Isolation and characterisation of promoter regions from Streptococcus thermophilus

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.     

嗜热链球菌高密度发酵过程的优化方案探讨

本研究介绍了嗜热链球菌（Streptococcus thermophilus）高密度发酵过程的优化方案。首先从培养基优化方案入手,讨论了单因素法与正交实验法在培养基优化过程中的利弊。第二,介绍了培养条件以及发酵过程的优化,包括pH、培养温度、搅拌转速、接种量和培养时间。第三,介绍了菌体的后处理过程,包括降温处理、超声解链、离心冻干。     

Characterization of a bacteriocin, Thermophilin 1277, produced by Streptococcus thermophilus SBT1277

AIMS: To assess the inhibitory activity and the influence of culture condition on the growth and bacteriocin, Thermophilin 1277, production by Streptococcus thermophilus SBT1277. METHODS AND RESULTS: Thermophilin 1277, which was produced by S. thermophilus SBT1277, showed an antimicrobial activity against several lactic acid bacteria and food spoilage bacteria including Clostridium butylicum, C. sprogenes and Bacillus cereus. Thermophilin 1277 was inactivated by proteinase K. Heating treatment did not affect the antimicrobial activity. The partially purified Thermophilin 1277 had an apparent molecular mass of 3.7 kDa. N-terminal sequence analysis revealed 15 amino acid residues that correspond with amino acid sequence of the lantibiotics bovicin HJ50 produced by Streptococcus bovis HJ50. The effects of culture condition for the bacteriocin production by S. thermophilus SBT1277 were studied. During the batch fermentation, Thermophilin 1277 was produced in M17 broth, but no bacteriocin production occurred in the sucrose-tryptone (ST) broth. Bacteriocin production was detected in pH controlled ST broth at pH values of 5.5-6.5. CONCLUSIONS: Thermophilin 1277 production from S. thermophilus strain depended on the culture conditions. Some characters and N-terminal amino acid sequence of Thermophilin 1277 differed from bacteriocins produced by S. thermophilus reported previously. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptococcus thermophilus SBT1277 or its bacteriocin which has a wide inhibitory spectrum has a potential use as a biopreservative in dairy products.     

Characterisation of Streptococcus thermophilus CNRZ1205 and its cured and re-lysogenised derivatives

Streptococcus thermophilus CNRZ1205 is the lysogenic host for the temperate phage phi O1205. A derivative of CNRZ1205 was isolated which was cured of phi O1205 and this strain was used to construct a re-lysogenised derivative. Pulse field gel electrophoresis and sequencing of the attachment site regions confirmed that excision and re-integration of the phage was a site-specific event. Interestingly, cells from the cured, as well as its re-lysogenised derivative, were found to have a very long chain length.     

粘性嗜热链球菌ST-1产胞外多糖发酵条件优化

应用响应面法对一株粘性嗜热链球菌ST-1的产胞外多糖的发酵条件进行了优化.根据单因素的实验结果,选取接种量,发酵温度,发酵时间作为考察因素,以胞外多糖的产量作为响应值,利用Box-Behnken实验设计方法,建立了胞外多糖产量与三个考察因素之间的回归方程并得到最佳发酵条件为:接种量5%,发酵时间14 h,发酵温度42℃...     

Genotyping of Streptococcus thermophilus strains isolated from traditional Egyptian dairy products by sequence analysis of the phosphoserine phosphatase (serB) gene with phenotypic characterizations of the strains

Aims: To develop a new, simplified genotyping method for examining the genetic diversity of Streptococcus thermophilus strains isolated from traditional Egyptian fermented dairy products and to characterize phenotypic traits of those strains related to their potential use in bioprocessing applications. Methods and Results: A novel, simplified approach was developed for genotyping Strep. thermophilus involving the analysis of nucleotide sequence variations within a housekeeping gene encoding the phosphoserine phosphatase (SerB). Using this method, it was possible to identify ten genotypes involving diverse serB alleles among 54 Strep. thermophilus isolates cultured from Egyptian dairy products. These isolates harboured five de novo serB alleles that have not been detected in other Strep. thermophilus strains, deposited in a multilocus sequence typing (MLST) database. To assess distinct genotypes of the organism with phenotypic traits relevant to their potential use in industry, Strep. thermophilus strains were all subjected to a series of phenotypic characterizations. The strains were found to exhibit phenotypic diversity in terms of their ability to ferment lactose and galactose, express urease activity, produce exopolysaccharides and develop acidity. Conclusions: The analysis of nucleotide sequence variations within the serB gene could serve as a suitable tool for probing diverse genotypes of Strep. thermophilus. Streptococcus thermophilus isolates associated with traditional Egyptian dairy products show high degree of genetic and phenotypic diversity. Significance and Impact of the Study: This study presents a novel, simplified procedure based on serB nucleotide sequencing for genotyping Strep. thermophilus. It also provides a pool of phenotypically diverse Strep. thermophilus cultures, from which certain strains could be selected for use in bioprocessing applications including the preparation of fermented dairy products.     

High-frequency deletion involving closely spaced rRNA gene sets in Streptococcus thermophilus

Abstract Homologous rDNA probes were used to study the number of rRNA genes in a five-generation genealogy of Streptococcus thermophilus CNRZ368. Whereas the CNRZ368 strain contained six rRNA loci, four independant mutants with five rrn loci were obtained. The deletion frequency was 5 × 10⁻². Molecular analysis provided identical hybridization patterns for all the deletion mutants. The deletion was shown to occur within the two close rRNA loci, rrnD and rrnE , probably due to a homologous recombination event and to give rise to a hybrid rrnD/E locus.     

Multifactorial experimental design for optimizing transformation: Electroporation of Streptococcus thermophilus

A multifactorial process was used to optimize transformation of Streptococcus thermophilus by electroporation. Simple experimental designs were applied to study three, four, or five factors in eight experiments. Four qualitative factors, growth and recovering media, and plasmid and bacterial strains, were studied empirically. Eight quantitative factors, including electrical, physiological, and chemical parameters, were studied by fractional factorial designs. Effects of individual parameters as well as interactions between them were investigated and optimized. Optimization was performed for one S. thermophilus strain, ST11, and proved to work for all other tested strains of the same species. Transformation efficiencies of 9 x 10(2) to 6 x 10(5) transformants per microgram DNA were achieved, depending on the strains and vectors used. (c) 1994 John Wiley & Sons, Inc.     

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

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.     

Identification of four loci isolated from two Streptococcus thermophilus phage genomes responsible for mediating bacteriophage resistance

Sequence data derived from the Streptococcus thermophilus phages phiO1205 and phi7201 indicated that each of these phages contains a distinct DNA region dedicated to replication. Southern blotting experiments showed that phages infecting S. thermophilus may be divided into at least two groups, each containing the presumptive replication functions of either φO1205 (group I) or φ7201 (group II). Specific regions from the putative replication module of each of the two phages were examined for their ability to provide phage resistance.     

Inhibition of microbial adhesion to silicone rubber treated with biosurfactant from Streptococcus thermophilus A

Microbial adhesion of four bacterial and two yeast strains isolated from explanted voice prostheses to silicone rubber before and after conditioning with a biosurfactant obtained from the probiotic bacterium Streptococcus thermophilus A was investigated in a parallel plate flow chamber. The silicone rubber with and without an adsorbed biosurfactant layer was characterized using contact angle measurements. Water contact angles indicated that the silicone rubber surface with adsorbed biosurfactant was more hydrophilic (58 degrees) than bare silicone rubber (109 degrees). The results obtained showed that the biosurfactant was effective in decreasing the initial deposition rates, and the number of bacterial cells adhering after 4 h, for all microorganisms tested. A decrease in the initial deposition rate was observed for Rothia dentocariosa GBJ 52/2B and Staphylococcus aureus GB 2/1 from 1937+/-194 to 179+/-21 microorganisms cm(-2) s(-1) and from 1255+/-54 to 233+/-26 microorganisms cm(-2) s(-1), respectively, accounting for an 86% reduction of the initial deposition rate for both strains. The number of bacterial cells adhering to the silicone rubber with preadsorbed biosurfactant after 4 h was further reduced by 89% and 97% by the two strains, respectively. The two yeast strains tested showed less reduction in adhesion after 4 h, to values between 67% and 70%. Such a pretreatment with surface-active compounds may constitute a promising strategy to reduce the microbial colonization rate of silicone rubber voice prostheses.     

Isolation and characterization of a type II restriction endonuclease from Streptococcus thermophilus

The alpha-toxin (phospholipase C) of Clostridium perfringens has been reported to contain catalytically essential zinc ions. We report here that histidine residues are essential for the co-ordination of these ion(s). Incubation of alpha toxin with diethylpyrocarbonate, a histidine modifying reagent, did not result in the loss of phospholipase C activity unless the protein was first incubated with EDTA, suggesting that zinc ions normally protect the susceptible histidine residues. When the amino acid sequences of three phospholipase C's were aligned, essential zinc binding histidine residues in the non-toxic B. cereus phospholipase C were found in similar positions in the toxic C. perfringens enzyme and the weakly toxic C. bifermentans phospholipase C.     

Carbamoylphosphate synthetase activity is essential for the optimal growth of Streptococcus thermophilus in milk

Aim:  The aim of the study was to study the role of carbon dioxide metabolism in Streptococcus thermophilus through investigation of the phenotype of a carbamoylphosphate synthetase-negative mutant.
Methods and results:  The effect of carbon dioxide on the nutritional requirements of Strep. thermophilus DSM20617^T and its derivative, carbamoylphosphate synthetase-negative mutant A17( ΔcarB ), was investigated by cultivating the strain in a chemically defined medium under diverse gas compositions and in milk. The results obtained revealed that CO₂ depletion or carB gene inactivation determined the auxotrophy of Strep. thermophilus for l -arginine and uracil. In addition, the parent strain grew faster than the mutant, even when milk was supplemented with uracil or arginine.
Conclusions:  Milk growth experiments underlined that carbamoylphosphate synthetase activity was essential for the optimal growth of Strep. thermophilus in milk.
Significance and impact of the study:  The study of the carbon dioxide metabolism in Strep. thermophilus revealed new insights with regard to the metabolism of this species, which could be useful for the optimization of dairy fermentation processes.     

A study of Streptococcus thermophilus proteome by integrated analytical procedures and differential expression investigations

Streptococcus thermophilus is a Gram-positive bacterium belonging to the group of lactic acid bacteria, among which several genera play an essential role in manufacture of food products. Recently, a genomic consortium sequenced and annotated its entire genome, which has been demonstrated to contain 1900 coding sequences. In this study, we have revealed the expression products of almost 200 different genes using a proteomic strategy combining 2-DE plus MALDI-TOF PMF and differential 1-DE plus muLC-ESI-IT-MS/MS. Thus, a number of cellular pathways related to important physiological processes were described at the proteomic level. Almost 50 genes were related to multiple electrophoretic species, whose heterogeneity was mainly due to variability in pI values. A 2-DE reference map obtained for lactose-grown cells was compared with those obtained after heat, cold, acid, oxidative and starvation stresses. Protein up/down-regulation measurements demonstrated that adaptation to different environmental challenges may involve the contribution of unique as well as combined physiological mechanisms. Common regulatory sites in the promoter region of genes whose expression was induced after stress were identified. These results provide a better comprehension of biochemical processes related to stress resistance in S. thermophilus, allowing defining the molecular bases of adaptative responses or markers for the identification of strains with potential industrial applications.     

Gene cluster for biosynthesis of thermophilin 1277--a lantibiotic produced by Streptococcus thermophilus SBT1277, and heterologous expression of TepI, a novel immunity peptide

Aims: To identify genes cluster for thermophilin 1277 produced by Streptococcus thermophilus SBT1277. Methods and Results: To identify genes for thermophilin 1277 production, the chromosomal DNA region surrounding the structural gene, tepA, was sequenced using a primer‐walking method. The thermophilin 1277 biosynthesis gene locus (tep) is a 9·9‐kb region, which consists of at least ten open reading frames (ORFs) in the following order: tepAMTFEGKRI and ORF4. Homology analysis showed high similarity to genes involved in bovicin HJ50 production by Streptococcus bovis HJ50. tepI encodes a novel, small, positively charged hydrophobic peptide of 52 amino acids, which contains a putative transmembrane segment. By heterologous expression in Lactococcus lactis ssp. cremoris MG1363, the TepI‐expressing strain exhibited at least 1·3 times higher resistance to thermophilin 1277. Conclusions: Thermophilin 1277 biosynthesis genes were encoded by a 9·9‐kbp region containing at least ten ORFs. TepI is a novel immunity peptide, which protected Strep. thermophilus SBT1277 against thermophilin 1277 in addition to TepFEG, a putative ABC transporter. Significance and Impact of the Study: This is the first report regarding a lantibiotic gene cluster produced by Strep. thermophilus strain.     

Possible promoter regions within the proteolytic system in Streptococcus thermophilus and their interaction with the CodY homolog

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

A species-specific DNA probe obtained from Streptococcus salivarius subsp. thermophilus detects strain restriction polymorphism

Genomic polymorphism in Streptococcus salivarius subsp. thermophilus was revealed by DNA restriction pattern analysis. A 4.2-kb variable DNA fragment was cloned from strain NST7 and hybridised with the DNA of 25 strains allowing an easy detection of intraspecific RFLP. Strong and weak hybridisation signals were observed and the latter were specifically revealed by a 2.1-kb fragment of the probe. Probe specificity was demonstrated by the absence of homology with DNA of strains belonging to 10 other species, with the exception of S. salivarius subsp. salivarius, confirming a close relationship between S. salivarius and S. thermophilus.     

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

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.