U.S. patent application number 16/469784 was filed with the patent office on 2020-03-19 for methods and strain.
The applicant listed for this patent is DUPONT NUTRITION BIOSCIENCES APS. Invention is credited to David Blandine, Patrick Boyaval, Laetitia Fontaine, Christophe Fremaux, Pascal Hols, Philippe Horvath, Amandine Radziejwoski, Frederic Toussaint.
Application Number | 20200087686 16/469784 |
Document ID | / |
Family ID | 57614155 |
Filed Date | 2020-03-19 |
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United States Patent
Application |
20200087686 |
Kind Code |
A1 |
Fremaux; Christophe ; et
al. |
March 19, 2020 |
METHODS AND STRAIN
Abstract
The present invention relates to a method for transforming a
strain of the Lactococcus genus through natural competence. The
present invention further relates to strains obtained or obtainable
by said method. The present invention also relates to a method for
identifying a strain of the Lactococcus genus which is
transformable through natural competence.
Inventors: |
Fremaux; Christophe;
(Poitiers, FR) ; Horvath; Philippe;
(Chatellerault, FR) ; Boyaval; Patrick; (La
Meziere, FR) ; Hols; Pascal; (Vedrin, BE) ;
Blandine; David; (Floreffe, BE) ; Radziejwoski;
Amandine; (Sterrebeek, BE) ; Fontaine; Laetitia;
(Louvain-la-Neuve, BE) ; Toussaint; Frederic;
(Rixensart, BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DUPONT NUTRITION BIOSCIENCES APS |
Copenhagen K |
|
DK |
|
|
Family ID: |
57614155 |
Appl. No.: |
16/469784 |
Filed: |
December 19, 2017 |
PCT Filed: |
December 19, 2017 |
PCT NO: |
PCT/EP2017/083601 |
371 Date: |
June 14, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 7/00 20130101; C12N
15/87 20130101; C12N 15/902 20130101; C12R 1/46 20130101; C12Q 1/02
20130101 |
International
Class: |
C12N 15/90 20060101
C12N015/90; C07K 7/00 20060101 C07K007/00; C12Q 1/02 20060101
C12Q001/02 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 19, 2016 |
EP |
16205055.3 |
Claims
1. A method for transforming a strain of the Lactococcus genus with
an exogenous DNA polynucleotide comprising the steps of: (a)
providing a strain of the Lactococcus genus, wherein said strain is
transformable through natural competence; (b) modulating the
production of a ComX protein in said strain; (c) contacting said
strain of step (b) with an exogenous DNA polynucleotide in a medium
and incubating the resulting mixture for integration of the
exogenous DNA polynucleotide into the genome of said strain; and
(d) selecting a strain which has integrated the exogenous DNA
polynucleotide into its genome.
2. A method according to claim 1, wherein the step of modulating
the production of a ComX protein is performed by expressing a comX
gene in said strain or increasing the expression of a comX gene in
said strain.
3. A method according to claim 2, wherein said comX gene is an
exogenous comX gene.
4. A method according to claim 3, wherein said exogenous comX gene
is transferred into said strain by conjugation, transduction, or
transformation.
5. A method according to claim 2, wherein said comX gene is the
endogenous comX gene of said strain.
6. A method according to claim 5, wherein the method comprises
carrying out step (b) and then carrying out step (c) or comprises
carrying out step (b) and step (c) simultaneously.
7. A method according to claim 1, wherein said ComX protein has:
the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22; an amino acid
sequence having at least 90% identity to the amino acid sequence of
SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,
SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID
NO:20 or SEQ ID NO:22; or an amino acid sequence having at least
90% similarity to the amino acid sequence of SEQ ID NO: 2, SEQ ID
NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID
NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID
NO:22.
8. A method according to claim 1, wherein said comX gene has: the
nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ
ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ
ID NO:17, SEQ ID NO:19, or SEQ ID NO:21; or a nucleotide sequence
having at least 90% identity to the nucleotide sequence of SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or
SEQ ID NO:21.
9. A method according to claim 1, wherein said medium of step (c)
is a chemically defined medium.
10. A method according to claim 1, wherein prior to step (c) said
strain is incubated in a pre-culture medium.
11. A method according to claim 1, wherein said strain is incubated
with the exogenous DNA polynucleotide for around 4-8 hours at
around 30.degree. C. and said medium of step (c) is supplemented
with an osmo-stablizer.
12. A method according to claim 1, wherein said strain of the
Lactococcus genus of step (a) is a strain of the Lactococcus
raffinolactis species or a strain of the Lactococcus lactis
species.
13. A method according to claim 1, wherein said exogenous DNA
polynucleotide used in step (c) is obtained from a strain of the
same species as the strain provided in step (a).
14. A strain of the Lactococcus genus obtained by the method of
claim 1.
15. A method for identifying a strain of the Lactococcus genus
which is transformable through natural competence comprising the
steps of: (a) providing a strain of the Lactococcus genus; (b)
transforming said strain with a plasmid expressing a comX gene
having at least 90% identity to the endogenous comX gene of said
strain; (c) contacting said strain obtained in step (b) with an
exogenous DNA polynucleotide encoding a marker gene in a medium and
incubating the resulting mixture for integration of the exogenous
DNA polynucleotide into the genome of said strain; and (d)
determining the rate of recombination events; wherein a rate of at
least 1.times.10.sup.-6 transformants per .mu.g of DNA is
indicative of a strain which is transformable through natural
competence.
16. A method according claim 1, wherein said strain of step (a) is
identified using a method for identifying a strain of the
Lactococcus genus which is transformable through natural competence
comprising: (a) providing a strain of the Lactococcus genus; (b)
transforming said strain with a plasmid expressing a comX gene
having at least 90% identity to the endogenous comX gene of said
strain; (c) contacting said strain obtained in step (b) with an
exogenous DNA polynucleotide encoding a marker gene in a medium and
incubating the resulting mixture for integration of the exogenous
DNA polynucleotide into the genome of said strain; and (d)
determining the rate of recombination events; wherein a rate of at
least 1.times.10.sup.-6 transformants per .mu.g of DNA is
indicative of a strain which is transformable through natural
competence.
17. A method according to claim 1, wherein said strain of step (a)
is identified using assay A, which is performed as follows: i)
providing a strain of the Lactococcus genus; ii) transforming the
strain with a plasmid expressing a comX gene having at least 90%
identity to the endogenous comX gene of the strain; iii)
pre-culturing the transformed strain overnight in a complex medium
supplemented with glucose; iv) diluting about 1.5 mL of the
pre-culture in 8.5 mL of fresh medium; v) after 2 hr of further
growth at 30.degree. C., washing the cells twice with distilled
water and adjusting the OD.sub.600 to 0.05 in a chemically defined
medium comprising 5 .mu.g mL.sup.-1 erythromycin and an
osmo-stabilizer; vi) adding 5 .mu.g of exogenous DNA polynucleotide
bearing an antibiotic resistance gene to 300 al of the culture
medium; vii) incubating the resulting culture for 6 hr at
30.degree. C.; viii) plating the cells onto agar plates comprising
the complex medium supplemented with glucose and antibiotic
corresponding to the antibiotic resistance gene of the exogenous
DNA polynucleotide and incubating for 48 hr; ix) counting the
colony forming units and determining the transformation rate,
wherein: the transformation rate equals the number of
antibiotic-resistance colony forming units per mL divided by the
total number of viable colony forming units per mL, and a
transformation rate of at least 1.times.10.sup.-6 transformants per
.mu.g of DNA is indicative of a strain that is transformable
through natural competence.
18. A method according to claim 1, wherein said ComX protein has
the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22.
19. A method according to claim 1, wherein said comX gene has the
nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ
ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ
ID NO:17, SEQ ID NO:19, or SEQ ID NO:21.
20. A method according to claim 11, wherein the osmo-stabilizer is
glycerol or mannitol.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method for transforming a
strain of the Lactococcus genus through natural competence. The
present invention further relates to strains obtained or obtainable
by said method. The present invention also relates to a method for
identifying a strain of the Lactococcus genus which is
transformable through natural competence.
BACKGROUND TO THE INVENTION
[0002] Lactococcus lactis is one of the most important lactic acid
bacteria used in the dairy industry, in particular as a main dairy
starter species in various cheese preparations (e.g. gouda,
cheddar, brie, parmesan, roquefort) and fermented milk products
(e.g. buttermilk, sour cream). Other applications of L. lactis
bacteria include as a host for heterologous protein production or
as a delivery platform for therapeutic molecules. While the growth
and fermentation properties of L. lactis have been gradually
improved by selection and classical methods, there is great
potential for further improvement through natural processes or by
genetic engineering. Of particular interest are methods to
naturally transform L. lactis without the use of genetic
engineering, thereby generating new non-GMO strains with useful
industrial properties.
[0003] Lactococcus raffinolactis is present in a wide range of
environments, such as foods (meat, fish, milk, vegetable), animals,
and plant materials. In the dairy environment, this species has
been found in raw milks (cow, ewe, goat, and camel), natural dairy
starter cultures, and a great variety of cheeses. The prevalence of
this bacterium in foods even if with a "nondominant" status
compared to other lactococci could make it a candidate for future
development of starter cultures.
[0004] DNA acquisition by natural transformation is widespread
among prokaryotes and has been identified in over 80 species.
Various functions are attributed to competence for natural
transformation: genome plasticity, DNA repair, and/or nutrition. In
Gram-positive bacteria, competence for natural transformation has
been well-characterized in Bacillus subtilis and in various species
of the genus Streptococcus (e.g. S. pneumoniae, S. mutans, and S.
thermophilus).
[0005] In streptococci, competence for DNA transformation is
induced in response to secreted signalling peptides referred to as
competence pheromones/alarmones. The production of this class of
cell-to-cell communication molecules is initiated in response to
specific environmental stresses or conditions and allows the
coordination of physiological functions (e.g. competence,
predation, biofilm formation). Above a threshold concentration,
competence pheromones activate the master regulator ComX
(alternative sigma factor .sigma..sup.X), which ultimately leads to
a transcriptional reprogramming of cells (globally known as late
competence phase) including the induction of genes strictly
required for DNA transformation. ComX binds to a specific DNA
sequence named Com-box or Cin-box, which is located at least in the
vicinity of promoters of late competence (corn) genes/operons
responsible for DNA uptake (e.g.; comG, comF and comE operons), DNA
protection (e.g. ssb) and DNA recombination (e.g. recA, dprA,
coiA), and positively controls their expression.
[0006] The early steps leading to competence activation (early
competence phase) differs among bacteria. In streptococci, two
major peptide-based signaling pathways--i.e. ComCDE and ComRS--have
been identified so far. In mitis and anginosus groups of
streptococci (S. pneumoniae as paradigm), the competence signaling
peptide (CSP, or mature ComC) triggers a phosphorylation cascade
mediated by the two-component system ComD-ComE, leading to the
transcriptional activation of comX. In salivarius, mutans,
pyogenes, bovis and suis groups of streptococci, another regulation
mechanism is operational (S. thermophilus as paradigm). This system
involves the ComX-induction peptide (XIP, or mature ComS) which is
internalized by the oligopeptide transporter Opp, binds to and
activates the regulator ComR, and in turn induces comX
transcription.
[0007] Orthologues of comX and of all late corn genes essential for
natural transformation have been identified in the genome of L.
lactis, although some are present as putative pseudogenes in
different strains (Wydau et al., 2006).
[0008] Specific growth conditions have been reported to activate
corn genes in Lactococcus lactis. For example, the promoter of comX
was shown to be induced during cheese-making conditions in strain
MG5267 (an MG1363 derivative) which belongs to the subspecies
cremoris (Bachmann et al. 2010).
[0009] In the L. lactis subspecies (subsp.) lactis, carbon
starvation was shown to activate six late corn genes in strain
IL1403 of dairy origin (i.e. comX, comEA, comGA, comGB, radA, and
nucA) and most of the late essential corn genes in strain KF147 of
plant origin (i.e. comX, comC, coiA, and operons comG, comE, comF)
(Ercan et al., 2015). However, when the authors attempted to
validate functional natural transformation in KF147, they were
unsuccessful.
[0010] Wydau et al. reported that all the well-established late
genes/operons display an upstream and conserved Com-box, suggesting
that they are similarly controlled by ComX as reported in
streptococci. However, the authors did not comment on whether comX
over-expression in IL1403 induced natural competence. Indeed, the
authors neither report any experiment evaluating natural competence
in this strain nor suggest any experimental conditions appropriate
for inducing natural competence. Thus, as noted in the recent
literature (see Ercan et al., 2015) [i.e., 9 years after Wydau et
al.], there is no experimental evidence for successful
transformation of any species of the genus Lactococcus by natural
competence, and even less of IL1403.
[0011] Accordingly, there remains a need for a method for naturally
transforming Lactococcus strains using natural competence. In
addition, since some strains of the Lactococcus genus may not
encode a full set of functional late corn genes, there is a need
for a method for identifying Lactococcus strains which can be
transformed by natural competence.
SUMMARY OF THE INVENTION
[0012] In a first aspect, the present invention provides a method
for transforming a strain of the Lactococcus genus with an
exogenous DNA polynucleotide comprising the steps of: [0013] (a)
providing a strain of the Lactococcus genus, wherein said strain is
transformable through natural competence; [0014] (b) modulating the
production of a ComX protein in said strain; [0015] (c) contacting
said strain of step (b) with an exogenous DNA polynucleotide in a
medium and incubating the resulting mixture for integration of the
exogenous DNA polynucleotide into the genome of said strain; and
[0016] (d) selecting a strain which has integrated the exogenous
DNA polynucleotide into its genome.
[0017] In one embodiment, the step of modulating the production of
a ComX protein is performed by expressing a comX gene in said
strain or increasing the expression of a comX gene in said
strain.
[0018] In a further embodiment, the comX gene is an exogenous comX
gene. Said exogenous comX gene may be transferred into said strain
by conjugation, transduction, or transformation. Said exogenous
comX gene may be operably linked to transcription regulator(s).
[0019] In an alternative embodiment, said comX gene is the
endogenous comX gene of said strain.
[0020] In one embodiment, when said comX gene is the endogenous
comX gene of said strain, the method comprises: [0021] (a)
providing a strain of the Lactococcus genus, wherein said strain is
transformable through natural competence; [0022] (b) modulating the
production of a ComX protein, by expressing the endogenous comX
gene or increasing the expression of the endegenous comX of said
strain; [0023] (c) contacting said strain of step (b) with an
exogenous DNA polynucleotide in a medium and incubating the
resulting mixture for integration of the exogenous DNA
polynucleotide into the genome of said strain; and [0024] (d)
selecting a strain which has integrated the exogenous DNA
polynucleotide into its genome, [0025] wherein step (c) is carried
out after step (b) or wherein step (b) and step (c) are carried out
simultaneously.
[0026] In some embodiments, said ComX protein has the amino acid
sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ
ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,
SEQ ID NO:20, SEQ ID NO:22, or has at least 90% identity or at
least 90% similarity to the amino acid sequence of SEQ ID NO:2, SEQ
ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ
ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22.
In some embodiments, said ComX protein has the amino acid sequence
of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or has at least 90%
identity or at least 90% similarity to the amino acid sequence of
SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.
[0027] In some embodiments, said comX gene has the nucleotide
sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ
ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17,
SEQ ID NO:19, SEQ ID NO:21, or has at least 90% identity to the
nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ
ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ
ID NO:17, SEQ ID NO:19 or SEQ ID NO:21.
[0028] In some embodiments, said comX gene has the nucleotide
sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or has at least
90% identity to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3
or SEQ ID NO:5.
[0029] In some embodiments, the medium of step (c) is a chemically
defined medium. In a preferred embodiment the chemically defined
medium (CDM) comprises 0.5 g/L NH.sub.4Cl, 9.0 g/L
KH.sub.2PO.sub.4, 7.5 g/L K.sub.2HPO.sub.4, 0.2 g/L MgCl.sub.2, 5
mg/L FeCl.sub.2, 50 mg/L CaCl.sub.2), 5 mg/L ZnSO.sub.4, 2.5 mg/L
CoCl.sub.2, 0.05 g/L tyrosine, 0.1 g/L asparagine, 0.1 g/L
cysteine, 0.1 g/L glutamine, 0.1 g/L isoleucine, 0.1 g/L leucine,
0.1 g/L methionine, 0.1 g/L tryptophan, 0.1 g/L valine, 0.1 g/L
histidine, 0.2 g/L arginine, 0.2 g/L glycine, 0.2 g/L lysine, 0.2
g/L phenylalanine, 0.2 g/L threonine, 0.3 g/L alanine, 0.3 g/L
proline, 0.3 g/L serine, 10 mg/L paraaminobenzoic acid, 10 mg/L
biotin, 1 mg/L folic acid, 1 mg/L nicotinic acid, 1 mg/L
panthotenic acid, 1 mg/L riboflavin, 1 mg/L thiamine, 2 mg/L
pyridoxine, 1 mg/L cyanocobalamin, 5 mg/L orotic acid, 5 mg/L
2-deoxythymidine, 5 mg/L inosine, 2.5 mg/L dl-6,8-thioctic acid, 5
mg/L pyridoxamine, 10 mg/L adenine, 10 mg/L guanine, 10 mg/L
uracil, 10 mg/L xanthine, and 5 g/L glucose.
[0030] In some embodiments, prior to step (c) said strain is
incubated in a pre-culture medium, preferably wherein the
pre-culture medium is a complex medium, more preferably wherein the
pre-culture medium is M17G or THBG.
[0031] In some embodiments of the present invention, said strain is
incubated with the exogenous DNA polynucleotide for around 4 to 8
hours at around 30.degree. C. and said medium of step (c) is
supplemented with an osmo-stablizer, preferably wherein the
osmo-stablizer is glycerol or mannitol, more preferably wherein the
osmo-stabilizer is 5% [v/v] glycerol or 5% [w/v] mannitol.
[0032] In some embodiments, said exogenous DNA polynucleotide is
from a strain of the Lactococcus lactis species.
[0033] In some embodiments, said exogenous DNA polynucleotide is
from a strain of the Lactococcus raffinolactis species.
[0034] In some embodiments, said strain of step (a) is a
Lactoccocus lactis subsp. cremoris strain.
[0035] In another aspect, the present invention provides a strain
of the Lactococcus genus obtained or obtainable by the method of
the first aspect of the present invention.
[0036] In one embodiment, said strain of the Lactococcus genus is a
strain of the Lactococcus lactis or Lactococcus raffinolactis
species.
[0037] In a further aspect, the present invention provides a method
for identifying a strain of the Lactococcus genus which is
transformable through natural competence comprising the steps of:
[0038] (a) providing a strain of the Lactococcus genus species;
[0039] (b) transforming said strain with a plasmid expressing a
comX gene having at least 90% identity, preferably having 100%
identity, to the endogenous comX gene of said strain; [0040] (c)
contacting said strain obtained in step (b) with an exogenous DNA
polynucleotide encoding a marker gene in a medium and incubating
the resulting mixture for integration of the exogenous DNA
polynucleotide into the genome of said strain; and [0041] (d)
determining the rate of recombination events;
[0042] wherein a rate of at least 1.times.10.sup.-6 transformants
per .mu.g of DNA is indicative of a strain which is transformable
through natural competence.
[0043] In a particular embodiment of method for transforming a
strain of the Lactococcus genus of the present invention, said
strain of step (a) is identified using the method for identifying a
strain of the Lactococcus genus which is transformable through
natural competence according to the present invention. In some
embodiments of the present invention, said strain of step (a) is
identified using Assay A.
DESCRIPTION OF THE DRAWINGS
[0044] FIG. 1: Table showing the status of genes involved in
natural competence for L. lactis strains MG1363, SK11, KW2, IL1403,
SL12651 and SL12653.
[0045] Late corn genes in the complete genomes of strains MG1363,
SK11, and KW2 of Lactococcus lactis subsp. cremoris and of strain
IL1403, SL12651 and SL12653 of Lactococcus lactis subsp. lactis.
Origin is indicated above strain names. Gene-associated function in
DNA transformation is indicated on the left. Reg. denotes
regulation. The complete and incomplete status of late genes is
based on blastp and tblastn homology searches
(https://blast.ncbi.nlm.nih.gov/Blast.cgi) using orthologues of S.
pneumoniae TIGR4 and S. thermophilus LMD-9 and default parameters.
+denotes the presence of a complete gene; * denotes the presence of
an incomplete gene due to nucleotide(s) exchange, insertion or
deletion resulting in a premature stop codon; and Tn denotes a
disrupted gene by the insertion of at least one transposon.
[0046] FIG. 2: Graphs displaying the results of luciferase assays
which demonstrate the activation of a reporter construct comprising
the late promoter P.sub.comGA driven by constitutive comX
overexpression
[0047] (A) Maximum specific luciferase (Lux) activity (RLU
OD.sub.600.sup.-1) emitted by eight independent clones (cl01 to
cl08) of the KW2-derived reporter strain (BLD101,
P.sub.comGA[MG]-luxAB) carrying plasmid pGhP32comX.sub.MG compared
to the control strain (Ctl) carrying the empty vector
pG.sup.+host9. (B) Kinetics of specific Lux activity (solid line)
during growth (RLU/OD.sub.600; dotted line) for the control strain
(Ctl; black lines) and three selected clones (BLD101
[pGhP32comX.sub.MG], cl02, cl04 and cl05; gray lines). (C) Kinetics
of specific luciferase activity (closed symbols) during growth
(RLU/OD.sub.600; open symbols) of the
MG1363+pGhP32comX.sub.MG-P.sub.comGA[MG]-luc, grown in M17G at
30.degree. C. (D) Kinetics of specific luciferase activity (closed
symbols) during growth (RLU/OD.sub.600; open symbols) of
IL1403+pGhP32comX.sub.IO-P.sub.comGA[IO]-luc strains, grown in M17G
at 30.degree. C.
[0048] FIG. 3: Graphs displaying the results of luciferase assays
which demonstrate the impact of growth medium on P.sub.comGA
activation
[0049] Maximum specific Lux activity of BLD101 [pGhP32comX.sub.MG]
cl02 grown in different final culture media (CDM, THBG, and M17G)
according to preculture conditions (CDM, THBG, and M17G). Overnight
precultures were 10-fold diluted in the pre-culture medium and
grown for 2 hours. Then, cells were washed twice in distilled water
and the OD.sub.600 was adjusted to 0.05 in the final growth medium
before measuring growth and luciferase activity. One representative
experiment of two independent replicates.
[0050] FIG. 4: Results of a transformation assay implemented on a
L. lactis subsp. cremoris KW2 constitutively expressing comX
contacted with a DNA consisting of a mutated allele of the rpsL
gene as exogenous DNA polynucleotide
[0051] (A) Alignment of the rpsL gene sequences of strain MG1363, a
spontaneous streptomycin-resistant clone of strain MG1363, strain
KW2 and a KW2-derived transformant obtained using the method of the
invention (partial sequence). The arobase, pound and dollar signs
below the alignment indicate the positions of nucleotide
differences existing between the rpsL sequences. The dollar sign at
position 167 indicates the point mutation (A.fwdarw.T; strA1
allele) responsible for the streptomycin-resistance phenotype; the
pound sign at position 156 highlights a nucleotide that is
naturally different between MG1363 and KW2 (T in KW2, A in MG1363);
the arobase sign at position 39 indicates a silent nucleotide
substitution (T.fwdarw.G) which is found in the
streptomycin-resistant clone derived from MG1363. (B) DNA
transformation with the strA1 allele was assessed for L. lactis
strains constitutively expressing ComX. Transformation rate (white
bars) and maximum specific luciferase (Lux) activity (black
diamonds, RLU OD.sub.600.sup.-1, as reported in FIG. 2) of eight
clones (cl01 to cl08) of the reporter strain (BLD101,
P.sub.comGA[MG]-luxAB) carrying plasmid pGhP32comX.sub.MG compared
to the negative control strain (Ctl-) carrying the empty vector
(BLD101 [pG.sup.+host9]).
[0052] FIG. 5: Graphs displaying the results of transformation rate
of the KW2 derivative BLD101 [pGhP32comX.sub.MG] obtained with
overlap PCR products (comEC, mecA, ciaRH, covRS and clpC) and strA1
(rpsL*)-donor DNA.
[0053] The threshold represents the theoretical transformation rate
to obtain only one transformant.
[0054] FIG. 6: Graphs depicting the results of transformation
assays for a L. lactis subsp. cremoris deleted in its comEC gene
and constitutively expressing comX.
[0055] DNA transformation with the strA1 allele was assessed for L.
lactis strains constitutively expressing comX. Transformation rate
(white bars) and maximum specific luciferase (Lux) activity (RLU
OD.sub.600.sup.-1) of four clones (cl01 to cl04) of the
ComEC-deficient reporter strain (BLD102, P.sub.comGA[MG]-luxAB)
carrying plasmid pGhP32comX.sub.MG compared to the positive
(Ctl.sup.+, BLD101 [pGhP32comX.sub.MG] cl02) and negative
(Ctl.sup.-, BLD101 [pG.sup.+host9]) control strains.
Transformability was assessed according to the standard protocol
described in Materials and Methods using strA1-carrying PCR
products as donor DNA. ND denotes a transformation rate below the
detection level of spontaneous Str.sup.r mutants (<10.sup.-7).
One representative experiment of two independent replicates.
[0056] FIG. 7: Graphs displaying natural competence in Lactococcus
lactis subsp. lactis SL12651 and 12653 strains.
[0057] (A) Transformation rate of L. lactis subsp. lactis SL12651
and 12653 strains in M17G medium, with rpsL* donor DNA (+DNA) or
without donor DNA (-DNA); (B) DNA transformation with increasing
initial concentration of donor DNA assessed in SL12653 strain; (C)
Comparison of transformation rates between wild-type (WT) SL12653
strain and a SL12653 strain deleted for the comX gene (ComX-);
transformation rate of three clones of the ComX-deficient strain
compared to the WT strain, in presence (+DNA) or in absence (-DNA)
of donor DNA.
DETAILED DESCRIPTION
[0058] The present invention is based on the observation that
overexpression of ComX in a strain of the Lactococcus genus allowed
to transform this strain by natural competence. Using this approach
a L. lactis strain was generated by natural transformation with an
exogenous DNA polynucleotide. Importantly, these results are the
first demonstration of transformation of a L. lactis strain by
natural competence. Further, existence of natural competence in the
Lactococcus genus has been confirmed in two strains of the
Lactococcus raffinolactis species and two Lactococcus lactis
species.
[0059] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
biochemistry, microbiology, bacteriology, and related fields, which
are within the capabilities of a person of ordinary skill in the
art. Such techniques are explained in the literature.
[0060] Thus, the present invention provides a method for
transforming a strain of the Lactococcus genus with an exogenous
DNA polynucleotide comprising the steps of: [0061] (a) providing a
strain of the Lactococcus genus, wherein said strain is
transformable through natural competence; [0062] (b) modulating the
production of a ComX protein in said strain; [0063] (c) contacting
said strain of step (b) with an exogenous DNA polynucleotide in a
medium and incubating the resulting mixture for integration of the
exogenous DNA polynucleotide into the genome of said strain; and
[0064] (d) selecting a strain which has integrated the exogenous
DNA polynucleotide into its genome.
[0065] As detailed below, step (b) and step c) can be carried out
sequentially [i.e., step (b) and then step (c)] or in another
embodiment step (b) and step (c) can be carried out
simultaneously.
Lactococcus Genus
[0066] The present invention relates to a method for transforming a
strain of the Lactococcus genus, a Gram-positive bacterium.
Lactococcus strains are known as lactic acid bacteria (LAB) for
their ability to convert carbohydrate to lactic acid. A strain of
the Lactococcus genus and Lactococcus strain are used herein
interchangeably.
[0067] The Lactococcus genus comprises, but is not limited to the
following species: Lactococcus chungangensis, Lactococcus
fujiensis, Lactococcus garvieae, Lactococcus lactis, Lactococcus
piscium, Lactococcus plantarum and Lactococcus raffinolactis. Any
strain of one of these species may be used in the current
invention, provided that this strain is transformable through
natural competence as defined herein.
[0068] In a particular embodiment, said strain of the Lactococcus
genus of step a) is a strain of the Lactococcus lactis species or a
strain of the Lactococcus raffinolactis species.
Lactococcus lactis
[0069] In a particular embodiment, said strain of the Lactococcus
genus of step a) is a strain of the Lactococcus lactis species. The
species Lactococcus lactis comprises several subspecies. Thus, when
the strain of the Lactococcus genus of step a) is a strain of the
Lactococcus lactis species, said strain is selected in the group
consisting of Lactococcus lactis subsp. cremoris, Lactococcus
lactis subsp. hordniae, Lactococcus lactis subsp. lactis and
Lactococcus lactis subsp. tructae. As used herein a strain of the
Lactococcus lactis species is understood to be a genetic variant or
subtype of any L. lactis species or subspecies. The different
Lactococcus lactis subspecies disclosed here, and in particular the
lactis and the cremoris subspecies, are defined herein based on DNA
sequences coding for 16S ribosomal RNA [Ward et al., 1998].
[0070] In a particular aspect, the present invention provides a
method for transforming a strain of the Lactococcus lactis species
with an exogenous DNA polynucleotide comprising the steps of:
[0071] (a) providing a strain of the Lactococcus lactis species,
wherein said strain is transformable through natural competence;
[0072] (b) modulating the production of a ComX protein in said
strain; [0073] (c) contacting said strain of step (b) with an
exogenous DNA polynucleotide in a medium and incubating the
resulting mixture for integration of the exogenous DNA
polynucleotide into the genome of said strain; and [0074] (d)
selecting a strain which has integrated the exogenous DNA
polynucleotide into its genome.
[0075] In a preferred embodiment, the strain of step (a) is a
Lactococcus lactis subsp. cremoris strain or a Lactococcus lactis
subsp. lactis strain. Both subspecies have been identified and
characterised with full genome sequences see, e.g., Wegmann et al.
(2007) J. Bacteriol. 189:3256-3270 and Bolotin et al. (2001) Genome
Res. 11:731-753. With regards to the dairy industry, L. lactis
subsp. lactis (previously known as Streptococcus lactis) is
preferred for making soft cheese while L. lactis subsp. cremoris
(previously known as Streptococcus cremoris) is preferred for hard
cheese production.
[0076] In a preferred embodiment, the strain of step (a) is
Lactococcus lactis subsp. cremoris strain.
[0077] In another preferred embodiment, the strain of step (a) is
Lactococcus lactis subsp. lactis strain.
Lactococcus raffinolactis
[0078] In a particular embodiment, said strain of the Lactococcus
genus of step a) is a strain of the Lactococcus raffinolactis
species.
[0079] In a particular aspect, the present invention provides a
method for transforming a strain of the Lactococcus raffinolactis
species with an exogenous DNA polynucleotide comprising the steps
of: [0080] (a) providing a strain of the Lactococcus raffinolactis
species, wherein said strain is transformable through natural
competence; [0081] (b) modulating the production of a ComX protein
in said strain; [0082] (c) contacting said strain of step (b) with
an exogenous DNA polynucleotide in a medium and incubating the
resulting mixture for integration of the exogenous DNA
polynucleotide into the genome of said strain; and [0083] (d)
selecting a strain which has integrated the exogenous DNA
polynucleotide into its genome. Lactococcus plantarum
[0084] In a particular embodiment, said strain of the Lactococcus
genus of step a) is a strain of the Lactococcus plantarum
species.
[0085] In a particular aspect, the present invention provides a
method for transforming a strain of the Lactococcus plantarum
species with an exogenous DNA polynucleotide comprising the steps
of: [0086] (a) providing a strain of the Lactococcus plantarum
species, wherein said strain is transformable through natural
competence; [0087] (b) modulating the production of a ComX protein
in said strain; [0088] (c) contacting said strain of step (b) with
an exogenous DNA polynucleotide in a medium and incubating the
resulting mixture for integration of the exogenous DNA
polynucleotide into the genome of said strain; and [0089] (d)
selecting a strain which has integrated the exogenous DNA
polynucleotide into its genome. Lactococcus piscium
[0090] In a particular embodiment, said strain of the Lactococcus
genus of step a) is a strain of the Lactococcus piscium
species.
[0091] In a particular aspect, the present invention provides a
method for transforming a strain of the Lactococcus piscium species
with an exogenous DNA polynucleotide comprising the steps of:
[0092] (a) providing a strain of the Lactococcus piscium species,
wherein said strain is transformable through natural competence;
[0093] (b) modulating the production of a ComX protein in said
strain; [0094] (c) contacting said strain of step (b) with an
exogenous DNA polynucleotide in a medium and incubating the
resulting mixture for integration of the exogenous DNA
polynucleotide into the genome of said strain; and [0095] (d)
selecting a strain which has integrated the exogenous DNA
polynucleotide into its genome. Lactococcus garvieae
[0096] In a particular embodiment, said strain of the Lactococcus
genus of step a) is a strain of the Lactococcus garvieae
species.
[0097] In a particular aspect, the present invention provides a
method for transforming a strain of the Lactococcus garvieae
species with an exogenous DNA polynucleotide comprising the steps
of: [0098] (a) providing a strain of the Lactococcus garvieae
species, wherein said strain is transformable through natural
competence; [0099] (b) modulating the production of a ComX protein
in said strain; [0100] (c) contacting said strain of step (b) with
an exogenous DNA polynucleotide in a medium and incubating the
resulting mixture for integration of the exogenous DNA
polynucleotide into the genome of said strain; and [0101] (d)
selecting a strain which has integrated the exogenous DNA
polynucleotide into its genome. Lactococcus fujiensis
[0102] In a particular embodiment, said strain of the Lactococcus
genus of step a) is a strain of the Lactococcus fujiensis
species.
[0103] In a particular aspect, the present invention provides a
method for transforming a strain of the Lactococcus fujiensis
species with an exogenous DNA polynucleotide comprising the steps
of: [0104] (a) providing a strain of the Lactococcus fujiensis
species, wherein said strain is transformable through natural
competence; [0105] (b) modulating the production of a ComX protein
in said strain; [0106] (c) contacting said strain of step (b) with
an exogenous DNA polynucleotide in a medium and incubating the
resulting mixture for integration of the exogenous DNA
polynucleotide into the genome of said strain; and [0107] (d)
selecting a strain which has integrated the exogenous DNA
polynucleotide into its genome. Lactococcus chungangensis
[0108] In a particular embodiment, said strain of the Lactococcus
genus of step a) is a strain of the Lactococcus chungangensis
species.
[0109] In a particular aspect, the present invention provides a
method for transforming a strain of the Lactococcus chungangensis
species with an exogenous DNA polynucleotide comprising the steps
of: [0110] (a) providing a strain of the Lactococcus chungangensis
species, wherein said strain is transformable through natural
competence; [0111] (b) modulating the production of a ComX protein
in said strain; [0112] (c) contacting said strain of step (b) with
an exogenous DNA polynucleotide in a medium and incubating the
resulting mixture for integration of the exogenous DNA
polynucleotide into the genome of said strain; and [0113] (d)
selecting a strain which has integrated the exogenous DNA
polynucleotide into its genome.
DNA Acquisition
[0114] Bacteria may naturally acquire exogenous DNA via one of
three possible mechanisms: transformation, conjugation, or
transduction.
[0115] As used herein the term "transformation" refers to the
uptake of exogenous genetic material (e.g. a DNA polynucleotide)
from the external medium. Since transformation requires that
genetic material cross the bacterial cell wall and membrane and the
uptake of exogenous genetic material is energetically costly, the
process is tightly regulated. Accordingly, bacterial cells may only
be transformed under certain conditions. Bacterial cells which are
in a transformable state are said to be competent.
[0116] Competence may be artificially induced in the laboratory,
e.g. by electroporation or exposure to divalent cations (e.g.
CaCl.sub.2)) and heat shock. Alternatively, some species of
bacteria express a proteinaceous machinery that provides natural
competence; this system of natural competence has been widely
studied in streptococci.
[0117] As used herein the term "conjugation" refers to the transfer
of genetic material between bacterial cells.
[0118] As used herein the term "transduction" refers to the
transfer of genetic material from a virus (e.g. a bacteriophage) or
a viral vector into bacterial cell.
ComX Protein
[0119] The method of the present invention comprises the step of
modulating the production of a ComX protein in said strain.
[0120] ComX protein is an alternative sigma factor, also known as
.sigma..sup.x, which acts as master regulator for the late corn
genes and is responsible for transcriptional reprogramming of cells
including the induction of genes strictly required for DNA
transformation (Lee et al., 1989; Petersen et al. 2004).
[0121] ComX may bind to a specific target sequence (or box) termed
the Com-box (or Cin-box). Com-boxes are located in the vicinity of
the promoters of late competence (corn) genes/operons responsible
for DNA uptake (e.g., comG, comF, and comE operons), DNA protection
(e.g. ssb) and DNA recombination (e.g. recA, dprA, coiA), and
positively controls their expression (Campbell et al., 1998; Luo
and Morrison, 2003).
[0122] The production of the ComX protein in a strain of interest
may be increased relatively to an appropriate control strain, i.e.,
the Lactococcus strain in which the production of the ComX protein
has not been modulated. ComX protein may be produced (expressed)
following modulation as compared to an appropriate control strain,
i.e., the Lactococcus strain in which the ComX protein is not
produced.
[0123] In some embodiments, the production of the ComX protein is
constitutive or inducible.
[0124] The production of ComX protein may be monitored using any
method known in the art. For example, by western blotting using an
antibody specific for the ComX protein. Alternatively, comX gene
mRNA transcript levels may be measured by qPCR.
[0125] Alternatively, the ComX protein may be monitored using a
reporter construct polynucleotide, e.g. as described in the Example
1 and Materials and Methods. The reporter construct polynucleotide
may comprise genes encoding one or more reporter proteins,
preferably the genes encoding the reporter proteins are operably
linked to a promoter comprising a Com-box sequence. The reporter
proteins may be LuxAB or Luc. Accordingly, ComX expression (and
activity) may be detected and measured using a luciferase assay
(Fontaine et al., 2010).
[0126] In some embodiments of the method of the present invention,
the step of modulating the production of a ComX protein is
performed by expressing a comX gene in said strain or increasing
the expression of a comX gene in said strain. In a particular
embodiment, the step of modulating the production of a ComX protein
is performed by expressing a comX gene in said strain in some
growth conditions, whereas said strain does not express the ComX
protein outside of these growth conditions. In a particular
embodiment, the step of modulating the production of a ComX protein
is performed by increasing the expression of a comX gene in said
strain in some growth conditions.
[0127] The comX gene may be an exogenous comX gene. As used herein
an "exogenous comX gene" is understood to be a comX gene which is
brought into the cytoplasm of the Lactococcus strain of step a), in
order to be expressed. The exogenous comX gene may have the same
sequence as the comX gene found in the genome of the Lactococcus
strain of step a) or may have a different sequence from the comX
gene found in the genome of the Lactococcus strain of step a). When
different, the comX gene may be derived from a strain of a
different species, a different subspecies or a different strain of
Lactococcus.
[0128] The exogenous comX gene may be integrated within the genome
of said Lactococcus strain.
[0129] Alternatively, the exogenous comX gene may be located within
a vector. The vector may be selected from a plasmid, a viral vector
(e.g. a phage), a cosmid, or a bacterial artificial chromosome.
[0130] Said plasmid may be transferred into said Lactococcus strain
by conjugation, transformation or transduction. Said plasmid may be
auto-replicative in the transformed Lactococcus strain or not.
[0131] The exogenous comX gene may be operably linked to
transcription regulator(s). The exogenous comX gene may be located
in a linear or circular polynucleotide.
[0132] Alternatively, in some embodiments of the method of the
present invention, the comX gene is the endogenous comX gene of
said Lactococcus strain. As used herein "the endogenous comX gene
of said strain" is understood to be a comX gene that is naturally
present in the genome of said strain.
[0133] In some embodiments, said comX gene is a Lactococcus comX
gene. In an embodiment, said comX gene is a Lactococcus lactis comX
gene. In a particular embodiment, said comX gene is a Lactococcus
lactis subsp. lactis comX gene. In a particular embodiment, said
comX gene is a Lactococcus lactis subsp. cremoris comX gene.
[0134] The comX gene may comprise or consist of a nucleotide
sequence selected from the group consisting of: [0135] SEQ ID NO:1,
SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,
SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19; SEQ ID
NO:21; [0136] a nucleotide sequence having at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, or at least 99% identity to
the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15,
SEQ ID NO:17, SEQ ID NO:19; SEQ ID NO:21; [0137] a variant of SEQ
ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ
ID NO:21 encoding respectively a ComX protein of SEQ ID NO:2, SEQ
ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ
ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22;
and [0138] a variant of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ
ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ
ID NO:17, SEQ ID NO:19, SEQ ID NO:21 encoding respectively a
functional ComX protein having at least 90% identity or at least
90% similarity to a ComX protein of SEQ ID NO:2, SEQ ID NO:4, SEQ
ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ
ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22.
TABLE-US-00001 [0138] [SEQ ID NO: 1]
ATAACATATTACTTGGAAGAAGAGGATTTTGAAAATCTTTTTTCAGAAATGAAACCTATAGTTATGAA
ATTAATGAAACAAATTCGCATTAGAACATGGAAAATAGAGGATTATCTTCAAGAGGGGATGATTATTT
TACATCTTCTATTAGAAGAGCAGAACGATGGTCAAAAGCTGCATACAAAATTTAAGGTAAAGTATCAT
CAAAGATTAATAGATGAATTAAGACGAAGTTATGCAAAGAAACGAAGCCATGACCATTTTATAGGTTT
AGATGTTTATGAATGCTCAGACTGGATAAATTCAGGTGATACTAGTCCAGATAATGAAGTGGTCTTCA
ATCATTTGCTGGCAGAAGTATATGAAGGTTTGAGCGCACATTATCAAGACTTACTACTTCGACAAATG
CGAGGAGAAGAACTAACTCGCATGCAACGGTATCGCCTTCGTGAAAAAATAAAGGCCATCTTATTTTC
AGAAGACGAAGAGTGA [SEQ ID NO: 2]
MTYYLEEEDFENLFSEMKPIVMKLMKQIRIRTWKIEDYLQEGMIILHLLLEEQNDGQKLHTKFKVKYH
QRLIDELRRSYAKKRSHDHFIGLDVYECSDWINSGDTSPDNEVVFNHLLAEVYEGLSAHYQDLLLRQM
RGEELTRMQRYRLREKIKAILFSEDEE [SEQ ID NO: 3]
ATGACATATTACCTGGAAGAAAATGAATTCGAAGGTTTATTTTCTGGAATGAAACCAATCATCAGAAA
ATTGATGAAACAAATTCGAATCAAAGCATGGGACATAGAGGATTATTATCAAGAAGGAATGATTATTT
TGCATCACCTTTTAGAAGAAAATCACCCATCCACTAATATTTATACAAAGTTCAAAGTAAAATATCAT
CAACATTTGATTGATGAACTACGCCATAGCTACGCCAAAAAACGGCTTCATGACCATTTTGTAGGTCT
GGACATTTATGAATGTTCGGACTGGATAGATGCAGGAGGAAGTACCCCTGAAAGCGAGCTTGTGTTCA
ATCATCTTTTAGCAGAAGTTTATGAAGGATTGAGCGCCCACTATCAGGAATTACTCGTGCGTCAAATG
AGAGGAGAAGAACTCACGCGAATGGAACGCTATCGGCTAAGAGAAAAAATCAAAAATATACTATTTTC
TCGAGATGATGATTAA [SEQ ID NO: 4]
MTYYLEENEFEGLFSGMKPIIRKLMKQIRIKAWDIEDYYQEGMIILHHLLEENHPSTNIYTKFKVKYH
QHLIDELRHSYAKKRLHDHFVGLDIYECSDWIDAGGSTPESELVFNHLLAEVYEGLSAHYQELLVRQM
RGEELTRMERYRLREKIKNILFSRDDD [SEQ ID NO: 5]
ATGGATGACATTCAAGAAAAATACGGTTTAGAATTCAACGAATTATTCTCTGAGATGCGGCCGATAAT
TTATAAATTGATGAAGCAATTGCACATCAACACATGGGATTACGATGATTACTTCCAAGAGGGAATGA
TTACACTACATGAATTGCTGCAGAAAATTACAAATTTAGATCATGTACATACGAAATTTAAAGTGGCT
TACCATCAGCACTTAATTGACGAAATTCGCCATATTAAAGCACGAAAAAGAGGTTTTGATCAGCTCCA
TCCGATCAATGTTTATGACTGCGCAGATTGGATTGGCTCAAACCTTGCTACACCTGAAAGCGAGATAG
TTTTCAACCATCTACTAGAAGAAGTTTATGATAAACTTTCAACACACTATAAAGAACTGTTGGTAAAG
CAAATGCATGGGGAACATCTTACGAGAATGCAGAAGTATCGTTTAAAGGAAAAAATTAAAGCGATTTT
ATTTGATGAAGACTAA [SEQ ID NO: 6]
MDDIQEKYGLEFNELFSEMRPITYKLMKQLHINTWDYDDYFQEGMITLHELLQKITNLDHVHTKFKVA
YHQHLIDEIRHIKARKRGFDQLHPINVYDCADWIGSNLATPESEIVFNHLLEEVYDKLSTHYKELLVK
QMHGEHLTRMQKYRLKEKIKAILFDED [SEQ ID NO: 7]
ATGGATAAAATTGAAACCATACTTAAAAGTATTGAACCGATTATTATGAACTGTCGGAAAAAAACTAA
AATTCCTTCCTGGGAATTAGACGACTATATGCAGGAAGGGATGATTATTGCTTTAGAGATGTACCATC
AACTCTTATTAGATCCACCAGATGATGACTTTAACTTCTATGTCTATTTCAAAGTCAGGTATTCTTGT
TTCTTAATTGATCACTATCGCAAAGCTATGGCAGTCAAGAGAAAATTCGACCAGCTTGACTATTGTGA
ACTTTCTGAGTCTGTTAATCTTTTTGATCACAAACAAAATGTGTCTGAAAACGTCATGTATAACTTGT
TGTGTCAAGAAATACACTTGGTTTTATCCCCGGAGGAGCTCAAGCTTTTTGAGGCACTTATTTGA
[SEQ ID NO: 8]
MDKIETILKSIEPIIMNCRKKTKIPSWELDDYMQEGMIIALEMYHQLLLDPPDDDFNFYVYFKVRYSC
FLIDHYRKAMAVKRKFDQLDYCELSESVNLFDHKQNVSENVMYNLLCQEIHLVLSPEELKLFEALI
[SEQ ID NO: 9]
ATGGATAGCATAGAAATGATGCTTCAAAATATTGAGCCAATTATTATGAATTGTAGTAAAACAACTAG
GATTCCATCTTGGGAGCTAGATGATTACATGCAGGAGGGGATGATTATTGCACTGGAAATGTATCAAA
ATAGACATAACATCAATAACGGTAACGCGTTTAATTTCTATGTCTATTTTAAAGTCAGGTATTCCTGT
TACCTGATAGATAGTTTTAGAAAGGCTAACGCATATAAAAGAAAATTTGATCAACCATTATATTGTGA
AATATCTGAAGCCTTCAACCTTTATGATCACCACCAAAATGTTGCAGACAATGTCTGTTATCAGCTAT
TGCAAGTTGAAATTCTTGAGATATTAACACCAGATGAAGCTGATTTATTTATGACCTTGAAAAATGGT
GGGAAAGTAGAGAGAAATAAAAAGTATAGATTAAAGAAAAAAATTATTGATTATCTTAAAGACATGTT
ATGA [SEQ ID NO: 10]
MDSIEMMLQNIEPIIMNCSKTTRIPSWELDDYMQEGMIIALEMYQNRHNINNGNAFNFYVYFKVRYSC
YLIDSFRKANAYKRKFDQPLYCEISEAFNLYDHHQNVADNVCYQLLQVEILEILTPDEADLFMTLKNG
GKVERNKKYRLKKKIIDYLKDML [SEQ ID NO: 11]
ATGGAGACTTTAGAAGCCATGCTCAAAAACATTGAACCTATTATTATGAATTGTCAAAAGATGGCAAA
AATACCTTCCTGGGATATTGACGATTATATGCAGGAGGGGAGGATCATTGCATTAGACTTGTATAATC
AGCTAGCAGAAAGAATGGAGACGGATGAGGTGAACTTTTACGTCTACTTCAAAGTCAGATATACCTGT
TTCTTGATTGATACTTACCGTAAGACAAATGCCTTTAAAAGAAAATTTGACCAACCGATTTACTTAGA
TGTATCCGAAGCATTTAATCTGTATGATCATAAGCAGAATGTCGCTGATAATGTCATGTATACTTTAT
TGCATCAGGAGATTCTAGACATCTTAACGCCTGTAGAAATTCAAACGCTAAACGCACTAAAAAGGGGA
GAAAAGGTCGACCGCAATAAAAAATTTAGGATTAAAAAGAAGATTATCAACTATATTAATCAGATTTT
CTAG [SEQ ID NO: 12]
METLEAMLKNIEPIIMNCQKMAKIPSWDIDDYMQEGRIIALDLYNQLAERMETDEVNFYVYFKVRYTC
FLIDTYRKTNAFKRKFDQPIYLDVSEAFNLYDHKQNVADNVMYTLLHQEILDILTPVEIQTLNALKRG
EKVDRNKKFRIKKKIINYINQIF [SEQ ID NO: 13]
ATGGAGCATAATTTAGATATGGAGCAGCTGGAAGAAATTTTTCATTCTGTCCAACATATTGTGTGGAA
GAACAGTCGTTTGATTCCGATAAATTTTTGGACGTTTGATGACTATCAGCAGGAAGGGCGCTTGGTAT
TATACGATTTGCTGGGAGATGGTGTGACGCAAAGGAACTTATTTTGCCATTTTAAGGTACGCTATAAG
CAGAGACTTATTGATATTAAAAGAAGGGAGCGGGCTTTTAAAAGGGGTTTTGATTGCGGGACTGGCTT
AGATATATACGAATATTCTGATGCTCTAAAGGGGAAAGCAGCCAGTCCAGAACATATCCTGATTTCTG
GAAGTTTACTTGAAGAAGTTTTTGAAAACTTAAATTTACGCTACCGACGGCTCCTCAAAAGTTACCTC
GCCGGCGATGAATTGCACCGTATGGAAAAGTATCGTTTGAAGGAAAAAATAACGAATATATTATATGA
ACAGCAGTGA [SEQ ID NO: 14]
MEHNLDMEQLEEIFHSVQHIVWKNSRLIPINFWTFDDYQQEGRLVLYDLLGDGVTQRNLFCHFKVRYK
QRLIDIKRRERAFKRGFDCGTGLDIYEYSDALKGKAASPEHILISGSLLEEVFENLNLRYRRLLKSYL
AGDELHRMEKYRLKEKITNILYEQQ [SEQ ID NO: 15]
ATGGCAGAAAATAATTTAGATAAAGAACAGCTTGAAGAGTTATTCCATTCACTTCAACATATTGTTTG
GAAGAACAGTCATTTAATTAAAATAAATTTTTGGACAATGGATGATTATCAGCAAGAAGGGCGACTGG
TTTTATACCAGTTACTTGAAGATGGCGTGACACAGGAAAAACTATTTTGCCATTTTAAAGTGCGATAT
AAGCAACGGTTGATTGATATAAAAAGACGAGAAAGAGCATTTAAGCGGGGTTTTGATTGTGGGGCTGG
TTTAGATATATATGAGTATTCTGATGCCCTGAAAGGCAAAGCTACCAGTCCTGAATATAACTTAATTT
CAGTTACTTTACTTGAAGAGGTTCATCAAAGTTTGAGTTTGAGATACCGCAATTTATTGGAGAATCAT
CTGTCAGGAGTGGAGTTGCATCGAATGGAAAAATACCGTTTAAAGGAAAAAATCAAGAGAATACTCTA
TGAAGAAGAATGA [SEQ ID NO: 16]
MAENNLDKEQLEELFHSLQHIVWKNSHLIKINFWTMDDYQQEGRLVLYQLLEDGVTQEKLFCHFKVRY
KQRLIDIKRRERAFKRGFDCGAGLDIYEYSDALKGKATSPEYNLISVTLLEEVHQSLSLRYRNLLENH
LSGVELHRMEKYRLKEKIKRILYEEE [SEQ ID NO: 17]
ATGGAGCATAATTTAGATATGGAGCAGCTGGAAGAGATATTTCATTCTGTTCAACATATTGTATGGAA
GAATAGTCGTTTGATTCCGATAAATTTTTGGACGATAGATGACTATCAGCAGGAAGGGCGTTTGGTAT
TATATGATTTACTTGAGGATGGTGTGACACAAAGAAAACTTTTTTGCCATTTTAAAGTACGTTATAAG
CAGAGACTTATTGATATTAAAAGAAGGGAGCGGGCTTTTAAAAGGGGTTTTGACTGTGGGACTGGGCT
AGATATTTACGAATATTCAGATGCTTTAAAAGGAAAAGTAGCCAGTCCAGAACATACTCTGATTTCTG
GCAGTTTGCTTGAAGAAGTTTTAGAAAACTTAAATTTACGCTACCGTGCTCTTCTTAAAAGTTACCTT
GCTGGTGATGAACTGCATCGAATGGAAAAACATCGTTTGAAAGAAAAAATAATAAAAATATTATATGA
TGAACAGTGA [SEQ ID NO: 18]
MEHNLDMEQLEEIFHSVQHIVWKNSRLIPINFWTIDDYQQEGRLVLYDLLEDGVTQRKLFCHFKVRYK
QRLIDIKRRERAFKRGFDCGTGLDIYEYSDALKGKVASPEHTLISGSLLEEVLENLNLRYRALLKSYL
AGDELHRMEKHRLKEKIIKILYDEQ [SEQ ID NO: 19]
TTGAAACCGATCGTTTCAAAATCTATGAGAACATTAAAAATCAATTTTTGGACTACAGAGGATTATCA
TCAAGAGGGTCTAATTACATTAAATGAAATATTAAATTCAGGATGTAAGGAGTCACAACTATACATTC
ACTTTAAAGTCAAATATCGACAAAAGCTAATAGACGTGATTAGAAAATCACAGGCGCAAAAAAGAATC
TGGGATAATGCAGAGAGTATTGATGTTTACGAATCTGAAAATCAAATTAATTCCAGTAACTCAAACCC
CGAAGACATAATAGTCTATGACAGTCTTGTAAAGGAAGTAATAACAAAATTAACACCTTCATACCGGA
AACTACTGAAACGACATCTAAGAGGTGAGGATGTGACAAGGATGGAAAAATACAGACTGAAGGAACGA
ATCAAACAAATTTTATTTGATGGTGATTGA [SEQ ID NO: 20]
MKPIVSKSMRTLKINFWTTEDYHQEGLITLNEILNSGCKESQLYIHFKVKYRQKLIDVIRKSQAQKRI
WDNAESIDVYESENQINSSNSNPEDIIVYDSLVKEVITKLTPSYRKLLKRHLRGEDVTRMEKYRLKER
IKQILFDGD [SEQ ID NO: 21]
ATGGATAAGATTGAAACCATACTTAAAAATATTGAACCGATTATCATGAACTGTCGAAAAAAAACTAA
CATCCCTTCCTGGCAATTAGACGACTATCTCCAGGAAGGCATGATTATTGCTCTAGAGATGTATCATC
AACTTTTATTAGACCCACCAGATGATGACTTTAACTTCTATGTTTATTTCAAAGTGAGATATTCTTGT
TTCTTGATTGATCAGTATCGGAGAAACATGGCTGTCAAAAGAAAATTCGACCAGATTGACTATTGTGA
ACTATCTGAGGCGTTTTATCTTTTTGATCAAAATCAAGATGTCTCTGAAAACGTCATGTATAATTTGT
TATGTCAAGAAATACACTTGCTTCTATCTCCTGAAGAACGAGAGCTTTTTGAGGCACTTAAAAATGGA
CAGAAGATTGACCGTAATCAAAAGTTTCGTATCAAGAAGAAAATTATTGAATATATTAAGAGGTTTTG
GTGA [SEQ ID NO: 22]
MDKIETILKNIEPIIMNCRKKTNIPSWQLDDYLQEGMIIALEMYHQLLLDPPDDDFNFYVYFKVRYSC
FLIDQYRRNMAVKRKFDQIDYCELSEAFYLFDQNQDVSENVMYNLLCQEIHLLLSPEERELFEALKNG
QKIDRNQKFRIKKKIIEYIKRFW
[0139] In some embodiments, said comX gene has the nucleotide
sequence of SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or has at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least
99% identity to the nucleotide sequence of SEQ ID NO:1 or SEQ ID
NO:3 or SEQ ID NO:5 or is a variant of SEQ ID NO:1 or SEQ ID NO:3
or SEQ ID NO:5 encoding respectively the ComX protein of SEQ ID
NO:2, SEQ ID NO:4 or SEQ ID NO:6 or is a variant of SEQ ID NO:1 or
SEQ ID NO:3 or SEQ ID NO:5 encoding respectively a functional ComX
protein having at least 90% identity or at least 90% similarity to
a ComX protein of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. In a
particular embodiment, said comX gene is used when the Lactococcus
strain in step a) is a Lactococcus lactis strain.
[0140] In a particular embodiment, when the strain of step a) is a
Lactococcus lactis subsp. lactis strain, the comX gene comprises
the nucleotide sequence of SEQ ID NO:1, any sequence having at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least
99% identity to SEQ ID NO:1, a variant of SEQ ID NO:1 encoding the
ComX protein of SEQ ID NO:2 or a variant of SEQ ID NO:1 encoding a
functional ComX protein having at least 90% identity or at least
90% similarity to a ComX protein of SEQ ID NO:2.
[0141] In a particular embodiment, when the strain of step a) is a
Lactococcus lactis subsp. cremoris strain, the comX gene comprises
the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:5, any sequence
having at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
or at least 99% identity to SEQ ID NO:3 or SEQ ID NO:5 or a variant
of SEQ ID NO:3 or SEQ ID NO:5 encoding respectively the ComX
protein of SEQ ID NO:4 or SEQ ID NO:6 or a variant of SEQ ID NO:3
or SEQ ID NO:5 encoding respectively a functional ComX protein
having at least 90% identity or at least 90% similarity to a ComX
protein of SEQ ID NO:4 or SEQ ID NO:6.
[0142] In some embodiments, said comX gene has the nucleotide
sequence of SEQ ID NO:7 or has at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, or at least 99% identity to the nucleotide
sequence of SEQ ID NO:7 or is a variant of SEQ ID NO:7 encoding the
ComX protein of SEQ ID NO:8, or is a variant of SEQ ID NO:7
encoding a functional ComX protein having at least 90% identity or
at least 90% similarity to a ComX protein of SEQ ID NO:8. In a
particular embodiment, said comX gene is used when the Lactococcus
strain in step a) is a Lactococcus raffinolactis strain.
[0143] In some embodiments, said comX gene has the nucleotide
sequence of SEQ ID NO:9 or has at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, or at least 99% identity to the nucleotide
sequence of SEQ ID NO:9 or is a variant of SEQ ID NO:9 encoding the
ComX protein of SEQ ID NO:10, or is a variant of SEQ ID NO:9
encoding a functional ComX protein having at least 90% identity or
at least 90% similarity to a ComX protein of SEQ ID NO:10. In a
particular embodiment, said comX gene is used when the Lactococcus
strain in step a) is a Lactococcus plantarum strain.
[0144] In some embodiments, said comX gene has the nucleotide
sequence of SEQ ID NO:11 or has at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, or at least 99% identity to the
nucleotide sequence of SEQ ID NO:11 or is a variant of SEQ ID NO:11
encoding the ComX protein of SEQ ID NO:12, or is a variant of SEQ
ID NO:11 encoding a functional ComX protein having at least 90%
identity or at least 90% similarity to a ComX protein of SEQ ID
NO:12. In a particular embodiment, said comX gene is used when the
Lactococcus strain in step a) is a Lactococcus piscium strain.
[0145] In a particular embodiment, said comX gene has the
nucleotide sequence of SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID
NO:17, any sequence having at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, or at least 99% identity to SEQ ID NO:13
or SEQ ID NO:15 or SEQ ID NO:17 or a variant of SEQ ID NO:13 or SEQ
ID NO:15 or SEQ ID NO:17 encoding respectively the ComX protein of
SEQ ID NO:14 or SEQ ID NO:16 or SEQ ID NO:18 or a variant of SEQ ID
NO:13 or SEQ ID NO:15 or SEQ ID NO:17 encoding respectively a
functional ComX protein having at least 90% identity or at least
90% similarity to a ComX protein of SEQ ID NO:14 or SEQ ID NO:16 or
SEQ ID NO:18. In a particular embodiment, said comX gene is used
when the Lactococcus strain in step a) is a Lactococcus garvieae
strain.
[0146] In some embodiments, said comX gene has the nucleotide
sequence of SEQ ID NO:19 or has at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, or at least 99% identity to the
nucleotide sequence of SEQ ID NO:19 or is a variant of SEQ ID NO:19
encoding the ComX protein of SEQ ID NO:20, or is a variant of SEQ
ID NO:19 encoding a functional ComX protein having at least 90%
identity or at least 90% similarity to a ComX protein of SEQ ID
NO:20. In a particular embodiment, said comX gene is used when the
Lactococcus strain in step a) is a Lactococcus fujiensis
strain.
[0147] In some embodiments, said comX gene has the nucleotide
sequence of SEQ ID NO:21 or has at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, or at least 99% identity to the
nucleotide sequence of SEQ ID NO:21 or is a variant of SEQ ID NO:21
encoding the ComX protein of SEQ ID NO:22, or is a variant of SEQ
ID NO:21 encoding a functional ComX protein having at least 90%
identity or at least 90% similarity to a ComX protein of SEQ ID
NO:22. In a particular embodiment, said comX gene is used when the
Lactococcus strain in step a) is a Lactococcus chungangensis
strain.
[0148] By way of example and for the avoidance of doubt, in
particular embodiments, where a comX gene is specified as having a
particular nucleotide sequence, it is understood that the comX gene
comprises said nucleotide sequence. In particular other
embodiments, where a comX gene is specified as having a particular
nucleotide sequence, it is understood that the comX gene consists
of said nucleotide sequence.
[0149] In some embodiments, variants as defined herein of comX
genes are selected from the list of DNA sequences disclosed in
Table 1 below:
TABLE-US-00002 TABLE 1 Strain Accession number Position of comX,
from start to stop Lactococcus lactis Al06 CP009472.1 From 2260881
to 2261372 (reverse) Lactococcus lactis Bpl1 JRFX01000055.1 From
34668 to 35159 (forward) Lactococcus lactis Ll1596 LDEK01000015.1
From 33401 to 33892 (forward) Lactococcus lactis subsp. cremoris
A17 JQIC01000009.1 From 6445 to 6936 (reverse) Lactococcus lactis
subsp. cremoris A76 CP003132.1 From 2293232 to 2293723 (reverse)
Lactococcus lactis subsp. cremoris AM2 LITE01000081.1 From 9954 to
10444 (forward) Lactococcus lactis subsp. cremoris B40
LITC01000320.1 From 10186 to 10677 (forward) Lactococcus lactis
subsp. cremoris DPC6856 LAVW01000168.1 From 445 to 936 (reverse)
Lactococcus lactis subsp. cremoris GE214 AZSI01000020.1 From 186 to
677 (reverse) Lactococcus lactis subsp. cremoris HP JAUH01000192.1
From 40 to 531 (reverse) Lactococcus lactis subsp. cremoris IBB477
JMMZ01000035.1 From 92323 to 92814 (reverse) Lactococcus lactis
subsp. cremoris KW10 LIYF01000023.1 From 40421 to 40912 (forward)
Lactococcus lactis subsp. cremoris KW2 CP004884.1 From 2276371 to
2276862 (reverse) Lactococcus lactis subsp. cremoris LMG6897
LISZ01000238.1 From 10034 to 10525 (forward) Lactococcus lactis
subsp. cremoris Mast36 JZUI01000076.1 From 310 to 801 (reverse)
Lactococcus lactis subsp. cremoris MG1363 AM406671.1 From 2376782
to 2377273 (reverse) Lactococcus lactis subsp. cremoris NBRC 100676
BCVK01000073.1 From 9879 to 10370 (forward) Lactococcus lactis
subsp. cremoris NZ9000 CP002094.1 From 2377598 to 2378089 (reverse)
Lactococcus lactis subsp. cremoris SK11 CP000425.1 From 2283008 to
2283498 (reverse) Lactococcus lactis subsp. cremoris TIFN1
ASXF01000005.1 From 5621 to 6112 (forward) Lactococcus lactis
subsp. cremoris TIFN3 ATBE01000400.1 From 431 to 922 (reverse)
Lactococcus lactis subsp. cremoris TIFN5 ATBC01000090.1 From 315 to
809 (reverse) Lactococcus lactis subsp. cremoris TIFN6
ATBB01000278.1 From 265 to 756 (forward) Lactococcus lactis subsp.
cremoris TIFN7 ATBA01000081.1 From 5620 to 6111 (forward)
Lactococcus lactis subsp. cremoris UC509.9 CP003157.1 From 2107522
to 2108013 (reverse) Lactococcus lactis subsp. cremoris V4
LIYG01000005.1 From 8625 to 9116 (forward) Lactococcus lactis
subsp. hordniae NBRC 100931 BCVL01000030.1 From 70 to 561 (reverse)
Lactococcus lactis subsp. lactis 1AA59 AZQT01000035.1 From 118 to
609 (reverse) Lactococcus lactis subsp. lactis 511 JNLP01000001.1
From 1703029 to 1703520 (reverse) Lactococcus lactis subsp. lactis
A12 LT599049.1 From 2415707 to 2416198 (reverse) Lactococcus lactis
subsp. lactis ATCC 19435 LKLC01000004.1 From 32310 to 32801
(forward) Lactococcus lactis subsp. lactis bv. diacetylactis DRA4
LIWD01000119.1 From 147 to 638 (reverse) Lactococcus lactis subsp.
lactis CV56 CP002365.1 From 2213300 to 2213791 (reverse)
Lactococcus lactis subsp. lactis DPC6853 LAVD01000101.1 From 544 to
1035 (reverse) Lactococcus lactis subsp. lactis E34 LKLD01000014.1
From 197 to 688 (reverse) Lactococcus lactis subsp. lactis Il1403
AE005176.1 From 2223528 to 2224019 (reverse) Lactococcus lactis
subsp. lactis IO-1 DNA AP012281.1 From 2287126 to 2287617 (reverse)
Lactococcus lactis subsp. lactis JCM 7638 BBAP01000017.1 From 34164
to 34656 (forward) Lactococcus lactis subsp. lactis K231
LKLE01000041.1 From 32159 to 32650 (forward) Lactococcus lactis
subsp. lactis K337 LKLF01000041.1 From 34909 to 35400 (forward)
Lactococcus lactis subsp. lactis KF134 LKLJ01000010.1 From 34939 to
35430 (forward) Lactococcus lactis subsp. lactis KF147 CP001834.1
From 2446402 to 2446893 (reverse) Lactococcus lactis subsp. lactis
KF201 LKLM01000024.1 From 28747 to 29238 (forward) Lactococcus
lactis subsp. lactis KF24 LKLH01000011.1 From 34116 to 34607
(forward) Lactococcus lactis subsp. lactis KF282 LKLN01000033.1
From 170 to 661 (reverse) Lactococcus lactis subsp. lactis KLDS
4.0325 CP006766.1 From 2407603 to 2408094 (reverse) Lactococcus
lactis subsp. lactis LMG 7760 JQCM01000018.1 From 37736 to 38227
(forward) Lactococcus lactis subsp. lactis LMG8526 LKLQ01000046.1
From 38499 to 38993 (forward) Lactococcus lactis subsp. lactis NCDO
2118 CP009054.1 From 2402923 to 2403414 (reverse) Lactococcus
lactis subsp. lactis S0 CP010050.1 From 2359456 to 2359947
(reverse) Lactococcus lactis subsp. lactis UC317 LKLY01000004.1
From 36130 to 36621 (forward) Lactococcus lactis WG2 LXWJ01000007.1
From 37921 to 38412 (forward) Lactococcus raffinolactis NBRC 100932
BCVN01000102.1 From 139 to 617 (forward) Lactococcus piscium CNCM
I-4031 FLZT01000001.1 From 149 to 628 (forward) Lactococcus piscium
MKFS47 LN774769.1 From 1708720 to 1709199 (forward) Lactococcus
garvieae 122061 AP017373.1 From 1356405 to 1356890 (forward)
Lactococcus garvieae 8831 AFCD01000005.1 From 510 to 995 (forward)
Lactococcus garvieae Lg-ilsanpaik-gs201105 JPUJ01000002.1 From
180817 to 181302 (reverse) Lactococcus garvieae LG9 AGQY01000137.1
From 5631 to 6116 (reverse) Lactococcus garvieae M79 FOTJ01000023.1
From 3224 to 3709 (forward) Lactococcus garvieae NBRC 100934
BBJW01000010.1 From 105946 to 106431 (reverse) Lactococcus garvieae
PAQ102015-99 LXWL01000009.1 From 238437 to 238922 (reverse)
Lactococcus garvieae TB25 AGQX01000090.1 From 28088 to 28573
(reverse) Lactococcus garvieae TRF1 AVFE01000015.1 From 42141 to
42626 (reverse)
[0150] As used herein a comX gene is understood to be a gene that
encodes a functional ComX protein in the strain where it is
expressed. By "functional ComX protein" it is meant a protein which
induces or is able to induce the expression of genes regulated by
the Com-box, and at least one of the late competence genes selected
from comFA, comFA, comGA, dprA, coiA, ssbA, radA, radC, recA, and
recX.
[0151] The ComX protein may have the amino acid sequence of SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID
NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or
SEQ ID NO:22, or an amino acid sequence having at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identity
to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22, or an amino acid
sequence having at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, or at least 99% similarity to the amino acid sequence of
SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,
SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID
NO:20 or SEQ ID NO:22.
[0152] In some embodiments, the ComX protein may have the amino
acid sequence of SEQ ID NO:2, or an amino acid sequence having at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least
99% identity to the amino acid sequence of SEQ ID NO:2 or an amino
acid sequence having at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99% similarity to the amino acid sequence
of SEQ ID NO:2. In a particular embodiment, said ComX protein is
used when the Lactococcus strain in step a) is a Lactococcus lactis
subsp. lactis strain.
[0153] In some embodiments, the ComX protein may have the amino
acid sequence of SEQ ID NO:4 or SEQ ID NO:6, or an amino acid
sequence having at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, or at least 99% identity to the amino acid sequence of
SEQ ID NO:4 or SEQ ID NO:6 or an amino acid sequence having at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least
99% similarity to the amino acid sequence of SEQ ID NO:4 or SEQ ID
NO:6. In a particular embodiment, said ComX protein is used when
the Lactococcus strain in step a) is a Lactococcus lactis subsp.
cremoris strain.
[0154] In some embodiments, the ComX protein may have the amino
acid sequence of SEQ ID NO:8, or an amino acid sequence having at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least
99% identity to the amino acid sequence of SEQ ID NO:8 or an amino
acid sequence having at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99% similarity to the amino acid sequence
of SEQ ID NO:8. In a particular embodiment, said ComX protein is
used when the Lactococcus strain in step a) is a Lactococcus
raffinolactis strain.
[0155] In some embodiments, the ComX protein may have the amino
acid sequence of SEQ ID NO:10, or an amino acid sequence having at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least
99% identity to the amino acid sequence of SEQ ID NO:10 or an amino
acid sequence having at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99% similarity to the amino acid sequence
of SEQ ID NO:10. In a particular embodiment, said ComX protein is
used when the Lactococcus strain in step a) is a Lactococcus
plantarum strain.
[0156] In some embodiments, the ComX protein may have the amino
acid sequence of SEQ ID NO:12, or an amino acid sequence having at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least
99% identity to the amino acid sequence of SEQ ID NO:12 or an amino
acid sequence having at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99% similarity to the amino acid sequence
of SEQ ID NO:12. In a particular embodiment, said ComX protein is
used when the Lactococcus strain in step a) is a Lactococcus
piscium strain.
[0157] In some embodiments, the ComX protein may have the amino
acid sequence of SEQ ID NO:14, SEQ ID NO:16 or SEQ ID NO:18, or an
amino acid sequence having at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, or at least 99% identity to the amino acid
sequence of SEQ ID NO:14, SEQ ID NO:16 or SEQ ID NO:18 or an amino
acid sequence having at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99% similarity to the amino acid sequence
of SEQ ID NO:14, SEQ ID NO:16 or SEQ ID NO:18. In a particular
embodiment, said ComX protein is used when the Lactococcus strain
in step a) is a Lactococcus garvieae strain.
[0158] In some embodiments, the ComX protein may have the amino
acid sequence of SEQ ID NO:20, or an amino acid sequence having at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least
99% identity to the amino acid sequence of SEQ ID NO:20 or an amino
acid sequence having at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99% similarity to the amino acid sequence
of SEQ ID NO:20. In a particular embodiment, said ComX protein is
used when the Lactococcus strain in step a) is a Lactococcus
fujiensis strain.
[0159] In some embodiments, the ComX protein may have the amino
acid sequence of SEQ ID NO:22, or an amino acid sequence having at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least
99% identity to the amino acid sequence of SEQ ID NO:22 or an amino
acid sequence having at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99% similarity to the amino acid sequence
of SEQ ID NO:22. In a particular embodiment, said ComX protein is
used when the Lactococcus strain in step a) is a Lactococcus
chungangensis strain.
[0160] According to the invention, when a ComX protein is defined
by its amino acid sequence having a percentage of identity or
percentage of similarity to a specific SEQ ID, said ComX protein is
a functional ComX protein as defined herein.
[0161] By way of example and for the avoidance of doubt, in
particular embodiments, where a ComX protein is specified as having
a particular amino acid sequence, it is understood that the ComX
protein comprises said amino acid sequence. In particular other
embodiments, where a ComX protein is specified as having a
particular amino acid sequence, it is understood that the ComX
protein consists of said amino acid sequence.
[0162] In some embodiments, ComX proteins having percentage of
identity or percentage of similarity as defined herein are selected
from the list of protein sequences derived, after translation, from
the list of DNA sequences disclosed in Table 1 above.
[0163] Preferably, reference to a sequence which has a percentage
identity or similarity to any one of the SEQ ID NOs detailed herein
refers to a sequence which has the stated percent identity or
similarity with the SEQ ID NO referred to, over the entire length
of the two sequences. Percentage (%) sequence identity is defined
as the percentage of amino acids or nucleotides in a candidate
sequence that are identical to the amino acids or nucleotides in a
reference sequence, after aligning the sequences and introducing
gaps, if necessary, to achieve the maximum percent sequence
identity. Percentage (%) sequence similarity is defined as the
percentage of amino acids in a candidate sequence that are similar
to the amino acids in a reference sequence, after aligning the
sequences and introducing gaps, if necessary, to achieve the
maximum percent sequence similarity. Similarity between amino acids
is based on established amino acid substitution matrices such as
the PAM series (Point Accepted Mutation; e.g. PAM30, PAM70, and
PAM250) or the BLOSUM series (BLOck SUbstitution Matrix; e.g.
BLOSUM45, BLOSUM50, BLOSUM62, BLOSUM80, and BLOSUM90). Alignment
for purposes of determining percent sequence identity or similarity
can be achieved in various ways that are within the skill in the
art, for instance, using publicly available computer software such
as CLUSTALW, CLUSTALX, CLUSTAL Omega, BLAST, BLAST-2, ALIGN,
ALIGN-2 or Megalign (DNASTAR) software. In a particular embodiment,
similarity between amino acids is determined using the BLASTp
software with the BLOSUM62 matrix. Appropriate parameters for
measuring alignment, including any algorithms needed to achieve
maximal alignment over the full-length of the sequences being
compared, or gap penalties to be introduced, can be determined by
known methods.
[0164] In a particular embodiment, when the modulation in step b)
results from an exogenous comX gene, said exogenous comX gene is
(obtained) from a strain of the same species, in particular of the
same subspecies, as the strain provided in step a). In any case,
the exogenous comX gene needs to be functional, in particular needs
to encode a functional ComX protein, as defined herein in the
strain provided in step a).
Exogenous DNA Polynucleotide
[0165] The method of the present invention comprises the step of
contacting the strain of step b) with an exogenous DNA
polynucleotide in a medium and incubating the resulting mixture for
integration of the exogenous DNA into the genome of said strain
[step c].
[0166] As used herein the term "exogenous DNA polynucleotide"
refers to a DNA polynucleotide that is brought into the cytoplasm
of said strain, in order to be integrated into the genome of said
strain (target sequence).
[0167] In a particular embodiment, the method comprises carrying
out step (b) [ComX modulation] and then carrying out step (c)
[contact with the exogenous DNA polynucleotide] [i.e., that step
(c) is carried out on a strain obtained following step (b)]. Thus,
the method comprising the steps of: [0168] (a) providing a strain
of the Lactococcus genus, wherein said strain is transformable
through natural competence; [0169] (b) modulating the production of
a ComX protein in said strain; [0170] (c) contacting said strain
obtained in step (b) with an exogenous DNA polynucleotide in a
medium and incubating the resulting mixture for integration of the
exogenous DNA polynucleotide into the genome of said strain; and
[0171] (d) selecting a strain which has integrated the exogenous
DNA polynucleotide into its genome.
[0172] In another embodiment, the method comprises carrying out
simultaneously step (b) [ComX modulation] and step (c) [contact
with the exogenous DNA polynucleotide]. This option is appropriate
when the ComX modulation is the result of the expression of the
endogenous comX gene or of the increase of the expression of the
endogenous comX gene of said strain. Thus, the method comprising
the steps of: [0173] (a) providing a strain of the Lactococcus
genus, wherein said strain is transformable through natural
competence; [0174] (b) modulating the production of a ComX protein
in said strain; [0175] (c) contacting said strain with an exogenous
DNA polynucleotide in a medium and incubating the resulting mixture
for integration of the exogenous DNA polynucleotide into the genome
of said strain; and [0176] (d) selecting a strain which has
integrated the exogenous DNA polynucleotide into its genome; [0177]
wherein step (b) and step (c) are carried out simultaneously.
[0178] In a particular embodiment, the sequence of the exogenous
DNA polynucleotide used in step c) share some similarities or
identities with the genome of the Lactococcus strain to be
transformed (of step a). In a particular embodiment, the exogenous
DNA polynucleotide used in step c) is designed such that its 5'
part and its 3' part are identical or highly similar to parts of
the genome of the Lactococcus strain to be transformed (of step a),
while its central part can be different from the genome of the
Lactococcus strain to be transformed (of step a). The high
similarity of the arms with the regions surrounding the target
sequence can be determined by the person skilled in the art using
common general knowledge, in particular by reference to homologous
recombination.
[0179] Thus, to replace a target sequence by a mutated sequence or
a truncated sequence or a supplementary sequence in the genome of
the Lactococcus strain to be transformed (of step a), the exogenous
DNA polynucleotide used in step c) is designed such that: [0180]
its 5' part is identical or highly similar to the region of the
genome of the Lactococcus strain to be transformed which is on one
side of the target sequence; [0181] its central part contains the
replacing sequence (i.e., the mutated sequence or the truncated
sequence or the supplementary sequence); and [0182] its 3' part is
identical or highly similar to the region of the genome of the
Lactococcus strain to be transformed which is on the other side of
the target sequence.
[0183] The 5' part and 3' part are long enough to ensure efficient
recombination. In a particular embodiment, each of the 5' part and
3' part is from 0.5 to 5 kb in length. The size of the arms can be
determined by the person skilled in the art using common general
knowledge, in particular by reference to homologous
recombination.
[0184] In a particular embodiment, the exogenous DNA polynucleotide
used in step c) is (obtained) from a strain of the Lactococcus
genus.
[0185] In a particular embodiment, said exogenous DNA
polynucleotide used in step (c) is (obtained) from a strain of the
same species, in particular of the same subspecies, as the strain
provided in step (a).
[0186] In a particular embodiment, the exogenous DNA polynucleotide
used in step c) is from a strain of the Lactococcus lactis species.
In a particular embodiment, the exogenous DNA polynucleotide used
in step c) is from a strain of the same Lactococcus lactis
subspecies as the strain provided in step a). In a particular
embodiment, the exogenous DNA polynucleotide used in step c) is
from a strain of a Lactococcus lactis subspecies which is different
from the strain provided in step a).
[0187] In a particular embodiment, the exogenous DNA polynucleotide
used in step c) is from a strain of the Lactococcus raffinolactis
species
[0188] The exogenous DNA polynucleotide may encode part of a gene
sequence, a gene sequence, or a plurality of gene sequences. The
gene sequence may be operably linked to transcription regulator(s).
In a particular embodiment, the exogenous DNA polynucleotide is
linear. The exogenous DNA polynucleotide may be designed to
facilitate its incorporation within the genome of the L. lactis
strain by homologous recombination (e.g. the exogenous DNA
polynucleotide may comprise one or more recombination arms). The
exogenous DNA polynucleotide may be a single stranded linear
DNA.
[0189] The exogenous DNA polynucleotide, when incorporated into the
genome of said Lactococcus strain leads to genetic modification of
the strain such as gene replacement (to add or to remove a
mutation), gene addition (to add a new gene or to duplicate an
existing gene), gene deletion (to remove part or the totality of a
gene), modification of non-coding region (to modulate expression of
a gene). Typically, the exogenous DNA polynucleotide, when
incorporated into the genome of said Lactococcus strain confers an
interesting or useful phenotype, e.g. modified kinetic of
acidification, improved resistance to bacteriophage, modified
capability to grow in milk, modified texturing properties, improved
safety of the strain. For example, improved bacteriophage
resistance could be achieved by incorporating genes coding for a
restriction/modification system into the strain genome or by
introducing a mutation or a deletion into the pip gene.
[0190] As an example, growth of a L. lactis strain in milk could be
improved by inserting into the chromosome the prtP and prtM genes
that allow casein hydrolysis and better nitrogen nutrition;
alternatively, these genes could be inactivated to reduced milk
proteolysis in cheese. hisDC and tyrDC are genes known to be
responsible for biogenic amine production (histamine and tyramine,
respectively) in a diversity of lactic acid bacteria; disruption or
mutation of these genes could help to prevent safety issues related
to cheese consumption.
[0191] In a particular embodiment, the exogenous DNA polynucleotide
has a minimal size selected from the group consisting of 100 bp,
200 bp, 500 bp, 1 kb, 2 kb and 5 kb, and a maximal size selected
from the group consisting of 500 bp, 1 kb, 2 kb, 5 kb, 10 kb, 20 kb
and 50 kb. In a particular embodiment, the size of the exogenous
DNA polynucleotide may be between 100 bp and 50 kb, more preferably
between 500 bp to 20 kb, even more preferably between 1 kb to 10
kb.
[0192] The concentration of exogenous DNA polynucleotide in the
medium of step (c) may be between 0.5 mg/L and 1 g/L, preferably
between 1 mg/L and 500 mg/L, more preferably between 5 mg/L and 100
mg/L, even more preferably between 10 mg/L and 50 mg/L of
medium.
Selection of Transformed Strains
[0193] The method of the present invention comprises the step of
selecting a strain which has integrated the exogenous DNA
polynucleotide into its genome [step d)].
[0194] If needed, selection is carried out on some cells of
colonies that have been previously obtained by multiplying, in the
appropriate medium, cells obtained at the end of step c) (or at the
end of the simultaneous steps b) and c), when appropriate).
[0195] Various methods for the selection of transformed bacteria
are well known in the art (see, e.g. Sambrook et al.) and may be
routinely applied by the person skilled in the art, such as PCR,
DNA sequencing . . . .
[0196] For example, when the exogenous DNA polynucleotide used in
step c) provides a particular phenotype that the Lactococcus strain
of step a) does not display (either a new phenotype or restoring a
lost phenotype), it is possible to select strains which have
integrated the exogenous DNA polynucleotide into their genome by
selecting strains expressing the phenotype. This is the case for a
strain having integrated in its genome an exogenous DNA
polynucleotide mutated for the pip gene (that provides resistance
to some bacteriophages).
[0197] For example, when the exogenous DNA polynucleotide used in
step c) leads once integrated to a loss of a phenotype initially
displayed by the Lactococcus strain of step a), it is possible to
select strains which have integrated the exogenous DNA
polynucleotide into their genome by selecting strains which do not
display the phenotype any more. This is the case for an exogenous
DNA polynucleotide bearing a mutated hisDC or tyrDC gene, which
suppresses or decreases the production of histamine or tyramine,
respectively.
[0198] As a particular example, the exogenous DNA polynucleotide
may bear an antibiotic resistance gene. Accordingly, a Lactococcus
strain which has integrated the exogenous DNA polynucleotide into
its genome may be selected by plating onto a medium comprising said
antibiotic. Only strains that express the appropriate antibiotic
resistance gene, as a result of a successful transformation with
the exogenous DNA polynucleotide, will multiply.
Growth Conditions
[0199] As described in Example 3, a positive effect on natural
competence induction in L. lactis strains was observed when cells
were pre-cultured in a complex medium before transferring the cells
to a chemically defined medium (FIG. 3).
[0200] Accordingly, in some embodiments the medium of step (c) is a
chemically defined medium. As used herein, the term "chemically
defined medium" (CDM) refers to a medium for which the exact
chemical composition is known. Preferably, the CDM may have the
composition of the CDM set out in Sissler et al. (1999, Proc Natl
Acad Sci USA 96:8985-8990). Thus, in an embodiment, the chemically
defined medium (CDM) comprises 0.5 g/L NH.sub.4Cl, 9.0 g/L
KH.sub.2PO.sub.4, 7.5 g/L K.sub.2HPO.sub.4, 0.2 g/L MgCl.sub.2, 5
mg/L FeCl.sub.2, 50 mg/L CaCl.sub.2), 5 mg/L ZnSO.sub.4, 2.5 mg/L
CoCl.sub.2, 0.05 g/L tyrosine, 0.1 g/L asparagine, 0.1 g/L
cysteine, 0.1 g/L glutamine, 0.1 g/L isoleucine, 0.1 g/L leucine,
0.1 g/L methionine, 0.1 g/L tryptophan, 0.1 g/L valine, 0.1 g/L
histidine, 0.2 g/L arginine, 0.2 g/L glycine, 0.2 g/L lysine, 0.2
g/L phenylalanine, 0.2 g/L threonine, 0.3 g/L alanine, 0.3 g/L
proline, 0.3 g/L serine, 10 mg/L paraaminobenzoic acid, 10 mg/L
biotin, 1 mg/L folic acid, 1 mg/L nicotinic acid, 1 mg/L
panthotenic acid, 1 mg/L riboflavin, 1 mg/L thiamine, 2 mg/L
pyridoxine, 1 mg/L cyanocobalamin, 5 mg/L orotic acid, 5 mg/L
2-deoxythymidine, 5 mg/L inosine, 2.5 mg/L dl-6,8-thioctic acid, 5
mg/L pyridoxamine, 10 mg/L adenine, 10 mg/L guanine, 10 mg/L
uracil, 10 mg/L xanthine, and 5 g/L glucose..
[0201] In some embodiments, prior to step (c) said strain is
incubated in a pre-culture medium, preferably wherein the
pre-culture medium is a complex medium, more preferably wherein the
pre-culture medium is M17G (i.e., the M17 medium supplemented with
glucose) or THBG (i.e., the THB medium supplemented with
glucose).
[0202] The complex medium may be Todd Hewitt broth (THB) (Todd and
Hewitt, 1932; Updyke and Nickle, 1954) or M17 broth (Terzaghi and
Sandine, 1975). THB may comprise 500 g/L beef heart infusion, 20
g/L peptic digest of animal tissue, 2 g/L dextrose, 2 g/L sodium
chloride, 0.4 g/L sodium phosphate, 2.5 g/L sodium carbonate. M17
broth may comprise: 0.5 g/L ascorbic acid, 5 g/L lactose, 0.25 g/L
magnesium sulfate, 5 g/L meat extract, 2.5 g/L meat peptone
(peptic), 19 g/L sodium glycerophosphate, 5 g/L soya peptone
(papainic), 2.5 g/L tryptone, 2.5 g/L yeast extract.
Method for Identifying Strains Transformable by Natural
Competence
[0203] In another aspect, the present invention relates to a method
for identifying a strain of the Lactococcus genus which is
transformable through natural competence. Said method comprises the
following steps: [0204] (a) providing a strain of the Lactococcus
genus; [0205] (b) transforming said strain with a plasmid
expressing a comX gene having at least 90% identity, preferably
having 100% identity, to the endogenous comX gene of said strain;
[0206] (c) contacting said strain obtained in step (b) with an
exogenous marker DNA polynucleotide in a medium and incubating the
resulting mixture for integration of the exogenous DNA
polynucleotide into the genome of said strain; and [0207] (d)
determining the rate of recombination events;
[0208] wherein a rate of at least 1.times.10.sup.-6 transformants
per .mu.g of DNA is indicative of a strain which is transformable
through natural competence.
[0209] The term "rate of recombination events" may be used
interchangeably with the term "transformation rate". The rate of
recombination events is calculated by determining the ratio of the
number of cells having integrated the exogenous marker DNA
polynucleotide over the total number of viable cells. A rate of at
least 10.sup.-6 was selected as a threshold, based on the
observation that the level of spontaneous mutation in lactococci is
less than 10.sup.-6, typically around 10.sup.-7 mutants per .mu.g
of DNA [spontaneous means with no comX expression or
overexpression].
[0210] By "at least 90% identity to the endogenous comX gene of
said strain", it is meant--as particular embodiments of the
method--at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least
99% identity. In a particular embodiment, said comX gene has 100%
identity to the endogenous comX gene of said strain.
[0211] In a particular embodiment, said method is implemented with
a strain of the Lactococcus genus selected from the group
consisting of Lactococcus lactis, Lactococcus raffinolactis,
Lactococcus plantarum, Lactococcus piscium, Lactococcus garivieae,
Lactococcus fujiensis and Lactococcus chungangensis.
[0212] In a particular embodiment, said method is implemented with
a strain of the Lactococcus lactis species. Said method comprises
the following steps: [0213] (a) providing a strain of the
Lactococcus lactis species; [0214] (b) transforming said strain
with a plasmid expressing a comX gene having at least 90% identity
to the polynucleotide sequence of SEQ ID NO:1, 3 or 5; [0215] (c)
contacting said strain obtained in step (b) with an exogenous
marker DNA polynucleotide in a medium and incubating the resulting
mixture for integration of the exogenous DNA polynucleotide into
the genome of said strain; and [0216] (d) determining the rate of
recombination events;
[0217] wherein a rate of at least 1.times.10.sup.-6 transformants
per .mu.g of DNA is indicative of a strain of the Lactococcus
lactis species which is transformable through natural
competence.
[0218] In a particular embodiment, said method is implemented with
a strain of the Lactococcus raffinolactis species. Said method
comprises the following steps: [0219] (a) providing a strain of the
Lactococcus raffinolactis species; [0220] (b) transforming said
strain with a plasmid expressing a comX gene having at least 90%
identity to the polynucleotide sequence of SEQ ID NO:7; [0221] (c)
contacting said strain obtained in step (b) with an exogenous
marker DNA polynucleotide in a medium and incubating the resulting
mixture for integration of the exogenous DNA polynucleotide into
the genome of said strain; and [0222] (d) determining the rate of
recombination events; [0223] wherein a rate of at least
1.times.10.sup.-6 transformants per .mu.g of DNA is indicative of a
strain of the Lactococcus lactis species which is transformable
through natural competence.
[0224] In some embodiments, the comX gene is from a strain of the
same species, in particular of the same subspecies, as the strain
provided in step a). In some embodiments, the comX gene is
identical (100% identity) to the polynucleotide sequence of the
endogenous comX gene of the strain of step a).
[0225] In some embodiments, when the strain of step a) is a
Lactococcus lactis subsp. lactis strain, the comX gene has at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
or 100% identity to the polynucleotide sequence of SEQ ID NO:1.
[0226] In some embodiments, when the strain of step a) is a
Lactococcus lactis subsp. cremoris strain the comX gene has at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100% identity to the polynucleotide sequence of SEQ ID NO:3
or SEQ ID NO:5.
[0227] In some embodiments, when the strain of step a) is a
Lactococcus raffinolactis strain the comX gene has at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%
identity to the polynucleotide sequence of SEQ ID NO:7.
[0228] In some embodiments, when the strain of step a) is a
Lactococcus plantarum strain the comX gene has at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%
identity to the polynucleotide sequence of SEQ ID NO:9.
[0229] In some embodiments, when the strain of step a) is a
Lactococcus piscium strain the comX gene has at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100%
identity to the polynucleotide sequence of SEQ ID NO:11.
[0230] In some embodiments, when the strain of step a) is a
Lactococcus garvieae strain the comX gene has at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%
identity to the polynucleotide sequence of SEQ ID NO:13, SEQ ID
NO:15, or SEQ ID NO:17.
[0231] In some embodiments, when the strain of step a) is a
Lactococcus fujiensis strain the comX gene has at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%
identity to the polynucleotide sequence of SEQ ID NO:19.
[0232] In some embodiments, when the strain of step a) is a
Lactococcus chungangensis strain the comX gene has at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%
identity to the polynucleotide sequence of SEQ ID NO:21.
[0233] It is preferable to use, as an exogenous marker DNA
polynucleotide, a polynucleotide bearing a gene which is initially
not present in the Lactococcus strain of step a) [even as a mutated
version]. This would avoid that during step c) the Lactococcus
strain acquires a functional gene by other means than natural
competence, e.g. by spontaneous mutation of its genome.
[0234] As an example, the exogenous marker DNA polynucleotide bears
a gene encoding a luciferase gene. Accordingly, a Lactococcus
strain which has integrated the exogenous DNA polynucleotide into
its genome may be selected for expression of the luciferase. Only
strains that express the luciferase gene (i.e., integrated) will be
detectable by bioluminescence.
[0235] As another example, the exogenous marker DNA polynucleotide
bears an antibiotic resistance gene. Accordingly, a Lactococcus
strain which has integrated the exogenous DNA polynucleotide into
its genome may be selected by plating the cells onto a medium
comprising said antibiotic.
[0236] An example of a method for identifying a strain of the
Lactococcus genus which is transformable through natural competence
according to the present invention (Assay A) may be performed using
the following steps: [0237] i) Providing a strain of the
Lactococcus genus, in particular of the Lactococcus lactis species.
[0238] ii) Transforming said strain with a plasmid expressing a
comX gene having at least 90% identity, preferably having 100%
identity to the endogenous comX gene of said strain (e.g. the
pGhP32comX.sub.MG plasmid of Materials and Methods). [0239] iii)
Pre-culturing the transformed strain overnight in a complex medium
supplemented with glucose (e.g. M17G) at 30.degree. C. [0240] iv)
Diluting about 1.5 mL of the pre-culture in about 8.5 mL of fresh
medium. [0241] v) After about 2 hours further growth at 30.degree.
C., washing the cells twice with distilled water and adjusting the
OD.sub.600 to 0.05 in a chemically defined medium (e.g. CDM)
containing 5 .mu.g mL.sup.-1 erythromycin and an osmo-stabilizer
(e.g. 5% [v/v] glycerol or 5% [w/v] mannitol). [0242] vi) Adding 5
.mu.g of exogenous DNA polynucleotide bearing an antibiotic
resistance gene to 300 .mu.l of the culture medium (e.g. the 3.7 kb
PCR product generated from the pGEMrpsL plasmid as described in
Materials and Methods). [0243] vii) Incubating the resulting
culture for about 6 hours at 30.degree. C. [0244] viii) Plating the
cells onto agar plates comprising the complex medium supplemented
with glucose (e.g. M17G) and appropriate antibiotic (i.e.
corresponding to the antibiotic resistance gene of the exogenous
DNA polynucleotide) and incubating for about 48 hours. [0245] ix)
Counting the colony forming units (CFU) and determining the
transformation rate, wherein a transformation rate of at least
1.times.10.sup.-6 transformants per .mu.g of DNA is indicative of a
strain which is transformable through natural competence.
[0246] The transformation rate may be calculated as the number of
antibiotic-resistance CFU mL.sup.-1 divided by the total number of
viable CFU mL.sup.-1.
[0247] Various preferred features and embodiments of the present
invention will now be described by way of non-limiting
examples.
EXAMPLES
Example 1: Induction of the comGA Promoter by Constitutive comX
Expression in Various Strains of the Lactococcus Species
[0248] a) In Lactococcus lactis Subsp. Cremoris Strains (MG1363 and
KW2)
[0249] To test the ability of ComX to induce the late competence
genes in Lactococcus lactis subsp. cremoris strains, a constitutive
comX expression plasmid (pGhP32comX.sub.MG) was created by cloning
the comX gene from strain MG1363, under the control of the
lactococcal P.sub.32 promoter on the thermosensitive plasmid
pG.sup.+host9. The latter was introduced in strain KW2 that
contains a chromosomally-encoded P.sub.comGA[MG]-luxAB
transcriptional fusion (BLD101). The promoter of the late
competence gene comGA (P.sub.comGA) contains a putative
ComX-binding motif and is used here as proxy for competence
activation in the ComX strain.
[0250] As alternative for more resistant strains to
electro-transformation, and subsequently to chromosome integration,
a portable luminescent reporter system was also constructed. This
replicative plasmid carries the luminescent reporter
P.sub.comGA[MG]-luc with the P.sub.32-comX.sub.MG cassette. The
pGhP32comX.sub.MG-P.sub.comGA[MG]-luc plasmid was transformed in
strain MG1363. Specific P.sub.comGA[MG]-luc/luxAB activities were
monitored for the different strains constructed. The luminescent
assays were performed in rich (M17G) and/or CDM media comparing the
luciferase activity between the overexpressing comX strain and its
related negative control (no additional comX copy).
[0251] In the KW2 strain containing the P.sub.comGA[MG]-luxAB
reporter as mono-copy in their chromosome, specific luciferase
activity was observed for KW2 containing the P.sub.32-comX cassette
allowing the constitutive production of ComX. This confirms that
comX expression can be carried out in various L. lactis subsp.
cremoris strains using an exogenous comX gene obtained from the
same strain or from a strain of the same subspecies. Eight
recombinant clones of the KW2 ComX.sup.+ reporter strain were
randomly selected and their specific luciferase activity was
monitored in CDM growth conditions. This medium was chosen because
it was shown to be permissive for competence development in various
streptococcal species. To ensure reproducibility of the assay,
exponentially-growing cells in complex medium (M17 conditions) were
washed and inoculated in fresh CDM before starting the experiment.
As expected, all tested ComX.sup.+ clones (cl01 to cl08) displayed
between 10.sup.1- and 10.sup.4-fold higher specific luciferase
(Lux) activity than the control strain carrying the empty vector
(FIGS. 2A and 2B).
[0252] Similar results were obtained with the portable luminescent
reporter systems in MG1363 (FIG. 2C).
[0253] These results strongly suggest that, in the L. lactis subsp.
cremoris strains MG1363 and KW2, ComX induces the comG operon.
Additionally, these observations validate these reporter fusions
(both chromosomal and plasmid-borne) as a tool to identify
conditions capable to activate the comG-operon which is essential
to natural transformation.
[0254] b) In a L. lactis Subsp. Lactis Strain (IL1403)
[0255] A constitutive comX expression plasmid (pGhP32comX.sub.IO)
was created by cloning the comX gene from strain IO-1, under the
control of the lactococcal P.sub.32 promoter on the thermosensitive
plasmid pG.sup.+host9. A portable luminescent reporter system was
also constructed; this replicative plasmid carries the luminescent
reporter P.sub.comGA[IO]-luc with the P.sub.32-comX.sub.IO
cassette. The promoter of the late competence gene comGA
(P.sub.comGA) contains a putative ComX-binding motif and is used
here as proxy for competence activation in the ComX.sup.+
strain.
[0256] This replicative plasmid
pGhP32comX.sub.IO-P.sub.comGA[IO]-luc was transformed in strain
IL1403 and specific P.sub.comGA[IO]-luc activities were monitored.
One of the IL1403 transformants produced specific
P.sub.comGA[IO]-luc activities confirming that ComX induces the
comG operon (FIG. 2D).
Example 2: Analysis of Essential Late Corn Genes Present in L.
lactis Genomes
[0257] Among L. lactis strains, genomic variability was previously
investigated for comX and dprA alleles (Wydau et al., 2006). While
all strains (31/31) display a complete version of comX, the dprA
content is variable among subspecies: 50% of the lactis strains
(10/20) contain nonsense mutations in dprA while all cremoris
strains (11/11) harbor an intact and potentially functional dprA
gene.
[0258] Since dprA is hypothesized to be important in the natural
competence mechanism, its integrity in L. lactis strains prompted
us to further analyze the minimal set of late corn genes (17
candidate genes including comX; FIG. 1) in the genomes of 3 subsp.
cremoris strains and 1 subsp. lactis strain which are publicly
available (strains MG1363, SK11, KW2 and IL1403). This in silico
analysis reveals that the genome of SK11 contains a high number of
pseudogenes in key competence genes (between 5 and 8 incomplete
late corn genes) due to transposon insertion or frameshifting
events (nucleotide(s) insertion or deletion). In particular, the
presence of transposable elements in comGA and/or comEC genes,
which are respectively essential for pilus assembly and DNA
transport, strongly suggests that natural transformation is no more
functional in those strains. Although the set of full-length
competence genes in the laboratory strain MG1363 is larger,
mutations in comEC (nucleotide insertion) and coiA (nonsense
mutation) probably impair its ability to transform DNA by
competence (Wegmann et al., 2007). Those mutations were also found
in the genome of its isogenic derivative NZ9000, which strongly
suggests that they do not result from DNA sequencing errors. As far
as the L. lactis subsp. lactis IL1403 strain is concerned, its dprA
gene contains nonsense mutations probably impairing its ability to
transform DNA by competence. In contrast, strain KW2 of plant
origin (corn fermentation) contains the whole set of known
essential late genes required to fulfil natural DNA transformation,
making it the best candidate to further study the functionality of
competence in the cremoris subspecies. Two other strains from our
collection, L. lactis subsp. lactis SL12651 and SL12653, were also
found to contain the whole set of known essential late genes (FIG.
1).
Example 3: Effect of Growth Conditions on ComX Activation
[0259] We investigated the effect of pre-culturing and culturing
conditions (M17G, THBG, and CDM) on the activation of the reporter
fusion in the ComX.sup.+ strain. For this purpose, clone 02 (FIG.
2A) was selected since it exhibits the strongest Lux activity.
Interestingly, more than a 20-fold variation in the maximum Lux
activity was dependent on the pre- and culturing medium which was
used (FIG. 3). Particularly, a positive impact of the transition of
pre-culture cells from a complex medium to a defined medium was
observed. The highest specific Lux activity
(.about.3.times.10.sup.6 RLU OD.sub.600.sup.-1) was obtained for a
switch from M17G to CDM, followed by THBG to CDM, while all other
combinations gave lower activities (between .about.1.5.times. and
5.5.times.10.sup.5 RLU OD.sub.600.sup.-1). This indicates that
first a chemically defined medium is superior for maximizing
activation of late corn genes of L. lactis KW2 than complex rich
media, but also that the switch from complex medium (e.g. M17G or
THBG) to defined medium is critical.
[0260] Together, these results show that ComX is functional in
strain KW2 when it is constitutively produced (i.e. expressed) and
that growth conditions have a significant impact on the activation
of late corn genes.
Example 4: Constitutive comX Expression Induces Natural
Transformation
[0261] a) Acquisition of Single Mutations in the KW2 Genome from
Exogenous DNA
[0262] We first tested the transfer of single point mutations in
the chromosome of the ComX.sup.+ KW2 strain. The transforming PCR
fragments used encompass the mutated rpsL allele of a spontaneous
streptomycin-resistant (Str.sup.r) clone of L. lactis subsp.
cremoris MG1363 (strA1 allele, also called rpsL*). This mutated
allele bears an A.fwdarw.T substitution at position 167 [resulting
in the altered ribosomal protein S12 with mutation K56I] as
compared to the sequence of the wild-type, streptomycin-sensitive
MG1363. In addition to this mutation, the two rpsL alleles differ
by a silent nucleotide substitution at position 39 (T.fwdarw.G).
The sequence of the rpsL (wild-type) and rpsL* (conferring
streptomycin resistance) alleles are disclosed respectively as SEQ
ID NO:23 and NO:24 (FIG. 4A). Independently of these two
substitutions located at positions 39 and 167, the rpsL alleles of
KW2 and MG1363 differ by a nucleotide substitution at position 156
(A in MG1363, T in KW2). The rpsL allele of KW2 is disclosed as SEQ
ID NO:25 (FIG. 4A). To ensure efficient recombination, the
transforming PCR product also contains upstream and downstream
recombination arms of .about.1.85 kb surrounding the strA1
mutation. Transformation assays were performed with the eight
previously selected clones of the ComX.sup.+ reporter strain
(BLD101 [pGhP32comX.sub.MG]) and the control strain (BLD101
[pG.sup.+host9], empty vector) using the standard protocol reported
in Material and Methods. Validation of natural transformation is
made by sequencing the rpsL region covering the point mutations
from the donor DNA conferring streptomycin resistance using primers
RpsL Univ UP and RpsL Univ DN.
[0263] Remarkably, the ComX.sup.+ clones 02 and 04 that displayed
the highest P.sub.comGA activation (.gtoreq.7.times.10.sup.5 RLU
OD.sub.600.sup.-1) yielded mutation frequencies .about.15-fold
higher than the background level of spontaneous mutation that was
calculated in the absence of DNA (FIG. 4B). After subtraction of
the background, a transformation rate of up to 4.times.10.sup.-5
transformants per .mu.g of DNA (.about.10.sup.4 transformants
ml.sup.-1) was obtained for clone 02 which displays the highest
P.sub.comGA activation. In contrast, the negative control strain
had a spontaneous mutation rate of .about.1.times.10.sup.-7
transformants per .mu.g of DNA.
[0264] The rpsL ORF of 10 Str.sup.r-derivatives of cl02 was
amplified by PCR and sequenced. In all cases, we observed the
co-transfer of strA1 (mutation A.fwdarw.T at position 167 of the
rpsL gene) and the closely-located T.fwdarw.A mutation at position
156. In some cases, the T.fwdarw.G mutation at position 39 was also
co-transferred with strA1. The chimeric nature of rpsL in some
Str.sup.r ComX.sup.+ derivatives of KW2 (i.e. presence of both
mutations at positions 156 and 167 without the mutation at position
39) ultimately demonstrates that a recombination process occurred
between the exogenous and chromosomal DNA (FIG. 4A). In contrast,
this rearrangement was not observed in the rpsL gene of spontaneous
Str.sup.r mutants obtained in the negative control experiments
(i.e. assays performed in absence of exogenous DNA, or with the
control strain carrying the empty vector in presence of exogenous
DNA). These results show that exogenous DNA can enter KW2 cells and
be integrated in their chromosome by homologous recombination when
a certain threshold of comX expression is reached.
[0265] b) Construction of Deletion Mutants by Natural Competence in
L. lactis Subsp. Cremoris KW2 Overexpressing comX
[0266] The previous result (Example 4, section a) strongly suggests
that DNA transfer occurs in L. lactis KW2. The 3 mutations
transferred by natural transformation are grouped on a 128-bp
fragment. If a longer DNA fragment could be similarly integrated in
the L. lactis chromosome remains to be determined.
[0267] We wondered if overlap PCR as donor DNA could equivalently
allow gene insertions or gene deletions. The idea was to replace
the target gene by an antibiotic resistance cassette, i.e. the
chloramphenicol resistance cassette P.sub.32-cat. For this purpose,
a DNA fragment was constructed by overlap PCR containing the
P.sub.32-cat cassette flanked by two homologous arms (minimum
.about.1.5 kb) containing the upstream and downstream regions of
the targeted gene.
[0268] To this end, exogenous DNA polynucleotides containing
P.sub.32-cat surrounded by KW2-specific recombination arms
(.about.1.5 kb) were assembled in vitro by overlapping PCR to
target the comEC, mecA, ciaRH, covRS or clpC gene (see Materials
and Methods for details) and transferred by natural transformation
in the ComX.sup.+ strain (cl02). Validation of natural
transformation is made by sequencing the targeted region (comEC,
mecA, ciaRH, covRS or clpC, which should contain the
chloramphenicol resistance cassette P.sub.32-cat) using primers
listed in Table 3.
[0269] The transformation rate observed for overlap PCR products
was .about.1.2.times.10.sup.-6 to 1.1.times.10.sup.-4 transformants
per .mu.g of DNA for the different overlap DNA fragments that were
tested (see FIG. 5). Compared to the transformation rate observed
for the exchange of a homologous DNA fragment containing only three
point mutations (rpsL* donor DNA; 8.times.10.sup.-4 transformants
per .mu.g of DNA), these rates are relatively high for DNA double
recombination deletion/replacement.
Example 5: A KW2 .DELTA.comEC Mutant is Unable of Natural
Competence Transformation
[0270] To confirm that the observed horizontal DNA transfer in
ComX.sup.+ KW2 cells was indeed mediated by natural competence, and
not by phage transduction or conjugation, we investigated the role
of the ComEC protein, which is essential for the uptake of
transforming DNA through the cell membrane (the comFA gene,
together with the comFA, comGA, dprA, coiA, ssbA, radA, radC, recA
and recX genes are preceded by a Com-box and have been found to be
activated in KW2 following constitutive comX expression; data not
shown).
[0271] To create the .DELTA.comEC strain, clone 02 of the
ComX.sup.+ reporter strain, which was tested above, was grown in
CDM conditions in presence of PCR products encompassing the comEC
gene disrupted by the insertion of the chloramphenicol resistance
cassette P.sub.32-cat (see Materials and Methods). Four mutants
with disrupted comEC (BLD102 [pGhP32comX.sub.MG] cl01 to cl04) were
validated by PCR for P.sub.32-cat insertion in comEC.
Transformation assays with the mutated rpsL allele showed that the
frequencies of appearance for Str.sup.r clones in all tested
.DELTA.comEC derivatives were similar to the background level of
spontaneous rpsL mutation frequencies (<10.sup.-7) (FIG. 6).
Although heterogeneity in P.sub.comGA activation was observed
between clones as previously reported for the WT ComX.sup.+
reporter strain, half of the .DELTA.comEC derivative clones (i.e.
cl01 and cl03) displayed maximum specific Lux activity similar to
the transformable WT strains (>1.0.times.10.sup.6 RLU
OD.sub.600.sup.-1) (FIG. 4B). This shows that the transformation
defect in these .DELTA.comEC clones does not result from a too low
production of ComX.
[0272] Taken together, these results demonstrate that natural DNA
transformation could be activated by ComX overexpression in L.
lactis subsp. lactis KW2. Moreover, to the best of our knowledge,
these data provide the first ever experimental evidence of
transformation of L. lactis by natural competence.
Example 6: Natural Competence in Two Strains of the L.
Raffinolactis Species
[0273] Following the positive results obtained regarding natural
competence in Lactococcus lactis strains, other strains of the
Lactococcus genus were tested. Two strains of L. raffinolactis were
able to capture plasmid pGhost-Core (15 .mu.g/300 .mu.l) used as
donor DNA: LMG13098 and LMG14164. These results suggest that these
two strains of L. raffinolactis are naturally competent for plasmid
transformation and that, in these strains, natural competence is
independent of artificial comX-overexpression.
[0274] The fact that another Lactococcus species could be
transformed by competence opens additional possibilities for
industrial applications.
Example 7: Transformation by Natural Competence in 2 Lactococcus
lactis Subsp. Lactis Strains
[0275] Two Lactococcus lactis subsp. lactis strains, SL12651 and
SL12653, carrying all the essential late corn genes (FIG. 1) were
tested. As donor DNA, PCR fragments which encompass the mutated
rpsL allele (rpsL*) of a spontaneous streptomycin-resistant
(Str.sup.r) clone of L. lactis subsp. lactis IL1403 was used. Cells
were pre-cultured overnight in a complex medium supplemented with
glucose (e.g. M17G) at 30.degree. C. Cells were washed twice with
distilled water and inoculated at an OD.sub.600 of 0.05 in 200
.mu.l M17G containing 25 .mu.g mL.sup.-1 donor DNA rpsL*. After 24
hours of culture at 30.degree. C., cells incubated or not with
donor DNA were spread onto agar plates comprising the complex
medium supplemented with glucose (e.g. M17G) and appropriate
antibiotic (i.e. streptomycin). CFUs were counted after 48 hours of
incubation at 30.degree. C. Remarkably, SL12651 and SL12653 yielded
a transformation rate of up to 1.times.10.sup.-6 of DNA when grown
in M17G rich medium (FIG. 7A; +DNA). In contrast, the negative
control in absence of donor DNA had a spontaneous mutation rate of
6.times.10.sup.-9 (FIG. 7A; -DNA). The transformants were validated
by sequencing the rpsL region covering the point mutation from the
donor DNA conferring streptomycin resistance.
[0276] Then, the SL12653 strain was assayed in the same conditions
with variable quantity of donor DNA (0.5, 2.5, 5 and 25 .mu.g
mL.sup.-1). It has been shown that the transformation rate obtained
is directly correlated to the initial quantity of donor DNA,
yielding up to a transformation rate of 5.times.10.sup.-6 (FIG.
7B).
[0277] Moreover, to confirm that the observed horizontal DNA
transfer was mediated by natural competence, the comX gene of
SL12653 was knocked-out (as described in example 5 above). Three
mutants of SL12653 with disrupted comX gene were designed by
inserting PCR products encompassing the comX gene disrupted by the
insertion of the chloramphenicol resistance cassette P.sub.32-cat
and validated by PCR for P.sub.32-cat insertion. Transformation
assays with rpsL* as donor DNA in all .DELTA.comX clones
(ComX.sup.-) showed that the frequencies of appearance of Str.sup.r
clones were similar to the background level of spontaneous mutation
frequencies (FIG. 7C). These results confirm that in SL12653, the
transformation is dependent on the expression of the endogenous
comX gene.
[0278] Finally, the transformability of the SL12653 strain was also
assayed by overexpressing the comX gene. Thus, an inducible comX
expression plasmid [pGhPxylTcomX.sub.IO] was constructed by cloning
the comX gene from strain L. lactis subsp. lactis IO-1 under the
control of the P.sub.xyIT promoter from strain IO-1 on the
thermosensitive plasmid pG.sup.+host9. This plasmid is a variant of
pGhPxylTcomX.sub.MG (pGIFPT001) described in David et al., 2017.
The transformation procedure described in David et al (2017) was
followed. In presence of xylose (1%), SL12653 [pGhPxylTcomX.sub.IO]
yielded a transformation rate at least 20-fold higher than in
absence of xylose, confirming that the overexpression of comX in
SL12653 increased its transformability by natural competence.
Materials and Methods
[0279] Bacterial Strains, Plasmids, and Growth Conditions
[0280] The bacterial strains and plasmids used in this application
are listed in Table 2.
TABLE-US-00003 TABLE 2 list of used bacterial strains and plasmids
Strain or plasmid Characteristics .sup.a Source or reference E.
coli TG1 supE hsd.DELTA.5 thi .DELTA.(lac-proAB) Sambrook, J., E.
F. Fritsch, and T. Maniatis. F'[traD36 proAB.sup.+ lacl.sup.q
lacZ.DELTA.M15] 1989. Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y. Km.sup.r, recA.sup.+; MC1000 containing a Law, J., G.
Buist, A. Haandrikman, J. Kok, G. EC1000 copy of the repA gene from
pWV01 Venema, and K. Leenhouts. 1995. J. in its chromosome
Bacteriol. 177: 7011-7018. L. lactis MG1363 Laboratory strain,
dairy origin Gasson, M. J. 1983. J. Bacteriol. 154: 1-9. KW2
Wild-type isolate from corn Kelly, W. J., E. Altermann, S. C.
Lambie, and fermentation S. C. Leahy. 2013. Front Microbiol. 4:
257. IL1403 Laboratory strain, dairy origin Chopin, A., M. C.
Chopin, A. Moillo-Batt, and P. Langella. 1984. Plasmid 11: 260-263
IO-1 Wild-type isolate from water in the Ishizaki A, Osajima K,
Nakamura K, Katsunori drain pit of a kitchen sink K, Hara T, and
Ezaki T. 1990. J. Gen. Appl. Microbiol., 36, 1-6 SL12651 Wild-type
isolate from plant DuPont/Danisco collection SL12653 material
(maize) BLD101 KW2 kw2_0563::P.sub.comGA[MG]-luxAB This application
BLD102 BLD101 comEC::P.sub.32-cat This application BLD107 BLD101
mecA::P.sub.32-cat This application BLD108 BLD101
ciaRH::P.sub.32-cat This application BLD109 BLD101
covRS::P.sub.32-cat This application BLD105 BLD101
clpC::P.sub.32-cat This application L. raffinolactis LMG13098
Wild-type isolate from garden LMG collection carrots LMG14164
Wild-type isolate from goose LMG collection Plasmids pGEM .RTM.-T
easy Apr; cloning vector Promega pG.sup.+host9 Em.sup.r Ts Maguin,
E., H. Prevost, S. D. Ehrlich, and A. Gruss. 1996. J. Bacteriol.
178: 931-935 pGhost-Core Em.sup.r Ts; pG.sup.+host9 derivative This
application containing the Core part of the resolution site IRS
recognized by the Tnpl from Tn4430 pMG36eT Em.sup.r; E. coli-L.
lactis shuttle vector Fontaine, L. and P. Hols. 2008. Appl.
Environ. containing the P.sub.32 constitutive Microbiol. 74:
1102-1110. promoter from L. lactis pJIM4900 Em.sup.r Ts;
pG.sup.+host9 derivative E. Guedon, (laboratory collection)
containing the luxAB genes of Photorhabdus luminescens pXL
Em.sup.r; pTRKH2 derivative containing Blomqvist T, Steinmoen H,
Havarstein L S. the luc reporter gene Appl Environ Microbiol. 2006.
Oct; 72(10): 6751-6. pSEUDOPusp45GFP Em.sup.r; suicide vector
containing the Overkamp, W., K. Beilharz, W. R. Detert
llmg_pseudo_10(kw2_0563)::P.sub.usp45- Oude, A. Solopova, H.
Karsens, A. Kovacs, J. gfp.sup.+ insertion cassette Kok, O. P.
Kuipers, and J. W. Veening. 2013. Appl. Environ. Microbiol. 79:
6481-6490. pUC18Cm Ap.sup.r Cm.sup.r: pUC18 derivative Goffin, P.,
F. Lorquet, M. Kleerebezem, and containing the P32-cat cassette P.
Hols. 2004. J. Bacteriol. 186: 6661-6666. pUC18Ery Ap.sup.r
Em.sup.r; pUC18 derivative van Kranenburg, R., J. D. Marugg, I. I.
van containing an erythromycin Swam, N. J. Willem, and W. M. de
Vos. 1997. resistance marker Mol. Microbiol. 24: 387-397. pNZ5319
Em.sup.r Cm.sup.r: pACYC184 derivative Lambert, J. M., R. S.
Bongers, and M. containing the P32-cat cassette Kleerebezem. 2007.
Appl. Environ. Microbial. surrounded by lox sites 73: 1126-1135.
pGhPcomGAluxAB Em.sup.r Ts; pG.sup.+host9 derivative This
application containing the llmg_pseudo_10
(kw2_0563)::P.sub.comGA[MG]-luxAB insertion cassette
pGhP32comX.sub.MG Em.sup.r Ts, pG.sup.+host9 derivative This
application carrying comX of strain MG1363 under the control of the
constitutive promoter P.sub.32 pGhP32comX.sub.IO Em.sup.r Ts,
pG.sup.+host9 derivative This application carrying comX of strain
IO-1 under the control of the constitutive promoter P.sub.32
pGhP32comX.sub.MG- pGhP32comX.sub.MG derivative carrying This
application P.sub.comGA[MG]-luc a P.sub.comGA[MG]-luc fusion
pGhP32comX.sub.IO- pGhP32comX.sub.IO derivative carrying This
application P.sub.comGA[IO]-luc a P.sub.comGA[IO]-luc fusion
pGEMrpsL* Ap.sup.r, pGEM .RTM.-T easy derivative This application
carrying the rpsL* gene (strA1 allele) pUCcomECcat Ap.sup.r
Em.sup.r Cm.sup.r, pUC18Ery derivative This application allowing
the insertion of P.sub.32-cat at the comEC locus
pGhPxylTcomX.sub.IO Em.sup.r Ts, pG.sup.+host9 derivative This
application carrying comX of strain IO-1 under the control of the
inducible promoter P.sub.xylT from IO-1 .sup.a Em.sup.r, Ap.sup.r,
Cm.sup.r and Ts: erythromycin, ampicillin, chloramphenicol
resistance and thermo-sensitive RepA protein, respectively.
[0281] Escherichia coli was grown with shaking at 37.degree. C. in
Lysogeny-Broth (LB) broth. Plasmids derived from pMG36e and
pG.sup.+host9 were constructed in E. coli strains TG1 and EC1000,
respectively. L. lactis and L. raffinolactis were cultivated in M17
(Becton, Dickinson, and Company), Todd Hewitt broth (THB) (Becton,
Dickinson, and Company) or CDM at 30.degree. C. without agitation.
M17 and THB were supplemented with 0.5% (w/v) of glucose (M17G and
THBG, respectively). Solid agar plates were prepared by adding 2%
(w/v) agar to the medium. When required, 5 .mu.g ml.sup.-1 of
erythromycin, 1 mg ml.sup.-1 of streptomycin, and/or 10 .mu.g
ml.sup.-1 of chloramphenicol were added to the medium for L. lactis
and L. raffinolactis; and 250 .mu.g ml.sup.-1 of erythromycin, 250
.mu.g ml.sup.-1 of ampicillin, 10 .mu.g ml.sup.-1 of
chloramphenicol for E. coli.
[0282] Detection of Absorbance and Luminescence.
[0283] Growth (OD.sub.600) and luciferase (Lux) activity were
monitored at 10-minutes intervals in a Varioskan Flash multi-mode
reader (ThermoFisher). The luciferase activity is expressed in
relative light units (RLU) and the specific luciferase activity in
RLU OD.sub.600.sup.-1.
[0284] DNA Techniques and Electrotransformation
[0285] General molecular biology techniques were performed
according to the instructions given by Sambrook et al. (1989).
Electrotransformation of E. coli and L. lactis was performed as
previously described. The electrotransformed cells of L. lactis
were immediately resuspended in 1 ml of M17G and incubated for 6
hours at 30.degree. C. Chromosomal DNAs of L. lactis were prepared
as previously described. PCRs were performed with Phusion DNA
polymerase (NEB) in a GeneAmp PCR system 2400 (Applied Biosystems).
The primers used in this application are listed in Table 3.
TABLE-US-00004 TABLE 3 list of primers Primer name Sequence (5'-3')
Primers used for the construction of the constitutive comX
expression plasmid pGhP32comX.sub.MG/IO: BID_ComXSDLLCup
AAAAGAGCTCAATTATGAAAAAGAGG BID_ComXSDLLCdown
AAAACTGCAGTTAATCATCATCTCG BID_ComXSDLLLup
AAAAGAGCTCATAAAAGGAGAACTTTCC BID_ComXSDLLLdown
AAAACTGCAGTCACTCTTCGTCTTC BID_pMGP32UpMfeI
ATATCAATTGGTCCTCGGGATATGATAAG BID_pMGTerDown GACTTTGAACCTCAACTCC
Primers used for the construction of the P.sub.comGA[MG]-luxAB
reporter strain BLD101: BID_LuxLLCf1
ATAGTCTCGAGTTTAAGCAATTGAATCGCTAG BID_LuxLLCr1
GCAAAAAGTTTCCAAATTTCATACTAGAATATACGCAATTTG BID_LuxLLCf2
CAAATTGCGTATATTCTAGTATGAAATTTGGAAACTTTTTGC BID_LuxLLCr2
GCGAAAGGATCCCTATTAGGTATATTCCATGTGG BID_P3pseudoLLC
GCTCCCTCGAGGGCGGCTCTGTTGGATTAATATATGG Primers used for the
construction of portable luc reporter vectors: BID_LucLLCr1
CTTTATGTTTTTGGCGGATCTCATACTAGAATATACGCAATTTG BID_LucLLCf2
CAAATTGCGTATATTCTAGTATGAGATCCGCCAAAAACATAAAG BID_LucLLCr2
GCGAAAGGATCCTTACAATTTGGGCTTTCCG BID_PcomGALLCF1*
AAAACCCGGGTTTAAGCAATTGAATCGCTAG BID_PcomGALLLF1* 5'
AAAACCCGGGAAATAAATGGCTACAAAATT BID_lucR1*
AAAACGGCCGTTACAATTTGGGCTTTCCG BID_luxLLLf1
ATAGTCTCGAGAAATAAATGGCTACAAAATT BID_lucLLLr1
CTTTATGTTTTTGGCGGATCTCATACTAGACTATACGCAAATAATC BID_lucLLLf2
GATTATTTGCGTATAGTCTAGTATGAGATCCGCCAAAAACATAAAG BID_lucLLLr2
GCGAAAGGATCCTTACAATTTGGGCTTTCCG Primers used for the construction
of pGhost-Core DD-pGhost-CoreUp
AGCTTCCTAATACAACACAATTAATATTGTGTTGTATTATTG DD-pGhost-CoreDW
AATTCAATAATACAACACAATATTAATTGTGTTGTATTAGGA Primers used for rpsL
sequencing: RpsL Univ UP ATGCCTACAATTAACCAAT RpsL Univ DN
CACCGTATTTAGAACGG LR_RpsL Univ UP ATGCCTACTATTAACCAAT LR_RpsL Univ
DN TACCGTATTTAGAACGG Primers used for rpsL amplification:
BID_LLcdacARpsL AGTAGTATCAGCACTGACAGC BID_LLIcfusARpsL
ACACCTTTGTTCTTGAAGG primers used for the construction of the comEC
disruption mutant: BID_ComECLLCUp AAAGAGCTCAAAATAAAAATGAAATTATGG
BID_ComECLLCDown AAAGCTAGCGGGAAAAAATTGTGAATTAC BID_CatUpSpeI
AAAAACTAGTGCAGTTTAAATTCGGTCCTCGG BID_CatDownSpeI
AAAAACTAGTGTACAGTCGGCATTATCTCAT Primers used for the construction
and validation of the mecA deletion mutant: BID_fgt01FmecArec
CTTTAATGATGGAATGATTG BID_fgt01RVmecArec
CTATTAATCTTATCATATCCCGAGGATCCATATAACTATATGAAACC BID_fgt02Fcat
TCCTCGGGATATGATAAGATTAATAG BID_fgt02RVcat
TCTCATATTATAAAAGCCAGTCATTAG BID_fgt03FmecArec
CTAATGACTGGCTTTTATAATATGAGACTTAGAAAAATCTAAATATGGTTG
BID_fgt03RVmecArec GAAGATTTTTAATTTCAAGTGTAG BID_mecAKOF
TCAGTACCGAAAAACGAATG BID_mecAKORV ATTTACCAGTTCCGTTAGG Primers used
for the construction and validation of the ciaRH deletion mutant:
BID_ciaRHUPF TAACAATGATACAGAAGATG BID_ciaRHUPRVRec
CTATTAATCTTATCATATCCCGAGGATATTTTTGTCTTGTACTAGG BID_fgt02Fcat
TCCTCGGGATATGATAAGATTAATAG BID_fgt02RVcat
TCTCATATTATAAAAGCCAGTCATTAG BID_ciaRHDownFRec
CTAATGACTGGCTTTTATAATATGAGAGAGAGAAAAAAATTACTGAC BID_ciaRHDownRV
AAAATCTGTTAGAACTGTTG BID_ciaRHKODiagF AAGATAAGGCAGTTGAAATG
BID_ciaRHKODiagRy TCACCATGTGAATAAAGTCC Primers used for the
construction and validation of the covRSdeletion mutant:
BID_covRSfgt01F CAAAAATGTGAAGCTTATC BID_covRSfgt01RVRec
CTATTAATCTTATCATATCCCGAGGATGCATAATTCGATTTC BID_fgt02Fcat
TCCTCGGGATATGATAAGATTAATAG BID_fgt02RVcat
TCTCATATTATAAAAGCCAGTCATTAG BID_covRSfgt03FRec
TAATGACTGGCTTTTATAATATGAGACTATTTATCTGCTCATTTC BID_covRSfgt03RV
GAGCTTTTTTCAAATCTTC BID_covRSKOFdiag GAAGTGATGAATGAGATG
BID_covRSKORVdiag CTTTCTCATCAATTGAGAC Primers used for the
construction and validation of the clpC deletion mutant:
BID_clpCUPF CTTTGGGTTCTAATTTATC BID_clpCUPRVRec
CTATTAATCTTATCATATCCCGAGGACGTTGGTGTATATTTTAC BID_fgt02Fcat
TCCTCGGGATATGATAAGATTAATAG BID_fgt02RVcat
TCTCATATTATAAAAGCCAGTCATTAG BID_clpCDownFRec
CTAATGACTGGCTTTTATAATATGAGATAGAAATAAAGGAAAGGAC BID_clpCDownRV
TTGCTTTAAGGATAGTTTC BID_clpCFdiag AGAAGCCAATAATGACGATG
BID_clpCRVdiag AGAATTCTGATGATGCACAGTC Primers used for the
construction of the inducible comX expression plasmid
pGhPxylTcomX.sub.IO: FT_ AGCGCCGCGGTGGGATCCTCTAGAGTC
pGhPxylcomXIOsacllrv FT_pGhPxylcomX CTGCAGGCATGCACATCATCAACTTGAAGGG
FT_PxylTIOsacllfw CCCACCGCGGTGGAGATACGAACAAATTAG FT_PxylTIOrv
GATAGTAACTCCTTAATTTTTATTTGC FT_comXIOrecfw
GCAAATAAAAATTAAGGAGTTACTATCATGACATATTACTTGGAAGAAGAGGAT TTTG
FT_comXIOrecrv CCTTCAAGTTGATGATGTGCATGCCTGCAGTCACTCTTCGTCTTC
Primers used for the construction and validation of the
SL12653-comX deletion mutant FT_comXlocusfw
TGACCATGTTACACAAGCCTATATCCT FT_comXrecrv
CGCCCTTATGGGATTTATCTTCCTTACTTCGTTTCTTTGCATAACTTCGTCTTA AT Uplox66
TAAGGAAGATAAATCCCATAAGG Dnlox71 TTCACGTTACTAAAGGGAATGTA
FT_comXrecfw TCTACATTCCCTTTAGTAACGTGAACCATGACCATTTTATAGGTTTAGATGTTT
ATG AR_comxDNspecR CGGTGTTCCTCCATATATCTACGC FT_PxylcomXfw
CGCTAAACTCAACAGGTGATCCGATTG
[0286] Construction of Plasmid pGhP32comX.sub.MG
[0287] As a representative of the cremoris subspecies, the comX
gene from the laboratory strain MG1363 was initially chosen. ComX
proteins of this subspecies are highly conserved with at least 98%
of identity. The comX gene was amplified by PCR using primers
BID_ComXSDLLCup/BID_ComXSDLLCdown and inserted into plasmid pMG36eT
under the control of the constitutive P.sub.32 promoter by
SacI/PstI cloning, yielding plasmid pMGP32comX.sub.MG. The
P.sub.32-comX.sub.MG fusion from pMGP32comX.sub.MG was amplified by
PCR with primers BID_pMGP32UpMfeI/BID_pMGTerDown, digested by
MfeI/KpnI, and cloned in the EcoRI/KpnI-digested thermosensitive
pG.sup.+host9 vector. The resulting plasmid was named
pGhP32comX.sub.MG.
[0288] Construction of Plasmid pGhP32comX.sub.IO
[0289] As a representative of the lactis subspecies, the comX gene
from the IO-1 strain was chosen. The comX gene was amplified by PCR
using primers BID_ComXSDLLLup/BID_ComXSDLLLdown and inserted into
plasmid pMG36eT under the control of the constitutive P.sub.32
promoter by SacI/PstI cloning, yielding plasmid pMGP32comX.sub.IO.
The P.sub.32-comX.sub.IO fusion from pMGP32comX.sub.IO was
amplified by PCR with primers BID_pMGP32UpMfeI/BID_pMGTerDown,
digested by MfeI/KpnI, and cloned in the EcoRI/KpnI-digested
thermosensitive pG.sup.+host9 vector. The resulting plasmid was
named pGhP32comX.sub.IO.
[0290] Construction of Plasmid pGhost-Core
[0291] The Core part of the resolution site (IRS) recognized by the
TnpI recombinase from Tn4430 was assembled by using the
complementary primers DD-pGhost-CoreUp/DD-pGhost-CoreDW. The
resulting DNA fragment was cloned between HindIII and EcoRI sites
in plasmid pG.sup.+host9. The resulting plasmid, named pGhost-Core,
was transformed in E. coli harbouring plasmid pGIV004 (TnpI.sup.+)
for obtaining multimeric forms (Vanhooff V, Galloy C, Agaisse H,
Lereclus D, Revet B, Hallet B. Mol Microbiol. 2006 May;
60(3):617-29).
[0292] Construction of P.sub.comGA[MG]-luxAB Reporter Strain
BLD101
[0293] The P.sub.comGA[MG] promoter was amplified by PCR from
chromosomal DNA of L. lactis MG1363 (identical nucleotide sequence
between MG1363 and KW2) with primers BID_LuxLLCf1/BID_LuxLLCr1
(PCR1 product). The luxAB genes were amplified by PCR from plasmid
pJIM4900 with primers BID_LuxLLCf2/BID_LuxLLCr2 (PCR2 product). The
P.sub.comGA[MG]-luxAB fusion was created by overlapping PCR using
PCR1 and PCR2 products and primers BID_LuxLLCf1/BID_LuxLLCr2. The
resulting fusion was cloned in plasmid pSEUDOPusp45GFP using
restriction enzymes XhoI and BamHI, yielding plasmid
pSEUDOPusp45PcomGAluxAB. In order to remove the P.sub.usp45
promoter, the entire vector except the P.sub.usp45 promoter was
amplified by inverse PCR with primers BID_P3pseudoLLC/BID_LuxLLCf1
and self-ligated after XhoI digestion, leading to plasmid
pSEUDOPcomGAluxAB. The insertion cassette
llmg_pseudo_10::P.sub.comGA[MG]-luxAB was excised from plasmid
pSEUDOPcomGAluxAB and cloned into the pG.sup.+host9 thermosensitive
vector using restriction enzymes KpnI/EagI. The resulting plasmid
pGhPcomGAluxAB was then electro-transformed in strain KW2 and used
to integrate the P.sub.comGA[MG]-luxAB cassette at locus kw2_0563
(llmg_pseudo_10 in MG1363) by double homologous recombination,
resulting in the reporter strain KW2
kw2_0563::P.sub.comGA[MG]-luxAB (strain BLD101).
[0294] Construction of Portable Luc Reporter Systems
[0295] The P.sub.comGA[MG] promoter was amplified by PCR from
chromosomal DNA of L. lactis MG1363 with primers
BID_LuxLLCf1/BID_LucLLCr1 (PCR1 product). The luc gene was
amplified by PCR from plasmid pXL with primers
BID_LucLLCf2/BID_LucLLCr2 (PCR2 product). The P.sub.comGA[MG]-luc
fusion was created by overlapping PCR using PCR1 and PCR2 products
and primers BID_LuxLLCf1/BID_LucLLCr2. The resulting fusion was
cloned in plasmid pSEUDOPusp45GFP using restriction enzymes XhoI
and BamHI, yielding plasmid pSEUDOPusp45PcomGAluc. In order to
remove the P.sub.usp45 promoter, the entire vector except the
P.sub.usp45 promoter was amplified by inverse PCR with primers
BID_P3pseudoLLC/BID_LuxLLCf1 and self-ligated after XhoI digestion,
leading to plasmid pSEUDOPcomGAluc. The reporter cassette
P.sub.comGA[MG]-luc was amplified by PCR from pSEUDOPcomGAluc
(primers BID_PcomGALLCF1*/BID_IucR1*) and cloned between XmaI and
EagI into the pGhP32comX.sub.MG plasmid. The resulting reporter
plasmid was named pGhP32comX.sub.MG-P.sub.comGA[MG]-luc.
[0296] The P.sub.comGA[IO] promoter was amplified from the IO-1
chromosome (primers BID_IuxLLLf1/BID_IucLLLr1) and the luciferase
gene (luc) was amplified from plasmid pXL (primers
BID_IucLLLf2/BID_IucLLLr2). The cassette P.sub.comGA[IO]-luc was
created by overlapping PCR with primers BID_IuxLLLf1/BID_IucLLLr2.
The cassette P.sub.comGA[IO]-luc was then amplified from the
overlapping PCR product with primers BID_PcomGALLLF1*/BID_IucR1*
for XmaI/EagI cloning into pGhP32comX.sub.IO. The resulting
reporter plasmid was named
pGhP32comX.sub.IO-P.sub.comGA[IO]-luc.
[0297] Isolation of a rpsL Mutant Conferring Resistance to
Streptomycin
[0298] Spontaneous streptomycin-resistant MG1363 clones were
isolated on 1 mg ml.sup.-1 streptomycin-containing plates. After
the sequencing of the rpsL gene with primers RpsL Univ UP/RpsL Univ
DN, one spontaneous mutant resulting in a mutation (K56I) into the
ribosomal protein S12 that was previously shown to confer
resistance to streptomycin was selected (FIG. 6). A 3.7-kb fragment
containing the rpsL mutated gene (strA1 allele) was amplified by
PCR with primers BID_LLcdacARpsL/BID_LLIcfusARpsL and cloned into
the pGEM.RTM.-T easy vector (Promega), yielding plasmid pGEMrpsL*.
This plasmid was used as template to generate the 3.7-kb PCR
product with primers BID_LLcdacARpsL/BID_LLIcfusARpsL that was used
as donor DNA in natural transformation assays of strain KW2.
[0299] Standard Natural Transformation Assay
[0300] The BLD101 reporter strain carrying the pGhP32comX.sub.MG
plasmid (BLD101 [pGhP32comX.sub.MG]) was grown overnight in M17G at
30.degree. C. Then, 1.5 ml of the pre-culture was diluted in 8.5 ml
of fresh M17G medium to restart the culture. After 2 hours of
growth, cells were washed twice in distilled water and OD.sub.600
was adjusted to 0.05 in CDM containing erythromycin (5 .mu.g
ml.sup.-1) and supplemented with either 5% (v/v) glycerol or 5%
(w/v) mannitol used as potential osmo-stabilizers. Typically, 5
.mu.g of DNA was added in 300 .mu.l of inoculated medium and the
culture was further incubated during 6 hours at 30.degree. C. Cells
were then spread on M17G agar plates supplemented with appropriate
antibiotics and CFUs were counted after 48 hours of incubation. The
transformation frequency was calculated as the number of
antibiotic-resistant CFU ml.sup.-1 divided by the total number of
viable CFU ml.sup.-1. In the case of streptomycin-resistant
transformants, antibiotic-resistant CFU ml.sup.-1 corresponds to
the number of transformants obtained in presence of DNA less the
number of spontaneous transformants obtained in conditions where no
DNA is added in the culture. The transfer of the mutation
conferring streptomycin resistance was confirmed by DNA sequencing
of the rpsL gene after its amplification by PCR using primers RpsL
Univ UP/RpsL Univ DN.
[0301] Disruption of comEC by Natural Transformation
[0302] A comEC-containing DNA fragment of .about.3.2 kb was
amplified by PCR with primers BID_ComECLLCUp/BID_ComECLLCDown.
Then, the PCR product was digested by SacI/NheI and cloned into the
SacI/XbaI-digested suicide plasmid pUC18Ery (van Kranenburg et al.,
1997), yielding plasmid pUCcomEC. To generate a comEC disruption
cassette that allows the selection of double crossing-over
recombinants, the P.sub.32-cat fusion conferring resistance to
chloramphenicol was cloned in the middle of the comEC gene. For
this purpose, the P.sub.32-cat cassette was amplified by PCR from
plasmid pNZ5319 (Lambert et al., 2007, Appl. Environ. Microbiol.
73:1126-1135) with primers BID_CatUpSpeI/BID_CatDownSpeI. The
amplification product was digested by SpeI and cloned into the
XbaI-digested pUCcomEC, yielding plasmid pUCcomECcat. This suicide
plasmid was used to generate high quantity of donor DNA by PCR
amplification for comEC disruption by natural transformation. The
insertion of the P.sub.32-cat cassette in the comEC gene of KW2
transformants was validated by PCR (primers in Table 3).
[0303] Deletion of mecA, ciaRH, covRS, and clpC Genes by Natural
Transformation
[0304] The mecA, ciaRH, covRS, and clpC genes were similarly
inactivated by the exchange of their ORFs by the P.sub.32-cat
cassette using double crossing-over events. For this purpose,
overlapping PCR products containing the P.sub.32-cat cassette
flanked by two recombination arms of .about.1.5 kb (upstream and
downstream homologous regions) were generated as previously
reported. Briefly, upstream, downstream, and P.sub.32-cat fragments
were separately amplified by PCR, purified, mixed in equimolar
concentration, and assembled by overlapping PCR by using the most
external primers (see list of primers in Table 3). 5 .mu.g of the
obtained overlapping PCR product was used as donor DNA for natural
transformation of strain BLD101 [pGhP32comX.sub.MG]. The correct
insertion of the P.sub.32-cat cassette in each targeted locus of
the KW2 transformants was validated by PCR (see list of primers in
Table 3). To obtain the final mutant strains, the thermosensitive
vector pGhP32comX.sub.MG was cured by growing the strains overnight
at 37.degree. C. without erythromycin. The cultures were
subsequently diluted and plated on M17G agar without erythromycin
at 30.degree. C. The resulting colonies were streaked in parallel
on M17G plates with and without erythromycin. Absence of plasmid
pGhP32comX.sub.MG in Ery.sup.S clones was validated by PCR.
[0305] Induction of Natural Competence in Lactococcus
raffinolactis
[0306] Wild-type Lactococcus raffinolactis (i.e., L. raffinolactis
strains which have not been previously engineered for the
overproduction of the comX gene) were grown overnight in M17G at
30.degree. C. 1.5 ml of the pre-culture was diluted in 8.5 ml of
fresh M17G medium to restart the culture. After 2 hours of growth,
cells were washed twice in distilled water and OD.sub.600 was
adjusted to 0.05 in CDM supplemented with either 5% (v/v) glycerol
or 5% (w/v) mannitol used as potential osmo-stabilizers. 15 .mu.g
of plasmid pGhost-Core was added in 300 .mu.l of inoculated medium
and the culture was further incubated during 6 hours at 30.degree.
C. Cells were then spread on M17G agar plates supplemented with
appropriate antibiotics and CFUs were counted after 48 hours of
incubation. The transformation frequency was calculated as the
number of antibiotic-resistant CFU ml.sup.-1 divided by the total
number of viable CFU ml.sup.-1.
[0307] Natural Competence in Lactococcus lactis Subsp Lactis
SL12651 and SL12653 Strains
[0308] The L. lactis subsp. lactis SL12653 and 12651 strains were
grown overnight at 30.degree. C. Cells were washed twice in
distilled water and OD.sub.600 was adjusted to 0.05 in M17G.
Typically, 5 .mu.g of donor DNA was added in 200 .mu.l of
inoculated medium (25 .mu.g/ml) and the culture was further
incubated during 24 hours at 30.degree. C. Cells were then spread
on M17G agar plates supplemented with appropriate antibiotics and
CFUs were counted after 48 hours of incubation at 30.degree. C. The
transformation frequency calculated exactly as described above (see
Standard natural transformation assay).
[0309] The same experiments were done in SL12653 with various
concentrations of donor DNA (0.5, 2.5, 5 and 25 .mu.g/ml)
[0310] Construction of Plasmid pGhPxylTcomXIO
[0311] As a representative of the lactis subspecies, the comX gene
and the promoter of the xylT gene from the IO-1 strain were chosen.
The comX gene was amplified by PCR using primers FT_comXIOrecfw and
FT_comXIOrecry (PCR1), both containing overlapping sequences. The
xylT promoter region was amplified by PCR using primers
FT_PxylTIOsacllfw and FT_PxylTIOrv (PCR2). The carrying vector was
amplified from plasmid pGhP32comX.sub.MG and amplified by PCR using
primers FT_pGhPxylcomXIOsacllrv and FT_pGhPxylcomX (PCR3). The
three PCR products were purified, mixed in an equimolar
concentration and assembled by overlapping PCR using the most
external primers, containing a SacII restriction site. The
amplification product was digested by SacI I and self-ligated. The
resulting plasmid was named pGhPxylTcomX.sub.IO.
[0312] Transformation Assay in SL12653 Mutants Deleted for the comX
Gene
[0313] The comX gene of SL12653 was inactivated by exchange of
their ORF by the P.sub.32-cat cassette using double crossing-over
events. For this purpose, overlapping PCR products containing the
P.sub.32-cat cassette flanked by two recombination arms of
.about.1.5 kb (upstream and downstream homologous regions) were
generated as previously reported. Briefly, upstream, downstream,
and the P.sub.32-cat fragments were separately amplified by PCR,
purified and mixed in equimolar concentration, and assembled by
overlapping PCR by using the most external primers (see primers in
Table 3). 5 .mu.g of the obtained PCR product was used as donor DNA
for natural transformation of strain SL12653 [pGhPxylTcomX.sub.IO]
(ComX.sup.+). The correct insertion of the P.sub.32-cat cassette in
the targeted locus of SL12653 transformants was validated by PCR
(see primers in Table 3). To obtain the final mutant strains, the
thermosensitive vector pGhPxylTcomX.sub.IO was cured by growing the
strains overnight at 37.degree. C. without erythromycin.
[0314] The cultures were subsequently diluted and plated on M17G
agar without erythromycin at 30.degree. C. The resulting colonies
were streaked in parallel on M17G plates with and without
erythromycin. Absence of plasmid pGhPxylTcomX.sub.IO in Ery.sup.S
clones was validated by PCR. Thus, 3 .DELTA.comX clones of SL12653
were obtained.
[0315] Xylose-Induced Natural Transformation in SL12653.
[0316] The L. lactis subsp. lactis SL12653 [pGhPxylTcomX.sub.IO]
was grown overnight at 30.degree. C. Cells were washed twice in
distilled water and OD600 was adjusted to 0.05 in M17 supplemented
with 1% (w/v) xylose. Typically, 5 .mu.g of DNA was added in 200
.mu.l of inoculated medium and the culture was further incubated
during 24 hours at 30.degree. C. Cells were then spread on M17G
agar plates supplemented with appropriate antibiotics and CFUs were
counted after 48 hours of incubation at 30.degree. C. The
transformation frequency was calculated exactly as described above
(see Standard natural transformation assay).
[0317] All publications mentioned in the above specification are
herein incorporated by reference. Various modifications and
variations of the described present invention will be apparent to
those skilled in the art without departing from the scope and
spirit of the present invention. Although the present invention has
been described in connection with specific preferred embodiments,
it should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention
which are obvious to those skilled in molecular biology,
biochemistry, microbiology, bacteriology, or related fields are
intended to be within the scope of the following claims.
REFERENCES
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Sequence CWU 1
1
961492DNALactococcus lactis 1ataacatatt acttggaaga agaggatttt
gaaaatcttt tttcagaaat gaaacctata 60gttatgaaat taatgaaaca aattcgcatt
agaacatgga aaatagagga ttatcttcaa 120gaggggatga ttattttaca
tcttctatta gaagagcaga acgatggtca aaagctgcat 180acaaaattta
aggtaaagta tcatcaaaga ttaatagatg aattaagacg aagttatgca
240aagaaacgaa gccatgacca ttttataggt ttagatgttt atgaatgctc
agactggata 300aattcaggtg atactagtcc agataatgaa gtggtcttca
atcatttgct ggcagaagta 360tatgaaggtt tgagcgcaca ttatcaagac
ttactacttc gacaaatgcg aggagaagaa 420ctaactcgca tgcaacggta
tcgccttcgt gaaaaaataa aggccatctt attttcagaa 480gacgaagagt ga
4922163PRTLactococcus lactis 2Met Thr Tyr Tyr Leu Glu Glu Glu Asp
Phe Glu Asn Leu Phe Ser Glu1 5 10 15Met Lys Pro Ile Val Met Lys Leu
Met Lys Gln Ile Arg Ile Arg Thr 20 25 30Trp Lys Ile Glu Asp Tyr Leu
Gln Glu Gly Met Ile Ile Leu His Leu 35 40 45Leu Leu Glu Glu Gln Asn
Asp Gly Gln Lys Leu His Thr Lys Phe Lys 50 55 60Val Lys Tyr His Gln
Arg Leu Ile Asp Glu Leu Arg Arg Ser Tyr Ala65 70 75 80Lys Lys Arg
Ser His Asp His Phe Ile Gly Leu Asp Val Tyr Glu Cys 85 90 95Ser Asp
Trp Ile Asn Ser Gly Asp Thr Ser Pro Asp Asn Glu Val Val 100 105
110Phe Asn His Leu Leu Ala Glu Val Tyr Glu Gly Leu Ser Ala His Tyr
115 120 125Gln Asp Leu Leu Leu Arg Gln Met Arg Gly Glu Glu Leu Thr
Arg Met 130 135 140Gln Arg Tyr Arg Leu Arg Glu Lys Ile Lys Ala Ile
Leu Phe Ser Glu145 150 155 160Asp Glu Glu3492DNALactococcus lactis
3atgacatatt acctggaaga aaatgaattc gaaggtttat tttctggaat gaaaccaatc
60atcagaaaat tgatgaaaca aattcgaatc aaagcatggg acatagagga ttattatcaa
120gaaggaatga ttattttgca tcacctttta gaagaaaatc acccatccac
taatatttat 180acaaagttca aagtaaaata tcatcaacat ttgattgatg
aactacgcca tagctacgcc 240aaaaaacggc ttcatgacca ttttgtaggt
ctggacattt atgaatgttc ggactggata 300gatgcaggag gaagtacccc
tgaaagcgag cttgtgttca atcatctttt agcagaagtt 360tatgaaggat
tgagcgccca ctatcaggaa ttactcgtgc gtcaaatgag aggagaagaa
420ctcacgcgaa tggaacgcta tcggctaaga gaaaaaatca aaaatatact
attttctcga 480gatgatgatt aa 4924163PRTLactococcus lactis 4Met Thr
Tyr Tyr Leu Glu Glu Asn Glu Phe Glu Gly Leu Phe Ser Gly1 5 10 15Met
Lys Pro Ile Ile Arg Lys Leu Met Lys Gln Ile Arg Ile Lys Ala 20 25
30Trp Asp Ile Glu Asp Tyr Tyr Gln Glu Gly Met Ile Ile Leu His His
35 40 45Leu Leu Glu Glu Asn His Pro Ser Thr Asn Ile Tyr Thr Lys Phe
Lys 50 55 60Val Lys Tyr His Gln His Leu Ile Asp Glu Leu Arg His Ser
Tyr Ala65 70 75 80Lys Lys Arg Leu His Asp His Phe Val Gly Leu Asp
Ile Tyr Glu Cys 85 90 95Ser Asp Trp Ile Asp Ala Gly Gly Ser Thr Pro
Glu Ser Glu Leu Val 100 105 110Phe Asn His Leu Leu Ala Glu Val Tyr
Glu Gly Leu Ser Ala His Tyr 115 120 125Gln Glu Leu Leu Val Arg Gln
Met Arg Gly Glu Glu Leu Thr Arg Met 130 135 140Glu Arg Tyr Arg Leu
Arg Glu Lys Ile Lys Asn Ile Leu Phe Ser Arg145 150 155 160Asp Asp
Asp5492DNALactococcus lactis 5atggatgaca ttcaagaaaa atacggttta
gaattcaacg aattattctc tgagatgcgg 60ccgataattt ataaattgat gaagcaattg
cacatcaaca catgggatta cgatgattac 120ttccaagagg gaatgattac
actacatgaa ttgctgcaga aaattacaaa tttagatcat 180gtacatacga
aatttaaagt ggcttaccat cagcacttaa ttgacgaaat tcgccatatt
240aaagcacgaa aaagaggttt tgatcagctc catccgatca atgtttatga
ctgcgcagat 300tggattggct caaaccttgc tacacctgaa agcgagatag
ttttcaacca tctactagaa 360gaagtttatg ataaactttc aacacactat
aaagaactgt tggtaaagca aatgcatggg 420gaacatctta cgagaatgca
gaagtatcgt ttaaaggaaa aaattaaagc gattttattt 480gatgaagact aa
4926163PRTLactococcus lactis 6Met Asp Asp Ile Gln Glu Lys Tyr Gly
Leu Glu Phe Asn Glu Leu Phe1 5 10 15Ser Glu Met Arg Pro Ile Ile Tyr
Lys Leu Met Lys Gln Leu His Ile 20 25 30Asn Thr Trp Asp Tyr Asp Asp
Tyr Phe Gln Glu Gly Met Ile Thr Leu 35 40 45His Glu Leu Leu Gln Lys
Ile Thr Asn Leu Asp His Val His Thr Lys 50 55 60Phe Lys Val Ala Tyr
His Gln His Leu Ile Asp Glu Ile Arg His Ile65 70 75 80Lys Ala Arg
Lys Arg Gly Phe Asp Gln Leu His Pro Ile Asn Val Tyr 85 90 95Asp Cys
Ala Asp Trp Ile Gly Ser Asn Leu Ala Thr Pro Glu Ser Glu 100 105
110Ile Val Phe Asn His Leu Leu Glu Glu Val Tyr Asp Lys Leu Ser Thr
115 120 125His Tyr Lys Glu Leu Leu Val Lys Gln Met His Gly Glu His
Leu Thr 130 135 140Arg Met Gln Lys Tyr Arg Leu Lys Glu Lys Ile Lys
Ala Ile Leu Phe145 150 155 160Asp Glu Asp7405DNALactococcus
raffinolactis 7atggataaaa ttgaaaccat acttaaaagt attgaaccga
ttattatgaa ctgtcggaaa 60aaaactaaaa ttccttcctg ggaattagac gactatatgc
aggaagggat gattattgct 120ttagagatgt accatcaact cttattagat
ccaccagatg atgactttaa cttctatgtc 180tatttcaaag tcaggtattc
ttgtttctta attgatcact atcgcaaagc tatggcagtc 240aagagaaaat
tcgaccagct tgactattgt gaactttctg agtctgttaa tctttttgat
300cacaaacaaa atgtgtctga aaacgtcatg tataacttgt tgtgtcaaga
aatacacttg 360gttttatccc cggaggagct caagcttttt gaggcactta tttga
4058134PRTLactococcus raffinolactis 8Met Asp Lys Ile Glu Thr Ile
Leu Lys Ser Ile Glu Pro Ile Ile Met1 5 10 15Asn Cys Arg Lys Lys Thr
Lys Ile Pro Ser Trp Glu Leu Asp Asp Tyr 20 25 30Met Gln Glu Gly Met
Ile Ile Ala Leu Glu Met Tyr His Gln Leu Leu 35 40 45Leu Asp Pro Pro
Asp Asp Asp Phe Asn Phe Tyr Val Tyr Phe Lys Val 50 55 60Arg Tyr Ser
Cys Phe Leu Ile Asp His Tyr Arg Lys Ala Met Ala Val65 70 75 80Lys
Arg Lys Phe Asp Gln Leu Asp Tyr Cys Glu Leu Ser Glu Ser Val 85 90
95Asn Leu Phe Asp His Lys Gln Asn Val Ser Glu Asn Val Met Tyr Asn
100 105 110Leu Leu Cys Gln Glu Ile His Leu Val Leu Ser Pro Glu Glu
Leu Lys 115 120 125Leu Phe Glu Ala Leu Ile 1309480DNALactococcus
plantarum 9atggatagca tagaaatgat gcttcaaaat attgagccaa ttattatgaa
ttgtagtaaa 60acaactagga ttccatcttg ggagctagat gattacatgc aggaggggat
gattattgca 120ctggaaatgt atcaaaatag acataacatc aataacggta
acgcgtttaa tttctatgtc 180tattttaaag tcaggtattc ctgttacctg
atagatagtt ttagaaaggc taacgcatat 240aaaagaaaat ttgatcaacc
attatattgt gaaatatctg aagccttcaa cctttatgat 300caccaccaaa
atgttgcaga caatgtctgt tatcagctat tgcaagttga aattcttgag
360atattaacac cagatgaagc tgatttattt atgaccttga aaaatggtgg
gaaagtagag 420agaaataaaa agtatagatt aaagaaaaaa attattgatt
atcttaaaga catgttatga 48010159PRTLactococcus plantarum 10Met Asp
Ser Ile Glu Met Met Leu Gln Asn Ile Glu Pro Ile Ile Met1 5 10 15Asn
Cys Ser Lys Thr Thr Arg Ile Pro Ser Trp Glu Leu Asp Asp Tyr 20 25
30Met Gln Glu Gly Met Ile Ile Ala Leu Glu Met Tyr Gln Asn Arg His
35 40 45Asn Ile Asn Asn Gly Asn Ala Phe Asn Phe Tyr Val Tyr Phe Lys
Val 50 55 60Arg Tyr Ser Cys Tyr Leu Ile Asp Ser Phe Arg Lys Ala Asn
Ala Tyr65 70 75 80Lys Arg Lys Phe Asp Gln Pro Leu Tyr Cys Glu Ile
Ser Glu Ala Phe 85 90 95Asn Leu Tyr Asp His His Gln Asn Val Ala Asp
Asn Val Cys Tyr Gln 100 105 110Leu Leu Gln Val Glu Ile Leu Glu Ile
Leu Thr Pro Asp Glu Ala Asp 115 120 125Leu Phe Met Thr Leu Lys Asn
Gly Gly Lys Val Glu Arg Asn Lys Lys 130 135 140Tyr Arg Leu Lys Lys
Lys Ile Ile Asp Tyr Leu Lys Asp Met Leu145 150
15511480DNALactococcus piscium 11atggagactt tagaagccat gctcaaaaac
attgaaccta ttattatgaa ttgtcaaaag 60atggcaaaaa taccttcctg ggatattgac
gattatatgc aggaggggag gatcattgca 120ttagacttgt ataatcagct
agcagaaaga atggagacgg atgaggtgaa cttttacgtc 180tacttcaaag
tcagatatac ctgtttcttg attgatactt accgtaagac aaatgccttt
240aaaagaaaat ttgaccaacc gatttactta gatgtatccg aagcatttaa
tctgtatgat 300cataagcaga atgtcgctga taatgtcatg tatactttat
tgcatcagga gattctagac 360atcttaacgc ctgtagaaat tcaaacgcta
aacgcactaa aaaggggaga aaaggtcgac 420cgcaataaaa aatttaggat
taaaaagaag attatcaact atattaatca gattttctag 48012159PRTLactococcus
piscium 12Met Glu Thr Leu Glu Ala Met Leu Lys Asn Ile Glu Pro Ile
Ile Met1 5 10 15Asn Cys Gln Lys Met Ala Lys Ile Pro Ser Trp Asp Ile
Asp Asp Tyr 20 25 30Met Gln Glu Gly Arg Ile Ile Ala Leu Asp Leu Tyr
Asn Gln Leu Ala 35 40 45Glu Arg Met Glu Thr Asp Glu Val Asn Phe Tyr
Val Tyr Phe Lys Val 50 55 60Arg Tyr Thr Cys Phe Leu Ile Asp Thr Tyr
Arg Lys Thr Asn Ala Phe65 70 75 80Lys Arg Lys Phe Asp Gln Pro Ile
Tyr Leu Asp Val Ser Glu Ala Phe 85 90 95Asn Leu Tyr Asp His Lys Gln
Asn Val Ala Asp Asn Val Met Tyr Thr 100 105 110Leu Leu His Gln Glu
Ile Leu Asp Ile Leu Thr Pro Val Glu Ile Gln 115 120 125Thr Leu Asn
Ala Leu Lys Arg Gly Glu Lys Val Asp Arg Asn Lys Lys 130 135 140Phe
Arg Ile Lys Lys Lys Ile Ile Asn Tyr Ile Asn Gln Ile Phe145 150
15513486DNALactococcus garvieae 13atggagcata atttagatat ggagcagctg
gaagaaattt ttcattctgt ccaacatatt 60gtgtggaaga acagtcgttt gattccgata
aatttttgga cgtttgatga ctatcagcag 120gaagggcgct tggtattata
cgatttgctg ggagatggtg tgacgcaaag gaacttattt 180tgccatttta
aggtacgcta taagcagaga cttattgata ttaaaagaag ggagcgggct
240tttaaaaggg gttttgattg cgggactggc ttagatatat acgaatattc
tgatgctcta 300aaggggaaag cagccagtcc agaacatatc ctgatttctg
gaagtttact tgaagaagtt 360tttgaaaact taaatttacg ctaccgacgg
ctcctcaaaa gttacctcgc cggcgatgaa 420ttgcaccgta tggaaaagta
tcgtttgaag gaaaaaataa cgaatatatt atatgaacag 480cagtga
48614161PRTLactococcus garvieae 14Met Glu His Asn Leu Asp Met Glu
Gln Leu Glu Glu Ile Phe His Ser1 5 10 15Val Gln His Ile Val Trp Lys
Asn Ser Arg Leu Ile Pro Ile Asn Phe 20 25 30Trp Thr Phe Asp Asp Tyr
Gln Gln Glu Gly Arg Leu Val Leu Tyr Asp 35 40 45Leu Leu Gly Asp Gly
Val Thr Gln Arg Asn Leu Phe Cys His Phe Lys 50 55 60Val Arg Tyr Lys
Gln Arg Leu Ile Asp Ile Lys Arg Arg Glu Arg Ala65 70 75 80Phe Lys
Arg Gly Phe Asp Cys Gly Thr Gly Leu Asp Ile Tyr Glu Tyr 85 90 95Ser
Asp Ala Leu Lys Gly Lys Ala Ala Ser Pro Glu His Ile Leu Ile 100 105
110Ser Gly Ser Leu Leu Glu Glu Val Phe Glu Asn Leu Asn Leu Arg Tyr
115 120 125Arg Arg Leu Leu Lys Ser Tyr Leu Ala Gly Asp Glu Leu His
Arg Met 130 135 140Glu Lys Tyr Arg Leu Lys Glu Lys Ile Thr Asn Ile
Leu Tyr Glu Gln145 150 155 160Gln15489DNALactococcus garvieae
15atggcagaaa ataatttaga taaagaacag cttgaagagt tattccattc acttcaacat
60attgtttgga agaacagtca tttaattaaa ataaattttt ggacaatgga tgattatcag
120caagaagggc gactggtttt ataccagtta cttgaagatg gcgtgacaca
ggaaaaacta 180ttttgccatt ttaaagtgcg atataagcaa cggttgattg
atataaaaag acgagaaaga 240gcatttaagc ggggttttga ttgtggggct
ggtttagata tatatgagta ttctgatgcc 300ctgaaaggca aagctaccag
tcctgaatat aacttaattt cagttacttt acttgaagag 360gttcatcaaa
gtttgagttt gagataccgc aatttattgg agaatcatct gtcaggagtg
420gagttgcatc gaatggaaaa ataccgttta aaggaaaaaa tcaagagaat
actctatgaa 480gaagaatga 48916162PRTLactococcus garvieae 16Met Ala
Glu Asn Asn Leu Asp Lys Glu Gln Leu Glu Glu Leu Phe His1 5 10 15Ser
Leu Gln His Ile Val Trp Lys Asn Ser His Leu Ile Lys Ile Asn 20 25
30Phe Trp Thr Met Asp Asp Tyr Gln Gln Glu Gly Arg Leu Val Leu Tyr
35 40 45Gln Leu Leu Glu Asp Gly Val Thr Gln Glu Lys Leu Phe Cys His
Phe 50 55 60Lys Val Arg Tyr Lys Gln Arg Leu Ile Asp Ile Lys Arg Arg
Glu Arg65 70 75 80Ala Phe Lys Arg Gly Phe Asp Cys Gly Ala Gly Leu
Asp Ile Tyr Glu 85 90 95Tyr Ser Asp Ala Leu Lys Gly Lys Ala Thr Ser
Pro Glu Tyr Asn Leu 100 105 110Ile Ser Val Thr Leu Leu Glu Glu Val
His Gln Ser Leu Ser Leu Arg 115 120 125Tyr Arg Asn Leu Leu Glu Asn
His Leu Ser Gly Val Glu Leu His Arg 130 135 140Met Glu Lys Tyr Arg
Leu Lys Glu Lys Ile Lys Arg Ile Leu Tyr Glu145 150 155 160Glu
Glu17486DNALactococcus garvieae 17atggagcata atttagatat ggagcagctg
gaagagatat ttcattctgt tcaacatatt 60gtatggaaga atagtcgttt gattccgata
aatttttgga cgatagatga ctatcagcag 120gaagggcgtt tggtattata
tgatttactt gaggatggtg tgacacaaag aaaacttttt 180tgccatttta
aagtacgtta taagcagaga cttattgata ttaaaagaag ggagcgggct
240tttaaaaggg gttttgactg tgggactggg ctagatattt acgaatattc
agatgcttta 300aaaggaaaag tagccagtcc agaacatact ctgatttctg
gcagtttgct tgaagaagtt 360ttagaaaact taaatttacg ctaccgtgct
cttcttaaaa gttaccttgc tggtgatgaa 420ctgcatcgaa tggaaaaaca
tcgtttgaaa gaaaaaataa taaaaatatt atatgatgaa 480cagtga
48618161PRTLactococcus garvieae 18Met Glu His Asn Leu Asp Met Glu
Gln Leu Glu Glu Ile Phe His Ser1 5 10 15Val Gln His Ile Val Trp Lys
Asn Ser Arg Leu Ile Pro Ile Asn Phe 20 25 30Trp Thr Ile Asp Asp Tyr
Gln Gln Glu Gly Arg Leu Val Leu Tyr Asp 35 40 45Leu Leu Glu Asp Gly
Val Thr Gln Arg Lys Leu Phe Cys His Phe Lys 50 55 60Val Arg Tyr Lys
Gln Arg Leu Ile Asp Ile Lys Arg Arg Glu Arg Ala65 70 75 80Phe Lys
Arg Gly Phe Asp Cys Gly Thr Gly Leu Asp Ile Tyr Glu Tyr 85 90 95Ser
Asp Ala Leu Lys Gly Lys Val Ala Ser Pro Glu His Thr Leu Ile 100 105
110Ser Gly Ser Leu Leu Glu Glu Val Leu Glu Asn Leu Asn Leu Arg Tyr
115 120 125Arg Ala Leu Leu Lys Ser Tyr Leu Ala Gly Asp Glu Leu His
Arg Met 130 135 140Glu Lys His Arg Leu Lys Glu Lys Ile Ile Lys Ile
Leu Tyr Asp Glu145 150 155 160Gln19438DNALactococcus fujiensis
19ttgaaaccga tcgtttcaaa atctatgaga acattaaaaa tcaatttttg gactacagag
60gattatcatc aagagggtct aattacatta aatgaaatat taaattcagg atgtaaggag
120tcacaactat acattcactt taaagtcaaa tatcgacaaa agctaataga
cgtgattaga 180aaatcacagg cgcaaaaaag aatctgggat aatgcagaga
gtattgatgt ttacgaatct 240gaaaatcaaa ttaattccag taactcaaac
cccgaagaca taatagtcta tgacagtctt 300gtaaaggaag taataacaaa
attaacacct tcataccgga aactactgaa acgacatcta 360agaggtgagg
atgtgacaag gatggaaaaa tacagactga aggaacgaat caaacaaatt
420ttatttgatg gtgattga 43820145PRTLactococcus fujiensis 20Met Lys
Pro Ile Val Ser Lys Ser Met Arg Thr Leu Lys Ile Asn Phe1 5 10 15Trp
Thr Thr Glu Asp Tyr His Gln Glu Gly Leu Ile Thr Leu Asn Glu 20 25
30Ile Leu Asn Ser Gly Cys Lys Glu Ser Gln Leu Tyr Ile His Phe Lys
35 40 45Val Lys Tyr Arg Gln Lys Leu Ile Asp Val Ile Arg Lys Ser Gln
Ala 50 55 60Gln Lys Arg Ile Trp Asp Asn Ala Glu Ser Ile Asp Val Tyr
Glu Ser65 70 75 80Glu Asn Gln Ile Asn Ser Ser Asn Ser Asn Pro Glu
Asp Ile Ile Val 85 90 95Tyr Asp Ser Leu Val Lys Glu Val Ile Thr Lys
Leu Thr Pro Ser Tyr 100 105 110Arg Lys Leu Leu Lys Arg His Leu Arg
Gly Glu Asp Val Thr Arg Met 115 120 125Glu Lys Tyr Arg Leu Lys Glu
Arg Ile Lys Gln Ile Leu Phe Asp Gly 130 135
140Asp14521480DNALactococcus chungangensis 21atggataaga ttgaaaccat
acttaaaaat attgaaccga ttatcatgaa ctgtcgaaaa 60aaaactaaca tcccttcctg
gcaattagac gactatctcc aggaaggcat gattattgct 120ctagagatgt
atcatcaact tttattagac ccaccagatg atgactttaa cttctatgtt
180tatttcaaag tgagatattc ttgtttcttg attgatcagt atcggagaaa
catggctgtc 240aaaagaaaat tcgaccagat tgactattgt gaactatctg
aggcgtttta tctttttgat 300caaaatcaag atgtctctga aaacgtcatg
tataatttgt tatgtcaaga aatacacttg 360cttctatctc ctgaagaacg
agagcttttt gaggcactta aaaatggaca gaagattgac 420cgtaatcaaa
agtttcgtat caagaagaaa attattgaat atattaagag gttttggtga
48022159PRTLactococcus chungangensis 22Met Asp Lys Ile Glu Thr Ile
Leu Lys Asn Ile Glu Pro Ile Ile Met1 5 10 15Asn Cys Arg Lys Lys Thr
Asn Ile Pro Ser Trp Gln Leu Asp Asp Tyr 20 25 30Leu Gln Glu Gly Met
Ile Ile Ala Leu Glu Met Tyr His Gln Leu Leu 35 40 45Leu Asp Pro Pro
Asp Asp Asp Phe Asn Phe Tyr Val Tyr Phe Lys Val 50 55 60Arg Tyr Ser
Cys Phe Leu Ile Asp Gln Tyr Arg Arg Asn Met Ala Val65 70 75 80Lys
Arg Lys Phe Asp Gln Ile Asp Tyr Cys Glu Leu Ser Glu Ala Phe 85 90
95Tyr Leu Phe Asp Gln Asn Gln Asp Val Ser Glu Asn Val Met Tyr Asn
100 105 110Leu Leu Cys Gln Glu Ile His Leu Leu Leu Ser Pro Glu Glu
Arg Glu 115 120 125Leu Phe Glu Ala Leu Lys Asn Gly Gln Lys Ile Asp
Arg Asn Gln Lys 130 135 140Phe Arg Ile Lys Lys Lys Ile Ile Glu Tyr
Ile Lys Arg Phe Trp145 150 15523414DNALactococcus lactis
23atgcctacaa ttaaccaatt ggtacgcaaa cctcgtcgtg ctcaagtgac taaatctaaa
60tcaccagcaa tgaacgttgg ctacaacagc cgtaaaaaag tacaaactaa acttgcaagc
120ccacaaaaac gtggagtagc aactcgtgtt ggtacaatga ctcctaaaaa
acctaactca 180gcgcttcgta aattcgcgcg tgtacgtctt tcaaacctta
tggaagtaac agcgtacatc 240ccaggtatcg gacacaacct ccaagaacac
agtgttgtac ttcttcgtgg tggacgtgta 300aaagaccttc caggggtacg
ttaccatatc gttcgtggtg cacttgatac agcaggtgtc 360gctgaccgta
aacaaagccg ttctaaatac ggtgctaaaa aaccaaaagc ttaa
41424414DNALactococcus lactis 24atgcctacaa ttaaccaatt ggtacgcaaa
cctcgtcggg ctcaagtgac taaatctaaa 60tcaccagcaa tgaacgttgg ctacaacagc
cgtaaaaaag tacaaactaa acttgcaagc 120ccacaaaaac gtggagtagc
aactcgtgtt ggtacaatga ctcctataaa acctaactca 180gcgcttcgta
aattcgcgcg tgtacgtctt tcaaacctta tggaagtaac agcgtacatc
240ccaggtatcg gacacaacct ccaagaacac agtgttgtac ttcttcgtgg
tggacgtgta 300aaagaccttc caggggtacg ttaccatatc gttcgtggtg
cacttgatac agcaggtgtc 360gctgaccgta aacaaagccg ttctaaatac
ggtgctaaaa aaccaaaagc ttaa 41425414DNALactococcus lactis
25atgcctacaa ttaaccaatt ggtacgcaaa cctcgtcgtg ctcaagtgac taaatctaaa
60tcaccagcaa tgaacgttgg ctacaacagc cgtaaaaaag tacaaactaa acttgcaagc
120ccacaaaaac gtggagtagc aactcgtgtt ggtactatga ctcctaaaaa
acctaactca 180gcgcttcgta aattcgcgcg tgtacgtctt tcaaacctta
tggaagtaac agcgtacatc 240ccaggtatcg gacacaacct ccaagaacac
agtgttgtac ttcttcgtgg tggacgtgta 300aaagaccttc caggggtacg
ttaccatatc gttcgtggtg cacttgatac agcaggtgtc 360gctgaccgta
aacaaagccg ttctaaatac ggtgctaaaa aaccaaaagc ttaa
4142626DNAArtificial SequenceOligonucleotide primer 26aaaagagctc
aattatgaaa aagagg 262725DNAArtificial SequenceOligonucleotide
primer 27aaaactgcag ttaatcatca tctcg 252828DNAArtificial
SequenceOligonucleotide primer 28aaaagagctc ataaaaggag aactttcc
282925DNAArtificial SequenceOligonucleotide primer 29aaaactgcag
tcactcttcg tcttc 253029DNAArtificial SequenceOligonucleotide primer
30atatcaattg gtcctcggga tatgataag 293119DNAArtificial
SequenceOligonucleotide primer 31gactttgaac ctcaactcc
193232DNAArtificial SequenceOligonucleotide primer 32atagtctcga
gtttaagcaa ttgaatcgct ag 323342DNAArtificial
SequenceOligonucleotide primer 33gcaaaaagtt tccaaatttc atactagaat
atacgcaatt tg 423442DNAArtificial SequenceOligonucleotide primer
34caaattgcgt atattctagt atgaaatttg gaaacttttt gc
423534DNAArtificial SequenceOligonucleotide primer 35gcgaaaggat
ccctattagg tatattccat gtgg 343637DNAArtificial
SequenceOligonucleotide primer 36gctccctcga gggcggctct gttggattaa
tatatgg 373744DNAArtificial SequenceOligonucleotide primer
37ctttatgttt ttggcggatc tcatactaga atatacgcaa tttg
443844DNAArtificial SequenceOligonucleotide primer 38caaattgcgt
atattctagt atgagatccg ccaaaaacat aaag 443931DNAArtificial
SequenceOligonucleotide primer 39gcgaaaggat ccttacaatt tgggctttcc g
314031DNAArtificial SequenceOligonucleotide primer 40aaaacccggg
tttaagcaat tgaatcgcta g 314130DNAArtificial SequenceOligonucleotide
primer 41aaaacccggg aaataaatgg ctacaaaatt 304229DNAArtificial
SequenceOligonucleotide primer 42aaaacggccg ttacaatttg ggctttccg
294331DNAArtificial SequenceOligonucleotide primer 43atagtctcga
gaaataaatg gctacaaaat t 314446DNAArtificial SequenceOligonucleotide
primer 44ctttatgttt ttggcggatc tcatactaga ctatacgcaa ataatc
464546DNAArtificial SequenceOligonucleotide primer 45gattatttgc
gtatagtcta gtatgagatc cgccaaaaac ataaag 464642DNAArtificial
SequenceOligonucleotide primer 46agcttcctaa tacaacacaa ttaatattgt
gttgtattat tg 424742DNAArtificial SequenceOligonucleotide primer
47aattcaataa tacaacacaa tattaattgt gttgtattag ga
424819DNAArtificial SequenceOligonucleotide primer 48atgcctacaa
ttaaccaat 194917DNAArtificial SequenceOligonucleotide primer
49caccgtattt agaacgg 175019DNAArtificial SequenceOligonucleotide
primer 50atgcctacta ttaaccaat 195117DNAArtificial
SequenceOligonucleotide primer 51taccgtattt agaacgg
175221DNAArtificial SequenceOligonucleotide primer 52agtagtatca
gcactgacag c 215319DNAArtificial SequenceOligonucleotide primer
53acacctttgt tcttgaagg 195430DNAArtificial SequenceOligonucleotide
primer 54aaagagctca aaataaaaat gaaattatgg 305529DNAArtificial
SequenceOligonucleotide primer 55aaagctagcg ggaaaaaatt gtgaattac
295632DNAArtificial SequenceOligonucleotide primer 56aaaaactagt
gcagtttaaa ttcggtcctc gg 325731DNAArtificial
SequenceOligonucleotide primer 57aaaaactagt gtacagtcgg cattatctca t
315820DNAArtificial SequenceOligonucleotide primer 58ctttaatgat
ggaatgattg 205947DNAArtificial SequenceOligonucleotide primer
59ctattaatct tatcatatcc cgaggatcca tataactata tgaaacc
476026DNAArtificial SequenceOligonucleotide primer 60tcctcgggat
atgataagat taatag 266127DNAArtificial SequenceOligonucleotide
primer 61tctcatatta taaaagccag tcattag 276251DNAArtificial
SequenceOligonucleotide primer 62ctaatgactg gcttttataa tatgagactt
agaaaaatct aaatatggtt g 516324DNAArtificial SequenceOligonucleotide
primer 63gaagattttt aatttcaagt gtag 246420DNAArtificial
SequenceOligonucleotide primer 64tcagtaccga aaaacgaatg
206519DNAArtificial SequenceOligonucleotide primer 65atttaccagt
tccgttagg 196620DNAArtificial SequenceOligonucleotide primer
66taacaatgat acagaagatg 206746DNAArtificial SequenceOligonucleotide
primer 67ctattaatct tatcatatcc cgaggatatt tttgtcttgt actagg
466847DNAArtificial SequenceOligonucleotide primer 68ctaatgactg
gcttttataa tatgagagag agaaaaaaat tactgac 476920DNAArtificial
SequenceOligonucleotide primer 69aaaatctgtt agaactgttg
207020DNAArtificial SequenceOligonucleotide primer 70aagataaggc
agttgaaatg 207120DNAArtificial SequenceOligonucleotide primer
71tcaccatgtg aataaagtcc 207219DNAArtificial SequenceOligonucleotide
primer 72caaaaatgtg aagcttatc 197342DNAArtificial
SequenceOligonucleotide primer 73ctattaatct tatcatatcc cgaggatgca
taattcgatt tc 427445DNAArtificial SequenceOligonucleotide primer
74taatgactgg cttttataat atgagactat ttatctgctc atttc
457519DNAArtificial SequenceOligonucleotide primer 75gagctttttt
caaatcttc 197618DNAArtificial SequenceOligonucleotide primer
76gaagtgatga atgagatg 187719DNAArtificial SequenceOligonucleotide
primer 77ctttctcatc aattgagac 197819DNAArtificial
SequenceOligonucleotide primer 78ctttgggttc taatttatc
197944DNAArtificial SequenceOligonucleotide primer 79ctattaatct
tatcatatcc cgaggacgtt ggtgtatatt ttac 448046DNAArtificial
SequenceOligonucleotide primer 80ctaatgactg gcttttataa tatgagatag
aaataaagga aaggac 468119DNAArtificial SequenceOligonucleotide
primer 81ttgctttaag gatagtttc 198220DNAArtificial
SequenceOligonucleotide primer 82agaagccaat aatgacgatg
208322DNAArtificial SequenceOligonucleotide primer 83agaattctga
tgatgcacag tc 228427DNAArtificial SequenceOligonucleotide primer
84agcgccgcgg tgggatcctc tagagtc 278531DNAArtificial
SequenceOligonucleotide primer 85ctgcaggcat gcacatcatc aacttgaagg g
318630DNAArtificial SequenceOligonucleotide primer 86cccaccgcgg
tggagatacg aacaaattag 308727DNAArtificial SequenceOligonucleotide
primer 87gatagtaact ccttaatttt tatttgc 278858DNAArtificial
SequenceOligonucleotide primer 88gcaaataaaa attaaggagt tactatcatg
acatattact tggaagaaga ggattttg 588945DNAArtificial
SequenceOligonucleotide primer 89ccttcaagtt gatgatgtgc atgcctgcag
tcactcttcg tcttc 459027DNAArtificial SequenceOligonucleotide primer
90tgaccatgtt acacaagcct atatcct 279156DNAArtificial
SequenceOligonucleotide primer 91cgcccttatg ggatttatct tccttacttc
gtttctttgc ataacttcgt cttaat 569223DNAArtificial
SequenceOligonucleotide primer 92taaggaagat aaatcccata agg
239323DNAArtificial SequenceOligonucleotide primer 93ttcacgttac
taaagggaat gta 239457DNAArtificial SequenceOligonucleotide primer
94tctacattcc ctttagtaac gtgaaccatg accattttat aggtttagat gtttatg
579524DNAArtificial SequenceOligonucleotide primer 95cggtgttcct
ccatatatct acgc 249627DNAArtificial SequenceOligonucleotide primer
96cgctaaactc aacaggtgat ccgattg 27
* * * * *
References