U.S. patent application number 11/263000 was filed with the patent office on 2006-04-13 for reducing branching and enhancing fragmentation in culturing filamentous microorganisms.
Invention is credited to Barend Kraal, Rudolf G.M. Luiten, Gilles P. van Wezel.
Application Number | 20060078981 11/263000 |
Document ID | / |
Family ID | 8233854 |
Filed Date | 2006-04-13 |
United States Patent
Application |
20060078981 |
Kind Code |
A1 |
van Wezel; Gilles P. ; et
al. |
April 13, 2006 |
Reducing branching and enhancing fragmentation in culturing
filamentous microorganisms
Abstract
The invention relates to the field of microorganisms and in
particular to the culturing of microorganisms. The invention
provides means and methods for enhancing the culturing properties
of filamentous microorganisms, in particular filamentous fungi. The
means and methods according to the invention comprise reducing
branching and/or enhancing the fragmentation of filamentous
microorganisms, whereby their liquid culturing properties are
improved. In one embodiment, this is achieved by providing the
microorganisms with activity capable of enhancing fragmentation
and/or reducing branching, such as the activity in which, e.g.,
Streptomyces griseus is encoded by ssgA.
Inventors: |
van Wezel; Gilles P.;
(Oegstegeest, NL) ; Kraal; Barend; (Leiden,
NL) ; Luiten; Rudolf G.M.; (Leiden, NL) |
Correspondence
Address: |
TRASK BRITT
P.O. BOX 2550
SALT LAKE CITY
UT
84110
US
|
Family ID: |
8233854 |
Appl. No.: |
11/263000 |
Filed: |
October 28, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09749185 |
Dec 26, 2000 |
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11263000 |
Oct 28, 2005 |
|
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PCT/NL99/00395 |
Jun 25, 1999 |
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09749185 |
Dec 26, 2000 |
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Current U.S.
Class: |
435/252.35 ;
435/471 |
Current CPC
Class: |
C07K 14/36 20130101 |
Class at
Publication: |
435/252.35 ;
435/471 |
International
Class: |
C12N 1/21 20060101
C12N001/21; C12N 15/74 20060101 C12N015/74 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 26, 1998 |
EP |
98202148.7 |
Claims
1. A method for producing a filamentous bacterium exhibiting
reduced branching and fragment septation during growth, said method
comprising: providing a filamentous bacterium, said filamentous
bacterium lacking significant endogenous SsgA-activity, with the
capability of having or expressing heterologous SsgA-activity,
which SsgA-activity, in Streptomyces griseus, is encoded by an ssgA
gene having at least the nucleotide sequence of SEQ ID NO: 1.
2. A method for producing a filamentous bacterium exhibiting
enhanced fragmentation during growth, said method comprising:
providing a filamentous bacterium, wherein said filamentous
bacterium lacks significant endogenous SsgA-activity, with the
capability of having or expressing heterologous SsgA-activity,
which SsgA-activity in Streptomyces griseus is encoded by an ssgA
gene having the nucleotide sequence of SEQ ID NO: 1.
3. The method according to claim 1, wherein said additional
SsgA-activity is provided by transfecting or transforming said
filamentous bacterium with additional genetic information encoding
said additional SsgA-activity.
4. The method according to claim 3, wherein said additional genetic
information comprises an ssgA gene or a derivative or fragment
thereof encoding similar SsgA-activity.
5. The method according to claim 4, wherein said ssgA gene is
derived from an actinomycete or a streptomycete.
6. The method according to claim 5, wherein said gene originates
from Streptomyoes griseus, Streptomyces collinus, Streptomyces
albus, Streptomyces goldeniensis or Streptomyces netropsis.
7. The method according to claim 3, wherein said additional genetic
information is integrated into the filamentous bacterium's
genome.
8. The method according to claim 3, wherein said additional genetic
information is part of an episomal element.
9. The method according to claim 3, wherein said filamentous
bacterium does not have significant endogenous SsgA-activity.
10. The method according to claim 3, wherein said SsgA-activity is
inducible or repressible with a signal.
11. The method according to claim 3, wherein said filamentous
bacterium is an Actinomyces or Streptomyces.
12. The method according to claim 3, wherein said filamentous
bacterium produces an antibiotic or a protein.
13. The method according to claim 12, wherein said protein is
heterologous to said filamentous bacterium.
14. The method according to claim 12, wherein said protein is
expressed from a vector encoding said protein present in said
filamentous bacterium.
15. The method according to claim 14, wherein said protein is
secreted by said filamentous bacterium.
16. A filamentous bacterium produced by the method according claim
3.
17. The filamentous bacterium of claim 16, wherein said filamentous
bacterium is an actinomycete.
18. A method for producing an antibiotic or a useful protein
comprising culturing the filamentous bacterium according to claim
15 in a culture and harvesting said antibiotic or protein from said
culture.
19. The method according to claim 18, wherein said culturing is
submerged culture.
20. The method according to claim 2, wherein said additional
SsgA-activity is provided by transfecting or transforming said
filamentous bacterium with additional genetic information encoding
said additional SsgA-activity.
21. The method according to claim 20, wherein said additional
genetic information comprises an ssgA gene.
22. The method according to claim 21, wherein said ssgA gene
originates from an actinomycete.
23. The method according to claim 21, wherein said ssgA gene
originates from a streptomycete.
24. The method according to claim 23, wherein said gene is derived
from Streptomyoes griseus, Streptomyces collinus, Streptomyces
albus, Streptomyces goldeniensis or Streptomyces netropsis.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/749,185, filed Dec. 26, 2000, pending,
which is a continuation of PCT International Patent Application No.
PCT/NL99/00395, filed on Jun. 25, 1999, designating the United
States of America, and published, in English, as PCT International
Publication No. WO 00/00613 on Jan. 6, 2000, which application
claims priority to European Patent Application Serial No.
98202148.7, filed Jun. 26, 1998, the contents of the entirety of
each of which are incorporated by this reference.
TECHNICAL FIELD
[0002] The invention relates to industrial microbiology, in
particular to fermentation technology and especially to
fermentation methods for filamentous microorganisms, in particular
filamentous bacteria, such as actinomycetes. The invention was made
in a research program into mechanisms of growth of
streptomycetes.
BACKGROUND
[0003] Streptomycetes are Gram-positive, aerobic, filamentous soil
bacteria, that belong to the order of actinomycetales. In an early
stage of Streptomyces growth on a solid medium, spores germinate,
and subsequently develop into a vegetative mycelium of
multinucleoidal and branching hyphae with occasional septums
(Chater and Losick, 1996). After environmental signals, such as
nutrient depletion, aseptate aerial hyphae are formed, growing on
the vegetative hyphae, the latter being used as a substrate.
Eventually, the aerial hyphae form uninucleoidal cells that develop
into hydrophobic spores, which are budded off from the tips of the
hyphae. One of the striking features of streptomycetes and other
members of the order of actinomycetales is their ability to produce
a wide variety of secondary metabolites, including many
antibiotics, which are produced in temporal relation to the onset
of morphological differentiation in surface-grown cultures (Chater,
1989; Miyadoh, 1993). The molecular processes regulating the events
that lead to differentiation of Streptomyces are presently only
superficially understood, although new and interesting insights
into the genetics of streptomycetes have come to light (reviewed in
Champness and Chater, 1993; Chater, 1993).
[0004] Most streptomycetes only sporulate on solid media, while
growth in liquid cultures is restricted to the formation of
vegetative mycelium. This typically develops into intricate
networks of hyphae, among others resulting in pellet formation,
with only the most outwardly oriented sections showing high
physiological activity, resulting in low yield of the desired
product per unit of biomass. Furthermore, because of their
filamentous morphology, high density fermentations of
biotechnologically interesting streptomycetes often are highly
viscous, resulting in a low biomass accumulation due to for
instance aeration and mixing problems. From this perspective it is
desirable that fragmentation of the mycelium in submerged cultures
is stimulated, that branching of the mycelium is reduced and that
in general the viscosity of the culture is reduced.
[0005] Cell division in all bacteria analyzed so far involves the
tubulin-like GTP-binding protein FtsZ, which polymerizes into a
ring at the prospected site of the septum, presumably forming the
physical scaffold for the assembly of the cell division apparatus
(reviewed in Lutkenhaus and Addinall, 1997). In Escherichia coli
and Bacillus species many factors have been identified that are
involved in cell division, but little is known about this process
in actinomycetes. Here septum formation does not lead to actual
cell division, and while in most bacteria ftsZ is essential, the
gene has been shown to be dispensable for mycelial growth in
Streptomyces coelicolor (McCormick et al., 1994).
[0006] In contrast to most actinomycetes, Streptomyces griseus
shows the ability to sporulate in submerged cultures over a short
time period, when grown in defined minimal media (Kendrick and
Ensign, 1983; Ensign, 1988). Kawamoto and Ensign (1995a, b)
identified a mutation in the gene ssgA that relieved repression of
sporulation in rich media. SsgA encodes an acidic protein with a
molecular mass of approximately 5 kDa that displays no significant
homology to any other known protein in the database; in the
sequenced genome of the actinomycetes Mycobacterium tuberculosis
and Mycobacterium leprea no ssgA has been found
(http//kiev.physchem.kth.se/mycdb). Overexpression of ssgA resulted
in fragmented growth and suppression of sporulation in submerged
cultures of S. griseus. Fragmented growth was also observed by
Kawamoto and Ensign (1995b) by overexpression of ssgA in S.
lividans, which was supposed to have an ssgA of its own on the
basis of weak signals on a Southern blot. In S. griseus, Western
blot analysis with polyclonal antibodies raised against SsgA
revealed that expression of SsgA directly correlates to the onset
of submerged sporulation, with the protein appearing shortly before
spore formation (Kawamoto et al., 1997). Importantly, although
sporulation and production of the antibiotic streptomycin are
apparently linked in S. griseus, no suppression of streptomycin
production was observed. Apparently, regulation of sporulation and
antibiotic biosynthesis occur via separate pathways.
DISCLOSURE OF THE INVENTION
[0007] The present inventors have shown that the activity of SsgA
from S. griseus is not limited to the organism in which it is
found. The activity can advantageously be transferred to other
organisms, thereby allowing more fragmented growth and/or reduced
branching and/or reduced viscosity of the culture of many
filamentous microorganisms, in particular actinomycetes and
streptomycetes. This special growth behavior is observed in a wide
variety of culture mediums. It is particularly surprising, that
organisms in which a significant endogenous ssgA-like activity is
not detectable still respond to the presence of the product of the
ssgA gene. Thus, we demonstrate that introduction of ssgA into
various bacteria, in particular actinomycetes that lack significant
endogenous ssgA activity results in suppressed branching and
enhanced fragmentation of the mycelium in liquid culture, resulting
in significantly lower viscosity of culture broths. In addition to
autonomously replicating plasmids containing constitutively
expressed ssgA, we devised a system that allows easy integration of
the gene in the chromosome, with the advantage of high stability
combined to that of independent regulation of ssgA.
[0008] Thus, the invention now provides a method for producing a
filamentous bacterium showing reduced branching during growth,
particularly growth in a liquid medium, comprising providing such a
bacterium with the capability of having or expressing heterologous
SsgA activity, which activity in Streptomyces Griseus is encoded by
an ssgA gene having the sequence of SEQ ID NO:1.
BRIEF DESCRIPTION OF THE FIGURES
[0009] FIG. 1. Some of the ssgA constructs. Arrows show direction
of ssgA. P.sub.ermE, ermE promoter; P.sub.T4, T7 promoter. Solid
lines represent ssgA DNA, broken lines represent plasmid DNA.
[0010] FIG. 2. Southern hybridization for the detection of ssgA in
actinomycetes. All numbered lanes contain BamHI/PstI-digested
chromomal DNA. Marker lanes (M) contain 1 kb DNA ladder. Blots were
hybridized with the 580 bp insert from pGWS5 as probe, and
subsequently with a small amount of radioactively labeled 1 kb
ladder.
[0011] FIG. 2A. Lanes: (1) S. coelicolor; (2) S. lividans 1326; (3)
S. lividans TK24; (4) S. griseofuscus; (5) S. fradiae; (6) S.
ramocissimus; (7) S. collinus; (8) S. kasugaensis; (9) S.
antibioticus; (10) Sacch. erythraea; (11) N. lactamdurans; (12) P.
rosea; (13) S. griseus.
[0012] FIG. 2B. Lanes: (1) S. albus; (2) S. ambofaciens; (3) S.
coelicolor; (4) S. clavuligerus; (5) S. collinus; (6) Sacch.
erythraea; (7) S. goldeniensis; (8) S. mobaraensis; (9) S.
netropsis; (10) P. rosea.
[0013] FIG. 3. Phase-contrast microscopy of S. coelicolor M145
containing pGWS2 (FIG. 3A), and pGWS3 (FIG. 3B) at 200.times.
magnification, S. coelicolor M145 (FIG. 3C) with chromosomally
integrated pGWS4 (magnification 500.times.); upper part, details
revealed by electron microscopy (magnification 10.000.times.).
[0014] FIG. 4. Phase-contrast microscopy of S. clavuligerus ATCC
27064. FIG. 4A: S. clavuligerus (wild-type); FIG. 4B Recombinant S.
clavuligerus containing pGWS4-SD.
[0015] FIG. 5. Sequences of different ssgA genes and proteins from
different strains and oligonucleotides.
DETAILED DESCRIPTION OF THE INVENTION
[0016] As explained above the presence of additional SsgA activity,
in particular heterologous SsgA-activity (meaning activity not in a
form as present in the microorganism in nature), irrespective of
the presence or absence of endogenous SsgA activity, leads to
enhanced fragmentation, reduced branching and, thus, reduced
viscosity in a wide range of culture mediums. The activity may be
provided in any suitable manner, but it is preferred that the
activity is provided by transfecting or transforming filamentous
bactrium with additional genetic information encoding the activity.
Examples of such methods are presented hereinbelow, but the art of
genetic engineering of bacteria is so well advanced that persons
skilled in the art will be able to come up with numerous methods
and variations thereof to provide an intended filamentous bacterium
with a gene encoding SsgA-like activity. SsgA-like activity is
functionally defined as the ability to enhance septation,
fragmentation and/or reduce branching in (typically) submerged
cultures of filamentous microorganisms, in particular bacteria,
more specifically actinomycetes. The activity of other ssgA-like
genes or fragments of ssgA genes or derivatives of ssgA genes that
are within the invention must be functionally the same, but that
does not mean that the amount of activity per molecule needs to be
the same. SsgA-like activity is thus defined as similar in kind,
though not necessarily in amount. Other genes encoding such SsgA
activity than the genes disclosed herein can be obtained without
departing from the invention by applying routine hybridization
and/or amplification techniques. Means and methods for expressing
such genes are well known in the art so that there is no need to go
into detail here regarding cloning vectors, expression vectors,
(inducible) promoters, enhancers, repressors, restriction enzymes,
etc. For stability of the presence of the added SsgA-activity to
the bacterium, in particular for application in large scale
fermentations, it is however preferred that the genetic information
encoding the additional SsgA activity is integrated into the host
cell genome. In this case typically the genetic information will be
in the form of DNA. However, neither RNA, heteroduplexes or even
PNAs are excluded from the present invention as means to provide
the additional genetic information to a microorganism. The
invention is preferably applied in the field of filamentous
bacteria, in particular actinomycetes and most specifically to
streptomycetes. In these embodiments in particular, it is preferred
to apply ssgA genes derived from actinomycetes, especially from
other actinomycetes than the one to be altered in growth
characteristics. This, of course, is automatically the case in a
bacterium that does not have SsgA activity to any significant
amount itself. Using a gene from a related organism enhances the
compatibility of the expression machinery of the host with the
gene. Thus, it is particularly preferred to provide a Streptomyces
with an ssgA (-like) gene from a different Streptomyces. SsgA genes
are found in Streptomyces griseus, Streptomyces collinus,
Streptomyces albus, Streptomyces goldeniensis and Streptomyces
netropsis. It is preferred to provide Streptomyces strains not
having significant endogenous SsgA activity with a gene from the
earlier mentioned strains.
[0017] It is useful to ensure that additional SsgA activity is
inducible or repressible with a signal. In this way the growth
characteristics of the bacteria can be modified at will. Of course,
the final goal of the present invention is to enhance the
production of useful products by the microorganisms by modifying
the microorganisms according to the invention. Useful products
produced by or through microorganisms according to the invention
include so called secondary metabolites, typically antibiotics or
antitumor agents, but also immunosuppressive agents,
hypocholesterolemic agents, enzyme inhibitors, antimigraine agents,
herbicides, antiparasitic agents, ruminant growth promoters,
bioinsecticides, receptor (ant)agonists, heterologous proteins or
even simple biomass. In the case of Streptomycetes such a useful
product is typically an antibiotic. It is thus, therefore,
preferred according to the invention to modify antibiotic producing
strains of Streptomyces, particularly those not displaying a
significant endogenous SsgA-like activity, with genetic information
encoding SsgA activity. On the other hand, the invention can also
be very suitably applied to Streptomycetes or other microorganisms
expressing heterologous proteins (or overexpressing
homologous/endogenous proteins).
[0018] For ease of production it is preferred that the useful
product, antibiotic or protein is secreted by the bacterium. The
protein to be expressed may very well be a protein involved in the
pathway of making a useful product, such as an antibiotic, so that
this production can be further enhanced on top of the improvement
by the reduced fragmentation, etc. In that case it would be very
suitable to combine the two genes on one vehicle for introduction
into the bacterium. The bacteria resulting from the methods
according to the invention are, of course, also part of the
invention. They have additional SsgA activity (or are capable of
expressing such activity) and they thereby will typically have
different growth characteristics than the unmodified microorganisms
when the SsgA activity is present. Thus, the invention also
provides a filamentous bacterium obtainable by a method according
to invention. Preferred microorganisms according to the invention
are actinomycetes and typically streptomycetes. As stated above it
is an important goal of the present invention to improve
fermentative production of useful products, such as antibiotics.
Thus, the invention also provides a method for producing an
antibiotic or a useful protein comprising culturing a filamentous
bacterium according to the invention and harvesting the antibiotic
or protein from the culture. The advantages of the invention are
most clear when the method of culturing is submerged culture. The
invention will be explained in more detail in the following
experimental part.
EXPERIMENTAL PROCEDURES
Bacterial Strains, Culture Conditions and Plasmids
[0019] E. coli K-12 strains JM109 (Messing et al., 1981), and
ET12567 (MacNeil, et al., 1992) were used for routine sub-cloning.
The strains were grown and transformed by standard procedures
(Sambrook et al., 1989); transformants were selected in L broth
containing 1% (w/v) glucose, and ampicillin at a final
concentration of 200 .mu.g ml.sup.-1. L broth with 1% glucose and
30 .mu.g ml.sup.-1 chloramphenicol was used to grow ET12567.
[0020] Streptomyces coelicolor A3(2) M145 and Streptomyces lividans
1326 (Hopwood et al., 1985) were used for transformation and
propagation of Streptomyces plasmids. Protoplast preparation and
transformation were performed as described by Hopwood et al.
(1985). SFM medium (mannitol, 20 g l.sup.-1; soya flour, 20 g
l.sup.-1; agar, 20 g l.sup.-1, dissolved in tap water) is a
modified version of that reported by Hobbs et al. (1989) and was
used to make spore suspensions. R2YE (Hopwood et al., 1985) was
used for regenerating protoplasts and, after addition of the
appropriate antibiotic, for selecting recombinants.
[0021] For liquid culturing of Streptomyces we used YEME medium
(Hopwood et al., 1985), Tryptone soy broth (Difco) containing 10%
sucrose (designated TSBS), or standard minimal medium (MM; Hopwood
et al.) with 1% mannitol as carbon source.
[0022] Strains used for screening of ssgA were Streptomyces albus G
(ATCC 3004), Streptomyces ambofaciens (ATCC 23877), Streptomyces
antibioticus (ATCC8663), Streptomyces clavuligerus (ATCC 27064),
Streptomyces coelicolor M145, Streptomyces collinus (DSM 40733),
Streptomyces fradiae (CBS 498.68), Streptomyces goldeniensis (ATCC
21386), Streptomyces griseus (ATCC 23345), Streptomyces kasugaensis
(DSM 40819), Streptomyces lividans, Streptomyces mobaraensis (ATCC
25365), Streptomyces netropsis (formerly Streptoverticilium
netropsis; ATCC 23940), Streptomyces ramocissimus (ATCC 27529), and
the actinomycetes Nocardia lactamdurans (ATCC 27382), Planobispora
rosea (ATCC 53773), Saccharopolyspora erythraea (NRRL 2338).
[0023] Plasmids pUC18 (Yanisch-Perron et al., 1985), pIJ2925
(Janssen and Bibb, 1993), and pSET152 (Bierman et al., 1992) were
used for cloning experiments. While pSET152 is a conjugative
shuttle plasmid, in the experiments described in this study the
plasmid and its derivatives were introduced by standard protoplast
transformation.
[0024] pIJ486 (Ward et al., 1986) and the E. coli/Streptomyces
shuttle vector pWHM3 (Vara et al.) as high copy-number vectors
(approximately 50-100 copies per chromosome) in S. coelicolor. An
expression vector, designated pWHM3-E, was constructed by cloning
the 300 bp EcoRI/BamHI fragment containing the ermE promoter (Bibb
et al., 1994) into pWHM3. Standard procedures were used to isolate
plasmid DNA from E. coli (Sambrook et al., 1989), and to isolate
plasmid and total DNA from Streptomyces (Hopwood et al., 1985).
PCR Conditions
[0025] Polymerase chain reactions (PCRs) were performed in a
minicycler (MJ Research, Watertown, Mass.), using Pfu polymerase
(Stratagene, La Jolla, La.), and the buffer provided by the
supplier, in the presence of 5% (v/v) DMSO and 200 mM dNTP. No
additional Mg.sup.++ was added to the reaction mixture. The
following PCR program was used: 30 cycles of 45 seconds melting at
94.degree. C., 1 minute annealing at 54.degree. C., and 90 seconds
extension at 72.degree. C., followed by an additional 10 minutes at
72.degree. C.
Constructs for Expression of ssgA
[0026] A 750 bp DNA fragment containing the ssgA gene (Accession
D50051) was amplified from the Streptomyces griseus chromosome by
PCR, using primers ssg1 and ssg2 (Table 1). The PCR fragment was
cloned as an EcoRI-BamHI fragment in pIJ2925, and further into
pWHM3, pWHM3-E, and pSET152, resulting in pGWS1, pGWS2, pGWS3, and
pGWS4, respectively (Table 1). For pGWS1 and pGWS3, see also FIG.
1. The S. coelicolor strain with pGWS4 integrated in the attP site
on the chromosome was designated S. coelicolor GSA1. For pGWS1,
pGWS3, and pGWS4 we also made derivatives in which the upstream
region of S. griseus ssgA was replaced by that of S. ramocissmus
tuf1 (Vijgenboom et al., 1994), which is known to be very
efficiently recognized by ribosomes and hence typically results in
higher expression; these were designated pGWS1-SD, pGWS3-SD, and
pGWS4-SD, respectively.
Southern Hybridization and Probes
[0027] Genomic DNAs used for Southern analysis were isolated
according to the method described by Hopwood et al. (1985). For
high-resolution hybridization experiments, to investigate the
presence of ssgA in various actinomycetes, genomic DNA was digested
with the appropriate enzymes and separated electrophoretically on a
0.7% agarose gel in TAE buffer, using the Gibco BRL 1 kb ladder as
DNA size markers. Agarose gels were pretreated and subsequently
blotted on Hybond-N.sup.+ nylon membranes (Amersham) using
20.times.SSC buffer as the transfer buffer, basically according to
Sambrook et al. (1989). Hybridization and washing conditions were
described previously (van Wezel et al., 1991). Stripping of blots
was done by 30 minutes incubation in 0.4 N NaOH at 65.degree. C.
and subsequent incubation in 0.1.times.SSC/0.25 M Tris (pH 6.5).
The total removal of the probe was checked by overnight exposure of
an X-ray film.
[0028] For recognition of ssgA in Southern hybridization
experiments the 580 bp insert from pGWS5 was [.sup.32P]-labeled by
the random-prime method (Feinberg and Vogelstein, 1983).
Northern Analysis
[0029] RNA samples (approximately 20 .mu.g) were glyoxylated, run
in a 1.2% agarose gel in 20 mM sodium phosphate buffer (pH 6.7),
and blotted onto Hybond N.sup.+ nylon membranes using 30 mM sodium
phosphate (pH 6.7) as the blotting buffer. Hybridization with the
S. netropsis ssgA gene was carried out in 5.times.SSC, 0.1% SDS,
and 1.times. Blocking reagent (Boehringer Mannheim), O/N at
65.degree. C. Washing occurred until the background was
sufficiently low.
Nuclease S1 Mapping
[0030] For nuclease S1 protection assays, 50 mmol of
.sup.32P-end-labeled probe (.apprxeq.10.sup.4 Cerenkov counts
min.sup.-1) was hybridized to 20 .mu.g of RNA in 3M Na-TCA at
45.degree. C. overnight after denaturation at 70.degree. C. All
subsequent steps were carried out as described previously (Strauch
et al., 1991).
Computer Analysis
[0031] The BLAST search engines BlastN, BlastP, and BlastX
(Altschul et al., 1990) were used to perform database searches, and
the Wisconsin GCG Package (Devereux et al., 1984) for sequence
alignments and protein analysis.
Results
[0032] SsgA is a unique protein that does not belong to any known
protein family.
[0033] Extensive searches with S. griseus SsgA of both the
translated nucleotide database and the protein database using the
BLAST search engines BLASTX and BLASTP resulted in one relevant
hit, namely a partial sequence of Streptomyces albus G DNA
(Accession M28303) that apparently encodes part of SsgA. This DNA
was identified upstream of a .beta.-lactamase gene (Dehottay et
al., 1987), and apparently encodes 67 residues of a putative
protein with 86% aa identity to aa 18-84 of S. griseus SsgA. The
lack of the C-terminal half of the gene suggests that the cloning
of this ssgA homologue was probably coincidental and the result of
a cloning artifact. The cloning and sequencing of the complete gene
is described below.
Cloning of S. griseus ssgA by PCR
[0034] The sequence of S. griseus ssgA was published by Kawamoto
and Ensign (1995b), and deposited in the EMBL/GENBANK database
(D50051). In a recent update the translational start codon was
proposed 30 nt downstream of the originally indicated start codon.
This ambiguity does not influence the outcome of our experiments.
On the basis of protein electrophoresis (SDS PAGE) experiments
using over-expressed SsgA and in view of the optimal spacing
between ribosome binding sequence and start codon, we believe that
the ATG of the 11.sup.th triplet of the originally proposed reading
frame represents the correct translational start codon (data not
shown). This is also supported by phylogenetic evidence from the
ssgA homologous mentioned below.
[0035] The 750 bp DNA fragment generated by PCR amplification of S.
griseus chromosomal DNA using oligonucleotides ssg1 and ssg2 was
cloned into pIJ2925, resulting in pGWS1 (Table 1). Restriction site
and sequence analysis confirmed that the fragment indeed contained
ssgA.
Southern Hybridization Reveals ssgA in a Limited Number of
Streptomycetes
[0036] Genomic DNAs isolated from several actinomycetes (see legend
to FIG. 2) was digested with BamHI and PstI, submitted to agarose
gel electrophoresis and hybridized with the 580 bp insert from
pGWS5 harboring S. griseus ssgA, under conditions of low stringency
to identify all genes with at least remote similarity to ssgA. One
hybridizing band was observed in the lanes containing S. collinus,
S. albus, S. goldeniensis, and S. griseus genomic DNAs, and two
bands of equal intensity in the lane containing S. netropsis DNA
(FIG. 2). Under stringency conditions allowing the detection of
genes with at least 65% homology to S. griseus ssgA, we failed to
detect a band corresponding to ssgA in all other Streptomyces
species, including S. coelicolor and S. lividans, in contrast to a
previous Southern analysis by Kawamoto and Ensign (1995b), who used
a probe that included ssgA flanking sequences from an unrelated
genomic DNA region. The duplicity of the signal corresponding to
ssgA in S. netropsis was due to a BamHI restriction site in the
gene, as can be deduced from the DNA sequence. We also could not
detect an ssgA homologue in any of the other actinomycetes checked,
namely Nocardia lactamdurans, Planobispora rosea, and
Saccharopolyspora erythraea.
Cloning and Sequencing of ssgA Homologues from Other
Streptomycetes
[0037] Genomic DNA fragments harboring ssgA homologues from three
streptomycetes, namely S. albus, S. goldeniensis, and S. netropsis,
were amplified by PCR, using oligonucleotides ssg3 and ssg4. These
fragments were cloned as EcoRI/BamHI fragments into pIJ2925, and
the DNA sequence was determined. Table 2 shows the similarities of
the ssgA genes and the deduced amino acid sequences. Interestingly,
the S. netropsis and S. griseus ssgA gene products share more than
86% identical amino acids (90% similar), which is high in
comparison to 79% (85%) for S. goldeniensis SsgA and, strikingly, a
poor 63% (71%) for S. albus SsgA.
S. griseus and S. netropsis Sporulate in Liquid Cultures
[0038] The morphology of the streptomycetes and actinomycetes
discussed in this paper was checked by various microscopic
techniques. To this purpose, the strains were grown in complex
(TSBS) or minimal (MM) liquid medium for three days, and growth
characteristics monitored. From these experiments it appeared that
only S. griseus and S. netropsis produced abundant spores in liquid
cultures, while S. goldeniensis and S. collinus showed unusual
thickening of the tips of the hyphae, but failed to sporulate under
the chosen conditions. Interestingly, while S. griseus sporulated
only in MM, as was already reported by Kendrick and Ensign (1983),
S. netropsis sporulated abundantly in TSBS as well as in MM. We
believe that the relation between sporulation and the expression of
SsgA is of particular interest.
Transcription Analysis
[0039] Transcription analysis by nuclease S1 mapping showed an
accumulation of ssgA transcripts in S. griseus and S. netropsis
after nutritional shift-down and at the onset of sporulation. S.
coelicolor did not sporulate under these conditions. Northern
analysis of RNA isolated from S. coelicolor M145 after nutritional
shift-down or normal growth was carried out, using the S. netropsis
ssgA gene as the probe. Expectedly, this did not reveal ssgA
transcripts in S. coelicolor.
Expression of ssgA in S. coelicolor M145 Results in Reduced
Branching of the Hyphae and Fragmented Growth
[0040] The insert of pGWS1 was cloned into pWHM3 and pWHM3-E,
multicopy shuttle vectors that replicate in E. coli and
Streptomyces. The resulting plasmids pGWS2 and pGWS3 (Table 1) were
introduced into S. coelicolor M145 and correct recombinants were
selected by checking the insert lengths of the plasmids. In a
control experiment we used pWHM3-E transformants.
[0041] Transformants containing pWHM3-E (without ssgA) or pGWS2
showed little or no altered morphology in the complex liquid media
TSBS, YEME, nor in minimal medium (MM), as judged by phase-contrast
microscopy (FIG. 3A). However, hyphae of transformants containing
pGWS3 showed strongly reduced branching in complex and minimal
medium cultures, resulting in clearly less dense mycelial lumps
(FIG. 3B). The vegetative hyphae not only show limited branching,
but many of the branches are less than a micron in length. When
pGWS3-SD was used instead of pGWS3, the effect was even stronger,
with small fragments appearing after approximately 30 hours, which
increased over time (FIG. 4). While MM cultures of S. coelicolor
typically result in very large mycelial lumps that sediment rapidly
(virtually all mycelium precipitates within one minute when shaking
was stopped), MM cultures containing pGWS3-SD transformants showed
significantly reduced sedimentation rates, with the majority of the
mycelium failing to sediment within five minutes after shaking of
the cultures was stopped.
Constitutive Expression of Chromosomally-Integrated ssgA Also
Results in Fragmented Growth
[0042] The insert of pGWS3 and pGWS3-SD was cloned in pSET152, a
conjugative E. coli/Streptomyces shuttle vector, resulting in pGWS4
and pGWS4-SD, respectively. These plasmids were introduced into S.
coelicolor M145 by standard protoplast transformation, and
transformants selected by overlay of the transformation plates with
apramycin. Chromosomal integration was checked by Southern
analysis, and presence of the complete gene confirmed by PCR using
oligonucleotides ssg1 and ssg2. The pGWS4 and pGWS4-SD integrants
were designated GSA1 and GSA2. S. coelicolor M145 harboring pSET152
without ssgA was used as control strain.
[0043] While recombinants containing pSET152 displayed wild-type
phenotype, with large mycelial lumps and very few smaller
fragments, GSA1 showed limited branching, while the phenotype of
GSA2 is much similar to that of S. coelicolor harboring pGWS3-SD,
with strongly limited branching, frequent septation and fragmented
growth (FIG. 3C). This shows that S. griseus ssgA integrated in the
S. coelicolor chromosome can be expressed at a level high enough to
allow fragmentation of S. coelicolor mycelium in complex and
minimal liquid cultures.
High Level Expression of ssgA in Other Actinomycetes
[0044] The ssgA expression vectors pGWS3-SD and pGWS4 were
introduced in S. lividans, S. clavuligerus, and Sacch. erythraea,
to test the effect of SsgA on the morphology of strains other than
S. coelicolor. Expression in S. lividans using pGWS3-SD or pGWS4
led to a phenotype much similar to that of S. coelicolor harboring
the same plasmids, as was expected since S. lividans and S.
coelicolor are strongly related streptomycetes. Interestingly,
expression of SsgA in both S. clavuligerus and Sacch. erythraea
also resulted in reduced branching and increased fragmentation in
liquid cultures (FIG. 4), even though morphology of these strains
is different from that of S. coelicolor.
[0045] Thus, it appears that overproduction of SsgA has a strong
effect on mycelium morphology in submerged cultures of
actinomycetes, irrespective of the presence or absence of
endogenous ssgA-like activities, with the vegetative hyphae showing
much enhanced septation and restricted branching. Furthermore, the
ageing cultures showed an increasing degree of fragmentation,
resulting in higher culture densities and lower viscosity of
recombinant streptomycetes expressing ssgA. Comparison of the
phenotypes of the two categories of Streptomyces strains, namely
those displaying ssgA activity and those without a significant
level, is currently in progress, and could give us more insight
into the role of SsgA in Streptomyces physiology.
[0046] Table 1. Oligonucleotides and ssgA constructs. Nucleotide
positions refer to the location of the primers in respect to the
first nucleotide (+1) of the ATG translational start codon of ssgA.
Underlined sequences indicate non-homologous sequences added to
create restriction sites (in italics) at the ends of the PCR
fragments.
[0047] Oligonucleotides TABLE-US-00001 Nucl. primer Pos. ssg1 5'
-194/-170 GGCGAATTCGAACAGCTACGTGGCGAAGTCGCCA 3' (SEQ ID NO: 10)
(nucleotides 1-9 are non-homologous, added nucleotides to create an
EcoRI site at nucleotides 5-9) ssg2 5'
GTGGGATCCGTGCTCGCGGCGCTGGTCGTCTC +539/+517 3' (SEQ ID NO: 11)
(nucleotides 1-9 are non-homologous, added nucleotides to create a
BamHI site at nucleotides 4-9) ssg3 5'
GGGAATTCCATATGCGCGAGTCGGTTCAAGCA -30/-10 3' (SEQ ID NO: 12)
(nucleotides 1-11 are non-homolo- gous, added nucleotides to create
an EcoRI and NdeI sites at nucleotides 3-14) ssg4 5'
CCGGTCAGCCGGCGTTCTGCTCCTC 3' +412/388 (SEQ ID NO: 13)
Plasmids [0048] pIJ2925 Derivative of pUC19, with BglII sites
flanking the Janssen and Bibb, slightly altered multiple cloning
site. 1993 [0049] pWHM3 Multi-copy E. coli/Streptomyces shuttle
vector. Carries Vara et al. thiostrepton resistance marker [0050]
pWHM3-E pWHM3 with the 300 bp fragment containing the constitutive
this study ermE promoter for gene expression [0051] pSET152 E.
coli/Streptomyces shuttle vector that allows integration in Bierman
et al., the _C31 attachment site on the Streptomyces chromosome.
1992 Carries apramycin resistance marker. [0052] pGWS1 pIJ2925
containing the 750 bp ssgA PCR (ssg1/ssg2) product this study
[0053] pGWS1-SD pGWS1 with the upstream region of ssgA replaced by
nt -1/-70 this study of S. ramocissimus tuf1 [0054] pGWS2 pWHM3
containing the EcoRI/HindIII insert from pGWS1 this study [0055]
pGWS3 pWHM3-E containing the BglII/HindIII insert from pGWS1 this
study [0056] pGWS3-SD pWHM3-E containing the BglII/HindIII insert
from pGWS1-SD this study [0057] pGWS4 pSET152 containing the
EcoRI/PstI insert from pGWS3 this study [0058] pGWS4-SD pSET152
containing the EcoRI/PstI insert from pGWS3-SD this study
[0059] pGWS5 pIJ2925 containing the 580 bp ssgA PCR (ssg3/ssg2)
product cloned EcoRI/BamHI. TABLE-US-00002 TABLE 2 DNA and deduced
protein sequence homologies of ssgA homologues. Above the diagonal:
DNA sequence identities (%). Below the diagonal: protein sequence
identities (similarities between brackets). S. S. albus
goldeniensis S. griseus S. netropsis S. albus X 75.2 74.5 72.3 S.
goldeniensis 71.3 (75.7) X 77.5 75.7 S. griseus 66.2 (71.3) 78.7
(85.3) X 83.3 S. netropsis 63.2 (70.6) 77.9 (83.8) 86.0 (90.4)
X
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Sequence CWU 1
1
13 1 438 DNA Streptomyces griseus 1 atgcgcgagt cggttcaagc
agaggtcatg atgagcttcc tcgtctccga ggagctctcg 60 ttccgtattc
cggtggagct ccgatacgag gtcggcgatc cgtatgccat ccggatgacg 120
ttccaccttc ccggcgatgc ccctgtgacc tgggcgttcg gccgcgagct gctgctggac
180 gggctcaaca gcccgagcgg cgacggcgat gtgcacatcg gcccgaccga
gcccgagggc 240 ctcggagatg tccacatccg gctccaggtc ggcgcggacc
gtgcgctgtt ccgggcgggg 300 acggcaccgc tggtggcgtt cctcgaccgg
acggacaagc tcgtgccgct cggccaggag 360 cacacgctgg gtgacttcga
cggcaacctg gaggacgcac tgggccgcat cctcgccgag 420 gagcagaacg ccggctga
438 2 407 DNA Streptomyces griseus CDS (1)..(405) 2 atg agc ttc ctc
gtc tcc gag gag ctc tcg ttc cgt att ccg gtg gag 48 Met Ser Phe Leu
Val Ser Glu Glu Leu Ser Phe Arg Ile Pro Val Glu 1 5 10 15 ctc cga
tac gag gtc ggc gat ccg tat gcc atc cgg atg acg ttc cac 96 Leu Arg
Tyr Glu Val Gly Asp Pro Tyr Ala Ile Arg Met Thr Phe His 20 25 30
ctt ccc ggc gat gcc cct gtg acc tgg gcg ttc ggc cgc gag ctg ctg 144
Leu Pro Gly Asp Ala Pro Val Thr Trp Ala Phe Gly Arg Glu Leu Leu 35
40 45 ctg gac ggg ctc aac agc ccg agc ggc gac ggc gat gtg cac atc
ggc 192 Leu Asp Gly Leu Asn Ser Pro Ser Gly Asp Gly Asp Val His Ile
Gly 50 55 60 ccg acc gag ccc gag ggc ctc gga gat gtc cac atc cgg
ctc cag gtc 240 Pro Thr Glu Pro Glu Gly Leu Gly Asp Val His Ile Arg
Leu Gln Val 65 70 75 80 ggc gcg gac cgt gcg ctg ttc cgg gcg ggg acg
gca ccg ctg gtg gcg 288 Gly Ala Asp Arg Ala Leu Phe Arg Ala Gly Thr
Ala Pro Leu Val Ala 85 90 95 ttc ctc gac cgg acg gac aag ctc gtg
ccg ctc ggc cag gag cac acg 336 Phe Leu Asp Arg Thr Asp Lys Leu Val
Pro Leu Gly Gln Glu His Thr 100 105 110 ctg ggt gac ttc gac ggc aac
ctg gag gac gca ctg ggc cgc atc ctc 384 Leu Gly Asp Phe Asp Gly Asn
Leu Glu Asp Ala Leu Gly Arg Ile Leu 115 120 125 gcc gag gag cag aac
gcc ggc tg 407 Ala Glu Glu Gln Asn Ala Gly 130 135 3 135 PRT
Streptomyces griseus 3 Met Ser Phe Leu Val Ser Glu Glu Leu Ser Phe
Arg Ile Pro Val Glu 1 5 10 15 Leu Arg Tyr Glu Val Gly Asp Pro Tyr
Ala Ile Arg Met Thr Phe His 20 25 30 Leu Pro Gly Asp Ala Pro Val
Thr Trp Ala Phe Gly Arg Glu Leu Leu 35 40 45 Leu Asp Gly Leu Asn
Ser Pro Ser Gly Asp Gly Asp Val His Ile Gly 50 55 60 Pro Thr Glu
Pro Glu Gly Leu Gly Asp Val His Ile Arg Leu Gln Val 65 70 75 80 Gly
Ala Asp Arg Ala Leu Phe Arg Ala Gly Thr Ala Pro Leu Val Ala 85 90
95 Phe Leu Asp Arg Thr Asp Lys Leu Val Pro Leu Gly Gln Glu His Thr
100 105 110 Leu Gly Asp Phe Asp Gly Asn Leu Glu Asp Ala Leu Gly Arg
Ile Leu 115 120 125 Ala Glu Glu Gln Asn Ala Gly 130 135 4 407 DNA
Streptomyces albus G CDS (1)..(405) 4 atg agc ttc ctc gtc tcc gag
gag ctc gcc ttc cgc atc ccg gtg gag 48 Met Ser Phe Leu Val Ser Glu
Glu Leu Ala Phe Arg Ile Pro Val Glu 1 5 10 15 ctg cgg tac gag acc
gtc gat ccg tac gcg gtg cgg ctg acg ttc cac 96 Leu Arg Tyr Glu Thr
Val Asp Pro Tyr Ala Val Arg Leu Thr Phe His 20 25 30 ctc ccc gga
gac gcc ccg gtc acc tgg gtc ttc ggg cgt gaa ctg ctg 144 Leu Pro Gly
Asp Ala Pro Val Thr Trp Val Phe Gly Arg Glu Leu Leu 35 40 45 gtc
gag gga gtc ctg gac gcc gcg ggc gac ggc gac gtc cgg gtc tgc 192 Val
Glu Gly Val Leu Asp Ala Ala Gly Asp Gly Asp Val Arg Val Cys 50 55
60 ccg gtg ggg cag acg gcc acc agg gag gtg cac atc acc ctc cag gtc
240 Pro Val Gly Gln Thr Ala Thr Arg Glu Val His Ile Thr Leu Gln Val
65 70 75 80 ggc tcc gag cag gcg ctc ttc cgc gtc ggc aag gcg ccg ctg
ctc gcc 288 Gly Ser Glu Gln Ala Leu Phe Arg Val Gly Lys Ala Pro Leu
Leu Ala 85 90 95 ttc ctc gac cgc acc gac cag ggc ttg tcg ctc ggc
agc gag cgg gca 336 Phe Leu Asp Arg Thr Asp Gln Gly Leu Ser Leu Gly
Ser Glu Arg Ala 100 105 110 cac gcc gac ttc gac agc cac ctc gac gac
gct ctg aac cgc agc ctc 384 His Ala Asp Phe Asp Ser His Leu Asp Asp
Ala Leu Asn Arg Ser Leu 115 120 125 gcc gag gag cag agc gcc ggc tg
407 Ala Glu Glu Gln Ser Ala Gly 130 135 5 135 PRT Streptomyces
albus G 5 Met Ser Phe Leu Val Ser Glu Glu Leu Ala Phe Arg Ile Pro
Val Glu 1 5 10 15 Leu Arg Tyr Glu Thr Val Asp Pro Tyr Ala Val Arg
Leu Thr Phe His 20 25 30 Leu Pro Gly Asp Ala Pro Val Thr Trp Val
Phe Gly Arg Glu Leu Leu 35 40 45 Val Glu Gly Val Leu Asp Ala Ala
Gly Asp Gly Asp Val Arg Val Cys 50 55 60 Pro Val Gly Gln Thr Ala
Thr Arg Glu Val His Ile Thr Leu Gln Val 65 70 75 80 Gly Ser Glu Gln
Ala Leu Phe Arg Val Gly Lys Ala Pro Leu Leu Ala 85 90 95 Phe Leu
Asp Arg Thr Asp Gln Gly Leu Ser Leu Gly Ser Glu Arg Ala 100 105 110
His Ala Asp Phe Asp Ser His Leu Asp Asp Ala Leu Asn Arg Ser Leu 115
120 125 Ala Glu Glu Gln Ser Ala Gly 130 135 6 407 DNA Streptomyces
goldeniensis CDS (1)..(405) 6 atg agc ttc ctc gtc tcg gaa gaa ctc
tcc ttc cgt att ccg gtg gag 48 Met Ser Phe Leu Val Ser Glu Glu Leu
Ser Phe Arg Ile Pro Val Glu 1 5 10 15 ctg cgt tac gag acc tgt gat
ccc tac gcc gtg cgg ctg acc ttt cat 96 Leu Arg Tyr Glu Thr Cys Asp
Pro Tyr Ala Val Arg Leu Thr Phe His 20 25 30 ctg ccc gga gat gcc
ccg gtg acc tgg gcg ttc ggg cgg gag ttg ctc 144 Leu Pro Gly Asp Ala
Pro Val Thr Trp Ala Phe Gly Arg Glu Leu Leu 35 40 45 atc gac gga
ggt ccg cgg ccg tgc ggg gac ggg gac gtc cac atc gcg 192 Ile Asp Gly
Gly Pro Arg Pro Cys Gly Asp Gly Asp Val His Ile Ala 50 55 60 ccc
gcc gac ccg gag acg ttc ggc gag gtc ctg atc cgc ctg cag gtg 240 Pro
Ala Asp Pro Glu Thr Phe Gly Glu Val Leu Ile Arg Leu Gln Val 65 70
75 80 ggg agc gac cag gcg atg ttc cgg gtc ggc acg gcg ccg ctg gtg
gcc 288 Gly Ser Asp Gln Ala Met Phe Arg Val Gly Thr Ala Pro Leu Val
Ala 85 90 95 ttc ctg gac cgc acg gac aag atc gtg ccg ctg ggg cag
gag cgt tcc 336 Phe Leu Asp Arg Thr Asp Lys Ile Val Pro Leu Gly Gln
Glu Arg Ser 100 105 110 ctc gcc gac ttc gac gcc ctg ctc gac gag gcg
ctg gac cgc atc ctg 384 Leu Ala Asp Phe Asp Ala Leu Leu Asp Glu Ala
Leu Asp Arg Ile Leu 115 120 125 gcc gag gag cag aac gcc ggc tg 407
Ala Glu Glu Gln Asn Ala Gly 130 135 7 135 PRT Streptomyces
goldeniensis 7 Met Ser Phe Leu Val Ser Glu Glu Leu Ser Phe Arg Ile
Pro Val Glu 1 5 10 15 Leu Arg Tyr Glu Thr Cys Asp Pro Tyr Ala Val
Arg Leu Thr Phe His 20 25 30 Leu Pro Gly Asp Ala Pro Val Thr Trp
Ala Phe Gly Arg Glu Leu Leu 35 40 45 Ile Asp Gly Gly Pro Arg Pro
Cys Gly Asp Gly Asp Val His Ile Ala 50 55 60 Pro Ala Asp Pro Glu
Thr Phe Gly Glu Val Leu Ile Arg Leu Gln Val 65 70 75 80 Gly Ser Asp
Gln Ala Met Phe Arg Val Gly Thr Ala Pro Leu Val Ala 85 90 95 Phe
Leu Asp Arg Thr Asp Lys Ile Val Pro Leu Gly Gln Glu Arg Ser 100 105
110 Leu Ala Asp Phe Asp Ala Leu Leu Asp Glu Ala Leu Asp Arg Ile Leu
115 120 125 Ala Glu Glu Gln Asn Ala Gly 130 135 8 407 DNA
Streptomyces netropsis CDS (1)..(405) 8 atg agc ttc ctc gtc tcc gag
gag ctc tcc ttc aag atc cca gtc gaa 48 Met Ser Phe Leu Val Ser Glu
Glu Leu Ser Phe Lys Ile Pro Val Glu 1 5 10 15 ctg cga tac gag acc
cgg gat ccc tac gcg gtg cgg atg acc ttc cac 96 Leu Arg Tyr Glu Thr
Arg Asp Pro Tyr Ala Val Arg Met Thr Phe His 20 25 30 ctc ccc gga
gac gcg cct gtg acc tgg gcg ttc ggc cgg gag ctg ctg 144 Leu Pro Gly
Asp Ala Pro Val Thr Trp Ala Phe Gly Arg Glu Leu Leu 35 40 45 ctc
gac ggg atc aac cgc ccg agc ggc gac ggc gac gtc cac atc gcc 192 Leu
Asp Gly Ile Asn Arg Pro Ser Gly Asp Gly Asp Val His Ile Ala 50 55
60 ccg acc gac ccc gag ggc ctg tcg gac gtc tcc atc cgg ctc cag gtg
240 Pro Thr Asp Pro Glu Gly Leu Ser Asp Val Ser Ile Arg Leu Gln Val
65 70 75 80 ggc gcg gac cgc gcc ctc ttc cgt gca ggc gcc ccg ccg ctg
gtc gcc 288 Gly Ala Asp Arg Ala Leu Phe Arg Ala Gly Ala Pro Pro Leu
Val Ala 85 90 95 ttc ctc gac cgc acg gac aag tcg gtg ccg ctc ggt
cag gaa cag act 336 Phe Leu Asp Arg Thr Asp Lys Ser Val Pro Leu Gly
Gln Glu Gln Thr 100 105 110 ctg ggt gac ttc gag gac agc ctg gag gcc
gcg ctc ggc aag atc ctc 384 Leu Gly Asp Phe Glu Asp Ser Leu Glu Ala
Ala Leu Gly Lys Ile Leu 115 120 125 gcc gag gag cag aac gcc ggc tg
407 Ala Glu Glu Gln Asn Ala Gly 130 135 9 135 PRT Streptomyces
netropsis 9 Met Ser Phe Leu Val Ser Glu Glu Leu Ser Phe Lys Ile Pro
Val Glu 1 5 10 15 Leu Arg Tyr Glu Thr Arg Asp Pro Tyr Ala Val Arg
Met Thr Phe His 20 25 30 Leu Pro Gly Asp Ala Pro Val Thr Trp Ala
Phe Gly Arg Glu Leu Leu 35 40 45 Leu Asp Gly Ile Asn Arg Pro Ser
Gly Asp Gly Asp Val His Ile Ala 50 55 60 Pro Thr Asp Pro Glu Gly
Leu Ser Asp Val Ser Ile Arg Leu Gln Val 65 70 75 80 Gly Ala Asp Arg
Ala Leu Phe Arg Ala Gly Ala Pro Pro Leu Val Ala 85 90 95 Phe Leu
Asp Arg Thr Asp Lys Ser Val Pro Leu Gly Gln Glu Gln Thr 100 105 110
Leu Gly Asp Phe Glu Asp Ser Leu Glu Ala Ala Leu Gly Lys Ile Leu 115
120 125 Ala Glu Glu Gln Asn Ala Gly 130 135 10 34 DNA Primer ssg1
10 ggcgaattcg aacagctacg tggcgaagtc gcca 34 11 32 DNA Primer ssg2
11 gtgggatccg tgctcgcggc gctggtcgtc tc 32 12 32 DNA Primer ssg3 12
gggaattcca tatgcgcgag tcggttcaag ca 32 13 25 DNA Primer ssg4 13
ccggtcagcc ggcgttctgc tcctc 25
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