Role of the Sortase A in the Release of Cell-Wall Proteinase PrtS in the Growth Medium of Streptococcus thermophilus 4F44.
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SUBMITTER: Awussi AA
PROVIDER: S-EPMC8623714 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Streptococcus thermophilus CNRZ 385 expresses a cell envelope proteinase (PrtS), which is characterized in the present work, both at the biochemical and genetic levels. Since PrtS is resistant to most classical methods of extraction from the cell envelopes, we developed a three-step process based on loosening of the cell wall by cultivation of the cells in the presence of glycine (20 mM), mechanical disruption (with alumina powder), and enzymatic treatment (lysozyme). The pure enzyme is a serine proteinase highly activated by Ca(2+) ions. Its activity was optimal at 37 degrees C and pH 7.5 with acetyl-Ala-Ala-Pro-Phe-paranitroanilide as substrate. The study of the hydrolysis of the chromogenic and casein substrates indicated that PrtS presented an intermediate specificity between the most divergent types of cell envelope proteinases from lactococci, known as the PI and PIII types. This result was confirmed by the sequence determination of the regions involved in substrate specificity, which were a mix between those of PI and PIII types, and also had unique residues. Sequence analysis of the PrtS encoding gene revealed that PrtS is a member of the subtilase family. It is a multidomain protein which is maturated and tightly anchored to the cell wall via a mechanism involving an LPXTG motif. PrtS bears similarities to cell envelope proteinases from pyogenic streptococci (C5a peptidase and cell surface proteinase) and lactic acid bacteria (PrtP, PrtH, and PrtB). The highest homologies were found with streptococcal proteinases which lack, as PrtS, one domain (the B domain) present in cell envelope proteinases from all other lactic acid bacteria.
| S-EPMC92378 | biostudies-literature
Project description:In Streptococcus thermophilus, gene transfer events and loss of ancestral traits over the years contribute to its high level of adaptation to milk environments. Biofilm formation capacity, a phenotype that is lost in the majority of strains, plays a role in persistence in dairy environments, such as milk pasteurization and cheese manufacturing plants. To investigate this property, we have studied S. thermophilus UC8547, a fast-acidifying dairy starter culture selected for its high capacity to form biofilm on stainless steel under environmental conditions resembling the dairy environment. Using a dynamic flow cell apparatus, it was shown that S. thermophilus UC8547 biofilm formation on stainless steel depends on the presence of milk proteins. From this strain, which harbors the prtS gene for the cell wall protease and shows an aggregative phenotype, spontaneous mutants with impaired biofilm capacity can be isolated at high frequency. These mutants lack the PrtS expendable island, as confirmed by comparison of the genome sequence of UC8547Δ3 with that of the parent strain. The prtS island excision occurs between two 26-bp direct repeats located in the two copies of the ISSth1 flanking this genomic island. The central role of PrtS was confirmed by analyzing the derivative strain UC8547Δ16, whose prtS gene was interrupted by an insertional mutation, thereby making it incapable of biofilm formation. PrtS, acting as a binding substance between the milk proteins adhered to stainless steel and S. thermophilus cell envelopes, mediates biofilm formation in dairy environments. This feature provides S. thermophilus with an ecological benefit for its survival and persistence in this environment.IMPORTANCE The increased persistence of S. thermophilus biofilm has consequences in the dairy environment: if, on the one hand, the release of this microorganism from biofilm can promote the fermentation of artisanal cheeses, under industrial conditions it may lead to undesirable contamination of dairy products. The study of the molecular mechanism driving S. thermophilus biofilm formation provides increased knowledge on how an ancestral trait affects relevant phenotypes, such as persistence in the environment and efficiency of growth in milk. This study provides insight into the genetic factors affecting biofilm formation at dairy plants.
| S-EPMC5377502 | biostudies-literature
Project description:BackgroundIn industrial fermentation processes, the rate of milk acidification by Streptococcus thermophilus is of major technological importance. The cell-envelope proteinase PrtS was previously shown to be a key determinant of the milk acidification activity in this species. The PrtS enzyme is tightly anchored to the cell wall via a mechanism involving the typical sortase A (SrtA) and initiates the breakdown of milk casein into small oligopeptides. The presence or absence of PrtS divides the S. thermophilus strains into two phenotypic groups i.e. the slow and the fast acidifying strains. The aim of this study was to improve the milk acidification rate of slow S. thermophilus strains, and hence optimise the fermentation process of dairy products.ResultsIn the present work, we developed for the first time a strategy based on natural transformation to confer the rapid acidification phenotype to slow acidifying starter strains of S. thermophilus. First, we established by gene disruption that (i) prtS, encoding the cell-envelope proteinase, is a key factor responsible for rapid milk acidification in fast acidifying strains, and that (ii) srtA, encoding sortase A, is not absolutely required to express the PrtS activity. Second, a 15-kb PCR product encompassing the prtS genomic island was transferred by natural transformation using the competence-inducing peptide in three distinct prtS-defective genetic backgrounds having or not a truncated sortase A gene. We showed that in all cases the milk acidification rate of transformants was significantly increased, reaching a level similar to that of wild-type fast acidifying strains. Furthermore, it appeared that the prtS-encoded activity does not depend on the prtS copy number or on its chromosomal integration locus.ConclusionWe have successfully used natural competence to transfer the prtS locus encoding the cell-envelope proteinase in three slow acidifying strains of S. thermophilus, allowing their conversion into fast acidifying derivatives. The efficient protocol developed in this article will provide the dairy industry with novel and optimised S. thermophilus starter strains.
| S-EPMC3231928 | biostudies-literature
Project description:Streptococcus thermophilus is widely used for producing fermented dairy products such as yogurt and cheese. Some S. thermophilus strains possessing the cell-wall protease PrtS show high proteolytic activity and fast acidification properties, which are very useful in industrial starters. However, few S. thermophilus strains possessing the prtS gene have been isolated from the environment. To clarify whether or not S. thermophilus strains possessing the prtS gene are present in Japan, we isolated S. thermophilus from raw milk collected in Japan from 2011 to 2017 and investigated the strains for the presence of prtS by PCR. A total of 172 S. thermophilus strains were isolated, and 59 strains were confirmed to possess prtS. We measured fermentation times of 59 prtS-positive strains in skim milk broth and found that 53 strains showed fast acidification properties, finishing fermentation within 10 hr. However, the remaining 6 prtS-positive strains showed slow acidification properties, and they had several amino acid mutations in PrtS compared with fast acidifying S. thermophilus LMD-9 and 4F44. These results demonstrate that S. thermophilus strains possessing prtS are prevalent in Japan and that some prtS-positive strains could lose their fast acidifying properties through mutations in PrtS.
| S-EPMC7392909 | biostudies-literature
Project description: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.
| S-EPMC2805209 | biostudies-literature
Project description:Receptors on the cell surfaces of bacterial hosts are essential during the infection cycle of bacteriophages. To date, the phage receptors of the industrial relevant dairy starter bacterium Streptococcus thermophilus remain elusive. Thus, we set out to identify cell surface structures that are involved in host recognition by dairy streptococcal phages. Five industrial S. thermophilus strains sensitive to different phages (pac type, cos type, and the new type 987), were selected to generate spontaneous bacteriophage-insensitive mutants (BIMs). Of these, approximately 50% were deselected as clustered regularly interspaced short palindromic repeat (CRISPR) mutants, while the other pool was further characterized to identify receptor mutants. On the basis of genome sequencing data, phage resistance in putative receptor mutants was attributed to nucleotide changes in genes encoding glycan biosynthetic pathways. Superresolution structured illumination microscopy was used to visualize the interactions between S. thermophilus and its phages. The phages were either regularly distributed along the cells or located at division sites of the cells. The cell wall structures mediating the latter type of phage adherence were further analyzed via phenotypic and biochemical assays. Altogether, our data suggested that phage adsorption to S. thermophilus is mediated by glycans associated with the bacterial cell surface. Specifically, the pac-type phage CHPC951 adsorbed to polysaccharides anchored to peptidoglycan, while the 987-type phage CHPC926 recognized exocellular polysaccharides associated with the cell surface.IMPORTANCE Streptococcus thermophilus is widely used in starter cultures for cheese and yoghurt production. During dairy fermentations, infections of bacteria with bacteriophages result in acidification failures and a lower quality of the final products. An understanding of the molecular factors involved in phage-host interactions, in particular, the phage receptors in dairy bacteria, is a crucial step for developing better strategies to prevent phage infections in dairy plants.
| S-EPMC6238053 | biostudies-literature
Project description:BACKGROUND:Exopolysaccharides (EPSs) produced by lactic acid bacteria are important for the texture of fermented foods and have received a great deal of interest recently. However, the low production levels of EPSs in combination with the complex media used for growth of the bacteria have caused problems in the accurate analysis of the EPS. The purpose of this study was to find a growth medium for physiological studies of the lactic acid bacterium Streptococcus thermophilus, and to develop a simple method for qualitative and quantitative analysis of EPSs produced in this medium. RESULTS:A semi-defined polysaccharide medium was developed and evaluated on six strains of Streptococcus thermophilus. The EPSs were analysed using a novel protocol incorporating ultracentrifugation for the removal of interfering sugars, hydrolysis and analysis of the monomer composition by High Performance Anion-Exchange Chromatography with pulsed amperometric detection. The medium and analysis method allowed accurate quantification and monomer analysis of 0.5 ml samples of EPSs from tube cultures. CONCLUSIONS:The presented medium should be useful for physiological studies of S. thermophilus, and, in combination with the method of analysis of EPS, will allow downscaling of physiological studies and screening for EPSs.
| S-EPMC57807 | biostudies-literature
Project description:Sortases are a family of Gram-positive bacterial transpeptidases that anchor secreted proteins to bacterial cell surfaces. These include many proteins that play critical roles in the virulence of Gram-positive bacterial pathogens such that sortases are attractive targets for development of novel antimicrobial agents. All Gram-positive pathogens express a "housekeeping" sortase that recognizes the majority of secreted proteins containing an LPXTG wall-sorting motif and covalently attaches these to bacterial cell wall peptidoglycan. Many Gram-positive pathogens also express additional sortases that link a small number of proteins, often with variant wall-sorting motifs, to either other surface proteins or peptidoglycan. To better understand the mechanisms of catalysis and substrate recognition by the housekeeping sortase produced by the important human pathogen Streptococcus pyogenes, the crystal structure of this protein has been solved and its transpeptidase activity established in vitro. The structure reveals a novel arrangement of key catalytic residues in the active site of a sortase, the first that is consistent with kinetic analysis. The structure also provides a complete description of residue positions surrounding the active site, overcoming the limitation of localized disorder in previous structures of sortase A-type proteins. Modification of the active site Cys through oxidation to its sulfenic acid form or by an alkylating reagent supports a role for a reactive thiol/thiolate in the catalytic mechanism. These new insights into sortase structure and function could have important consequences for inhibitor design.
| S-EPMC2652338 | biostudies-literature
Project description:Recent work by our group has shown that an exopolysaccharide (EPS)-producing starter pair, Streptococcus thermophilus MR-1C and Lactobacillus delbrueckii subsp. bulgaricus MR-1R, can significantly increase moisture retention in low-fat mozzarella (D. B. Perry, D. J. McMahon, and C. J. Oberg, J. Dairy Sci. 80:799-805, 1997). The objectives of this study were to determine whether MR-1C, MR-1R, or both of these strains are required for enhanced moisture retention and to establish the role of EPS in this phenomenon. Analysis of low-fat mozzarella made with different combinations of MR-1C, MR-1R, and the non-EPS-producing starter culture strains S. thermophilus TA061 and Lactobacillus helveticus LH100 showed that S. thermophilus MR-1C was responsible for the increased cheese moisture level. To investigate the role of the S. thermophilus MR-1C EPS in cheese moisture retention, the epsE gene in this bacterium was inactivated by gene replacement. Low-fat mozzarella made with L. helveticus LH100 plus the non-EPS-producing mutant S. thermophilus DM10 had a significantly lower moisture content than did cheese made with strains LH100 and MR-1C, which confirmed that the MR-1C capsular EPS was responsible for the water-binding properties of this bacterium in cheese. Chemical analysis of the S. thermophilus MR-1C EPS indicated that the polymer has a novel basic repeating unit composed of D-galactose, L-rhamnose, and L-fucose in a ratio of 5:2:1.
| S-EPMC106291 | biostudies-literature
Project description:The important human pathogen Streptococcus pyogenes (group A streptococcus GAS), requires several surface proteins to interact with its human host. Many of these are covalently linked by a sortase enzyme to the cell wall via a C-terminal LPXTG motif. This motif is followed by a hydrophobic region and charged C terminus, which are thought to retard the protein in the cell membrane to facilitate recognition by the membrane-localized sortase. Previously, we identified two sortase enzymes in GAS. SrtA is found in all GAS strains and anchors most proteins containing LPXTG, while SrtB is present only in some strains and anchors a subset of LPXTG-containing proteins. We now report the presence of a third sortase in most strains of GAS, SrtC. We show that SrtC mediates attachment of a protein with a QVPTGV motif preceding a hydrophobic region and charged tail. We also demonstrate that the QVPTGV sequence is a substrate for anchoring of this protein by SrtC. Furthermore, replacing this motif with LPSTGE, found in the SrtA-anchored M protein of GAS, leads to SrtA-dependent secretion of the protein but does not lead to its anchoring by SrtA. We conclude that srtC encodes a novel sortase that anchors a protein containing a QVPTGV motif to the surface of GAS.
| S-EPMC516832 | biostudies-literature