U.S. patent application number 11/238976 was filed with the patent office on 2006-10-12 for gram-positive bacteria deprived of htra protease activity and their uses.
This patent application is currently assigned to Institut National de la Recherche Agronomique. Invention is credited to Alexandre Bolotine, Alexandra Gruss, Isabelle Poquet, Alexei Sorokine.
Application Number | 20060228377 11/238976 |
Document ID | / |
Family ID | 9534523 |
Filed Date | 2006-10-12 |
United States Patent
Application |
20060228377 |
Kind Code |
A1 |
Poquet; Isabelle ; et
al. |
October 12, 2006 |
Gram-positive bacteria deprived of HtrA protease activity and their
uses
Abstract
The invention concerns bacteria strains, obtained from
gram-positive bacteria whereof the genome size is not more than 3.2
Mb, and wherein the HtrA surface protease is inactive. Said strains
are useful for expressing exported proteins of interest.
Inventors: |
Poquet; Isabelle; (Paris,
FR) ; Gruss; Alexandra; (Orsay, FR) ;
Bolotine; Alexandre; (Nancy, FR) ; Sorokine;
Alexei; (Gif-Sur-Yvette, FR) |
Correspondence
Address: |
MORGAN LEWIS & BOCKIUS LLP
1111 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
20004
US
|
Assignee: |
Institut National de la Recherche
Agronomique
|
Family ID: |
9534523 |
Appl. No.: |
11/238976 |
Filed: |
September 30, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09869106 |
Oct 9, 2001 |
6994997 |
|
|
PCT/FR99/03270 |
Dec 23, 1999 |
|
|
|
11238976 |
Sep 30, 2005 |
|
|
|
Current U.S.
Class: |
424/234.1 ;
424/93.4; 435/252.3; 435/252.31; 435/69.1; 530/350 |
Current CPC
Class: |
C12P 21/02 20130101;
C12N 15/74 20130101; C12N 9/52 20130101; A61K 35/74 20130101 |
Class at
Publication: |
424/234.1 ;
424/093.4; 435/252.3; 435/252.31; 435/069.1; 530/350 |
International
Class: |
A61K 35/74 20060101
A61K035/74; C12P 21/06 20060101 C12P021/06; A61K 39/02 20060101
A61K039/02; C12N 1/21 20060101 C12N001/21; C07K 14/195 20060101
C07K014/195 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 24, 1998 |
FR |
98/16462 |
Claims
1-10. (canceled)
11. A method of producing a fermented product, comprising:
culturing a bacterial strain with a fermentation substrate under
conditions suitable to produce a fermented product, wherein the
bacterial strain does not express a functional HtrA.
12. A method of producing a dietetic food, comprising: culturing a
bacterial strain with a substrate under conditions suitable to
produce a dietetic food, wherein the bacterial strain does not
express a functional HtrA.
13. A method of producing a medicinal product, comprising:
culturing a bacterial strain with a suitable substrate under
conditions effective to produce a medicinal product, wherein the
bacterial strain does not express a functional HtrA.
14. A method according to claim 13, wherein the medicinal product
is a vaccine.
Description
[0001] This application is a divisional of copending U.S.
application Ser. No. 09/869,106 filed on Jun. 22, 2001, which is a
U.S. National Phase Application of International Application No.
PCT/FR99/03270, filed on Dec. 23, 1999, which claims the benefit of
French Application No. 98/16462, filed Dec. 24, 1998, all of which
are herein incorporated by reference in their entirety.
[0002] The invention relates to the production, in Gram-positive
bacteria, of exported proteins.
[0003] The general term "exported proteins" denotes proteins which
are transported across the cytoplasmic membrane. In the case of
Gram-positive bacteria, this transport results in the secretion of
the protein into the medium, or its association with the cell
surface.
[0004] One of the main problems which arises during the production
of exported proteins of interest by host bacteria lies in the
degradation of these proteins during and/or after their
exportation, at the cell envelope or at the cell surface. This
degradation often leads to a decrease in the yield, and/or a
modification of the structure and of the activity of the
protein.
[0005] The enzymes responsible for this degradation of exported
proteins are bacterial proteases, themselves exported in the
envelope; they are "housekeeping" proteases, one of the main
functions of which is normally a role of degradation of abnormal or
incorrectly folded exported proteins which accumulate in the medium
or in the envelope, in particular under conditions of stress, and
the role of which is also the recycling of exported proteins.
[0006] Heterologous proteins, which are often incompletely
recognized by the chaperone proteins involved in protein folding in
the host bacterium, are particularly sensitive to attack by these
proteases.
[0007] The oldest characterized exported housekeeping protease is
the E. coli serine protease HtrA/DegP. It is a protease which as a
periplasmic location, and which is expressed under the control of a
promoter which is inducible at high temperature; Beckwith and
Strauch (Proc. Natl. Acad. Sci. USA 85:1576-1580, 1988) have
observed that it is involved in the proteolysis of proteins made
from fusion between exported proteins of E. coli and the PhoA
exportation reporter. They have proposed the inactivation of this
protease in E. coli in order to limit the degradation of the
heterologous exported proteins.
[0008] Mutant E. coli strains, in which the gene encoding the
HtrA/DegP protease has been inactivated, have thus been obtained
[Beckwith and Strauch, abovementioned publication, and PCT
application WO 88/05821]; however, it has been noted that this
inactivation results in a slowing down of the kinetics of
degradation, but is not sufficient to abolish it because of the
existence, in the envelope, of other proteases which degrade the
exported proteins.
[0009] In E. coli several envelope housekeeping proteases, which
carry out functions similar to those of HtrA/DegP, have been
characterized: they are in particular the HhoA/DegQ and HhoB/DegS
proteases, which are structurally homologous to HtrA/DegP, and
proteases which are structurally different but functionally
comparable (ApeA/proteaseI, OmpT, OmpP, Prc/Tsp, SppA/proteaseIV,
PrtIII and SohB).
[0010] Studies relating to other bacteria have also made it
possible to demonstrate the existence, in each species studied, of
several exported housekeeping proteases. For example, a large
number of bacterial species have several proteases of the HtrA
family (Pallen and Wren, Mol. Microbiol. 19:209-21, 1997); three
homologues of HtrA have been identified in B. subtilis (YyxA, YkdA
and YvtB/Yirf), Synechocystis (HtrA, HhoA and HhoB), Pseudomonas
aeruginosa and Aquifex aeolicus, two in Hemophilus influenzae (HtoA
and HhoB), Campylobacter jejuni, Brucella abortus and Yersinia
enterolitica, and four in Mycobacterium tuberculosis. Various
Gram-positive bacteria also have serine proteases considered to be
related to the HtrA family on the basis of homology in the
catalytic domain: EtA, EtB and V8/StsP of S. aureus, GseP of
Bacillus licheniformis and Spro of Mycobacterium paratuberculosis
(Koonin et al., Chap 117 in Escherichia coli and Salmonella
typhimurium, 2203-17, 1997). Finally, exported proteases which are
not related to HtrA have also been demonstrated, for example in B.
subtilis (Margot and Karamata, Microbiology, 142:3437-44, 1996;
Stephenson and Harwood Appl. Environn. Microbiol. 64:2875-2881,
1998; Wu et al. J. Bacteriol. 173:4952-58, 1991).
[0011] It has therefore been proposed to combine mutations
affecting several exported proteases in order to obtain an
effective decrease in the degradation of heterologous exported
proteins.
[0012] For example, an E. coli strain mutated in the degP/htrA,
ompT, prt and prc genes (Meerman and Georgiou, Bio/technology
12:1107-10, 1994), and a B. subtilis strain deficient in the six
extracellular proteases (Wu et al., 1991, abovementioned
publication), have been constructed with this aim. However, the use
of these strains does not make it possible to completely eliminate
the proteolysis of the exported proteins. For example, in the case
of the B. subtilis strain described by Wu et al., although the
residual extracellular protease activity is negligible (<1%),
degradation of the heterologous exported proteins remains
significant. In order to overcome this problem, that same team has
carried out further modifications to this strain in order to make
it overproduce various chaperones (Wu et al., J. Bacteriol.
180:2830-35, 1998). Furthermore, although the inactivation of the
gene of one of these exported housekeeping proteases does not have
any notable consequences for the bacterium, the accumulation of
mutations may affect strain viability; Meerman and Georgiou (1994,
abovementioned publication) thus observe a decrease in growth rate
which can range up to 50%.
[0013] In lactic acid bacteria, only a few exported proteases have
been studied; the most well characterized at the present time is
the protease named PrtP (Kok, FEMS Microbiol. Reviews 87:15-42,
1990), which is located at the cell surface, where it is anchored
to the peptidoglycan. This protease is present in many lactic acid
bacteria, in particular Lactococcus lactis, and is located on a
plasmid. It contributes to the nitrogen-based nutrition of bacteria
by degrading milk caseins. Other surface proteases have been
purified from two species of lactic acid bacteria, Lactobacillus
delbrueckeii subsp. bulgaricus and Lactobacillus helveticus, but
their function has not been determined (Stefanitsi et al., FEMS
Microbiol. Lett. 128:53-8, 1995; Stefanitsi and Garel, Lett. Appl.
Microbiol. 24:180-84, 1997; Yamamoto, et al., J. Biochem.
114:740-45, 1993). A stress-induced gene encoding a protein which
is highly homologous to the proteases of the HtrA family has
recently been revealed in Lactobacillus helveticus (Smeds et al.,
J. Bacteriol. 180:6148-53, 1998). It has been observed that this
gene is necessary for survival at high temperature; a mutant
Lactobacillus helveticus strain in which the htrA gene has been
inactivated by insertion of a reporter gene (gusA, encoding
.beta.-glucuronidase) under the control of the htrA promoter, was
constructed. The study of the expression of the gusA gene in this
mutant made it possible to demonstrate induction of the
transcription of this gene under the same conditions as that of the
htrA gene in the wild-type strains; on the other hand, no
.beta.-glucuronidase activity was observed.
[0014] In previous investigations directed towards studying
exported proteins of Lactococcus lactis by studying proteins fused
with the .DELTA..sub.SPNuc exportation reporter (Poquet et al., J.
Bacteriol. 180:1904-12, 1998), the team of inventors has observed
significant extracellular proteolysis even though the experiments
were carried out in an L. lactis subsp. cremoris strain free of any
plasmid and therefore, in particular, of that which carries
prtP.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1. FIG. 1 shows the nucleotide and amino acid sequence
of the gene HtrA.sub.L1 (SEQ ID NO: 1). The amino acids of the
catalytic domain and transmembrane domain are box framed. The
positions of primers F, G, and A are indicated by arrows.
[0016] FIGS. 2 A and B. FIGS. 2 A and B show the growth curves of
the htrA.sup.+/htrA strain, htrA strain, and the wild-type IL1403
strain. The growth was monitored by measuring the OD.sub.600 at the
indicated time intervals. FIG. 2A shows the growth of
htrA.sup.+/htrA, htrA, and IL1403 strains at 30.degree. C. FIG. 2B
shows the growth of htrA.sup.+/htrA, htrA, and IL1403 strains at
37.degree. C.
[0017] FIG. 3. FIG. 3 shows the effect of HtrA.sub.L1 mutation on
the stability of Nuc protein. The degradation profiles of Nuc
protein in IL1403 (first three wells), htrA (three central wells)
and htrA.sup.+/htrA (last three wells) strains are shown. The
immunological detection of the Nuc protein was carried out on the
protein samples extracted from the total culture (T), cells alone
(C) and from the medium (M) of each strain.
[0018] FIG. 4. FIG. 4 shows the effect of HtrA.sub.L1 mutation on
the stability of Usp-.sub.spNuc protein. The degradation profiles
of Usp-.sub.spNuc protein in IL1403 (first three wells), htrA
(three central wells) and htrA.sup.+/htrA (last three wells)
strains are shown. The immunological detection for the
Usp-.sub.spNuc protein was carried out on the protein samples
extracted from the total culture (T), cells alone (C) and from the
medium (M) of each strain.
[0019] FIG. 5. FIG. 5 shows the effect of HtrA.sub.L1 mutation on
the stability of N1p4-.sub.spNuc protein. The degradation profiles
of N1p4-.sub.spNuc protein in IL1403 (first well), htrA (central
well) and htrA.sup.+/htrA (last well) strains are shown.
[0020] FIG. 6. FIG. 6 shows the effect of HtrA.sub.L1 mutation on
the stability of Exp5-.sub.spNuc protein. The degradation profiles
of Exp5-.sub.spNuc protein in IL1403 (first well), htrA (central
well) and htrA.sup.+/htrA (last well) strains are shown.
[0021] FIG. 7. FIG. 7 shows a zymogram of the bacteriolysin
activity of AcmA protein. The degradation profiles of AcmA protein
in IL1403 (first three wells), htrA (three central wells) and
htrA.sup.+/htrA (last three wells) strains are shown. The detection
of the AcmA protein was carried out on the protein samples
extracted from the total culture (T), cells alone (C) and from the
medium (M) of each strain.
[0022] The inventors undertook to investigate extracellular
proteases responsible for this proteolysis.
[0023] They have thus discovered, in L. lactis, the existence of a
gene of the htrA family.
[0024] This gene, detected in the genome of the IL1403 strain of L.
lactis subsp. lactis, encodes a 408 amino acid protein, hereinafter
named HtrA.sub.L1, the nucleotide sequence and the amino acid
sequence of which are represented on FIG. 1, and appear in the
attached sequence listing (SEQ ID NO: 1). This protein is very
homologous to E. coli HtrA, and to various other known members of
the HtrA family, as shown in table I below, which illustrates the
percentages of identity and of similarity between HtrA.sub.L1 and
various proteins of the HtrA family: TABLE-US-00001 TABLE I Protein
Organism % identity % similarity HtrA/DepP/Do protease E. coli 31.5
38.2 HhoA/DegQ E. coli 34.0 40.8 HhoB/DegS E. coli 29.9 37.3 HtrA
S. typhimurium 32.4 39.1 HtoA H. influenzae 31.9 39.2 HhoB/DegS H.
influenzae 31.2 40.0 spHtrA S. pneumoniae 55.6 62.0 HtrA Lb.
helveticus 46.9 54.1 YyxA B. subtilis 43.5 52.0 YkdA B. subtilis
42.5 49.4
[0025] The HtrA protein of the IL1403 strain of L. lactis subsp.
lactis has the three amino acids Ser, His and Asp, which define the
catalytic site characteristic of serine proteases related to
trypsin, among which is the HtrA family; in addition, it has,
around these three amino acids, the following three motifs:
DAYVVTNYH.sub.127VI, D.sub.157LAVLKIS, and
GNS.sub.239GGALINIEGQVIGIT, which correspond to the consensus
regions defined by Pallen and Wren (Mol. Microbiol. 19:209-21,
1997) for the catalytic domain of the HtrA proteases: -GY-TN-HV-,
D-AV- and GNSGG-L-N-G-IGIN.
[0026] At its N-terminal end, it has a hydrophobic amino acid
sequence L.sub.10LTGVVGGAIALGGSAI.sub.26 corresponding to a
putative transmembrane segment. The HtrA.sub.L1 protein of L.
lactis subsp. lactis is therefore thought to be an integral protein
of the cytoplasmic membrane. According to the "positive inside"
rule concerning the topology of these proteins (Von Heijne, Nature,
341:456-8, 1989), it topology corresponds to the "C-out" type, i.e.
its C-terminal portion, which comprises in particular its catalytic
site, would be exposed to the outside of the plasma membrane. Like
the HtrA protease of E. coli, L. lactis subsp. lactis HtrA.sub.L1
therefore appears to be an envelope protease which can degrade
exported proteins. The amino acids of the catalytic domain and of
the transmembrane domaine are framed on FIG. 1.
[0027] The inventors have inactivated this gene by mutation; at
optimum temperature (30.degree. C.), the mutant L. lactis subsp.
lactis strain thus obtained is viable and grows normally; on the
other hand, its growth and viability are affected at higher
temperatures (from 37.degree. C.), both on plates and in liquid
medium.
[0028] In addition, the inventors have studied the effect of this
mutation on the exportation of various fusion proteins, and have
noted that the inactivation of the HtrA.sub.L1 protease in L.
lactis is sufficient to completely abolish the degradation of the
exported proteins; this effect is surprising given the residual
proteolysis observed previously in other bacteria after
inactivation on proteases of the HtrA family.
[0029] A subject of the present invention is a process for
producing a protein of interest, characterized in that it comprises
culturing a bacterial strain which expresses said protein of
interest, and which can be obtained from a Gram-positive bacterium,
the size of the genome of which bacterium is at most equal to 3.2
Mb, preferably at most equal to 3 Mb, and advantageously at most
equal to 2.5 Mb, by mutation which inactivates the HtrA surface
protease of said bacterium;
[0030] and producing said protein of interest exported by said
strain.
[0031] According to a preferred embodiment of the present
invention, the starting Gram-positive bacterium is chosen from
bacteria of the group consisting of the Streptococcaceae, and
Lactobacillaceae. Advantageously, it is chosen from lactococci.
[0032] It may be also be chosen from bacteria belonging to the
group consisting of the Bacillaceae, for example to the Listeria
genus, and the Enterococcaceae, in particular of the Enterococcus
genus.
[0033] Advantageously, said bacterial strain may also comprise one
or more other modifications of its genome, directed toward
improving the production and/or secretion of proteins expressed in
said bacterium, and/or toward avoiding their degradation. Depending
on the type of protein intended to be produced, it is possible, for
example, to use a bacterial strain in which the PrtP protease
activity has been inactivated, and/or a bacterial strain which
overproduces a protein allowing the stabilization of exported
proteins, such as the Nlp4 protein of Lactococcus lactis, or a
homologue thereof (Poquet et al. 1998, abovementioned
publication).
[0034] A subject of the present invention is also any bacterial
strain which can be obtained from a Gram-positive bacterium, the
size of the genome of which bacterium is at most equal to 3.2 Mb,
as defined above, by mutation which inactivates the HtrA surface
protease of said bacterium, and which also comprises at least one
cassette for expressing a gene of interest, with the exception of a
Lactobacillus helveticus strain comprising a single expression
cassette consisting of the sequence encoding the gusA reporter gene
inserted into the htrA gene of said strain, under the
transcriptional control of the promoter of said gene.
[0035] The term "expression cassette" is intended to mean any
recombinant DNA construct comprising a gene of interest, the
expression of which is desired, or a site allowing the insertion of
said gene, placed under the control of regulatory sequences for
transcription (promoter, terminator), which are functional in the
host bacterium under consideration.
[0036] For the purpose of the present invention, the term "HtrA
protease" is intended to mean any serine protease of the trypsin
type, having functional and structural similarities with the HtrA
protease of E. coli which are sufficient for it to be included in
the same family, i.e.:
[0037] a catalytic site formed by the three amino acids Ser, His
and Asp;
[0038] the presence, around this catalytic site, of the consensus
regions: -GY-TN-HV-, D-AV- and GNSGG-L-N-G-IGIN;
[0039] an exportation signal enabling the protease to be
transported to the cell surface of the bacterium, (it may, for
example, be a signal peptide, a transmembrane domain, a signal for
anchorage to the wall, etc.).
[0040] In order to implement the present invention, mutant bacteria
lackiing HtrA activity can be produced by carrying out one or more
mutations, in particular in the sequence encoding the HtrA protease
and/or in the regulatory sequences allowing the expression of the
htrA gene, so as to prevent the expression of a functional HtrA
protease. These mutations can be carried out conventionally, by
deletion, insertion or replacement of at least one nucleotide or
one nucleotide sequence in the htrA gene; they can result either in
the absence of production of HtrA, or in the production of an HtrA
protease in which at least one amino acid required for activity has
been deleted or replaced.
[0041] The suitable mutagenesis techniques are known per se;
advantageously, use will be made of site-directed mutagenesis
techniques, since the data available on the proteases of the HtrA
family make it possible, even though more precise information on
the specific sequence of the gene whose inactivation is desired is
not available, to target the mutation(s) on conserved domains which
are required for activity (for example the catalytic domain).
[0042] The present invention can be implemented in many
domains.
[0043] Firstly, it can be used in the domain of the production of
proteins of interest (for example enzymes, human proteins, etc.) by
genetic engineering, using cultures of bacteria transformed with a
gene of interest. In this domain, the present invention makes it
possible to improve the yield of exported proteins (and in
particular secreted proteins), and to avoid their contamination
with inactive proteolytic products: this makes it possible to
purify them easily and less expensively.
[0044] For this application, use will preferably be made of the
mutant strains produced from nonpathogenic bacteria, such as
Lactococcus spp. or Lactobacillus spp., and also food streptococci,
Streptococcus thermophilus.
[0045] The mutant strains produced from bacteria conventionally
used in the agro-foods industry, such as lactic acid bacteria (in
particular lactococci, lactobacilli and thermophilic streptococci),
can advantageously be used in this domain. For example, they can be
used in the composition of ferments, in order to produce
heterologous proteins making it possible to improve the quality of
the finished fermented product; thus, the exportation of foreign
enzymes produced by a mutant L. lactis strain in accordance with
the invention, within cheeses fermented with L. lactis, may improve
their maturing and their organoleptic qualities.
[0046] These mutant strains can also be used for producing dietetic
products or medicinal products. In this domain, mutant strains in
accordance with the invention can, for example, be used in order to
express, prior to the ingestion of the product and/or after its
ingestion, proteins with a prophylactic or therapeutic effect, such
as enzymes (for facilitating digestion, for example), proteins for
stimulating the immune system, immunization antigens, etc. In most
cases, for use in this domain, and in order to guarantee maximum
innocuity, mutant strains produced from nonpathogenic bacteria and,
advantageously, from bacteria conventionally used for food will be
preferred. However, in the context of uses for immunization, mutant
strains produced from pathogenic bacteria (in particular
streptococci, staphylococci, enterococci or listeria), and
preferably from variants of these bacteria already carrying one or
more mutations which attenuate their pathogenic power, can be used;
the inactivation of the HtrA protein, in limiting the capacities of
survival of these bacteria under conditions of stress, may
contribute to attenuating their virulence, as previously observed
in the case of certain Gram-negative bacteria.
[0047] In the context of certain applications, in which the host
bacterium must be viable and capable of producing proteins at
temperatures of about 35 to 40.degree. C., for example the
production, in a fermentor, of certain proteins, or the production,
after ingestion, in the digestive tract of humans or animals, of
proteins with therapeutic or prophylactic activity, mutant strains
produced from thermophilic bacteria, such as Streptococcus
thermophilus, will advantageously be used.
[0048] The present invention will be more clearly understood with
the aid of the continuation of the description which follows, which
refers to nonlimiting examples illustrating the production of L.
lactis mutants in which the HtrA surface protease is inactive, and
the properties of these mutants.
EXAMPLE 1
Inactivation of the hrtA Gene of L. lactis
[0049] htrA gene, carried by the chromosome of the IL1403 strain
(Chopin et al. Plasmid, 11, 260-263, 1984) of L. lactis subsp.
lactis, was inactivated by integration of a suicide plasmid
carrying a 665 bp internal fragment of the gene (FA).
[0050] As a positive control for integration, a suicide plasmid
carrying a 902 bp fragment truncated in the 3' region (GA), the
integration of which onto the chromosome restores a wild-type copy
of the gene, was used.
[0051] These fragments were obtained beforehand by PCR
amplification from the genomic DNA of the IL1403 strain of L.
lactis subsp. lactis, using the pairs of primers F/A and G/A:
TABLE-US-00002 - F[5'-GGAGCCA(G/T) (A/C/T)GC(A/G/C/T) (C/T)T
(A/G/T)GG-3']
[0052] located downstream of the ATG initiation codon
TABLE-US-00003 - G[5'-GTTTCCACTTTTCTGTGG-3']
[0053] located upstream of the htrA promoter TABLE-US-00004 -
A[5'-TT(A/T)CC(A/T)GG(A/G)TT(A/G/T)AT(A/G/C/T) GC-3']
located upstream of the serine codon of the catalytic site. The
positioning of the F, G and A primers is indicated on FIG. 1.
[0054] The amplification was carried out under the following
conditions:
[0055] reaction mixture: 0.2 mM of each dNTP, 5 .mu.M of each
oligonucleotide, approximately 500 ng of chromosomal DNA, 2 mM of
MgCl.sub.2 and 1.25 units of Taq-DNA-pol (Boehringer Mannheim), in
the Taq buffer provided by the manufacturer;
[0056] temperature conditions: 5 min 94.degree. C., 30 cycles (30
sec at 94.degree. C., 30 sec at 46.degree. C., and 30 sec at
72.degree. C.), and 4.degree. C.
[0057] The amplified fragments were ligated to the linear
pGEM.sup.T plasmid (Promega). After transformation of E. coli TG1
with the ligation products, the clones which are resistant to
ampicillin and lack .beta.-galactosidase activity are selected. The
plasmids obtained, bearing the FA and GA fragments, respectively,
are named pES1.1 and pES2.1.
[0058] The FA and GA inserts were subcloned into a suicide vector
carrying a chloramphenicol resistance gene. Since this vector is
incapable of replicating alone in the absence of the RepA protein
which is required for initiating its replication, co-integrants
were created by ligation between each of the pES1.1 and pES2.1
plasmids and the suicide vector, linearized beforehand.
[0059] After transformation of the E. coli TG1 strain, and
selection of the chloramphenical-resistant clones, the pGEM.sup.T
portion of the co-integrants was deleted and the vectors were
re-circularized. The plasmids obtained are multiplied in the TG1
repA.sup.+ strain of E. coli; after selection of the
chloramphenicol-resistant clones, the suicide plasmids named pVS6.1
and pVS7.4 are obtained.
[0060] pVS6.1 contains the FA fragment, and pVS7.4 contains the GA
fragment, of the htrA.sub.L1 gene of the IL1403 strain of L. lactis
subsp. lactis.
[0061] These plasmids were used to transform the IL1403 strain of
L. lactis subsp. lactis; the clones which had integrated these
plasmids at the htrA locus on the chromosome were selected in the
presence of chloramphenicol.
[0062] In both cases, several independent chloramphenicol-resistant
clones were obtained. Five clones of each class termed A to E in
the case of the integration of pVS6.1, and 17 to 22 in the case of
the integration of pVS7.4, were chosen for analysis.
[0063] For each of these clones, the integration at the htrA locus
was confirmed by Southern transfer.
[0064] Two clones, A and 17, were chosen for the following
analyses; they constitute the two prototypes of the mutant strains,
which hereinafter will be named:
[0065] htrA (null mutation of the htrA.sub.L1 gene, Cm.sup.R); this
strain does not express any active HtrA protease;
[0066] htrA.sup.+/htrA (wild-type copy+truncated copy of the
htrA.sub.L1 gene, Cm.sup.R); this strain expresses an active
Htra.sub.L1 protease.
EXAMPLE 2
Role of the htrA.sub.L1 Gene of L. lactis in Survival at High
Temperature
[0067] The two strains htrA and htrA.sup.+/htrA are cultured, in
liquid culture, under the conventional conditions for growth of L.
lactis, i.e. at 30.degree. C. and in the presence of oxygen, but
without stirring, and in the presence of chloramphenicol.
The behavior of the htrA strain of L. lactis subsp. lactis at
30.degree. C. and at 37.degree. C. was studied using the
htrA.sup.+/htrA strain and also the IL403 parent-strain (cultured
in the absence of chloramphenicol) as control.
[0068] The bacteria were cultured overnight at room temperature, in
an M17 medium containing 1% of glucose (+2.5 .mu.g/ml of
chloramphenicol for both the htrA strain and the htrA.sup.+/htrA
strain). The cultures were diluted 100-fold in the morning, in the
same medium, and divided into two batches placed in
semi-anaerobiosis at 30.degree. C. or at 37.degree. C. The growth
was monitored by measuring the OD.sub.600.
[0069] The results are illustrated in FIG. 2.
[0070] At 30.degree. C. (FIG. 2A), it is noted that the
htrA.sup.+/htrA strain (.box-solid.), the htrA strain
(.diamond-solid.), and the wild-type IL1403 strain
(.diamond-solid.) have very close generation times: 65 min for the
wild-type strain, 70 min for htrA.sup.+/htrA and 75 min for htrA;
finally, for the 3 cultures, the OD.sub.600 values corresponding to
the stationary phase are very comparable (OD.sub.600=2.1 to
2.2).
[0071] These results indicate that there is no significant
difference in growth between these three strains at 30.degree.
C.
[0072] At 37.degree. C. (FIG. 2B), the htrA.sup.+/htrA strain
(.box-solid.) has a generation time of 100 min and the OD.sub.600
of the stationary phase is less than at 30.degree. C.
(OD.sub.600=1.25). Less growth at 37.degree. C. than at 30.degree.
C. is also observed for the wild-type IL1403 strain
(.diamond-solid.); the generation time is 65 min, but the
OD.sub.600 of the stationary phase is less than at 30.degree. C.
(OD.sub.600=1.9). In the case of the htrA strain (.diamond-solid.),
the growth is very slight, or even zero, and the OD.sub.600 does
not exceed 0.1, even after culturing for 7 h.
[0073] It emerges from these results that the htrA strain of L.
lactis subsp. lactis is heat-sensitive and that the htrA mutation
is lethal at 37.degree. C.
EXAMPLE 3
Role of the htrA.sub.L1 Gene of L. LACTIS in Surface
Proteolysis
[0074] The effect of the htrA.sub.L1 mutation on the stability of
five exported proteins was tested. These proteins are:
[0075] i) a heterologous protein, the secreted nuclease of S.
aureus, Nuc; this protein is expressed by the plasmid pNuc3 (Le
Loir et al., J. Bacteriol. 176:5135-5139, 1994; Le Loir et al., J.
Bacteriol. 180:1895-903, 1998);
[0076] ii) three hybrid proteins (Usp-.DELTA..sub.SPNuc,
Nlp4-.DELTA..sub.SPNuc and Exp5-.DELTA..sub.SPNuc) resulting from
the fusion between the .DELTA..sub.SPNuc reporter and fragments of
exported proteins of L. lactis: the secreted protein Usp45 (Van
Asseldonk et al., Gene 95:155-60, 1990), the lipoprotein Nlp4 and
the protein Exp5 (which is, itself, a protein made from fusion
between an exported protein and a cytoplasmic protein); these
proteins, and also the plasmids pVE8009, pVE8024 and pVE8021 which
express them, respectively, are described by Poquet et al. (1998,
abovementioned publication);
[0077] iii) a naturally exported protein of L. lactis, AcmA.
[0078] In the wild-type MG1363 strain of L. lactis subsp. cremoris,
Usp-.DELTA..sub.SPNuc is secreted and Nlp4-.DELTA..sub.SPNuc is
associated with the cells; for these two proteins, various
degradation products, among which the NucA peptide originating from
the .DELTA..sub.SPNuc portion of the fusion, are detected in the
medium, along with the mature form; with regard to the
Exp5-.DELTA..sub.SPNuc tripartite fusion, it is very unstable and
the mature form is not detected in the medium, only the degradation
products, including the NucA peptide. The mature form, and also the
degradation products of these three hybrid proteins, can be
detected using anti-NucA antibodies.
[0079] The naturally exported protein of L. lactis chosen is the
bacteriolysin AcmA (Buist et al., J. Bacteriol. 177:1554-1563,
1995). This protein, which degrades peptidoglycan, is both secreted
and associated with the surface, probably by affinity with its
substrate. It provides, both in the MG1363 strain of L. lactis
subsp. cremoris and the IL1403 strain of L. lactis subsp. lactis,
proteolysis products which are active and therefore detectable,
like the intact protein, by zymogram.
[0080] The strains transformed with the plasmids expressing these
various proteins are cultured at 30.degree. C. for several hours,
at least up to the middle of the exponential phase or up to the
start of the stationary phase.
[0081] For each plasmid, cultures of the three strains IL1403, hrtA
and htrA.sup.+/htrA, which had reached comparable OD.sub.600
values, were used to extract protein samples: a) from the total
culture, b) from the cells and c) from the medium, according to the
protocol described by Poquet et al. (1998, abovementioned
publication).
[0082] These samples are subjected to electrophoresis (SDS-PAGE) on
denaturing gel.
[0083] In order to detect the Nuc, USP-.DELTA..sub.SPNuc,
Nlp4-.DELTA..sub.SPNuc and Exp5-.DELTA..sub.SPNuc proteins and
their degradation products, the proteins are transferred onto a
membrane, followed by immunological revelation using anti-NucA
antibodies, which are detected using a protein G/peroxidase
conjugate (BIO-RAD) and a chemiluminescence kit (Dupont-Nen).
[0084] AcmA is detected by zymogram (Buist et al., 1995,
abovementioned publication): micrococci, in which the wall is
sensitive to AcmA, are included in the electrophoresis gel at the
concentration of 0.2%, which makes it opaque; after
electrophoresis, the gel is treated at 37.degree. C. overnight in a
buffer containing 50 mM of Tris/HCl at pH 7 and 0.1% of Triton
X100, which allows lysis of the micrococci by AcmA or its active
proteolytic products. The gel is then colored with methylene blue
at 0.1% in 0.01% KOH: the bands corresponding to the AcmA activity
appear as transparent hydrolysis halos on a blue background.
[0085] For each protein, the degradation profiles in the IL1403,
htrA and htrA.sup.+/htrA strains were compared by observing the
protein content accumulated during culturing for several hours.
[0086] FIGS. 3 to 6 show, respectively, the results of
immunological detection for the Nuc, Usp-.DELTA..sub.SPNuc,
Nlp4-.DELTA..sub.SPNuc and Exp5-.DELTA..sub.SPNuc proteins. For the
Nuc (FIG. 3) and Usp-.DELTA..sub.SPNuc (FIG. 4) proteins,
[lacuna]
[0087] FIG. 7 represents a zymogram of the bacteriolysin activity
of AcmA; the detection was carried out on the total culture (T),
the cells alone (C) or the medium (M).
In the IL1403 Strain:
[0088] For the secreted proteins Nuc and Usp-.DELTA..sub.SPNuc
(FIGS. 3 and 4: first three wells), and for the lipoprotein
Nlp4-.DELTA..sub.SPNuc (FIG. 5: first well), a three-band profile
is detected, as previously observed in the MG1363 strain (Le Loir
et al., 1994; Poquet et al., 1998, abovementioned
publications):
[0089] a) the band with the highest molecular weight is the
precursor from which the signal peptide has not been cleaved, which
is confirmed by its presence exclusively in the cells (FIGS. 3 and
4);
[0090] b) the intermediate band is the mature form after cleavage
of the signal peptide, and, in the case of the secreted proteins
Nuc and Usp-.DELTA..sub.SPNuc (FIGS. 3 and 4), it is present
exclusively in the medium;
[0091] c) the band with the lowest molecular weight is the NucA
peptide which practically comigrates with the commercial NucA form
purified from S. aureus (the slight difference in migration being
due to the different cleavage specificities in S. aureus and L.
lactis), and which is both released into the medium and associated
with the cells.
[0092] For the Exp5-.DELTA..sub.SPNuc protein (FIG. 6: first well),
two forms are detected only with great difficulty, one having a
high molecular weight and one having a low molecular weight, NucA,
which practically comigrates with the purified commercial form;
there is, therefore, practically total proteolysis in IL1403.
[0093] For the AcmA protein (FIG. 7: the first three wells), a
four-band profile, as previously observed in the MG1363 strain
(Buist et al., 1995, abovementioned publication), is detected:
[0094] a) the band with the highest molecular weight is the
precursor from which the signal peptide has not been cleaved, which
is present exclusively in the cells;
[0095] b) the band with a slightly lower molecular weight is the
mature form after cleavage of the signal peptide, which is both
secreted into the medium and associated with the surface of the
cells by affinity for its substrate;
[0096] c and d) the two bands of lower molecular weight are active
proteolytic products, both secreted into the medium and associated
with the surface of the cells by affinity for their substrate.
In the htrA.sup.+/htrA Strain:
[0097] (FIGS. 3 and 4: last three wells, FIGS. 5 and 6: last well,
and FIG. 7: last three wells). The profiles observed are absolutely
identical to those observed in the wild-type strain. The
htrA.sup.+/htrA strain therefore exhibits a wild-type proteolytic
phenotype which is explained by the wild-type copy of the
htrA.sub.L1 gene which it possesses.
In the htrA Strain:
[0098] (FIGS. 3 and 4: three central wells, FIGS. 5 and 6: central
well, and FIG. 7: three central wells).
[0099] In all cases, none of the proteolytic products are detected;
simultaneously, the amount of mature protein (or of high molecular
weight protein in the case of Exp5-.DELTA..sub.SPNuc)
increases.
[0100] These results show that the product of the htrA.sub.L1 gene
is clearly responsible for the degradation of the secreted
proteins, and that its inactivation leads to the complete abolition
of this degradation.
Sequence CWU 1
1
2 1 1740 DNA Lactococcus lactis CDS (230)..(1453) 1 aaacaagatg
aaaacatgat ttatcaacat ttttttactt ttttccactt ttctgtggaa 60
aactttatta aaatatccac ttatcctcat taatttttag attatccaca aaaatgtgga
120 gaaactatat tagtttgatt tttgttacta ttaaggtatt attaagtgag
agtagatata 180 attacatcat agaaatgcta caaagattaa taattgaaag
gaattattt atg gca aaa 238 Met Ala Lys 1 gct aat ata gga aaa ttg cta
tta aca ggt gtc gtg ggc gga gcc atc 286 Ala Asn Ile Gly Lys Leu Leu
Leu Thr Gly Val Val Gly Gly Ala Ile 5 10 15 gca ctt gga gga agt gca
atc tat caa agc act aca aat caa tcg gca 334 Ala Leu Gly Gly Ser Ala
Ile Tyr Gln Ser Thr Thr Asn Gln Ser Ala 20 25 30 35 aat aat agt cgt
tca aat aca act agt aca aag gtt agt aac gtt tcg 382 Asn Asn Ser Arg
Ser Asn Thr Thr Ser Thr Lys Val Ser Asn Val Ser 40 45 50 gta aat
gtc aat acc gat gtt acc tct gca att gaa aaa gtt tca aat 430 Val Asn
Val Asn Thr Asp Val Thr Ser Ala Ile Glu Lys Val Ser Asn 55 60 65
tct gtc gtt tct gtt atg aat tat caa aaa gat aac tca caa agt agt 478
Ser Val Val Ser Val Met Asn Tyr Gln Lys Asp Asn Ser Gln Ser Ser 70
75 80 gac ttc agt tca att ttt ggt gga aat agc ggt tca agt tca tcg
act 526 Asp Phe Ser Ser Ile Phe Gly Gly Asn Ser Gly Ser Ser Ser Ser
Thr 85 90 95 gat ggc tta cag ctt tct agt gaa ggc tct ggt gtc atc
tac aaa aaa 574 Asp Gly Leu Gln Leu Ser Ser Glu Gly Ser Gly Val Ile
Tyr Lys Lys 100 105 110 115 tct ggt ggt gat gcc tac gtt gta act aac
tac cac gtt att gct ggt 622 Ser Gly Gly Asp Ala Tyr Val Val Thr Asn
Tyr His Val Ile Ala Gly 120 125 130 aat agc tca ctt gat gtt ctg ctt
tct ggt gga caa aaa gtc aaa gat 670 Asn Ser Ser Leu Asp Val Leu Leu
Ser Gly Gly Gln Lys Val Lys Asp 135 140 145 tct gtg gtt ggt tat gat
gaa tac aca gac ctt gct gtt ctt aaa atc 718 Ser Val Val Gly Tyr Asp
Glu Tyr Thr Asp Leu Ala Val Leu Lys Ile 150 155 160 agt tct gaa cat
gtc aaa gat gtg gcg aca ttc gct gat tct agt aaa 766 Ser Ser Glu His
Val Lys Asp Val Ala Thr Phe Ala Asp Ser Ser Lys 165 170 175 tta aca
att ggt gaa cct gcc att gcc gtt ggc tca cct tta ggt agt 814 Leu Thr
Ile Gly Glu Pro Ala Ile Ala Val Gly Ser Pro Leu Gly Ser 180 185 190
195 caa ttt gca aac acc gca act gaa gga att tta tct gca aca agc cgt
862 Gln Phe Ala Asn Thr Ala Thr Glu Gly Ile Leu Ser Ala Thr Ser Arg
200 205 210 caa gtg act ttg acc caa gaa aat ggt caa aca act aat atc
aat gca 910 Gln Val Thr Leu Thr Gln Glu Asn Gly Gln Thr Thr Asn Ile
Asn Ala 215 220 225 att caa aca gat gct gcc att aac cct ggt aac tct
gga ggg gct ttg 958 Ile Gln Thr Asp Ala Ala Ile Asn Pro Gly Asn Ser
Gly Gly Ala Leu 230 235 240 att aat att gaa gga caa gtt att gga att
act caa agt aaa att aca 1006 Ile Asn Ile Glu Gly Gln Val Ile Gly
Ile Thr Gln Ser Lys Ile Thr 245 250 255 aca act gaa gat ggt tct act
tct gtc gaa ggt tta gga ttt gcg att 1054 Thr Thr Glu Asp Gly Ser
Thr Ser Val Glu Gly Leu Gly Phe Ala Ile 260 265 270 275 cct tct aat
gat gtc gta aat atc att aat aaa ctt gaa gat gat ggt 1102 Pro Ser
Asn Asp Val Val Asn Ile Ile Asn Lys Leu Glu Asp Asp Gly 280 285 290
aag att tca cgc cct gct tta ggt atc cga atg gtt gac ctt tca caa
1150 Lys Ile Ser Arg Pro Ala Leu Gly Ile Arg Met Val Asp Leu Ser
Gln 295 300 305 tta tca aca aat gac agt tct caa ttg aaa tta cta agc
agt gta aca 1198 Leu Ser Thr Asn Asp Ser Ser Gln Leu Lys Leu Leu
Ser Ser Val Thr 310 315 320 ggt ggg gtt gtt gtt tac tcc gtc caa tct
gga ctt cct gct gcc tca 1246 Gly Gly Val Val Val Tyr Ser Val Gln
Ser Gly Leu Pro Ala Ala Ser 325 330 335 gct ggt ttg aaa gct gga gat
gta att aca aag gtt ggc gat aca gca 1294 Ala Gly Leu Lys Ala Gly
Asp Val Ile Thr Lys Val Gly Asp Thr Ala 340 345 350 355 gta acc tct
tca aca gac ttg caa agt gct ctt tac tca cac aat atc 1342 Val Thr
Ser Ser Thr Asp Leu Gln Ser Ala Leu Tyr Ser His Asn Ile 360 365 370
aat gat aca gta aaa gtt act tat tat cgt gat ggt aaa tca aat aca
1390 Asn Asp Thr Val Lys Val Thr Tyr Tyr Arg Asp Gly Lys Ser Asn
Thr 375 380 385 gca gat gtt aaa ctt tct aaa tca acc agt gac tta gaa
aca agc agt 1438 Ala Asp Val Lys Leu Ser Lys Ser Thr Ser Asp Leu
Glu Thr Ser Ser 390 395 400 cca tct tct tct aat taataactta
ataatttaat aaaagtcttc tgtaaataga 1493 Pro Ser Ser Ser Asn 405
aggctttttt catactaaag tctgaaattt ttaaaaataa taaatttcca tttttctttt
1553 attgatttat ggtaaaataa agttaagcat gaaaatttta ctttacttag
aagccgaaca 1613 atttttgagt cattcaggaa ttggtcgtgc aatgaaacat
caacaacgcg cccttgattt 1673 aatgggcatt gactggacaa aaaatcctga
ggatgattac gatatcctcc atttaaatac 1733 ttatggc 1740 2 408 PRT
Lactococcus lactis 2 Met Ala Lys Ala Asn Ile Gly Lys Leu Leu Leu
Thr Gly Val Val Gly 1 5 10 15 Gly Ala Ile Ala Leu Gly Gly Ser Ala
Ile Tyr Gln Ser Thr Thr Asn 20 25 30 Gln Ser Ala Asn Asn Ser Arg
Ser Asn Thr Thr Ser Thr Lys Val Ser 35 40 45 Asn Val Ser Val Asn
Val Asn Thr Asp Val Thr Ser Ala Ile Glu Lys 50 55 60 Val Ser Asn
Ser Val Val Ser Val Met Asn Tyr Gln Lys Asp Asn Ser 65 70 75 80 Gln
Ser Ser Asp Phe Ser Ser Ile Phe Gly Gly Asn Ser Gly Ser Ser 85 90
95 Ser Ser Thr Asp Gly Leu Gln Leu Ser Ser Glu Gly Ser Gly Val Ile
100 105 110 Tyr Lys Lys Ser Gly Gly Asp Ala Tyr Val Val Thr Asn Tyr
His Val 115 120 125 Ile Ala Gly Asn Ser Ser Leu Asp Val Leu Leu Ser
Gly Gly Gln Lys 130 135 140 Val Lys Asp Ser Val Val Gly Tyr Asp Glu
Tyr Thr Asp Leu Ala Val 145 150 155 160 Leu Lys Ile Ser Ser Glu His
Val Lys Asp Val Ala Thr Phe Ala Asp 165 170 175 Ser Ser Lys Leu Thr
Ile Gly Glu Pro Ala Ile Ala Val Gly Ser Pro 180 185 190 Leu Gly Ser
Gln Phe Ala Asn Thr Ala Thr Glu Gly Ile Leu Ser Ala 195 200 205 Thr
Ser Arg Gln Val Thr Leu Thr Gln Glu Asn Gly Gln Thr Thr Asn 210 215
220 Ile Asn Ala Ile Gln Thr Asp Ala Ala Ile Asn Pro Gly Asn Ser Gly
225 230 235 240 Gly Ala Leu Ile Asn Ile Glu Gly Gln Val Ile Gly Ile
Thr Gln Ser 245 250 255 Lys Ile Thr Thr Thr Glu Asp Gly Ser Thr Ser
Val Glu Gly Leu Gly 260 265 270 Phe Ala Ile Pro Ser Asn Asp Val Val
Asn Ile Ile Asn Lys Leu Glu 275 280 285 Asp Asp Gly Lys Ile Ser Arg
Pro Ala Leu Gly Ile Arg Met Val Asp 290 295 300 Leu Ser Gln Leu Ser
Thr Asn Asp Ser Ser Gln Leu Lys Leu Leu Ser 305 310 315 320 Ser Val
Thr Gly Gly Val Val Val Tyr Ser Val Gln Ser Gly Leu Pro 325 330 335
Ala Ala Ser Ala Gly Leu Lys Ala Gly Asp Val Ile Thr Lys Val Gly 340
345 350 Asp Thr Ala Val Thr Ser Ser Thr Asp Leu Gln Ser Ala Leu Tyr
Ser 355 360 365 His Asn Ile Asn Asp Thr Val Lys Val Thr Tyr Tyr Arg
Asp Gly Lys 370 375 380 Ser Asn Thr Ala Asp Val Lys Leu Ser Lys Ser
Thr Ser Asp Leu Glu 385 390 395 400 Thr Ser Ser Pro Ser Ser Ser Asn
405
* * * * *