Transfer of the Integrative and Conjugative Element ICESt3 of Streptococcus thermophilus in Physiological Conditions Mimicking the Human Digestive Ecosystem.

Metagenome analyses of the human microbiome suggest that horizontal gene transfer (HGT) is frequent in these rich and complex microbial communities. However, so far, only a few HGT studies have been conducted in vivo. In this work, three different systems mimicking the physiological conditions encountered in the human digestive tract were tested, including (i) the TNO gastro-Intestinal tract Model 1 (TIM-1) system (for the upper part of the intestine), (ii) the ARtificial COLon (ARCOL) system (to mimic the colon), and (iii) a mouse model. To increase the likelihood of transfer by conjugation of the integrative and conjugative element studied in the artificial digestive systems, bacteria were entrapped in alginate, agar, and chitosan beads before being placed in the different gut compartments. The number of transconjugants detected decreased, while the complexity of the ecosystem increased (many clones in TIM-1 but only one clone in ARCOL). No clone was obtained in a natural digestive environment (germfree mouse model). In the human gut, the richness and diversity of the bacterial community would offer more opportunities for HGT events to occur. In addition, several factors (SOS-inducing agents, microbiota-derived factors) that potentially increase in vivo HGT efficiency were not tested here. Even if HGT events are rare, expansion of the transconjugant clones can happen if ecological success is fostered by selecting conditions or by events that destabilize the microbial community. IMPORTANCE The human gut microbiota plays a key role in maintaining normal host physiology and health, but its homeostasis is fragile. During their transit in the gastrointestinal tract, bacteria conveyed by food can exchange genes with resident bacteria. New traits acquired by HGT (e.g., new catabolic properties, bacteriocins, antibiotic resistance) can impact the gut microbial composition and metabolic potential. We showed here that TIM-1, a system mimicking the upper digestive tract, is a useful tool to evaluate HGT events in conditions closer to the physiological ones. Another important fact pointed out in this work is that Enterococcus faecalis is a good candidate for foreign gene acquisition. Due to its high ability to colonize the gut and acquire mobile genetic elements, this commensal bacterium could serve as an intermediate for HGT in the human gut.

Identification of New Factors Modulating Adhesion Abilities of the Pioneer Commensal Bacterium Streptococcus salivarius.

Biofilm formation is crucial for bacterial community development and host colonization by Streptococcus salivarius, a pioneer colonizer and commensal bacterium of the human gastrointestinal tract. This ability to form biofilms depends on bacterial adhesion to host surfaces, and on the intercellular aggregation contributing to biofilm cohesiveness. Many S. salivarius isolates auto-aggregate, an adhesion process mediated by cell surface proteins. To gain an insight into the genetic factors of S. salivarius that dictate host adhesion and biofilm formation, we developed a screening method, based on the differential sedimentation of bacteria in semi-liquid conditions according to their auto-aggregation capacity, which allowed us to identify twelve mutations affecting this auto-aggregation phenotype. Mutations targeted genes encoding (i) extracellular components, including the CshA surface-exposed protein, the extracellular BglB glucan-binding protein, the GtfE, GtfG and GtfH glycosyltransferases and enzymes responsible for synthesis of cell wall polysaccharides (CwpB, CwpK), (ii) proteins responsible for the extracellular localization of proteins, such as structural components of the accessory SecA2Y2 system (Asp1, Asp2, SecA2) and the SrtA sortase, and (iii) the LiaR transcriptional response regulator. These mutations also influenced biofilm architecture, revealing that similar cell-to-cell interactions govern assembly of auto-aggregates and biofilm formation. We found that BglB, CshA, GtfH and LiaR were specifically associated with bacterial auto-aggregation, whereas Asp1, Asp2, CwpB, CwpK, GtfE, GtfG, SecA2 and SrtA also contributed to adhesion to host cells and host-derived components, or to interactions with the human pathogen Fusobacterium nucleatum. Our study demonstrates that our screening method could also be used to identify genes implicated in the bacterial interactions of pathogens or probiotics, for which aggregation is either a virulence trait or an advantageous feature, respectively.

Impact of Cell Surface Molecules on Conjugative Transfer of the Integrative and Conjugative Element ICESt3 of Streptococcus thermophilus.

Integrative conjugative elements (ICEs) are chromosomal elements that are widely distributed in bacterial genomes, hence contributing to genome plasticity, adaptation, and evolution of bacteria. Conjugation requires a contact between both the donor and the recipient cells and thus likely depends on the composition of the cell surface envelope. In this work, we investigated the impact of different cell surface molecules, including cell surface proteins, wall teichoic acids, lipoteichoic acids, and exopolysaccharides, on the transfer and acquisition of ICESt3 from Streptococcus thermophilus The transfer of ICESt3 from wild-type (WT) donor cells to mutated recipient cells increased 5- to 400-fold when recipient cells were affected in lipoproteins, teichoic acids, or exopolysaccharides compared to when the recipient cells were WT. These mutants displayed an increased biofilm-forming ability compared to the WT, suggesting better cell interactions that could contribute to the increase of ICESt3 acquisition. Microscopic observations of S. thermophilus cell surface mutants showed different phenotypes (aggregation in particular) that can also have an impact on conjugation. In contrast, the same mutations did not have the same impact when the donor cells, instead of recipient cells, were mutated. In that case, the transfer frequency of ICESt3 decreased compared to that with the WT. The same observation was made when both donor and recipient cells were mutated. The dominant effect of mutations in donor cells suggests that modifications of the cell envelope could impair the establishment or activity of the conjugation machinery required for DNA transport.IMPORTANCE ICEs contribute to horizontal gene transfer of adaptive traits (for example, virulence, antibiotic resistance, or biofilm formation) and play a considerable role in bacterial genome evolution, thus underlining the need of a better understanding of their conjugative mechanism of transfer. While most studies focus on the different functions encoded by ICEs, little is known about the effect of host factors on their conjugative transfer. Using ICESt3 of S. thermophilus as a model, we demonstrated the impact of lipoproteins, teichoic acids, and exopolysaccharides on ICE transfer and acquisition. This opens up new avenues to control gene transfer mediated by ICEs.

Study of Streptococcus thermophilus population on a world-wide and historical collection by a new MLST scheme.

We analyzed 178 Streptococcus thermophilus strains isolated from diverse products, from around the world, over a 60-year period with a new multilocus sequence typing (MLST) scheme. This collection included isolates from two traditional cheese-making sites with different starter-use practices, in sampling campaigns carried out over a three years period. The nucleotide diversity of the S. thermophilus population was limited, but 116 sequence types (ST) were identified. Phylogenetic analysis of the concatenated sequences of the six housekeeping genes revealed the existence of groups confirmed by eBURST analysis. Deeper analyses performed on 25 strains by CRISPR and whole-genome analysis showed that phylogenies obtained by MLST and whole-genome analysis were in agreement but differed from that inferred by CRISPR analysis. Strains isolated from traditional products could cluster in specific groups indicating their origin, but also be mixed in groups containing industrial starter strains. In the traditional cheese-making sites, we found that S. thermophilus persisted on dairy equipment, but that occasionally added starter strains may become dominant. It underlined the impact of starter use that may reshape S. thermophilus populations including in traditional products. This new MLST scheme thus provides a framework for analyses of S. thermophilus populations and the management of its biodiversity.

Commensal Streptococcus salivarius Modulates PPARγ Transcriptional Activity in Human Intestinal Epithelial Cells.

The impact of commensal bacteria in eukaryotic transcriptional regulation has increasingly been demonstrated over the last decades. A multitude of studies have shown direct effects of commensal bacteria from local transcriptional activity to systemic impact. The commensal bacterium Streptococcus salivarius is one of the early bacteria colonizing the oral and gut mucosal surfaces. It has been shown to down-regulate nuclear transcription factor (NF-кB) in human intestinal cells, a central regulator of the host mucosal immune system response to the microbiota. In order to evaluate its impact on a further important transcription factor shown to link metabolism and inflammation in the intestine, namely PPARγ (peroxisome proliferator-activated receptor), we used human intestinal epithelial cell-lines engineered to monitor PPARγ transcriptional activity in response to a wide range of S. salivarius strains. We demonstrated that different strains from this bacterial group share the property to inhibit PPARγ activation independently of the ligand used. First attempts to identify the nature of the active compounds showed that it is a low-molecular-weight, DNase-, proteases- and heat-resistant metabolite secreted by S. salivarius strains. Among PPARγ-targeted metabolic genes, I-FABP and Angptl4 expression levels were dramatically reduced in intestinal epithelial cells exposed to S. salivarius supernatant. Both gene products modulate lipid accumulation in cells and down-regulating their expression might consequently affect host health. Our study shows that species belonging to the salivarius group of streptococci impact both host inflammatory and metabolic regulation suggesting a possible role in the host homeostasis and health.

Genomics of Streptococcus salivarius, a major human commensal.

The salivarius group of streptococci is of particular importance for humans. This group consists of three genetically similar species, Streptococcus salivarius, Streptococcus vestibularis and Streptococcus thermophilus. S. salivarius and S. vestibularis are commensal organisms that may occasionally cause opportunistic infections in humans, whereas S. thermophilus is a food bacterium widely used in dairy production. We developed Multilocus sequence typing (MLST) and comparative genomic analysis to confirm the clear separation of these three species. These analyses also identified a subgroup of four strains, with a core genome diverging by about 10%, in terms of its nucleotide sequence, from that of S. salivarius sensu stricto. S. thermophilus species displays a low level of nucleotide variability, due to its recent emergence with the development of agriculture. By contrast, nucleotide variability is high in the other two species of the salivarius group, reflecting their long-standing association with humans. The species of the salivarius group have genome sizes ranging from the smallest (∼ 1.7 Mb for S. thermophilus) to the largest (∼ 2.3 Mb for S. salivarius) among streptococci, reflecting genome reduction linked to a narrow, nutritionally rich environment for S. thermophilus, and natural, more competitive niches for the other two species. Analyses of genomic content have indicated that the core genes of S. salivarius account for about two thirds of the genome, indicating considerable variability of gene content and differences in potential adaptive features. Furthermore, we showed that the genome of this species is exceptionally rich in genes encoding surface factors, glycosyltransferases and response regulators. Evidence of widespread genetic exchanges was obtained, probably involving a natural competence system and the presence of diverse mobile elements. However, although the S. salivarius strains studied were isolated from several human body-related sites (all levels of the digestive tract, skin, breast milk, and body fluids) and included clinical strains, no genetic or genomic niche-specific features could be identified to discriminate specific group.

Anti-inflammatory properties of Streptococcus salivarius, a commensal bacterium of the oral cavity and digestive tract.

Streptococcus salivarius is one of the first colonizers of the human oral cavity and gut after birth and therefore may contribute to the establishment of immune homeostasis and regulation of host inflammatory responses. The anti-inflammatory potential of S. salivarius was first evaluated in vitro on human intestinal epithelial cells and human peripheral blood mononuclear cells. We show that live S. salivarius strains inhibited in vitro the activation of the NF-κB pathway on intestinal epithelial cells. We also demonstrate that the live S. salivarius JIM8772 strain significantly inhibited inflammation in severe and moderate colitis mouse models. These in vitro and in vivo anti-inflammatory properties were not found with heat-killed S. salivarius, suggesting a protective response exclusively with metabolically active bacteria.

Complete genome sequence of the pigmented Streptococcus thermophilus strain JIM8232.

Streptococcus thermophilus is a dairy species commonly used in the manufacture of cheese and yogurt. Here, we report the complete sequence of S. thermophilus strain JIM8232, isolated from milk and which produces a yellow pigment, an atypical trait for this bacterium.

Complete genome sequence of the commensal Streptococcus salivarius strain JIM8777.

The commensal bacterium Streptococcus salivarius is a prevalent species of the human oropharyngeal tract with an important role in oral ecology. Here, we report the complete 2.2-Mb genome sequence and annotation of strain JIM8777, which was recently isolated from the oral cavity of a healthy, dentate infant.

Complete genome sequence of the clinical Streptococcus salivarius strain CCHSS3.

Streptococcus salivarius is a commensal species commonly found in the human oral cavity and digestive tract, although it is also associated with human infections such as meningitis, endocarditis, and bacteremia. Here, we report the complete sequence of S. salivarius strain CCHSS3, isolated from human blood.

Inhibition of the NF-kappaB pathway in human intestinal epithelial cells by commensal Streptococcus salivarius.

Streptococcus salivarius exhibited an anti-inflammatory effect on intestinal epithelial cells (IECs) and monocytes. Strains were screened using a reporter clone, HT-29/kB-luc-E, induced by tumor necrosis factor alpha (TNF-α). Supernatant from each strain downregulated NF-κB activation. The two most efficient strains produced an active metabolite (<3 kDa) which was able to downregulate the secretion of the proinflammatory chemokine interleukin-8 (IL-8).

Enterotypes of the human gut microbiome.

Our knowledge of species and functional composition of the human gut microbiome is rapidly increasing, but it is still based on very few cohorts and little is known about variation across the world. By combining 22 newly sequenced faecal metagenomes of individuals from four countries with previously published data sets, here we identify three robust clusters (referred to as enterotypes hereafter) that are not nation or continent specific. We also confirmed the enterotypes in two published, larger cohorts, indicating that intestinal microbiota variation is generally stratified, not continuous. This indicates further the existence of a limited number of well-balanced host-microbial symbiotic states that might respond differently to diet and drug intake. The enterotypes are mostly driven by species composition, but abundant molecular functions are not necessarily provided by abundant species, highlighting the importance of a functional analysis to understand microbial communities. Although individual host properties such as body mass index, age, or gender cannot explain the observed enterotypes, data-driven marker genes or functional modules can be identified for each of these host properties. For example, twelve genes significantly correlate with age and three functional modules with the body mass index, hinting at a diagnostic potential of microbial markers.

Emergence of a cell wall protease in the Streptococcus thermophilus population.

Streptococcus thermophilus is perceived as a recently emerged food bacterium that evolved from a commensal ancestor by loss and gain of functions. Here, we provide data allowing a better understanding of this evolutionary scheme. A multilocus sequence typing approach that we developed showed that S. thermophilus diverges significantly from its potential ancestors of the salivarius group and displays a low level of allelic variability, confirming its likely recent emergence. An analysis of the origin and dissemination of the prtS gene was carried out within this evolutionary scheme. This gene encodes a protease that allows better growth in milk by facilitating casein breakdown to supply amino acids. The S. thermophilus protease exhibits 95% identity to the animal Streptococcus suis protein PrtS. Genomic analysis showed that prtS is part of an island flanked by two tandem insertion sequence elements and containing three other genes which present the best identities and synteny with the S. suis genome. These data indicate a potential origin for this "ecological" island in a species closely related to S. suis. The analysis of the distribution of the prtS gene in S. thermophilus showed that the gene is infrequent in historical collections but frequent in recent industrial ones. Moreover, this "ecological" island conferring an important metabolic trait for milk adaptation appears to have disseminated by lateral transfer in the S. thermophilus population. Taken together, these data support an evolutionary scheme of S. thermophilus where gene acquisition and selection by food producers are determining factors. The source and impact of genes acquired by horizontal gene transfer on the physiology and safety of strains should be addressed.

Characterisation of thermotolerant cocci from indigenous flora of 'leben' in algerian arid area and DNA identification of atypical Lactococcus lactis strains.

Lactic acid bacteria (LAB) are widely used in food industry and their growth performance is important for the quality of the fermented product. By combining results from conventional isolation methods and molecular investigation of 16S rRNA gene and lactococcal/enterococcal specific genes, we identify at species level catalase negative gram positive thermoresistant cocci isolated from traditional 'leben', a 24-h fermented milk in arid area of west Algeria. Forty strains phenotypically related to cocci LAB were identified as belonging to the species Lactococcus lactis ssp. lactis, Enterococcus faecalis, Enterococcus faecium, and other Enterococcus species. No Streptococcus thermophilus strain was isolated. Ten different phenotype groups were recognized, and the species content of these groups were in some cases different from the expected features usually given in genus and species descriptions. In particular, atypical lactococci, able to grow in 6.5% NaCl, at pH 9.5 and showing high resistance to thermal stresses were isolated. Lactococci, but also enterococci isolated from traditional 'leben' produced in the desert area, may be therefore of interest in milk fermentation. Further studies to assess this source of diversity within the wild microbial population should provide starter new strains for product innovation.

Safety assessment of dairy microorganisms: Streptococcus thermophilus.

Streptococcus thermophilus is a major dairy starter used in yogurt and cheese production. In Streptococcus genus, S. thermophilus is the only one food species among commensal and opportunistic pathogen species. Comparative genomics suggest that this species recently emerged and evolved by combination of loss-of-function and horizontal gene transfer events. These gene transfer events detected in S. thermophilus have originated from other dairy species and might contribute to its adaptation to the milk environment.

Control of EpsE, the phosphoglycosyltransferase initiating exopolysaccharide synthesis in Streptococcus thermophilus, by EpsD tyrosine kinase.

We studied the roles of Streptococcus thermophilus phosphogalactosyltransferase (EpsE) (the priming enzyme), tyrosine kinase (EpsD), phosphatase (EpsB), and a membrane-associated protein with no known biochemical function (EpsC) in exopolysaccharide (EPS) synthesis. These proteins are well-conserved among bacteria and are usually encoded by clustered genes. Exopolysaccharide synthesis took place in the wild-type strain and a mutant lacking EpsB but not in mutants lacking EpsC, EpsD, or EpsE. The three mutants unable to synthesize EPS lacked the EpsE phosphogalactosyltransferase activity, while the two EPS-synthesizing strains possessed this activity, showing that EpsC and EpsD are required for EpsE function. An EpsD phosphorylated form was found in all strains except the epsC mutant, indicating that EpsC is necessary for EpsD phosphorylation. Moreover, the phosphorylated form of EpsD, a supposedly cytoplasmic protein, was found to be associated with the plasma membrane, possibly due to interaction with EpsC. Finally, the EpsD and EpsE elution profiles in a gel filtration chromatography assay were similar, suggesting that these two proteins colocalize in the membrane. Mutation of Tyr200, predicted to be a phosphorylation site and present in a conserved motif in bacterial phosphoglycosyltransferases, led to EpsE inactivation. In contrast, mutation of Tyr162 or Tyr199 had no effect. Taken together, these data show that EpsD controls EpsE activity. Possible mechanisms for this control are discussed.

Extent of horizontal gene transfer in evolution of Streptococci of the salivarius group.

The phylogenetically closely related species Streptococcus salivarius and Streptococcus vestibularis are oral bacteria that are considered commensals, although they can also be found in human infections. The relationship between these two species and the relationship between strains isolated from carriers and strains responsible for invasive infections were investigated by multilocus sequence typing and additional sequence analysis. The clustering of several S. vestibularis alleles and the extent of genomic divergence at certain loci support the conclusion that S. salivarius and S. vestibularis are separate species. The level of sequence diversity in S. salivarius alleles is generally high, whereas that in S. vestibularis alleles is low at certain loci, indicating that the latter species might have evolved recently. Cluster analysis indicated that there has been genetic exchange between S. salivarius and S. vestibularis at three of the nine loci investigated. Horizontal gene transfer between streptococci belonging to the S. salivarius group and other oral streptococci was also detected at several loci. A high level of recombination in S. salivarius was revealed by allele index association and split decomposition sequence analyses. Commensal and infection-associated S. salivarius strains could not be distinguished by cluster analysis, suggesting that the pathogen isolates are opportunistic. Taken together, our results indicate that there is a high level of gene exchange that contributes to the evolution of two streptococcal species from the human oral cavity.

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.

Intracellular effectors regulating the activity of the Lactococcus lactis CodY pleiotropic transcription regulator.

CodY is a pleiotropic transcriptional regulator conserved in low-G+C Gram-positive bacteria. Two distinct signals have been shown independently to influence the activity of this regulator: the level of intracellular GTP in Bacillus subtilis and the level of intracellular branched-chain amino acids (BCAA) isoleucine, leucine and valine in Lactococcus lactis. Measurement of BCAA and GTP levels in several environmental conditions showed that L. lactis CodY responded to the intracellular BCAA concentrations but not to physiological fluctuations in intracellular GTP. In addition, we demonstrated that CodY from L. lactis did not respond to intracellular GTP even when complementing CodY activity in B. subtilis. However, L. lactis CodY activity could still be modulated in B. subtilis by adding a rich nitrogen source to the growth media. This finding suggests that only BCAA are sensed by L. lactis CodY, whereas both GTP and BCAA signals may be integrated by B. subtilis CodY. The difference in the function of CodY from B. subtilis and L. lactis seems to reflect the difference in the physiology of these two bacteria.