1980 3282 5.7 4
3281 4280 35.0 5 4284 5253 34.1
[0267] The representations of the probabilities that these open
reading frames are coding frames, with those observed on the other
frames (5 in total) in parallel, are given in FIG. 9. These five
frames are encoded by the same strand. Four of them (open reading
frames 1 to 4) display the characteristics of coding frames in
translational coupling (Normak et al., 1983), namely, the
translation initiation codon of frame x+1 overlaps the translation
termination codon of frame x, or else these codons are very
close.
[0268] 3.2. Eight frames are characterised for the 8.7-kb
EcoRI-EcoRI fragment. They are designated frames 6 to 13 and their
positions in the sequence of the 8.7-kb fragment are given in the
table below. Table: Probable open reading frames of the 8.7-kb
EcoRI fragment. The positions in the sequence correspond to the
positions in the sequence described in FIG. 8; in this figure, the
coding strand is the upper strand. TABLE-US-00002 Molecular weight
Translation Initiation Stop in kD of the frame number codon codon
encoded protein 6 736 1519 28.9 7 1620 2997 46.7 8 3002 3632 22.0 9
3631 4366 25.8 10 4365 5127 27.1 11 5126 5867 26.8 12 5862 7101
42.9 13 7172 7931 26.8
[0269] The representations of the probabilities of these open
reading frames, with those observed on the other frames (6 frames
in total) in parallel, are given in FIG. 10. With the exception of
frame 11, these eight frames are encoded by the same strand. Four
of them (from 7 to 10) display the characteristics of coding frames
in translational coupling (Normark et al., 1983), namely, the
translation initiation codon of frame x+1 overlaps the translation
termination codon of frame x, or else these codons are very
close.
[0270] 3.3. Four open reading frames are characterised for the
4.8-kb SalI-SalI-SalI-SalI-SalI-BalI fragment. They are designated
phases 14 to 17 and their positions in the sequence of the 4.8-kb
fragment are as follows (in the 5'-3' sequence from the SalI sites
to the BglI site): Table: Probable open reading frames of the
4.8-kb SalI-SalI-SalI-SalI-SalI-BglI fragment. The positions in the
sequence corresponds to the positions described in FIG. 32, where
the upper strand is given in its 5'- 3' orientation. Frames 15, 16
and 17 are encoded by the upper strand, in contrast to frame 14.
TABLE-US-00003 Translation Molecular weight Frame initiation Stop
in kD of the number codon codon encoded protein 14 660 379 10286 15
925 1440 18941 16 1512 2510 36983 17 2616 4511 70335
[0271] The representations of the probabilities that these open
reading frames are coding, with those observed on the other frames
(4 in total) in parallel, are given in FIG. 34. Frames 15, 16 and
17 are encoded by the same strand, frame 14 by the complementary
strand.
[0272] 3.4. Four frames are characterised for the 3.9-kb
SstI-SstI-BamHI fragment. They are designated 18 to 21 and their
positions in the sequence of the 3.9-kb fragment are given in the
table below. Table: Probable open reading frames of the 3.9-kb
SstI-SstI-BamHI fragment. The positions in the sequence correspond
to the positions described in FIG. 33, where the polarity of the
upper strand is 5'- 3'. Frames 18 and 19 are encoded by the lower
strand, in contrast to frames 20 and 21. TABLE-US-00004 Translation
Molecular weight Frame initiation Stop in kD of the number codon
codon encoded protein 18 809 108 25148 19 1971 1063 30662 20 2099
3115 34682 21 3344 3757 14802
[0273] The representations of the probabilities that these open
reading frames are coding, with those observed on the other frames
(4 in total) in parallel, are given in FIG. 35. Frames 19 and 20
are transcribed in a differing manner.
[0274] 3.5. Nine open reading frames are characterised for the
13.1-kb EcoRI-BglII-EcoRI-BglII fragment. They are designated
frames 22 to 30 and their positions in the sequence of the 13.1-kb
fragment are as follows (in the 5'-3' sequence from the EcoRI site
to the BglII site): Table: Probable open reading frames of the
13.1-kb EcoRI-BglII-EcoRI-BgIII fragment. The positions in the
sequence correspond to the positions described in FIG. 43, where
the upper strand is given in its 5'- 3' orientation. The frames 22,
23, 24, 25, 26, 27 and 29 are encoded by the upper strand, in
contrast to the frames 28 and 30. TABLE-US-00005 Molecular weight
Translation Initiation Stop in kD of the frame number codon codon
encoded protein 22 429 1884 51982 23 3364 3886 19442 24 3892 4954
38121 25 5060 8885 138055 26 9034 9676 24027 27 9678 10101 14990 28
10835 10306 21057 29 11656 12181 19183 30 13059 12368 24321
[0275] The representations of the probabilities that open reading
frames 22, 23, 24, 25 and 26 are coding, with those observed on the
other frames (5 in total) in parallel, are given in FIG. 45. These
5 frames are encoded by the same strand.
EXAMPLE 4
Genetic Studies on the DNA Fragments Carrying cob Genes
[0276] This example shows the relationship which exists between the
different open reading frames identified above and the genes
involved in the biosynthesis of cobalamins and/or cobamides carried
by these same fragments. These genes are identified by a genetic
study as described below.
[0277] 4.1--Genetic Study of the 5.4-kb Fragment
[0278] Plasmid pXL723 is plasmid pRK290 (Ditta et al., 1980)
containing the 2264-bp EcoRI-HindIII fragment corresponding to the
right-hand portion of the fragment studied, cloned at the EcoRI
site of pRK290 (FIG. 11). The construction of the other plasmids
used in this study (pXL302, pXL1397, pXL545, pXL545.OMEGA., pXL556
and pXL1500) is described in the legend to FIGS. 11 and 12.
[0279] Insertions were obtained in plasmid pXL723 using the
technique of de Bruijn and Lupski, 1984. Insertions of transposon
Tn5 into plasmid pXL723 were selected and then mapped in the 5.4-kb
fragment (FIG. 12). pXL723 complements the Cob mutant G572 of
Pseudomonas putida and the Cob mutant G634 of Agrobacterium
tumefaciens. These insertions are classified in two groups of
inactivating insertions: either those which no longer permit
complementation of the Cob mutant G572, or those which abolish the
complementation of the Cob mutant G634 (FIG. 12). Insertions which
inactivate the complementation of mutant G572 are mapped in open
reading frame 4 (these are insertions 15, 27, 68, 81 and 97); open
reading frame 4 hence corresponds to a cob gene. The latter is
designated cobC. Insertions which inactivate the complementation of
mutant G634 are mapped in frame 5 (these are insertions 66 and 107,
FIG. 12); open reading frame 5 hence corresponds to a cob gene. The
latter is designated cobD. Moreover, insertions with a transposon
Tn5Sp' were produced. Transposon Tn5Sp' was constructed in the
laboratory by cloning a BamHI cassette, containing the
spectinomycin resistance gene originating from plasmid pHP45.OMEGA.
(Prentki and Krisch, 1984), at the BamHI site of transposon Tn5
(Jorgensen et al., 1979). These insertions were made in the
chromosome of Pseudonomas denitrificans strain SBL27 Rif'. Strain
SBL27 is a strain of Pseudomonas denitrificans from which SC510 is
derived by several mutageneses. SBL27 produces 10-fold less
cobalamins than SC510 on PS4 medium. Of 10,000 clones of strain
SBL27 Rif' each carrying an insertion of transposon, more than 30
had lost the capacity to synthesise cobalamins. Some of these
clones possessed an insertion in the fragment studied in this
example. These insertions were mapped by restriction analysis
according to Southern's method (Southern, 1975). The sites of
insertions of the transposon in these different mutants are given
in FIG. 12. One of these insertions, number 2639, occurs in the
cobC gene; this insertion is complemented by plasmid pXL302, which
carries a fragment containing the cobC gene (FIG. 12). Two
insertions, designated 2636 and 2638, are in open reading frame 3.
These mutants are blocked in the biosynthesis of cobalamins, and
they are complemented by plasmid pXL1397 which contains only open
reading frame 3, but non-complemented by plasmid pXL302 which
contains the cobC and cobD genes (FIG. 12). Both of these
insertions are hence in another gene. With open reading frame 3, we
associate the cobB gene. An insertion 2933 is placed in open
reading frame 2; it is complemented by plasmid pXL1500 which
contains open reading frame 2; this insertion is non-complemented
by plasmid pXL1397, which contains the cobB gene and which
complements the two insertions in cobB. In this instance, the
insertion is hence in another gene; with open reading frame 2, we
associate a gene designated cobA.
[0280] A kanamycin resistance cassette originating from plasmid
pUC4K (Barany et al., 1985) was introduced at the NotI site of the
ClaI (position 0 in the sequence)-RsaI (position 1686 in the
sequence) fragment cloned into a plasmid pUC8 (Viera and Messing,
1982); the NotI site in question is located at position 771 in
frame 1 (see the sequence in FIG. 7); two insertions were adopted,
each corresponding to a different orientation of the resistance
cassette. These fragments, each carrying an insertion of the
resistance cassette, were cloned into plasmid pRK404 (Ditta and
al.) to give plasmids pXL1630 and 1631. These plasmids were
introduced by conjugative transfer into Pseudomonas denitrificans
strain SC510 Rif', and then, by a series of cultures/dilutions in
the absence of the selective antibiotic for the plasmid
(tetracycline), double recombinants which had exchanged the plasmid
fragment with the chromosomal fragment and had lost the plasmid
were found. Two strains were thereby characterised:
[0281] i) one is designated SC510:1631 Rif'; in this strain, the
kanamycin resistance cassette is inserted in the chromosome at the
NotI site (occurring in frame 1); the polarities of the
transcriptions of the kanamycin resistance gene and that of open
frame 1 are opposite,
[0282] ii) the other insertion is designated SC510:1630 Rif'; the
resistance cassette is inserted at the same site, but the
transcription of the resistance gene has the same polarity as that
of the complete open reading frame 1.
[0283] These two strains both have a rate of synthesis of
cobalamins at least 100-fold lower than that of SC510.
[0284] Plasmid pXL545.OMEGA. corresponds to plasmid pXL545 into
which the spectinomycin resistance cassette of plasmid pHP45.OMEGA.
has been inserted at the BamHI site. This plasmid (FIG. 12), which
contains the 814-bp ClaI-HindIII fragment (in which only open
reading frame 1 is complete) complements only mutant SC510:1630
Rif'. This suffices to define a new gene, since this mutant is
complemented by a plasmid which only contains the complete open
reading frame 1. Open reading frame 1 corresponds to a gene of the
pathway of biosynthesis of cobalamins and/or cobamides. This gene
is designated cobE. The absence of complementation of mutant
SC510:1631 Rif' by plasmid pXL545.OMEGA. is possibly due to the
fact that the cobA, cobB, cobC, cobD and cobE genes, or a part of
them, belong to the same operon, and that the insertion in cobE
which preserves a transcription in the direction of transcription
of the operon may be complemented only by trans expression of the
cobE gene. In contrast, mutant SC510:1631 Rif', for its part, can
be complemented only by a plasmid which permits trans expression of
the cobA to cobE genes.
[0285] The 5.4-kb ClaI-HindIII-HindIII-HindIII fragment hence
contains five cob genes designated cobA, cobB, cobC, cobD and
cobE.
[0286] 4.2--Genetic Studies of the 8.7-kb Fragment
[0287] Plasmid pXL367 is pRK290 (Ditta et al., 1980) containing the
8.7-kb EcoRI fragment cloned at the EcoRI site (FIG. 13).
[0288] Insertions of transposon Tn5 into plasmid pXL367 were
selected using the technique already described (de Bruijn and
Lupski, 1984). The insertions in the 8.7-kb fragment were mapped.
In the same manner, insertions of transposon Tn3lacZ were obtained
according to the method already described (Stachel et al., 1985)
and mapped. 29 insertions of transposon Tn5 and 13 insertions of
transposon Tn3lacZ were thus mapped. The precise position of these
insertions in the 8.7-kb fragment is given in FIG. 14. Plasmids
each carrying a single insertion in the 8.7-kb fragment were
introduced by conjugative transfers into the Cob mutants of
Agrobacterium tumefaciens G164, G609, G610, G611, G612, G613, G614,
G615, G616, G620 and G638. These mutants are all complemented by
pXL367. Insertions which no longer permit the complementation of
the different mutants were sought. They correspond to an insertion
in the gene responsible for complementation of the corresponding
mutant. The results of the complementations of the different
mutants for their character of production of cobalamins (Cob
phenotype) are given in FIG. 14. If the recombinant mutant produces
less than threefold less cobalamins than are produced by the same
mutant with plasmid pXL367, it is considered to be
non-complemented. Of the mutants studied, G164, G609, G610, G611,
G612, G613, G614, G615, G616, G620 and G638, eight different
classes of inactivating insertions of transposons leading to a
mutated phenotype are observed. These classes characterise
insertions by the absence of complementation of one or more mutants
by plasmids pXL367 carrying these same insertions. Each class hence
corresponds to a mutated gene. It is observed that the insertions
belonging to the same class are positioned beside one another.
Eight classes of insertions are thus observed, which enable eight
genes to be defined. Each class of insertions defines a minimum
fragment which must be contained in the corresponding gene. FIG. 14
demonstrates a perfect correlation between the regions bounded by
each class, in respect of the restriction map, and the open reading
frames described above (Example 3). It is found, in effect, that,
for each class of insertions, the transposons are always inserted
in a portion of the 8.7-kb fragment which is contained in a single
open reading frame. Each class of insertions is hence associated
with one, and only one, open reading frame. The open reading frames
indicated above hence each code for a protein involved in the
pathway of biosynthesis of cobalamins and/or cobamides. The open
reading frames each correspond to genes involved in the
biosynthesis of cobalamins and/or cobamides. These open reading
frames are referred to as cobF, cobG, cobH, cobI, cobJ, cobK, cobL
and cobM for frames 6 to 13, respectively. The position of these
genes relative to the restriction map is shown in FIG. 14.
[0289] 4.3--Genetic Study of the 4.8-kb Fragment
[0290] Plasmid pXL1558 is plasmid pRK290 (Ditta et al., 1980)
containing the 12-kb HindIII-HindIII fragment of pXL154 (Cameron et
al., 1989) cloned at the EcoRI site of pRK290 (FIG. 36). The
construction of the other plasmids used in this study (pXL233 and
pXL843) is described in the legend to FIG. 36.
[0291] Tn5Sp insertions were obtained in plasmid pXL1558. First, a
strain containing a transposon Tn5Sp was constructed; this was done
by transforming strain C2110 (Stachel et al., 1985) using plasmid
pRK2013Tn5Sp (Blanche et al., 1989); since it has a ColEl origin of
replication, plasmid pRK2013Tn5Sp does not replicate in strain
C2110, which is polA-. The colonies obtained after transformation,
which are resistant to spectinomycin, hence have transposon Tn5Sp
in their chromosome; a colony is then reisolated, after which the
insertion of the transposon is then transduced using phage P1 in
strain MC1060 as described previously (Miller, 1972). Strain MC1060
Tn5Sp is transformed with plasmid pXL1558; plasmid pXL1558 is then
mobilised by conjugation using pRK2013 in C600 Rif'. Conjugants
resistant to tetracycline (for plasmid pXL1558) and to
spectinomycin (for the transposon) are then selected. Such
conjugants must contain plasmid pXL1558 in which transposon Tn5Sp
has been inserted. Insertions carried in plasmid pXL1558, and more
precisely in the 12-kb fragment, are then mapped by restriction
digestion; 23 insertions are thereby obtained and mapped on the
12-kb fragment; the position of these different insertions in the
fragment is presented in FIG. 37. These 23 insertions were
introduced into the chromosome of strain SC510 Rif' after
conjugative transfer of p-XL1558::Tn5Sp, followed by introduction
of plasmid pR751. Plasmid pR751 is a trimethoprim-resistant plasmid
of the same incompatibility group as pXL1558 (incP, Thomas and
Smith, 1987). By culturing non-selectively for pXL1558 (absence of
tetracycline) but selectively for pR751 and the transposon
(presence of trimethoprim and of spectinomycin), the exchange of
the mutation carried by pXL1558::Tn5Sp with the chromosome and also
the segregation of pXL1558 are obtained by the technique of marker
exchange by double homologous recombination, as already described
(Schell et al., 1988). The strains thereby selected carry the
transposon in their chromosome. The double homologous recombination
is verified by Southern's method (Southern, 1975). In this way, 23
SC510 Rif'::Tn5Sp strains in the 12-kb fragment were
identified.
[0292] Furthermore, another Tn5Sp insertion obtained by random
mutagenesis of transposon Tn5Sp in strain SBL27 Rif' (Blanche et
al., 1989) was mapped on the 12-kb fragment by restriction analysis
according to Southern's method (Southern, 1975), see FIG. 37; this
strain is designated SBL27 Rif'::Tn5Sp 1480.
[0293] The level of cobalamin synthesis is determined for these 24
strains cultured in PS4 medium according to the protocol already
described (Cameron et al., 1989), and the Cob-phenotype is assigned
to strains producing at least 1000 (or 100) times less vitamin
B.sub.12 than the parent strain SC510 Rif' (or SBL27 Rif'), FIG.
37. It is thus observed that 6 of these chromosomal insertions lead
to a Cob-phenotype in P. denitrificans; they are the insertions
31.1, 41.3, 45, 55, 22.1 and 1480.
[0294] Three plasmids pxL233, pXL837 (Cameron et al.) and pXL843
are introduced by conjugative transfers into three strains
possessing the Cob-phenotype, namely SC510 Rif'::Tn5Sp 31.1, SC510
Rif'::Tn5Sp 45 and SBL27 Rif'::Tn5Sp 1480. These three mutants each
have a different complementation profile for cobalamine synthesis.
In effect, SBL27 Rif'::Tn5Sp 1480 is complemented by pXL837 and
pXL843 but not by pXL233; the mutant SC510 Rif'::Tn5Sp 45 is
complemented only by pXL843; the mutant SC510 Rif'::Tn5Sp 31.1 is
complemented by plasmid pXL843 and also by plasmid pXL233 (see FIG.
37). The data presented hence enable it to be concluded, on the
basis of the results of the complementations of the three mutants,
that the three mutants are different and that, for each of them,
transposon Tn5Sp has been inserted into a different cob gene.
[0295] Furthermore, plasmids pXL1558::Tn5Sp 41.3, pXL1558::Tn5Sp 45
and pXL1558::Tn5Sp 22.1 are introduced by conjugative transfers
into strain G2035 (Cameron et al., 1989), and do not complement it.
Plasmid pXL1558 complements this mutant, in contrast to plasmid
pXL1558::Tn5Sp 31.1.
[0296] The phenotype and complementation data enable us to define 3
classes of insertions; each of these classes is represented by the
following insertions : 31.1, class 1; 45, 41.3, 55 and 22.1, class
2; 1480, class 3.
[0297] For each class of insertions, the transposons are always
inserted in a portion of the 4.8-kb fragment which is contained in
a single open reading frame (ORF14, ORF16 and ORF17, as defined in
Example 3). Each class of insertions is associated with a single
open reading frame. The open reading frames indicated above hence
code for a protein involved in the pathway of biosynthesis of
cobalamins and/or cobinamides. These open reading frames are
referred to as cobX, cobS and cobT for frames 14, 16 and 17. The
position of these genes relative to the restriction map is shown in
FIG. 37. Open reading frame 15 is not a gene involved in the
biosynthesis of coenzyme B.sub.12.
[0298] 4.4--Genetic Studies of the 3.9-kb Fragment
[0299] Plasmid pXL1557 is plasmid pRK290 (Ditta et al., 1980)
containing the 9-kb HindIII-BamHI fragment of pXL519 cloned at the
EcoRI site of pRK290 (FIG. 38). The construction of the other
plasmids used in this study (pXL1286, pXL1303, pXL1324) is
described in the legend to FIG. 38. Moreover, the 2-kb BglII-XhoI
fragment (positions in the sequence presented in FIG. 33: 251 and
2234) of plasmid pXL519 is cloned at the BamHI-SalI sites of
plasmid pXL435 (Cameron et al) to generate plasmid pXL699.
[0300] Tn5Sp insertions were obtained in plasmid pXL1557 according
to the technique described in Example 4.3. Insertions of transposon
Tn5Sp into plasmid pXL1557, then designated pXL1557::Tn5Sp, were
selected. Those which are mapped in the 9-kb fragment (FIG. 39)
were introduced into the chromosome of strain SC510 Rif' after
conjugative transfer of pXL1557::Tn5Sp and marker exchange by
double homologous recombination as described in 4.3.
[0301] The double homologous recombination is verified by
Southern's method (Southern, 1975). In this way, 20 SC510
Rif'::Tn5Sp strains were identified.
[0302] Furthermore, two other Tn5Sp insertions obtained by random
mutagenesis of transposon Tn5Sp in strain SBL27 Rif' (Blanche et
al., 1989) were mapped on the 9-kb fragment by restriction analysis
according to Southern's method (Southern, 1975), see the insertions
1003 and 1147 in FIG. 39.
[0303] The level of cobalamin synthesis is determined for these 22
strains cultured in PS4 medium according to the protocol already
described (Cameron et al., 1989), and the Cob-phenotype is assigned
to strains producing 1000 (or 100) times less vitamin B.sub.12 than
the parent strain SC510 Rif' (or SBL27 Rif'), FIG. 39. Only the 4
insertions 1, 1003, 23 and 1147 result in a Cob-phenotype in P.
denitrificans.
[0304] Four plasmids pXL699, pXL1286, pXL1303 and pXL1324 are
introduced by conjugative transfers into the four strains
possessing the cob-phenotype, namely SC510 Rif'::Tn5Sp 1, SBL27
Rif'::Tn5Sp 1003, SC510 Rif'::Tn5Sp 23 and SBL27 Rif'::Tn5Sp 1147.
Plasmid pXL699 complements the first two mutants (SC510 Rif'::Tn5Sp
1, SBL27 Rif'::Tn5Sp 1003), but plasmid pXL1303 does not complement
them, plasmid pXL1324 complements the other two mutants (SC510
Rif'::Tn5Sp 23 and SBL27 Rif'::Tn5Sp 1147) but plasmid pXL1286 does
not complement them.
[0305] Furthermore, plasmid pXL1557::Tn5Sp 1, is introduced by
conjugative transfer into strain G2040, and does not complement it,
whereas plasmids pXL1557, pXL1557::Tn5Sp 6A, pXL1557::Tn5Sp 54,
pXL1557::Tn5Sp 48, pXL1557::Tn5Sp 21, pXL1557::Tn5Sp 8,
pXL1557::Tn5Sp 23, also introduced by conjugative transfers,
complement it (see FIG. 39).
[0306] The phenotype and complementation data enable 2 classes of
insertions to be defined. For each class of insertions, the
transposons are always inserted in a portion of the 3.9-kb fragment
which is contained in a single open reading frame (ORF19 and ORF20
as defined in Example 3).
[0307] Each class of insertions is associated with a single open
reading frame. The open reading frames indicated above code for a
protein involved in the pathway of biosynthesis of cobalamins
and/or cobinamides. These open reading frames are referred to as
cobV and cobU for frames 19 and 20. Frames 18 and 21 are not genes
involved in the pathway of biosynthesis of coenzyme B.sub.12. The
position of these genes relative to the restriction map is shown in
FIG. 39. The insertions 48, 21 and 8 are mapped between the cobU
and cobV genes.
[0308] 4.5--Genetic Studies of the 13.4-kb Fragment
[0309] 4.5.1. Studies on the 4327-bp EcoRI-BqII Fragment.
[0310] Plasmid pXL189 (Cameron et al., 1989), which contains at
least one cob gene, carries a 3.1-kb insert which, except for 300
bp, corresponds to a 4.26-kb EcoRI-ClaI fragment (see FIG. 45).
pXL189 was subjected to a mutagenesis with transposon Tn5, as
described previously (De Bruijn and Lupski (1984)). 13 insertions
were thereby mapped in the insert of pXL189, as presented in FIG.
46. These 13 mutant plasmids, as well as pXL189, were conjugated in
two A. tumefaciens mutants, G632 and G633, which are mutants
complemented by pXL189 (Cameron et al., 1989). Only the insertion
58 proved to be an inactivating insertion. This result shows that
the two mutants G632 and G633 correspond to a mutation in the same
gene, and that, moreover, the only gene of P. denitrificans which
could be responsible for their complementation corresponds to open
reading frame 26 (see FIG. 46), since insertion 58 is mapped in
this open reading frame; in addition, it is the only insertion of
the 13 which is mapped in this open reading frame. A cob gene,
designated cobO, is hence associated with open reading frame
26.
[0311] To know whether the four open reading frames (open reading
frames 27 to 30) identified in this fragment correspond to cob
genes, a spectinomycin resistance cassette from plasmid
pHP45.OMEGA. (Prentki and Krisch, 1984) was specifically inserted
into each of these genes, and then introduced into the chromosome
of P. denitrificans SC510 Rif' by homologous recombination so as to
obtain mutants of insertions in each of these open reading frames.
For this purpose, the EcoRI-ClaI fragment (respective positions
8818 and 13082 in the sequence presented in FIG. 43) was used. This
fragment, which carries the open reading frames 27 to 30, was
purified from pXL157 (Cameron et al., 1989); an EcoRI linker was
added to the ClaI end after the latter had been filled in with the
Klenow fragment of E. coli DNA polymerase. This fragment was then
cloned into plasmid pUC13 (Viera et al., 1982) at the EcoRI site.
The plasmid thus constructed was referred to as pXL332. Insertions
of the spectinomycin resistance cassette from plasmid pHP45.OMEGA.
(Prentki and Krisch, 1984) were carried out on pXL332. These
insertions were done separately at the SmaI (position 9868, open
reading frame 27), BamHI (position 10664, open reading frame 28),
ClaI (position 11687, open reading frame 29) and NcoI (position
12474, open reading frame 30) sites by total or partial digestions
of pXL332 with the corresponding enzymes, and then, if necessary,
filling-in of these ends with the Klenow fragment of E. coli DNA
polymerase, followed by ligation with the 2-kb SmaI fragment of
pHP45.noteq. (Prentki and Krisch, 1984) containing a spectinomycin
resistance gene; these insertions are designated .OMEGA.2,
.OMEGA.1, .OMEGA.3 and .OMEGA.4, respectively, as presented in FIG.
46. The EcoRI fragments carrying these different insertions were
then cloned into pRK404 (Ditta et al., 1985) at one of the two
EcoRI sites. The 4 plasmids carrying these different insertions
were then introduced by conjugation in SC510 Rif', as described
above. Plasmid pR751 (Thomas and Smith, 1987) was then introduced
into the transconjugants. The exchange of mutations carried by the
4 different derivatives of pRK404 and the chromosome of SC510 Rif'
could be selected as described (see Example 4.3). 4 strains were
thereby obtained. These strains each carry an insertion of the
resistance cassette in one of the four open reading frames 27 to
30. These insertions were verified by analysis of the genomic DNA
by Southern blotting (Southern, 1975). The cobalamin production of
these different strains was studied. They all showed a Cob+
phenotype on culturing in PS4 medium. This result indicates that
these four open reading frames do not participate in the
biosynthesis of coenzyme B.sub.12. However, it is possible that one
or more of these frames code for proteins which participate, e.g.,
in the conversion of coenzyme B.sub.12 to methylcobalamin for
example, i.e. the synthesis of another cobalamin or even of another
corrinoid.
[0312] 4.5.2. Study of the 9.1-kb EcoRI-EcoRI Fragment.
[0313] Various plasmids are used in this study; plasmid pXL1560 is
plasmid pRK290 (Ditta et al., 1980) containing the 9.1-kb
EcoRI-EcoRI fragment of pXL156 (Example 1) cloned at the EcoRI site
of pRK290 (see FIG. 46). The construction of the other plasmids
used in this study (pXL618, pXL593, pXL623, pXL1909, pXL1938,
pXL1908, pXL221, pXL208, pXL297) is described in the legend to FIG.
45.
[0314] Tn5Sp insertions were obtained in plasmid pXL1560. Strain
MC1060 Tn5Sp transformed with plasmid pXL1560 was used to obtain
insertions of transposon Tn5Sp into the pXL1560 fragment; 27
insertions were thereby obtained and mapped on the 9.1-kb fragment;
the position of these different insertions in the fragment is
presented in FIG. 4. These 27 insertions were introduced into the
chromosome of strain SC510 Rif' after conjugated transfer of
pXL1560::Tn5Sp, followed by introduction of plasmid pR751. Plasmid
pR751 is a trimethoprim-resistant plasmid of the same
incompatibility group as pXL1560 (incP, Thomas and Smith, 1987). By
culturing non-selectively for pXL1560 (absence of tetracycline) but
selectively for pR751 and the transposon (presence of trimethoprim
and of spectinomycin), the exchange of the mutation carried by
pXL1560::Tn5Sp with the chromosome and also the segregation of
pXL1560 are obtained; this technique of marker exchange by double
homologous recombination is equivalent to that already described by
Schell et al., 1988. The strains thus selected carry the transposon
in their chromosome.
[0315] The double homologous recombination is verified by
Southern's method (Southern, 1975). In this way, 27 SC510
Rif'::Tn5Sp strains each possessing a different insertion of
transposon Tn5Sp in the 9.1-kb fragment were identified.
[0316] The level of cobalamin synthesis is determined for these 27
strains cultured in PS4 medium, and the Cob-phenotype is assigned
to strains producing at least 1000 times less vitamin B.sub.12 than
the parent strain SC510 Rif', FIG. 46. It is thus observed that 18
out of the 27 of these chromosomal insertions lead to a
Cob-phenotype in P. denitrificans, as shown in FIG. 46.
[0317] The insertions 19, 32, 24, 27, 37, 39, 26, 11 and 14 are
mapped in open reading frame 22 (see FIG. 46). All these insertions
are complemented by plasmid pXL618, which contains only open
reading frame 22. We deduce from this that open reading frame 22
corresponds to a cob gene, which we referred to as cobO. No
insertion was obtained in open reading frame 23; however, plasmid
pXL623, which contains only this open reading frame (see FIG. 46),
complements two cob mutants of Agrobacterium tumefaciens, G642 and
G2043 (Cameron et al., 1989). Open reading frame 23 hence
corresponds to a cob gene designated cobP. The insertions 23, 13,
12, 30, 22, 40, 35, 10 and 17 which are mapped in open reading
frames 24 and 25 lead to a Cob-phenotype in SC510 Rif'. There hence
appear to be two open reading frames whose product is involved in
the biosynthesis of cobalamins. However, it cannot be ruled out
that these insertions have polar effects on the genes positioned on
the 3' side, such as cobO. It is hence appropriate to study the
complementation of these mutants in order to determine whether the
Cob-phenotype does not result from a polar effect.
[0318] The Cob mutants of Agrobacterium tumefaciens, G622, G623 and
G630, complemented by pXL156, were studied. These mutants are not
complemented by plasmid pXL189 (Cameron et al., 1989), which
contains cobO as the only cob gene. In contrast, they are
complemented by plasmid pXL1908, which contains cobO and open
reading frame 25 in addition to the open reading frames 27 to 30
(see FIG. 45). The latter frames cannot be responsible for the
complementation of these mutants, since the proteins for which they
code do not participate in the coenzyme B.sub.12 pathway. Hence,
the observed complementations can only result from open reading
frame 25. In addition, the SC510 Rif' Tn5Sp mutants mapped in this
same open reading frame (these are the mutants 22, 40, 35, 10 and
17) are complemented by plasmid pXL1908, see FIG. 46, (carrying
cobO and frame 25), whereas at least two of them are not
complemented by pXL189, which contains only cobO as a cob gene.
These results show clearly that open reading frame 25 is a cob
gene; this cob gene is designated cobN.
[0319] The SC510 Rif' Tn5Sp mutants 23, 13 and 12, which have the
Cob-phenotype, are mapped in open reading frame 24. These mutants
are not complemented by plasmid pXL623, which contains only the
cobP gene. In contrast, these mutants are complemented by plasmid
pXL593 which contains cobP and open reading frame 24, thereby
indicating that open reading frame 24 is responsible for their
complementation. Open reading frame 24 is hence a cob gene, which
is designated cobW.
EXAMPLE 5
Genes and Proteins
[0320] 5.1-5.4-kb Fragment
[0321] Five genes (cobA, cobB, cobC, cobD and cobE) are hence
defined on the 5.4-kb ClaI-HindIII-HindIII-HindIII fragment. They
code, respectively, for the following COB proteins: COBA, COBB,
COBC, COBD and COBE. The coding portions of the genes (cobA to
cobE) are described in FIG. 15, as well as the sequences of the
COBA to COBE proteins. Properties of each of these proteins are
also presented (amino acid composition, isoelectric point, index of
polarity and hydrophilicity profile).
[0322] 5.2-8.7-kb Fragment
[0323] Eight genes are hence defined on the 8.7-kb fragment. These
cobF to cobM genes code, respectively, for the following COB
proteins: COBF, COBG, COBH, COBI, COBJ, COBK, COBL, and COBM. The
coding portions of the genes (cobF to cobM) are described in FIG.
16, as well as the sequences of the COBF to COBM proteins.
Properties of each of these proteins are also presented (amino acid
composition, molecular weight, isoelectric point, index of polarity
and hydrophilicity profile).
[0324] 5.3-4.8-kb Fragment
[0325] Three genes (cobX, cobS, cobT) are defined on the 4.8-kb
SalI-SalI-SalI-SalI-SalI-BglI fragment. They code, respectively,
for the following proteins: COBX, COBS and COBT. The coding
portions of these genes are described in FIG. 40, as well as the
sequences of the COBX, COBS and COBT proteins. Arbitrarily, the ATG
at position 1512 of cobs has been chosen as the initiation codon,
rather than that located at position 1485 (see FIG. 32). Properties
of each of these proteins are also shown (amino acid composition,
isoelectric point, index of polarity and hydrophobicity profile).
COBT possesses a hydrophilic pocket corresponding to amino acids
214 to 305.
[0326] 5.4-3.9-kb Fragment
[0327] Two genes (cobU and cobV) are defined on the 3.9-kb
SstI-SstI-BamHI fragment. They code, respectively, for the
following proteins: COBU and COBV. The coding portions of these
genes are described in FIG. 41, as well as the sequences of the
COBU to COBV proteins. Properties of each of these proteins is also
shown (amino acid composition, isoelectric point, index of polarity
and hydrophobicity profile).
[0328] 5.5-13.4-kb Fragment
[0329] Five cob genes are defined on the 13.4-kb fragment (cobQ,
cobP, cobW, cobN and cobO and cobV). They code, respectively, for
the following proteins: COBQ, COBP, COBW, COBN and COBO. The coding
portions of these genes (cobQ, cobP, cobW, cobN and cobO) are
described in FIG. 46, as well as the sequences of COBQ, COBP, COBW,
COBN and COBO proteins. Properties of each of these proteins are
also shown (amino acid composition, isoelectric point, index of
polarity and hydrophobicity profile).
[0330] From the hydrophilicity profiles, which were produced
according to the programmes of Hopp and Woods (1981), all the COB
proteins with the exception of COBV are probably soluble proteins,
as opposed to membrane proteins, since the absence of large
hydrophobic domains is noted. COBV is either a membrane protein,
since 4 long hydrophobic domains are noted (see FIG. 41), or a
cytoplasmic protein having large hydrophobic domains.
[0331] For all the amino acid sequences of the COB proteins, a
methionine is indicated as the first amino acid at the
NH.sub.2-terminal position. It is understood that this methionine
may be excised in vivo (Ben Bassat and Bauer, 1984). Rules relating
to the in vivo excision of NH.sub.2-terminal methionine by
methionine aminopeptidase are known to have been proposed. (Hirel
et al., 1989).
[0332] Moreover, these protein sequences were compared with the
Genpro proteins, Genpro being a Genbank protein extraction (version
59) augmented by putative coding portions larger than 200 amino
acids, according to the programme of Kanehisa (1984). No
significant homology could be demonstrated with the parameters used
on Genbank version 59, except for COBT. In effect, the COBT protein
possesses a "core of acidic amino acids" between (amino acid)
positions 224 and 293 (see FIG. 40); in this portion of the
protein, more than one amino acid out of 2 is a glutamic or
aspartic acid residue; this core of acidic amino acids renders the
protein homologous over this region, according to the programme of
Kanehisa (1984), with other proteins also having such an acidic
core. The most homologous proteins are: GARP protein of Plasmodium
falciparum (Triglia et al., 1988), rat cardiac troponin T (Jin and
Lin, 1989), human and rat prothymosin (Eschenfeld and Berger,
1986), an androgen-dependant rat protein that binds to spermine
(Chang et al., 1987), and the human, rat and chicken "mid-size
neurofilament subunit", proteins (Myers et al., 1987, Levy et al.,
1987, Zopf et al., 1987). The function of these cores rich in
acidic residues is unknown; however, this acidic core should either
permit the binding of metal cations such as Co.sup.++, which would
give the COBT protein the role of a cobalt metallothionein, or else
permit interactions with other proteins.
EXAMPLE 6
Enzymatic Studies
[0333] 6.1--Identification of COB Proteins and Their Genes from
Purified Enzymatic Activities
[0334] This example describes how, from a purified protein, after
its NH.sub.2-terminal sequence has been established, it is possible
to find the corresponding structural gene among sequenced cob
genes.
[0335] 6.1.1. Identification of the COBA Protein Encoded by the
cobA Gene
[0336] The purification of Pseudomonas denitrificans SUMT has been
described (F. Blanche et al., 1989). The NH.sub.2-terminal sequence
of the protein thus purified could be determined according to the
technique described above. The first ten amino acids were
identified: TABLE-US-00006 1 2 3 4 5 6 7 8 9 10 Met Ile Asp Asp Leu
Phe Ala Gly Leu Pro
[0337] The NH.sub.2 terminal sequence of the COBA protein (FIG. 15)
corresponds exactly to this sequence. The molecular weight of the
purified SUMT, estimated by 12.5% SDS-PAGE electrophoresis, is
30,000. The COBA protein has a molecular weight deduced from its
sequence of 29,234 (FIG. 15). The correspondences between the
NH.sub.2-terminal sequences and the molecular weights indicate
clearly that the COBA protein corresponds to SUMT. The cobA gene is
the SUMT structural gene.
[0338] 6.1.2. Identification of the COBB Protein Encoded by the
cobB Gene
a) Assay of Cobyrinic Acid a,c-Diamide Synthase Activity
[0339] This example illustrates the assay of an activity of the
pathway of biosynthesis of corrinoids which has never yet been
described. The enzyme in question is cobyrinic acid a,c-diamide
synthase (CADAS), which catalyses the amidation of two carboxylic
acid functions of the corrin or decobalt-ocorrin ring-system at
positions a and c (FIG. 17). The donor of the NH.sub.2 group is
L-glutamine, and the reaction consumes 1 molecule of ATP per
amidation of each carboxylic acid function. The assay which is
described below applies to the diamidation reaction of cobyrinic
acid; with a few modifications (detection in HPLC at 330 nm in
particular), it applies to the diamidation reaction of
hydrogenobyrinic acid.
[0340] The incubation mixture (0.1 M Tris-HCl pH 8 (250 .mu.l))
containing ATP (1 mM), MgCl.sub.2 (2.5 mM), glut-amine (100 .mu.m),
cobyrinic acid (50 .mu.M) or hydrogeno-byrinic acid (50 .mu.M) and
cobyrinic a,c-diamide synthase (approximately 1 unit of activity)
is incubated for 1 hour at 30.degree. C. At the end of the
incubation, an aqueous solution (125 .mu.l) of KCN (2.6 g/l) and
0.2 M HCl (125 .mu.l) are added to the mixture, which is then
heated to 80.degree. C. for 10 minutes and thereafter centri-fuged
for 5 minutes at 5,000 g. An aliquot (50 .mu.l) of the
centrifugation supernatant is analysed in HPLC. It is injected onto
a 25-cm Nucleosil 5-C.sub.18 column and eluted with a gradient from
0 to 100% of buffer B in A in the course of 30 minutes; buffer A:
0.1 M potassium phosphate pH 6.5, 10 mM KCN; buffer B: 0.1 M
potassium phosphate pH 8, 10 mM KCN/acetonitrile (1:1). The
corr-inoids are detected by means of their UV absorption at 371 nm.
The unit of enzymatic activity is defined as the quantity of enzyme
necessary for synthesising 1 nmol of amide groups per hour under
the conditions described.
b) Purification of Pseudomonas Denitrificans Cobyrinic Acid
a,c-Diamide Synthase Activity
[0341] This experiment illustrates how a Pseudomonas denitrificans
protein participating in the pathway of biosynthesis of cobalamins
may be purified.
[0342] Using the assay described in Example 6.1.2 a), the
purification of Pseudomonas denitrificans cobyrinic acid
a,c-diamide synthase is carried out as described below.
[0343] In a typical purification experiment, wet cells (7 g) of
strain SC 510 Rif', into which plasmid pXL1500 has been introduced
(see Example 4.1. for the description of pXL1500, as well as FIG.
12), are suspended in 0.1 M Tris-HCl pH 7.7 (30 ml) and sonicated
for 15 minutes at 4.degree. C. The crude extract is then recovered
by centrifugation for 1 hour at 50,000 g, and a portion (10 ml) of
this extract is injected onto a Mono Q HR 10/10 column equilibrated
with the same buffer. The proteins are eluted with a linear KCl
gradient (0 to 0.5 M). The fractions containing the enzymatic
activity (demonstrated by means of the test described in Example
6.2 b)) are combined and concentrated to 2.5 ml. After dilution
with 25 mM Tris-HCl pH 7.7 (1 ml), the proteins are fractionated on
a Mono Q HR 5/5 using the above KCl gradient (0 to 0.5 M). The
active fractions are combined, and 0.1 M Tris-HCl pH 7.7 (1 ml)
containing 1.7 M ammonium sulphate is added to the sample, which is
then chromatographed on a Phenyl-Superose (Pharmacia) column with a
decreasing ammonium sulphate gradient (1.0 M to 0 M). The fractions
containing the desired activity are combined and chromatographed on
a Bio-Gel HPHT (Bio-Rad) column with a potassium phosphate gradient
(0 to 0.35 M).
[0344] After this step, the enzyme is more than 95% pure. It shows
no contaminant protein in SDS-PAGE. The purity of the protein is
confirmed by the uniqueness of the NH.sub.2-terminal sequence. Its
molecular weight in this technique is 45,000. The different steps
of purifi-cation of CADAS, with their purification factor and their
yield, are given in the table below. TABLE-US-00007 TABLE
Purification of CADAS Sp. activity Purification Vol Proteins (u/mg
of Purification step (ml) (mg) proteins) Yield factor.sup.1 Crude
extract 10 200 8.5 -- -- MonoQ 10/10 12 15.1 108 96 12.7 MonoQ 5/5
3 3.75 272 60 32 Phenyl- 1 0.865 850 43 100 Superose Bio-Gel HPHT 2
0.451 1320 35 155 .sup.1This factor is calculated from the increase
in the specific activity of the fractions during the
purification.
c) NH.sub.2-terminal Sequence of Pseudomonas Denitrificans
Cobyrinic Acid a,c-Diamide Synthase and Identification of the
Pseudomonas Denitrificans Structural Gene Coding for this
Activity
[0345] This example illustrates how the NH.sub.2-terminal sequence
of a protein which participates in the pathway of biosynthesis of
cobalamins enables the structural gene which codes for this protein
to be identified.
[0346] The NH.sub.2-terminal sequence of Pseudomonas denitrificans
cobyrinic acid a,c-diamide synthase, purified as described in
Example 6.1.2 b), was determined as described above. 15 residues
were identified: TABLE-US-00008 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Ser Gly Leu Leu Ile Ala Ala Pro Ala Ser Gly Ser Gly Lys Thr
[0347] The NH.sub.2-terminal sequence of the COBB protein (FIG. 15)
corresponds exactly to this sequence except that, in the sequence
presented in FIG. 15, a methionine precedes the peptide sequence
determined by direct sequencing. It follows from this that the
amino-terminal methionine is definitely excised in vivo by
methionine aminopeptidase (Ben Bassat and Bauer, 1987). The
molecular weight of the purified CADAS, estimated by 12.5% SDS-PAGE
electrophoresis, is 45,000. The COBB protein has a molecular weight
deduced from its sequence of 45,676 (FIG. 15). The correspondences
between the NH.sub.2-terminal sequences and the molecular weights
indicate clearly that the COBB protein corresponds to CADAS. The
cobB gene is the CADAS structural gene.
[0348] 6.1.3. Identification of the COBI Protein Encoded by the
cobI Gene
a) Assay of an S-Adenosyl-L-Methionine:Precorrin-2
Methyltransferase Activity
[0349] This example illustrates the assay of an enzymatic activity
of the pathway of bipsynthesis of corrinoids which has never yet
been described. The enzyme in question is S-adenosyl-L-methionine:
precorrin-2 methyltransferase (SP.sub.2MT), which catalyses the
transfer of a methyl group from S-adenosyl-L-methionine (SAM) to
precorrin-2 to give precorrin-3 (FIG. 18). Factors II and III,
oxidation products of precorrin-2 and precorrin-3, respectively,
have already been purified from cell extracts of Propionibacterium
shermanii (Battersby and MacDonald, 1982, Scott et al., 1984);
precorrin-2 and precorrin-3 are recognised as presumed
intermediates of coenzyme B.sub.12 biosynthesis, but they have
never been purified as such. For this reason, the corresponding
activity has never been either assayed or purified beforehand. The
substrate of the enzymatic reaction, precorrin-2, is a very labile
molecule which it is not possible to store, since it oxidises
spontaneously in the presence of even infinitesimal traces of
oxygen (Battersby and MacDonald, 1982). The principle of this
enzymatic test hence lies in the possibility of generating
precorrin-2 from SAM and .delta.-aminolevulinic acid at the
required moment using an enzymatic extract of strain SC510 Rif'
into which plasmid pXL1500 has been introduced. The incubation must
be performed under strictly anaerobic conditions.
[0350] The fractions containing SP.sub.2MT are incubated in 0.1 M
Tris-HCl pH 7.7 (1 ml) in the presence of 5 mM DTT, 1 mM EDTA, 100
.mu.M [methyl-.sup.3H]SAM (1 .mu.Ci), 0.8 mM .delta.-aminolevulinic
acid and crude enzyme extract (6 mg) of Pseudomonas denitrificans
strain SC510 Rif' pXL1500 for 3 hours at 30.degree. C. Strain SC510
Rif' pXL1500 contains a strong SUMT activity (F. Blanche et al.,
1989). The tetrapyrrole compounds produced during the incubation
are bound to a DEAE-Sephadex anion exchange column and esterified
in methanol containing 5% of sulphuric acid in the absence of
oxygen. The dimethylated and trimethylated derivatives of uro'gen
III are then separated by thin-layer chromatography on silica using
dichloromethane/methanol (98.3:1.7) as an eluent system (F. Blanche
et al., 1989). The SP.sub.2MT activity is expressed as the ratio of
the quantity of trimethylated derivatives obtained to the total of
(di- and tri-) methylated derivatives produced, referred to the
quantity of protein. The SC510 Rif' pXL1500 extract introduced in
the test does not display detectable SP.sub.2MT activity under the
assay conditions (the ratio of precorrin-3 produced to precorrin-2
produced during the test is less than 0.05).
b) Purification of Pseudomonas Denitrificans
S-Adenosyl-L-Methionine:Precorrin-2 Methyltransferase
[0351] This experiment illustrates how a Pseudomonas denitrificans
protein participating in the pathway of biosynthesis of cobalamins
may be purified when an assay for the activity in question
exists.
[0352] The protein is purified from SC510 Rif' cells containing
plasmid pXL253. This is plasmid pKT230 into which the 8.7-kb EcoRI
fragment has been inserted (FIG. 13). In a typical purification
experiment, wet cells (50 g) of strain SC150 Rif' into which
plasmid pXL253 has been introduced are suspended in 0.1 M potassium
phosphate pH 7.7, 5 mM DTT (250 ml) and sonicated for 15 minutes at
4.degree. C. After centrifugation at 50,000 g for 1 hour, the
supernatant is passed through a DEAE-Sephadex column (10 ml of gel)
to remove the tetrapyrrole compounds. The pH of the crude extract
thereby obtained is adjusted to pH 7.7 with 0.1 M KOH. The proteins
precipitating at between 33% and 45% ammonium sulphate saturation
are collected and dissolved in 0.1 M Tris-HCl pH 7.7, 5 mM DTT (40
ml). This solution is passed through a Sephadex G-25 column eluted
with 10 mM Tris-HCl pH 7.7, 5 mM DTT, and the proteins collected
are injected onto a DEAE-Trisacryl-M column. The proteins are
eluted with a linear gradient of 0 to 0.25 M KCl, and the fractions
containing the SP.sub.2MT activity are combined and passed a second
time through a Sephadex G-25 column as above. The protein fraction
is injected onto an Ultrogel HA (IBF) column equilibrated in 10 mM
Tris-HCl pH 7.7, 5 mM DTT. The proteins are eluted with a linear
gradient of 0 to 50 mM potassium phosphate pH 7.8 containing 5 mM
DTT. The fractions containing the desired activity are combined and
injected onto a MonoQ HR 5/5 (Pharmacia) column equilibrated with
50 mM Tris-HCl pH 7.7, 5 mM DTT. The SP.sub.2MT is eluted with a
linear gradient (0 to 0.25 M) of KCl. At emergence from the MonoQ
step, 12.5% SDS-PAGE electrophoresis with staining with silver
salts reveals the enzyme is more than 99% pure. This is confirmed
by the uniqueness of the NH.sub.2-terminal sequence of the protein.
The molecular weight calculated from the electrophoresis under
denaturing conditions (12.5% SDS-PAGE) is 26,500. The steps of
purification of SP.sub.2MT with their yields are described in the
table below. TABLE-US-00009 TABLE Purification of SP.sub.2MT
Purification Vol Proteins Purification step (ml) (mg) factor.sup.1
Crude extract 300 6000 -- Precipitation 40 1530 3.9 (33-45%)
DEAE-Tris- 57 355 16.9 acryl-M Ultrogel HA 30 71 85 MonoQ HR 5/5 12
33.5 179 .sup.1This factor is calculated from the yield of
protein.
c) NH.sub.2-Terminal Sequence of SP.sub.2MT and Identification of
the Structural Gene Coding for this Activity
[0353] This example illustrates how the NH.sub.2-terminal sequence
of a protein participating in the biosynthetic pathway enables the
structural gene which codes for this protein to be identified. In
the present example, the structural gene in question is that for
SP.sub.2MT.
[0354] The NH.sub.2-terminal sequence of the purified protein was
determined as described above. The first 15 amino acids were
identified: TABLE-US-00010 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Ser
Gly Val Gly Val Gly Arg Leu Ile Gly Val Gly Thr Gly Pro
[0355] The NH.sub.2-terminal sequence of the COBI protein (FIG. 16)
corresponds exactly to this sequence except that, in the sequence
presented in FIG. 16, a methionine precedes the peptide sequence
deduced from the nucleotide sequence. It follows from this that the
amino-terminal methionine is definitely excised in vivo by
methionine aminopeptidase (Ben Bassat and Bauer, 1987). The
molecular weight of the purified SP.sub.2MT, estimated by 12.5%
SDS-PAGE electrophoresis, is 26,500. The COBI protein has a
molecular weight deduced from its amino acid sequence of 25,878
(FIG. 16). The correspondences between the NH.sub.2-terminal
sequences and the molecular weights indicate clearly that the COBI
protein corresponds to SP.sub.2MT. The cobI gene is the SP.sub.2MT
structural gene.
[0356] 6.1.4. Identification of the COBH Protein Encoded by the
cobH Gene
a) Assay of Precorrin-8x Mutase Activity
[0357] This example illustrates the assay of an enzymatic activity
of the pathway of biosynthesis of cobalamine which has never been
described hitherto. The enzyme in question is precorrin-8x mutase.
This enzyme catalyses the transfer of the methyl group from
position C-11 to position C-12 during the conversion of
precorrin-8x to hydrogenobyrinic acid (see the nomenclature of the
carbon atoms in FIG. 19; PL 68). More generally, it is the enzyme
which catalyses the transfer of the methyl group C-11 to C-12,
thereby leading to the corrin ring-system. The enzyme is referred
to here as a mutase, although it has not been formally demonstrated
that the transfer of the methyl group is intramolecular, even
though this is very probable.
[0358] The enzymatic activity is demonstrated by the conversion of
precorrin-8x (5 .mu.M) to hydrogenobyrinic acid during incubations
in the presence of enzyme fractions in 0.1 M Tris-HCl pH 7.7, 1 mM
EDTA, at 30.degree. C. for 1 h. At the end of the incubation, the
reaction is stopped by heating to 80.degree. C. for 10 min and,
after centrifugation at 3000.times.g for 10 min, the
hydrogenobyrinic acid formed, present in the supernatant, is
analysed by HPLC (see Example 6.1.2.a).
b) Purification of Precorrin-8x Mutase.
[0359] The purification of Pseudomonas denitrificans precorrin-8x
mutase is carried out as described below.
[0360] During this purification, all the buffer solutions are
adjusted to pH 7.7.
[0361] In a typical purification experiment, cells (50 g) of strain
SC510 Rif', carrying plasmid pXL253 (plasmid pKT230 into which the
8.7-kb fragment has been cloned at the EcoRI site, FIG. 13) and
obtained after culture in PS4 medium, are resuspended in 0.1 M
potassium phosphate buffer (200 ml) and sonicated for 12 minutes.
After centrifugation at 50,000 g for 1 hour, the supernatant is
passed through a DEAE-Sephadex column (10 ml of gel) to remove the
tetrapyrrole compounds. The pH of the solution is immediately
adjusted to 7.7 with 1 M KOH solution. The protein fraction
precipitating at between 40 and 60% ammonium sulphate saturation is
collected by centrifugation and dissolved in 0.1 M Tris-HCl (50
ml). This sample is then injected onto an Ultrogel AcA 54 (IBF,
France) column (gel volume 1,000 ml) and the proteins are eluted at
a flow rate of 60 ml/h with 50 mM Tris-HCl. The fractions
containing the activity are pooled and injected onto a
DEAE-Trisacryl M (IBF, France) column equilibrated with 50 mM
Tris-HCl, and the proteins are eluted with a gradient of 0 to 0.2 M
KCl. The fractions containing the protein to be purified are pooled
and passed through a Sephadex G-25 column equilibrated in 10 mM
Tris-HCl. The protein fraction is injected onto an Ultrogel HA
(IBF, France) column equilibrated with 10 mM Tris-HCl, the proteins
are eluted with a gradient of 0 to 0.1 M potassium phosphate, and
the active fraction is then chromatographed on a Phenyl-Sepharose
CL (Pharmacia) 4B column in 10 mM potassium phosphate, the column
being eluted with a gradient of 0.65 to 0 M ammonium sulphate. The
active fractions are pooled. The protein thereby obtained is more
than 95% pure (according to the results of 12.5% SDS-PAGE
electrophoresis and staining with silver salts). The purity of the
protein is confirmed by the uniqueness of the N-terminal sequence.
Its molecular weight calculated using this technique is 22,000. The
steps of purification of precorrin-8x mutase with their
purification yields are described in the table below.
TABLE-US-00011 TABLE Purification of precorrin-8x mutase
Purification Vol Proteins Purification step (ml) (mg) factor.sup.1
Crude extract 250 6000 -- Precipitation 50 2350 2.6 (40-60%)
Ultrogel ACA 54 70 655 9.2 DEAE-Tris- 30 271 22 acryl-M Ultrogel HA
22 93 65 Phenyl-Sepharose 12 31 194 .sup.1This factor is calculated
from the yield of protein.
c) NH.sub.2-Terminal Sequence of Precorrin-8x Mutase and
Identification of its Structural Gene
[0362] This example illustrates how the NH.sub.2-terminal sequence
of a protein participating in the biosynthetic pathway enables the
structural gene which codes for this protein to be identified.
[0363] The NH.sub.2-terminal sequence of this protein was
determined as described above. 15 residues were identified:
TABLE-US-00012 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Pro Glu Tyr Asp
Tyr Ile Arg Asp Gly Asn Ala Ile Tyr Glu Arg
[0364] The NH.sub.2-terminal sequence of the COBH protein (FIG. 16)
corresponds exactly to this sequence except that, in the sequence
presented in FIG. 16, a methionine precedes the peptide sequence
determined by the sequencing described above. It follows from this
that the amino-terminal methionine is definitely excised in vivo by
methionine aminopeptidase (Ben Bassat and Bauer, 1987). Since the
second residue is a proline, this excision is in keeping with the
rules already stated (Hirel et al., 1989). The molecular weight of
the purified precorrin-8x mutase, estimated by 12.5% SDS-PAGE
electrophoresis, is 22,000. The COBH protein has a molecular weight
deduced from its sequence of 22,050 (FIG. 16). The correspondences
between the NH.sub.2-terminal sequences and the molecular weights
of these proteins indicate clearly that the COBH protein
corresponds to precorrin-8x mutase. cobH is the precorrin-8x mutase
structural gene.
d) Preparation, Isolation and Identification of Precorrin-8x.
[0365] In a typical experiment for preparation of precorrin-8x, a
crude enzyme extract of strain SC510 Rif' pXL253 (1000 mg of
proteins) is incubated anaerobically for 20 h at 30.degree. C. in
0.1 M Tris-HCl buffer pH 7.7 (100 ml) with
trimethylisobacteriochlorin (1000 nmol) prepared as described
previously (Battersby et al., 1982), EDTA (1 mM), ATP (100
.mu.mol), MgCl.sub.2 (250 .mu.mol), NADH (50 .mu.mol), NADPH (50
.mu.mol), SAM (50 .mu.mol) and hydrogenobyrinic acid (20 .mu.mol).
At the end of the incubation, precorrin-8x is the preponderant
tetrapyrrole product formed. It is isolated and purified by HPLC on
a .mu.Bondapak C18 (Waters) column using a linear elution gradient
of 0 to 50% of acetonitrile in a potassium phosphate buffer pH 5.8.
The mass of precorrin-8x (m/z=880) and the mass of its methyl ester
derivative (m/z=978) indicate that it is a compound having the same
empirical formula as hydrogenobyrinic acid. The UV/visible and
fluorescence characteristics are very different from those of
hydrogenobyrinic acid, and indicate that the molecule possesses two
separate chromophors. Since the only enzymatic isomerisation
reaction between precorrin-6x (Thibaut et al., 1990) and
hydrogenobyrinic acid is the migration of the methyl from C-11 to
C-12, precorrin-8x is the last intermediate before hydrogenobyrinic
acid, and the corresponding reaction is the migration of the methyl
from C-11 to C-12, catalysed by precorrin-8x mutase.
[0366] 6.1.5. Identification of the COBU Protein Encoded by the
cobU Gene
[0367] a) Assay of nicotinate-nucleotide:dimethylbenzimidazole
phosphoribosyltransferase activity (FIG. 5, reaction 5). This
example illustrates the assay of an enzymatic activity directly
linked to the pathway of biosynthesis of cobalamins. The enzyme in
question is nicotinate-nucleotide:dimethylbenzimidazole
phosphoribosyl-transferase (NN:DMBI PRT) (EC 2.4.2.21). The
fractions containing NN:DMBI PRT activity (approximately 5 units)
are incubated at 30.degree. C. for 8 min in 0.1 M glycine-NaOH
buffer pH 9.7 (500 .mu.l) in the presence of 1 mM NaMN (nicotinic
acid mononucleotide) and 10 .mu.M DMBI. The reaction is then
stopped by heating to 80.degree. C. for 10 min, the reaction
mixture is diluted with water (4 volumes) and this solution (100
.mu.l) is injected onto a 15-cm Nucleosil 5-C8 HPLC column eluted
with a 0.1 M potassium phosphate pH 2.9/acetonitrile (93:7) mixture
at a flow rate of 1 ml/min. The .alpha.-ribazole 5'-phosphate is
detected and quantified by fluorimetry (excitation: 260 nm;
emission>370 nm). The unit of enzymatic activity is defined as
the quantity of enzyme necessary for generating 1 nmol of
.alpha.-ribazole 5'-phosphate per hour under these conditions.
[0368] b) Purification of Pseudomonas denitrificans NN:DMBI PRT
activity. This experiment illustrates how a P. denitrificans
protein participating in the pathway of biosynthesis of cobalamins
may be purified. Using the assay described in Example 6.1.5.a), the
purification of Pseudomonas denitrificans NN:DMBI PRT is carried
out as described below. In a typical purification experiment, wet
cells (10 g) of strain SC510 Rif', into which plasmid pXL1490B has
been introduced as described above, are used. Plasmid pXL1490B is
described in FIG. 38; this plasmid was obtained by cloning the
3.85-kb BamHI-SstI-SstI fragment of pXL519 (see FIG. 38). This
plasmid hence carries the cobU and cobV genes of P. denitrificans.
The cells, cultured in PS4 medium supplemented with lividomycin, as
described previously, are harvested after 96 hours of culture in
PS4 medium. They are resuspended in 0.1M Tris-HCl buffer pH 7.2 (25
ml) and sonicated for 15 min at 4.degree. C. The crude extract is
then recovered by centrifugation for 1 h at 50,000 g, and
thereafter passed through a DEAE-Trisacryl M (IBF, France) column
equilibrated with the same buffer. 10% of the eluate (120 mg of
proteins) is fractionated on a mono Q HR 10/10 column using a KCl
gradient (0 to 0.6 M). The active fractions are pooled and
concentrated to 2 ml by ultrafiltration, and then, after mixing
with 30 mM Tris-HCl buffer pH 7.2 (one volume), the sample is
fractionated a second time on a Mono Q HR 5/5 column as before. The
active fractions are pooled, and the sample is then brought to a
molarity of 1 M using ammonium sulphate and chromatographed on a
Phenyl-Superose HR 5/5 column eluted with a decreasing ammonium
sulphate gradient (1 M to 0 M). The fractions containing the
desired activity are pooled, concentrated by ultrafiltration and
chromatographed on a Bio-Sil 250 gel permeation column eluted with
20 mM sodium phosphate/50 mM sodium sulphate pH 6.8.
[0369] After this step, the enzyme is more than 95% pure. It shows
no contaminant protein in SDS-PAGE. This purity is confirmed by the
uniqueness of the NH.sub.2-terminal sequence. Its molecular weight
in this technique is 35,000. The different steps of purification of
the NN:DMBI PRT are given in the table below. TABLE-US-00013 TABLE
Purification of P. denitrificans NN:DMBI PRT Sp. activity
Purification Vol Proteins (u/mg of Purification Step (ml) (mg)
proteins Yield factor.sup.1 Crude extract 6.0 120 2650 -- -- MonoQ
10/10 6.0 12.7 13515 51.3 5.1 MonoQ 5/5 3.0 6.19 20140 39.2 7.6
Phenyl-Superose 1.5 2.60 35510 29.0 13.4 Bio-Sil 250 1.2 1.92 39750
24.0 15.0
[0370] c) NH.sub.2-terminal sequence of P. denitrificans NN:DMBI
PRT and identification of the Pseudomonas denitrificans structural
gene coding for this activity. The NH.sub.2-terminal sequence of
Pseudomonas denitrificans NN:DMBI PRT, purified as described in
Example 6.1.5b), was carried out according to the technique
described above. The first 15 residues-were identified:
TABLE-US-00014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Ser Ala Ser Gly
Leu Pro Phe Asp Asp Phe Arg Glu Leu Leu Arg
[0371] The NH.sub.2-terminal sequence of the COBU protein (FIG. 41)
corresponds to this sequence except that, in the sequence presented
in FIG. 41, a methionine precedes the first amino acid of the
peptide sequence determined by direct sequencing. It follows from
this that the amino-terminal methionine is definitely excised in
vivo by methionine aminopeptidase (Ben Bassat and Bauer, 1987). The
molecular weight of the purified N-transglycosidase, estimated by
12.5% SDS-PAGE electrophoresis, is 35,000. The COBU protein has a
molecular weight deduced from its sequence of 34,642 (FIG. 41). The
correspondences between the NH.sub.2-terminal sequences and the
molecular weights clearly indicate that the COBU protein
corresponds to NN:DMBI PRT. The cobU gene is the NN:DMBI PRT
structural gene.
[0372] d) Specificity of NN:DBI PRT for DBI. This example
illustrates how a study of the specificity of P. denitrificans
NN:DMBI PRT enables P. denitrificans to be made to biosynthesise
various cobamides, using the catalytic properties of P.
denitrificans NN:DMBI PRT to perform the synthesis of the
nucleotide base in question.
[0373] The enzyme substrate for synthesising cobalamine is
5,6-dimethylbenzimidazole. Benzimidazole and 5-methylbenzimidazole,
respectively, are substrates for the reaction with reaction rates
of 157% and 92%, respectively, compared to the natural substrate
(5,6-dimethylbenzimidazole), the NaMN concentration being fixed at
2 mM. The specificity of P. denitrificans NN:DMBI PRT is hence low
for substrates containing a benzimidazole ring-system. It is hence
possible to use P. denitrificans strain SC510 Rif' (Cameron et al.,
1989), and to culture it in PS4 medium in which
5,6-dimethylbenzimidazole is replaced by benzimidazole or
5-dimethylbenzimidazole, respectively, in order to make the
bacterium synthesise
Co.alpha.-(benzimidazolyl)-Co.beta.-cyanocobamide or
Co.alpha.-(5-methylbenzimidazolyl)-Co.beta.-cyanocobamide,
respectively. There is no doubt that other cobamides could be
synthesised in this way.
[0374] 6.1.6. Identification of the COBV Protein Encoded by the
cobV Gene
[0375] This example illustrates how the assay of an activity of the
pathway of biosynthesis of coenzyme B.sub.12 in P. denitrificans,
and then the partial purification of this activity, can enable the
structural gene for this enzyme to be identified in P.
denitrificans.
[0376]
[0377] a) Assay of GDP-cobinamide:.alpha.-ribazole-5'-phosphate
cobinamidephosphotransferase (or cobalamin-5'-phosphate synthase)
activity. This example illustrates the assay of an activity
directly linked to the pathway of biosynthesis of cobalamines. The
enzyme in question is cobalamin-5'-phosphate synthase. The
fractions containing the activity (approximately 5 to 10 units) are
incubated in darkness at 30.degree. C. in 0.3 Tris-HCl buffer pH
9.0 (500 .mu.l) in the presence of 1 mM EDTA, 12.5 MM MgCl.sub.2,
50 .mu.M .alpha.-ribazole 5'-phosphate and 20 .mu.M GDP-cobinamide
[in 5'-deoxy-5'-adenosyl (Ado) or coenzyme form]. After 15 min of
incubation, 20 mM potassium cyanide (500 .mu.l) is added and the
solution is heated to 80.degree. C. for 10 min. After
centrifugation to remove the precipitated matter, the vitamin
B.sub.12 5'-phosphate present in the supernatant is assayed as
described in Example 9. One unit of cobalamin-5'-phosphate synthase
is defined as the quantity of enzyme necessary for generating 1
nmol of cobalamine 5'-phosphate per h under the conditions
described above.
[0378] Ado-GDP-cobinamide is obtained by incubation of
Ado-cobinamide phosphate (Blanche et al., 1989) with a SC510 Rif'
pXL623 extract under the conditions of assay of cobinamidephosphate
guanylyltransferase (see 6.1.11.b). The .alpha.-ribazole and
.alpha.-ribazole 5'-phosphate are isolated from SC510 Rif' cultures
and purified by HPLC under the assay conditions described in
Example 6.1.5a).
b) Partial Purification of Cobalamin-5'-Phosphate Synthase
[0379] This experiment illustrates how a P. denitrificans enzymatic
activity participating in the pathway of biosynthesis of
cobalamines of P. denitrificans can be partially purified. Using
the assay described above, the purification of cobalamin
5'-phosphate synthase is carried out. For this purpose, in a
typical purification experiment, wet cells (10 g) of strain SC510
Rif', into which plasmid pXL1490B has been introduced as described
previously, are used. Plasmid pXL1490B is described in FIG. 38:
this plasmid corresponds to the 3.85-kb SstI-SstI-BamHI fragment
cloned into PKT230. This plasmid carries the P. denitrificans cobU
and cobV genes. The presence of this plasmid in P. denitrificans
SC510 Rif' leads to an amplification of the cobalamin-5'-phosphate
synthase activity by a factor of approximately 100; it is hence
probable that the insert carried by plasmid pXL1490B contains the
structural gene for this enzyme; hence this gene can be only cobU
or cobV. The SC510 Rif' pXL1490B cells are obtained by culture in
PS4 medium supplemented with lividomycin, as described above. The
cells are centrifuged and then resuspended in 0.1 M Tris-HCl (pH
8.3)/1 mM EDTA buffer (buffer A) (25 ml) and sonicated for 15 min
at 4.degree. C. The crude extract is then recovered by
centrifugation for 1 h at 50,000 g and passed through a Sephadex
G-25 column equilibrated with buffer A. The protein fraction is
recovered and injected in 300-.mu.l fractions (7.5 mg of proteins)
onto a Superose 12 HR 10/30 column eluted in buffer A. The excluded
fraction is recovered, mixed with an equal volume of buffer A/1.0 M
ammonium sulphate and chromatographed on a Phenyl-Superose HR 5/5
column. The proteins are eluted with a decreasing ammonium sulphate
gradient (0.5 M to 0 M) in buffer A, followed by a plateau at 0 M
ammonium sulphate with the object of eluting the
cobalamin-5'-phosphate synthase activity. The partial purification
of this enzyme is described in the table below, on the basis of 75
mg of proteins introduced at the start in the purification process.
TABLE-US-00015 TABLE Partial purification of P. denitrificans
cobalamin-5'-phosphate synthase Sp. activity Purification Vol
Proteins (u/mg of Purification step (ml) (mg) proteins) Yield
factor.sup.1 Crude extract 3.0 75 325 -- -- Superose 12 HR 50.0 2.9
6,810 81 21 Phenyl-Superose 4.5 0.35 17,850 26 55
[0380] c) Specificity of cobalamin-5'-phosphate synthase. The Km
for (Ado)GDP-cobinamide is 0.9 .mu.m. However, the enzyme possesses
the same affinity and a virtually identical reaction rate for the
(CN, aq) form of the substrate. The Km of the enzyme for
.alpha.-ribazole 5'-phosphate is approximately 2.7 .mu.M. In
addition, the purest preparations of cobalamine-5'-phosphate
synthase catalyse the reaction of Ado-GDP-cobinamide with
.alpha.-ribazole to give coenzyme B.sub.12 and, under these
conditions, no accumulation of cobalamin 5'-phosphate is observed.
The Km of the enzyme for .alpha.-ribazole is 7.8 .mu.M.
Intracellular .alpha.-ribazole 5'-phosphate and .alpha.-ribazole
concentrations of 30 and 700 .mu.M, respectively, were measured by
HPLC during the production of cobalamins by SC510 Rif' in PS4
medium under the culture conditions described in Example 6.1.5a).
This shows that coenzyme B.sub.12 may be generated directly from
Ado-GDP-cobinamide by cobalamin-5'-phosphate synthase without the
participation of a cobalamin 5'-phosphatase.
[0381] The absence of accumulation or the presence of traces of
cobalamin 5'-phosphate in the P. dinitrificans SC510 Rif' cultures
confirms that coenzyme B.sub.12 is produced by the direct reaction
of Ado-GDP-cobinamide with .alpha.-ribazole in vivo.
[0382] This direct reaction has already been observed and described
in vitro in Propionibacterium shermanii (Ronzio et al., 1967; Renz,
1968). As the cobalamin-5'-phosphate synthase structural gene can
be only cobU or cobV, since the amplication in P. denitrificans of
a fragment carrying these two P. denitrificans cob genes leads to
an increase in cobalamin-5'-phosphate synthase activity by a factor
of 100, and since the cobU gene is the NN:DMBI PRT structural gene,
cobV is hence the cobalamin-5'-phosphate synthase structural
gene.
[0383] 6.1.7. Identification of the COBK Protein Encoded by the
cobK Gene
a) Assay of Precorrin-6x Reductase Activity.
[0384] This example illustrates the assay of a novel enzymatic
activity directly linked to the pathway biosynthesis of cobalamins.
The enzyme in question is precorrin-6x reductase.
[0385] The fractions containing precorrin-6x reductase activity
(approximately 0.05 unit, U) are incubated at 30.degree. C. for 60
min in 0.1 M Tris-HCl buffer pH 7.7 (250 .mu.l) in the presence of
1 mM EDTA, 500 .mu.M NADPH, 25 .mu.M [methyl-.sup.3H]SAM (80
.mu.Ci/.mu.mol), 4 .mu.M precorrin-6x (Thibaut et al., 1990) and
partially purified dihydroprecorrin-6x methylase (0.5 U) (see
preparation below). The reaction is then stopped by heating to
80.degree. C. for 5 min and, after centrifugation at 5000.times.g
for 5 min, the supernatant is injected onto a DEAE-Sephadex column
(containing 200 .mu.l of gel). The column is then washed
extensively with the Tris-HCl buffer, and the compounds bound are
eluted with 1 M HCl (4 ml). The radio activity in this eluent is
counted by liquid scintillation counting. The unit of enzymatic
activity is defined as the quantity of enzyme necessary for
reducing 1 nmol of precorrin-6x per h under these conditions.
[0386] Dihydroprecorrin-6x methylase is partially purified from a
crude extract of SC510 Rif' pXL253 on a Mono Q HR 5/5 (Pharmacia)
anion exchange column. The column is eluted with a linear gradient
of 0 to 0.4 M KCl in 0.1 M Tris-HCl buffer pH 7.7. The enzymatic
activity is eluted at 0.35 M KCl. This activity is detected and
quantified by means of the precorrin-6x reductase activity test
defined above (in the presence of precorrin-6x reductase (0.5 U) in
the incubation medium). After the Mono Q step, the fractions
containing dihydroprecorrin-6x methylase activity are completely
devoid of precorrin-6x reductase activity. The unit of methylase
activity is defined as the quantity of enzyme necessary for
transferring 1 nmol of methyl groups to dihydroprecorrin-6x per h
under the conditions described above.
b) Purification of Precorrin-6x Reductase Activity
[0387] Using the assay described above, the purification of
Pseudomonas denitrificans precorrin-6x reductase is carried out as
described below.
[0388] In a typical purification experiment, wet cells (100 g) of
strain SC510 Rif', into which plasmid pXL253 (plasmid pKT230 into
which the 8.7-kb fragment has been cloned at the EcoRI site, FIG.
13) has been introduced, are suspended in 0.1 M Tris-HCl pH 7.7/1
mM EDTA buffer (buffer A) (200 ml) and sonicated for 15 min at
4.degree. C. The crude extract is then recovered by centrifugation
at 1 h at 50,000.times.g and passed in three portions through a
Sephadex G-25 column equilibrated with buffer A. The three
fractions excluded from the gel are pooled and adjusted to 1 l with
buffer A. The proteins precipitating at between 25 and 40% ammonium
sulphate saturation are collected by centrifugation and resuspended
in buffer A (50 ml), and this solution is desalted through a
Sephadex G-25 column equilibrated with buffer B (25 mM Tris-HCl/500
.mu.M DTT/15% glycerol). The protein solution is then injected at
2.5 ml/min onto a Q Sepharose Fast Flow (Pharmacia) column
equilibrated with buffer B, and the proteins are eluted with a
buffer B/0.2 M KCl mixture. This fraction is desalted on a Sephadex
G-25 column equilibrated with buffer C (50 mM Tris-HCl/500 .mu.M
DTT/15% glycerol). The protein solution is then fractionated (100
mg of proteins at each chromatographic run) on a Mono Q HR 10/10
(Pharmacia) column using a gradient of 0 to 0.4 M KCl in buffer C,
and the fraction containing the activity is thereafter
chromatographed on a Phenyl-Superose HR 10/10 (Pharmacia) column in
a linear decreasing ammonium sulphate gradient (1 to 0 M). The
active fraction is desalted and the precorrin-6x reductase is
repurified on a Mono Q HR 5/5 column. It is eluted in 50 mM
Tris-HCl pH 8.1/500 .mu.M DTT/15% glycerol buffer with a gradient
of 0 to 0.2 M KCl. To complete the purification, the protein is
finally chromatographed on a Bio-Sil 250 (Bio-Rad) column eluted
with 20 mM potassium phosphate/50 mM sodium sulphate pH 6.8/500
.mu.M DTT/15% glycerol. After this step, the enzyme is more than
95% pure. It shows no contaminant protein in SDS-PAGE, the proteins
being visualised with silver nitrate. This degree of purity is
confirmed by the uniqueness of the NH.sub.2-terminal sequence. Its
molecular weight in this technique is 31,000. The different steps
of purification of precorrin-6x reductase with their purification
factor and their yield, are given in the table below.
TABLE-US-00016 TABLE Purification of precorrin-6x reductase Sp.
activity Purification Vol Proteins (u/mg of Purification step (ml)
(mg) proteins) Yield factor.sup.1 Crude extract 270 9600 0.535 --
-- A.S. 25 40% 100 4160 1.14 92 2.1 Q Sepharose 150 1044 3.64 74
6.8 Mono Q 10/10 55 67 24.5 32 46 Phenyl-Superose 10 2.2 325 14 607
Mono Q 5/5 2.5 0.082 5750 9.2 10750 Bio-sil 250 1.0 0.055 7650 8.2
14300
c) NH.sub.2-Terminal Sequence and Partial Internal Sequences of
Pseudomonas Denitrificans Precorrin-6x Reductase and Identification
of the Pseudomonas Denitrificans Structural Gene Coding for this
Activity
[0389] The NH.sub.2-terminal sequence of Pseudomonas denitrificans
precorrin-6x reductase, purified as described above, was determined
as described before. Six residues were identified:
Ala-Gly-Ser-Leu-Phe-Asp
[0390] Similarly, after tryptic digestion and separation of the
fragments by HPLC on a C-18 reversed-phase column, three internal
sequences were obtained: TABLE-US-00017 Ile-Gly-Gly-Phe-Gly-G
ly-Ala-Asp-Gly-Leu Arg-Pro-Glu-Trp-Val-Pro-Leu-Pro-Gly-Asp-Arg
Val-Phe-Leu-Ala-Ile-Gly
[0391] The NH.sub.2-terminal sequence of the COBK protein (FIG. 16)
corresponds exactly to the NH.sub.2-terminal sequence of
precorrin-6x reductase except that, in the sequence presented in
FIG. 16, a methionine precedes the peptide sequence determined by
direct sequencing. It follows from this that the amino-terminal
methionine is definitely excised in vivo by methionine
aminopeptidase (Ben Bassat and Bauer, 1987). Similarly, the three
internal sequences correspond to the three sequences 60 to 69, 112
to 122 and 143 to 148 of the COBK protein. The molecular weight of
the purified precorrin-6x reductase is estimated by SDS-PAGE
electrophoresis at 31,000. The COBK protein has a molecular weight
deduced from its sequence of 28,000 (FIG. 16). The correspondences
between the internal NH.sub.2-terminal sequences and the molecular
weights indicate clearly that the COBK protein corresponds to
precorrin-6x reductase. The cobK gene is the precorrin-6x reductase
structural gene.
d) Reaction Catalysed by Precorrin-6x Reductase
[0392] The enzymatic reaction of reduction of precorrin-6x is
strictly NADPH-dependant in P. denitrificans. NADPH cannot be
replaced by NADH. When the purified enzyme (or an active fraction
during purification, or even a crude enzyme extract) is incubated
under the conditions of the assay of activity, but in the absence
of SAM and of dihydroprecorrin-6x methylase, the product of the
reaction can then be purified by HPLC in the system described for
the purification of precorrin-6x (see Example 6.1.4.d). After
desalting and esterification (4% methanolic sulphuric acid,
20.degree. C., 24 h, argon atmosphere), the corresponding ester has
a mass m/z=1008. The product of the reaction catalysed by
precorrin-6x reductase is hence dihydroprecorrin-6x, also known as
precorrin-6y.
[0393] 6.1.8. Identification of the COBQ Protein Encoded by the
cobQ Gene
a) Assay of Cobyric Acid Synthase Activity
[0394] This example illustrates the assay of an enzymatic activity
of the pathway of biosynthesis of cobalamins which has never been
described hitherto. The enzyme in question is cobyric acid
synthase. This enzyme catalyses the amidation of the peripheral
carboxylic acid functions at positions b, d, e and g on the corrin
ring-system (see FIG. 19; PL. 68). The NH.sub.2-group donor is
L-glutamine, and each amidation reaction is accompanied by the
consumption of one ATP molecule.
[0395] The fraction to be assayed is incubated in darkness at
30.degree. C. for 60 min in 0.1 M Tris hydrochloride buffer pH 7.5
(250 .mu.l) containing 1 mM DTT, 1 mM EDTA, 1 mM ATP, 2.5 mM
MgCl.sub.2, 1 mM glutamine and 10 .mu.M Ado-cobyrinic acid di- or
pentaamide. The reaction is stopped by adding 0.1 M aqueous
potassium cyanide solution (25 .mu.l). After heating to 80.degree.
for 10 min and centrifugation at 3000.times.g for 10 min, the
compounds formed, present in the supernatant, are analysed by HPLC.
The unit of activity is defined as the quantity of enzyme necessary
for generating 1 nmol of amide functions per h under these
conditions.
[0396] 5'-Deoxy-5'-adenosyl(Ado)-cobyrinic acid diamide and
pentaamide are isolated from cultures of strain SC510 in PS4
medium, using the method the principle of which is described in
Example 9.
b) Purification of Cobyric Acid Synthase
[0397] Using the assay described in Example 6.1.8 a), purification
of Pseudomonas denitrificans cobyric acid synthase is carried out
as described below.
[0398] In a typical purification experiment, wet SC510 Rif' cells
(6 g), into which strain plasmid pXL618 (see Example 4.5.2) has
been introduced, are sonicated in 0.1 M Tris-HCl pH 7.7, 1 mM DTT,
1 mM EDTA buffer (15 ml). After centrifugation (50,000.times.g for
1 h), the extract is brought to 20% of glycerol (vol/vol). 10 mM
Tris-HCl, 1 mM DTT, 20% glycerol buffer (24 ml) are added to the
crude extract (8.5 ml; 203.5 mg of proteins). The solution is
injected onto Mono Q HR 10/10 (Pharmacia) at 2 ml/min, equilibrated
with 50 mM Tris-HCl pH 7.7, 1 mM DTT, 20% glycerol buffer. The
proteins are eluted with a linear gradient of 0.5 M NaCl and the
active fractions are pooled and brought to 1 mM EDTA. The solution
is brought to 0.85 M with respect to ammonium sulphate and injected
onto a Phenyl-Superose HR 5/5 (Pharmacia) column equilibrated in
Tris-HCl pH 7.7, 1 mM DTT, 0.85 M ammonium sulphate buffer, and the
proteins are eluted with a linear decreasing gradient of 0.85 M to
0 M ammonium sulphate. The fractions are immediately brought to 20%
of glycerol. The active fraction is concentrated to 2.5 ml by
ultrafiltration and chromatographed on a PD 10 (Pharmacia) column
equilibrated and eluted with 50 mM Tris-HCl pH 8.3, 1 mM DTT, 20%
glycerol (vol/vol) buffer. The protein fraction is collected and
injected onto a mono Q HR 5/5 column equilibrated with the same
buffer, and the proteins are eluted with a linear gradient of 0.5 M
NaCl. Gel permeation chromatography on Bio-Sil 250 (Bio-Rad) gel in
50 mM Tris-HCl pH 7.5, 1 mM DTT, 20% glycerol, 0.1 M NaCl buffer
medium finally enables a protein which is more than 97% pure to be
obtained. It shows no contaminant protein in SDS-PAGE. This purity
is confirmed by the uniqueness of the NH.sub.2-terminal sequence.
Its molecular weight in this technique is 57,000. The different
steps of purification of cobyric acid synthase with their
purification factor and their yield are given in the table below.
TABLE-US-00018 TABLE Purification of cobyric acid synthase Sp.
activity U/mg Purification Vol Proteins a b Purification step (ml)
(mg) A B Yield* factor.sup.1 Crude extract 8.5 203 114 / 118 -- --
Mono Q 10/10 8.0 35.5 388 / 425 60 3.4 Phenyl- 8.0* 3.23 1988 /
2021 28 17 Superose Mono Q 5/5 1.0 1.20 4549 / 4085 24 40 Bio-Sil
250 0.75 0.88 4992 / N.D. 19 44 a / with Ado-cobyrinic acid
a,c-diamide as substrate b / with Ado-cobyrinic acid pentaamide as
substrate ND = Not Determined
[0399] The very high degree of purity of the purified protein,
together with the constancy of the ratio of the activities of
amidation of cobyrinic acid diamide and pentaamide throughout the
process of purification of the protein (see table above), indicate
unambiguously that one and the same protein is responsible for the
four activities of amidation of the corrin ring-system at positions
b, d, e and g.
c) NH.sub.2-Terminal Sequence of Pseudomonas Denitrificans Cobyric
Acid Synthase and Identification of the Pseudomonas Denitrificans
Structural Gene Coding for this Activity
[0400] The NH.sub.2-terminal sequence of Pseudomonas denitrificans
cobyric acid synthase was determined as described above. Sixteen
residues were identified: TABLE-US-00019
Thr-Arg-Arg-Ile-Met-Leu-Gln-Gly-Thr-Gly-Ser-Asp-
Val-Gly-Lys-Ser
[0401] The NH.sub.2-terminal sequence of the COBQ protein (FIG. 47)
corresponds exactly to this sequence except that, in the sequence
presented in FIG. 47, a methionine precedes the peptide sequence
determined by direct sequencing. It follows from this that the
amino-terminal methionine is definitely excised in vivo by
methionine aminopeptidase (Ben Bassat and Bauer, 1987). The
molecular weight of the purified cobyric acid synthase is estimated
by SDS-PAGE electrophoresis at 57,000. The COBQ protein has a
molecular weight deduced from its sequence of 52,000 (FIG. 47). The
correspondences between the NH.sub.2-terminal sequences and the
molecular weights indicate clearly that the COBQ protein
corresponds to cobyric acid synthase. The cobQ gene is the cobyric
acid synthase structural gene.
[0402] 6.1.9. Identification of the COBO Protein Encoded by the
cobO Gene
a) Assay of cob(I)alamin Adenosyltransferase (EC 2.5.1.17)
Activity
[0403] This example illustrates the assay of an enzymatic activity
directly linked to the pathway of biosynthesis of cobalamins. The
enzyme in question is cob(I)alamin adenosyltransferase (EC
2.5.1.17). This enzyme was demonstrated in bacterial cells (Ohta et
al., 1976, Brady et al. , 1962) and animal cells (Fenton et al.,
1978). It was purified from Clostridium tetanomorphum (Vitols et
al., 1966).
[0404] The fractions containing cob(I)alamin adenosyltransferase
activity (approximately 20 units) are incubated anaerobically at
30.degree. C. for 15 min protected from light in 0.2 M Tris-HCl
buffer pH 8.0 (1 ml) in the presence of 5 mM DTT, 400 .mu.M
[8-.sup.14C]-ATP (2.5 .mu.Ci/.mu.mol), 800 MM MnCl.sub.2, 50 .mu.M
hydroxocobalamin or diaquacobinamide and KBH.sub.4 (3 mg). The
reaction is then stopped by heating to 80.degree. C. for 10 min
and, after centrifugation at 15000.times.g for 5 min, the
supernatant (200 .mu.l) is analysed by HPLC (Gimsing et al., 1986,
Jacobsen et al., 1986).
[0405] The unit of enzymatic activity is defined as the quantity of
enzyme necessary for generating 1 nmol of adenosylcorrinoid per min
under these conditions.
b) Purification of cob(I)alamin Adenosyltransferase Activity
[0406] Using the assay described in Example 6.1.9 a), the
purification of Pseudomonas denitrificans cob(I)alamin
adenosyltransferase is carried out as described below.
[0407] In a typical purification experiment, wet cells (10 g) of
strain SC510 Rif' in which the cobO gene has been amplified are
suspended in 0.2 M Tris-HCl buffer pH 8.0 (20 ml) and sonicated for
40 min at 4.degree. C. The crude extract is then recovered by
centrifugation for 1 h at 50,000.times.g and desalted on PD10
(Pharmacia) columns equilibrated with 50 mM Tris-HCl pH 8.0, 5 mM
DTT buffer (buffer A). The protein solution is then fractionated
(280 mg of proteins at each chromatographic run) on a Mono Q HR
10/10 (Pharmacia) column using a gradient of 0 to 0.5 M KCl in
buffer A, and the fractions containing the activity are then
pooled, concentrated by ultrafiltration and chromatographed on a
Phenyl-Superose HR 10/10 (Pharmacia) column in a linear decreasing
ammonium sulphate gradient (1.7 to 0 M), the column being
equilibrated in 0.1 M Tris-HCl pH 8.0, 5 mM DTT buffer. To complete
the purification, the protein is finally chromatographed, after
concentration by ultrafiltration, on a Bio-Sil 250 (Bio-Rad) column
eluted with 50 mM Tris-HCl pH 7.5, 0.1 M NaCl, 5 mM DTT buffer.
[0408] After this step, the enzyme is more than 95% pure. It does
not show any contaminant protein in SDS-PAGE. Its molecular weight
in this technique is 28,000. This degree of purity is confirmed by
the uniqueness of the NH.sub.2-terminal sequence. The different
steps of purification of cob(I)alamin adenosyltransferase, with
their purification factor and their yield, are given in the table
below for the following two substrates: diaquacobinamide (a) and
hydroxocobalamin (b). These results demonstrate the absence of
specificity of this enzyme for the nature of the corrinoid
substrate. Moreover, all corrinoids of the biosynthetic pathway
between cobyrinic acid diamide and B.sub.12 have been isolated
(Blanche et al., unpublished results) in their native form, and
have proved to be in coenzyme form. This demonstrates that the
natural substrate of cob(I)alamin adenosyltransferase is cobyrinic
acid a,c-diamide. TABLE-US-00020 TABLE Purification of cob(I)alamin
adenosyltransferase Sp. activity U/mg Purification Vol Proteins a b
Purification step (ml) (mg) A B Yield* factor.sup.1 Crude
extract.sup.c 100 1400 5.4 / 3.4 -- -- Mono Q 10/10 90 140 34.9 /
14.1 65 6.5 Phenyl- 30 15.9 84.5 / 49.5 18 16 Superose Bio-Sil 250
6.5 2.9 182.4 / 88.7 7.0 34 .sup.cafter desalting on PD10
c) NH.sub.2-Terminal Sequence of Pseudomonas Denitrificans
cob(I)alamin Adenosyltransferase and Identification of the
Pseudomonas Denitrificans Structural Gene Coding for this
Activity.
[0409]
[0410] The NH.sub.2-terminal sequence of Pseudomonas denitrificans
cob(I)alamin adenosyltransferase, purified as described in Example
6.1.9 b), was determined as described above. 13 residues were
identified:
Ser-Asp-Glu-Thr-?-Val-Gly-Gly-Glu-Ala-Pro-Ala-Lys-Lys
[0411] The NH.sub.2-terminal sequence of the COBO protein (FIG. 47)
corresponds exactly to the NH.sub.2-terminal sequence of
cob(I)alamin adenosyltransferase except that, in the sequence
presented in FIG. 47, a methionine precedes the peptide sequence
determined by direct sequencing. It follows from this that the
amino-terminal methionine is definitely excised in vivo by
methionine aminopeptidase (Ben Bassat and Bauer, 1987). The
molecular weight of the purified cob(I)alamin adenosyltransferase
is estimated by SDS-PAGE electrophoresis at 28,000. The COBO
protein has a molecular weight deduced from its sequence of 24,000
(FIG. 47). The correspondences between NH.sub.2-terminal sequences
and the molecular weights indicate clearly that the COBO protein
corresponds to cob(I)alamin adenosyltransferase. The cobO gene is
the cob(I)alamin adenosyltransferase structural gene.
[0412] 6.1.10. Identification of the COBN Protein Encoded by the
cobN Gene
a) Demonstration of the Activity of Conversion of Hydrogenobyrinic
Acid a,c-Diamide to Cobyrinic Acid a,c-Diamide
[0413] This example illustrates the demonstration of an enzymatic
activity directly linked to the pathway of biosynthesis of
cobalamins which has never been described hitherto. The activity in
question is that of conversion of hydrogenobyrinic acid a,c-diamide
to cobyrinic acid a,c-diamide.
[0414] This activity is demonstrated, inter alia, by the following
typical experiment. A crude extract of strain SC510 Rif' is
obtained by sonication of wet cells (10 g) in 0.2 M Tris-HCl buffer
pH 8.0 (20 ml), followed by removal of the cell debris by
centrifugation for 1 h at 50,000.times.g. Proteins (1000 mg) of
this extract are incubated for 1 h at 30.degree. C. with
carbon-14-labelled hydrogenobyrinic acid diamide (32 nmol; 50
.mu.Ci/.mu.mol) in 0.2 M Tris-HCl buffer pH 8.0 (40 ml) containing
7 mM ATP and 200 .mu.M CoCl.sub.2. The reaction is stopped by
adding 1 M KH.sub.2PO.sub.4 (7.5 ml) and 0.3 M KCN (6 ml), followed
by heating for 10 min at 80.degree. C. After centrifugation at
15000.times.g for 50 min, HPLC analysis of the supernatant shows:
(1) the formation during the incubation of cobyrinic acid
a,c-diamide (19.2 nmol) having the same specific radioactivity as
the starting hydrogenobyrinic acid a,c-diamide, and (2) the
disappearance of a corresponding quantity of the latter. To confirm
that the product is indeed cobyrinic acid a,c-diamide, the product
is purified by HPLC and then esterified in methanol containing 5%
of sulphuric acid (18 h, 20.degree. C.). The authenticity of the
cobyrinic acid a,c-diamide pentamethyl ester produced is
demonstrated by TLC (relative to a reference sample) and mass
spectrometry. It should be noted that, under similar incubation
conditions in which the radioactive labelling is introduced, not
into the hydrogenobyrinic acid a,c-diamide, but into the cobalt
(using cobalt-57), cobalt-57-labelled cobyrinic acid a,c-diamide is
biosynthesised and the same conclusions could be drawn.
[0415] Carbon-14-labelled hydrogenobyrinic acid a,c-diamide is
obtained in the following manner: hydrogenobyrinic acid is
biosynthesised in vitro using [methyl-.sup.14C] SAM, then converted
to hydrogenobyrinic acid a,c-diamide and purified by HPLC as
described in Example 6.1.2.
[0416] This study demonstrates that the insertion of cobalt takes
place at hydrogenobyrinic acid a,c-diamide level in P.
denitrificans. Under the conditions described, hydrogenobyrinic
acid is not a substrate for enzymatic chelation with cobalt.
b) Assay and Purification of a Protein of Strain SC510 Rif'
Involved in the Conversion of Hydrogenobyrinic Acid a,c-Diamide to
Cobyrinic Acid a,c-Diamide
[0417] The fraction to be assayed (0.5 to 2 units) is incubated for
60 min at 30.degree. C. with crude extract (50 .mu.l) of strain
SC510 Rif' obtained as described above, 7 mM ATP, 200 .mu.M
CoCl.sub.2, and 7 .mu.M carbon-14-labelled hydrogenobyrinic acid
a,c-diamide (50 .mu.Ci/.mu.mol) in 0.1 M Tris-HCl buffer pH 8.0
(400 .mu.l). The reaction is stopped by adding 1 M KH.sub.2PO.sub.4
(75 .mu.l) and 0.3 M KCN (60 .mu.l), followed by heating for 10 min
at 80.degree. C. After centrifugation at 15000.times.g for 15 min,
the supernatant is analysed by HPLC in order to quantify the
cobyrinic acid a,c-diamide formed (see Example 9). The unit of
enzymatic activity is defined as the quantity of enzyme necessary
for generating 1 nmol of cobyrinic acid a,c-diamide per h under
these conditions. Under these conditions, it is apparent that
extracts of strain SC510 Rif' into which plasmid pXL1909 has been
introduced (see Example 4.5.2) possess an activity between 20 and
50 times as high as extracts of strain SC510 Rif'. It is on this
basis that a protein which is alone responsible for this
amplication of activity is purified.
[0418] In a typical purification experiment, wet cells (10 g) of
strain SC510 Rif', into which plasmid pXL1909 has been introduced,
are suspended in 0.2 M Tris-HCl buffer pH 8.0 (20 ml) and sonicated
for 30 min at 4.degree. C. The crude extract is then recovered by
centrifugation for 1 h at 50,000.times.g and desalted on PD10
(Pharmacia) columns equilibrated with 0.1 M Tris-HCl buffer pH 8.0
(buffer A). The protein solution is then fractionated (213 mg of
proteins at each chromatographic run) on a Mono Q HR 10/10
(Pharmacia) column using a gradient of 0 to 0.5 M KCl in buffer A,
and the fractions containing the activity are then pooled,
concentrated by ultrafiltration, desalted on PD10 (Pharmacia)
columns equilibrated with 0.1 M Tris-HCl buffer pH 7.2 (buffer B)
and chromatographed on a Mono Q HR 10/10 (Pharmacia) column using a
gradient of 0 to 0.5 M KCl in buffer B. The fractions containing
the activity are pooled, concentrated by ultrafiltration, desalted
on PD10 (Pharmacia) columns equilibrated with buffer B and
chromatographed on a Mono Q HR 5/5 (Pharmacia) column using a
gradient of 0 to 0.5 M KCl in buffer B. To complete the
purification, the protein is finally chromatographed on a Bio-Sil
250 (Bio-Rad) column eluted with 20 mM potassium phosphate/50 mM
sodium sulphate pH 6.8.
[0419] After this step, the enzyme is more than 95% pure. It does
not show any contaminant protein in SDS-PAGE. Its molecular weight
in this technique is 135,000. This degree of purity is confirmed by
the uniqueness of the NH.sub.2-terminal sequence. The different
steps of purification of the protein of strain SC510 Rif' involved
in the conversion of hydrogenobyrinic acid a,c-diamide to cobyrinic
acid a,c-diamide, with their purification factor and their yield,
are given in the table below. TABLE-US-00021 TABLE Purification of
a protein of strain SC510 Rif.sup.r involved in the conversion of
hydrogenobyrinic acid a,c-diamide cobyrinic acid a,c-diamide Sp.
activity Purification Vol Proteins (u/mg of Purification step (ml)
(mg) proteins) Yield factor.sup.1 Crude extract 31.5 1278 0.23 --
-- Mono Q 10/10 44 79.2 2.4 64 10 Mono Q 10/10 21 33.6 6.8 78 30
Mono Q 5/5 3 6.6 16.0 36 70 Bio-Sil 250 1.8 5.9 16.3 33 71
[0420] c) NH.sub.2-terminal sequence of the Pseudomonas
denitrificans protein involved in the conversion of
hydrogenobyrinic acid a,c-diamide to cobyrinic acid a,c-diamide,
and identification of the Pseudomonas denitrificans structural gene
coding for this activity
[0421] The NH.sub.2-terminal sequence of this protein, purified as
described in Example 6.1.10b), was determined as described above.
Six residues were identified:
[0422] His-Leu-Leu-Leu-Ala-Gln
[0423] The NH.sub.2-terminal sequence of the COBN protein (FIG. 47)
corresponds exactly to the NH.sub.2-terminal sequence of the
purified protein except that, in the sequence presented in FIG. 47,
a methionine precedes the peptide sequence determined by direct
sequencing. It follows from this that the amino-terminal methionine
is definitely excised in vivo by methionine aminopeptidase (Ben
Bassat and Bauer, 1987). The molecular weight of the purified
protein is estimated by SDS-PAGE electrophoresis at 135,000. The
COBN protein has a molecular weight deduced from its sequence of
138,000 (FIG. 47). The correspondences between the
NH.sub.2-terminal sequences and the molecular weights indicated
clearly that the COBN protein corresponds to the protein involved
in the conversion of hydrogenobyrinic acid a,c-diamide to cobyrinic
acid a,c-diamide. The cobN gene is hence the structural gene for
this protein.
[0424] 6.1.11. Identification of the COBP Protein Encoded by the
cobP Gene
a) Assay of Cobinamide Kinase Activity
[0425] This example illustrates the assay of an enzymatic activity
of the pathway of biosynthesis of cobalamins which has never been
studied hitherto. The activity in question is that of cobinamide
kinase. It catalyses the ATP-dependent phosphorylation of the
hydroxyl group of the (R)-1-amino-2-propanol residue of
Ado-cobinamide to generate cobinamide phosphate.
[0426] The fraction to be assayed is incubated in darkness at
30.degree. C. for 60 min in 0.1 M Tris-HCl buffer pH 8.8 (500
.mu.l) containing 1 mM EDTA, 1 mM ATP, 2.5 mM MgCl.sub.2 16 .mu.M
Ado-cobinamide (Blanche et al., 1989). The reaction is stopped by
adding 20 mM aqueous potassium cyanide solution (500 .mu.l). After
heating to 80.degree. C. for 10 min and centrifugation at
5,000.times.g for 10 min, the cobinamide phosphate formed, present
in the supernatant, is assayed by HPLC (see Example 9) using the
following simplified linear gradient: 25% to 30% of B in A in the
course of 15 min, then 30% to 100% of B in the course of 12 min,
and 3 min at 100% of B.
[0427] The unit of activity is defined as the quantity of enzyme
necessary for generating 1 nmol of cobinamide phosphate from
cobinamide per h under these conditions.
b) Assay of Cobinamidephosphate Guanylyltransferase Activity
[0428] This example illustrates the assay of an enzymatic activity
of the pathway of biosynthesis of cobalamins which has never been
studied hitherto. The activity in question is that of
cobalamidephosphate guanylyltransferase. It catalyses the addition
of the GMP portion of a GTP molecule to Ado-cobinamide phosphate,
thereby generating one molecule of GDP-cobinamide and liberating
one molecule of pyrophosphate.
[0429] This activity is assayed under the same conditions as
cobinamide kinase, except that Ado-cobinamide phosphate (16 .mu.M)
(Blanche et al., 1989) and GTP (2 mM) replace Ado-cobinamide and
ATP, respectively, during the incubation.
[0430] The unit of activity is defined as the quantity of enzyme
necessary for generating 1 nmol of GDP-cobinamide from cobinamide
phosphate per h under these conditions.
c) Purification of Cobinamide Kinase
[0431] Using the assay described in Example 6.1.11a), the
purification of Pseudomonas denitrificans kinase is carried as
described below.
[0432] In a typical purification experiment, wet SC510 Rif' cells
(5 g), into which strain plasmid pXL623 has been introduced (see
Example 4.5.2) are sonicated in 0.1 M Tris buffer pH 7.6 (buffer A)
(20 ml). After centrifugation (50,000.times.g for 1 h) and dialysis
for 4 h against buffer A, the retentate (4.5 ml) is injected onto
Mono Q HR 10/10 (Pharmacia) equilibrated with buffer A. The
proteins are eluted with a linear gradient of 0.4 M NaCl, and the
pooled active fractions are passed through a PD-10 (Pharmacia)
column equilibrated in 30 mM Tris-HCl/5 mM potassium phosphate/5
.mu.M calcium chloride pH 7.6 (buffer B). The protein solution is
fractionated on a Bio-Gel HPHT (Bio-Rad) column equilibrated in
buffer B and eluted with a gradient of 5 to 350 mM potassium
phosphate. The active fractions are pooled and brought to 500 mM
with respect to ammonium sulphate, and then fractionated on a
Phenyl-Superose HR 5/5 (Pharmacia) column eluted with a decreasing
ammonium sulphate gradient. The fraction containing the activity is
finally repurified on a Mono Q HR 5/5 column in Tris-HCl at pH 7.3.
After this step, the protein is more than 97% pure. It shows no
contaminant protein in SDS-PAGE. This purity is confirmed by the
uniqueness of the NH.sub.2-terminal sequence. Its molecular weight
in this technique is 20,000. The different steps of purification of
cobinamide kinase, with their purification factor and their yield,
are given in Table A.
[0433] The fractions containing cobinamide kinase activity also
possess cobinamidephosphate guanylyltransferase activity. Moreover,
as shown by the results presented in the table above, the ratio of
these two activities remains constant in the fractions throughout
the purification. Lastly, the purified protein possesses a very
high degree of purity, exceeding 97%. These results collectively
hence indicate unambiguously that one and the same protein is
responsible for both successive activities, namely cobinamide
kinase and cobinamidephosphate guanylyltransferase of the pathway
of biosynthesis of cobalamins in Pseudomonas denitrificans.
[0434] d) NH.sub.2-Terminal sequence of Pseudomonas denitrificans
cobinamide kinase/cobinamidephosphate gunaylyltransferase, and
identification of the Pseudomonas denitrificans structural gene
coding for this activity
[0435] The NH.sub.2-terminal sequence of Pseudomonas denitrificans
cobinamide kinase/cobinamidephosphate guanylyltransferase was
determined as described above. Ten residues were identified:
[0436] Ser-Ser-Leu-Ser-Ala-Gly-Pro-Val-Leu-Val
[0437] The NH.sub.2-terminal sequence of the COBP protein (FIG. 47)
corresponds exactly to this sequence except that, in the sequence
presented in FIG. 47, a methionine precedes the peptide sequence
determined by direct sequencing. It follows from this that the
amino terminal methionine is definitely excised in vivo by
methionine aminopeptidase (Ben Bassat and Bauer, 1987). The
molecular weight of the purified cobinamide
kinase/cobinamidephosphate guanylyltransferase is estimated by
SDS-PAGE electrophoresis at 20,000. The COBP protein has a
molecular weight deduced from its sequence of 19,500 (FIG. 47). The
correspondences between the NH.sub.2-terminal sequences and the
molecular weights indicate clearly that the COBP protein
corresponds to cobinamide kinase/cobinamidephosphate
guanylyltransferase. The cobP gene is the cobinamide
kinase/cobinamidephosphate guanylyltransferase structural gene.
[0438] 6.2--Determination of the Properties of COB Proteins by
Measurement of Accumulated Biosynthesis Intermediates
[0439] This example illustrates how it is possible to assign an
enzymatic activity to a COB protein of Pseudomonas denitrificans.
This activity is assigned on the basis of data obtained relating to
accumulated biosynthesis intermediates in the Cob mutant or mutants
blocked in the step in question. In effect, if a mutant accumulates
a biosynthesis intermediate, it is very probable that this mutant
is blocked in the step which has the intermediate in question as
its substrate.
[0440] 6.2.1. Properties of the COBC and COBD Proteins
[0441] The Cob mutants G643 (Agrobacterium tumefaciens) and G572
(Pseudomonas putida) already described in Examples 1 and 4 are
blocked in the step corresponding to the COBC protein. In effect,
these two mutants are not complemented by the inactivating
insertions of transposons Tn5 which occur in the cobC gene. The two
strains G643 and G572, as well as the unmutated parent strains
[C58-C9 Rif' and KT 2440 Rif' (Cameron et al., 1989)), were
cultured in PS4' medium for A. tumefaciens and PS4'' medium for P.
putida (PS4' and PS4'' correspond to PS4 medium containing 100-fold
and 1000-fold, respectively, less cobalt than PS4 described above)
for 3 days as described above. .sup.57CoCl.sub.2 was added to the
cultures (2.5 .mu.Ci/0.1 .mu.m for a 25-ml culture). The
intracellular corrinoids were isolated in their native form and
identified by their HPLC behaviour. The parent strains do not
accumulate corrinoids other than coenzyme B.sub.12. The two mutants
G643 and G572 accumulate adenosylated cobyric acid in respective
proportions of 11% and 6%. These % proportions are calculated
relative to the level of coenzyme B.sub.12 synthesised by the
parent strain. Apart from cobyric acid, mutant G643 accumulates
cobyrinic acid pentaamide in a proportion of 2%; cobyrinic acid
pentaamide is the intermediate which precedes cobyric acid. A study
of these mutants brings out the fact that they are blocked after
cobyric acid. All these Cob mutants are blocked either between
uro'gen III and cobinamide, or between cobinamide and the
cobalamins. The mutants G643 and G572 are blocked between uro'gen
III and cobinamide. Now, if these mutants are blocked before
cobinamide, and both accumulate cobyric acid, the proteins for
which they code can participate only in the enzymatic step
(referred to as cobinamide synthase) which catalyses the amidation
of cobyric acid with an aminopropanol residue to give cobinamide;
they can also possibly participate in the synthesis of the
substrate of the reaction which provides aminopropanol, if not
aminopropanol itself. The cobC gene codes for a protein which is
either cobinamide synthase or one of its subunits.
[0442] The Cob mutant G634 of Agrobacterium tumefaciens which is
blocked in the step corresponding to the cobD gene was analysed in
the same manner. This mutant is not complemented by the
inactivating insertions in the cobD gene (Example 4.1). The only
intracellular corrinoid found in this mutant is adenosylated
cobyric acid. Like the above mutants, this mutant codes for a
protein participating in the conversion of cobyric acid to
cobinamide, or else possibly in the synthesis of the other
substrate of the reaction.
[0443] These two different genes (cobC and cobD) code for two
proteins which participate in the same step.
[0444] 6.2.2. Properties of the COBF to COBM Proteins
[0445] The Agrobacterium tumefaciens mutants already described were
studied, the study described in Example 4.2 having shown in which
genes each of these mutants is blocked. They are the following
mutants: G612 (cobF), G615 (cobG), G616 (cobH), G613 (cobI), G611
(cobJ), G620 (cobK), G638 (cobL) and G609 (cobM); we have shown in
brackets the Pseudomonas denitrificans gene responsible for the
complementation of these mutants (Example 5), which hence
corresponds to the gene mutated in this mutant. These mutants were
cultured in PS4 medium as described above with labelled cobalt.
After four days' incubation, the mutants were analysed for their
intracellular content of corrinoids and decobaltocorrinoids (see
Examples 6.1.2 and 9). Table: Intermediates accumulated by
Agrobacterium tumefaciens mutants blocked in the genes of the
8.7-kb fragment of Pseudomonas denitrificans TABLE-US-00022
Intracellular Intracellular Mu- decobaltocorrinoids in %.sup.1
corrinoids as tated Strain HBA HBAM HBAD % of cobalamins gene
C58-C9* 100 100 100 coenzyme B.sub.12 100 -- G612 <5 <5 64
cobinamide 2.2 cobF coenzyme B.sub.12 34 G615 <5 <5 84
coenzyme B.sub.12 17 cobG G616 35 <10 <10 coenzyme B.sub.12
13 cobH G613 <5 <5 57 coenzyme B.sub.12 <1 cobI G611 <5
<5 65 coenzyme B.sub.12 <1 cobJ G620 12 <5 <10 coenzyme
B.sub.12 <1 cobK G638 <5 <5 47 coenzyme B.sub.12 <1
cobL G609 <5 <5 33 coenzyme B.sub.12 <1 cobM HBA:
hydrogenobyrinic acid HBAM: hydrogenobyrinic acid monoamide HBAD:
hydrogenobyrinic acid diamide *in fact, this is strain C58-C9
Rif.sup.rNal.sup.r already described (Cameron et al., 1989)
.sup.1the values are expressed as % of the same intermediates
accumulated in the unmutated parent strain C58-C9
Rif.sup.rNal.sup.r.
[0446] These results show that none of the mutants accumulate any
corrinoid (with the exception of the mutant inactivated in the cobF
gene, G612, which, for its part, accumulates cobinamide but at a
low level equivalent to 2.2% of the cobalamins synthesised by the
unmutated strain). However, some mutants (G612, G615 and G616) have
levels of cobalamins which represent more than 10% of the cobalamin
level of the parent strain. It is probable that all these mutants
are blocked at least before cobyrinic acid diamide. All the mutants
accumulate hydrogenobyrinic acid and hydrogenobyrinic acid diamide
in smaller quantities than the unmutated strain; they are hence
very probably blocked before hydrogenobyrinic acid. It may be
concluded that all the cobF to cobG genes code for proteins which
participate before hydrogenobyrinic acid. Mutant G613 is known to
be mutated in the cobI gene which codes for SP.sub.2MT,
participating well before hydrogenobyrinic acid. For this mutant,
the results of the present example relating to the accumulation of
intermediates are in complete agreement with the step inactivated
in this mutant, namely, this mutant accumulates no intermediate
after hydrogenobyrinic acid at a level higher than that observed
with the unmutated strain. This result is, for the cobF, cobJ,
cobL, and cobM genes, consistent with those of Example 6.4, where
it proposed that these genes code for proteins which catalyse
SAM-dependent transfers of methyl and hence which participate
before hydrogenobyrinic acid. With the exception of cobI, which is
the SP.sub.2MT structural gene, these genes participate after
precorrin-3. In effect, since they are neither the structural genes
for SUMT nor for SP.sub.2MT, they inevitably participate later,
that is to say after precorrin-3 (all the cob genes described in
the present invention participate between uro'gen III and the
cobalamins). These cobF to cobH and cobJ to cobM genes code for
enzymes which participate between precorrin-3 and hydrogenobyrinic
acid.
[0447] 6.2.3. Properties of the COBS and COBT Proteins
[0448] The mutant G2035 described in Examples 1 and 4.3 is blocked
in the step corresponding to the COBS protein. The mutant G2037
described in Example 1 is blocked in this step corresponding to the
COBT protein. These strains, as well as the parent strain
(Agrobacterium tumefaciens C58C9Rif'), are cultured in PS4' medium
(this is PS4 medium in which the cobalt chloride concentration is
100-fold lower than in PS4 medium) in the presence of radioactive
cobalt 57CoCl.sub.2 for 3 days, and their intracellular content of
decobaltocorrinoids is analysed, as is the corrinoid content, as
already described above (see Example 6.2.2). The strains G2035 and
G2037 do not accumulate corrinoids, and large concentrations
(greater than those observed with the parent strain) of
hydrogenobyrinic acid and hydrogenobyrinic acid mono- and diamide
are present only with strain G2035. This mutant is probably blocked
in a step located after hydrogenobyrinic acid diamide and before
cobyrinic acid diamide. Consequently, the cobs gene is considered
to code for one of the enzymes involved in the conversion of
hydrogenobyrinic acid diamide to cobyrinic acid diamide; this
protein may hence participate either in the insertion of cobalt, or
in the reduction of the cobalt of unadenosylated cobyrinic acid
a,c-diamide. In contrast, the mutant G2037 is considered to be
blocked in a step located upstream of hydrogenobyrinic acid. The
cobT gene is considered to code for a protein involved in an
enzymatic step upstream of hydrogenobyrinic acid and downstream of
precorrin-3 (other structural genes coding for enzymes involved
downstream of precorrin-3 have already been identified). Another
possibility for the COBT protein is that it participates, as
proposed in Example 5, as a cobalt-binding protein and/or as a
protein which interacts with other protein(s) via its acidic
portion.
[0449] 6.2.4. Properties of the COBV Protein
[0450] The mutants G2039 and G2040 described in Examples 1 and 4.4
are blocked in the step corresponding to the COBV protein. These
strains, as well as the parent strain, are cultured in PS4' medium
for 3 days in the presence of radioactive cobalt .sup.57CoCl.sub.2,
and their intracellular content of decobaltocorrinoids is then
analysed and the corrinoid content is determined as described in
Example 9. Strains G2039 and G2040 accumulate cobyric acid,
cobinamide, cobinamide phosphate and GDP-cobinamide. These mutants
are probably blocked in an enzymatic step downstream of
GDP-cobinamide. The cobV gene is considered to code for an enzyme
involved in the conversion of GDP-cobinamide to cobalamin, see FIG.
5. This result is in complete agreement with the
cobalamin-5'-phosphate synthase activity of the COBV protein which
possesses Ado-GDP-cobinamide as a substrate.
[0451] 6.3--Determination of the Activity of COB Proteins by
Studies of Affinity for SAM
[0452] This example illustrates how it is possible, using COB
proteins purified from Pseudomonas denitrificans, to demonstrate in
vitro a SAM-binding activity. If a COB protein possesses such an
activity, it means that this COB protein is a methyltransferase of
the pathway, and that it participates in one of the transfers of
the eight methyl groups which occur between the uro'gen III and
cobyrinic acid.
[0453] 6.3.1. Test of Affinity for SAM on a Purified Protein
[0454] The test is based on the principle according to which methyl
transferases of the pathway of biosynthesis of cobalamins
definitely have an SAM-binding site. This site must be demonstrated
by a higher affinity of SAM than for any protein which does not
specifically bind SAM. After incubation of the protein under study
in the presence of an excess of radioactive SAM, the latter is
separated from the free SAM by gel permeation chromatography. The
radioactivity appearing in the fraction having the molecular weight
of the protein corresponds to the SAM bound during the incubation.
The chromatography is performed at 2.degree. C. in order to limit
to the maximum the release of bound SAM during the separation.
[0455] The protein (approximately 10 .mu.g) is incubated for 10
minutes at 30.degree. C. in 0.1 M Tris-HCl pH 7.7 (200 .mu.l) with
[methyl-.sup.3H]SAM (5 nmol; 1 .mu.Ci). After incubation, a portion
(100 .mu.l) of the mixture is immediately injected onto a TSK-125
(Bio-Rad) column eluted at 1 ml/minute with the 50 mM sodium
sulphate/20 mM sodium dihydrogen phosphate mixture, pH 6.8,
recommended by the distributor of this column. 0.5-ml fractions are
collected and subjected to liquid scintillation counting. The
retention times of the protein and the SAM are obtained directly
from the recording of the absorbance of the eluate at 280 nm.
[0456] 6.3.2. In vitro Study of the Binding of SAM to the COBA and
COBF Proteins of Pseudomonas denitrificans
[0457] a) Purification of the COBF and COBA Proteins
[0458] The COBF protein of Pseudomonas denitrificans is purified as
described below. In a typical purification experiment, wet cells (5
g) of strain SC150 Rif' into which plasmid pXL1546 has been
introduced (see Ex. 7.3), obtained after culturing in PS4 medium,
are resuspended in 0.1 M Tris-HCl pH 7.7 (30 ml) and sonicated for
15 minutes at 4.degree. C. The crude extract is then recovered by
centrifugation for 1 hour at 50,000 g, and the supernatant is
passed through a DEAE-Sephadex column (1 ml of gel) to remove the
tetrapyrrole compounds present. Proteins (10 mg; 0.7 ml) of this
extract are then injected onto a MonoQ HR 5/5 column equilibrated
with the same buffer. The proteins are eluted with a linear KCl
gradient (0 to 0.25 M). The COBF protein is eluted with 0.20 M KCl.
It is diluted twofold with 0.1 M Tris-HCl pH 7.7 and purified a
second time on a MonoQ HR 5/5. SDS-PAGE electrophoresis with
visualisation with Coomassie blue is used to reveal the protein.
This technique shows, moreover, that COBF is approximately 95% pure
after this purification step. The NH.sub.2-terminal sequence of the
purified protein was determined as described above. Two
NH.sub.2-terminal sequences appear at the same time in each
degradation cycle; they are the following sequences, in the
proportions indicated: TABLE-US-00023 Sequence 1 (abundance 34%) 1
2 3 4 5 6 7 8 9 10 11 Ala Glu Ala Gly Met Arg Lys Ile Leu Ile Ile
Sequence 2 (abundance 66%) 1 2 3 4 5 6 7 8 9 10 11 Met Arg Lys Ile
Leu Ile Ile Gly Ile Gly Ser
[0459] Sequence 1 corresponds to the NH.sub.2-terminal sequence of
the COBF protein which is given in FIG. 16, except that the
amino-terminal methionine is excised according to rules already
stated (Hirel et al., 1989) by methionine aminopeptidase (Ben
Bassat and Bauer, 1989). Sequence 2, present in the larger amount,
corresponds to the same protein but having its translation
initiation apparently done not at the translation initiation ATG
codon we had assumed, but at that located 5 codons downstream on
the coding frame (FIG. 16). In effect, the amino acids of this
sequence are exactly those which are found in the sequence of the
COBF protein starting from the second methionine (amino acid No. 6)
of this sequence (FIG. 16). In this case, the amino-terminal
methionine is not excised, which confirms the rules already stated
(Hirel et al., 1989). In strain SC510 Rif' carrying plasmid
pXL1546, there are two translation initiations, on the one hand
that corresponding to the methionine codon positioned at the
correct distance, in our construction, from the Shine-Dalgarno
sequence, and on the other hand that which is carried out at the
second methionine codon occurring in the sequence of the cobF gene
presented in FIG. 16. It emerges from this that the COBF protein
proably begins not at the methionine indicated in FIG. 16, but at
that occurring 5 amino acids further on.
[0460] At all events, this result shows that the COBF protein is,
indeed, the one expressed, and that the latter is expressed in a
form elongated by 4 amino acids. During purification, both protein
forms are purified. In this example, the mixture of these two
purified proteins is referred to by us as purified COBF
protein.
[0461] The COBA protein of Pseudomonas denitrificans is purified as
described above (Blanche et al., 1989).
[0462] b) Binding of SAM
[0463] The binding of SAM to these two proteins is studied as
described above in Example 6.3 a). Bovine serum albumin and the
purified COBH protein are used as negative controls. For the COBA
and COBF proteins, a peak of radioactivity is observed at emergence
from the TSK-125 column at the emergence time of these proteins
(FIG. 20). In this test, the COBI protein displays the same
property of binding of SAM. In contrast, there are no such peaks of
radioactivity with BSA and the COBH protein. This test demonstrates
the in vitro binding of SAM to the COBA, COBI and COBF proteins.
These results show that COBA, COBI and COBF are SAM
methyltransferases. This result is in complete agreement with the
COBA and COBI activities, since they are the SUMT and the
SP.sub.2MT, respectively, of Pseudomonas denitrificans. The COBF
protein is hence probably an SAM methyltransferase of the pathway
of biosynthesis of cobalamins. This test confirms that COBF is a
methyltransferase.
[0464] 6.4--Determination of the Activity of COB Proteins by
Sequence Homology Studies
[0465] This example illustrates how it is possible to find the COB
proteins which are SAM methyl-transferases of the pathway of
biosynthesis of cobalamins by comparisons between the sequences of
various COB proteins of Pseudomonas denitrificans.
[0466] The COBI and COBA proteins are both SAM methyltransferases
of the biosynthetic pathway. These two proteins were compared
according to the programme of Kanehisa, 1984. This comparison
brings out three regions of strong homology (FIG. 21). In each of
these regions, there is more than 45% strict homology between the
two proteins. Three regions of strong homology between COBA and
CYSG are also presented (FIG. 22); they are the same regions of
COBA which display a strong homology with COBI. These regions of
strong homologies between COBA, CYSG and COBI display homology with
other COB proteins. The proteins in question are COBF, COBJ, COBL
and COBM (FIG. 23). As regards the region 1, the COBF, COBL and
COBM proteins display significant homologies with respect to all
the Genpro proteins, Genpro being a Genbank (version 59) protein
extraction augmented by putative coding portions larger than 200
amino acids, according to the programme of Kanehisa (1984). As
regards the region 2, the COBJ, COBL and COBM proteins display
significant homologies with respect to all the Genpro (version 59)
proteins. As regards the third region of homology, COBJ, COBL and
COBM display significant homologies with respect to all the Genpro
(version 59) proteins. The sequence comparisons hence enable it to
be demonstrated that four proteins, COBF, COBJ, COBL and COBM,
display significant homologies with the conserved regions of the
sequences of three types of methyltransferases, COBA, COBI and
COBF. The COBG, COBH and COBK proteins do not display significant
homologies with the conserved regions of the methylases. The COBF
protein displays a significant homology with the other proteins
only in the region 1. These homologies must probably correspond to
the fact that all these proteins are methyltransferases. This
result ties up with the biological data described for COBF,
relating to the capacity possessed by this protein for binding SAM
in vitro (Example 6.3). These homologies on the one hand enable it
to be confirmed that COF is an SAM methyltransferase of the pathway
of biosynthesis of cobalamins, and on the other hand demonstrate
that COBJ, COBL and COBM could be SAM methyltransferases of the
pathway of biosynthesis of cobalamins. These results also show the
homology existing between the COB proteins of P. denitrificans and
the isofunctional proteins of other microorganisms.
EXAMPLE 6(B)
Purification and Cloning of the Methanobacterium ivanovii SUMT
Structural Gene
[0467] This example illustrates how it is possible to obtain, in
other microorganisms, COB enzymes and cob genes corresponding to
those identified in P. denitrificans.
[0468] 6(B).1. Purification of Methanobacterium ivanovii SUMT
[0469] This example describes the purification of Methanobacterium
ivanovii SUMT and a study of its catalytic properties.
[0470] Methanobacterium ivanovii strain DSM2611 is cultured as
described (Souillard et al., 1988). Wet cells (12 g) are obtained.
The latter are resuspended in 0.1 M Tris-HCl buffer pH 7.6 (80 ml)
containing 5 mM DTT and 1 mM EDTA, and sonicated for 1 h 30 min at
4.degree. C. and then centrifuged for 1 h at 50,000 g. Free
tetrapyrrole compounds are then cleared from the extract by passage
through a small DEAE-Sephadex A25 column set up in the same buffer.
The proteins precipitating at between 55 and 75% ammonium sulphate
saturation are solubilised in a 0.1 M Tris-HCl pH 7.5, 0.5 mM DTT,
1.7 M ammonium sulphate buffer and injected onto a Phenyl-Superose
HR 10/10 (Pharmacia France/SA) column eluted with a decreasing
gradient (1.7 M to 0 M with respect to ammonium sulphate). The
active fractions are passed through a Sephadex G-25 column
equilibrated with 0.1 M Tris-HCl pH 7.5, 0.5 mM DTT, 25% glycerol
buffer (buffer A), then injected onto a Mono Q HR 5/5 (Pharmacia
France SA) column equilibrated with buffer A and eluted with a KCl
gradient of 0 to 0.3 M; this step is repeated a second time under
the same conditions. Gel permeation chromatography of the active
fraction of the preceding step on Bio-Sil TSK-250 (BioRad France
SA) enables a protein which is homogeneous in SDS-PAGE and in
RP-HPLC (C-18 .mu.Bondapak) to be obtained. The different steps of
purification, with their yield, as well as their purification
factor, are described in the table below.
[0471] As shown in this table, the total purification factor is
more than 4,500. Some properties of the pure enzyme have been
studied according to methods already described (Blanche et al.,
1989). This enzyme does indeed have SUMT activity, i.e. it does
indeed catalyse the SAM-dependant transfer of two methyl groups at
C-2 and at C-7 of uro'gen III. The molecular weight of the enzyme
estimated by gel permeation is 60,000.+-.1,500, while by SDS-PAGE
it is 29,000, which shows clearly that it is a homodimeric enzyme.
Under conditions already described (Blanche et al., 1989), the
enzyme has a Km for uro'gen III of 52.+-.8 nM. In addition, this
enzyme does not show inhibition by substrate at concentrations
below 20 .mu.M, whereas Pseudomonas denitrificans SUMT shows an
inhibition by uro'gen III at a concentration above 2 .mu.M (Blanche
et al., 1989). TABLE-US-00024 TABLE Purification of M. ivanovii
SUMT Sp. activity Purification Vol Proteins (u/mg of Purification
step (ml) (mg) proteins) Yield factor.sup.1 Crude extract 92 731
0.337 -- -- 55-75% AS 7.1 153 1.215 76 3.6 Phenyl-Superose 9.5 8.34
15.35 52 46 Mono Q 5/5 1.0 0.252 422 43 1252 Bio-Sil TSK 1.0 0.061
1537 38 4561 .sup.1calculated from the yield of proteins.
[0472] The Vmax of M. ivanovii SUMT was determined. It is 1537 U/mg
of proteins. This value is greater than that found for P.
denitrificans SUMT, already determined under optimal conditions for
the reaction (taking account of its inhibition by uro'gen III), 489
U/mg of proteins (Blanche et al., 1989).
[0473] 6(B).2. Cloning of the M. ivanovii SUMT Structural Gene in
E. coli
[0474] 6(B).2.1. Cloning of a fragment internal to the M. ivanovii
SUMT structural gene. For this purpose, the procedure is as
follows: 200 picomols of M. ivanovii SUMT are used for the
NH.sub.2-terminal sequencing of the protein as described above. In
addition, a peptide fragment obtained by tryptic digestion of the
protein is likewise subjected to a sequencing of its
NH.sub.2-terminal portion. The sequences obtained are presented in
FIG. 48. The sense and antisense oligonucleotides 946, 923 and 947,
respectively (see FIG. 48) are synthesised as described above;
these oligonucleotides contain a restriction site at their 5' end,
which is either EcoRI for the sense oligonucleotides or HindIII for
the antisense oligonucleotide. These oligonucleotides are used for
an enzymatic DNA amplification experiment (Saiki et al., 1988) as
shown diagrammatically in FIG. 48.B.
[0475] M. ivanovii genomic DNA is prepared in the following manner:
M. ivanovii (DSM 2611) cells (0.4 g) are washed with 0.15 M NaCl
solution. The cells are then incubated in a 25% sucrose, 50 mM
Tris-HCl pH 8, lysozyme (40 mg) solution (4 ml), and thereafter for
2 to 3 h at 50.degree. C. after the addition of proteinase K (40
mg) and a 0.2% SDS, 0.1 M EDTA pH 8 solution (5 ml). The DNA is
then extracted with phenol/chloroform (50%/50%) twice and then
twice with chloroform, and thereafter precipitated with isopropanol
and taken up in TNE (10 mM Tris-HCl pH 8, 1 mM EDTA, 100 mM NaCl)
(3 ml).
[0476] Enzymatic amplification of M. ivanovii DNA is performed
according to the protocol of Saiki et al., 1988, in a volume of 0.1
ml with M. ivanovii genomic DNA (600 ng), using the primers 946 and
947 (reaction 1) or 923 and 947 (reaction 2). The buffer used for
this reaction is 1 mM MgCl.sub.2, 50 mM KCl, 0.001% gelatin and
each dNTP at a concentration of 0.2 mM; for each amplification
reaction, 10 mg of each oligonucleotide are used, as well as Taq
DNA polymerase (2.5 units) (Cetus Corporation). Amplification is
carried out over 30 cycles in the Perkin-Elmer Cetus DNA
Amplication system; during each cycle, the DNA is denatured for 1
min at 95.degree. C., the oligonucleotide primers are hybridysed
with single-stranded DNA for 2 min at 38.degree. C. and the newly
formed strands are polymerised for 3 min at 72.degree. C. The
amplification products are then extracted with chloroform and
thereafter undergo ethanol precipitation; they can then be
visualised after migration on acrylamide gel, and thereafter be
digested with restriction enzymes such as EcoRI and HindIII.
[0477] In the case of reaction 1, two fragments are observed: at
615 bp as well as at 240 bp. As regards reaction 2, two fragments
are also observed: at 630 and 170 bp. The whole of the product of
an enzymatic amplification reaction between the oligonucleotides
946-947 is separated by migration on acrylamide gel; the 615-bp
fragment is purified as described above. This fragment is then
digested with EcoRi and HindIII in order to make the ends of the
fragment cohesive. This fragment is then ligated with the DNA of
the replicative form of phage M13mp19. The ligation is transformed
into E. coli TG1. Six recombinant clones containing a 615-bp insert
are analysed by sequencing with the universal primer-20 (Pharmacia
SA, France). As shown in FIG. 49, when the single-standed DNA of
the recombinant phages which contain 615-bp insert is sequenced,
there must be observed, downstream of the EcoRI site, a
non-degenerate sequence corresponding to that of the
oligonucleotide 946 followed, in the same frame, by a sequence
coding for the amino acids LITLKAVNVLK?ADVVL (? means that, at this
position, the residue could not be determined); this sequence
corresponds to that which, in the NH.sub.2-terminal sequence of
SUMT, follows the amino acids corresponding to the oligonucleotide
946 (see FIG. 48). For two clones, there was actually observed,
after the EcoRI site, a sequence able to code for the
NH.sub.2-terminal region of Methanobacterium ivanovii SUMT, this
sequence beginning with the arrangement Pro-Gly-Asp-Pro-Glu-Leu
which are the amino acids encoded by a sequence containing the
oligonucleotide 946. This observation shows that these two
recombinant replicative forms contain an insert which corresponds
to a fragment internal to the Methanobacterium ivanovii SUMT
structural gene. The replicative form carrying this fragment
internal to the M. ivanovii structural gene is referred to as pG10.
6(B).2.2. Cloning of the Methanobacterium ivanovii SUMT structural
gene
[0478] Methanobacterium ivanovii genomic DNA is digested with
several restriction enzymes (single or double digestions). After
digestion, the fragments are separated by agarose gel
electrophoresis and are then transferred onto a nylon membrane as
described above. After denaturation of the fragments thus
transferred and prehybridisation, a hybridisation is performed with
the replicative form pG10 as a .sup.32P-labelled probe, as
described above. It is thus found that a 3.2-kb fragment emanating
from an EcoRI-BqlII digestion of Methanobacterium ivanovii
hybridises with the probe (see FIG. 50). Genomic DNA (40 .mu.g) of
M. ivanovii are then digested with EcoRI and BqlII and thereafter
separated by migration on agarose gel. The fragments having a size
of between 3 and 3.5 kb are electroeluted as described above. The
fragments thus purified are ligated with the vector pBKS+
(Stratagene Cloning Systems, La Jolla) digested with BamHI-EcoRI.
The ligation is transformed into E. coli DH5.alpha. (Gibco BRL).
The transformants are selected on LB medium supplemented with
ampicillin and X-gal. 800 white colonies are subcultured on
filters; after growth and then lysis of the bacteria, a colony
hybridisation is performed according to the technique of Grunstein
and Hogness (1975). The probe used is the replicative form pG10
labelled with .sup.32p A single positive clone after this
hybridisation test with the probe is found. The plasmid DNA of this
clone is referred to as pXL1809 (see FIG. 56). A digestion of this
DNA with EcoRI-XbaI enables a 3.2-kb insert to be visualised, as
expected. Plasmid pXL1809 is sequenced on both strands by the
technique of Chen and Seeburg (1985). A sequence of 955 bases is
obtained (FIG. 51). An analysis of the open reading frames leads us
to identify an open reading frame from base 34 (ATG) to base 729
(TGA). This open reading frame codes for a protein whose sequence
is presented in FIG. 52. This protein has a molecular weight of
24,900 (see FIG. 53), which is close to the molecular weight of the
protein purified from M. ivanovii. The NH.sub.2-terminal sequence
of this protein is exactly that determined for purified M. ivanovii
SUMT (see FIG. 48 and FIG. 52). These observations establish
unambiguously that the cloned and sequenced gene is indeed the M.
ivanovii SUMT structural gene. Since this activity is assumed to
participate in the biosynthesis of corrinoids in all bacteria, this
gene is designated corA gene, and the protein encoded by this same
gene CORA protein. The hydrophobicity profile of the CORA protein
of M. ivanovii, produced from the programme of Hopp and Woods
(1981), shows that it is, as expected, a hydrophilic protein, as
presented in FIG. 54. The CORA protein of M. ivanovii shows a
degree of strict homology of more than 40% with respect to COBA of
P. denitrificans (FIG. 53). This homology extends over practically
the whole of both proteins, since it relates to residues 3 to 227
of CORA of M. ivanovii and residues 17 to 251 of COBA of P.
denitrificans. This homology reflects the structural homologies
existing between two proteins that catalyse the same reaction. The
regions which are most highly conserved between CORA and COBA of P.
denitrificans are the same ones as are conserved between COBA of P.
denitrificans and CYSG of E. coli (FIG. 22).
EXAMPLE 7
Expression of COB Proteins
[0479] 7.1--Expression in Pseudomonas denitrificans
[0480] This example illustrates that the amplification of a
structural gene for a COB protein of Pseudomonas denitrificans in
Pseudomonas denitrificans leads to amplification of the activity of
the COB protein.
[0481] 7.1.1--Expression of the COBA Protein
[0482] Plasmid pXL557 corresponds to plasmid pXL59 into which the
2.4-kb BqlII-EcoRV fragment (at positions 80 and 2394,
respectively, in the sequence of FIG. 7) of the 5.4-kb fragment has
been cloned. This fragment contains the cobA and cobE genes.
[0483] Plasmid pXL545 contains only the cobE gene. Its construction
has been described in Example 4.1.
[0484] These two plasmids were introduced by conjugative transfer
into SC510 Rif'. Strains SC510 Rif', SC510 Rif' pXL59, SC510 Rif'
pXL557 and SC510 Rif' pXL545 were cultured in PS4 medium. At 4
days, culturing was stopped and the SUMT activities were assayed
according to a standard protocol already described (F. Blanche et
al., 1989). The activities are given below. TABLE-US-00025 TABLE
SUMT activity of SC510 Rif.sup.r and of some of its derivatives
SUMT assayed nmol/h/mg of Strain protein SC510 Rif.sup.r 0.05 SC510
Rif.sup.r pXL59 0.04 SC510 Rif.sup.r pXL557 2.10 SC510 Rif.sup.r
pXL545 0.05
[0485] It emerges clearly from these results that only plasmid
pXL557 brings about a marked increase in SUMT activity (a factor of
50) in SC510 Rif'. This increase results from the amplification of
cobA and not of cobE, since plasmid pXL545, which permits the
amplification of only cobE, does not produce an increase in SUMT
activity. This result confirms that cobA is the structural gene for
SUMT of Pseudomonas denitrificans. This result shows that it is
possible to obtain an amplification of the SUMT activity in
Pseudomonas denitrificans by amplification of the structural gene
for SUMT of Pseudomonas denitrificans.
[0486] 7.1.2--Expression of the COBI Protein
[0487] A fragment originating from the 8.7-kb DNA fragment
containing the structural gene for SP.sub.2MT (cobI) is cloned into
a plasmid having a broad host range in Gram-negative bacteria, and
this plasmid is then introduced by conjugation into Pseudomonas
denitrificans SC510 Rif'. The S-adenosyl-L-methionine:precorrin-2
methyltransferase activity of the strain is then measured relative
to that of the strain carrying the vector.
[0488] The 1.9-kb BamHI-BamHI-SstI-SstI fragment containing the
cobH and cobI genes is purified from the 8.7-kb fragment. XbaI and
EcoRI linkers are placed at the BamHI and SstI ends, respectively,
after the latter have been filled in with bacteriophage T4 DNA
polymerase. The fragment is then inserted between the XbaI and
EcoRI sites of the broad host range plasmid pXL59. It carries
kanamycin resistance. The plasmid thereby obtained is designated
pXL1148 (FIG. 24).
[0489] Separately, a related plasmid was constructed: the 1.5-kb
BamHI-BamHI-SstI fragment containing only the whole cobH gene and
the 5' portion of the cobI gene was purified from the 8.7-kb
fragment. XbaI and EcoRI linkers were added at the BamHI and SstI
sites, respectively, after the latter had been filled in or
digested with phage T4 DNA polymerase. This fragment was then
inserted between the EcoRI and XbaI sites of pXL59 to give plasmid
pXL1149. Plasmids pXL1148 and pXL1149 differ only in the presence
in pXL1148 of the 0.3-kb SstI-SstI fragment which contains the 3'
end of the cobI gene. pXL1148 possesses the whole structural gene
for cobI, in contrast to pXL1149. Both plasmids contain the cobH
gene.
[0490] These two plasmids were introduced by conjugation into SC510
Rif'. Strains SC510 Rif', SC510 Rif' pXL59, SC510 Rif' pXL1148 and
SC510 Rif' pXL1149 are cultured in PS4 medium. After 4 days of
culture, the cells are harvested and the SP.sub.2MT activities are
assayed as described in Example 6.1.3 a).
[0491] The result of these assays is given below, with the
SP.sub.2MT activities defined as in Example 6.1.3 a).
TABLE-US-00026 TABLE SP.sub.2MT activities of various strains
derived from Pseudomonas denitrificans SP.sub.2MT activity.sup.1
Strain in % SC510 Rif.sup.r <5 SC510 Rif.sup.r pXL59 <5 SC510
Rif.sup.r pXL1148 75 SC510 Rif.sup.r pXL1149 <5 .sup.1per 500
.mu.g of crude extract introduced in the test.
[0492] The activity is expressed in % as defined in Example 6.1.3
a).
[0493] Only plasmid pXL1148 brings about a substantial increase in
SP.sub.2MT activity. In contrast, plasmid pXL1149 does not give
results different from those observed with the controls SC510 Rif'
and SC510 Rif' pXL59. pXL1148 is the only plasmid to contain the
cobI gene, and it is the only one to amplify SP.sub.2MT activity;
this result confirms that the structural gene for SP.sub.2MT of
Pseudomonas denitrificans is the cobI gene. Furthermore, if the
total proteins of these different strains are separated by
electrophoresis under denaturing conditions (SDS-PAGE with 10% of
acrylamide), the presence of a band which corresponds to a protein
having a molecular weight of 25,000 is observed specifically in the
case of pXL1148 (FIG. 25). The molecular weight of this protein
corresponds to that of the COBI protein. Plasmid pXL1148 enables
overproduction of the COBI protein to be obtained in
Pseudomonas Denitrificans.
[0494] 7.1.3--Expression of COBF
[0495] The expression is obtained by positioning the Ptrp promoter
of E. coli and the ribosome-binding site of the cII gene of
bacteriophage lambda upstream of the cobF gene. The expression
thereby obtained is much higher than that observed by simple gene
amplification using the same multicopy plasmid.
[0496] The 2-kb EcoRI-BamHI-BamHI fragment of pXL1496 (Example
7.2.1 below) is purified (FIG. 26). This fragment contains the Ptrp
promoter of E. coli and the ribosome-binding site of the cII gene
of bacteriophage lambda upstream of the cobF gene. Dowstream of the
cobF gene, there is the terminator of the rrnB operon of E. coli.
This fragment is cloned at the EcoRI-BamHI sites of plasmid pKT230
to give pXL1546 (FIG. 26). pKT230 is a plasmid of the
incompatibility group Q which replicates in almost all
Gram-negative bacteria (Bagdasarian et al., 1981); this plasmid
carries kanamycin resistance. Plasmid pXL1546 and pKT230 are
introduced by conjugation into SC510 Rif'. Strains Sc510 Rif',
SC510 Rif' pKT230 and SC510 Rif' pXL1546 are cultured in PS4 medium
as described above. After four days of culture, the total proteins
of the different strains are analysed in 10% SDS-PAGE. As shown in
FIG. 27, a protein of molecular weight 32,000 which is
overexpressed is observed in the extract of SC510 Rif' pXL1546;
this protein comigrates with the protein which is overexpressed by
E. coli B pXL1496 (Example 7.2.1 below). Furthermore, this protein
is specifically expressed in strain SC510 Rif' containing pXL1546,
where it represents at least 20% of the total proteins. In
contrast, this protein is not observed in the total proteins of
strains SC510 Rif' and SC510Rif' pKT230. This overexpressed protein
is hence the COBF protein.
[0497] 7.1.4--Expression of COBH
[0498] This example describes the amplification of a DNA fragment
of Pseudomonas denitrificans containing the cobH gene. The protein
which is encoded by this gene is purified; it is the COBH protein.
Plasmid pXL1149, described in Example 7.1.2, contains in the DNA
insert originating from the 8.7-kb fragment only the whole cobH
gene. In SC510 Rif', this plasmid, in contrast to the vector,
brings about the overexpression of a protein of molecular weight
22,000 (FIG. 25).
[0499] 7.1.5--Expression of COBV
[0500] This example describes the amplification of
cobalamin-5'-phosphate synthase activity by a plasmid carrying only
cobV (pXL699, see FIG. 38). The cobalamin-5'-phosphate synthase
activity is amplified in SC877 Rif' by plasmid pXL699 by a factor
of 50 relative to the same strain with the vector pXL435, pXL1303,
pXL1324 or pKT230. This plasmid contains in its insert only the
whole of cobV plus the 5'-terminal portions of ORF18 and of cobU.
In such a strain (SC877Rif' pXL699), the COBV protein is definitely
overexpressed; this overexpression is by a factor of 50 relative to
the expression of strain SC877Rif'.
[0501] 7.1.6--Expression of the CORA Protein
[0502] The 1.5-kb EcoRI-BamHI-BamHI fragment of pXL1832 (see
Example 7.2.4), containing the Ptrp promotor and then the RBS cII
of bacteriophage .lamda., the M. ivanovii SUMT structural gene and
the terminator region of the rrnB operon of E. coli, is cloned at
the EcoRI-BamHI sites of pKT230 (Bagdasarian et al., 1981). In this
manner, plasmid pXL1841 is obtained (see FIG. 56). This plasmid is
mobilised in P. denitrificans SC510 Rif' as described above. A
transconjugant is studied in greater detail. This strain is
cultured in PS4 medium, and the SUMT activity of the bacterial
extracts is assayed at the same time as that of the control strain
SC510 Rif' pXL435 (Cameron et al., 1989). The activities of these
strains are presented below.
[0503] Strain SUMT specific activity in pmol/h/mg of proteins
[0504] SC510 Rif'pXL435 50-100
[0505] SC510 Rif'pXL1841 1700
[0506] This result shows clearly that there is expression of the
SUMT activity of M. ivanovii in P. denitrificans as a result of
plasmid pXL1841, since the SUMT activity of strain SC510 Rif'
pXL1841 is markedly greater than that of SC510 Rif' pXL435.
[0507] 7.2--Expression in E. coli
[0508] This example illustrates how a COB protein of Pseudomonas
denitrificans can be overproduced in E. coli.
[0509] 7.2.1--Expression of COBF
[0510] The 2250-bp EcoRI-XhoI fragment of the 8.7-kb EcoRI fragment
(at the respective positions 0 and 2250 in the sequence presented
in FIG. 8) was cloned into phage M13mp19 (Norrander et al., 1983)
between the EcoRI and SalI sites. The plasmid thereby constructed
is designated pXL1405. An NdeI site was introduced by directed
mutagenesis so that the last three bases (ATG) of this restriction
site constitute the translation initiation site of the cobF gene.
This amounts to modifying the three bases which precede the ATG of
the cobF gene, GAA (the G is at position 733 in the sequence
presented in FIG. 8), to CAT. The NdeI-SphI-SphI fragment (FIG. 26)
containing the cobF gene is then purified; this 1.5-kb fragment is
then cloned between the NdeI-SphI sites of plasmid pXL694 (Denefle
et al., 1987). The plasmid thereby constructed is designated
pXL1496 (FIG. 26). Signals for regulation of genetic expression in
E. coli are present in the 120-bp EcoRI-NdeI fragment (which
originates from pXL694) which precedes the cobF gene. These signals
consist of the [-40+1] region of the Ptrp promoter of E. coli, and
then of 73 bp which contain the ribosome-binding site of the cII
gene of bacteriophage .lamda. (Denefle et al., 1987). Downstream of
the cobF gene, there are the terminators of the rrnB operon of E.
coli (in the HindIII-BamHI fragment). Plasmid pXL1496 was
introduced by transformation into the E. coli strain (Monod and
Wollman, 1947). Expression of the cobF gene was studied as already
described (Denefle et al., 1987) under conditions where the Ptrp
promoter is either repressed (in the presence of tryptophan) or not
repressed (absence of tryptophan). The medium in which the
expression was carried out is M9 minimum medium (Miller, 1972)
supplemented with 0.4% of glucose, 0.4% of casamino acids, 10 mM
thiamine and 40 .mu.g/ml of tryptophan in the case where it is
desired to repress the Ptrp promoter. E. coli strain B pXL1496 was
cultured at 37.degree. C. in the medium described above with
ampicillin (100 .mu.g). As shown in FIG. 28, the absence of
tryptophan brings about the expression of a protein of molecular
weight 32,000. In effect, in the extract of total proteins of E.
coli B pXL1496 analysed in SDS-PAGE (FIG. 28), a protein of
molecular weight 32,000 D which represents between 1 and 4% of the
total proteins is clearly observed. This protein is present in
markedly smaller quantities in the extract of the total proteins of
E. coli B pXL1496 cultured under the same conditions but in the
presence of tryptophan. The molecular weight of the protein which
is expressed under these conditions is close to the molecular
weight of the COBF protein deduced from the amino acid sequence of
the protein, which is 28,927 (FIG. 16). The protein which is thus
expressed in E. coli is the COBF protein.
[0511] 7.2.2--Expression of COBT
[0512] Overproduction is obtained by fusing the lac promotor and
the first three codons of lacZ of E. coli to the 5' end of the cob
gene.
[0513] The EcoRI site located at position 2624 in the sequence
presented in FIG. 32 of the 4.8-kb fragment contains the fourth
codon of the cobT gene. The 3.5-kb EcoRI-XbaI fragment of pXL837
(see FIG. 36) is cloned at the EcoRI and XbaI sites of pTZ18R or
pTZ19R (Pharmacia) to generate pXL1874 or pXL1875, respectively;
these two plasmids differ in the orientation of the truncated cobT
gene with respect to the promoter of the lactose operon of E. coli
(Plac). Plac is upstream of cobT in pXL1874 while the opposite is
true in pXL1875. Cloning of the EcoRI-XbaI fragment of pXL837 at
the EcoRI-XbaI sites of pTZ18R enables a protein fusion to be
carried out between the first 4 amino acids of E. coli
.beta.-galactosidase and the cobT gene from its 4.sup.th codon.
Expression of this lacZ' 'cobT gene is under the control of the
expression signals of lacZ. Plasmids pXL1874, pXL1875 and pTZ18R
are introduced by transformation into E. coli strain BL21.
Expression of the cobT gene is studied as already described
(Maniatis et al., 1989).
[0514] As shown in FIG. 42B, a protein of molecular weight 72,000
is expressed only with pXL1874 and represents, in the extract of
total proteins of BL21, pXL1874 analysed in SDS-PAGE, 1 to 4% of
the total proteins. The molecular weight of the protein which is
expressed under these conditions is close to the molecular weight
of the COBT protein deduced from the amino acid sequence, which is
70,335, in FIG. 40. This experiment shows clearly that, from the
EcoRI site located in the fourth codon of the cobT gene, an open
reading frame compatible with that found for the cobT gene can be
expressed.
[0515] 7.2.3--Expression of a Truncated COBS Protein
[0516] A BamHI site is located at the 45th codon of the COBS gene.
The 1.2-kb BamHI-BamHI fragment containing the 3' portion of the
cobs gene and sequences downstream of this gene is excised from
pXL843 and cloned at the BamHI site of plasmid pET-3b (Rosenberg et
al., 1987) to generate pXL1937. The BamHI fragment is oriented in
such a way that the truncated portion of the cobS gene is fused, in
frame, with the first 12 codons of the major capsid protein of
bacteriophage T7 or gene 10 (Rosenberg et al., 1987). This hydbrid
gene is under the control of the .PHI.10 promotor of bacteriophage
T7. Plasmid pXL1937 and also pET-3b are introduced by
transformation into E. coli BL21 pLysS (W. Studier, personal
communication). After reisolation on selective medium, both strains
are cultured in L liquid medium to an OD at 610 nm of 1; at this
stage, the medium is adjusted to an IPTG (isopropyl
.beta.-thiogalactoside) concentration of 1 mM in order to induce
expression of the polymerase of bacteriophage T7 (Rosenberg et al.,
1987). The culture is then incubated for 3 h at 37.degree. C. and
bacterial lysates are thereafter prepared. The total proteins of
the bacteria thus cultured are separated by PAGE under denaturing
conditions. As seen in FIG. 42A, there is specifically
overexpression of a 33,000 protein with the culture BL21 pLysS
pXL1937. This molecular weight is entirely compatible with the
expected molecular weight for the fusion protein (33 kD). This
experiment shows clearly that, from the BamHI site located at the
45th codon of the cobs gene, an open reading frame compatible with
that found for the cobs gene can be overexpressed.
[0517] 7.2.4. Expression of the CORA Protein
[0518] The following oligonucleotides were synthesised as described
above: TABLE-US-00027 oligonucleotide 1277 5' GGC CGA ATT CAT ATG
GTA GTT TAT TTA 3' ------- 1 2 3 4 5 (1 to 5 first 5 EcoRI
.sub.---------------------- codons of M. ivanovii NdeI SUMT)
oligonucleotide 1278 5' GGC CGA GCT CTA TTA CAT AAT T
.sub.----------------=============== SstI
(=sequence appearing in FIG. 51, positions 926 to 915, in the
strand complementary to the coding strand)
[0519] Oligonucleotide 1277 possesses the recognition sequences for
the restriction enzymes EcoRI and NdeI. The last three bases of the
NdeI site (ATG), which corresponds to a translation initiation
codon, are directly followed by codons 2 to 5 of the M. ivanovii
SUMT structural gene as appear in the sequence presented in FIG.
52. The oligonucleotide 1278 contains the recognition sequence for
SstI, followed directly by the sequence TATTACATAATT which
corresponds to a sequence present in the 955-bp fragment containing
the corA gene presented in FIG. 51; this sequence occurs at
position 926 to 915 (see FIG. 51) in the strand complementary to
the strand coding of the CORA protein. The two oligonucleotides
1277 and 1278 hence contain sequences in their 3' portion
corresponding, respectively, to the coding strand of the corA gene
and to the complementary strand downstream of this gene. These two
oligonucleotides may be used to carry out an enzymatic
amplification experiment with plasmid pXL1809 as template. This
experiment makes it possible to obtain a 910-bp fragment containing
the corA gene of M. ivanovii possessing an NdeI site at the ATG of
the corA gene, and an SstI site at the other end of the fragment
after the end of the corA gene. Enzymatic amplification is carried
out as described above for the enzymatic amplification performed on
the genomic DNA of M. ivanovii, except that the template consists
of DNA (10 ng) of plasmid pXL1809; the temperatures used are the
same, but only 20 amplification cycles are carried out. As
described above, the amplification products are digested with NdeI
and SstI before being separated by migration on agarose gel. As
expected, a fragment 910 bp in size is indeed visualised. This
fragment is purified as already described. This fragment is cloned
at the NdeI and SstI sites of pXL694 (Denefle et al., 1987). The
resulting plasmid, designated pXL1832, is described in FIG. 56. In
this plasmid, in the same way as described in Example 7.2, the M.
ivanovii SUMT structural gene is preceded by the ribosome binding
site of the cII gene of bacteriophage .lamda.. Upstream of this RBS
there is the Ptrp promotor. Plasmid pXL832 is introduced into E.
coli B5548, which is an E. coli strain carrying the mutation cysG44
(Cossart and Sanzey, 1982) by transformation. The SUMT activities
of the strains E. coli B5548 pUC13 and E. coli B5548 pXL1832 are
assayed on extracts obtained from cells cultured in LB medium
supplemented with ampicillin. The assay of SUMT activity is carried
out as already described (Blanche et al., 1989). The results of
this assay are given below. TABLE-US-00028 SUMT specific activity
Strain in pmol/h/mg of proteins E. coli B5548 pUC13 5.9 E. coli
B5548 pXL1832 310
[0520] The results presented in the table above show clearly that
there is expression of a SUMT activity in E. coli strain B5548 when
the latter contains a plasmid pXL1832 which expresses M. ivanovii
SUMT. The M. ivanovii SUMT can hence be expressed in E. coli.
EXAMPLE 8
Amplification of the Production of Cobalamins by Recombinant DNA
Techniques
[0521] 8.1--Amplification in P. denitrificans
[0522] This example illustrates how an improvement in the
production of cobalamins is obtained in Pseudomonas denitrificans
SC510 Rif' by amplification of cob genes of Pseudomonas
denitrificans SC510.
[0523] 8.1.1 Improvement in the production of Cobalamins in
Pseudomonas denitrificans by Removal of a Limiting Step in the
Biosynthesis of Cobalamins
[0524] This example illustrates how the productivity of cobalamins
in Pseudomonas denitrificans strains may be improved by
amplification of cob genes of Pseudomonas denitrificans. This
improvement results from the removal of a limiting step of the
biosynthetic pathway.
[0525] Plasmid pXL367 is described in Example 4.2 (FIG. 13). This
plasmid corresponds to pRK290 (Ditta et al., 1981) into which the
8.7-kb EcoRI fragment has been inserted. This plasmid pXL367
effects an improvement in the biosynthesis of cobalamins in strain
SC510 Rif'. Strains SC510 Rif', SC510 Rif' pRK290 and SC510 Rif'
pXL367 are cultured in an Erlenmeyer in PS4 medium according to the
conditions described in the experimemntal protocols. An improvement
in the production titre due to the presence of plasmid pXL367 is
observed. In effect, strain SC510 Rif' pXL367 produces 30% more
cobalamins than strains SC510 Rif' and SC510 Rif' pRK290. This
improvement is not due to the amplification of unspecified genes of
Pseudomonas denitrificans, but to the specific amplification of the
genes carried by the 8.7-kb EcoRI fragment. In effect, plasmid
pXL723 described in FIG. 11 gives no improvement, and the same
production titre is observed with this plasmid as with strains
SC510 Rif' and SC510 Rif' pRK290.
[0526] 8.1.2 Improvement in the production of coenzyme B1.sub.2 in
Pseudomonas denitrificans by removal of two limiting steps in the
biosynthesis of cobalamins
[0527] This example illustrates how the productivity of cobalamins
in strains of Pseudomonas denitrificans may be improved by
amplification of cob genes of Pseudomonas denitrificans. This
improvement results from the removal of two limiting steps of the
biosynthetic pathway.
[0528] The 2.4-kb ClaI-Eco RV fragment derived from the 5.4-kb
fragment (containing the cobA and cobE genes) is cocloned with the
8.7-kb EcoRI fragment into the broad host range plasmid pXL203. The
plasmid thereby constructed is referred to as pXL525 (FIG. 29).
This plasmid is introduced into SC510 Rif' by conjugation. Strain
SC510 Rif' pXL525 produces 20% more cobalamins than SC510 Rif'
pXL367. Amplification of the cobA and cobE genes enables a further
limiting step in SC510 Rif' in the biosynthesis of cobalamins to be
removed. Pseudomonas denitrificans strain SC510 Rif' is improved in
the present example by the successive removal of two limiting
steps. This example shows that the removal of two limiting steps in
the biosynthesis of cobalamins can lead to further improvements in
production.
[0529] 8.2--Improvement in the Productivity of Cobalamins in
Agrobacterium tumefaciens
[0530] This example illustrates the improvement in the production
of cobalamins in a strain productive of cobalamins by amplification
of the cob genes of Pseudomonas denitrificans SC510.
[0531] The strain used is a strain of a Gram-negative bacterium; it
is a strain of Agrobacterium tumefaciens.
[0532] The plasmids described in Examples 4.2 and 8.1, pXL367 and
pXL525, as well as the vector pRK290 (Ditta et al., 1981) and
plasmid pXL368 (FIG. 29), are introduced by conjugative transfer
into Agrobacterium tumefaciens strain C58-C9 Rif' (Cameron et al.,
1989). Strains C58-C9 Rif', C58-C9 Rif' pRK290, C58-C9 Rif' pXL367,
C58-C9 Rif' pXL368 and C58-C9 Rif' pXL525 are cultured in PS4
medium at 30.degree. C. as described above. The cobalamins produced
are assayed as described above. The production titres are given in
the table below. TABLE-US-00029 TABLE Titres of vitamin B.sub.12
produced by different recombinant strains of Agrobacterium
tumefaciens Vitamin B.sub.12 Strain in mg/l C58-C9 Rif.sup.r 0.4
C58-C9 Rif.sup.r pRK290 0.4 C58-C9 Rif.sup.r pXL367 0.8 C58-C9
Rif.sup.r pXL368 0.8 C58-C9 Rif.sup.r pXL525 1.2
[0533] As is clearly apparent in the above table, the production of
cobalamins is improved in the Agrobacterium tumefaciens strain
used. Two different plasmids improve the production of cobalamins
in the Agrobacterium tumefaciens strain used: pXL367 and pXL368.
These plasmids contain the 8.7-kb EcoRI fragment (cobF to cobM
genes) and the 2.4-kb ClaI-EcoRV fragment (cobE and cobA gene),
respectively. Separately, they improve the production of cobalamins
by Agrobacterium tumefaciens C58-C9 Rif' by a factor of 2; this
result shows that it is possible to improve the production of
cobalamins by a strain of Agrobacterium tumefaciens by amplifying
fragments carrying cob genes of Pseudomonas denitrificans. In the
present case, it is possible to speak of heterologous improvment,
that is to say improvement of the production of cobalamins by one
strain by means of the amplification of cob genes of another
strain.
[0534] The improvements in production of cobalamins provided by the
different Pseudomonas denitrificans fragments containing cob genes
are capable of cumulation, i.e., by putting into the same plasmid
the two fragments which are separately cloned into pXL367 and
pXL368, additive improvements are observed, in the sense that
plasmid pXL525 provides in Agrobacterium tumefaciens C58-C9 Rif' an
improvement in the production greater than that provided by each of
the fragments cloned separately into the same vector.
[0535] 8.3--Improvement in the Productivity of Cobalamins in
Rhizobium meliloti
[0536] This example describes the improvement in the production of
cobalamins by another strain productive of cobalamins.
[0537] The plasmid described in Example 8.2, pXL368, as well as the
vector pRK290 (Ditta et al., 1981), are introduced by conjugative
transfer into Rhizobium meliloti strain 102F34 Rif' (Leong et al.,
1982). The transconjugants, namely 102F34 Rif', 102F34 Rif' pRK290
and 102F34 Rif' pXL368, are cultured in PS4 medium at 30.degree. C.
as described above. The cobalamins produced are assayed as
described above. The production titres are given in the table
below. TABLE-US-00030 TABLE Titres of cobalamins produced by
different recombinant strains of Rhizobium meliloti Vitamin
B.sub.12 Strain in mg/l 102F34 Rif.sup.r 0.4 102F34 Rif.sup.r
pRK290 0.4 102F34 Rif.sup.r pXL368 0.8
[0538] As is clearly apparent in the above table, the production of
cobalamins is improved in the Rhizobium meliloti strain used.
Plasmid pXL368 improves the production of cobalamins by the
Rhizobium meliloti strain used. This plasmid contains the 2.4-kb
ClaI-EcoRV fragment (cobA and cobE genes); it improves the
production of cobalamins by Rhizobium meliloti 102F34 Rif' by a
factor of 2. This result shows that it is possible to improve the
production of cobalamins by a strain of Rhizobium meliloti by
amplifying fragments carrying cob genes of Pseudomonas
denitrificans. In the present case, it is possible to speak of
heterologous improvement, that is to say improvement of the
production of cobalamins by one strain by means of the
amplification of cob genes of another strain.
EXAMPLE 9
Assay of Corrinoids and Decobaltocorrinoids in Musts and Cells of
Strains Productive of Corrinoids
[0539] This example illustrates how it is possible to identify and
assay the different corrinoids and decobaltocorrinoids produced by
different strains productive of cobalamins. This assay makes it
possible, inter alia, to assay coenzyme B.sub.12.
[0540] The musts (or the cells alone) are cyanide-treated as
already described (Renz, 1971). After centrifugation, an aliquot of
the supernatant is passed through a DEAE-Sephadex column which is
then washed with 0.1 M phosphate buffer. The collected fractions
are combined and desalted on a Sep-Pak C-18 (Waters) cartridge.
After evaporation and resuspension in water (100 .mu.l to 1 ml
depending on the quantity of corrinoids present), the corrinoids
are identified and assayed by HPLC on a Nucleosil C-18 column
(Macherey-Nagel). The column is eluted at 1 ml/min with an
acetonitrile gradient (from 0% to 100%) in 0.1 M potassium
phosphate buffer containing 10 mM KCN.
[0541] The corrinoids are visualised by UV detection at 371 nm
and/or by specific detection of .sup.57Co (if culturing has been
performed in the presence of .sup.57CoCl.sub.2) using a Berthold LB
505 detector. They are hence identified by comparison of their
retention times with standards. Similarly, the "metal-free
corrinoids" (hydrogenobyrinic acid, hydrogenobyrinic acid monoamide
and hydrogenobyrinic acid diamide) are visualised by UV detection
at 330 nm. By this technique, the following intermediates are
separated: cobyrinic acid, cobyrinic acid monoamide, cobyrinic acid
diamide, cobyrinic acid triamide, cobyrinic acid tetraamide,
cobyrinic acid pentaamide, cobyric acid, cobinamide, cobinamide
phosphate, GDP-cobinamide, B.sub.12 phosphate and vitamin B.sub.12.
The adenosylated forms of these products are also separated and
assayed by this technique. For this purpose, the initial step of
the cyanide treatment is cut out and the HPLC column is eluted with
buffer devoid of KCN. FIG. 31 gives the retention times of
different standards separated by this system and identified at
emergence from the column by UV absorbance.
[0542] A sample of strain SC510 Rif' was deposited on 30th Jan.
1990 at the Centraal Bureau voor Schimmelcultures at Baarn
(Netherlands), where it was registered under reference CBS 103.90.
Sequence CWU 1
1
* * * * *