Caractérisation de la variabilité du système protéolytique de surface de la bactérie lactique Streptococcus thermophilus

The variability of the cell envelope-associated proteolytic system was studied in 30 strains of St. thermophilus. Variations in strains consist in the presence or absence of the gene prtS, the presence of two allelic forms of prtS, the presence of an anchored and/or soluble form of the protease PrtS and in the variable expression of the gene prtS and other genes involved mainly in nitrogen metabolism, thus in the variability of the regulation genetic of this system. Expression of the genes prtS, pepX, pepC, pepN, amiA1CDEF, dtpT, livJHMGF, ilvC, ilvDBN, bcaT, ackA, ldh, codY and relA was quantified in the PB302 and PB18O strains. The strain PB302 is representative of strains which exhibit a rapid growth in milk. The strain PB18O is representative those with intermediate growth in milk. In M17 medium, where both strains have similar growth, little difference in the expression of genes tested was observed. Conversely, the two strains did not express the selected genes in the same way when grown in milk. Overall, the difference in growth observed between strains in milk could result from variable proteolytic activities and variable expression of genes encoding, for example, the proteinase PrtS, the regulator CodY, transporters of oligo- or di-tri- peptides (Ami or DtpT) or branched chain amino acids, or BCAA (LivJ) and enzymes in the biosynthetic pathway of BCAA (IlvC, IlvB et BcaT) which are necessary for growth in milk. All these genes have a potential CodY box at the upstream of their promoter and could therefore belong to the regulon CodY.La variabilité du système protéolytique de surface a été étudiée chez 30 souches de St. thermophilus. Cette variabilité consiste en la présence ou l'absence du gène prtS, en la présence de deux allèles différents de ce gène, en la présence d'une protéase PrtS ancrée et/ou soluble et enfin en l'expression variable, due à une variabilité de la régulation du système protéolytique, du gène prtS et d'autres gènes qui interviennent, pour la plupart, dans le métabolisme azoté. L'expression des gènes prtS, pepX, pepC, pepN, amiA1CDEF, dtpT, livJHMGF, ilvC, ilvDBN, bcaT, ackA, ldh, codY et relA a été quantifiée chez les souches PB302 et PB18O en lait et en milieu M17. La souche PB302 est représentative des souches qui se développent rapidement en lait alors que la souche PB18O l'est de celles qui ont une croissance intermédiaire dans ce milieu. Alors que l'expression des gènes étudiés est peu différente en milieu M17 où les deux souches ont une croissance similaire, cette expression diverge lorsque les deux souches sont cultivées en lait.Globalement, la différence de croissance observée en lait entre les deux souches pourrait résulter d'une variabilité de la capacité protéolytique et de l'expression, entre autres, des gènes codant PrtS, le régulateur CodY, les transporteurs des oligopeptides (Ami), des di-tripeptides (DtpT) et des acides aminés ramifiés (LivJ) et de ceux codant des enzymes impliquées dans la voie de biosynthèse des acides aminés ramifiés (IlvC, IlvB et BcaT), ces derniers étant nécessaires pour la croissance en lait. Tous ces gènes possèdent en amont de leur promoteur une boîte CodY potentielle et pourraient donc appartenir au régulon CodY

Characterization of the variability of the proteolytic system of the lactic acid bacteria Streptococcus thermophilus

La variabilité du système protéolytique de surface a été étudiée chez 30 souches de St. thermophilus. Cette variabilité consiste en la présence ou l’absence du gène prtS, en la présence de deux allèles différents de ce gène, en la présence d'une protéase PrtS ancrée et/ou soluble et enfin en l'expression variable, due à une variabilité de la régulation du système protéolytique, du gène prtS et d'autres gènes qui interviennent, pour la plupart, dans le métabolisme azoté. L’expression des gènes prtS, pepX, pepC, pepN, amiA1CDEF, dtpT, livJHMGF, ilvC, ilvDBN, bcaT, ackA, ldh, codY et relA a été quantifiée chez les souches PB302 et PB18O en lait et en milieu M17. La souche PB302 est représentative des souches qui se développent rapidement en lait alors que la souche PB18O l’est de celles qui ont une croissance intermédiaire dans ce milieu. Alors que l’expression des gènes étudiés est peu différente en milieu M17 où les deux souches ont une croissance similaire, cette expression diverge lorsque les deux souches sont cultivées en lait.Globalement, la différence de croissance observée en lait entre les deux souches pourrait résulter d'une variabilité de la capacité protéolytique et de l’expression, entre autres, des gènes codant PrtS, le régulateur CodY, les transporteurs des oligopeptides (Ami), des di-tripeptides (DtpT) et des acides aminés ramifiés (LivJ) et de ceux codant des enzymes impliquées dans la voie de biosynthèse des acides aminés ramifiés (IlvC, IlvB et BcaT), ces derniers étant nécessaires pour la croissance en lait. Tous ces gènes possèdent en amont de leur promoteur une boîte CodY potentielle et pourraient donc appartenir au régulon CodYThe variability of the cell envelope-associated proteolytic system was studied in 30 strains of St. thermophilus. Variations in strains consist in the presence or absence of the gene prtS, the presence of two allelic forms of prtS, the presence of an anchored and/or soluble form of the protease PrtS and in the variable expression of the gene prtS and other genes involved mainly in nitrogen metabolism, thus in the variability of the regulation genetic of this system. Expression of the genes prtS, pepX, pepC, pepN, amiA1CDEF, dtpT, livJHMGF, ilvC, ilvDBN, bcaT, ackA, ldh, codY and relA was quantified in the PB302 and PB18O strains. The strain PB302 is representative of strains which exhibit a rapid growth in milk. The strain PB18O is representative those with intermediate growth in milk. In M17 medium, where both strains have similar growth, little difference in the expression of genes tested was observed. Conversely, the two strains did not express the selected genes in the same way when grown in milk. Overall, the difference in growth observed between strains in milk could result from variable proteolytic activities and variable expression of genes encoding, for example, the proteinase PrtS, the regulator CodY, transporters of oligo- or di-tri- peptides (Ami or DtpT) or branched chain amino acids, or BCAA (LivJ) and enzymes in the biosynthetic pathway of BCAA (IlvC, IlvB et BcaT) which are necessary for growth in milk. All these genes have a potential CodY box at the upstream of their promoter and could therefore belong to the regulon Cod

Caractérisation de la variabilité du système protéolytique de surface de la bactérie lactique Streptococcus thermophilus

La variabilité du système protéolytique de surface a été étudiée chez 30 souches de St. thermophilus. Cette variabilité consiste en la présence ou l absence du gène prtS, en la présence de deux allèles différents de ce gène, en la présence d'une protéase PrtS ancrée et/ou soluble et enfin en l'expression variable, due à une variabilité de la régulation du système protéolytique, du gène prtS et d'autres gènes qui interviennent, pour la plupart, dans le métabolisme azoté. L expression des gènes prtS, pepX, pepC, pepN, amiA1CDEF, dtpT, livJHMGF, ilvC, ilvDBN, bcaT, ackA, ldh, codY et relA a été quantifiée chez les souches PB302 et PB18O en lait et en milieu M17. La souche PB302 est représentative des souches qui se développent rapidement en lait alors que la souche PB18O l est de celles qui ont une croissance intermédiaire dans ce milieu. Alors que l expression des gènes étudiés est peu différente en milieu M17 où les deux souches ont une croissance similaire, cette expression diverge lorsque les deux souches sont cultivées en lait.Globalement, la différence de croissance observée en lait entre les deux souches pourrait résulter d'une variabilité de la capacité protéolytique et de l expression, entre autres, des gènes codant PrtS, le régulateur CodY, les transporteurs des oligopeptides (Ami), des di-tripeptides (DtpT) et des acides aminés ramifiés (LivJ) et de ceux codant des enzymes impliquées dans la voie de biosynthèse des acides aminés ramifiés (IlvC, IlvB et BcaT), ces derniers étant nécessaires pour la croissance en lait. Tous ces gènes possèdent en amont de leur promoteur une boîte CodY potentielle et pourraient donc appartenir au régulon CodYThe variability of the cell envelope-associated proteolytic system was studied in 30 strains of St. thermophilus. Variations in strains consist in the presence or absence of the gene prtS, the presence of two allelic forms of prtS, the presence of an anchored and/or soluble form of the protease PrtS and in the variable expression of the gene prtS and other genes involved mainly in nitrogen metabolism, thus in the variability of the regulation genetic of this system. Expression of the genes prtS, pepX, pepC, pepN, amiA1CDEF, dtpT, livJHMGF, ilvC, ilvDBN, bcaT, ackA, ldh, codY and relA was quantified in the PB302 and PB18O strains. The strain PB302 is representative of strains which exhibit a rapid growth in milk. The strain PB18O is representative those with intermediate growth in milk. In M17 medium, where both strains have similar growth, little difference in the expression of genes tested was observed. Conversely, the two strains did not express the selected genes in the same way when grown in milk. Overall, the difference in growth observed between strains in milk could result from variable proteolytic activities and variable expression of genes encoding, for example, the proteinase PrtS, the regulator CodY, transporters of oligo- or di-tri- peptides (Ami or DtpT) or branched chain amino acids, or BCAA (LivJ) and enzymes in the biosynthetic pathway of BCAA (IlvC, IlvB et BcaT) which are necessary for growth in milk. All these genes have a potential CodY box at the upstream of their promoter and could therefore belong to the regulon CodYNANCY-INPL-Bib. électronique (545479901) / SudocSudocFranceF

Acquisition of PrtS in Streptococcus thermophilus is not enough in certain strains to achieve rapid milk acidification

International audienceAbstractThe acquisition of prtS by Streptococcus thermophilus strains allowed hydrolysis of caseins into peptides and then to increase their growth in milk. This leads to faster milk acidification, which is important in dairy industry. However, some strains harboring the same allele of prtS present different acidification rates, which could be explained by a difference in the regulation of prtS expression. We chose two strains with the same allele of prtS (including the same promoter region): one, PB302, is with high acidification rate while the other, PB18O, is without. They exhibited similar growth in M17, but not in milk, where PB302 showed better growth. The expression of prtS and activity of PrtS were lower in PB18O, in the two media tested. We demonstrated that other genes known to be involved in carbon and nitrogen metabolism were overexpressed in PB302. Interestingly, these genes were overexpressed in milk compared to M17. Nearly all these genes possessed a putative CodY-box in their promoter region. Taken together, difference of gene expression detected in PB302 between milk (low-peptide medium) and M17 (rich-peptide medium) and presence of a putative CodY-box is a feature of the transcriptional pattern of CodY-regulated genes. Altogether, our results propose that acquisition of prtS is not enough in certain strains to achieve rapid milk acidification. High transcriptional level of dtpT, amiF, ilvC, ilvB, bcaT, livJ, ackA, codY, and prtS in fast acidifying strain suggests that this transcriptional pattern could be required for fast milk acidification in Streptococcus thermophilus

Relative Weight of Organic Waste Origin on Compost and Digestate 16S rRNA Gene Bacterial Profilings and Related Functional Inferences

International audienceEven though organic waste (OW) recycling via anaerobic digestion (AD) and composting are increasingly used, little is known about the impact of OW origin (fecal matters and food and vegetable wastes) on the end products’ bacterial contents. The hypothesis of a predictable bacterial community structure in the end products according to the OW origin was tested. Nine OW treatment plants were selected to assess the genetic structure of bacterial communities found in raw OW according to their content in agricultural and urban wastes and to estimate their modifications through AD and composting. Two main bacterial community structures among raw OWs were observed and matched a differentiation according to the occurrences of urban chemical pollutants. Composting led to similar 16S rRNA gene OTU profiles whatever the OW origin. With a significant shift of about 140 genera (representing 50% of the bacteria), composting was confirmed to largely shape bacterial communities toward similar structures. The enriched taxa were found to be involved in detoxification and bioremediation activities. This process was found to be highly selective and favorable for bacterial specialists. Digestates showed that OTU profiles differentiated into two groups according to their relative content in agricultural (manure) and urban wastes (mainly activated sludge). About one third of the bacterial taxa was significantly affected by AD. In digestates of urban OW, this sorting led to an enrichment of 32 out of the 50 impacted genera, while for those produced from agricultural or mixed urban/agricultural OW (called central OW), a decay of 54 genera over 60 was observed. Bacteria from activated sludge appeared more fit for AD than those of other origins. Functional inferences showed AD enriched genera from all origins to share similar functional traits, e.g., chemoheterotrophy and fermentation, while being often taxonomically distinct. The main functional traits among the dominant genera in activated sludge supported a role in AD. Raw OW content in activated sludge was found to be a critical factor for predicting digestate bacterial contents. Composting generated highly predictable and specialized community patterns whatever the OW origin. AD and composting bacterial changes were driven by functional traits selected by physicochemical factors such as temperature and chemical pollutants

Characterization of a new peptide transport system in Streptococcus thermophilus

In silk analysis of the genome of Streptococcus thermophilus LMD-9 revealed that this strain has a potential new peptide/nickel ABC transporter. We named this system OTS for Oligopeptide Transporter of S. thermophilus. It is composed of a peptide/nickel binding protein OtsA, two permeases OtsB and OtsC and a double ATPase OtsD. This system was presumably acquired by horizontal transfer from Actinobacteria or distant species like Lactococcus raffinolactis or Enterococcus asini may be via an intermediate like Lactococcus lactis or its ancestor. RT-PCR experiments proved that OTS gene cluster is transcribed and that at least the otsB, otsC, and otsD genes constitute an operon. A mutant LMD-9 Delta ots partially deleted for the otsA and otsB genes was constructed. Growth of LMD-9 and LMD-9 Delta ots strains was monitored in the presence of different nitrogen sources and in the presence of urea and nickel. Results revealed that OTS is not implicated in nickel transport but constitutes a new characterized transporter of peptides of small size, possibly di- and tripeptides in S. thermophilus. (C) 2016 Published by Elsevier Ltd

Strand-specific transcriptomes of Enterohemorrhagic Escherichia coli in response to interactions with ground beef microbiota: interactions between microorganisms in raw meat

France GenomiqueInternational audienceBackground: Enterohemorrhagic Escherichia coli (EHEC) are zoonotic agents associated with outbreaks worldwide. Growth of EHEC strains in ground beef could be inhibited by background microbiota that is present initially at levels greater than that of the pathogen E. coli. However, how the microbiota outcompetes the pathogenic bacteria is unknown. Our objective was to identify metabolic pathways of EHEC that were altered by natural microbiota in order to improve our understanding of the mechanisms controlling the growth and survival of EHECs in ground beef.Results: Based on 16S metagenomics analysis, we identified the microbial community structure in our beef samples which was an essential preliminary for subtractively analyzing the gene expression of the EHEC strains. Then, we applied strand-specific RNA-seq to investigate the effects of this microbiota on the global gene expression of EHEC O26(21765) and O157(EDL933) strains by comparison with their behavior in beef meat without microbiota. In strain O26(21765), the expression of genes connected with nitrate metabolism and nitrite detoxification, DNA repair, iron and nickel acquisition and carbohydrate metabolism, and numerous genes involved in amino acid metabolism were down-regulated. Further, the observed repression of ftsL and murF, involved respectively in building the cytokinetic ring apparatus and in synthesizing the cytoplasmic precursor of cell wall peptidoglycan, might help to explain the microbiota's inhibitory effect on EHECs. For strain O157(EDL933), the induced expression of the genes implicated in detoxification and the general stress response and the repressed expression of the peR gene, a gene negatively associated with the virulence phenotype, might be linked to the survival and virulence of O157:H7 in ground beef with microbiota.Conclusion: In the present study, we show how RNA-Seq coupled with a 16S metagenomics analysis can be used to identify the effects of a complex microbial community on relevant functions of an individual microbe within it. These findings add to our understanding of the behavior of EHECs in ground beef. By measuring transcriptional responses of EHEC, we could identify putative targets which may be useful to develop new strategies to limit their shedding in ground meat thus reducing the risk of human illnesses

New Insights into the Proteolytic System of Streptococcus thermophilus: Use of Isracidin To Characterize Cell-Associated Extracellular Peptidase Activities

International audienceThe influence on the hydrolysis of isracidin of cell-associated extracellular aminopeptidase and X-prolyl dipeptidyl peptidase activities in addition to protease PrtS of Streptococcus thermophilus strains was investigated. S. thermophilus LMD-9 (PrtS(+) phenotype) efficiently hydrolyzed the isracidin mainly through the PrtS activity, whereas strain CNRZ1066 (PrtS(-) phenotype) and two mutant strains LMD-9-Delta prtS and LMD-9-Delta prtS-Delta htrA also displayed substrate hydrolysis, but different from that of the wild type strain LMD-9. Identification by mass spectrometry of breakdown products of isracidin revealed the existence of novel cell-associated extracellular carboxypeptidase and peptidyl dipeptidase activities in all PrtS(-) strains, besides known cell-associated extracellular aminopeptidase and X-prolyl dipeptidyl peptidase activities. Both aminopeptidase and peptidyl dipeptidase activities were not able to cleave the isracidin at peptide bonds with proline residues. No hydrolysis of isracidin was detected in cell free filtrate for all the strains studied, indicating that no cell lysis had occurred. Taken together, these results suggested the presence of cell-associated extracellular peptidase activities in S. thermophilus strains that could be vital for the growth of PrtS(-) strains

Milk Fat Globules Hamper Adhesion of Enterohemorrhagic Escherichia coli to Enterocytes: In Vitro and in Vivo Evidence

International audienceEnterohemorrhagic Escherichia coli (EHEC; E. coli) are food-borne agents associated with gastroenteritis, enterocolitis, bloody diarrhea and the hemolytic-uremic syndrome (HUS). Bovine milk glycans have been shown to contain oligosaccharides which are similar to host epithelial cell receptors and can therefore prevent bacterial adhesion. This study aimed to describe interactions between EHEC O157:H7 EDL933 and O26:H11 21765 and milk fat globules (MFGs) in raw milk and raw milk cheese, and the impact of MFGs on EHEC strains adhesion to the intestinal tract in vitro and in vivo. Both EHEC serotypes clearly associated with native bovine MFGs and significantly limited their adhesion to a co-culture of intestinal cells. The presence of MFGs in raw milk cheese had two effects on the adhesion of both EHEC serotypes to the intestinal tracts of streptomycin-treated mice. First, it delayed and reduced EHEC excretion in mouse feces for both strains. Second, the prime implantation site for both EHEC strains was 6 cm more proximal in the intestinal tracts of mice fed with contaminated cheese containing less than 5% of fat than in those fed with contaminated cheese containing 40% of fat. Feeding mice with 40% fat cheese reduced the intestinal surface contaminated with EHEC and may therefore decrease severity of illness