[Metagenomics of the intestinal microbiota: potential applications]. / Métagénomique du microbiote intestinal: les applications potentielles.

A major challenge in the human metagenomics field is to identify associations of the bacterial genes and human phenotypes and act to modulate microbial populations in order to improve human health and wellbeing. MetaHIT project addresses this ambitious challenge by developing and integrating a number of necessary approaches within the context of the gut microbiome. Among the first results is the establishment of a broad catalog of the human gut microbial genes, which was achieved by an original application of the new generation sequencing technology. The catalog contains 3.3 million non-redundant genes, 150-fold more than the human genome equivalent and includes a large majority of the gut metagenomic sequences determined across three continents, Europe, America and Asia. Its content corresponds to some 1000 bacterial species, which likely represent a large fraction of species associated with humankind intestinal tract. The catalog enables development of the gene profiling approaches aiming to detect associations of bacterial genes and phenotypes. These should lead to the speedy development of diagnostic and prognostic tools and open avenues to reasoned approaches to the modulation of the individual's microbiota in order to optimize health and well-being.

New insights in the molecular biology and physiology of Streptococcus thermophilus revealed by comparative genomics.

Streptococcus thermophilus is a major dairy starter used for the manufacture of yoghurt and cheese. The access to three genome sequences, comparative genomics and multilocus sequencing analyses suggests that this species recently emerged and is still undergoing a process of regressive evolution towards a specialised bacterium for growth in milk. Notably, S. thermophilus has maintained a well-developed nitrogen metabolism whereas its sugar catabolism has been subjected to a high level of degeneracy due to a paucity of carbon sources in milk. Furthermore, while pathogenic streptococci are recognised for a high capacity to expose proteins at their cell surface in order to achieve cell adhesion or to escape the host immune system, S. thermophilus has nearly lost this unique feature as well as many virulence-related functions. Although gene decay is obvious in S. thermophilus genome evolution, numerous small genomic islands, which were probably acquired by horizontal gene transfer, comprise important industrial phenotypic traits such as polysaccharide biosynthesis, bacteriocin production, restriction-modification systems or oxygen tolerance.

Conservation of key elements of natural competence in Lactococcus lactis ssp.

Natural competence is active in very diverse species of the bacterial kingdom and probably participates in horizontal gene transfer. Recently, the genome sequence of various species, including Lactococcus lactis, revealed the presence of homologues of competence genes in bacteria, which were not previously identified as naturally transformable. We investigated the conservation among lactococcal strains of key components of the natural competence process in streptococci: (i) comX which encodes a sigma factor, allowing the expression of the late competence genes involved in DNA uptake, (ii) its recognition site, the cin-box and (iii) dprA which encodes a protein shown to determine the fate of incoming DNA. The comX and dprA genes and the cin-box appeared conserved among strains, although some L. lactis ssp. lactis strains presented an inactivated dprA gene. We established that ComX controls the expression of the late competence genes in L. lactis. In conclusion, our work strongly suggests that ComX has the same role in streptococci and L. lactis, i.e. the regulation of late competence genes. It also allowed the identification of a set of L. lactis strains and the construction of a comX overexpression system, which should facilitate the investigation of the natural competence activity in lactococci.

Requirement for RecFOR-mediated recombination in priA mutant.

Restart of arrested replication forks is an important process and PriA, the main Escherichia coli replication restart protein, is essential for viability under any condition that increases the frequency of fork arrest. In priA mutant, replication forks are arrested by spontaneously occurring roadblocks and blocked replication forks persist as a result of the defect in replication restart. In the present work, we analysed how recombination proteins contribute to the viability of the priA mutant. RecFOR-mediated homologous recombination occurs in a large fraction of priA mutant cells, indicating a frequent formation of DNA single strand gaps and their recombinational repair. This high level of homologous recombination renders the proteins that resolve Holliday junctions recombination intermediates essential for viability. When homologous recombination is blocked at early steps by recFOR or recA inactivation, exonuclease V-mediated DNA degradation is required for full viability of priA mutants, indicating that unrepaired gaps are broken and that DNA degradation of the broken DNA allows the formation of viable cells. Models for the formation of single strand DNA gaps consequently to a replication restart defect and for gap processing are proposed.

sbcB sbcC null mutations allow RecF-mediated repair of arrested replication forks in rep recBC mutants.

We have proposed previously that, in Escherichia coli, blockage of replication forks can lead to the reversal of the fork. Annealing of the newly synthesized strands creates a double-stranded end adjacent to a Holliday junction. The junction is migrated away from the DNA end by RuvAB and can be cleaved by RuvC, while RecBCD is required for the repair of the double-stranded tail. Consequently, the rep mutant, in which replication arrests are frequent and fork reversal occurs, requires RecBCD for growth. We show here that the combination of sbcB sbcCD null mutations restores the viability to rep recBC mutants by activation of the RecF pathway of recombination. This shows that the proteins belonging to the RecF pathway are able to process the DNA ends made by the replication fork reversal into a structure that allows recombination-dependent replication restart. However, we confirm that, unlike sbcB null mutations, sbcB15, which suppresses all other recBC mutant defects, does not restore the viability of rep recBC sbcCD strains. We also show that ruvAB inactivation suppresses the lethality and the formation of double-stranded breaks (DSBs) in a rep recBC recF strain, totally deficient for homologous recombination, as well as in rep recBC mutants. This confirms that RuvAB processing of arrested replication forks is independent of the presence of recombination intermediates.