The lactic acid bacterium Streptococcus thermophilus is widely used for the manufacture of yogurt and cheese. This dairy species of major economic importance is phylogenetically close to pathogenic ...streptococci, raising the possibility that it has a potential for virulence. Here we report the genome sequences of two yogurt strains of S. thermophilus. We found a striking level of gene decay (10% pseudogenes) in both microorganisms. Many genes involved in carbon utilization are nonfunctional, in line with the paucity of carbon sources in milk. Notably, most streptococcal virulence-related genes that are not involved in basic cellular processes are either inactivated or absent in the dairy streptococcus. Adaptation to the constant milk environment appears to have resulted in the stabilization of the genome structure. We conclude that S. thermophilus has evolved mainly through loss-of-function events that remarkably mirror the environment of the dairy niche resulting in a severely diminished pathogenic potential.
Biofilm formation is crucial for bacterial community development and host colonization by
, 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
isolates auto-aggregate, an adhesion process mediated by cell surface proteins. To gain an insight into the genetic factors of
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
. 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.
Surface proteins of Gram-positive bacteria play crucial roles in bacterial adhesion to host tissues. Regarding commensal or probiotic bacteria, adhesion to intestinal mucosa may promote their ...persistence in the gastro-intestinal tract and their beneficial effects to the host. In this study, seven Lactococcus lactis strains exhibiting variable surface physico-chemical properties were compared for their adhesion to Caco-2 intestinal epithelial cells. In this test, only one vegetal isolate TIL448 expressed a high-adhesion phenotype. A nonadhesive derivative was obtained by plasmid curing from TIL448, indicating that the adhesion determinants were plasmid-encoded. Surface-exposed proteins in TIL448 were analyzed by a proteomic approach consisting in shaving of the bacterial surface with trypsin and analysis of the released peptides by LC-MS/MS. As the TIL448 complete genome sequence was not available, the tryptic peptides were identified by a mass matching approach against a database including all Lactococcus protein sequences and the sequences deduced from partial DNA sequences of the TIL448 plasmids. Two surface proteins, encoded by plasmids in TIL448, were identified as candidate adhesins, the first one displaying pilin characteristics and the second one containing two mucus-binding domains. Inactivation of the pilin gene abolished adhesion to Caco-2 cells whereas inactivation of the mucus-binding protein gene had no effect on adhesion. The pilin gene is located inside a cluster of four genes encoding two other pilin-like proteins and one class-C sortase. Synthesis of pili was confirmed by immunoblotting detection of high molecular weight forms of pilins associated to the cell wall as well as by electron and atomic force microscopy observations. As a conclusion, surface proteome analysis allowed us to detect pilins at the surface of L. lactis TIL448. Moreover we showed that pili appendages are formed and involved in adhesion to Caco-2 intestinal epithelial cells.
Streptococcus agalactiae (Group B streptococcus, GBS) is a leading cause of infections in neonates and an emerging pathogen in adults. The Lancefield Group B carbohydrate (GBC) is a ...peptidoglycan-anchored antigen that defines this species as a Group B Streptococcus. Despite earlier immunological and biochemical characterizations, the function of this abundant glycopolymer has never been addressed experimentally. Here, we inactivated the gene gbcO encoding a putative UDP-N-acetylglucosamine-1-phosphate:lipid phosphate transferase thought to catalyze the first step of GBC synthesis. Indeed, the gbcO mutant was unable to synthesize the GBC polymer, and displayed an important growth defect in vitro. Electron microscopy study of the GBC-depleted strain of S. agalactiae revealed a series of growth-related abnormalities: random placement of septa, defective cell division and separation processes, and aberrant cell morphology. Furthermore, vancomycin labeling and peptidoglycan structure analysis demonstrated that, in the absence of GBC, cells failed to initiate normal PG synthesis and cannot complete polymerization of the murein sacculus. Finally, the subcellular localization of the PG hydrolase PcsB, which has a critical role in cell division of streptococci, was altered in the gbcO mutant. Collectively, these findings show that GBC is an essential component of the cell wall of S. agalactiae whose function is reminiscent of that of conventional wall teichoic acids found in Staphylococcus aureus or Bacillus subtilis. Furthermore, our findings raise the possibility that GBC-like molecules play a major role in the growth of most if not all beta-hemolytic streptococci.
Summary
Bacteria such as Lactococcus lactis have d‐aspartate (d‐Asp) or its amidated derivative d‐asparagine (d‐Asn), in their peptidoglycan (PG) interpeptide crossbridge. We performed a subtractive ...genome analysis to identify L. lactis gene yxbA, orthologues of which being present only in bacteria containing d‐amino acids in their PG crossbridge, but absent from those that instead insert l‐amino acids or glycine. Inactivation of yxbA required a complementing Streptococcus pneumoniae murMN genes, which express enzymes that incorporate l‐Ser‐l‐Ala or l‐Ala‐l‐Ala in the PG crossbridge. Our results show that (i) yxbA encodes d‐Asp ligase responsible for incorporation of d‐Asp in the PG crossbridge, and we therefore renamed it as aslA, (ii) it is an essential gene, which makes its product a potential target for specific antimicrobials, (iii) the absence of d‐Asp may be complemented by l‐Ser‐l‐Ala or l‐Ala‐l‐Ala in the L. lactis PG, indicating that the PG synthesis machinery is not selective for the side‐chain residues, and (iv) lactococcal strains having l‐amino acids in their PG crossbridge display defects in cell wall integrity, but are able to efficiently anchor cell wall proteins, indicating relative flexibility of lactococcal transpeptidation reactions with respect to changes in PG side‐chain composition.
Control of cell size and morphology is of paramount importance for bacterial fitness. In the opportunistic pathogen Enterococcus faecalis, the formation of diplococci and short cell chains ...facilitates innate immune evasion and dissemination in the host. Minimisation of cell chain size relies on the activity of a peptidoglycan hydrolase called AtlA, dedicated to septum cleavage. To prevent autolysis, AtlA activity is tightly controlled, both temporally and spatially. Here, we show that the restricted localization of AtlA at the septum occurs via an unexpected mechanism. We demonstrate that the C-terminal LysM domain that allows the enzyme to bind peptidoglycan is essential to target this enzyme to the septum inside the cell before its translocation across the membrane. We identify a membrane-bound cytoplasmic protein partner (called AdmA) involved in the recruitment of AtlA via its LysM domains. This work reveals a moonlighting role for LysM domains, and a mechanism evolved to restrict the subcellular localization of a potentially lethal autolysin to its site of action.
Endogenous peptidoglycan (PG)-hydrolyzing enzymes, the autolysins, are needed to relax the rigid PG sacculus to allow bacterial cell growth and separation. PGs of pathogens and commensal bacteria may ...also be degraded by hydrolases of animal origin (lysozymes), which act as antimicrobials. The genetic mechanisms regulating PG resistance to hydrolytic degradation were dissected in the Gram-positive bacterium Lactococcus lactis. We found that the ability of L. lactis to counteract PG hydrolysis depends on the degree of acetylation. Overexpression of PG O-acetylase (encoded by oatA) led to bacterial growth arrest, indicating the potential lethality of oatA and a need for its tight regulation. A novel regulatory factor, SpxB (previously denoted as YneH), exerted a positive effect on oatA expression. Our results indicate that SpxB binding to RNA polymerase constitutes a previously missing link in the multistep response to cell envelope stress, provoked by PG hydrolysis with lysozyme. We suggest that the two-component system CesSR responds to this stress by inducing SpxB, thus favoring its interactions with RNA polymerase. Induction of PG O-acetylation by this cascade renders it resistant to hydrolysis.
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