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Many food fermentations are performed using mixed cultures of lactic acid bacteria. Interactions between strains are of key importance for the performance of these fermentations. Yogurt fermentation by Streptococcus thermophilus and Lactobacillus bulgaricus (basonym, Lactobacillus delbrueckii subsp. bulgaricus) is one of the best-described mixed-culture fermentations. These species are believed to stimulate each other's growth by the exchange of metabolites such as folic acid and carbon dioxide. Recently, postgenomic studies revealed that an upregulation of biosynthesis pathways for nucleotides and sulfur-containing amino acids is part of the global physiological response to mixed-culture growth in S. thermophilus, but an in-depth molecular analysis of mixed-culture growth of both strains remains to be established. We report here the application of mixed-culture transcriptome profiling and a systematic analysis of the effect of interaction-related compounds on growth, which allowed us to unravel the molecular responses associated with batch mixed-culture growth in milk of S. thermophilus CNRZ1066 and L. bulgaricus ATCC BAA-365. The results indicate that interactions between these bacteria are primarily related to purine, amino acid, and long-chain fatty acid metabolism. The results support a model in which formic acid, folic acid, and fatty acids are provided by S. thermophilus. Proteolysis by L. bulgaricus supplies both strains with amino acids but is insufficient to meet the biosynthetic demands for sulfur and branched-chain amino acids, as becomes clear from the upregulation of genes associated with these amino acids in mixed culture. Moreover, genes involved in iron uptake in S. thermophilus are affected by mixed-culture growth, and genes coding for exopolysaccharide production were upregulated in both organisms in mixed culture compared to monocultures. The confirmation of previously identified responses in S. thermophilus using a different strain combination demonstrates their generic value. In addition, the postgenomic analysis of the responses of L. bulgaricus to mixed-culture growth allows a deeper understanding of the ecology and interactions of this important industrial food fermentation process.

Bacterial communities of fermented foods are usually investigated by culture-dependent methods. Real-time quantitative PCR (qPCR) and reverse transcription (RT)-qPCR offer new possibilities to quantify the populations present and their metabolic activity. The aim of this work was to develop qPCR and RT-qPCR methods to assess the metabolic activity and the stress level of the two species used as ripening cultures in Emmental cheese manufacture, Propionibacterium freudenreichii and Lactobacillus paracasei. Three small scale (1/100) microbiologically controlled Emmental cheeses batches were manufactured and inoculated with Lactobacillus helveticus, Streptococcus thermophilus, P. freudenreichii and L. paracasei. At 12 steps of cheese manufacture and ripening, the populations of P. freudenreichii and L. paracasei were quantified by numerations on agar media and by qPCR. 16S, tuf and groL transcript levels were quantified by RT-qPCR. Sampling was carried out in triplicate. qPCR and RT-qPCR assessments were specific, efficient and linear. The quantification limit was 10(3) copies of cells or cDNA/g of cheese. Cell quantifications obtained by qPCR gave similar results than plate count for P. freudenreichii growth and 0.5 to 1 log lower in the stationary phase. Bacterial counts and qPCR quantifications showed that L. paracasei began to grow during the pressing step while P. freudenreichii began to grow from the beginning of ripening (in the cold room). Tuf cDNA quantification results suggested that metabolic activity of L. paracasei reached a maximum during the first part of the ripening (in cold room) and decreased progressively during ripening (in the warm room). Metabolic activity of P. freudenreichii was maximum at the end of cold ripening room and was stable during the first two weeks in warm room. After lactate exhaustion (after two weeks of warm room), the number of tuf cDNA decreased reflecting reduced metabolic activity. For L. paracasei, groL cDNA were stable during ripening. For P. freudenreichii, groL1 gene was highly-expressed during acidification, while groL2 gene highly expression was only observed at the end of the ripening stage after lactate (carbon substrate of P. freudenreichii) exhaustion. The potential use of 16S and tuf genes for the normalization of cDNA quantification throughout an Emmental cheese manufacture is discussed. For the first time, specific gene expression was performed by RT-qPCR yielding metabolic activity and stress response evaluation for L. paracasei and P. freudenreichii in cheese., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Bacteria and Archaea have developed several defence strategies against foreign nucleic acids such as viral genomes and plasmids. Among them, clustered regularly interspaced short palindromic repeats (CRISPR) loci together with cas (CRISPR-associated) genes form the CRISPR/Cas immune system, which involves partially palindromic repeats separated by short stretches of DNA called spacers, acquired from extrachromosomal elements. It was recently demonstrated that these variable loci can incorporate spacers from infecting bacteriophages and then provide immunity against subsequent bacteriophage infections in a sequence-specific manner. Here we show that the Streptococcus thermophilus CRISPR1/Cas system can also naturally acquire spacers from a self-replicating plasmid containing an antibiotic-resistance gene, leading to plasmid loss. Acquired spacers that match antibiotic-resistance genes provide a novel means to naturally select bacteria that cannot uptake and disseminate such genes. We also provide in vivo evidence that the CRISPR1/Cas system specifically cleaves plasmid and bacteriophage double-stranded DNA within the proto-spacer, at specific sites. Our data show that the CRISPR/Cas immune system is remarkably adapted to cleave invading DNA rapidly and has the potential for exploitation to generate safer microbial strains.

This study characterized a glycoside hydrolase family 42 (GH42) β-galactosidase of Lactobacillus acidophilus (LacA) and compared lactose hydrolysis, hydrolysis of oNPG, pNPG and pNPG-analogues and galactooligosaccharides (GOSs) formation to GH2 β-galactosidases of Streptococcus thermophilus (LacZ type), Lactobacillus plantarum and Leuconostoc mesenteroides subsp. cremoris (both LacLM type). Beta-galactosidases were heterologously expressed in Lactococcus lactis using a p170 derived promoter; experiments were performed with L. lactis crude cell extract (CCE). The novel GH42 β-galactosidase of Lb. acidophilus had lower activity on lactose, oNPG and pNPG but higher relative activity on pNP analogues compared to GH2 β-galactosidases, and did not transgalactosylate at high lactose concentrations. Temperature and pH optima for lactose hydrolysis varied between GH2 β-galactosidases. oNPG and pNPG were the preferred substrates for hydrolysis; in comparison, activity on pNPG-analogues was less than 1.5%. GH2 β-galactosidases formed structurally similar GOS with varying preferences. The diversity of lactic acid bacteria β-galactosidase activity in L. lactis CCE can be exploited in future nutritional or therapeutic applications., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Histamine, a toxic compound that is formed by the decarboxylation of histidine through the action of microbial decarboxylases, can accumulate in fermented food products. From a total of 69 Streptococcus thermophilus strains screened, two strains, CHCC1524 and CHCC6483, showed the capacity to produce histamine. The hdc clusters of S. thermophilus CHCC1524 and CHCC6483 were sequenced, and the factors that affect histamine biosynthesis and histidine-decarboxylating gene (hdcA) expression were studied. The hdc cluster began with the hdcA gene, was followed by a transporter (hdcP), and ended with the hdcB gene, which is of unknown function. The three genes were orientated in the same direction. The genetic organization of the hdc cluster showed a unique organization among the lactic acid bacterial group and resembled those of Staphylococcus and Clostridium species, thus indicating possible acquisition through a horizontal transfer mechanism. Transcriptional analysis of the hdc cluster revealed the existence of a polycistronic mRNA covering the three genes. The histidine-decarboxylating gene (hdcA) of S. thermophilus demonstrated maximum expression during the stationary growth phase, with high expression levels correlated with high histamine levels. Limited expression was evident during the lag and exponential growth phases. Low-temperature (4 degrees C) incubation of milk inoculated with a histamine-producing strain showed lower levels of histamine than did inoculated milk kept at 42 degrees C. This reduction was attributed to a reduction in the activity of the HdcA enzyme itself rather than a reduction in gene expression or the presence of a lower cell number.

The gene encoding beta-galactosidase from dairy Streptococcus thermophilus strain LMD9 was cloned, sequenced and expressed in Escherichia coli. The recombinant enzyme was purified and showed high specific activity of 464 U/mg. This protein displays a homotetrameric arrangement composed of four 118 kDa monomers. Monitoring of the activity showed that this enzyme was optimally active at a wide range of temperatures (25-40 degrees C) and at pH from 6.5 to 7.5. Immobilization of the recombinant E. coli in alginate beads clearly enhanced the enzyme activity at various temperatures, including 4 and 50 degrees C, and at pH values from 4.0 to 8.5. Stability studies indicated that this biocatalyst has high stability within a broad range of temperatures and pH. This stability was improved not only by addition of 1 mM of Mn(2+) and 1.2 mM Mg(2+), but essentially through immobilization. The remarkable bioconversion rates of lactose in milk and whey at different temperatures revealed the attractive catalytic efficiency of this enzyme, thus promoting its use for lactose hydrolysis in milk and other dairy products., (Copyright 2010 Elsevier Masson SAS. All rights reserved.)

Objective: To develop a PCR method for identifying the 16S rRNA of Lactobacillus and Streptococcus thermophilus at the species level., Methods: Optimizing the method for DNA extraction and the conditions for PCR amplification. Joining the PCR amplification products from 16S rRNA to plasmid puc18-T and detecting the sequence., Results: All 50 isolates recovered from yoghourt products were characterized by 16S rRNA sequence analysis and 7 groups were identified as L. bulgaricus (24 strains), S. thermophilus (12 strains), L. acidophilus (7 strains), L. casei (3 strains), L. delbrueckii (2 strains), L. fermentum (1 strain) and S. lutetiensis (1 strain)., Conclusion: 16S rRNA PCR method developed in this research is a sensitive and reliable method for the identification of both Lactobacillus and Streptococcus thermophilus.

The cumulative effect of peptidase and protease activities associated with cells of Streptococcus thermophilus (ST) and Lactobacillus delbrueckii subsp. bulgaricus (LB) was evaluated on the milk protein-based antimicrobial peptides casocidin and isracidin. Reaction mixtures of casocidin or isracidin and nonproliferating mid-log cells of these essential yogurt starter cultures were individually incubated for up to 4 h at pH 4.5 and 7.0, and samples removed at various time points were analyzed by reverse phase-high performance liquid chromatography (RP-HPLC) and MALDI-TOF/TOF-MS. Both casocidin and isracidin remained largely unchanged following exposure to cell suspensions of ST or LB strains at pH 4.5. Casocidin was extensively degraded by both ST and LB strains at pH 7.0, whereas isracidin remained largely intact after incubation for 4 h with ST strains but was degraded by exposure to LB strains. The results showed the feasibility of using the bovine casein-based peptides casocidin and isracidin as food grade antimicrobial supplements to impart fermented dairy foods additional protection against bacterial contamination. The structural integrity and efficacy of these biodefensive peptides may be preserved by timing their addition near the end of the fermentation of yogurt-like dairy foods (at or below pH 4.5), when conditions for bacterial proteolytic activity become unfavorable.

Aims: To test whether a single vector, nisin-controlled expression (NICE) system could be used to regulate expression of the pediocin operon in Streptococcus thermophilus, Lactococcus lactis subsp. lactis and Lactobacillus casei., Methods and Results: The intact pediocin operon was cloned immediately into pMSP3535 downstream of the nisA promoter (PnisA). The resulting vector, pRSNPed, was electrotransformed into Strep. thermophilus ST128, L. lactis subsp. lactis ML3 and Lact. casei C2. Presence of the intact vector was confirmed by PCR, resulting in the amplification of a 0.8-kb DNA fragment, and inhibition zones were observed for all lactic acid bacteria (LAB) transformants following induction with 50 ng ml(-1) nisin, when Listeria monocytogenes Scott A was used as the target bacterium. Using L. monocytogenes NR30 as target, the L. lactis transformants produced hazy zones of inhibition, while the Lact. casei transformants produced clear zones of inhibition. Zones of inhibition were not observed when the Strep. thermophilus transformants were tested against NR30., Conclusions: The LAB hosts were able to produce enough pediocin to inhibit the growth of L. monocytogenes Scott A; the growth of L. monocytogenes NR30 was effectively inhibited only by the Lact. casei transformants., Significance and Impact of the Study: This is the first time that the NICE system has been used to express the intact pediocin operon in these LAB hosts. This system could allow for the in situ production of pediocin in fermented dairy foods supplemented with nisin to prevent listeria contamination.

We previously reported the strong immunostimulatory effects of a CpG oligodeoxynucleotide (ODN), designated MsST, from the lacZ gene of Streptococcus (S.) thermophilus ATCC19258. Here we show that 24h of stimulation with MsST in mouse splenocytes and peritoneal macrophages strongly induces expression of interleukin (IL)-33, a cytokine in the IL-1 superfamily. Other IL-1 superfamily members, including IL-1alpha, IL-1beta and IL-18, are down-regulated after 24h of stimulation of MsST. We also found that MsST-induced IL-33 mRNA expression is inhibited by the suppressive ODN A151, which can inhibit Toll-like receptor 9 (TLR9)-mediated responses. This is the first report to show that IL-33 can be induced by CpG ODNs. The strong induction of IL-33 by MsST suggests that it may be a potential therapeutic ODN for the treatment of inflammatory disease. The presence of a strong CpG ODN in S. thermophilus also suggests that the bacterium may be a good candidate as a starter culture for the development of new physiologically functional foods., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Aims: An efficient approach for generation of bacteriophage-insensitive mutants (BIMs) of Streptococcus thermophilus starters was described in our laboratory [Mills et al. (2007) J Microbiol Methods70, 159-164]. The aim of this study was to analyse the phage resistance mechanism responsible for BIM formation., Methods and Results: Three clustered regularly interspaced short palindromic repeat (CRISPR) regions have been identified in Strep. thermophilus, and Strep. thermophilus can integrate novel spacers into these loci in response to phage attack. Characterization of three sets of BIMs indicated that two sets had altered CRISPR1 and/or CRISPR3 loci. A range of BIMs of yoghurt starter CSK938 were generated with the same phage in different phage challenge experiments, and each acquired unique spacer regions ranging between one and four new spacers in CRISPR1. In addition, the BIM that acquired only one new spacer in CRISPR1 also acquired an additional spacer in CRISPR3. A fourth BIM, generated with a different phage, had two spacers deleted from CRISPR1 but acquired two spacers in CRISPR3. Analysis of the Mozzarella starter CSK939 and its associated BIMs indicated that formation of second generation BIMs does not lead to increases in spacer number but to alterations in spacer regions. BIMs of an exopolysaccharide (EPS)-producing strain that lost the ability to produce EPS did not harbour an altered CRISPR, suggesting that phage sensitivity may be related to the EPS-producing phenotype., Conclusions: Acquisition/deletion of new spacers in CRISPR loci in response to phage attack generates distinctly individual variants. It also demonstrates that other modifications may be responsible for the phage resistance of Strep. thermophilus BIMs., Significance and Impact of the Study: Isolation of individual BIMs that have unique spacers towards the leader region of the CRISPR locus may be a very useful approach for rotation strategies with the same starter backbone. Upon phage infection, BIMs 'in reserve' can be slotted into the rotation scheme.

Aims: To develop a general method for site-directed mutagenesis in the dairy starter strain Streptococcus thermophilus LMG 18311 which does not depend on antibiotic-resistance genes or other selection markers for the identification of transformants., Methods and Results: In a previous study, we demonstrated that Strep. thermophilus LMG 18311 can be made competent for natural genetic transformation by overexpression of the alternative sigma factor ComX. In the present study, we wanted to investigate whether the natural transformation mechanism of Strep. thermophilus LMG 18311 is efficient enough to make it feasible to perform site-directed mutagenesis in this strain without the use of a selection marker. Competent bacteria were mixed with a DNA fragment engineered to contain a nonsense and a frameshift mutation in the middle of the target gene (lacZ) and subsequently seeded on agar plates. By performing colony-lift hybridization using a digoxigenin-labelled oligonucleotide probe, we succeeded in identifying transformants containing the sought after mutation., Conclusions: By exploiting the natural transformability of Strep. thermophilus LMG 18311 and standard molecular methods, we have demonstrated that the genome of this bacterium can be altered at preselected sites without introduction of any foreign DNA., Significance and Impact of the Study: A food-grade site-directed mutagenesis system has been developed for Strep. thermophilus LMG 18311 that can be used by the dairy industry to construct starter strains with novel and/or improved properties.

Forty-three samples of kurut, a kind of traditional naturally-fermented yak milk, were collected in Qinghai, China. One hundred and forty-eight lactic acid bacterial (LAB) strains were isolated and identified from the kurut samples according to phenotypic characterization and 16S rRNA gene sequence analysis. Among them, 52 isolates belonged to the Lactobacillus strains. Ninety-six isolates were resolved to coccoid LAB. The results showed that these isolates belonged to five genera and thirteen different species and subspecies. Moreover, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus were the predominant population in these samples. This paper systematically studied the composition of LAB in kurut, which may provide raw data for further study involving probiotics strain selection and starter culture design for industrialization production of traditional fermented milk in the future.

Aims: To investigate the effect of an absence of aminopeptidase PepS on the growth of Streptococcus thermophilus on different media and at different temperatures., Methods and Results: Using gene interruption, a negative mutant of the Strep. thermophilus CNRZ385 strain was constructed for the aminopeptidase PepS (strain DeltapepS). Checks were first of all made using biochemical assays that the DeltapepS strain lacks the peptide hydrolase activity of aminopeptidase PepS. It was demonstrated that the absence of the aminopeptidase PepS exerted a negative effect on growth whatever the culture medium (M17, chemically defined medium, milk). The role of aminopeptidase PepS in growth was enhanced at a high temperature (45 degrees C vs 37 degrees C). The DeltapepS strain was more resistant to lysozyme than the wild-type strain., Conclusions: We were able to demonstrate that aminopeptidase PepS probably plays a pleiotropic role through its involvement in growth via nitrogen nutrition, as well as via other cellular functions/metabolisms (such as peptidoglycane metabolism)., Significance and Impact of the Study: This study constitutes the first report on the role of a member of the M29 MEROPS family of metallopeptidases (http://merops.sanger.ac.uk/).

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.

Folate, a water-soluble vitamin, includes naturally occurring food folate and synthetic folic acid in supplements and fortified foods. Mammalian cells cannot synthesize folate and its deficiency has been implicated in a wide variety of disorders. A number of reviews have dwelt up on the health benefits associated with increased folate intakes and many countries possess mandatory folate enrichment programs. Lately, a number of studies have shown that high intakes of folic acid, the chemically synthesized form, but not natural folates, can cause adverse effects in some individuals such as the masking of the hematological manifestations of vitamin B(12) deficiency, leukemia, arthritis, bowel cancer, and ectopic pregnancies. As fermented milk products are reported to contain even higher amounts of folate produced by the food-grade bacteria, primarily lactic acid bacteria (LAB), the focus has primarily shifted toward the natural folate, that is, folate produced by LAB and levels of folate present in foods fermented by/or containing these valuable microorganisms. The proper selection and use of folate-producing microorganisms is an interesting strategy to increase "natural" folate levels in foods. An attempt has been made through this review to share information available in the literature on wide ranging aspects of folate, namely, bioavailability, analysis, deficiency, dietary requirements, and health effects of synthetic and natural folate, dairy and nondairy products as a potential source of folate, microorganisms with special reference to Streptococcus thermophilus as prolific folate producer, and recent insight on modulation of folate production levels in LAB by metabolic engineering.

Aims: To study the transcriptional analysis of glutamate dehydrogenase gene, involved in the amino acid conversion to aroma compound in Streptococcus thermophilus., Methods and Results: Analysis of the gdhA gene nucleotide sequence of S. thermophilus CNRZ1066 revealed that the coding region is 1353 nucleotides long. The deduced amino acids sequence exhibits the putative GDH active site and some conserved domains characteristic of family I of hexameric GDHs. Phylogenetic analysis revealed that the gdh gene of S. thermophilus clustered with the orthologues of other streptococci such as Streptococcus mutans, Streptococcus agalactiae and Streptococcus infantarius. Studying the structural organization of the gdhA locus the amino acid similarity of GDHs was higher than 87%, but the locus organization was not conserved. A dominant transcript of approximately 1.4 kbp was revealed by Northern blot hybridization, suggesting that gdhA mRNA is monocystronic. Primer extension showed that transcription start point of gdhA was localized 43 bp upstream of the potential start codon (ATG)., Conclusions: The gdhA represents a monocistronic operon highly conserved in phylogenetic-related bacteria., Significance and Impact of the Study: A deeper knowledge of gdh transcriptional mechanisms could lead to develop S. thermophilus industrial starter cultures with optimized aromatic properties.

Immunostimulatory sequences of oligodeoxynucleotides (ODNs), such as CpG ODNs, are potent stimulators of innate immunity. Here, we identified a strong immunostimulatory CpG ODN, which we named MsST, from the lac Z gene of Streptococcus (S.) thermophilus ATCC19258, and we evaluated its immune functions. In in vitro studies, MsST had a similar ability as the murine prototype CpG ODN 1555 to induce inflammatory cytokine production and cell proliferation. In mouse splenocytes, MsST increased the number of CD80+CD11c+and CD86+CD11c+ dendritic cells and CD4+CD25+ regulatory T cells. We also analyzed the effects of MsST on the expression of regulatory cytokines by real-time quantitative PCR. MsST was more potent at inducing interleukin-10 expression than the ODN control 1612, indicating that MsST can augment the regulatory T cell response via Toll-like receptor 9, which plays an important role in suppressing T helper type 2 responses. These results suggest that S. thermophilus, whose genes include a strong Immunostimulatory sequence-ODN, is a good candidate for a starter culture to develop new physiologically functional foods and feeds.

Streptococcus thermophilus is a lactic acid bacteria (LAB) widely used in milk fermentation processes as a starter culture. In this work the genetic diversity of S. thermophilus isolates from different sources was analyzed using Amplified Fragment Length Polymorphism fingerprinting (AFLP). Since this is the first report that indicates the application of AFLP in order to study genotypic polymorphism in S. thermophilus species, an optimization of experimental conditions was carried out to decide the optimal AFLP analysis protocol. Furthermore the fingerprinting resolutions of AFLP and RAPD (Random Amplified Polymorphic DNA) were evaluated and compared. The overall data suggest that genotypic characterization performed by AFLP provide a better view of microbial diversity of S. thermophilus, indicating that RAPD is less discriminating than AFLP. The successful use of AFLP analysis in the characterization of S. thermophilus strains reported in this study suggests the potential uses for this technique to define the whole-genome diversity of each specific strain, as an alternative to the fingerprinting methods used till now.

Possible promoter regions preceding 14 genes belonging to the proteolytic system of Streptococcus thermophilus KLDS 3.0503 were predicted by a promoter analysis software nnpp. The 14 genes included an extracellular protease gene prtS, an oligopeptide ABC transport system gene amiA1, and 12 genes, respectively, encoding peptidases pepA, pepS, pepN, pepC, pepB, pepQ, pepV, pepT, pepM, pepXP, pepP, and pepO. These predicted promoter sequences were cloned and inserted into the upstream of a promoterless Escherichia coli gusA (beta-glucuronidase) gene in a promoter probe vector pNZ273. The resulting vectors were, respectively, introduced into S. thermophilus KLDS 3.0503 and all 14 predicted promoter sequences were able to drive gusA expression, which indicated that these sequences were functional promoters. These promoters were able to interact with the S. thermophilus CodY homolog in an in vitro DNA binding assay but they did not contain a conserved CodY-box sequence identified in Lactococcus lactis. These results were useful for further studies on the regulation of protein metabolism in S. thermophilus.

Aim: The aim of the study was to study the role of carbon dioxide metabolism in Streptococcus thermophilus through investigation of the phenotype of a carbamoylphosphate synthetase-negative mutant., Methods and Results: The effect of carbon dioxide on the nutritional requirements of Strep. thermophilus DSM20617(T) and its derivative, carbamoylphosphate synthetase-negative mutant A17(DeltacarB), was investigated by cultivating the strain in a chemically defined medium under diverse gas compositions and in milk. The results obtained revealed that CO(2) depletion or carB gene inactivation determined the auxotrophy of Strep. thermophilus for l-arginine and uracil. In addition, the parent strain grew faster than the mutant, even when milk was supplemented with uracil or arginine., Conclusions: Milk growth experiments underlined that carbamoylphosphate synthetase activity was essential for the optimal growth of Strep. thermophilus in milk., Significance and Impact of the Study: The study of the carbon dioxide metabolism in Strep. thermophilus revealed new insights with regard to the metabolism of this species, which could be useful for the optimization of dairy fermentation processes.

In this report, we describe the amino acid metabolism and amino acid dependency of the dairy bacterium Streptococcus thermophilus LMG18311 and compare them with those of two other characterized lactic acid bacteria, Lactococcus lactis and Lactobacillus plantarum. Through the construction of a genome-scale metabolic model of S. thermophilus, the metabolic differences between the three bacteria were visualized by direct projection on a metabolic map. The comparative analysis revealed the minimal amino acid auxotrophy (only histidine and methionine or cysteine) of S. thermophilus LMG18311 and the broad variety of volatiles produced from amino acids compared to the other two bacteria. It also revealed the limited number of pyruvate branches, forcing this strain to use the homofermentative metabolism for growth optimization. In addition, some industrially relevant features could be identified in S. thermophilus, such as the unique pathway for acetaldehyde (yogurt flavor) production and the absence of a complete pentose phosphate pathway.

Lactobacillus bulgaricus and Streptococcus thermophilus, used in yogurt starter cultures, are well known for their stability and protocooperation during their coexistence in milk. In this study, we show that a close interaction between the two species also takes place at the genetic level. We performed an in silico analysis, combining gene composition and gene transfer mechanism-associated features, and predicted horizontally transferred genes in both L. bulgaricus and S. thermophilus. Putative horizontal gene transfer (HGT) events that have occurred between the two bacterial species include the transfer of exopolysaccharide (EPS) biosynthesis genes, transferred from S. thermophilus to L. bulgaricus, and the gene cluster cbs-cblB(cglB)-cysE for the metabolism of sulfur-containing amino acids, transferred from L. bulgaricus or Lactobacillus helveticus to S. thermophilus. The HGT event for the cbs-cblB(cglB)-cysE gene cluster was analyzed in detail, with respect to both evolutionary and functional aspects. It can be concluded that during the coexistence of both yogurt starter species in a milk environment, agonistic coevolution at the genetic level has probably been involved in the optimization of their combined growth and interactions.

Tetracycline-resistant Streptococcus thermophilus isolates from soft cheeses harbored the genes tet(S), tet(M), and tet(L). Molecular analysis of these genes revealed their expression, localization on plasmids or Tn916-Tn1545 family transposons, and their similarity with published sequences. The study highlights the importance of an accurate safety assessment of using S. thermophilus as a starter culture.

An integrative vector was constructed for inserting heterologous genes within a non-functional open reading frame (ORF) on the chromosome of Streptococcus thermophilus. The vector, pINTRS, contained a temperature sensitive origin of replication and an erythromycin resistance gene for initial selection in S. thermophilus. The region of the vector containing unique cloning sites, for insertion of recombinant genes, was flanked by homologous DNA sequences corresponding to a pseudogene in S. thermophilus to facilitate chromosomal integration. The gene encoding green fluorescent protein, regulated by a plasmid borne hsp promoter of S. thermophilus, was cloned into pINTRS to demonstrate proper functioning of the vector.

Integrative and conjugative elements (ICEs), also called conjugative transposons, are genomic islands that excise, self-transfer by conjugation, and integrate in the genome of the recipient bacterium. The current investigation shows the intraspecies conjugative transfer of the first described ICEs in Streptococcus thermophilus, ICESt1 and ICESt3. Mitomycin C, a DNA-damaging agent, derepresses ICESt3 conjugative transfer almost 25-fold. The ICESt3 host range was determined using various members of the Firmicutes as recipients. Whereas numerous ICESt3 transconjugants of Streptococcus pyogenes and Enterococcus faecalis were recovered, only one transconjugant of Lactococcus lactis was obtained. The newly incoming ICEs, except the one from L. lactis, are site-specifically integrated into the 3' end of the fda gene and are still able to excise in these transconjugants. Furthermore, ICESt3 was retransferred from E. faecalis to S. thermophilus. Recombinant plasmids carrying different parts of the ICESt1 recombination module were used to show that the integrase gene is required for the site-specific integration and excision of the ICEs, whereas the excisionase gene is required for the site-specific excision only.

Streptococcus thermophilus is one of the most widely used lactic acid bacteria in the dairy industry, in particular in yoghurt manufacture, where it is associated with Lactobacillus delbrueckii subsp. bulgaricus. This bacterial association, known as a proto-cooperation, is poorly documented at the molecular and regulatory levels. We thus investigate the kinetics of the transcriptomic and proteomic modifications of S. thermophilus LMG 18311 in response to the presence of L. delbrueckii subsp. bulgaricus ATCC 11842 during growth in milk at two growth stages. Seventy-seven different genes or proteins (4.1% of total coding sequences), implicated mainly in the metabolism of nitrogen (24%), nucleotide base (21%), and iron (20%), varied specifically in coculture. One of the most unpredicted results was a significant decrease of most of the transcripts and enzymes involved in purine biosynthesis. Interestingly, the expression of nearly all genes potentially encoding iron transporters of S. thermophilus decreased, whereas that of iron-chelating dpr as well as that of the fur (perR) regulator genes increased, suggesting a reduction in the intracellular iron concentration, probably in response to H(2)O(2) production by L. bulgaricus. The present study reveals undocumented nutritional exchanges and regulatory relationships between the two yoghurt bacteria, which provide new molecular clues for the understanding of their associative behavior.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated CRISPR-associated sequence (CAS) proteins constitute a novel antiviral defence system that is widespread in prokaryotes. Repeats are separated by spacers, some of them homologous to sequences in mobile genetic elements. Although the whole process involved remains uncharacterized, it is known that new spacers are incorporated into CRISPR loci of the host during a phage challenge, conferring specific resistance against the virus. Moreover, it has been demonstrated that such interference is based on small RNAs carrying a spacer. These RNAs would guide the defence apparatus to foreign molecules carrying sequences that match the spacers. Despite this essential role, the spacer uptake mechanism has not been addressed. A first step forward came from the detection of motifs associated with spacer precursors (proto-spacers) of Streptococcus thermophilus, revealing a specific recognition of donor sequences in this species. Here we show that the conservation of proto-spacer adjacent motifs (PAMs) is a common theme for the most diverse CRISPR systems. The PAM sequence depends on the CRISPR-CAS variant, implying that there is a CRISPR-type-specific (motif-directed) choice of the spacers, which subsequently determines the interference target. PAMs also direct the orientation of spacers in the repeat arrays. Remarkably, observations based on such polarity argue against a recognition of the spacer precursors on transcript RNA molecules as a general rule.

Cell separation is dependent on cell wall hydrolases that cleave the peptidoglycan shared between daughter cells. In Streptococcus thermophilus, this step is performed by the Cse protein whose depletion resulted in the formation of extremely long chains of cells. Cse, a natural chimeric enzyme created by domain shuffling, carries at least two important domains for its activity: the LysM expected to be responsible for the cell wall-binding and the CHAP domain predicted to contain the active centre. Accordingly, the localization of Cse on S. thermophilus cell surface has been undertaken by immunogold electron and immunofluorescence microscopies using of antibodies raised against the N-terminal end of this protein. Immunolocalization shows the presence of the Cse protein at mature septa. Moreover, the CHAP domain of Cse exhibits a cell wall lytic activity in zymograms performed with cell walls of Micrococcus lysodeikticus, Bacillus subtilis and S. thermophilus. Additionally, RP-HPLC analysis of muropeptides released from B. subtilis and S. thermophilus cell wall after digestion with the CHAP domain shows that Cse is an endopeptidase. Altogether, these results suggest that Cse is a cell wall hydrolase involved in daughter cell separation of S. thermophilus.

Aims: To identify the chemical structure of a bacteriocin, thermophilin 1277, produced by Streptococcus thermophilus SBT1277., Methods and Results: Thermophilin 1277 was purified and partial N-terminal sequence analysis revealed 6 unidentified amino acids amongst 31 amino acids residues. A 2.7-kbp region containing the thermophilin 1277 structural gene (tepA) encoding 58 amino acids was cloned and sequenced. Mature thermophilin 1277 (33 amino acids) was preceded by a 25-amino acid putative leader peptide containing a double glycine cleavage motif. Peptide sequence analysis following chemical modification of thermophilin 1277 revealed that the Cys21 and Cys29 residues form a disulfide bridge and that Thr8 or Thr10 forms two 3-methyllanthionines with Cys13 or Cys32 via thioether bridges. Antimicrobial activity was disrupted by ethanethiol or reductive agent treatments, indicating that the internal amino acid modifications are crucial for the activity., Conclusions: Thermophilin 1277 from Strep. thermophilus SBT1277 belongs to the class of AII-type lantibiotics that has a disulfide and two thioether bridges., Significance and Impact of the Study: This is the first report of a lantibiotic produced by a GRAS species of Strep. thermophilus; thermophilin 1277 has a unique structure containing both a disulfide bridge and two thioether bridges that are crucial for its activity.

To prevent textural defects in low-fat and fat-free yogurts, fat substitutes are routinely added to milk. In situ production of exopolysaccharides (EPS) by starter cultures is an acknowledged alternative to the addition of biothickeners. With the aim of increasing in situ EPS production, a recombinant galactose-positive EPS(+) Streptococcus thermophilus strain, RD-534-S1, was generated and compared with the parent galactose-negative EPS(+) strain RD-534. The RD-534-S1 strain produced up to 84 mg/L of EPS during a single-strain milk fermentation process, which represented 1.3 times more than the EPS produced by strain RD-534. Under conditions that mimic industrial yogurt production, the starter culture consisting of RD-534-S1 and (EPS(-)) Lactobacillus bulgaricus L210R strain (RD-534-S1/L210R) led to an EPS production increase of 1.65-fold as compared with RD-534-S1 alone. However, the amount of EPS produced did not differ from that found in yogurts produced using an isogenic starter culture that included the parent S. thermophilus strain RD-534 and Lb. bulgaricus L210R (RD-534/L210R). Moreover, the gel characteristics of set-style yogurt and the rheological properties of stirred-style yogurt produced using RD-534-S1/L210R were similar to the values obtained for yogurts made with RD-534/L210R. In conclusion, it is possible to increase the production of EPS by ropy S. thermophilus strains through genetic engineering of galactose metabolism. However, when used in combination with Lb. bulgaricus for yogurt manufacture, the EPS overproduction of recombinant strain is not significant.

The identification of 5 probiotic strains of lactic acid bacteria has been conducted by the method of analysis of physiological properties, API-testing as well as by molecular-genetic methods. It has been confirmed that 4 strains belong to Lactobacillus plantarum, Enterococcus faecium and Streptococcus thermophilus species. S. thermophilus strain has been reidentified as E. durans. The importance of clear identification of microorganism species which is used in probiotics has been demonstrated.

Yoghurt and starter culture producers are still searching strains of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus to produce healthier yogurt with longer shelf life, better texture, taste and quality. However, selective identification of Lb. delbrueckii subsp. bulgaricus and Strep. thermophilus from a mixed population using microbiological and biochemical methods is difficult, time consuming and may not be accurate. In this study, a quick, sensitive and accurate method is proposed to identify both Lb. delbrueckii subsp. bulgaricus and Strep. thermophilus using PCR. The method is comprised of two parts. In the first part, methionine biosynthesis genes, known to be present in both species were partially amplified by designed primers (cysmet2F and cysmet2R). Partial amplification of the methionine biosynthesis gene which gives 700 bp fragment resulted in selective identification of Lb. bulgaricus and Strep. thermophilus. All 16 Lb. bulgaricus and 6 Strep. thermophilus isolates assessed by this method gave the expected amplification. On the other hand, further analysis of other closely related species with the same primers have indicated that the same product was also amplified in two more lactobacilli namely, Lb. delbrueckii subsp. lactis and Lb. helveticus species. Thus, in the second part of the method, further differentiation of Lb. delbrueckii subsp. bulgaricus and Strep. thermophilus from each other and these species was achieved using restriction analysis of 16S rRNA gene with EcoRI.

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.

A DNA microarray platform based on 2,200 genes from publicly available sequences was designed for Streptococcus thermophilus. We determined how single-nucleotide polymorphisms in the 65- to 75-mer oligonucleotide probe sequences affect the hybridization signals. The microarrays were then used for comparative genome hybridization (CGH) of 47 dairy S. thermophilus strains. An analysis of the exopolysaccharide genes in each strain confirmed previous findings that this class of genes is indeed highly variable. A phylogenetic tree based on the CGH data showed similar distances for most strains, indicating frequent recombination or gene transfer within S. thermophilus. By comparing genome sizes estimated from the microarrays and pulsed-field gel electrophoresis, the amount of unknown DNA in each strain was estimated. A core genome comprised of 1,271 genes detected in all 47 strains was identified. Likewise, a set of noncore genes detected in only some strains was identified. The concept of an industrial core genome is proposed. This is comprised of the genes in the core genome plus genes that are necessary in an applied industrial context.

The lactose transporter and beta-galactosidase from Streptococcus thermophilus, encoded by the lacSZ operon, were introduced into the lactose-negative strain Lactococcus lactis MG1363 and the expression of the lacSZ operon was modulated by substitution of the native promoter with randomized synthetic promoters. A series of strains with various expression levels of lacSZ were examined for their fermentation of lactose. Strains with a high expression level were found to metabolize lactose in a similar manner to S. thermophilus, i.e. the galactose moiety of lactose was excreted to the growth medium and only glucose was metabolized in glycolysis. Interestingly, strains with low expression of the operon showed a mixed acid metabolism and co-metabolism of galactose and glucose. The lactose flux increased gradually with increasing expression of the lacSZ operon until an optimum was observed at intermediate beta-galactosidase activities of 2000-3000 Miller units. At higher expression levels, the flux decreased. These strains had a glycolytic flux comparable with those of reference strains with the standard lactococcal PTS(lac) (lactose phosphotransferase transport system) lactose transporter, which indicates that lactose transport is not rate-limiting for glycolysis in Lactococcus. Finally, an additional ATP drain was introduced into the fastest growing strain, CS2004, to test whether the ATP demand controlled glycolysis under these conditions, but in fact no increase in glycolytic flux was observed.

The enzymatic breakdown of milk proteins releases bioactive peptides. Two such peptides are the 11-residue antimicrobial peptide from bovine lactoferrin (BL-11) and the 12-residue hypotensive peptide from alpha(s1)-casein (C-12). These two peptides have now been cloned in Streptococcus thermophilus to develop strains that enhance the functionality and nutritional value of dairy food products. Nucleic acid sequences encoding the peptides were generated by overlapping PCR and were subsequently cloned into a new expression vector under control of the ST(2201) promoter. S. thermophilus transformants were successfully identified using GFP as a selectable marker. The presence of the synthetic gene constructs in S. thermophilus was confirmed by PCR.

Using Streptococcus thermophilus phages, plasmid transduction in Lactococcus lactis was demonstrated. The transduction frequencies were 4 orders of magnitude lower in L. lactis than in S. thermophilus. These results are the first evidence that there is phage-mediated direct transfer of DNA from S. thermophilus to L. lactis. The implications of these results for phage evolution are discussed.

Aims: To determine whether the presence and type of exopolysaccharides (EPS), slime-EPS or capsular, and the structural characteristics of the polymers produced by Streptococcus thermophilus strains could interfere with or be involved in phage adsorption., Methods and Results: Phage-host interactions between eight EPS-producing Strep. thermophilus strains (CRL419, 638, 804, 810, 815, 817, 821, 1190) and five streptococcus specific phages (phiYsca, phi3, phi5, phi6, phi8) isolated from Argentinean faulty fermentation failed yoghurts were evaluated. No relationship was found between the EPS chemical composition and the phage sensitivity/resistance phenotype. In general, the capsular-producing strains were more sensitive to phage attacks than the noncapsular-producing strains. Streptococcus thermophilus CRL1190 (capsular-producing) was the only strain sensitive to all bacteriophages and showed the highest efficiency of plating. Phage adsorption to a capsular-negative, EPS low-producing mutant of strain CRL1190 was reduced, especially for phiYcsa and phi8., Conclusions: The presence of capsular polysaccharide surrounding the cells of Strep. thermophilus strains could play a role in the adsorption of specific phages to the cells., Significance and Impact of the Study: Capsular-producing Strep. thermophilus strains should be evaluated for their bacteriophage sensitivity if they are included in starter cultures for the fermented food industry.

This work aimed to investigate the effects of thermal treatments and yeast extract addition on the composition of the microbial community of natural whey starters for Grana Padano cheese. Different natural whey starter samples were held at 4 degrees C for 24 h (cooling treatment), or at -20 degrees C for 24 h (freezing treatment) to evaluate the possibility of conservation, or at 54 degrees C for 1 h (heat treatment) to evaluate the effect of the temperature commonly used during curd cooking. Separately, another set of samples was enriched with 0.3, 0.5, and 1.0% (wt/vol) of yeast extract to study its effect on the growth of lactic acid bacteria (LAB) in the starter. The new approach in this study is the use of 2 culture-independent methods: length heterogeneity (LH)-reverse transcription (RT)-PCR and fluorescence microscopy. These techniques allowed us to easily, quickly, and reproducibly assess metabolically active LAB in the control and treated samples. The LH-RT-PCR technique distinguished microorganisms based on natural variations in the length of 16S rRNA amplified by RT-PCR, as analyzed by using an automatic gene sequencer. Fluorescence microscopy counts were performed by using a Live/Dead BacLight bacterial viability kit. The repeatability of LH-RT-PCR showed that this technique has great potential to reveal changes in the microbial community of natural whey starters for Grana Padano cheese. All species showed low sensitivity to cold (4 degrees C). However, after the freezing (-20 degrees C) and heating (54 degrees C) treatments, different behaviors of the species were reported, with significant changes in their viability and relative composition. Heating treatment during curd cooking profoundly affected the viability and composition of the community that remained in the cheese and that consequently modified the microbial population. At the same time, this treatment produced the selection of LAB in whey and could be considered as the first step in natural whey starter production. Addition of yeast extract stimulated the growth of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. lactis to the detriment of Lactobacillus helveticus species. Because the yeast extract altered the microflora balance, whey starter conservation at -20 degrees C and yeast extract addition cannot be suggested as technological innovations.

The blp(St) cluster of Streptococcus thermophilus LMD-9 was recently shown to contain all the genetic information required for the production of bacteriocins active against other S. thermophilus strains. In this study, we further investigated the antimicrobial activity of S. thermophilus LMD-9 by testing the susceptibility of 31 bacterial species (87 strains). We showed that LMD-9 displays an inhibitory spectrum targeted toward related gram-positive bacteria, including pathogens such as Listeria monocytogenes. Using deletion mutants, we investigated the contribution of the three putative bacteriocin-encoding operons blpD(St)-orf2, blpU(St)-orf3, and blpE(St)-blpF(St) (bac(St) operons) and of the blpG(St) gene, which encodes a putative modification protein, to the inhibitory spectrum and immunity of strain LMD-9. Our results present evidence that the blp(St) locus encodes a multipeptide bacteriocin system called thermophilin 9. Among the four class II bacteriocin-like peptides encoded within the bac(St) operons, BlpD(St) alone was sufficient to inhibit the growth of most thermophilin 9-sensitive species. The blpD(St) gene forms an operon with its associated immunity gene(s), and this functional bacteriocin/immunity module could easily be transferred to Lactococcus lactis. The remaining three Bac(St) peptides, BlpU(St), BlpE(St), and BlpF(St), confer poor antimicrobial activity but act as enhancers of the antagonistic activity of thermophilin 9 by an unknown mechanism. The blpG(St) gene was also shown to be specifically required for the antilisteria activity of thermophilin 9, since its deletion abolished the sensitivities of most Listeria species. By complementation of the motility deficiency of Escherichia coli dsbA, we showed that blpG(St) encodes a functional thiol-disulfide oxidase, suggesting an important role for disulfide bridges within thermophilin 9.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated genes are linked to a mechanism of acquired resistance against bacteriophages. Bacteria can integrate short stretches of phage-derived sequences (spacers) within CRISPR loci to become phage resistant. In this study, we further characterized the efficiency of CRISPR1 as a phage resistance mechanism in Streptococcus thermophilus. First, we show that CRISPR1 is distinct from previously known phage defense systems and is effective against the two main groups of S. thermophilus phages. Analyses of 30 bacteriophage-insensitive mutants of S. thermophilus indicate that the addition of one new spacer in CRISPR1 is the most frequent outcome of a phage challenge and that the iterative addition of spacers increases the overall phage resistance of the host. The added new spacers have a size of between 29 to 31 nucleotides, with 30 being by far the most frequent. Comparative analysis of 39 newly acquired spacers with the complete genomic sequences of the wild-type phages 2972, 858, and DT1 demonstrated that the newly added spacer must be identical to a region (named proto-spacer) in the phage genome to confer a phage resistance phenotype. Moreover, we found a CRISPR1-specific sequence (NNAGAAW) located downstream of the proto-spacer region that is important for the phage resistance phenotype. Finally, we show through the analyses of 20 mutant phages that virulent phages are rapidly evolving through single nucleotide mutations as well as deletions, in response to CRISPR1.

Streptococcus thermophilus is unable to metabolize the galactose moiety of lactose. In this paper, we show that a transformant of S. thermophilus SMQ-301 expressing Streptococcus salivarius galK and galM was able to grow on galactose and expelled at least twofold less galactose into the medium during growth on lactose.

Clustered regularly interspaced short palindromic repeats (CRISPR) are hypervariable loci widely distributed in prokaryotes that provide acquired immunity against foreign genetic elements. Here, we characterize a novel Streptococcus thermophilus locus, CRISPR3, and experimentally demonstrate its ability to integrate novel spacers in response to bacteriophage. Also, we analyze CRISPR diversity and activity across three distinct CRISPR loci in several S. thermophilus strains. We show that both CRISPR repeats and cas genes are locus specific and functionally coupled. A total of 124 strains were studied, and 109 unique spacer arrangements were observed across the three CRISPR loci. Overall, 3,626 spacers were analyzed, including 2,829 for CRISPR1 (782 unique), 173 for CRISPR2 (16 unique), and 624 for CRISPR3 (154 unique). Sequence analysis of the spacers revealed homology and identity to phage sequences (77%), plasmid sequences (16%), and S. thermophilus chromosomal sequences (7%). Polymorphisms were observed for the CRISPR repeats, CRISPR spacers, cas genes, CRISPR motif, locus architecture, and specific sequence content. Interestingly, CRISPR loci evolved both via polarized addition of novel spacers after exposure to foreign genetic elements and via internal deletion of spacers. We hypothesize that the level of diversity is correlated with relative CRISPR activity and propose that the activity is highest for CRISPR1, followed by CRISPR3, while CRISPR2 may be degenerate. Globally, the dynamic nature of CRISPR loci might prove valuable for typing and comparative analyses of strains and microbial populations. Also, CRISPRs provide critical insights into the relationships between prokaryotes and their environments, notably the coevolution of host and viral genomes.

The integrative and conjugative elements (ICEs) excise by site-specific recombination between attL and attR flanking sites, self-transfer the resulting circular form and integrate into the genome of the recipient cell. Two putative ICEs, ICESt1 and ICESt3, are integrated in the same locus in 2 strains of Streptococcusthermophilus. ICESt1 is a composite element harbouring an internal recombination site, attL'. The recombination between attL' and attR leads to the excision of a shorter putative ICE, ICESt2. ICESt1/ICESt2 and ICESt3 carry related regulation modules sharing the open reading frame arp1 that encodes a protein related to the cI repressor of the phage lambda. The repressors belonging to this family autoproteolyse in the presence of damaged DNA. Treatments with mitomycin C induce an increase in the excision of ICESt1, ICESt2 and ICESt3. Furthermore, the arp1 deletion leads to a 1,000-fold increase in the excision of ICESt1 and ICESt2 and to a decrease in the excision induction by mitomycin C. Thus, all together, these results suggest that the autocleavage of the arp1 repressor is involved in derepression of the S. thermophilus putative ICE excision by mitomycin C.

Background: Yogurt has traditionally been considered a probiotic-carrier food with health-promoting effects. Despite the universal assumption of this assertion, several researchers have evaluated the real capability of the yogurt bacteria Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus to survive and proliferate in the human intestine and have found contradictory results., Objective: This double-blind crossover study assessed the qualitative and quantitative effects of fresh and heat-treated yogurt on bacterial intestinal microbiota from healthy subjects., Design: The subjects were divided into experimental (n=63) and control (n=16) groups. The experimental group consumed fresh and heat-treated yogurt for 15 d according to a crossover design with a washout period of 2 wk. Three different fecal samples per individual were recovered: at baseline, after fresh yogurt intake, and after heat-treated yogurt intake. Qualitative changes in microbiota were studied by denaturing gel gradient electrophoresis (DGGE) with universal and lactic acid bacteria (LAB) 16S-rRNA primers. Quantitative changes in LAB, Clostridium coccoides, Clostridium perfringens, and Bacteroides groups were analyzed by real-time polymerase chain reaction., Results: A particular DGGE stable band pattern was observed in each sample. No significant qualitative differences were detected in any fecal sample. However, a significantly higher density of LAB and C. perfringens and a significant decrease in the density of Bacteroides was observed after consumption of both types of yogurt. Microbiota density was not significantly different between the fresh and heat-treated yogurt groups, except for LAB, which was significantly greater in the fresh yogurt group., Conclusion: The main change in human microbiota observed after yogurt consumption was an increase in the density of LAB and C. perfringens to the detriment of Bacteroides. Bacterial changes were not different after the consumption of fresh and heat-treated yogurt.

Gram-positive bacteria secrete a variety of peptides that are often subjected to posttranslational modifications and that are either antimicrobials or pheromones involved in bacterial communication. Our objective was to identify peptides secreted by Streptococcus thermophilus, a nonpathogenic bacterium widely used in dairy technology in association with other bacteria, and to understand their potential roles in cell-cell communication. Using reverse-phase liquid chromatography, mass spectrometry, and Edman sequencing, we analyzed the culture supernatants of three S. thermophilus strains (CNRZ1066, LMG18311, and LMD-9) grown in a medium containing no peptides. We identified several peptides in the culture supernatants, some of them found with the three strains while others were specific to the LMD-9 strain. We focused our study on a new modified peptide secreted by S. thermophilus LMD-9 and designated Pep1357C. This peptide contains 9 amino acids and lost 2 Da in a posttranslational modification, most probably a dehydrogenation, leading to a linkage between the Lys2 and Trp6 residues. Production of Pep1357C and transcription of its encoding gene depend on both the medium composition and the growth phase. Furthermore, we demonstrated that transcription of the gene coding for Pep1357C is drastically decreased in mutants inactivated for the synthesis of a short hydrophobic peptide, a transcriptional regulator, or the oligopeptide transport system. Taken together, our results led us to deduce that the transcription of the Pep1357C-encoding gene is controlled by a new quorum-sensing system.

Molecular features of the 4139-bp plasmid pER13 found in the dairy fermentation bacterium Streptococcus thermophilus ST113 include five open reading frames (ORFs). ORF1, ORF2 and ORF3 encode proteins for transcriptional repression (CopG), replication (RepB) and mobilization (Mob) that share homology with corresponding proteins of the pMV158 plasmid family, while ORF4 and ORF5 encode putative proteins with unspecified functions. Sequence homologies shared with plasmids found in group B and group D streptococci imply the possibility for genetic exchange with the food-grade S. thermophilus. The structural features of pER13 may be useful in designing strategies for gene transfer in lactic fermentation bacteria.