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1. Wong  WY, Su  P, Allison  GE, Liu  CQ, Dunn  NW,     ( 2003 )

A potential food-grade cloning vector for Streptococcus thermophilus that uses cadmium resistance as the selectable marker.

Applied and environmental microbiology 69 (10)
PMID : 14532023  :   DOI  :   10.1128/aem.69.10.5767-5771.2003     PMC  :   PMC201215    
Abstract >>
A potential food-grade cloning vector, pND919, was constructed and transformed into S. thermophilus ST3-1, a plasmid-free strain. The vector contains DNAs from two different food-approved organisms, Streptococcus thermophilus and Lactococcus lactis. The 5.0-kb pND919 is a derivative of the cloning vector pND918 (9.3 kb) and was constructed by deletion of the 4.3-kb region of pND918 which contained DNA from non-food-approved organisms. pND919 carries a heterologous native cadmium resistance selectable marker from L. lactis M71 and expresses the Cd(r) phenotype in S. thermophilus transformants. With the S. thermophilus replicon derived from the shuttle vector pND913, pND919 is able to replicate in the two S. thermophilus industrial strains tested, ST3-1 and ST4-1. Its relatively high retention rate in S. thermophilus further indicates its usefulness as a potential food-grade cloning vector. To our knowledge, this is the first report of a replicative potential food-grade vector for the industrially important organism S. thermophilus.
KeywordMeSH Terms
Genetic Vectors
2. Monnet  C, Nardi  M, Hols  P, Gulea  M, Corrieu  G, Monnet  V,     ( 2003 )

Regulation of branched-chain amino acid biosynthesis by alpha-acetolactate decarboxylase in Streptococcus thermophilus.

Letters in applied microbiology 36 (6)
PMID : 12753249  :  
Abstract >>
To demonstrate the presence of an active alpha-acetolactate decarboxylase in Streptococcus thermophilus and to investigate its physiological function. Streptococcus thermophilus CNRZ385 contains a gene encoding an alpha-acetolactate decarboxylase. Comparison of the production of alpha-acetolactate and its decarboxylation products, by the parent strain and an alpha-acetolactate decarboxylase-deficient mutant, demonstrated the presence of a control of the pool of alpha-acetolactate by valine, leucine and isoleucine. This control occurs via an allosteric activation of the alpha-acetolactate decarboxylase. Cell-free extracts of S. thermophilus were not able to decarboxylate the isoleucine precursor alpha-acetohydroxybutyrate. These results strongly suggest that one of the physiological functions of the alpha-acetolactate decarboxylase in S. thermophilus is to regulate leucine and valine biosynthesis by diverting the flux of alpha-acetolactate towards acetoin when the branched-chain amino acids are present at a high concentration. Regulation of branched-chain amino acid biosynthesis by alpha-acetolactate decarboxylase may occur in several other micro-organisms and explain some of their growth properties.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
3. Cochu  A, Vadeboncoeur  C, Moineau  S, Frenette  M,     ( 2003 )

Genetic and biochemical characterization of the phosphoenolpyruvate:glucose/mannose phosphotransferase system of Streptococcus thermophilus.

Applied and environmental microbiology 69 (9)
PMID : 12957931  :   DOI  :   10.1128/aem.69.9.5423-5432.2003     PMC  :   PMC194979    
Abstract >>
In most streptococci, glucose is transported by the phosphoenolpyruvate (PEP):glucose/mannose phosphotransferase system (PTS) via HPr and IIAB(Man), two proteins involved in regulatory mechanisms. While most strains of Streptococcus thermophilus do not or poorly metabolize glucose, compelling evidence suggests that S. thermophilus possesses the genes that encode the glucose/mannose general and specific PTS proteins. The purposes of this study were to determine (i) whether these PTS genes are expressed, (ii) whether the PTS proteins encoded by these genes are able to transfer a phosphate group from PEP to glucose/mannose PTS substrates, and (iii) whether these proteins catalyze sugar transport. The pts operon is made up of the genes encoding HPr (ptsH) and enzyme I (EI) (ptsI), which are transcribed into a 0.6-kb ptsH mRNA and a 2.3-kb ptsHI mRNA. The specific glucose/mannose PTS proteins, IIAB(Man), IIC(Man), IID(Man), and the ManO protein, are encoded by manL, manM, manN, and manO, respectively, which make up the man operon. The man operon is transcribed into a single 3.5-kb mRNA. To assess the phosphotransfer competence of these PTS proteins, in vitro PEP-dependent phosphorylation experiments were conducted with purified HPr, EI, and IIAB(Man) as well as membrane fragments containing IIC(Man) and IID(Man). These PTS components efficiently transferred a phosphate group from PEP to glucose, mannose, 2-deoxyglucose, and (to a lesser extent) fructose, which are common streptococcal glucose/mannose PTS substrates. Whole cells were unable to catalyze the uptake of mannose and 2-deoxyglucose, demonstrating the inability of the S. thermophilus PTS proteins to operate as a proficient transport system. This inability to transport mannose and 2-deoxyglucose may be due to a defective IIC domain. We propose that in S. thermophilus, the general and specific glucose/mannose PTS proteins are not involved in glucose transport but might have regulatory functions associated with the phosphotransfer properties of HPr and IIAB(Man).
KeywordMeSH Terms
4. Broadbent  JR, McMahon  DJ, Welker  DL, Oberg  CJ, Moineau  S,     ( 2003 )

Biochemistry, genetics, and applications of exopolysaccharide production in Streptococcus thermophilus: a review.

Journal of dairy science 86 (2)
PMID : 12647947  :   DOI  :   10.3168/jds.S0022-0302(03)73619-4    
Abstract >>
Many strains of Streptococcus thermophilus synthesize extracellular polysaccharides. These molecules may be produced as capsules that are tightly associated with the cell, or they may be liberated into the medium as a loose slime (i.e., "ropy" polysaccharide). Although the presence of exopolysaccharide does not confer any obvious advantage to growth or survival of S. thermophilus in milk, in situ production by this species or other dairy lactic acid bacteria typically imparts a desirable "ropy" or viscous texture to fermented milk products. Recent work has also shown that exopolysaccharide-producing S. thermophilus can enhance the functional properties of Mozzarella cheese, but they are not phage-proof. As our understanding of the genetics, physiology, and functionality of bacterial exopolysaccharides continues to improve, novel applications for polysaccharides and polysaccharide-producing cultures are likely to emerge inside and outside the dairy industry. This article provides an overview of biochemistry, genetics, and applications of exopolysaccharide production in S. thermophilus.
KeywordMeSH Terms
5. Geis  A, El Demerdash  HA, Heller  KJ,     ( 2003 )

Sequence analysis and characterization of plasmids from Streptococcus thermophilus.

Plasmid 50 (1)
PMID : 12826058  :  
Abstract >>
The nucleotide sequences of eight plasmids isolated from seven Streptococcus thermophilus strains have been determined. Plasmids pSt04, pER1-1, and pJ34 are related and replicate via a rolling circle mechanism. Plasmid pJ34 encodes for a replication initiation protein (RepA) and a small polypeptide with unknown function. Plasmids pSt04 and pER1-1 carry in addition to repA genes coding for small heat shock proteins (sHsp). Expression of these proteins is induced at elevated temperatures or low pH and increases the thermo- and acid resistance. Plasmids pER1-2 and pSt22-2 show identical sequences with five putative open reading frames (ORFs). The gene products of ORF1 and ORF4 reveal some similarities to transposon encoded proteins of Bacillus subtilis and Tn916. ORF1 of plasmid pSt106 encodes a protein similar to resolvases of different Gram-positive bacteria. Integrity of ORF2 and 3, encoding a putative DNA primase and a replication protein, is essential for replication. ORF1 to 3 of plasmid pSt08, which are organized in a tricistronic operon, encode a RepA protein, an adenosine-specific methyltransferase, and a type II restriction endonuclease. Another type II restriction-modification (R/M) system is encoded on plasmid pSt0 which is highly similar to those encoded on lactococcal plasmid pHW393 and B. subtilis plasmid pXH13. Plasmid-free derivatives of strains St0 and St08 show increased phage sensitivity, indicating that in the wild-type strains the R/M systems are functionally expressed. Recombinant plasmids based on the replicons of plasmids pSt04, pJ34, pSt106, pSt08, and pSt0, are able to replicate in Lactococcus lactis and B. subtilis, respectively, whereas constructs carrying pER1-2 only replicate in S. thermophilus.
KeywordMeSH Terms
Sequence Analysis, DNA
6. Varcamonti  M, Graziano  MR, Pezzopane  R, Naclerio  G, Arsenijevic  S, De Felice  M,     ( 2003 )

Impaired temperature stress response of a Streptococcus thermophilus deoD mutant.

Applied and environmental microbiology 69 (2)
PMID : 12571059  :   DOI  :   10.1128/aem.69.2.1287-1289.2003     PMC  :   PMC143660    
Abstract >>
An insertional deoD mutant of Streptococcus thermophilus strain SFi39 had a reduced growth rate at 20 degrees C and an enhanced survival capacity to heat shock compared to the wild type, indicating that the deoD product is involved in temperature shock adaptation. We report evidence that ppGpp is implicated in this dual response.
KeywordMeSH Terms
Heat-Shock Response
Hot Temperature
Mutation
7. Su  P, Jury  K, Allison  GE, Wong  WY, Kim  WS, Liu  CQ, Vancov  T, Dunn  NW,     ( 2002 )

Cloning vectors for Streptococcus thermophilus derived from a native plasmid.

FEMS microbiology letters 216 (1)
PMID : 12423750  :   DOI  :   10.1111/j.1574-6968.2002.tb11412.x    
Abstract >>
A 3.5-kb native plasmid (pND103) was identified in Streptococcus thermophilus ST2-1. Preliminary sequence analysis indicated that pND103 belongs to group I S. thermophilus plasmids. A region of approximately 2 kb appears to contain three components: a plus origin of replication (ori) typical of plasmids that replicate via rolling circle replication; a gene encoding a replication protein (rep); and a gene encoding a small heat shock protein (hsp). pND103 was then used to construct S. thermophilus/Escherichia coli hybrid cloning vectors by ligating different portions of pND103 to an origin-probe vector (pND330) composed of pUC19 and a Gram-positive erythromycin resistance gene. The shuttle vectors (pND913, pND914 and pND915) were successfully introduced back into plasmid-free S. thermophilus ST3-1 as well as to Lactococcus lactis LM0230 and E. coli JM109. Segregational and structural stability study indicated that these vectors can be maintained in these hosts. The results indicated that pND913, pND914 and pND915 are potential shuttle cloning vectors for S. thermophilus.
KeywordMeSH Terms
Genetic Vectors
8. Ventura  M, Bruttin  A, Canchaya  C, Brüssow  H,     ( 2002 )

Transcription analysis of Streptococcus thermophilus phages in the lysogenic state.

Virology 302 (1)
PMID : 12429513  :   DOI  :   10.1006/viro.2002.1571    
Abstract >>
The transcription of prophage genes was studied in two lysogenic Streptococcus thermophilus cells by Northern blot and primer-extension experiments. In the lysogen containing the cos-site phage Sfi21 only two gene regions of the prophage were transcribed. Within the lysogeny module an 1.6-kb-long mRNA started at the promoter of the phage repressor gene and covered also the next two genes, including a superinfection exclusion (sie) gene. A second, quantitatively more prominent 1-kb-long transcript was initiated at the promoter of the sie gene. Another prophage transcript of 1.6-kb length covered a group of genes without database matches that were located between the lysin gene and the right attachment site. The rest of the prophage genome was transcriptionally silent. A very similar transcription pattern was observed for a S. thermophilus lysogen containing the pac-site phage O1205 as a prophage. Prophages from pathogenic streptococci encode virulence genes downstream of the lysin gene. We speculate that temperate phages from lactic streptococci also encode nonessential phage genes ("lysogenic conversion genes") in this region that increase the ecological fitness of the lysogen to further their own evolutionary success. A comparative genome analysis revealed that many temperate phages from low GC content Gram-positive bacteria encode a variable number of genes in that region and none was linked to known phage-related function. Prophages from pathogenic streptococci encode toxin genes in this region. In accordance with theoretical predictions on prophage-host genome interactions a prophage remnant was detected in S. thermophilus that had lost most of the prophage DNA while transcribed prophage genes were spared from the deletion process.
KeywordMeSH Terms
Transcription, Genetic
9. Schirawski  J, Hagens  W, Fitzgerald  GF, Van Sinderen  D,     ( 2002 )

Molecular characterization of cadmium resistance in Streptococcus thermophilus strain 4134: an example of lateral gene transfer.

Applied and environmental microbiology 68 (11)
PMID : 12406744  :   DOI  :   10.1128/aem.68.11.5508-5516.2002     PMC  :   PMC129935    
Abstract >>
Two genes (cadC(St) and cadA(St) [subscript St represents Streptococcus thermophilus]), located on the chromosome of S. thermophilus 4134, were shown to constitute a cadmium/zinc resistance cassette. The genes seem to be organized in an operon, and their transcription is cadmium dependent in vivo. The proposed product of the cadA open reading frame (CadA(St)) is highly similar to P-type cadmium efflux ATPases, whereas the predicted protein encoded by cadC(St) (CadC(St)) shows high similarity to ArsR-type regulatory proteins. The observed homologies and G+C content of this cassette and surrounding regions suggest that this DNA was derived from Lactococcus lactis and may have been introduced relatively recently into the S. thermophilus 4134 genome by a lateral gene transfer event. The complete cassette confers cadmium and zinc resistance to both S. thermophilus and L. lactis, but expression of cadA(St) alone is sufficient to give resistance. By using electrophoretic mobility shift assays it was shown that the CadC(St) protein is a DNA binding protein that binds specifically to its own promoter region, possibly to two copies of an inverted repeat, and that this CadC(St)-DNA interaction is lost in the presence of cadmium. Using lacZ fusion constructs it was shown that the cadmium-dependent expression of CadA(St) is mediated by the negative regulator CadC(St). A model for the regulation of the expression of cadmium resistance in S. thermophilus is discussed.
KeywordMeSH Terms
10. Crispie  F, Anba  J, Renault  P, Ehrlich  D, Fitzgerald  G, van Sinderen  D,     ( 2002 )

Identification of a phosphofructokinase-encoding gene from Streptococcus thermophilus CNRZ1205--a novel link between carbon metabolism and gene regulation?

Molecular genetics and genomics : MGG 268 (4)
PMID : 12471447  :   DOI  :   10.1007/s00438-002-0766-2    
Abstract >>
In order to isolate genes encoding so-called Two-Component Regulatory Systems from the lactic acid bacterium Streptococcus thermophilus, a cloning strategy was employed based on suppression of the alkaline phosphatase-negative phenotype displayed by the Escherichia coli strain ANCC22. Several suppressing clones were obtained which were shown to produce alkaline phosphatase activity. Sequence analysis of four of these clones revealed the presence of overlapping DNA inserts representing two ORFs, designated pfkT and pykT, whose deduced protein products exhibit significant similarity to phosphofructokinases and pyruvate kinases, respectively, from a variety of bacteria. A plasmid bearing pfkT was shown to complement a phosphofructokinase-negative mutant of E. coli, showing that this gene indeed specifies phosphofructokinase activity. It was shown that suppression of the alkaline phosphatase-negative phenotype of E. coli ANCC22 due to the presence of pfkT is caused by modulation of the intracellular level of acetyl phosphate.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Genes, Bacterial
11. Burrus  V, Pavlovic  G, Decaris  B, Guédon  G,     ( 2002 )

The ICESt1 element of Streptococcus thermophilus belongs to a large family of integrative and conjugative elements that exchange modules and change their specificity of integration.

Plasmid 48 (2)
PMID : 12383726  :  
Abstract >>
The 34,734-bp element ICESt1 from Streptococcus thermophilus CNRZ368 is site-specifically integrated into the 3(') end of the gene fda. ICESt1 encodes integrative functions and putative transfer functions. Six proteins of the putative conjugative system of ICESt1 are related to those encoded by the conjugative transposon Tn916 from Enterococcus faecalis. A comparison of these proteins with those encoded by the complete or partial genome sequences of various low G+C bacteria including Bacillus subtilis, Clostridium difficile, E. faecalis, Listeria monocytogenes, Staphylococcus aureus, and Streptococcus mutans revealed the presence of numerous putative site-specific integrative conjugative elements and/or conjugative transposons within these genomes. Sequence comparisons revealed that these elements possess a modular structure and that exchanges of unrelated or distantly related modules and genes have occurred between these elements, and also plasmids and prophages. These exchanges have probably led to modifications in the site specificity of integration of these elements. Therefore, a distinction between low specificity integrative conjugative elements (i.e., conjugative transposons) and site-specific integrative conjugative elements does not appear to be relevant. We propose to call all the conjugative elements that excise by site-specific recombination and integrate by recombination between a specific site of a circular intermediate and another site, "Integrative and Conjugative Elements" (ICEs), irrespective of the integration specificity.
KeywordMeSH Terms
12. Anastasiou  R, Papadelli  M, Georgalaki  MD, Kalantzopoulos  G, Tsakalidou  E,     ( 2002 )

Cloning and sequencing of the gene encoding X-prolyl-dipeptidyl aminopeptidase (PepX) from Streptococcus thermophilus strain ACA-DC 4.

Journal of applied microbiology 93 (1)
PMID : 12067374  :  
Abstract >>
To clone and sequence the pepX gene from Streptococcus thermophilus. Three pairs of primers were used in polymerase chain reactions using as template the total DNA from Strep. thermophilus ACA-DC 4 in order to amplify, clone and sequence the pepX gene. Sequence analysis revealed an open reading frame of 2268 nucleotides encoding a protein of 755 amino acids. The calculated molecular mass of 85 632 Da agreed well with the apparent molecular mass of 80 000 Da previously determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and gel filtration for the monomeric form of the purified enzyme. The pepX gene from Strep. thermophilus ACA-DC 4 was cloned and sequenced. The PepX protein showed significant sequence similarity with PepX enzymes from other lactic acid bacteria and contained a motif which was almost identical with the active site motif of the serine-dependent PepX family. There are economic and technological incentives for accelerating and controlling the process of cheese ripening. To achieve this, starters may be modified by introducing appropriate genes from other food-grade bacteria. New or additional peptidase activities may alter or improve the proteolytic properties of lactic acid bacteria.
KeywordMeSH Terms
13. Vaillancourt  K, Moineau  S, Frenette  M, Lessard  C, Vadeboncoeur  C,     ( 2002 )

Galactose and lactose genes from the galactose-positive bacterium Streptococcus salivarius and the phylogenetically related galactose-negative bacterium Streptococcus thermophilus: organization, sequence, transcription, and activity of the gal gene products.

Journal of bacteriology 184 (3)
PMID : 11790749  :   DOI  :   10.1128/jb.184.3.785-793.2002     PMC  :   PMC139519    
Abstract >>
Streptococcus salivarius is a lactose- and galactose-positive bacterium that is phylogenetically closely related to Streptococcus thermophilus, a bacterium that metabolizes lactose but not galactose. In this paper, we report a comparative characterization of the S. salivarius and S. thermophilus gal-lac gene clusters. The clusters have the same organization with the order galR (codes for a transcriptional regulator and is transcribed in the opposite direction), galK (galactokinase), galT (galactose-1-P uridylyltransferase), galE (UDP-glucose 4-epimerase), galM (galactose mutarotase), lacS (lactose transporter), and lacZ (beta-galactosidase). An analysis of the nucleotide sequence as well as Northern blotting and primer extension experiments revealed the presence of four promoters located upstream from galR, the gal operon, galM, and the lac operon of S. salivarius. Putative promoters with virtually identical nucleotide sequences were found at the same positions in the S. thermophilus gal-lac gene cluster. An additional putative internal promoter at the 3' end of galT was found in S. thermophilus but not in S. salivarius. The results clearly indicated that the gal-lac gene cluster was efficiently transcribed in both species. The Shine-Dalgarno sequences of galT and galE were identical in both species, whereas the ribosome binding site of S. thermophilus galK differed from that of S. salivarius by two nucleotides, suggesting that the S. thermophilus galK gene might be poorly translated. This was confirmed by measurements of enzyme activities.
KeywordMeSH Terms
Escherichia coli Proteins
Genes, Bacterial
Monosaccharide Transport Proteins
Symporters
14. Levander  F, Svensson  M, Rådström  P,     ( 2002 )

Enhanced exopolysaccharide production by metabolic engineering of Streptococcus thermophilus.

Applied and environmental microbiology 68 (2)
PMID : 11823219  :   DOI  :   10.1128/aem.68.2.784-790.2002     PMC  :   PMC126717    
Abstract >>
It is possible that the low levels of production of exopolysaccharides (EPSs) by lactic acid bacteria could be improved by altering the levels of enzymes in the central metabolism that influence the production of precursor nucleotide sugars. To test this hypothesis, we identified and cloned the galU gene, which codes for UDP glucose pyrophosphorylase (GalU) in Streptococcus thermophilus LY03. Homologous overexpression of the gene led to a 10-fold increase in GalU activity but did not have any effect on the EPS yield when lactose was the carbon source. However, when galU was overexpressed in combination with pgmA, which encodes phosphoglucomutase (PGM), the EPS yield increased from 0.17 to 0.31 g/mol of carbon from lactose. A galactose-fermenting LY03 mutant (Gal(+)) with increased activities of the Leloir enzymes was also found to have a higher EPS yield (0.24 g/mol of carbon) than the parent strain. The EPS yield was further improved to 0.27 g/mol of carbon by overexpressing galU in this strain. However, the highest EPS yield, 0.36 g/mol of carbon, was obtained when pgmA was knocked out in the Gal(+) strain. Measurements of the levels of intracellular metabolites in the cultures revealed that the Gal(+) strains had considerably higher glucose 1-phosphate levels than the other strains, and the strain lacking PGM activity had threefold-higher levels of glucose 1-phosphate than the other Gal(+) strains. These results show that it is possible to increase EPS production by altering the levels of enzymes in the central carbohydrate metabolism.
KeywordMeSH Terms
15. Thibessard  A, Fernandez  A, Gintz  B, Leblond-Bourget  N, Decaris  B,     ( 2002 )

Effects of rodA and pbp2b disruption on cell morphology and oxidative stress response of Streptococcus thermophilus CNRZ368.

Journal of bacteriology 184 (10)
PMID : 11976312  :   DOI  :   10.1128/jb.184.10.2821-2826.2002     PMC  :   PMC135019    
Abstract >>
Insertional mutagenesis was used to isolate clones from Streptococcus thermophilus CNRZ368 that were modified in their abilities to tolerate oxidative stress. During this process, two menadione-sensitive clones (6G4 and 18C3) were found to display abnormal cell morphologies and distorted chain topologies and were further studied. Molecular characterization of both 6G4 and 18C3 mutants indicated that they were disrupted in open reading frames homologous to rodA and pbp2b, respectively. Both genes encoded proteins in Escherichia coli that were described as being implicated in peptidoglycan synthesis during the process of cell elongation and to function in determining the rod shape of the cell. This work reports a possible connection between peptidoglycan biosynthesis and oxidative stress defense in S. thermophilus CNRZ368.
KeywordMeSH Terms
Aminoacyltransferases
Escherichia coli Proteins
Hexosyltransferases
Membrane Proteins
Oxidative Stress
Peptidyl Transferases
16. Garault  P, Le Bars  D, Besset  C, Monnet  V,     ( 2002 )

Three oligopeptide-binding proteins are involved in the oligopeptide transport of Streptococcus thermophilus.

The Journal of biological chemistry 277 (1)
PMID : 11602593  :   DOI  :   10.1074/jbc.M107002200    
Abstract >>
The functions necessary for bacterial growth strongly depend on the features of the bacteria and the components of the growth media. Our objective was to identify the functions essential to the optimum growth of Streptococcus thermophilus in milk. Using random insertional mutagenesis on a S. thermophilus strain chosen for its ability to grow rapidly in milk, we obtained several mutants incapable of rapid growth in milk. We isolated and characterized one of these mutants in which an amiA1 gene encoding an oligopeptide-binding protein (OBP) was interrupted. This gene was a part of an operon containing all the components of an ATP binding cassette transporter. Three highly homologous amiA genes encoding OBPs work with the same components of the ATP transport system. Their simultaneous inactivation led to a drastic diminution in the growth rate in milk and the absence of growth in chemically defined medium containing peptides as the nitrogen source. We constructed single and multiple negative mutants for AmiAs and cell wall proteinase (PrtS), the only proteinase capable of hydrolyzing casein oligopeptides outside the cell. Growth experiments in chemically defined medium containing peptides indicated that AmiA1, AmiA2, and AmiA3 exhibited overlapping substrate specificities, and that the whole system allows the transport of peptides containing from 3 to 23 residues.
KeywordMeSH Terms
17. Germond  JE, Delley  M, D'Amico  N, Vincent  SJ,     ( 2001 )

Heterologous expression and characterization of the exopolysaccharide from Streptococcus thermophilus Sfi39.

European journal of biochemistry 268 (19)
PMID : 11589707  :   DOI  :   10.1046/j.0014-2956.2001.02450.x    
Abstract >>
The genes responsible for exopolysaccharide (EPS) synthesis in Streptococcus thermophilus Sfi39 were identified on a 20-kb genomic fragment. The two genes, epsE and epsG, were shown to be involved in EPS synthesis as their disruption lead to the loss of the ropy phenotype. Several naturally selected nonropy mutants were isolated, one acquired an insertion sequence (IS)-element (IS905) in the middle of the eps gene cluster. The eps gene cluster was cloned and transferred into a nonEPS-producing heterologous host, Lactococcus lactis MG1363. The EPS produced was shown by chemical analysis and NMR spectroscopy to be identical to the EPS produced by S. thermophilus Sfi39. This demonstrated first that all genes needed for EPS production and export were present in the S. thermophilus Sfi39 eps gene cluster, and second that the heterologous production of an EPS was possible by transfer of the complete eps gene cluster alone, provided that the heterologous host possessed all necessary genetic information for precursor synthesis.
KeywordMeSH Terms
18. Labarre  C, Schirawski  J, van der Zwet  A, Fitzgerald  GF, van Sinderen  D,     ( 2001 )

Insertional mutagenesis of an industrial strain of Streptococcus thermophilus.

FEMS microbiology letters 200 (1)
PMID : 11410354  :   DOI  :   10.1111/j.1574-6968.2001.tb10697.x    
Abstract >>
Random mutagenesis of an industrial strain of Streptococcus thermophilus was achieved through an adapted version of a two-plasmid system. The mutagenesis strategy is based on random integration of derivatives of the non-replicative (Rep(-)) plasmid pORI19 by means of homologous recombination following a temperature shift that eliminates replication of the temperature-sensitive (Rep(ts)) helper plasmid pVE6007. In this way mutants were generated which were affected in bacteriophage sensitivity or sucrose metabolism. Homologues were identified of a protein related to folate metabolism from a bacteriophage-resistant mutant and of two subunits of an oligopeptide transport system from a mutant deficient in sucrose utilisation.
KeywordMeSH Terms
Industrial Microbiology
Mutagenesis, Insertional
19. Levander  F, Rådström  P,     ( 2001 )

Requirement for phosphoglucomutase in exopolysaccharide biosynthesis in glucose- and lactose-utilizing Streptococcus thermophilus.

Applied and environmental microbiology 67 (6)
PMID : 11375188  :   DOI  :   10.1128/AEM.67.6.2734-2738.2001     PMC  :   PMC92932    
Abstract >>
To study the influence of phosphoglucomutase (PGM) activity on exopolysaccharide (EPS) synthesis in glucose- and lactose-growing Streptococcus thermophilus, a knockout PGM mutant and a strain with elevated PGM activity were constructed. The pgmA gene, encoding PGM in S. thermophilus LY03, was identified and cloned. The gene was functional in Escherichia coli and was shown to be expressed from its own promoter. The pgmA-deficient mutant was unable to grow on glucose, while the mutation did not affect growth on lactose. Overexpression of pgmA had no significant effect on EPS production in glucose-growing cells. Neither deletion nor overexpression of pgmA changed the growth or EPS production on lactose. Thus, the EPS precursors in lactose-utilizing S. thermophilus are most probably formed from the galactose moiety of lactose via the Leloir pathway, which circumvents the need for a functional PGM.
KeywordMeSH Terms
20. Turgeon  N, Moineau  S,     ( 2001 )

Isolation and characterization of a Streptococcus thermophilus plasmid closely related to the pMV158 family.

Plasmid 45 (3)
PMID : 11407913  :   DOI  :   10.1006/plas.2001.1517    
Abstract >>
Twenty-two Streptococcus thermophilus strains used for milk fermentations were analyzed for their plasmid content and 13 of them (59%) were found to contain one or two plasmids. Fifteen S. thermophilus plasmids were divided into four groups using DNA homology. Ten plasmids were classified within group A and they shared homologies with all the previously sequenced S. thermophilus plasmids. Three plasmids (group B) hybridized with each other and two plasmids only hybridized with themselves (groups C and D). Single-stranded DNA was detected within strains containing plasmids of groups A, C, and D, indicating that they replicate via a rolling-circle mode. The only plasmid of group C, named pSMQ172, was further characterized. This 4230-bp plasmid replicates in Escherichia coli, Lactococcus lactis, and Streptococcus salivarius and does not confer phage resistance. Comparisons with databases showed that pSMQ172 was related to pMV158 of Streptococcus agalactiae and to pSSU1 of Streptococcus suis. These results suggest that genetic exchanges may have occurred between pathogenic and nonpathogenic streptococci.
KeywordMeSH Terms
21. Burrus  V, Bontemps  C, Decaris  B, Guédon  G,     ( 2001 )

Characterization of a novel type II restriction-modification system, Sth368I, encoded by the integrative element ICESt1 of Streptococcus thermophilus CNRZ368.

Applied and environmental microbiology 67 (4)
PMID : 11282600  :   DOI  :   10.1128/AEM.67.4.1522-1528.2001     PMC  :   PMC92764    
Abstract >>
A novel type II restriction and modification (R-M) system, Sth368I, which confers resistance to phiST84, was found in Streptococcus thermophilus CNRZ368 but not in the very closely related strain A054. Partial sequencing of the integrative conjugative element ICESt1, carried by S. thermophilus CNRZ368 but not by A054, revealed a divergent cluster of two genes, sth368IR and sth368IM. The protein sequence encoded by sth368IR is related to the type II endonucleases R.LlaKR2I and R.Sau3AI, which recognize and cleave the sequence 5'-GATC-3'. The protein sequence encoded by sth368IM is very similar to numerous type II 5-methylcytosine methyltransferases, including M.LlaKR2I and M.Sau3AI. Cell extracts of CNRZ368 but not A054 were found to cleave at the GATC site. Furthermore, the C residue of the sequence 5'-GATC-3' was found to be methylated in CNRZ368 but not in A054. Cloning and integration of a copy of sth368IR and sth368IM in the A054 chromosome confers on this strain phenotypes similar to those of CNRZ368, i.e., phage resistance, endonuclease activity of cell extracts, and methylation of the sequence 5'-GATC-3'. Disruption of sth368IR removes resistance and restriction activity. We conclude that ICESt1 encodes an R-M system, Sth368I, which recognizes the sequence 5'-GATC-3' and is related to the Sau3AI and LlaKR2I restriction systems.
KeywordMeSH Terms
DNA Transposable Elements
22. Vaughan  EE, van den Bogaard  PT, Catzeddu  P, Kuipers  OP, de Vos  WM,     ( 2001 )

Activation of silent gal genes in the lac-gal regulon of Streptococcus thermophilus.

Journal of bacteriology 183 (4)
PMID : 11157930  :   DOI  :   10.1128/JB.183.4.1184-1194.2001     PMC  :   PMC94991    
Abstract >>
Streptococcus thermophilus strain CNRZ 302 is unable to ferment galactose, neither that generated intracellularly by lactose hydrolysis nor the free sugar. Nevertheless, sequence analysis and complementation studies with Escherichia coli demonstrated that strain CNRZ 302 contained structurally intact genes for the Leloir pathway enzymes. These were organized into an operon in the order galKTE, which was preceded by a divergently transcribed regulator gene, galR, and followed by a galM gene and the lactose operon lacSZ. Results of Northern blot analysis showed that the structural gal genes were transcribed weakly, and only in medium containing lactose, by strain CNRZ 302. However, in a spontaneous galactose-fermenting mutant, designated NZ302G, the galKTE genes were well expressed in cells grown on lactose or galactose. In both CNRZ 302 and the Gal(+) mutant NZ302G, the transcription of the galR gene was induced by growth on lactose. Disruption of galR indicated that it functioned as a transcriptional activator of both the gal and lac operons while negatively regulating its own expression. Sequence analysis of the gal promoter regions of NZ302G and nine other independently isolated Gal(+) mutants of CNRZ 302 revealed mutations at three positions in the galK promoter region, which included substitutions at positions -9 and -15 as well as a single-base-pair insertion at position -37 with respect to the main transcription initiation point. Galactokinase activity measurements and analysis of gusA reporter gene fusions in strains containing the mutated promoters suggested that they were gal promoter-up mutations. We propose that poor expression of the gal genes in the galactose-negative S. thermophilus CNRZ 302 is caused by naturally occurring mutations in the galK promoter.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Genes, Bacterial
Regulon
23. Almirón-Roig  E, Mulholland  F, Gasson  MJ, Griffin  AM,     ( 2000 )

The complete cps gene cluster from Streptococcus thermophilus NCFB 2393 involved in the biosynthesis of a new exopolysaccharide.

Microbiology (Reading, England) 146 (Pt 11) (N/A)
PMID : 11065358  :   DOI  :   10.1099/00221287-146-11-2793    
Abstract >>
The cpsFGHIJKL genes from the cps cluster of Streptococcus thermophilus NCFB 2393 involved in the biosynthesis of EPS were identified, cloned and nucleotide sequenced. The complete cps cluster is contained on an approximately 11.2 kb chromosomal region which contains 12 ORFs, including the previously cloned cpsABCDE genes. Functions were assigned to some of the predicted gene products on the basis of homology to known sequences as follows: cpsK encodes a protein thought to be involved in the polymerization and export of the polysaccharide; cpsE, cpsF, cpsG, cpsH, cpsI and cpsJ encode putative sugar transferases. Two insertion sequences, IS1193 and ISS1, were identified within and flanking the 3' end of the cps cluster respectively. Analysis of the expression of the cpsE gene in Escherichia coli demonstrated that it encodes a glucose-1-phosphate transferase; the enzyme which catalyses the first step in EPS biosynthesis in S. thermophilus NCFB 2393.
KeywordMeSH Terms
Genes, Bacterial
Multigene Family
24. Garault  P, Letort  C, Juillard  V, Monnet  V,     ( 2000 )

Branched-chain amino acid biosynthesis is essential for optimal growth of Streptococcus thermophilus in milk.

Applied and environmental microbiology 66 (12)
PMID : 11097879  :   DOI  :   10.1128/aem.66.12.5128-5133.2000     PMC  :   PMC92433    
Abstract >>
Lactic acid bacteria are nutritionally demanding bacteria which need, among other things, amino acids for optimal growth. We identified the branched-chain amino acid (BCAA) biosynthesis pathway as an essential pathway for optimal growth of Streptococcus thermophilus in milk. Through random insertional mutagenesis, we isolated and characterized two mutants for which growth in milk is affected as a consequence of ilvB and ilvC gene interruptions. This situation demonstrates that the BCAA biosynthesis pathway is active in S. thermophilus. BCAA biosynthesis is necessary but not sufficient for optimal growth of S. thermophilus and is subject to retro-inhibition processes. The specificity of the BCAA biosynthesis pathway in S. thermophilus lies in the independent transcription of the ilvC gene encoding a keto acid reductoisomerase acting on acetolactate at the junction of the BCAA and acetoin biosynthesis pathways. The possible advantages for S. thermophilus of keeping this biosynthesis pathway active could be linked either to adaptation of the organism to milk, which is different than that of other dairy bacteria, or to the role of the pathway in maintaining the internal pH.
KeywordMeSH Terms
25. van den Bogaard  PT, Kleerebezem  M, Kuipers  OP, de Vos  WM,     ( 2000 )

Control of lactose transport, beta-galactosidase activity, and glycolysis by CcpA in Streptococcus thermophilus: evidence for carbon catabolite repression by a non-phosphoenolpyruvate-dependent phosphotransferase system sugar.

Journal of bacteriology 182 (21)
PMID : 11029416  :   DOI  :   10.1128/jb.182.21.5982-5989.2000     PMC  :   PMC94730    
Abstract >>
Streptococcus thermophilus, unlike many other gram-positive bacteria, prefers lactose over glucose as the primary carbon and energy source. Moreover, lactose is not taken up by a phosphoenolpyruvate-dependent phosphotransferase system (PTS) but by the dedicated transporter LacS. In this paper we show that CcpA plays a crucial role in the fine-tuning of lactose transport, beta-galactosidase (LacZ) activity, and glycolysis to yield optimal glycolytic flux and growth rate. A catabolite-responsive element (cre) was identified in the promoter of the lacSZ operon, indicating a possible role for regulation by CcpA. Transcriptional analysis showed a sevenfold relief of repression in the absence of a functional CcpA when cells were grown on lactose. This CcpA-mediated repression of lacSZ transcription did not occur in wild-type cells during growth on galactose, taken up by the same LacS transport system. Lactose transport during fermentation was increased significantly in strains carrying a disrupted ccpA gene. Moreover, a ccpA disruption strain was found to release substantial amounts of glucose into the medium when grown on lactose. Transcriptional analysis of the ldh gene showed that expression was induced twofold during growth on lactose compared to glucose or galactose, in a CcpA-dependent manner. A reduced rate of glycolysis concomitant with an increased lactose transport rate could explain the observed expulsion of glucose in a ccpA disruption mutant. We propose that CcpA in S. thermophilus acts as a catabolic regulator during growth on the preferred non-PTS sugar lactose. In contrast to other bacteria, S. thermophilus possesses an overcapacity for lactose uptake that is repressed by CcpA to match the rate-limiting glycolytic flux.
KeywordMeSH Terms
Bacterial Proteins
26. Fernandez-Espla  MD, Garault  P, Monnet  V, Rul  F,     ( 2000 )

Streptococcus thermophilus cell wall-anchored proteinase: release, purification, and biochemical and genetic characterization.

Applied and environmental microbiology 66 (11)
PMID : 11055922  :   DOI  :   10.1128/aem.66.11.4772-4778.2000     PMC  :   PMC92378    
Abstract >>
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.
KeywordMeSH Terms
Bacterial Proteins
Serine Endopeptidases
27. Lucchini  S, Sidoti  J, Brüssow  H,     ( 2000 )

Broad-range bacteriophage resistance in Streptococcus thermophilus by insertional mutagenesis.

Virology 275 (2)
PMID : 10998327  :   DOI  :   10.1006/viro.2000.0499    
Abstract >>
Streptococcus thermophilus is a lactic acid bacterium used in industrial milk fermentation. To obtain phage-resistant starters, S. thermophilus strain Sfi1 was submitted to mutagenesis with the thermolabile insertional vector pG(+)host9:ISS1 followed by a challenge with the lytic S. thermophilus phage Sfi19. Vector insertions into four distinct sites led to a phage-resistance phenotype. Three mutants were characterized further. They were protected against the homologous challenging phage and 14 heterologous phages. All three mutants adsorbed phages. No intracellular phage DNA synthesis was observed in mutants R7 and R71, while mutant R24 showed a delayed and diminished phage DNA synthesis compared to the parental Sfi1 strain. In mutant R7 a short deletion occurred next to the insertion site which removed the upstream sequences and the 15 initial codons from orf 394, encoding a likely transmembrane protein. Analogy with other phage systems suggests an involvement of this protein in the phage DNA injection process. In mutant R24 the vector was inserted into orf 269 predicting an oxido-reductase. When the vector sequence was removed via homologous recombination across the duplicated insertion elements, mutant R24 returned to the phage susceptibility of the parental strain. This observation suggested that inactivation of orf 269 was not crucial for the resistance phenotype. A gene encoding a likely restriction subunit of a type I restriction-modification system was located directly downstream of the insertion site in mutant R24. hsdM and hsdS genes encoding the modification and specificity subunits of a type I R-M system and biological evidence for an active R-M system were detected in strain Sfi1, suggesting involvement of a type I R-M system in the resistance phenotype of R24.
KeywordMeSH Terms
28. Chavagnat  F, Meyer  J, Casey  MG,     ( 2000 )

Purification, characterisation, cloning and sequencing of the gene encoding oligopeptidase PepO from Streptococcus thermophilus A.

FEMS microbiology letters 191 (1)
PMID : 11004403  :   DOI  :   10.1111/j.1574-6968.2000.tb09322.x    
Abstract >>
The oligopeptidase PepO from Streptococcus thermophilus A was purified to protein homogeneity by a five-step chromatography procedure. It was estimated to be a serine metallopeptidase of 70 kDa, with maximal activity at pH 6.5 and 41 degrees C. PepO has endopeptidase activity on oligopeptides composed of between five and 30 amino acids. PepO was demonstrated to be active and stable at the pH, temperature and salt concentrations found in Swiss-type cheese during ripening. Using a battery of PCR techniques, the pepO gene was amplified, subcloned and sequenced, revealing an open reading frame of 1893 nucleotides. The amino acid sequence analysis of the pepO gene-translation product shows homology with PepO enzymes from other lactic acid bacteria and contains the signature sequence of the metallopeptidase family.
KeywordMeSH Terms
Bacterial Proteins
Cloning, Molecular
29. Mengaud  J, Benbadis  L, Husson-Kao  C,     ( 2000 )

Mur1, a Streptococcus thermophilus peptidoglycan hydrolase devoid of a specific cell wall binding domain.

FEMS microbiology letters 187 (1)
PMID : 10828403  :   DOI  :   10.1111/j.1574-6968.2000.tb09139.x    
Abstract >>
The gene encoding Mur1, a Streptococcus thermophilus peptidoglycan hydrolase, was cloned by homology with acmA, the Lactococcus lactis major autolysin gene. Mur1 is a 24.7-kDa protein endowed with a putative signal peptide. Sequence analysis evidenced that Mur1 encompasses exactly the AcmA region containing the catalytic domain, but lacks the one containing amino acid repeats involved in cell wall binding. Mur1 appears to be expressed and cell-associated in S. thermophilus, as revealed by immunoblot analysis. These results suggest that the cell wall attachment mode of Mur1 differs from that of most peptidoglycan hydrolases described so far.
KeywordMeSH Terms
Bacterial Proteins
30. Roussel  Y, Burrus  V,     ( 2000 )

Characterization of a novel integrative element, ICESt1, in the lactic acid bacterium Streptococcus thermophilus.

Applied and environmental microbiology 66 (4)
PMID : 10742276  :   DOI  :   10.1128/aem.66.4.1749-1753.2000     PMC  :   PMC92057    
Abstract >>
The 35.5-kb ICESt1 element of Streptococcus thermophilus CNRZ368 is bordered by a 27-bp repeat and integrated into the 3' end of a gene encoding a putative fructose-1,6-biphosphate aldolase. This element encodes site-specific integrase and excisionase enzymes related to those of conjugative transposons Tn5276 and Tn5252. The integrase was found to be involved in a site-specific excision of a circular form. ICESt1 also encodes putative conjugative transfer proteins related to those of the conjugative transposon Tn916. Therefore, ICESt1 could be or could be derived from an integrative conjugative element.
KeywordMeSH Terms
DNA Transposable Elements
Viral Proteins
31. Dixon-Fyle  SM,     ( 1999 )

Characterization in vitro and in vivo of a new HU family protein from Streptococcus thermophilus ST11.

Plasmid 42 (3)
PMID : 10545259  :   DOI  :   10.1006/plas.1999.1423    
Abstract >>
Streptococcus thermophilus is a thermophilic gram-positive bacterium belonging to the lactic acid group. We report the isolation and characterization of a new 9.6-kDa DNA-binding protein, HSth, belonging to the HU family of nucleoid-associated proteins. The hsth gene was isolated in a 2.5-kb genomic region, upstream of a gene with strong homology to Lactococcus lactis pyrD. It is transcribed from a single E. coli sigma(70)-like promoter. Based on its high level of sequence similarity to B. subtilis and E. coli HU, HSth appears to be an HU homologue. The HSth protein shows biochemical and functional properties typical of HU proteins from gram-positive bacteria, being heat-stable, acid-soluble, and homodimeric. When expressed in HU-deficient E. coli cells, HSth supported the growth of bacteriophage Mu as efficiently as E. coli HU homo- and heterodimeric proteins. It did not, however, display any IHF-specific functions. Finally, we show that HSth binds to linear DNA with no apparent specificity, forming protein-DNA complexes similar but not identical to those observed with E. coli HU proteins.
KeywordMeSH Terms
32. Guimont  C, Bracquart  P, Perrin  C,     ( 1999 )

Expression of a new cold shock protein of 21.5 kDa and of the major cold shock protein by Streptococcus thermophilus after cold shock.

Current microbiology 39 (6)
PMID : 10525839  :  
Abstract >>
Streptococcus thermophilus is widely used in food fermentations; it commonly suffers diverse stress challenges during manufacturing. This study investigated the cold shock response of S. thermophilus when the cell culture temperature shifted from 42 degrees C to 15 degrees C or 20 degrees C. The growth of cells was affected more drastically after cold shock at 15 degrees C than at 20 degrees C. The generation time was increased by a factor of 19 when the temperature was lowered from 42 degrees to 20 degrees C, and by a factor of 72 after a cold shock at 15 degrees C. The two-dimensional electrophoretic protein patterns of S. thermophilus under cold shock conditions were compared with the reference protein pattern when cells were grown at optimal temperature. Two proteins of 21.5 and 7.5 kDa synthesized in response to cold shock were characterized. N-terminal sequencing and sequence homology searches have shown that the 7.5-kDa protein belonged to the family of the major cold shock proteins, while no homology was found for the new cold shock protein of 21.5 kDa.
KeywordMeSH Terms
Cold Temperature
33. Rombouts  FM, de Vos  WM, Wouters  JA,     ( 1999 )

Cold shock proteins and low-temperature response of Streptococcus thermophilus CNRZ302.

Applied and environmental microbiology 65 (10)
PMID : 10508072  :   PMC  :   PMC91590    
Abstract >>
Low-temperature adaptation and cryoprotection were studied in the thermophilic lactic acid bacterium Streptococcus thermophilus CNRZ302. S. thermophilus actively adapts to freezing during a pretreatment at 20 degrees C, resulting in an approximately 1, 000-fold increased survival after four freeze-thaw cycles compared to mid-exponential-phase cells grown at an optimal temperature of 42 degrees C. No adaptation is observed when cells are exposed to a temperature (10 degrees C) below the minimal growth temperature of the strain (just below 15 degrees C). By two-dimensional gel electrophoresis several 7-kDa cold-induced proteins were identified, which are the major induced proteins after a shift to 20 degrees C. These cold shock proteins were maximally expressed at 20 degrees C, while the induction level was low after cold shock to 10 degrees C. To confirm the presence of csp genes in S. thermophilus, a PCR strategy was used which yielded products of different sizes. Sequence analysis revealed csp-like sequences that were up to 95% identical to those of csp genes of S. thermophilus ST1-1, Streptococcus dysgalactiae, Streptococcus pyogenes, and Lactococcus lactis. Northern blot analysis revealed a seven- to ninefold induction of csp mRNA after a temperature shift to 20 degrees C, showing that this thermophilic bacterium indeed contains at least one cold-inducible csp gene and that its regulation takes place at the transcriptional level.
KeywordMeSH Terms
Cold Temperature
34. Rul  F,     ( 1999 )

PepS from Streptococcus thermophilus. A new member of the aminopeptidase T family of thermophilic bacteria.

European journal of biochemistry 263 (2)
PMID : 10406960  :   DOI  :   10.1046/j.1432-1327.1999.00528.x    
Abstract >>
The proteolytic system of lactic acid bacteria is essential for bacterial growth in milk but also for the development of the organoleptic properties of dairy products. Streptococcus thermophilus is widely used in the dairy industry. In comparison with the model lactic acid bacteria Lactococcus lactis, S. thermophilus possesses two additional peptidases (an oligopeptidase and the aminopeptidase PepS). To understand how S. thermophilus grows in milk, we purified and characterized this aminopeptidase. PepS is a monomeric metallopeptidase of approximately 45 kDa with optimal activity in the range pH 7.5-8.5 and at 55 degrees C on Arg-paranitroanilide as substrate. PepS exhibits a high specificity towards peptides possessing arginine or aromatic amino acids at the N-terminus. From the N-terminal protein sequence of PepS, we deduced degenerate oligonucleotides and amplified the corresponding gene by successive PCR reactions. The deduced amino-acid sequence of the PepS gene has high identity (40-50%) with the aminopeptidase T family from thermophilic and extremophilic bacteria; we thus propose the classification of PepS from S. thermophilus as a new member of this family. In view of its substrate specificity, PepS could be involved both in bacterial growth by supplying amino acids, and in the development of dairy products' flavour, by hydrolysing bitter peptides and liberating aromatic amino acids which are important precursors of aroma compounds.
KeywordMeSH Terms
Bacterial Proteins
Metalloendopeptidases
35. Guédon  G, Pluvinet  A, Gintz  B, Decaris  B,     ( 1999 )

Are horizontal transfers involved in the evolution of the Streptococcus thermophilus exopolysaccharide synthesis loci?

Gene 233 (1��2��)
PMID : 10375631  :   DOI  :   10.1016/s0378-1119(99)00144-4    
Abstract >>
A 32.5kb variable locus of the Streptococcus thermophilus CNRZ368 chromosome, the eps locus, contains 25 ORF and seven insertion sequences (IS). The putative products of 17 ORF are related to proteins involved in the synthesis of polysaccharides in various bacteria. The two distal regions and a small central region of the eps locus are constant and present in all or almost all of the S. thermophilus strains tested. The other regions are variable and present in only some S. thermophilus strains tested, particularly in the closely related strains CNRZ368 and A054. A 13.6kb variable region of the eps locus of S. thermophilus CNRZ368 contains two ORF that are almost identical to epsL and orfY of the eps locus of Lactococcus lactis NIZOB40 and seven IS belonging to four different families, ISS1, IS981, IS1193 and IS1194. Five of these sequences were probably acquired by horizontal transfer from L. lactis (Bourgoin, F., et al., 1996. Gene 178, 15-23). Three probes of this 13.6kb region hybridized with the DNA of several L. lactis strains tested. A specific probe for another sequence within the S. thermophilus eps locus, epsF, hybridized with the DNA of one of the L. lactis strains tested. Sequence comparisons also suggest that five ORF of the eps locus have a mosaic structure and probably result from recombinations between sequences that are 10 to 50% divergent. The chimeric structure of the eps locus suggests a very complex evolution. This evolution probably involves both the acquisition of the 13.6kb region from L. lactis by horizontal transfer and exchanges within the S. thermophilus species.
KeywordMeSH Terms
Evolution, Molecular
Gene Transfer, Horizontal
36. Casey  MG,     ( 1999 )

Purification, characterization, gene cloning, sequencing, and overexpression of aminopeptidase N from Streptococcus thermophilus A.

Applied and environmental microbiology 65 (7)
PMID : 10388695  :   PMC  :   PMC91448    
Abstract >>
The general aminopeptidase PepN from Streptococcus thermophilus A was purified to protein homogeneity by hydroxyapatite, anion-exchange, and gel filtration chromatographies. The PepN enzyme was estimated to be a monomer of 95 kDa, with maximal activity on N-Lys-7-amino-4-methylcoumarin at pH 7 and 37 degrees C. It was strongly inhibited by metal chelating agents, suggesting that it is a metallopeptidase. The activity was greatly restored by the bivalent cations Co2+, Zn2+, and Mn2+. Except for proline, glycine, and acidic amino acid residues, PepN has a broad specificity on the N-terminal amino acid of small peptides, but no significant endopeptidase activity has been detected. The N-terminal and short internal amino acid sequences of purified PepN were determined. By using synthetic primers and a battery of PCR techniques, the pepN gene was amplified, subcloned, and further sequenced, revealing an open reading frame of 2,541 nucleotides encoding a protein of 847 amino acids with a molecular weight of 96,252. Amino acid sequence analysis of the pepN gene translation product shows high homology with other PepN enzymes from lactic acid bacteria and exhibits the signature sequence of the zinc metallopeptidase family. The pepN gene was cloned in a T7 promoter-based expression plasmid and the 452-fold overproduced PepN enzyme was purified to homogeneity from the periplasmic extract of the host Escherichia coli strain. The overproduced enzyme showed the same catalytic characteristics as the wild-type enzyme.
KeywordMeSH Terms
CD13 Antigens
37. O'Sullivan  T, van Sinderen  D,     ( 1999 )

Structural and functional analysis of pCI65st, a 6.5 kb plasmid from Streptococcus thermophilus NDI-6.

Microbiology (Reading, England) 145 (Pt 1) (N/A)
PMID : 10206690  :   DOI  :   10.1099/13500872-145-1-127    
Abstract >>
The 6.5 kb cryptic plasmid pCI65st from Streptococcus thermophilus NDI-6, a strain isolated from the Indian fermented milk dahi, was subcloned and sequenced. Five putative ORFs were identified. ORF1 could encode a 315 aa polypeptide almost identical to the RepA protein of previously sequenced S. thermophilus plasmids, indicating that pCI65st is one of the pC194 group of small gram-positive rolling-circle plasmids. ORFs 2 and 4 were virtually identical and could specify proteins of approximately 150 aa with significant similarity to the small heat-shock proteins described from a variety of gram-positive bacteria. ORF3 could encode a 415 aa protein similar to enolase, an enzyme involved in glycolysis and gluconeogenesis. ORF5 could encode a 412 aa protein which had high similarity to the HsdS (specificity) proteins of type I restriction-modification systems. Variants of strain NDI-6 which lacked pCI65st were readily isolated after subculture of the parent strain at 32 degrees C. The plasmid-bearing parent culture was significantly more resistant to a temperature shift from 42 degrees C to 62 degrees C than its plasmid-free variant and expressed proteins which corresponded with the predicted translation products from ORF2 and ORF4. In addition, plasmid-free mutants were lysed in broth by bacteriophages to which the parent culture was resistant.
KeywordMeSH Terms
Cloning, Molecular
DNA Helicases
DNA-Binding Proteins
Trans-Activators
38. Girard  SL, Moineau  S,     ( 2007 )

Analysis of two theta-replicating plasmids of Streptococcus thermophilus.

Plasmid 58 (2)
PMID : 17507093  :   DOI  :   10.1016/j.plasmid.2007.03.003    
Abstract >>
We report the characterization of two new theta-replicating plasmids of Streptococcus thermophilus (pSMQ-312b and pSMQ-316) as well as the further analysis of pSMQ-308. The nucleotide sequences of pSMQ-312b and pSMQ-316 were determined and both contained 6710 bp. In fact, the two sequences were identical, despite that the plasmids were isolated from two different S. thermophilus strains as demonstrated by pulsed-field gel electrophoresis. Comparative analyses indicated that the two plasmids were highly related to the previously characterized S. thermophilus plasmid pSMQ-308 (8144 bp). Plasmid stability tests showed that pSMQ-312b/316 was more stable in LM17 medium while pSMQ-308 was the most stable in milk. The presence of the plasmids did not modify the acidification profile of the S. thermophilus strains during growth in milk and under time-temperature conditions mimicking an industrial process. These theta-replicating plasmids are unique genetic material for the construction of stable cloning vectors for industrially relevant strains of S. thermophilus.
KeywordMeSH Terms
39. Van der Meulen  R, Grosu-Tudor  S, Mozzi  F, Vaningelgem  F, Zamfir  M, de Valdez  GF, De Vuyst  L,     ( 2007 )

Screening of lactic acid bacteria isolates from dairy and cereal products for exopolysaccharide production and genes involved.

International journal of food microbiology 118 (3)
PMID : 17716765  :   DOI  :   10.1016/j.ijfoodmicro.2007.07.014    
Abstract >>
A total of 174 lactic acid bacteria (LAB) strains isolated from dairy and cereal products were screened for the production of exopolysaccharides (EPS). Therefore, a rapid screening method was developed based on ultrafiltration and gel permeation chromatography. Furthermore, a screening through the polymerase chain reaction (PCR) was performed with primer pairs targeting different genes involved in EPS production. Nine isolates produced a homopolysaccharide of the glucan type, whereas only one strain produced a heteropolysaccharide. The production of a glucan by a strain of Lactococcus lactis and the production of a heteropolysaccharide by a strain of Lactobacillus curvatus are reported for the first time. The PCR screening revealed many positive strains. For three of the ten EPS-producing strains, no corresponding genes could be detected. Furthermore, a lot of strains possessed one or more eps genes but did not produce an EPS. Therefore, a screening on the molecular level should always be accompanied by another screening method that is able to distinguish true EPS producer strains from non-producing ones. Statistical analysis did not reveal any relationship between the type and origin of the strains, the presence or absence of a capsular polysaccharide or EPS, and the presence or absence of eps genes.
KeywordMeSH Terms
40. Petrova  PM, Gouliamova  DE,     ( 2006 )

Rapid screening of plasmid-encoded small hsp-genes in Streptococcus thermophilus.

Current microbiology 53 (5)
PMID : 17066336  :   DOI  :   10.1007/s00284-006-0175-6    
Abstract >>
A new rapid procedure for detection of small heat shock protein genes (shsp) was developed. Using PCR-based molecular approach, single colonies of 49 Streptococcus thermophilus industrial and artisanal starters were examined. Five strains contained plasmid-encoded shsp. The nucleotide sequence analysis revealed that the genes are very conservative, as only a few nucleotide substitutions were noticed. It was shown that all new isolated plasmids belong to the pC194 family of rolling circle replicating (RCR) plasmids. We concluded that shsp genes are always inherited together with pC194-type replicative region. The viability of plasmid-bearing and plasmid-free derivatives of S. thermophilus strain ST2980 under heat shock condition was studied.
KeywordMeSH Terms
Plasmids
41. Mozzi  F, Vaningelgem  F, Hébert  EM, Van der Meulen  R, Foulquié Moreno  MR, Font de Valdez  G, De Vuyst  L,     ( 2006 )

Diversity of heteropolysaccharide-producing lactic acid bacterium strains and their biopolymers.

Applied and environmental microbiology 72 (6)
PMID : 16751563  :   DOI  :   10.1128/AEM.02780-05     PMC  :   PMC1489642    
Abstract >>
Thirty-one lactic acid bacterial strains from different species were evaluated for exopolysaccharide (EPS) production in milk. Thermophilic strains produced more EPS than mesophilic ones, but EPS yields were generally low. Ropiness or capsular polysaccharide formation was strain dependent. Six strains produced high-molecular-mass EPS. Polymers were classified into nine groups on the basis of their monomer composition. EPS from Enterococcus strains were isolated and characterized.
KeywordMeSH Terms
42. Poolman  B, Royer  TJ, Mainzer  SE, Schmidt  BF,     ( 1990 )

Carbohydrate utilization in Streptococcus thermophilus: characterization of the genes for aldose 1-epimerase (mutarotase) and UDPglucose 4-epimerase.

Journal of bacteriology 172 (1��7��)
PMID : 1694527  :   DOI  :   10.1128/jb.172.7.4037-4047.1990     PMC  :   PMC213390    
Abstract >>
The complete nucleotide sequences of the genes encoding aldose 1-epimerase (mutarotase) (galM) and UDPglucose 4-epimerase (galE) and flanking regions of Streptococcus thermophilus have been determined. Both genes are located immediately upstream of the S. thermophilus lac operon. To facilitate the isolation of galE, a special polymerase chain reaction-based technique was used to amplify the region upstream of galM prior to cloning. The galM protein was homologous to the mutarotase of Acinetobacter calcoaceticus, whereas the galE protein was homologous to UDPglucose 4-epimerase of Escherichia coli and Streptomyces lividans. The amino acid sequences of galM and galE proteins also showed significant similarity with the carboxy-terminal and amino-terminal domains, respectively, of UDPglucose 4-epimerase from Kluyveromyces lactis and Saccharomyces cerevisiae, suggesting that the yeast enzymes contain an additional, yet unidentified (mutarotase) activity. In accordance with the open reading frames of the structural genes, galM and galE were expressed as polypeptides with apparent molecular masses of 39 and 37 kilodaltons, respectively. Significant activities of mutarotase and UDPglucose 4-epimerase were detected in lysates of E. coli cells containing plasmids encoding galM and galE. Expression of galE in E. coli was increased 300-fold when the gene was placed downstream of the tac promoter. The gene order for the gal-lac gene cluster of S. thermophilus is galE-galM-lacS-lacZ. The flanking regions of these genes were searched for consensus promoter sequences and further characterized by primer extension analysis. Analysis of mRNA levels for the gal and lac genes in S. thermophilus showed a strong reduction upon growth in medium containing glucose instead of lactose. The activities of the lac (lactose transport and beta-galactosidase) and gal (UDPglucose 4-epimerase) proteins of lactose- and glucose-grown S. thermophilus cells matched the mRNA levels.
KeywordMeSH Terms
Carbohydrate Metabolism
Genes, Bacterial
43. Borges  F, Layec  S, Fernandez  A, Decaris  B, Leblond-Bourget  N,     ( 2006 )

High genetic variability of the Streptococcus thermophilus cse central part, a repeat rich region required for full cell segregation activity.

Antonie van Leeuwenhoek 90 (3)
PMID : 16902754  :   DOI  :   10.1007/s10482-006-9079-5    
Abstract >>
The cse gene of Streptococcus thermophilus encodes an extracytoplasmic protein involved in cell segregation. The Cse protein consists of two putative domains: a cell wall attachment LysM domain and a catalytic CHAP domain. These two domains are spaced by an interdomain linker, known as Var-Cse, previously reported to be highly divergent between two S. thermophilus strains. The aim of this study was to assess the extent of this intraspecific variability and the functional involvement of the var-cse region in cell segregation. Analysis of the var-cse sequence of 19 different strains allowed detection of 11 different alleles, varying from 390 bp to 543 bp, all containing interspersed and tandem nucleotides repeats. Overall, 11 different repeat units were identified and some series of these small repeats, named supermotifs, form large repeats. Results suggested that var-cse evolved by deletion of all or part of the repeats and by duplication of repeats or supermotifs. Moreover, sequence analysis of the whole cse locus revealed that the cse ORF is mosaic suggesting that var-cse polymorphism resulted from horizontal transfer. The partial deletion of the var-cse region of the S. thermophilus strain CNRZ368 led to the lengthening of the number of cells per streptococcal chain, indicating that this region is required for full cell segregation in S. thermophilus strain CNRZ368.
KeywordMeSH Terms
44. Fernandez  A, Borges  F, Gintz  B, Decaris  B, Leblond-Bourget  N,     ( 2006 )

The rggC locus, with a frameshift mutation, is involved in oxidative stress response by Streptococcus thermophilus.

Archives of microbiology 186 (3)
PMID : 16847652  :   DOI  :   10.1007/s00203-006-0130-8    
Abstract >>
In Streptococcus thermophilus, the locus rggC contains a frameshift mutation and thus consists of two open reading frames (ORFs), rggC (1) and rggC (2), which encode proteins exhibiting similarity with the Rgg transcriptional regulator family. In this work, mutants showing a partial deletion of rggC (1) and rggC (2)were constructed and their response to menadione, a superoxide-generating compound, was analysed. These mutants exhibited different behaviour to this oxidative stress compared with the wild-type strain. Analysis of this locus among 21 strains of S. thermophilus showed a polythymine tract length variability and a strain-dependant adenine residue could be found upstream of this repeat. This interstrain polymorphism supports evidence for the hypothesis that the rggC locus is phase variable.
KeywordMeSH Terms
Oxidative Stress
45. Itoh  Y, Kawamura  Y, Kasai  H, Shah  MM, Nhung  PH, Yamada  M, Sun  X, Koyana  T, Hayashi  M, Ohkusu  K, Ezaki  T,     ( 2006 )

dnaJ and gyrB gene sequence relationship among species and strains of genus Streptococcus.

Systematic and applied microbiology 29 (5)
PMID : 16487673  :   DOI  :   10.1016/j.syapm.2005.12.003    
Abstract >>
The dnaJ and gyrB nucleotide sequences were determined for members of the genus Streptococcus. The average similarity between the species tested was 76.4% (69.7-100%) for dnaJ and 75.9 (70.1-98.7%) for gyrB. These data indicated that the dnaJ and gyrB genes are more divergent and more discriminatory than the 16S rDNA gene. Furthermore, the variation in the dnaJ nucleotide sequences among the mitis group was greater than that of the gyrB nucleotide sequences, especially between Streptococcus pneumoniae and Streptococcus mitis. Subsequently, the high discrimination power of dnaJ within the mitis group was confirmed. Thus, we conclude that the dnaJ and gyrB genes are efficient alternative targets for the classification of the genus Streptococcus, and that dnaJ is suitable for phylogenetic analysis of closely related Streptococcus strains.
KeywordMeSH Terms
46. Monnet  C, Mora  D, Corrieu  G,     ( 2005 )

Glutamine synthesis is essential for growth of Streptococcus thermophilus in milk and is linked to urea catabolism.

Applied and environmental microbiology 71 (6)
PMID : 15933046  :   DOI  :   10.1128/AEM.71.6.3376-3378.2005     PMC  :   PMC1151851    
Abstract >>
Growth of a glutamine synthetase-deficient mutant of Streptococcus thermophilus was compared to that of the parent strain in milk that was not supplemented or was supplemented with ammonium chloride, glutamine, or the urease inhibitor flurofamide. It was concluded that one of the functions of urease is to supply ammonia for the synthesis of glutamine.
KeywordMeSH Terms
47. Sperisen  P, Schmid  CD, Bucher  P, Zilian  O,     ( 2005 )

Stealth proteins: in silico identification of a novel protein family rendering bacterial pathogens invisible to host immune defense.

PLoS computational biology 1 (1��6��)
PMID : 16299590  :   DOI  :   10.1371/journal.pcbi.0010063     PMC  :   PMC1285062    
Abstract >>
There are a variety of bacterial defense strategies to survive in a hostile environment. Generation of extracellular polysaccharides has proved to be a simple but effective strategy against the host's innate immune system. A comparative genomics approach led us to identify a new protein family termed Stealth, most likely involved in the synthesis of extracellular polysaccharides. This protein family is characterized by a series of domains conserved across phylogeny from bacteria to eukaryotes. In bacteria, Stealth (previously characterized as SacB, XcbA, or WefC) is encoded by subsets of strains mainly colonizing multicellular organisms, with evidence for a protective effect against the host innate immune defense. More specifically, integrating all the available information about Stealth proteins in bacteria, we propose that Stealth is a D-hexose-1-phosphoryl transferase involved in the synthesis of polysaccharides. In the animal kingdom, Stealth is strongly conserved across evolution from social amoebas to simple and complex multicellular organisms, such as Dictyostelium discoideum, hydra, and human. Based on the occurrence of Stealth in most Eukaryotes and a subset of Prokaryotes together with its potential role in extracellular polysaccharide synthesis, we propose that metazoan Stealth functions to regulate the innate immune system. Moreover, there is good reason to speculate that the acquisition and spread of Stealth could be responsible for future epidemic outbreaks of infectious diseases caused by a large variety of eubacterial pathogens. Our in silico identification of a homologous protein in the human host will help to elucidate the causes of Stealth-dependent virulence. At a more basic level, the characterization of the molecular and cellular function of Stealth proteins may shed light on fundamental mechanisms of innate immune defense against microbial invasion.
KeywordMeSH Terms
Computational Biology
48. Naser  S, Thompson  FL, Hoste  B, Gevers  D, Vandemeulebroecke  K, Cleenwerck  I, Thompson  CC, Vancanneyt  M, Swings  J,     ( 2005 )

Phylogeny and identification of Enterococci by atpA gene sequence analysis.

Journal of clinical microbiology 43 (5)
PMID : 15872246  :   DOI  :   10.1128/JCM.43.5.2224-2230.2005     PMC  :   PMC1153757    
Abstract >>
The relatedness among 91 Enterococcus strains representing all validly described species was investigated by comparing a 1,102-bp fragment of atpA, the gene encoding the alpha subunit of ATP synthase. The relationships observed were in agreement with the phylogeny inferred from 16S rRNA gene sequence analysis. However, atpA gene sequences were much more discriminatory than 16S rRNA for species differentiation. All species were differentiated on the basis of atpA sequences with, at a maximum, 92% similarity. Six members of the Enterococcus faecium species group (E. faecium, E. hirae, E. durans, E. villorum, E. mundtii, and E. ratti) showed > 99% 16S rRNA gene sequence similarity, but the highest value of atpA gene sequence similarity was only 89.9%. The intraspecies atpA sequence similarities for all species except E. faecium strains varied from 98.6 to 100%; the E. faecium strains had a lower atpA sequence similarity of 96.3%. Our data clearly show that atpA provides an alternative tool for the phylogenetic study and identification of enterococci.
KeywordMeSH Terms
49. Borges  F, Layec  S, Thibessard  A, Fernandez  A, Gintz  B, Hols  P, Decaris  B, Leblond-Bourget  N,     ( 2005 )

cse, a Chimeric and variable gene, encodes an extracellular protein involved in cellular segregation in Streptococcus thermophilus.

Journal of bacteriology 187 (8)
PMID : 15805520  :   DOI  :   10.1128/JB.187.8.2737-2746.2005     PMC  :   PMC1070363    
Abstract >>
The isolation of a Streptococcus thermophilus CNRZ368 mutant displaying a long-chain phenotype allowed us to identify the cse gene (for cellular segregation). The N terminus of Cse exhibits high similarity to Streptococcus agalactiae surface immunogenic protein (SIP), while its C terminus exhibits high similarity to S. thermophilus PcsB. In CNRZ368, deletion of the entire cse open reading frame leads to drastic lengthening of cell chains and altered colony morphology. Complementation of the Deltacse mutation with a wild-type allele restored both wild-type phenotypes. The central part of Cse is a repeat-rich region with low sequence complexity. Comparison of cse from CNRZ368 and LMG18311 strains reveals high variability of this repeat-rich region. To assess the impact of this central region variability, the central region of LMG18311 cse was exchanged with that of CNRZ368 cse. This replacement did not affect chain length, showing that divergence of the central part does not modify cell segregation activity of Cse. The structure of the cse locus suggests that the chimeric organization of cse results from insertion of a duplicated sequence deriving from the pcsB 3' end into an ancestral sip gene. Thus, the cse locus illustrates the module-shuffling mechanism of bacterial gene evolution.
KeywordMeSH Terms
Cell Compartmentation
50. Cochu  A, Roy  D, Vaillancourt  K, Lemay  JD, Casabon  I, Frenette  M, Moineau  S, Vadeboncoeur  C,     ( 2005 )

The doubly phosphorylated form of HPr, HPr(Ser~P)(His-P), is abundant in exponentially growing cells of Streptococcus thermophilus and phosphorylates the lactose transporter LacS as efficiently as HPr(His~P).

Applied and environmental microbiology 71 (3)
PMID : 15746339  :   DOI  :   10.1128/AEM.71.3.1364-1372.2005     PMC  :   PMC1065139    
Abstract >>
In Streptococcus thermophilus, lactose is taken up by LacS, a transporter that comprises a membrane translocator domain and a hydrophilic regulatory domain homologous to the IIA proteins and protein domains of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The IIA domain of LacS (IIALacS) possesses a histidine residue that can be phosphorylated by HPr(His~P), a protein component of the PTS. However, determination of the cellular levels of the different forms of HPr, namely, HPr, HPr(His~P), HPr(Ser-P), and HPr(Ser-P)(His~P), in exponentially lactose-growing cells revealed that the doubly phosphorylated form of HPr represented 75% and 25% of the total HPr in S. thermophilus ATCC 19258 and S. thermophilus SMQ-301, respectively. Experiments conducted with [32P]PEP and purified recombinant S. thermophilus ATCC 19258 proteins (EI, HPr, and IIALacS) showed that IIALacS was reversibly phosphorylated by HPr(Ser-P)(His~P) at a rate similar to that measured with HPr(His~P). Sequence analysis of the IIALacS protein domains from several S. thermophilus strains indicated that they can be divided into two groups on the basis of their amino acid sequences. The amino acid sequence of IIALacS from group I, to which strain 19258 belongs, differed from that of group II at 11 to 12 positions. To ascertain whether IIALacS from group II could also be phosphorylated by HPr(His~P) and HPr(Ser-P)(His~P), in vitro phosphorylation experiments were conducted with purified proteins from Streptococcus salivarius ATCC 25975, which possesses a IIALacS very similar to group II S. thermophilus IIALacS. The results indicated that S. salivarius IIALacS was phosphorylated by HPr(Ser-P)(His~P) at a higher rate than that observed with HPr(His~P). Our results suggest that the reversible phosphorylation of IIALacS in S. thermophilus is accomplished by HPr(Ser-P)(His~P) as well as by HPr(His~P).
KeywordMeSH Terms
51. Vidal  L, Calveras  J, Clapés  P, Ferrer  P, Caminal  G,     ( 2005 )

Recombinant production of serine hydroxymethyl transferase from Streptococcus thermophilus and its preliminary evaluation as a biocatalyst.

Applied microbiology and biotechnology 68 (4)
PMID : 15726349  :   DOI  :   10.1007/s00253-005-1934-1    
Abstract >>
The glyA gene encoding a serine hydroxymethyl transferase (SHMT) with threonine aldolase activity was isolated from Streptococcus thermophilus YKA-184 chromosomal DNA. This aldolase is a pyridoxal 5'-phosphate-dependent enzyme that stereospecifically catalyzes the interconversion of L-threonine to glycine and acetaldehyde. The enzyme was overexpressed in Escherichia coli M15 as a recombinant protein of 45 kDa with a His6-tag at its N-terminus. The recombinant enzyme was purified to homogeneity by a single chromatographic step using Ni-nitrilotriacetic acid affinity, obtaining a high activity-recovery yield (83%). Lyophilized and precipitated enzymes were stable at least for 10 weeks when stored at -20 degrees C and 4 degrees C. It was observed that the Km for L-allo-threonine was 38-fold higher than that for L-threonine, suggesting this enzyme can be classified as a specific L-allo-threonine aldolase. The optimum pH range of threonine aldolase activity for the recombinant SHMT was pH 6-7. When tested for aldol addition reactions with non-natural aldehydes, such as benzyloxyacetaldehyde and (R)-N-Cbz-alaninal, two possible beta-hydroxy-alpha-amino acid diastereoisomers were produced, but with moderate stereospecificity. The enzyme showed potential as a biocatalyst for the stereoselective synthesis of beta-hydroxy-alpha-amino acids.
KeywordMeSH Terms
52. Ercolini  D, Fusco  V, Blaiotta  G, Coppola  S,     ( 2005 )

Sequence heterogeneity in the lacSZ operon of Streptococcus thermophilus and its use in PCR systems for strain differentiation.

Research in microbiology 156 (2)
PMID : 15748980  :   DOI  :   10.1016/j.resmic.2004.09.005    
Abstract >>
Sequences of the lacSZ operon of 29 Streptococcus thermophilus strains from different dairy products were determined. Differences in sequence among the strains were detected within LacS more often than in the LacZ gene. The sequences were aligned and compared and it was possible to gather the strains into three groups of similarity on the basis of the LacS gene sequence. The dairy environment of origin did not seem to be related to the lacSZ operon sequence and thus to the similarity shown. Nucleotide variability was investigated and a total of 139 nucleotide changes were found in the LacS gene while 40 nucleotide changes were found in the sequences of the LacZ gene. Moreover, the influence of the nucleotide changes on the amino acid sequence of the LacS transporter and of the beta-galactosidase enzyme were discussed. Sequence variability within the region upstream from the LacS gene was used to develop group-specific PCR systems capable of distinguishing S. thermophilus at the strain level. A strain-specific primer set was designed allowing the specific detection of 11 out of 29 strains of S. thermophilus. Moreover, LacS-PCR-SSCP analysis of the 29 strains provided 2 different profiles, whereas 4 strain-specific profiles were detected by LacS-PCR-DGGE, indicating the potential to use these techniques for profiling and monitoring population of strains of S. thermophilus in food products. The results are discussed with reference to the potential of these PCR methods for ascertaining strain dominance and starter fitness in dairy processes.
KeywordMeSH Terms
Bacterial Typing Techniques
Genetic Variation
53. Mora  D, Monnet  C, Parini  C, Guglielmetti  S, Mariani  A, Pintus  P, Molinari  F, Daffonchio  D, Manachini  PL,     ( 2005 )

Urease biogenesis in Streptococcus thermophilus.

Research in microbiology 156 (9)
PMID : 16024230  :   DOI  :   10.1016/j.resmic.2005.04.005    
Abstract >>
Urease biogenesis was monitored in the lactic acid bacterium Streptococcus thermophilus during the growth cycle using in-gel detection and a phenol-hypochloride assay. Zymogram analysis, performed in a non-denaturing polyacrylamide gel, enabled visualization of a complex profile of bands whose number and intensity were dependent on the growth phase and culture conditions. The monitoring of urease biogenesis in batch fermentations revealed the onset of enzyme synthesis starting from the mid-exponential growth phase, with a maximum reached during the late exponential phase. Urease activity strongly increased at acidic pH but to a lesser extent when urea and nickel ions were added to the culture medium. When S. thermophilus cells were cultured with pH maintained at a neutral value, urease activity was detectable only in gel with extremely low signals. Evaluation of beta-glucuronidase activity in strain DSM 20617(T) harboring a transcriptional fusion between a DNA fragment containing the putative urease promoter and the gusA reporter evidenced significant expression at neutral pH that strongly increased in an acidic environment. Further experiments carried out on p(ureI)-gusA recombinant strain revealed that expression of ure genes was not affected by carbohydrates, nickel or urea availability. The presence of consistent expression of ure genes at neutral pH and the absence of induction of expression by carbohydrate availability demonstrated that the transcription of ure genes in S. thermophilus is regulated differently compared with that of the closely related S. salivarius. These differences are discussed taking into consideration the different habitats colonized by the two bacterial species.
KeywordMeSH Terms
54. de Vin  F, Rådström  P, Herman  L, De Vuyst  L,     ( 2005 )

Molecular and biochemical analysis of the galactose phenotype of dairy Streptococcus thermophilus strains reveals four different fermentation profiles.

Applied and environmental microbiology 71 (7)
PMID : 16000774  :   DOI  :   10.1128/AEM.71.7.3659-3667.2005     PMC  :   PMC1168995    
Abstract >>
Lactose-limited fermentations of 49 dairy Streptococcus thermophilus strains revealed four distinct fermentation profiles with respect to galactose consumption after lactose depletion. All the strains excreted galactose into the medium during growth on lactose, except for strain IMDOST40, which also displayed extremely high galactokinase (GalK) activity. Among this strain collection eight galactose-positive phenotypes sensu stricto were found and their fermentation characteristics and Leloir enzyme activities were measured. As the gal promoter seems to play an important role in the galactose phenotype, the galR-galK intergenic region was sequenced for all strains yielding eight different nucleotide sequences (NS1 to NS8). The gal promoter played an important role in the Gal-positive phenotype but did not determine it exclusively. Although GalT and GalE activities were detected for all Gal-positive strains, GalK activity could only be detected for two out of eight Gal-positive strains. This finding suggests that the other six S. thermophilus strains metabolize galactose via an alternative route. For each type of fermentation profile obtained, a representative strain was chosen and four complete Leloir gene clusters were sequenced. It turned out that Gal-positive strains contained more amino acid differences within their gal genes than Gal-negative strains. Finally, the biodiversity regarding lactose-galactose utilization among the different S. thermophilus strains used in this study was shown by RAPD-PCR. Five Gal-positive strains that contain nucleotide sequence NS2 in their galR-galK intergenic region were closely related.
KeywordMeSH Terms
55. Monnet  C, Pernoud  S, Sepulchre  A, Fremaux  C, Corrieu  G,     ( 2004 )

Selection and properties of Streptococcus thermophilus mutants deficient in urease.

Journal of dairy science 87 (6)
PMID : 15453477  :   DOI  :   10.3168/jds.S0022-0302(04)73318-4    
Abstract >>
Natural variations of the urea content of milk have a detrimental effect on the regularity of acidification by Streptococcus thermophilus strains used in dairy processes. The aim of the present study was to select urease-deficient mutants of S. thermophilus and to investigate their properties. Using an improved screening medium on agar plates, mutants were selected from 4 different parent strains after mutagen treatment and by spontaneous mutation. Most mutants were stable and had a phage sensitivity profile similar to that of their parent strain. Some of them contained detrimental secondary mutations, as their acidifying activity was lower than that of the parent strain cultivated in the presence of the urease inhibitor flurofamide. The proportion of this type of mutant was much lower among spontaneous mutants than among mutants selected after mutagen treatment. Utilization of urease-deficient mutants in dairy processes may have several advantages, such as an increase in acidification, an improved regularity of acidification, and a lower production of ammonia in whey.
KeywordMeSH Terms
56. Fernandez  A, Thibessard  A, Borges  F, Gintz  B, Decaris  B, Leblond-Bourget  N,     ( 2004 )

Characterization of oxidative stress-resistant mutants of Streptococcus thermophilus CNRZ368.

Archives of microbiology 182 (5)
PMID : 15378231  :   DOI  :   10.1007/s00203-004-0712-2    
Abstract >>
During industrial processes, the dairy organism Streptococcus thermophilus is exposed to stress conditions. Its ability to survive and grow in an aerobic environment indicates that it must possess defensive mechanisms against reactive oxygen species. To identify the genes involved in oxidative stress defence, a collection of mutants was generated by random insertional mutagenesis and screened for menadione sensitivity and resistance. Results obtained for resistant clones allowed the identification of eight loci. The insertions affected genes whose homologues in other bacteria were previously identified as being involved in stress response(deoB, gst) or transcription regulation (rggC) and five ORFs of unknown function. The tolerance of the eight mutants to air-exposure, methyl viologen and H2O2 was studied. Real-time quantitative PCR was used to analyse the transcript level of mutated genes and revealed that most were down-regulated during oxidative stress.
KeywordMeSH Terms
Drug Resistance, Bacterial
Mutation
Oxidative Stress
57. Hill  JE, Penny  SL, Crowell  KG, Goh  SH, Hemmingsen  SM,     ( 2004 )

cpnDB: a chaperonin sequence database.

Genome research 14 (8)
PMID : 15289485  :   DOI  :   10.1101/gr.2649204     PMC  :   PMC509277    
Abstract >>
Type I chaperonins are molecular chaperones present in virtually all bacteria, some archaea and the plastids and mitochondria of eukaryotes. Sequences of cpn60 genes, encoding 60-kDa chaperonin protein subunits (CPN60, also known as GroEL or HSP60), are useful for phylogenetic studies and as targets for detection and identification of organisms. Conveniently, a 549-567-bp segment of the cpn60 coding region can be amplified with universal PCR primers. Here, we introduce cpnDB, a curated collection of cpn60 sequence data collected from public databases or generated by a network of collaborators exploiting the cpn60 target in clinical, phylogenetic, and microbial ecology studies. The growing database currently contains approximately 2000 records covering over 240 genera of bacteria, eukaryotes, and archaea. The database also contains over 60 sequences for the archaeal Type II chaperonin (thermosome, a homolog of eukaryotic cytoplasmic chaperonin) from 19 archaeal genera. As the largest curated collection of sequences available for a protein-encoding gene, cpnDB provides a resource for researchers interested in exploiting the power of cpn60 as a diagnostic or as a target for phylogenetic or microbial ecology studies, as well as those interested in broader subjects such as lateral gene transfer and codon usage. We built cpnDB from open source tools and it is available at http://cpndb.cbr.nrc.ca.
KeywordMeSH Terms
58. Thibessard  A, Borges  F, Fernandez  A, Gintz  B, Decaris  B, Leblond-Bourget  N,     ( 2004 )

Identification of Streptococcus thermophilus CNRZ368 genes involved in defense against superoxide stress.

Applied and environmental microbiology 70 (4)
PMID : 15066816  :   DOI  :   10.1128/aem.70.4.2220-2229.2004     PMC  :   PMC383142    
Abstract >>
To better understand the defense mechanism of Streptococcus thermophilus against superoxide stress, molecular analysis of 10 menadione-sensitive mutants, obtained by insertional mutagenesis, was undertaken. This analysis allowed the identification of 10 genes that, with respect to their putative functions, were classified into five categories: (i) those involved in cell wall metabolism, (ii) those involved in exopolysaccharide translocation, (iii) those involved in RNA modification, (iv) those involved in iron homeostasis, and (v) those whose functions are still unknown. The behavior of the 10 menadione-sensitive mutants exposed to heat shock was investigated. Data from these experiments allowed us to distinguish genes whose action might be specific to oxidative stress defense (tgt, ossF, and ossG) from those whose action may be generalized to other stressful conditions (mreD, rodA, pbp2b, cpsX, and iscU). Among the mutants, two harbored an independently inserted copy of pGh9:ISS1 in two loci close to each other. More precisely, these two loci are homologous to the sufD and iscU genes, which are involved in the biosynthesis of iron-sulfur clusters. This region, called the suf region, was further characterized in S. thermophilus CNRZ368 by sequencing and by construction of DeltasufD and iscU(97) nonpolar mutants. The streptonigrin sensitivity levels of both mutants suggest that these two genes are involved in iron metabolism.
KeywordMeSH Terms
Genes, Bacterial
59. Mora  D, Maguin  E, Masiero  M, Parini  C, Ricci  G, Manachini  PL, Daffonchio  D,     ( 2004 )

Characterization of urease genes cluster of Streptococcus thermophilus.

Journal of applied microbiology 96 (1)
PMID : 14678176  :  
Abstract >>
The milk acidification rate of Streptococcus thermophilus strains can be affected by several factors, one of which is the hydrolysis of urea by the urease complex. To evaluate the technological suitability of S. thermophilus strains deprived of urease activity in milk fermentation, the genetic cluster related to urease enzymatic activity has been characterized in the type strain DSM 20167T. Amplification of the urease genes of S. thermophilus DSM 20167T was developed on the basis of the urease gene cluster of the phylogenetically related S. salivarius. Nucleotide sequencing revealed the presence of eight open reading frames, which were most homologous to ureABC (structural genes) and ureI, ureEFGD (accessory genes) of S. salivarius and other ureolytic bacteria. Reverse transcriptase PCR experiments were in agreement with an operon organization for the eight genes (ureIABCEFGD). A food grade mutant A16 (DeltaureC3) with a 693 bp in-frame deletion in ureC gene exhibited a urease negative (Ure-) phenotype. Unlike the wild-type strain, the acidification rate of the mutant in reconstituted skimmed milk was not affected by the presence of urea or nickel ions. A small-scale yoghurt fermentation trials were carried out using the wild-type or the Ure- mutant A16 (DeltaureC3) in co-culture with Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 in presence of urea. The result obtained underlines that when the Ure- mutant was used as a co-starter the acidification rate was higher than that obtained using the wild-type strain. The study provides the first genetic characterization and the technological implication of S. thermophilus DSM 20617T urease activity. The detrimental effect of ureolytic activity on the rate of milk acidification was evaluated and superseded using a food-grade Ure- recombinant strain. Small-scale yoghurt production trials highlighted the positive role of a Ure-S. thermophilus mutant as a co-starter in milk fermentations. Moreover, the vector pMI108 developed for the construction of the Ure- strain, should be considered as a potential tool for the generation of Ure- dairy S. thermophilus strains selected for other relevant technological properties but characterized by the undesirable ureolytic phenotype.
KeywordMeSH Terms
Genes, Bacterial
Multigene Family
60. Turgeon  N, Frenette  M, Moineau  S,     ( 2004 )

Characterization of a theta-replicating plasmid from Streptococcus thermophilus.

Plasmid 51 (1)
PMID : 14711526  :  
Abstract >>
Plasmids of Streptococcus thermophilus were previously classified, based on DNA homology, into at least four groups (A-D). Here, we report the characterization of plasmids of group B and D. The sequence analysis of pSMQ173b (group D) indicates that this plasmid contains 4449 bp, five open reading frames (ORFs) and replicates via the rolling-circle mechanism of the pGI3 family. The plasmid pSMQ308 (group B) contains 8144 bp and six ORFs. Two ORFs likely encode a primase/helicase and an integrase. Northern blot experiments demonstrate that these two orfs are transcribed within the three strains containing plasmids of group B. Two-dimensional agarose gel electrophoresis shows that pSMQ308 replicates via a theta mechanism. To our knowledge, this is the first report of a plasmid replicating via a theta mode in S. thermophilus. Finally, a classification of 20 sequenced S. thermophilus plasmids into six groups based on their mode of replication is proposed.
KeywordMeSH Terms
Plasmids
61. Gueneau de Novoa  P, Williams  KP,     ( 2004 )

The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts.

Nucleic acids research 32 (Database issue)
PMID : 14681369  :   DOI  :   10.1093/nar/gkh102     PMC  :   PMC308836    
Abstract >>
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
KeywordMeSH Terms
Databases, Nucleic Acid
Evolution, Molecular
Internet
62. Andrus  JM, Bowen  SW, Klaenhammer  TR, Hassan  HM,     ( 2003 )

Molecular characterization and functional analysis of the manganese-containing superoxide dismutase gene (sodA) from Streptococcus thermophilus AO54.

Archives of biochemistry and biophysics 420 (1)
PMID : 14622980  :   DOI  :   10.1016/j.abb.2003.09.007    
Abstract >>
This report describes the isolation, sequencing, and functional analysis of the sodA gene, encoding Mn-superoxide dismutase, from Streptococcus thermophilus AO54. The gene was found to encode a 201 amino acid polypeptide with 88 and 83% identity to SodA from Streptococcus mutans and Streptococcus agalacticae, respectively. Primer extension analysis revealed a transcriptional start site 27 nucleotides upstream of initiation codon. The gene was expressed in Escherichia coli and was able to rescue the growth of a sodAsodB mutant in a minimal-medium containing 10(-6)M paraquat. A sodA mutant of S. thermophilus was constructed and found to be more sensitive to aerobic growth than its parent strain. Supplementing the medium with MnCl(2) improved the growth of the mutant, only under microaerophilic conditions. The results suggest that sodA is essential for the aerobic growth of S. thermophilus. In the absence of functional SodA, manganese ions may provide partial protection against oxygen toxicity.
KeywordMeSH Terms
Sequence Alignment
63. Petrova  P, Miteva  V, Ruiz-Masó  JA, del Solar  G,     ( 2003 )

Structural and functional analysis of pt38, a 2.9kb plasmid of Streptococcus thermophilus yogurt strain.

Plasmid 50 (3)
PMID : 14597007  :  
Abstract >>
The cryptic plasmid pt38 (2911 bp) of Streptococcus thermophilus ST2783, a strain isolated from Bulgarian yogurt, was subcloned and sequenced. Five ORFs (ORF1 to ORF5) were identified, although putative transcription initiation and termination signals, and Shine-Dalgarno sequence could only be localized for three of them (ORF1, ORF2, and ORF5). ORF2 would specify a 142-amino acid protein sharing a high degree of homology with plasmid-born low-molecular-weight heat stress proteins described in a variety of S. thermophilus strains. On the other hand, ORF1 would encode a 311-residue protein, which was found to be almost identical to the putative Rep proteins of previously sequenced S. thermophilus rolling circle-replicating plasmids. Intracellular single-stranded pt38 DNA was detected, showing that, in fact, the plasmid replicates via a rolling circle mechanism. A putative double-strand origin with significant homology to that of pC194, and a ssoA-type single-strand origin were also identified on the nucleotide sequence of pt38. A DNA region that can be transcribed into a small RNA (ctRNA) complementary to the leader segment of the rep (ORF1) mRNA is proposed to be involved in the control of plasmid replication. In vitro synthesis of this ctRNA was observed, and this constitutes the first report on the existence of such antisense RNAs, likely acting as regulatory elements, in S. thermophilus plasmids.
KeywordMeSH Terms
DNA-Binding Proteins
64. Sapranauskas  R, Gasiunas  G, Fremaux  C, Barrangou  R, Horvath  P, Siksnys  V,     ( 2011 )

The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli.

Nucleic acids research 39 (21)
PMID : 21813460  :   DOI  :   10.1093/nar/gkr606     PMC  :   PMC3241640    
Abstract >>
The CRISPR/Cas adaptive immune system provides resistance against phages and plasmids in Archaea and Bacteria. CRISPR loci integrate short DNA sequences from invading genetic elements that provide small RNA-mediated interference in subsequent exposure to matching nucleic acids. In Streptococcus thermophilus, it was previously shown that the CRISPR1/Cas system can provide adaptive immunity against phages and plasmids by integrating novel spacers following exposure to these foreign genetic elements that subsequently direct the specific cleavage of invasive homologous DNA sequences. Here, we show that the S. thermophilus CRISPR3/Cas system can be transferred into Escherichia coli and provide heterologous protection against plasmid transformation and phage infection. We show that interference is sequence-specific, and that mutations in the vicinity or within the proto-spacer adjacent motif (PAM) allow plasmids to escape CRISPR-encoded immunity. We also establish that cas9 is the sole cas gene necessary for CRISPR-encoded interference. Furthermore, mutation analysis revealed that interference relies on the Cas9 McrA/HNH- and RuvC/RNaseH-motifs. Altogether, our results show that active CRISPR/Cas systems can be transferred across distant genera and provide heterologous interference against invasive nucleic acids. This can be leveraged to develop strains more robust against phage attack, and safer organisms less likely to uptake and disseminate plasmid-encoded undesirable genetic elements.
KeywordMeSH Terms
65. Sinkunas  T, Gasiunas  G, Fremaux  C, Barrangou  R, Horvath  P, Siksnys  V,     ( 2011 )

Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system.

The EMBO journal 30 (7)
PMID : 21343909  :   DOI  :   10.1038/emboj.2011.41     PMC  :   PMC3094125    
Abstract >>
Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase(+)/nuclease(-) and ATPase(-)/nuclease(+)) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade-crRNA complex targeting foreign DNA.
KeywordMeSH Terms
Repetitive Sequences, Nucleic Acid
66. Delorme  C, Bartholini  C, Bolotine  A, Ehrlich  SD, Renault  P,     ( 2010 )

Emergence of a cell wall protease in the Streptococcus thermophilus population.

Applied and environmental microbiology 76 (2)
PMID : 19915034  :   DOI  :   10.1128/AEM.01018-09     PMC  :   PMC2805209    
Abstract >>
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.
KeywordMeSH Terms
67. Glazunova  OO, Raoult  D, Roux  V,     ( 2010 )

Partial recN gene sequencing: a new tool for identification and phylogeny within the genus Streptococcus.

International journal of systematic and evolutionary microbiology 60 (Pt 9)
PMID : 19880633  :   DOI  :   10.1099/ijs.0.018176-0    
Abstract >>
Partial sequences of the recN gene (1249 bp), which encodes a recombination and repair protein, were analysed to determine the phylogenetic relationship and identification of streptococci. The partial sequences presented interspecies nucleotide similarity of 56.4-98.2 % and intersubspecies similarity of 89.8-98 %. The mean DNA sequence similarity of recN gene sequences (66.6 %) was found to be lower than those of the 16S rRNA gene (94.1 %), rpoB (84.6 %), sodA (74.8 %), groEL (78.1 %) and gyrB (73.2 %). Phylogenetically derived trees revealed six statistically supported groups: Streptococcus salivarius, S. equinus, S. hyovaginalis/S. pluranimalium/S. thoraltensis, S. pyogenes, S. mutans and S. suis. The 'mitis' group was not supported by a significant bootstrap value, but three statistically supported subgroups were noted: Streptococcus sanguinis/S. cristatus/S. sinensis, S. anginosus/S. intermedius/S. constellatus (the 'anginosus' subgroup) and S. mitis/S. infantis/S. peroris/S. oralis/S. oligofermentans/S. pneumoniae/S. pseudopneumoniae. The partial recN gene sequence comparison highlighted a high percentage of divergence between Streptococcus dysgalactiae subsp. dysgalactiae and S. dysgalactiae subsp. equisimilis. This observation is confirmed by other gene sequence comparisons (groEL, gyrB, rpoB and sodA). A high percentage of similarity was found between S. intermedius and S. constellatus after sequence comparison of the recN gene. To study the genetic diversity among the 'anginosus' subgroup, recN, groEL, sodA, gyrB and rpoB sequences were determined for 36 clinical isolates. The results that were obtained confirmed the high genetic diversity within this group of streptococci.
KeywordMeSH Terms
Phylogeny
68. Glazunova  OO, Raoult  D, Roux  V,     ( 2009 )

Partial sequence comparison of the rpoB, sodA, groEL and gyrB genes within the genus Streptococcus.

International journal of systematic and evolutionary microbiology 59 (Pt 9)
PMID : 19620365  :   DOI  :   10.1099/ijs.0.005488-0    
Abstract >>
Phylogenetic analysis and species identification of members of the genus Streptococcus were carried out using partial sequence comparison of the 16S rRNA gene (1468-1478 bp), rpoB, encoding the beta subunit of RNA polymerase (659-680 bp), sodA, encoding the manganese-dependent superoxide dismutase (435-462 bp), groEL, encoding the 60 kDa heat-shock protein (757 bp), and gyrB, encoding the Beta subunit of DNA gyrase (458-461 bp). For the first time, most species within the genus Streptococcus were represented in the study (65 strains, representing 58 species and nine subspecies). Phylogenies inferred from rpoB, sodA, gyrB and groEL sequence comparisons were more discriminative than those inferred from 16S rRNA gene sequence comparison, and showed common clusters. The minimal interspecies divergence was 0.3, 2.7, 0, 2.5 and 3.4 % for the 16S rRNA gene, rpoB, sodA, gyrB and groEL, respectively. In general, groEL partial gene sequence comparison represented the best tool for identifying species and subspecies and for phylogenetic analysis.
KeywordMeSH Terms
69. Schroeder  CJ, Robert  C, Lenzen  G, McKay  LL, Mercenier  A,     ( 1991 )

Analysis of the lacZ sequences from two Streptococcus thermophilus strains: comparison with the Escherichia coli and Lactobacillus bulgaricus beta-galactosidase sequences.

Journal of general microbiology 137 (2)
PMID : 1901904  :   DOI  :   10.1099/00221287-137-2-369    
Abstract >>
The lacZ gene from Streptococcus thermophilus A054, a commercial yogurt strain, was cloned on a 7.2 kb PstI fragment in Escherichia coli and compared with the previously cloned lacZ gene from S. thermophilus ATCC 19258. Using the dideoxy chain termination method, the DNA sequences of both lacZ structural genes were determined and found to be 3071 bp in length. When the two sequences were more closely analysed, 21 nucleotide differences were detected, of which only nine resulted in amino acid changes in the proteins, the remainder occurring in wobble positions of the respective codons. Only three bases separated the termination codon for the lacS gene from the initiation codon for lacZ, suggesting that the lactose utilization genes are organized as an operon. The amino acid sequence of the beta-galactosidase, derived from the DNA sequence, corresponds to a protein with a molecular mass of 116860 Da. Comparison of the S. thermophilus amino acid sequences with those from Lactobacillus bulgaricus, E. coli and Klebsiella pneumoniae showed 48, 35 and 32.5% identity respectively. Although little sequence homology was observed at the DNA level, many regions conserved in the amino acid sequence were identified when the beta-galactosidase proteins from S. thermophilus, E. coli and L. bulgaricus were compared.
KeywordMeSH Terms
Lac Operon
70. Slos  P, Bourquin  JC, Lemoine  Y, Mercenier  A,     ( 1991 )

Isolation and characterization of chromosomal promoters of Streptococcus salivarius subsp. thermophilus.

Applied and environmental microbiology 57 (5)
PMID : 1854195  :   PMC  :   PMC182951    
Abstract >>
A promoter probe vector, pTG244, was constructed with the aim of isolating transcription initiation signals from Streptococcus thermophilus (Streptococcus salivarius subsp. thermophilus). pTG244 is based on the Escherichia coli-streptococcus shuttle vector pTG222, into which the promoterless chloramphenicol acetyltransferase gene of Bacillus pumilus (cat-86) was cloned. Random Sau3A fragments from the S. thermophilus A054 chromosomal DNA were cloned upstream of the cat-86 gene by using E. coli as the host. The pool of recombinant plasmids were introduced into S. thermophilus and Lactococcus lactis subsp. lactis in order to search for promoter activity in these hosts. For S. thermophilus, it was necessary to first select erythromycin-resistant transformants and then to screen for chloramphenicol resistance among these. Direct selection of chloramphenicol-resistant clones was, however, possible in L. lactis subsp. lactis. Six fragments exhibiting promoter activity were characterized in S. thermophilus by measuring the levels of cat-86 transcription and/or chloramphenicol acetyltransferase specific activity. Three of the promoter-carrying fragments were sequenced. The 5' ends of their corresponding mRNAs were determined by S1 mapping and shown to correspond to a purine residue in all cases. Upstream from these potential transcription start points, sequences homologous to the E. coli sigma 70 and the Bacillus subtilis vegetative sigma 43 (or sigma A) consensus promoters were identified.
KeywordMeSH Terms
Chromosomes, Bacterial
Promoter Regions, Genetic
71. Sirén  N, Salonen  K, Leisola  M, Nyyssölä  A,     ( 2008 )

A new and efficient phosphate starvation inducible expression system for Lactococcus lactis.

Applied microbiology and biotechnology 79 (5)
PMID : 18431568  :   DOI  :   10.1007/s00253-008-1484-4    
Abstract >>
A new expression system for Lactococcus lactis was developed. The system is based on a phosphate starvation inducible pstF promoter of L. lactis MG1363. Intracellular beta-galactosidase and secreted alpha-amylase were produced using this tightly regulated system. No evidence of regulatory sites in regions of the 5'-end of the pstF coding sequence was found. High expression levels of the beta-galactosidase gene were obtained using the original pstF RBS in a phosphate-depleted medium. The results suggested that with the phosphate starvation inducible system, it is possible to achieve expression levels comparable to the ones obtained with the widely used nisin-controlled gene expression system (NICE). A specific beta-galactosidase activity of 670 microkat g(-1) using a phosphate-depleted medium and an alpha-amylase activity of 3.6 microkat l(-1) in a bioreactor cultivation were produced. The advantages of the current expression system include that no prior removal of phosphate from the medium in bioreactor scale is required, and no additions of inducing agents are needed. Furthermore, the system can be operated in L. lactis without introduction of regulatory genes into the host.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
72. Kazlauskiene  M, Tamulaitis  G, Kostiuk  G, Venclovas  ?, Siksnys  V,     ( 2016 )

Spatiotemporal Control of Type III-A CRISPR-Cas Immunity: Coupling DNA Degradation with the Target RNA Recognition.

Molecular cell 62 (2)
PMID : 27105119  :   DOI  :   10.1016/j.molcel.2016.03.024    
Abstract >>
Streptococcus thermophilus (St) type III-A CRISPR-Cas system restricts MS2 RNA phage and cuts RNA in vitro. However, the CRISPR array spacers match DNA phages, raising the question: does the St CRISPR-Cas system provide immunity by erasing phage mRNA or/and by eliminating invading DNA? We show that it does both. We find that (1) base-pairing between crRNA and target RNA activates single-stranded DNA (ssDNA) degradation by StCsm; (2) ssDNase activity is confined to the HD-domain of Cas10; (3) target RNA cleavage by the Csm3 RNase suppresses Cas10 DNase activity, ensuring temporal control of DNA degradation; and (4) base-pairing between crRNA 5'-handle and target RNA 3'-flanking sequence inhibits Cas10 ssDNase to prevent self-targeting. We propose that upon phage infection, crRNA-guided StCsm binding to the emerging transcript recruits Cas10 DNase to the actively transcribed phage DNA, resulting in degradation of both the transcript and phage DNA, but not the host DNA.
KeywordMeSH Terms
CRISPR-Cas Systems
73. Yu  J, Sun  Z, Liu  W, Xi  X, Song  Y, Xu  H, Lv  Q, Bao  Q, Menghe  B, Sun  T,     ( 2015 )

Multilocus sequence typing of Streptococcus thermophilus from naturally fermented dairy foods in China and Mongolia.

BMC microbiology 15 (N/A)
PMID : 26497818  :   DOI  :   10.1186/s12866-015-0551-0     PMC  :   PMC4620635    
Abstract >>
Streptococcus thermophilus is a major dairy starter used for manufacturing of dairy products. In the present study, we developed a multilocus sequence typing (MLST) scheme for this important food bacterium. Sequences of 10 housekeeping genes (carB, clpX, dnaA, murC, murE, pepN, pepX, pyrG, recA, and rpoB) were obtained for 239 S. thermophilus strains, which were isolated from home-made fermented dairy foods in 18 different regions of Mongolia and China. All 10 genes of S. thermophilus were sequenced, aligned, and defined sequence types (STs) using the BioNumerics Software. The nucleotide diversity was calculated by START v2.0. The population structure, phylogenetic relationships and the role of recombination were inferred using ClonalFrame v1.2, SplitsTree 4.0 and Structure v2.3. The 239 S. thermophilus isolates and 18 reference strains could be assigned into 119 different STs, which could be further separated into 16 clonal complexes (CCs) and 38 singletons. Among the 10 loci, a total of 132 polymorphic sites were detected. The standardized index of association (IAS=0.0916), split-decomposition and �l/�c (relative frequency of occurrence of recombination and mutation) and r/m value (relative impact of recombination and mutation in the diversification) confirms that recombination may have occurred, but it occurred at a low frequency in these 10 loci. Phylogenetic trees indicated that there were five lineages in the S. thermophilus isolates used in our study. MSTree and ClonalFrame tree analyses suggest that the evolution of S. thermophilus isolates have little relationship with geographic locality, but revealed no association with the types of fermented dairy product. Phylogenetic analysis of 36 whole genome strains (18 S. thermophilus, 2 S. vestibularis and 16 S. salivarius strains) indicated that our MLST scheme could clearly separate three closely related species within the salivarius group and is suitable for analyzing the population structure of the other two species in the salivarius group. Our newly developed MLST scheme improved the understanding on the genetic diversity and population structure of the S. thermophilus, as well as provided useful information for further studies on the genotyping and evolutionary research for S. thermophilus strains with global diversity.
KeywordMeSH Terms
Food Microbiology
Genetic Variation
74. Poolman  B, Royer  TJ, Mainzer  SE, Schmidt  BF,     ( 1989 )

Lactose transport system of Streptococcus thermophilus: a hybrid protein with homology to the melibiose carrier and enzyme III of phosphoenolpyruvate-dependent phosphotransferase systems.

Journal of bacteriology 171 (1)
PMID : 2644191  :   DOI  :   10.1128/jb.171.1.244-253.1989     PMC  :   PMC209579    
Abstract >>
The gene responsible for the transport of lactose into Streptococcus thermophilus (lacS) was cloned in Escherichia coli as a 4.2-kilobase fragment from an EcoRI library of chromosomal DNA by using the vector pKK223-3. From deletion analysis, the gene for lactose transport mapped to two HindIII fragments with a total size of 2.8 kilobases. The gene was transcribed in E. coli from its own promoter. Functional expression of lactose transport activity was shown by assaying for the uptake and exchange of lactose both in intact cells and in membrane vesicles. The nucleotide sequence of lacS and 200 to 300 bases of 3' and 5' flanking regions were determined. The gene was 1,902 base pairs long, encoding a 69,454-dalton protein with an NH2-terminal hydrophobic region and a COOH-terminal hydrophilic region. The NH2-terminal end was homologous with the melibiose carrier of E. coli (23% similarity overall; greater than 50% similarity for regions with at least 16 amino acids), whereas the COOH-terminal end showed 34 to 41% similarity with the enzyme III (domain) of three different phosphoenolpyruvate-dependent phosphotransferase systems. Among the conserved amino acids were two histidyl residues, of which one has been postulated to be phosphorylated by HPr. Since sugars are not phosphorylated during translocation by the lactose transport system, it is suggested that the enzyme III-like region serves a regulatory function in this protein. The lacS gene also appears similar to the partially sequenced lactose transport gene of Lactobacillus bulgaricus (lacL; greater than 60% similarity). Furthermore, the 3' flanking sequence of the S. thermophilus lactose transport gene showed approximately 50% similarity with the N-terminal portion of the beta-galactosidase gene of L. bulgaricus. In both organisms, the lactose transport gene and the beta-galactosidase appear to be separated by a 3-base-pair intercistronic region.
KeywordMeSH Terms
Escherichia coli Proteins
Genes
Genes, Bacterial
Monosaccharide Transport Proteins
Symporters
75. Tamulaitis  G, Kazlauskiene  M, Manakova  E, Venclovas  ?, Nwokeoji  AO, Dickman  MJ, Horvath  P, Siksnys  V,     ( 2014 )

Programmable RNA shredding by the type III-A CRISPR-Cas system of Streptococcus thermophilus.

Molecular cell 56 (4)
PMID : 25458845  :   DOI  :   10.1016/j.molcel.2014.09.027    
Abstract >>
Immunity against viruses and plasmids provided by CRISPR-Cas systems relies on a ribonucleoprotein effector complex that triggers the degradation of invasive nucleic acids (NA). Effector complexes of type I (Cascade) and II (Cas9-dual RNA) target foreign DNA. Intriguingly, the genetic evidence suggests that the type III-A Csm complex targets DNA, whereas biochemical data show that the type III-B Cmr complex cleaves RNA. Here we aimed to investigate NA specificity and mechanism of CRISPR interference for the Streptococcus thermophilus Csm (III-A) complex (StCsm). When expressed in Escherichia coli, two complexes of different stoichiometry copurified with 40 and 72 nt crRNA species, respectively. Both complexes targeted RNA and generated multiple cuts at 6 nt intervals. The Csm3 protein, present in multiple copies in both Csm complexes, acts as endoribonuclease. In the heterologous E. coli host, StCsm restricts MS2 RNA phage in a Csm3 nuclease-dependent manner. Thus, our results demonstrate that the type III-A StCsm complex guided by crRNA targets RNA and not DNA.
KeywordMeSH Terms
Clustered Regularly Interspaced Short Palindromic Repeats
RNA Cleavage
76. Couvigny  B, Thérial  C, Gautier  C, Renault  P, Briandet  R, Guédon  E,     ( 2015 )

Streptococcus thermophilus Biofilm Formation: A Remnant Trait of Ancestral Commensal Life?

PloS one 10 (6)
PMID : 26035177  :   DOI  :   10.1371/journal.pone.0128099     PMC  :   PMC4452758    
Abstract >>
Microorganisms have a long history of use in food production and preservation. Their adaptation to food environments has profoundly modified their features, mainly through genomic flux. Streptococcus thermophilus, one of the most frequent starter culture organisms consumed daily by humans emerged recently from a commensal ancestor. As such, it is a useful model for genomic studies of bacterial domestication processes. Many streptococcal species form biofilms, a key feature of the major lifestyle of these bacteria in nature. However, few descriptions of S. thermophilus biofilms have been reported. An analysis of the ability of a representative collection of natural isolates to form biofilms revealed that S. thermophilus was a poor biofilm producer and that this characteristic was associated with an inability to attach firmly to surfaces. The identification of three biofilm-associated genes in the strain producing the most biofilms shed light on the reasons for the rarity of this trait in this species. These genes encode proteins involved in crucial stages of biofilm formation and are heterogeneously distributed between strains. One of the biofilm genes appears to have been acquired by horizontal transfer. The other two are located in loci presenting features of reductive evolution, and are absent from most of the strains analyzed. Their orthologs in commensal bacteria are involved in adhesion to host cells, suggesting that they are remnants of ancestral functions. The biofilm phenotype appears to be a commensal trait that has been lost during the genetic domestication of S. thermophilus, consistent with its adaptation to the milk environment and the selection of starter strains for dairy fermentations.
KeywordMeSH Terms
77. Payne  KA, Fisher  K, Sjuts  H, Dunstan  MS, Bellina  B, Johannissen  L, Barran  P, Hay  S, Rigby  SE, Leys  D,     ( 2015 )

Epoxyqueuosine Reductase Structure Suggests a Mechanism for Cobalamin-dependent tRNA Modification.

The Journal of biological chemistry 290 (46)
PMID : 26378237  :   DOI  :   10.1074/jbc.M115.685693     PMC  :   PMC4646009    
Abstract >>
Queuosine (Q) is a hypermodified RNA base that replaces guanine in the wobble positions of 5'-GUN-3' tRNA molecules. Q is exclusively made by bacteria, and the corresponding queuine base is a micronutrient salvaged by eukaryotic species. The final step in Q biosynthesis is the reduction of the epoxide precursor, epoxyqueuosine, to yield the Q cyclopentene ring. The epoxyqueuosine reductase responsible, QueG, shares distant homology with the cobalamin-dependent reductive dehalogenase (RdhA), however the role played by cobalamin in QueG catalysis has remained elusive. We report the solution and structural characterization of Streptococcus thermophilus QueG, revealing the enzyme harbors a redox chain consisting of two [4Fe-4S] clusters and a cob(II)alamin in the base-off form, similar to RdhAs. In contrast to the shared redox chain architecture, the QueG active site shares little homology with RdhA, with the notable exception of a conserved Tyr that is proposed to function as a proton donor during reductive dehalogenation. Docking of an epoxyqueuosine substrate suggests the QueG active site places the substrate cyclopentane moiety in close proximity of the cobalt. Both the Tyr and a conserved Asp are implicated as proton donors to the epoxide leaving group. This suggests that, in contrast to the unusual carbon-halogen bond chemistry catalyzed by RdhAs, QueG acts via Co-C bond formation. Our study establishes the common features of Class III cobalamin-dependent enzymes, and reveals an unexpected diversity in the reductive chemistry catalyzed by these enzymes.
KeywordMeSH Terms
RNA modification
crystal structure
electron paramagnetic resonance (EPR)
enzyme mechanism
iron sulfur protein
nucleic acid enzymology
queuosine biosynthesis
transfer RNA (tRNA)
vitamin B12
78. Zhang  C, Xin  Y, Wang  Y, Guo  T, Lu  S, Kong  J,     ( 2015 )

Identification of a Novel Dye-Decolorizing Peroxidase, EfeB, Translocated by a Twin-Arginine Translocation System in Streptococcus thermophilus CGMCC 7.179.

Applied and environmental microbiology 81 (18)
PMID : 26092460  :   DOI  :   10.1128/AEM.01300-15     PMC  :   PMC4542251    
Abstract >>
Streptococcus thermophilus is a facultative anaerobic bacterium that has the ability to grow and survive in aerobic environments, but the mechanism for this remains unclear. In this study, the efeB gene, encoding a dye-decolorizing peroxidase, was identified in the genome of Streptococcus thermophilus CGMCC 7.179, and purified EfeB was able to decolorize reactive blue 5. Strikingly, genes encoding two components (TatA and TatC) of the twin-arginine translocation (TAT) system were also found in the same operon with the efeB gene. Knocking out efeB or tatC resulted in decreased growth of the strain under aerobic conditions, and complementation of the efeB-deficient strains with the efeB gene enhanced the biomass of the hosts only in the presence of the tatC gene. Moreover, it was proved for both S. thermophilus CGMCC 7.179 and Escherichia coli DE3 that EfeB could be translocated by the TAT system of S. thermophilus. In addition, the transcriptional levels of efeB and tatC increased when the strain was cultured under aerobic conditions. Overall, these results provide the first evidence that EfeB plays a role in protecting cells of S. thermophilus from oxidative stress, with the assistance of the TAT system.
KeywordMeSH Terms
79. Arioli  S, Guglielmetti  S, Amalfitano  S, Viti  C, Marchi  E, Decorosi  F, Giovannetti  L, Mora  D,     ( 2014 )

Characterization of tetA-like gene encoding for a major facilitator superfamily efflux pump in Streptococcus thermophilus.

FEMS microbiology letters 355 (1)
PMID : 24766488  :   DOI  :   10.1111/1574-6968.12449    
Abstract >>
Efflux pumps are membrane proteins involved in the active extrusion of a wide range of structurally dissimilar substrates from cells. A multidrug efflux pump named TetA belonging to the major facilitator superfamily (MFS) of transporters was identified in the Streptococcus thermophilus DSM 20617(T) genome. The tetA-like gene was found in the genomes of a number of S. thermophilus strains sequenced to date and in Streptococcus macedonicus ACA-DC 198, suggesting a possible horizontal gene transfer event between these two Streptococcus species, which are both adapted to the milk environment. Flow cytometry (single-cell) analysis revealed bistable TetA activity in the S. thermophilus population, and tetA-like gene over-expression resulted in a reduced susceptibility to ethidium bromide, tetracycline, and other toxic compounds even when the efflux pump was over-expressed in a strain naturally lacking tetA-like gene.
KeywordMeSH Terms
MFS efflux pump
Streptococcus thermophilus
antibiotic resistance
lactic acid bacteria
80. Zuo  F, Yu  R, Feng  X, Khaskheli  GB, Chen  L, Ma  H, Chen  S,     ( 2014 )

Combination of heterogeneous catalase and superoxide dismutase protects Bifidobacterium longum strain NCC2705 from oxidative stress.

Applied microbiology and biotechnology 98 (17)
PMID : 24903816  :   DOI  :   10.1007/s00253-014-5851-z    
Abstract >>
Bifidobacteria are generally sensitive to oxidative stress caused by reactive oxygen species (ROS). To improve oxidative-stress tolerance, the superoxide dismutase (SOD) gene from Streptococcus thermophilus (StSodA) and the heme-dependent catalase (KAT) gene from Lactobacillus plantarum (LpKatL) were heterologously expressed in Bifidobacterium longum strain NCC2705. Three types of strain NCC2705 transformants were obtained: with transgenic SOD expression, with transgenic KAT expression, and with coexpression of the two genes. Intracellular expression of the genes and their functional role in oxidative-stress resistance were evaluated. In response to oxidative stress, B. longum NCC2705/pDP401-LpKatL (expressing LpKatL) and NCC2705/pDP-Kat-Sod (coexpressing LpKatL and StSodA) rapidly degraded exogenous H2O2 and the peroxides generated as a byproduct of aerobic cultivation, preventing oxidative damage to DNA and RNA. Individual expression of StSodA or LpKatL both improved B. longum NCC2705 cell viability. Survival rate of strain NCC2705 was further improved by combining SOD and KAT expression. The two enzymes played complementary roles in ROS-scavenging pathways, and coexpression led to a synergistic beneficial effect under conditions of intensified oxidative stress. Our results illustrate that heterogeneous expression of heme-dependent KAT and Mn(2+)-dependent SOD is functional in the B. longum oxidative-stress response, and synergistic protection is achieved when their expressions are combined.
KeywordMeSH Terms
Oxidative Stress
81. Henry  R, Bruneau  E, Gardan  R, Bertin  S, Fleuchot  B, Decaris  B, Leblond-Bourget  N,     ( 2011 )

The rgg0182 gene encodes a transcriptional regulator required for the full Streptococcus thermophilus LMG18311 thermal adaptation.

BMC microbiology 11 (N/A)
PMID : 21981946  :   DOI  :   10.1186/1471-2180-11-223     PMC  :   PMC3199253    
Abstract >>
Streptococcus thermophilus is an important starter strain for the production of yogurt and cheeses. The analysis of sequenced genomes of four strains of S. thermophilus indicates that they contain several genes of the rgg familly potentially encoding transcriptional regulators. Some of the Rgg proteins are known to be involved in bacterial stress adaptation. In this study, we demonstrated that Streptococcus thermophilus thermal stress adaptation required the rgg0182 gene which transcription depends on the culture medium and the growth temperature. This gene encoded a protein showing similarity with members of the Rgg family transcriptional regulator. Our data confirmed that Rgg0182 is a transcriptional regulator controlling the expression of its neighboring genes as well as chaperones and proteases encoding genes. Therefore, analysis of a �Grgg0182 mutant revealed that this protein played a role in the heat shock adaptation of Streptococcus thermophilus LMG18311. These data showed the importance of the Rgg0182 transcriptional regulator on the survival of S. thermophilus during dairy processes and more specifically during changes in temperature.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
82.     ( 1996 )

Proline biosynthesis in Streptococcus thermophilus: characterization of the proBA operon and its products.

Microbiology (Reading, England) 142 (Pt 11) (N/A)
PMID : 8969524  :   DOI  :   10.1099/13500872-142-11-3275    
Abstract >>
The presence of proline in the medium was not essential for growth of Streptococcus thermophilus, indicating that there is a proline biosynthetic pathway in this organism. Genetic and biochemical analysis identified and characterized this pathway. Two genes, designated proB and proA, were cloned, sequenced and characterized. Biochemical analysis of the proB- and proA-encoded enzymes showed that the proline biosynthetic pathway of S. thermophilus is similar to the one previously described in Escherichia coli. The deduced amino acid sequence of a 2-408 kb DNA region containing the genes revealed the similarity of the S. thermophilus gene products to ProB and ProA of E. coli and Serratia marcescens, and to the corresponding N- and C-terminal domains of the bifunctional plant enzyme delta 1-pyrroline-5-carboxylate synthetase of Vigna aconitifolia. Northern blot analysis showed that the two genes in S. thermophilus are organized in a single operon with proB proximal and proA distal to the promoter; primer extension analysis indicated that proBA transcription is not under repressive control by exogenously supplied proline.
KeywordMeSH Terms
Genes, Bacterial
Operon
83.     ( 1997 )

Isolation and characterization of transcription signal sequences from Streptococcus thermophilus.

Current microbiology 34 (4)
PMID : 9058540  :  
Abstract >>
Streptococcus thermophilus (ST) chromosomal DNA fragments generated by partial Sau3A digestion were cloned into the unique BamHI site upstream from the promoterless chloramphenicol acetyltransferase (cat) gene of the Escherichia coli (EC)promoter-probe vector pKK520-3. Recombinant plasmids containing ST sequences with transcription-activation activity were isolated from chloramphenicol-resistant (CmR) EC transformants. A promoterless Streptomyces antibioticus melanin biosynthesis operon (melC) was inserted immediately downstream from the ST sequence to identify DNA with strong promoter activity. Several ST transcription-activation sequences, termed STPs, were isolated and subcloned, and their nucleic acid sequences determined. The -10 and -35 consensus sequences were identified in these putative ST promoters. Detailed analysis of STP3306 sequence data revealed two partial open reading frames (ORFs) with high degrees of homology to prokaryotic GTP-binding protein and DNA repair enzyme, thus providing valuable information for further study on DNA maintenance in this important lactic acid bacterium.
KeywordMeSH Terms
84.     ( 1997 )

The site-specific integration system of the temperate Streptococcus thermophilus bacteriophage phiSfi21.

Virology 237 (1)
PMID : 9344917  :   DOI  :   10.1006/viro.1997.8769    
Abstract >>
The temperate bacteriophage phiSfi21 integrates its DNA into the chromosome of Streptococcus thermophilus strains via site-specific recombination. Nucleotide sequencing of the attachment sites identified a 40-bp identity region which surprisingly overlaps both the 18-terminal bp of the phage integrase gene and the 11-terminal bp of a host tRNAArg gene. A 2.4-kb phage DNA segment, covering attP, the phage integrase, and a likely immunity gene contained all the genetic information for faithful integration of a nonreplicative plasmid into the attB site. A deletion within the int gene led to the loss of integration proficiency. A number of spontaneous deletions were observed in plasmids containing the 2.4-kb phage DNA segment. The deletion sites were localized to the tRNA side of the identity region and to phage or vector DNA with 3- to 6-bp-long repeats from the border region. A similar type of deletion was previously observed in a spontaneous phage mutant.
KeywordMeSH Terms
Recombination, Genetic
Virus Integration
85.     ( 1997 )

Thermophilin 13, a nontypical antilisterial poration complex bacteriocin, that functions without a receptor.

The Journal of biological chemistry 272 (22)
PMID : 9162062  :   DOI  :   10.1074/jbc.272.22.14277    
Abstract >>
A novel broad host range antimicrobial substance, Thermophilin 13, has been isolated and purified from the growth medium of Streptococcus thermophilus. Thermophilin 13 is composed of the antibacterial peptide ThmA (Mr of 5776) and the enhancing factor ThmB (Mr of 3910); the latter peptide increased the activity of ThmA approximately 40 x. Both peptides are encoded by a single operon, and an equimolar ratio was optimal for Thermophilin 13 activity. Despite the antilisterial activity of Thermophilin 13, neither ThmA nor ThmB contain the YGNGV-C consensus sequence of Listeria-active peptides, and post-translational modifications comparable to that in the lantibiotics are also absent. Mass spectrometry did reveal the apparent oxidation of methionines in ThmA, which resulted in a peptide that could not be enhanced any longer by ThmB, whereas the intrinsic bactericidal activity was normal. Thermophilin 13 dissipated the membrane potential and the pH gradient in liposomes, and this activity was independent of membrane components from a sensitive strain (e.g. lipid or proteinaceous receptor). Models of possible poration complexes formed are proposed on the basis of sequence comparisons, structure predictions, and the functional analysis of Thermophilin 13.
KeywordMeSH Terms
Anti-Bacterial Agents
Bacterial Proteins
86.     ( 1997 )

A 16 kDa protein family overexpressed by Streptococcus thermophilus PB18 in acid environments.

Microbiology (Reading, England) 143 (Pt 5) (N/A)
PMID : 9168610  :   DOI  :   10.1099/00221287-143-5-1587    
Abstract >>
The one- and two-dimensional protein patterns of Streptococcus thermophilus PB18 in the exponential and stationary phases of growth were analysed. One-dimensional SDS-PAGE showed that a 16 kDa protein was overexpressed in stationary phase as well as 2 h after an acid shock, and that it was not expressed when the bacteria reached the stationary phase in medium with limiting lactose concentrations (5 or 10 g l-1), in which the pH (5.5) was not as acid as in control cultures (pH 4.7, lactose 20 g l-1). The results support the idea that this protein is expressed in response to the acidic environment and not in response to the growth phase. Two-dimensional PAGE showed that nine proteins were expressed only during the exponential phase and ten others only during the stationary phase. The 16 kDa band seen in one-dimensional SDS-PAGE corresponded to a 16 kDa protein family observed on two-dimensional SDS-PAGE/IEF gels, whose expression was increased 8.5-fold when the extracellular pH reached a critical value below 5.0. The N-terminal sequences of proteins from two spots on the two-dimensional gels (members of the 16 kDa family) were determined and found to be identical. The physiological role of this protein family has not yet been elucidated.
KeywordMeSH Terms
87.     ( 1997 )

Analysis of the genetic polymorphism between three Streptococcus thermophilus strains by comparing their physical and genetic organization.

Microbiology (Reading, England) 143 (Pt 4) (N/A)
PMID : 9141697  :   DOI  :   10.1099/00221287-143-4-1335    
Abstract >>
The physical maps of Streptococcus thermophilus CNRZ368 and NST2280 strains were constructed by analysing PFGE patterns obtained with the low-frequency-cutting enzymes SmaI, BssHII and SfiI. Their chromosomes are 1864 and 1840 kb circular molecules, respectively. Comparison of their physical maps with that of the reference A054 strain revealed a relatively conserved organization of the restriction sites. Three variable regions were detected with the map of CNRZ368 whereas 15 were found with the map of NST2280. To construct the genetic maps, probes corresponding to 10 single-copy genes, the rrn genes and the insertion sequences IS1191, IS981 and ISS1 were hybridized to Southern blots of chromosomal DNA digested with the different mapping enzymes. Comparison of the genetic maps of the three strains showed a conserved location of the mapped single-copy genes. However, six rrn loci were present in the chromosome of A054 and CNRZ368 whereas five were present in the NST2280 chromosome. A polymorphism was also found in the copy number of the insertion sequences between the three strains.
KeywordMeSH Terms
Chromosome Mapping
Chromosomes, Bacterial
Polymorphism, Genetic
88.     ( 1996 )

Disruption of the gene encoding penicillin-binding protein 2b (pbp2b) causes altered cell morphology and cease in exopolysaccharide production in Streptococcus thermophilus Sfi6.

Molecular microbiology 22 (2)
PMID : 8930919  :   DOI  :   10.1046/j.1365-2958.1996.00121.x    
Abstract >>
Genetic and biochemical analysis of exopolysaccharide (EPS) production in lactic acid bacteria has been a growing field of interest in the food industry. We previously identified and characterized a gene cluster composed of 13 genes (epsA to epsM) responsible for EPS production in Streptococcus thermophilus Sfi6. Here we report one further gene, pbp2b, that is connected to EPS production. Mutants with a gene disruption in pbp2b were no longer able to produce EPS, exhibited a reduced growth-rate, and their cell morphology was altered. The predicted gene product showed significant homology to the class B penicillin-binding proteins 2b of Streptococcus pneumoniae, Streptococcus sanguis and Streptococcus mitis involved in peptidoglycan synthesis. Upstream of pbp2b, we further identified two genes which showed significant homology to the E. coli folD and urfl, which is an unidentified open reading frame presumed to be involved in DNA repair. Downstream of pbp2b, we identified a gene that showed homology to the Bacillus subtilis and the Escherichia coli recM or recR which, respectively, are involved in the methyl-dependent DNA mismatch repair. In S. thermophilus, pbp2b and recM were transcribed from their own promoters as monocistronic mRNAs and are therefore organized as independent transcriptional units.
KeywordMeSH Terms
Aminoacyltransferases
Hexosyltransferases
Peptidyl Transferases
89.     ( 1996 )

Characterization of a mosaic ISS1 element and evidence for the recent horizontal transfer of two different types of ISS1 between Streptococcus thermophilus and Lactococcus lactis.

Gene 178 (1��2��)
PMID : 8921885  :   DOI  :   10.1016/0378-1119(96)00316-2    
Abstract >>
A 12-kb region of the Streptococcus thermophilus CNRZ368 chromosome was found to contain two copies of IS981 (one complete and one truncated) and three copies of ISS1 (two complete, ISS1SA and ISS1SC, and one truncated, delta ISS1SB). Comparison of the nucleotide sequences of these ISS1 elements with those of previously identified iso-ISS1 elements from Lactococcus lactis and the Enterococcus genus indicated that the ISS1 group is divided into three distinct subgroups which we have named alpha, beta and gamma. Nucleotide sequences of elements belonging to the same subgroup share more than 97% identity whereas sequences of elements from different groups share only 75-85% identity. Sequence analysis of ISS1SA and delta ISS1SB showed that they are members of the alpha group. We found that ISS1SC from S. themophilus CNRZ368, an ISS1 from L. lactis IL964 and IS946 from L. lactis TEK1 resulted from recombinations between alpha and beta elements. In addition, ISS1W from L. lactis Wg2 resulted from a recombination event between a gamma element and an ISS1 belonging to an unidentified subgroup. ISS1 sequences belonging to the alpha and beta subgroups were found in both S. thermophilus and L. lactis and gamma sequences were found in both the Enterococcus genus and L. lactis. The quasi-identity of some ISS1 elements in S. thermophilus and L. lactis and the distribution of alpha and beta elements suggest that horizontal transfer of ISS1 elements recently took place from L. lactis to S. thermophilus, two lactic acid bacteria used in the manufacture of cheeses. Since the presence of IS981 in S. thermophilus CNRZ368 also probably resulted from a horizontal transfer from L. lactis [Gu?don et al. (1995) Mol. Microbiol. 16, 69-78], the 12-kb region bearing IS981 and ISS1 elements could be due to the integration of a lactococcal DNA fragment into the chromosome.
KeywordMeSH Terms
DNA Transposable Elements
90.     ( 1996 )

Identification and characterization of the eps (Exopolysaccharide) gene cluster from Streptococcus thermophilus Sfi6.

Journal of bacteriology 178 (6)
PMID : 8626297  :   DOI  :   10.1128/jb.178.6.1680-1690.1996     PMC  :   PMC177854    
Abstract >>
We report the identification and characterization of the eps gene cluster of Streptococcus thermophilus Sfi6 required for exopolysaccharide (EPS) synthesis. This report is the first genetic work concerning EPS production in a food microorganism. The EPS secreted by this strain consists of the following tetrasaccharide repeating unit:-->3)-beta-D-Galp-(1-->3)-[alpha-D-Galp-(1-->6)]-beta-D- D-Galp-(1-->3)-alpha-D-Galp-D-GalpNAc-(1-->. The genetic locus The genetic locus was identified by Tn916 mutagenesis in combination with a plate assay to identify Eps mutants. Sequence analysis of the gene region, which was obtained from subclones of a genomic library of Sfi6, revealed a 15.25-kb region encoding 15 open reading frames. EPS expression in the non-EPS-producing heterologous host, Lactococcus lactis MG1363, showed that within the 15.25-kb region, a region with a size of 14.52 kb encoding the 13 genes epsA to epsM was capable of directing EPS synthesis and secretion in this host. Homology searches of the predicted proteins in the Swiss-Prot database revealed high homology (40 to 68% identity) for epsA, B, C, D, and E and the genes involved in capsule synthesis in Streptococcus pneumoniae and Streptococcus agalactiae. Moderate to low homology (37 to 18% identity) was detected for epsB, D, F, and H and the genes involved in capsule synthesis in Staphylococcus aureus for epsC, D, and E and the genes involved in exopolysaccharide I (EPSI) synthesis in Rhizobium meliloti for epsC to epsJ and the genes involved in lipopolysaccharide synthesis in members of the Enterobacteriaceae, and finally for eps K and lipB of Neisseria meningitidis. Genes (epsJ, epsL, and epsM) for which the predicted proteins showed little or no homology with proteins in the Swiss-Prot database were shown to be involved in EPS synthesis by single-crossover gene disruption experiments.
KeywordMeSH Terms
Genes, Bacterial
Multigene Family
91.     ( 1994 )

Isolation and characterisation of promoter regions from Streptococcus thermophilus.

FEMS microbiology letters 122 (1��2��)
PMID : 7958782  :   DOI  :   10.1111/j.1574-6968.1994.tb07148.x    
Abstract >>
Four promoter regions required for the expression of a promoterless antibiotic resistance gene (cat194) in Streptococcus thermophilus were isolated by random chromosomal cloning experiments. These were shown to be functional in vivo, and their sequences were determined. Each region expressed different amounts of Cat protein as determined by enzyme activities. One region, STP10, was found to contain the 5' coding region of the large ribosomal subunit protein L20.
KeywordMeSH Terms
92.     ( 1995 )

Characterization of the gor gene of the lactic acid bacterium Streptococcus thermophilus CNRZ368.

Research in microbiology 146 (5)
PMID : 8525054  :  
Abstract >>
Cloning and characterization of the gor gene of the lactic acid bacterium Streptococcus thermophilus, encoding glutathione reductase, are described in this paper. This enzyme is a part of the enzymatic defences against oxidative stress in eukaryotic cells and in Gram-negative bacteria, but was never found in Gram-positive bacteria before this study. The amino acid sequence shares extensive similarities with glutathione reductases from other organisms, e.g. 62% amino acid identity with Escherichia coli protein. Northern blot analysis and glutathione reductase enzyme assays gave evidence that the gene is expressed in aerobically growing cells.
KeywordMeSH Terms
93. Ito  Y, Sasaki  T,     ( 1994 )

Cloning and nucleotide sequencing of L-lactate dehydrogenase gene from Streptococcus thermophilus M-192.

Bioscience, biotechnology, and biochemistry 58 (9)
PMID : 7765475  :  
Abstract >>
The gene encoding L-lactate dehydrogenase (LDH) was cloned from an industrial dairy strain of Streptococcus thermophilus M-192 using a synthetic oligonucleotide probe based on the N-terminal amino acid sequence of the purified enzyme, and its nucleotide sequence was determined. The enzyme was deduced to have 328 amino acid residues with a molecular weight of 35,428 and found to have high sequence similarity to LDHs from other lactic acid bacteria (89.0% to Streptococcus mutans, 76.3% to Lactococcus lactis subsp. lactis, 67% to Lactobacillus casei, and 60% to Lactobacillus plantarum). The gene contained a promoter-like sequence similar to the Escherichia coli promoter consensus, and expression of the S. thermophilus LDH gene was observed in E. coli cells.
KeywordMeSH Terms
94.     ( 1994 )

Gene cloning and characterization of PepC, a cysteine aminopeptidase from Streptococcus thermophilus, with sequence similarity to the eucaryotic bleomycin hydrolase.

European journal of biochemistry 224 (2)
PMID : 7925365  :   DOI  :   10.1111/j.1432-1033.1994.00497.x    
Abstract >>
Streptococcus thermophilus CNRZ 302 contains at least three general aminopeptidases able to hydrolyze Phe-beta-naphthylamide substrate. The gene encoding one of these aminopeptidases was cloned from a total DNA library of S. thermophilus CNRZ 302 constructed in Escherichia coli TG1 using pBluescript plasmid. The wild-type TG1 strain, although not deficient in aminopeptidase activity, is unable to hydrolyze the substrate Phe-beta-naphthylamide, and thus the library could be screened with an enzymic plate assay using this substrate. One clone was selected which was shown to express an aminopeptidase, identified as a PepC-like enzyme on the basis of cross-reactivity with polyclonal antibodies directed against the lactococcal PepC cysteine aminopeptidase. The gene was further subcloned and sequenced. A complete open reading frame coding for a 445-residue (50414 Da) polypeptide was identified. 70% identity was found between the deduced amino acid sequence and the sequence of PepC from Lactococcus lactis subspecies cremoris, confirming the identity of the cloned gene. High sequence similarity (38% identity) was also found with an eucaryotic enzyme, bleomycin hydrolase. In addition, the predicted amino acid sequence of the streptococcal PepC showed a region of strong similarity to the active site of cysteine proteinases with conservation of the residues involved in the catalytic site. The product of the cloned pepC gene was overproduced in E. coli and was purified from a cellular extract. Purification to homogeneity was achieved by two-step ion-exchange chromatography. Biochemical characterization of the pure recombinant enzyme confirms that the cloned peptidase is a thiol aminopeptidase possessing a broad specificity. The enzyme has a molecular mass of 300 kDa suggesting an hexameric structure. On the basis of sequence similarities as well as common biochemical and enzymic properties, the bacterial PepC-type enzymes and the eucaryotic bleomycin hydrolase constitute a new family of thiol aminopeptidases among the cysteine peptidases.
KeywordMeSH Terms
95. Szymczak  P, Filipe  SR, Covas  G, Vogensen  FK, Neves  AR, Janzen  T,     ( 2018 )

Cell Wall Glycans Mediate Recognition of the Dairy Bacterium Streptococcus thermophilus by Bacteriophages.

Applied and environmental microbiology 84 (23)
PMID : 30242010  :   DOI  :   10.1128/AEM.01847-18     PMC  :   PMC6238053    
Abstract >>
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.
KeywordMeSH Terms
Streptococcus thermophilus
adsorption
bacteriophages
cell wall
glycans
polysaccharides
receptors
96. You  L, Ma  J, Wang  J, Artamonova  D, Wang  M, Liu  L, Xiang  H, Severinov  K, Zhang  X, Wang  Y,     ( 2019 )

Structure Studies of the CRISPR-Csm Complex Reveal Mechanism of Co-transcriptional Interference.

Cell 176 (1��2��)
PMID : 30503210  :   DOI  :   10.1016/j.cell.2018.10.052    
Abstract >>
Csm, a type III-A CRISPR-Cas interference complex, is a CRISPR RNA (crRNA)-guided RNase that also possesses target RNA-dependent DNase and cyclic oligoadenylate (cOA) synthetase activities. However, the structural features allowing target RNA-binding-dependent activation of DNA cleavage and cOA generation remain unknown. Here, we report the structure of Csm in complex with crRNA together with structures of cognate or non-cognate target RNA bound Csm complexes. We show that depending on complementarity with the 5' tag of crRNA, the 3' anti-tag region of target RNA binds at two distinct sites of the Csm complex. Importantly, the interaction between the non-complementary anti-tag region of cognate target RNA and Csm1 induces a conformational change at the Csm1 subunit that allosterically activates DNA cleavage and cOA generation. Together, our structural studies provide crucial insights into the mechanistic processes required for crRNA-meditated sequence-specific RNA cleavage, RNA target-dependent non-specific DNA cleavage, and cOA generation.
KeywordMeSH Terms
CRISPR-Cas 9 system
Csm complex
type III-A
97.     ( 2013 )

crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus.

RNA biology 10 (5)
PMID : 23535272  :   DOI  :   10.4161/rna.24203     PMC  :   PMC3737341    
Abstract >>
The Cas9-crRNA complex of the Streptococcus thermophilus DGCC7710 CRISPR3-Cas system functions as an RNA-guided endonuclease with crRNA-directed target sequence recognition and protein-mediated DNA cleavage. We show here that an additional RNA molecule, tracrRNA (trans-activating CRISPR RNA), co-purifies with the Cas9 protein isolated from the heterologous E. coli strain carrying the S. thermophilus DGCC7710 CRISPR3-Cas system. We provide experimental evidence that tracrRNA is required for Cas9-mediated DNA interference both in vitro and in vivo. We show that Cas9 specifically promotes duplex formation between the precursor crRNA (pre-crRNA) transcript and tracrRNA, in vitro. Furthermore, the housekeeping RNase III contributes to primary pre-crRNA-tracrRNA duplex cleavage for mature crRNA biogenesis. RNase III, however, is not required in the processing of a short pre-crRNA transcribed from a minimal CRISPR array containing a single spacer. Finally, we show that an in vitro-assembled ternary Cas9-crRNA-tracrRNA complex cleaves DNA. This study further specifies the molecular basis for crRNA-based re-programming of Cas9 to specifically cleave any target DNA sequence for precise genome surgery. The processes for crRNA maturation and effector complex assembly established here will contribute to the further development of the Cas9 re-programmable system for genome editing applications.
KeywordMeSH Terms
CRISPR
DNA silencing
Type II CRISPR-Cas systems
Clustered Regularly Interspaced Short Palindromic Repeats
98. Kazlauskiene  M, Kostiuk  G, Venclovas  ?, Tamulaitis  G, Siksnys  V,     ( 2017 )

A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems.

Science (New York, N.Y.) 357 (6351)
PMID : 28663439  :   DOI  :   10.1126/science.aao0100    
Abstract >>
Type III CRISPR-Cas systems in prokaryotes provide immunity against invading nucleic acids through the coordinated degradation of transcriptionally active DNA and its transcripts by the Csm effector complex. The Cas10 subunit of the complex contains an HD nuclease domain that is responsible for DNA degradation and two Palm domains with elusive functions. In addition, Csm6, a ribonuclease that is not part of the complex, is also required to provide full immunity. We show here that target RNA binding by the Csm effector complex of Streptococcus thermophilus triggers Cas10 to synthesize cyclic oligoadenylates (cA n ; n = 2 to 6) by means of the Palm domains. Acting as signaling molecules, cyclic oligoadenylates bind Csm6 to activate its nonspecific RNA degradation. This cyclic oligoadenylate-based signaling pathway coordinates different components of CRISPR-Cas to prevent phage infection and propagation.
KeywordMeSH Terms
CRISPR-Cas Systems
RNA Stability
99.     ( 2013 )

Diversity of Streptococcus thermophilus in bacteriocin production; inhibitory spectrum and occurrence of thermophilin genes.

Food microbiology 35 (1)
PMID : 23628611  :   DOI  :   10.1016/j.fm.2013.02.006    
Abstract >>
The bacteriocin-producing Streptococcus thermophilus strains that can dominate in natural dairy ecosystems, may also enhance safety in products obtained from natural cultures. In this study, we sought to identify bacteriocin production and bacteriocin genes in 75 strains of dairy and plant origin. The strains were tested for antimicrobial activity against pathogens or pathogen models, spoiling bacteria, and lactic acid bacteria associated with dairy products. All strains moderately inhibited Staphylococcus aureus P310, none inhibited Listeria innocua LMG 11387(T) or Clostridium tyrobutyricum LMG 1285(T). In addition, 14 were active against one or more indicators in addition to S. aureus P310. Inhibition of other starter bacteria was more common than the inhibition of unwanted microorganisms. The involvement of a proteinaceous compound was ascertained in all cases. Results suggested that the selection of bacteriocinogenic S. thermophilus strains for use in biopreservation must take into account the effects exerted on other lactic acid bacteria. PCR detection of thermophilin genes proved unreliable in predicting antimicrobial activity. For S. thermophilus PRI36 and PRI45, with relevant inhibitory features, the identity of the bacteriocin genes present in the thermophilin 9 cluster was defined, thus revealing novel variants for this genome region.
KeywordMeSH Terms
Genetic Variation
100.     ( 2012 )

Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.

Proceedings of the National Academy of Sciences of the United States of America 109 (39)
PMID : 22949671  :   DOI  :   10.1073/pnas.1208507109     PMC  :   PMC3465414    
Abstract >>
Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide adaptive immunity against viruses and plasmids in bacteria and archaea. The silencing of invading nucleic acids is executed by ribonucleoprotein complexes preloaded with small, interfering CRISPR RNAs (crRNAs) that act as guides for targeting and degradation of foreign nucleic acid. Here, we demonstrate that the Cas9-crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system introduces in vitro a double-strand break at a specific site in DNA containing a sequence complementary to crRNA. DNA cleavage is executed by Cas9, which uses two distinct active sites, RuvC and HNH, to generate site-specific nicks on opposite DNA strands. Results demonstrate that the Cas9-crRNA complex functions as an RNA-guided endonuclease with RNA-directed target sequence recognition and protein-mediated DNA cleavage. These findings pave the way for engineering of universal programmable RNA-guided DNA endonucleases.
KeywordMeSH Terms
DNA Breaks, Double-Stranded
DNA Breaks, Single-Stranded
101.     ( 2012 )

Use of tuf as a target for sequence-based identification of Gram-positive cocci of the genus Enterococcus, Streptococcus, coagulase-negative Staphylococcus, and Lactococcus.

Annals of clinical microbiology and antimicrobials 11 (N/A)
PMID : 23181410  :   DOI  :   10.1186/1476-0711-11-31     PMC  :   PMC3533577    
Abstract >>
Accurate identification of isolates belonging to genus Enterococcus, Streptococcus, coagulase-negative Staphylococcus, and Lactococcus at the species level is necessary to provide a better understanding of their pathogenic potential, to aid in making clinical decisions, and to conduct epidemiologic investigations,especially when large blind samples must be analyzed. It is useful to simultaneously identify species in different genera using a single primer pair. We developed a primer pair based on the tuf gene (encoding elongation factor) sequence to identify 56 Gram-positive cocci isolates. The target sequences were amplified from all 56 samples. The sequencing results and the phylogenetic tree derived from the partial tuf gene sequences identified the isolates as three enterococcal species, two lactococcal species, two staphylococcal species, and six streptococcal species, as well as eight isolates that were novel species of the genus Streptococcus. Partial gene sequence analysis of the sodA, dnaK, and 16S RNA genes confirmed the results obtained by tuf gene sequencing. Based on the uniform amplification of the tuf gene from all samples and the ability to identify all isolates at both the genus and species levels, we conclude that the primer pair developed in this research provides a powerful tool for identifying these organisms in clinical laboratories where large blind samples are used.
KeywordMeSH Terms
102.     ( 1998 )

Structural and functional properties of the hsp16.4-bearing plasmid pER341 in Streptococcus thermophilus.

Plasmid 40 (1)
PMID : 9657935  :   DOI  :   10.1006/plas.1998.1352    
Abstract >>
The plasmid pER341 (2798 bp) of Streptococcus thermophilus ST134 was sequenced and its open reading frame (ORF) regions were characterized. Analysis of nucleotide sequences showed the putative translation product of ORF1 (rep) sharing a high level of homology with replication proteins of several small plasmids present in lactic acid bacteria and staphylococci. This and homology of regions of plus-strand (ORI) and minus-strand (ssoA) origin of replication with pC194-class plasmids indicated that pER341 replicates by the rolling-circle mechanism. ORF2 corresponded to a putative hsp gene that apparently encodes Hsp16.4, a 142-amino-acid heat stress protein. Hsp16.4 shared significant identity with other small, 18-kDa-class heat stress proteins from prokaryotic and eukaryotic sources. Hsp16.4 is apparently the first plasmidborne low-molecular-weight heat stress protein reported in dairy fermentation bacteria with a potential role in temperature-regulated functions in S. thermophilus.
KeywordMeSH Terms
103.     ( 1998 )

Characterization of a novel insertion sequence, IS1194, in Streptococcus thermophilus.

Plasmid 40 (1)
PMID : 9657932  :   DOI  :   10.1006/plas.1998.1337    
Abstract >>
A novel insertion sequence, IS1194, has been identified in the lactic acid bacterium Streptococcus thermophilus CNRZ368. This 1200-bp element has 16-bp imperfect terminal inverted repeats. The single large open reading frame of this element encodes a 332-amino-acid protein that displays similarities with transposases encoded by bacterial insertion sequences belonging to the IS5 group of the IS4 family. A single copy of IS1194 was detected by hybridization in only 2 of the 19 S. thermophilus strains tested and in 4 of the 13 Lactococcus lactis strains investigated. This suggests that this IS element was acquired by horizontal transfer. The unique IS1194 copy of S. thermophilus CNRZ368 is located in a region of at least 12 kb that was probably acquired by horizontal transfer from L. lactis. Furthermore, the IS1194 right end is identical to sequences found in a broad-host-range conjugative plasmid from Streptococcus pyogenes, pSM19035.
KeywordMeSH Terms
DNA Transposable Elements
104.     ( 1998 )

Characterization of a novel Streptococcus thermophilus rolling-circle plasmid used for vector construction.

Applied microbiology and biotechnology 50 (2)
PMID : 9763687  :  
Abstract >>
The complete nucleotide sequence of pER371, a native plasmid in Streptococcus thermophilus ST137, was determined. A putative open reading frame coding for a replication protein, Rep371, was identified. A characteristic promoter sequence and ribosome-binding site were found upstream of rep371. Rep371 (247 amino acid residues) does not show homology with RepA and RepS of the small S. thermophilus cryptic plasmids pST1-No.29 and pST1 respectively. The plus-origin sequence and Rep371 are highly homologous to the corresponding elements of the Staphylococcus aureus plasmids pC194 and pSK89. A novel 140-nucleotide palindromic minusorigin sequence, which is structurally similar but does not show sequence homology to the palA region of pC194, was identified in pER371. A palindromic sequence capable of forming a putative hairpin structure was identified and subsequently recognized as being highly conserved among several lactococcal rolling-circle plasmids. Cloning vectors derived from pER371 should provide valuable gene-delivery vehicles for the genetic engineering of lactic acid bacteria.
KeywordMeSH Terms
105.     ( 1998 )

Conservation of the major cold shock protein in lactic acid bacteria.

Current microbiology 37 (5)
PMID : 9767713  :  
Abstract >>
Primers designed from consensus regions of the major cold shock gene of different bacterial species were used in PCR amplification of Lactic Acid Bacteria (LAB). An appropriately-sized PCR product was obtained from Lactococcus lactis subsp. lactis LL43-1 and MG1363; Lactococcus lactis subsp. cremoris LC10-1, LC11-1, and LC12-1; Streptococcus thermophilus ST1-1; Enterococcus faecalis EF1-1; Lactobacillus acidophilus LA1-1; Lactobacillus helveticus LH1-1; Pediococcus pentosaceus PP1-1; and Bifidobacterium animalis BA1-1. The PCR products were cloned and sequenced. The deduced amino acid sequences displayed high sequence similarity with the major cold shock proteins of Escherichia coli and Bacillus subtilis and the human Y-box factor. The amino acid residues of the cold shock domain implicated in nucleic acid binding in several unrelated species were also highly conserved in the LAB strains. It is possible, therefore, that this protein in LAB may also act as a transcriptional enhancer to other cold shock genes and/or act as an RNA chaperone unwinding tightly folded RNA molecules.
KeywordMeSH Terms
Cold Temperature
106.     ( 1998 )

Identification of streptococci to species level by sequencing the gene encoding the manganese-dependent superoxide dismutase.

Journal of clinical microbiology 36 (1)
PMID : 9431917  :   PMC  :   PMC124804    
Abstract >>
We have used a PCR assay based on the use of degenerate primers in order to characterize an internal fragment (sodA(int)) representing approximately 85% of the genes encoding the manganese-dependent superoxide dismutase in various streptococcal type strains (S. acidominimus, S. agalactiae, S. alactolyticus, S. anginosus, S. bovis, S. constellatus, S. canis, S. cricetus, S. downei, S. dysgalactiae, S. equi subsp. equi, S. equi subsp. zooepidemicus, S. equinus, S. gordonii, S. iniae, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguis, S. pneumoniae, S. porcinus, S. pyogenes, S. salivarius, S. sanguis, S. sobrinus, S. suis, S. thermophilus, and S. vestibularis). Phylogenetic analysis of these sodA(int) fragments yields an evolutionary tree having a topology similar to that of the tree constructed with the 16S rRNA sequences. We have shown that clinical isolates could be identified by determining the positions of their sodA(int) fragments on the phylogenetic tree of the sodA(int) fragments of the type species. We propose this method for the characterization of strains that cannot be assigned to a species on the basis of their conventional phenotypic reactions.
KeywordMeSH Terms
107.     ( 1998 )

Role of Streptococcus thermophilus MR-1C capsular exopolysaccharide in cheese moisture retention.

Applied and environmental microbiology 64 (6)
PMID : 9603827  :   DOI  :   10113/24968     PMC  :   PMC106291    
Abstract >>
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.
KeywordMeSH Terms
108.     ( 1997 )

Sequence analysis and characterization of phi O1205, a temperate bacteriophage infecting Streptococcus thermophilus CNRZ1205.

Microbiology (Reading, England) 143 (Pt 11) (N/A)
PMID : 9387220  :   DOI  :   10.1099/00221287-143-11-3417    
Abstract >>
The complete nucleotide sequence of phi O1205, a temperate bacteriophage infecting Streptococcus thermophilus strain CNRZ1205, was determined. The phage genome has a unit length of 43,075 bp and appears to be packaged by the so-called headful mechanism. The genomic organization and structure of phi O1205 resemble those of several temperate lactococcal phages that display a life-cycle-specific organization, where ORFs believed to be involved in the lysogenic life-cycle are clustered and arranged in an orientation opposite to the ORFs supposedly involved in the lytic life-cycle. Database searches revealed putative functions for several identified ORFs and further indicated that phi O1205 is genetically related to a particular group of lactococcal phages. Three genes encoding the major structural proteins were identified on the phi O1205 genome. The phage attachment site attP, the bacterial attachment site attB, and the two phage/chromosome junctions attL and attR were identified and found to contain a 40 bp common core sequence.
KeywordMeSH Terms
Genome, Viral

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