Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Fusion of cells
Reexamination Certificate
2000-08-25
2002-09-10
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Fusion of cells
C435S243000, C435S252300, C435S320100, C435S471000, C536S024100
Reexamination Certificate
active
06448082
ABSTRACT:
This application is a 371 of PCT/FR99/03297, filed Dec. 12, 1999.
The present invention relates to novel DNA sequences capable of being transferred by conjugation, to plasmids containing these sequences, to bacteria containing these DNA sequences or these plasmids, and to the use of these bacteria.
Lactic acid bacteria are involved in the production and preservation of a large number of food products, such as cheese, butter, yogurt, sausage or sauerkraut, among which dairy products are of particular importance. The transformation of milk by lactic acid bacteria is being carried out in ever-larger vats. An understanding of the mechanisms of transfer of genetic material is essential for improving the strains of lactic acid bacteria used in these fermentations. The different mechanisms of transfer of genetic material are transformation, transduction, protoplast fusion and conjugation. Transformation consists in causing genetic material to enter a bacterium by natural competence, conversion of the cells to protoplasts or electropermeation of the cells.
Transduction consists in causing genetic material to enter a bacterium by means of a bacteriophage vector.
Protoplast fusion consists in converting two types of cells to protoplasts so that, after contact, genetic material passes from one strain into the other strain.
Conjugation consists in bringing two types of cells into contact so that genetic material passes from one strain into the other strain by virtue of natural conjugative genes.
Two distinct cases are possible: either the plasmid possesses all the genes involved in conjugation and said plasmid itself passes into the receptor strain, or the plasmid possesses only the genetic components sufficient for its transfer, the other components being present e.g. on another plasmid (Mobilization of the relaxable
Staphylococcus aureus
plasmid pC221 by the conjugative plasmid pG01 involves three pC221 loci, S. J. Projan and G. L. Archer, J. Bacteriol. 1989; 171: 1841-1845). The latter situation has an advantage: if the plasmid is transferred to a receptor strain without the conjugative plasmid, it will be unable to be transferred again, its dissemination thereby being prevented. This technique of conjugative plasmid transfer also has the advantage of introducing genetic material into strains in which this is difficult using the transfer techniques described above.
A few studies have already made it possible to demonstrate conjugative systems in lactic acid bacteria:
Genetic analysis of regions of the
Lactococcus lactis
subsp. lactis plasmid pRS01 involved in conjugative transfer, D. A. Mills, C. K. Choi, G. M. Dunny and L. L. McKay, Applied and Environ. Microbiol. 1994; 60 (12): 4413-4420;
Splicing of a group II intron involved in the conjugative transfer of pRS01 in lactococci, D. A. Mills, L. L. McKay and G. M. Dunny, J. Bacteriol. 1996; 178 (12): 3531-3538.
In the present patent application, a DNA sequence is described which comprises at least one conjugative transfer mechanism; this DNA sequence comprises a functional part of 5333 bp present in the strain
Lactococcus lactis
FL877 deposited on Sep. 30, 1998 in the CNCM (Collection Nationale de Cultures de Microorganismes) under no. I-2082.
This DNA sequence of 5333 bp (SEQ ID No: 1) was isolated from the plasmids contained in the strain FL877.
More precisely, the fragment of 5333 base pairs (bp) was isolated by total digestion, with the restriction enzyme EcoRI, of the plasmids contained in the strain
Lactococcus lactis
FL877. This fragment carries one or more conjugative transfer mechanisms and is capable of being transferred to another strain, especially another strain of
L. lactis, for example from the strain MG
1363 (GASSON M. J., J. Bacteriol. 1983; 154: 1-9). This fragment also carries a system of functional replication in
L. lactis.
From the nucleotide sequence SEQ ID No: 1, the Applicant then isolated a DNA sequence of 2590 bp, which alone confers on a plasmid the property of being transferred to another strain by conjugation, especially from the strain MG1363.
This sequence of 2590 bp (SEQ ID No: 2) can be obtained by the PCR (polymerase chain reaction) method with the aid of appropriate oligonucleotides.
Thus, according to a first feature, the present invention relates to a nucleic acid sequence capable of being transferred by conjugation, which comprises the sequence SEQ ID No: 2, its complementary strand or any sequence derived from said sequence or from its complementary strand by virtue of the degeneracy of the genetic code.
The invention preferably relates to a nucleic acid sequence capable of being transferred by conjugation, said sequence being selected from:
a) the nucleotide sequence of 5333 bp (SEQ ID No: 1) or its complementary strand;
b) any sequence hybridizing with the sequence a) under strict conditions; or
c) sequences derived from the sequences a) and b) by virtue of the degeneracy of the genetic code.
The invention relates more particularly to a nucleic acid sequence selected from:
a) the nucleotide sequence of 2590 bp (SEQ ID No: 2) or its complementary strand;
b) any sequence hybridizing with the sequence a) under strict conditions; or
c) sequences derived from the sequences a) and b) by virtue of the degeneracy of the genetic code.
According to the present invention, “hybridizing under strict conditions” is understood as meaning hybridization under the following stringency conditions: 42° C. in a 20 mM sodium phosphate buffer (pH 6.5) containing 50% of formamide, 5×SSC, 1×Denhardt's, 0.1% of SDS and 100 &mgr;g/ml of RNA, and then washing at 60° C. in a buffer containing 0.1×SSC and 0.1% of SDS.
The invention further relates to DNA sequences which have a high degree of homology with the above DNA sequences. A high degree of homology means a homology (ratio of the identical nucleotides to the total number of nucleotides) of at least 70%, preferably of at least 80% and particularly preferably of at least 90% of the nucleotide sequences when they are aligned according to the maximum homology by the optimum sequence alignment method of Needleman and Wunsch, J. Mol. Biol. 1970; 48: 443-453. This method is used especially in the UWGCG software of the University of Wisconsin: Devereux et al., Nucl. Ac. Res. 1984; 12: 8711-8721-option GAP.
The invention further relates to plasmids transformed with one of the DNA sequences according to the invention. These plasmids can be e.g. plasmid pLDP1 (PREVOTS F. et al., FEMS Microbiol. Lett. 1996; 142: 295-299) and plasmid pLAB510 derived from pPF107-3. (PREVOTS F. et al., FEMS Microbiol. Lett. 1998; 159: 331-336), into which one of the DNA sequences according to the invention has been cloned by the conventional techniques well known to those skilled in the art.
The invention further relates to bacteria, especially lactic acid bacteria, preferably belonging to the species Lactococcus lactis, which contain at least one DNA sequence or one plasmid as defined above.
These bacteria can be used for the conjugative transfer of genetic material, especially genetic material of industrial interest, to a strain of industrial interest. The conjugative transfer mechanism can be carried by a plasmid or by another part of the bacterial genome.
In particular, these bacteria can be used for the conjugative transfer of properties such as phage resistance, the ability to ferment lactose, proteolysis, peptidolysis and bacteriocin production, and of genes coding for proteins of pharmaceutical interest, to strains of industrial interest, particularly in the dairy industry, but also in the pharmaceutical industry.
The strains of industrial interest which can advantageously receive genetic material with the aid of the DNA sequences according to the invention, or a plasmid containing them, are e.g. the strains
L. lactis
ssp lactis and
L. lactis
ssp
cremoris.
The invention therefore also relates to these strains of industrial interest into which said genetic material has been integrated.
The invention will be understood more clearly with the aid of the Ex
Daloyau Marlene
Prevots Fabien
Antonelli Terry Stout & Kraus LLP
McKelvey Terry
SKW Biosystems
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