Induction of competence in streptococcus thermophilus
Résumé
Streptococcus thermophilus is dairy bacterium specie, phylogenetically close to pathogenic streptococci. Previous studies of genomic sequences from
S. thermophilus strains showed extreme adaptation to milk environment. This organism seems to have evolved through loss-of-function: pseudogenes occupy 10% of its genome and a proportion of its sequences were acquired from other lactic acid bacteria. In particular, the genome of S. thermophilus has lost its pathogenicity but carries genes for methionine biosynthesis, a rare amino acid in milk. This metabolic pathway likely originated
from Lactococcus bulgaricus. S. thermophilus is not supposedly able to incorporate foreign DNA into its genome. However, this food-processing bacterium seems to possess functional late competence genes, as pathogenic, competent streptococci do. A recent work shows that S. thermophilus is a major host for antibiotic resistance genes among cheese microbia. In order to evaluate the potential for antibiotic resistance gene transfer from
S. thermophilus to commensal streptococci, it appears important to study how this bacterium acquired such genes. We investigated the presence and
functionality of competence genes in S. thermophilus genome. Competent streptococci homology sequence studies allowed us to identify a series of genes homologous to late competence genes from S. pneumoniae, including all essential ones. These genes are responsible for intake DNA system and genome integration. Their expressions are induced by the alternative sigma factor ComX. This protein binds to a specific sequence, the com-box, in promoter regions of late competence genes. The com-box sequence from S. pneumoniae was degenerated in order to establish a S. thermophilus
com-box consensus sequence. It was then used to scan the whole genome. It was found not only upstream known late competence genes, but also up-stream novel ones, that could specifically belong to S. thermophilus ComX regulon. We are proposing to control ComX expression in S. thermophilus and determine which part of its genome belongs to the ComX regulon. In this organism, cloning tools are lacking in the perspective of genetic studies. We elaborated efficient expression systems for S. thermophilus protein studies. One was induced by an unmetabolized carbon source and the other is an adaptation of the Lactococcus lactis Pzn system to S. thermophilus. As this organism had a constitutive lactose operon expression, a LMG18311 strain lacking endogenous lacZ was generated, allowing the use of B-galactosidase assays to address the functionality of our expression systems.On the basis these tools, we will drive expression of the comX gene and analyze by quantitative PCR its ability to induce transcription of late competence genes. Their functionality will be addressed by visualization of foreign DNA intake and by measurement of selection marker integration in
S. thermophilus.