Three Distinct Proteases Are Responsible for Overall Cell Surface Proteolysis in Streptococcus thermophilus.

The lactic acid bacterium Streptococcus thermophilus was believed to display only two distinct proteases at the cell surface, namely, the cell envelope protease PrtS and the housekeeping protease HtrA. Using peptidomics, we demonstrate here the existence of an additional active cell surface protease, which shares significant homology with the SepM protease of Streptococcus mutans. Although all three proteases-PrtS, HtrA, and SepM-are involved in the turnover of surface proteins, they demonstrate distinct substrate specificities. In particular, SepM cleaves proteins involved in cell wall metabolism and cell elongation, and its inactivation has consequences for cell morphology. When all three proteases are inactivated, the residual cell-surface proteolysis of S. thermophilus is approximately 5% of that of the wild-type strain. IMPORTANCE Streptococcus thermophilus is a lactic acid bacterium used widely as a starter in the dairy industry. Due to its "generally recognized as safe" status and its weak cell surface proteolytic activity, it is also considered a potential bacterial vector for heterologous protein production. Our identification of a new cell surface protease made it possible to construct a mutant strain with a 95% reduction in surface proteolysis, which could be useful in numerous biotechnological applications.

Streptococcus thermophilus growth in soya milk: Sucrose consumption, nitrogen metabolism, soya protein hydrolysis and role of the cell-wall protease PrtS.

Societal demand for plant-based foods is increasing. In this context, soya products fermented using lactic acid bacteria (LAB) are appealing because of their potential health and nutritional benefits. The thermophilic LAB Streptococcus thermophilus is an essential starter species in the dairy industry. However, while its physiology is well characterized, little is known about its general metabolic activity or its techno-functional properties when it is grown in soya milk. In this study, S. thermophilus LMD-9 growth, sugar production, and lactic acid production in soya milk versus cow's milk were measured. Additionally, the main metabolic pathways used by the bacterium when growing in soya milk were characterized using a proteomic approach. Streptococcus thermophilus LMD-9 growth decreased soya milk pH, from 7.5 to 4.9, in 5 h. During fermentation, acidification thus occurred in tandem with lactate production and increasing population size (final population: 1.0 × 109 CFU/ml). As growth proceeded, sucrose was consumed, and fructose was produced. The proteomic analysis (LC-MS/MS) of the strain's cytosolic and cell envelope-associated proteins revealed that proteins related to amino acid transport and nitrogen metabolism were the most common among the 328 proteins identified (63/328 = 19.2% of total proteins). The cell-wall protease PrtS was present, and an LMD-9 deletion mutant was constructed by interrupting the prtS gene (STER_RS04165 locus). Acidification levels, growth levels, and final population size were lower in the soya milk cultures when the ΔprtS strain versus the wild-type (wt) strain was used. The SDS-PAGE profile of the soluble proteins in the supernatant indicated that soya milk proteins were less hydrolyzed by the ΔprtS strain than by the wt strain. It was discovered that S. thermophilus can grow in soya milk by consuming sucrose, can hydrolyze soya proteins, and can produce acidification levels comparable to those in cow's milk. This study comprehensively examined the proteomics of S. thermophilus grown in soya milk and demonstrated that the cell-wall protease PrtS is involved in the LAB's growth in soya milk and in the proteolysis of soya proteins, which are two novel findings. These results clarify how S. thermophilus adapts to soya milk and can help inform efforts to develop new fermented plant-based foods with better-characterized biochemical and microbiological traits.

Identification of Hanks-Type Kinase PknB-Specific Targets in the Streptococcus thermophilus Phosphoproteome.

Protein phosphorylation especially on serine/threonine/tyrosine residues are frequent in many bacteria. This post-translational modification has been associated with pathogenicity and virulence in various species. However, only few data have been produced so far on generally recognized as safe bacteria used in food fermentations. A family of kinases known as Hanks-type kinases is suspected to be responsible for, at least, a part of these phosphorylations in eukaryotes as in bacteria. The objective of our work was to establish the first phosphoproteome of Streptococcus thermophilus, a lactic acid bacterium widely used in dairy fermentations in order to identified the proteins and pathways tagged by Ser/Thr/Tyr phosphorylations. In addition, we have evaluated the role in this process of the only Hanks-type kinase encoded in the S. thermophilus genome. We have constructed a mutant defective for the Hanks type kinase in S. thermophilus and established the proteomes and phosphoproteomes of the wild type and the mutant strains. To do that, we have enriched our samples in phosphopeptides with titane beads and used dimethyl tags to compare phosphopeptide abundances. Peptides and phosphopeptides were analyzed on a last generation LC-MS/MS system. We have identified and quantified 891 proteins representing half of the theoretical proteome. Among these proteins, 106 contained phosphorylated peptides. Various functional groups of proteins (amino acid, carbon and nucleotide metabolism, translation, cell cycle, stress response, …) were found phosphorylated. The phosphoproteome was only weakly reduced in the Hanks-type kinase mutant indicating that this enzyme is only one of the players in the phosphorylation process. The proteins that are modified by the Hanks-type kinase mainly belong to the divisome.

Ruminococcin C, an anti-clostridial sactipeptide produced by a prominent member of the human microbiota Ruminococcus gnavus.

The human microbiota plays a central role in human physiology. This complex ecosystem is a promising but untapped source of bioactive compounds and antibiotics that are critical for its homeostasis. However, we still have a very limited knowledge of its metabolic and biosynthetic capabilities. Here we investigated an enigmatic biosynthetic gene cluster identified previously in the human gut symbiont Ruminococcus gnavus This gene cluster which encodes notably for peptide precursors and putative radical SAM enzymes, has been proposed to be responsible for the biosynthesis of ruminococcin C (RumC), a ribosomally synthesized and posttranslationally modified peptide (RiPP) with potent activity against the human pathogen Clostridium perfringens By combining in vivo and in vitro approaches, including recombinant expression and purification of the respective peptides and proteins, enzymatic assays, and LC-MS analyses, we determined that RumC is a sulfur-to-α-carbon thioether-containing peptide (sactipeptide) with an unusual architecture. Moreover, our results support that formation of the thioether bridges follows a processive order, providing mechanistic insights into how radical SAM (AdoMet) enzymes install posttranslational modifications in RiPPs. We also found that the presence of thioether bridges and removal of the leader peptide are required for RumC's antimicrobial activity. In summary, our findings provide evidence that production of the anti-Clostridium peptide RumC depends on an R. gnavus operon encoding five potential RumC precursor peptides and two radical SAM enzymes, uncover key RumC structural features, and delineate the sequence of posttranslational modifications leading to its formation and antimicrobial activity.

Mass Spectrometry Analysis of the Extracellular Peptidome of Lactococcus lactis: Lines of Evidence for the Coexistence of Extracellular Protein Hydrolysis and Intracellular Peptide Excretion.

We report here the use of a peptidomic approach to revisit the extracellular proteolysis of Lactococcus lactis. More than 1800 distinct peptides accumulate externally during growth of the plasmid-free protease-negative strain L. lactis IL1403 in a protein- and peptide-free medium. These peptides mainly originate from cell-surface- and cytoplasmic-located proteins, despite the fact that no cell lysis could be evidenced. Positioning each identified peptide on its parental protein sequence demonstrated the involvement of exo- and endopeptidase activities. The endopeptidases responsible for the release of surface and cytoplasmic peptides had distinct specificities. The membrane-anchored protease HtrA was responsible for the release of only a part of the surface peptides, and its preference for branched-chain amino acids in the N-terminal side of the cleaved bond was established in situ. Other yet uncharacterized surface proteases were also involved. Several lines of evidence suggest that surface and cytoplasmic peptides were produced by different routes, at least part of the latter being most likely excreted as peptides from the cells. The mechanism by which these cytoplasmic peptides are excreted remains an open question, as it is still the case for excreted cytoplasmic proteins.

A partial metabolic pathway enables group b streptococcus to overcome quinone deficiency in a host bacterial community.

Aerobic respiration metabolism in Group B Streptococcus (GBS) is activated by exogenous heme and menaquinone. This capacity enhances resistance of GBS to acid and oxidative stress and improves its survival. In this work, we discovered that GBS is able to respire in the presence of heme and 1,4-dihydroxy-2-naphthoic acid (DHNA). DHNA is a biosynthetic precursor of demethylmenaquinone (DMK) in many bacterial species. A GBS gene (gbs1789) encodes a homolog of the MenA 1,4-dihydroxy-2-naphthoate prenyltransferase enzyme, involved in the synthesis of demethylmenaquinone. In this study, we showed that gbs1789 is involved in the biosynthesis of long-chain demethylmenaquinones (DMK-10). The Δgbs1789 mutant cannot respire in the presence of heme and DHNA, indicating that endogenously synthesized DMKs are cofactors of the GBS respiratory chain. We also found that isoprenoid side chains from GBS DMKs are produced by the protein encoded by the gbs1783 gene, since this gene can complement an Escherichia coli ispB mutant defective for isoprenoids chain synthesis. In the gut or vaginal microbiote, where interspecies metabolite exchanges occur, this partial DMK biosynthetic pathway can be important for GBS respiration and survival in different niches.