Numerous microorganisms, including bacteria, yeasts, and molds, are present in cheeses, forming a complex ecosystem. Among these organisms, bacteria are responsible for most of the physicochemical and aromatic transformations that are intrinsic to the cheesemaking process. Identification of the bacteria that constitute the cheese ecosystem is essential for understanding their individual contributions to cheese production. We used temporal temperature gradient gel electrophoresis (TTGE) to identify different bacterial species present in several dairy products, including members of the genera Lactobacillus, Lactococcus, Leuconostoc, Enterococcus, Pediococcus, Streptococcus, and Staphylococcus. The TTGE technique is based on electrophoretic separation of 16S ribosomal DNA (rDNA) fragments by using a temperature gradient. It was optimized to reveal differences in the 16S rDNA V3 regions of bacteria with low-G؉C-content genomes. Using multiple control strains, we first set up a species database in which each species (or group of species) was characterized by a specific TTGE fingerprint. TTGE was then applied to controlled dairy ecosystems with defined compositions, including liquid (starter), semisolid (home-made fermented milk), and solid (miniature cheese models) matrices. Finally, the potential of TTGE to describe the bacterial microflora of unknown ecosystems was tested with various commercial dairy products. Subspecies, species, or groups of species of lactic acid bacteria were distinguished in dairy samples. In conclusion, TTGE was shown to distinguish bacterial species in vitro, as well as in both liquid and solid dairy products.

Bacteriophages (or phages) dominate the biosphere both numerically and in terms of genetic diversity. In particular, genomic comparisons suggest a remarkable level of horizontal gene transfer among temperate phages, favoring a high evolution rate. Molecular mechanisms of this pervasive mosaicism are mostly unknown. One hypothesis is that phage encoded recombinases are key players in these horizontal transfers, thanks to their high efficiency and low fidelity. Here, we associate two complementary in vivo assays and a bioinformatics analysis to address the role of phage encoded recombinases in genomic mosaicism. The first assay allowed determining the genetic determinants of mosaic formation between lambdoid phages and Escherichia coli prophage remnants. In the second assay, recombination was monitored between sequences on phage λ, and allowed to compare the performance of three different Rad52-like recombinases on the same substrate. We also addressed the importance of homologous recombination in phage evolution by a genomic comparison of 84 E. coli virulent and temperate phages or prophages. We demonstrate that mosaics are mainly generated by homology-driven mechanisms that tolerate high substrate divergence. We show that phage encoded Rad52-like recombinases act independently of RecA, and that they are relatively more efficient when the exchanged fragments are divergent. We also show that accessory phage genes orf and rap contribute to mosaicism. A bioinformatics analysis strengthens our experimental results by showing that homologous recombination left traces in temperate phage genomes at the borders of recently exchanged fragments. We found no evidence of exchanges between virulent and temperate phages of E. coli. Altogether, our results demonstrate that Rad52-like recombinases promote gene shuffling among temperate phages, accelerating their evolution. This mechanism may prove to be more general, as other mobile genetic elements such as ICE encode Rad52-like functions, and play an important role in bacterial evolution itself.

Temperate phages, the bacterial viruses able to enter in a dormant prophage state in bacterial genomes, are present in the majority of bacterial strains for which the genome sequence is available. Although these prophages are generally considered to increase their hosts’ fitness by bringing beneficial genes, studies demonstrating such effects in ecologically relevant environments are relatively limited to few bacterial species. Here, we investigated the impact of prophage carriage in the gastrointestinal tract of monoxenic mice. Combined with mathematical modelling, these experimental results provided a quantitative estimation of key parameters governing phage-bacteria interactions within this model ecosystem. We used wild-type and mutant strains of the best known host/phage pair, Escherichia coli and phage λ. Unexpectedly, λ prophage caused a significant fitness cost for its carrier, due to an induction rate 50-fold higher than in vitro, with 1 to 2% of the prophage being induced. However, when prophage carriers were in competition with isogenic phage susceptible bacteria, the prophage indirectly benefited its carrier by killing competitors: infection of susceptible bacteria led to phage lytic development in about 80% of cases. The remaining infected bacteria were lysogenized, resulting overall in the rapid lysogenization of the susceptible lineage. Moreover, our setup enabled to demonstrate that rare events of phage gene capture by homologous recombination occurred in the intestine of monoxenic mice. To our knowledge, this study constitutes the first quantitative characterization of temperate phage-bacteria interactions in a simplified gut environment. The high prophage induction rate detected reveals DNA damage-mediated SOS response in monoxenic mouse intestine. We propose that the mammalian gut, the most densely populated bacterial ecosystem on earth, might foster bacterial evolution through high temperate phage activity.

In the family Streptococcaceae, the genes encoding zinc ABC uptake systems (called zit or adc) are regulated by a coencoded MarR family member (i.e., ZitR or AdcR), whereas in the great majority of bacteria, these genes are regulated by Zur, the Fur-like zinc-responsive repressor. We studied the zit operon from Lactococcus lactis and its regulation in response to Zn(II) in vivo. zit transcription is repressed by Zn(II) in a wide concentration range starting from nontoxic micromolar levels and is derepressed at nanomolar concentrations. The level of zit promoter downregulation by environmental Zn(II) is correlated with the intracellular zinc content. The helix-turn-helix domain of ZitR is required for downregulation. In vitro, the purified protein is a dimer that complexes up to two zinc ligands per monomer and specifically binds two intact palindromic operator sites overlapping the ؊35 and ؊10 boxes of the zit promoter. DNA binding is abolished by the chelator EDTA or TPEN and fully restored by Zn(II) addition, indicating that the active repressor complexes Zn(II) with high affinity. These results suggest that derepression under starvation conditions could be an essential emergency mechanism for preserving Zn(II) homeostasis by uptake; under Zn(II)-replete conditions, the function of ZitR repression could be to help save energy rather than to avoid Zn(II) toxicity. The characterization of a MarR family zinc-responsive repressor in this report gives insight into the way Streptococcaceae efficiently adapt to Zn(II) fluctuations in their diverse ecological niches.

Enterococcus faecalis is an opportunistic pathogen that has emerged as a major cause of nosocomial infections worldwide. Many clinical strains are indeed resistant to last resort antibiotics and there is consequently a reawakening of interest in exploiting virulent phages to combat them. However, little is still known about phage receptors and phage resistance mechanisms in enterococci. We made use of a prophageless derivative of the well-known clinical strain E. faecalis V583 to isolate a virulent phage belonging to the Picovirinae subfamily and to the P68 genus that we named Idefix. Interestingly, most isolates of E. faecalis tested—including V583—were resistant to this phage and we investigated more deeply into phage resistance mechanisms. We found that E. faecalis V583 prophage 6 was particularly efficient in resisting Idefix infection thanks to a new abortive infection (Abi) mechanism, which we designated Abiα. It corresponded to the Pfam domain family with unknown function DUF4393 and conferred a typical Abi phenotype by causing a premature lysis of infected E. faecalis. The abiα gene is widespread among prophages of enterococci and other Gram-positive bacteria. Furthermore, we identified two genes involved in the synthesis of the side chains of the surface rhamnopolysaccharide that are important for Idefix adsorption. Interestingly, mutants in these genes arose at a frequency of ~10−4 resistant mutants per generation, conferring a supplemental bacterial line of defense against Idefix.

-Washed-curd cheeses manufactured with either Lactococcus lactis subsp. lactis IL416 or Lactococcus lactis subsp. cremoris AM2 were used to test the role of six Lactobacillus strains from the CNRZ collection as adjunct cultures. Proteolysis and volatile profile determinations were performed on all cheeses after 28 days of ripening; a trained panel assessed sensory characteristics. We observed that overall, the lactococcal starter strains had a major influence on cheese taste and texture. Control cheeses manufactured with the strain IL416 were mild. Lactobacilli adjunct addition to cheeses manufactured with the strain IL416 had a strong effect on their flavour attributes. In contrast, control cheeses manufactured with the strain AM2 had greater flavour intensities and were saltier and more acidic. These cheeses were more affected in their texture profile and overall flavour intensity, and to a lesser extent in their flavour profile by lactobacilli addition. In summary, while the major cheese characteristics are determined by the starter strain used, addition of lactobacilli adjuncts L. plantarum 1572, L. plantarum 1310 and L. casei 1227 can affect flavour and texture of washed-curd cheese products.Lactobacilli adjunct culture / starter / proteolysis / volatile compound / sensory property Résumé -Contribution du levain et d'une culture secondaire de lactobacilles à la protéolyse, au profil des composés volatils et aux caractéristiques sensorielles de fromages à pâte pressée. Des fromages à pâte pressée ont été fabriqués avec soit Lactococcus lactis subsp. lactis IL416, soit Lactococcus lactis subsp. cremoris AM2 pour tester le rôle de six souches de lactobacilles de la collection CNRZ en tant que culture secondaire. La protéolyse et le profil des composés volatils ont été déterminés sur tous les fromages après 28 jours d'affinage et un jury entraîné a établi leur profil sensoriel. Nous observons avant tout une influence majeure de la souche de levain lactocoque sur le goût et la texture des fromages. Les fromages témoins fabriqués avec la souche IL416 sont neutres et l'ajout d'une culture secondaire de lactobacilles a un fort impact sur les attributs de flaveur de ces fromages. Au contraire, les fromages témoins fabriqués avec la souche AM2 possèdent une plus forte intensité de flaveur, ils sont plus salés et plus acides. La texture et l'intensité globale de flaveur et, dans une moindre mesure, le profil de flaveur de ces fromages sont aussi davantage affectés par

-Ten strains of Lactobacillus were tested as adjunct cultures in combination with two different Lactocococcus lactis starters in miniature washed-curd cheeses manufactured under controlled bacteriological conditions. Growth of lactobacilli seemed to depend on the strain used, but was not influenced by the starter strain (either L. lactis subsp. lactis lL416 or L. lactis subsp. cremoris AM2). Lactococcal counts in miniature cheeses with AM2 starter and added lactobacilli were higher than in control cheeses without lactobacilli. Consistently good survival (~100% after four weeks) was observed for IL416, regardless of the presence of adjunct culture). In contrast, AM2 starter cell viability decreased slowly over the same time period. Gross composition and protein analyses were performed on the miniature cheeses. Our results indicate that production of soluble nitrogenous compounds was influenced by the lactobacilli adjunct, and depended on the starter strain. We conclude that the use of different combinations of starter and adjunct cultures can result in marked differences in bacterial populations and product properties. Such studies may be used to choose the combination of strains necessary to obtain a product with particular properties.Lactobacilli adjunct culture / starter / proteolysis / cheese Résumé -Influence du levain et d'une culture secondaire de lactobacilles sur l'affinage des fromages miniatures type pâte pressée. Dix souches de lactobacilles de la collection CNRZ ont été testées en tant que culture secondaire, en combinaison avec deux souches différentes de levain Lactococcus lactis, dans des fromages miniatures type pâte pressée fabriqués en conditions bactériologiques contrôlées. La croissance des lactobacilles semble dépendre de la souche utilisée, mais elle n'est pas influencée par la souche de levain (L. lactis subsp. lactis IL416 ou L. lactis subsp. cremoris AM2). La numération des lactocoques dans les fromages miniatures fabriqués avec la souche AM2 et addition de lactobacilles est plus élevée que dans les fromages témoins sans ajout de lactobacilles. Comme attendue, une bonne survie a été observée pour IL416 (~100 % après quatre semaines), et ce indépendamment de la présence d'une culture secondaire. Au contraire, la viabilité de AM2 a décru au cours de la même période. La composition globale et des analyses biochimiques ont été réalisées sur les fromages miniatures. Nos résultats indiquent que l'addition de lactobacilles influence la production de composés azotés solubles en liaison avec la souche de

Bacteriophages are remarkable for the wide diversity of proteins they encode to perform DNA replication and homologous recombination. Looking back at these ancestral forms of life may help understanding how similar proteins work in more sophisticated organisms. For instance, the Sak4 family is composed of proteins similar to the archaeal RadB protein, a Rad51 paralog. We have previously shown that Sak4 allowed single-strand annealing in vivo, but only weakly compared to the phage λ Redβ protein, highlighting putatively that Sak4 requires partners to be efficient. Here, we report that the purified Sak4 of phage HK620 infecting Escherichia coli is a poorly efficient annealase on its own. A distant homolog of SSB, which gene is usually next to the sak4 gene in various species of phages, highly stimulates its recombineering activity in vivo. In vitro, Sak4 binds single-stranded DNA and performs single-strand annealing in an ATP-dependent way. Remarkably, the single-strand annealing activity of Sak4 is stimulated by its cognate SSB. The last six C-terminal amino acids of this SSB are essential for the binding of Sak4 to SSB-covered single-stranded DNA, as well as for the stimulation of its annealase activity. Finally, expression of sak4 and ssb from HK620 can promote low-level of recombination in vivo, though Sak4 and its SSB are unable to promote strand exchange in vitro. Regarding its homology with RecA, Sak4 could represent a link between two previously distinct types of recombinases, i.e., annealases that help strand exchange proteins and strand exchange proteins themselves.