Regulation of urease gene expression by Streptococcus salivarius growing in biofilms

The metabolism of urea by urease enzymes of oral bacteria profoundly influences oral biofilm pH homeostasis and oral microbial ecology. The purpose of this study was to gain insight into the regulation of expression of the low pH-inducible urease genes in populations of Streptococcus salivarius growing in vitro in biofilms and to explore whether urease regulation or the levels of urease expression in biofilm cells differed significantly from planktonic cells. Two strains of S. salivarius harbouring urease promoter fusions to a chloramphenicol acetyltransferase (cat) gene were used: PurelCAT, containing a fusion to the full-length, pH-sensitive promoter; or Pureldelta100CAT, a constitutively derepressed deletion derivative of the urease gene promoter. The strains were grown in a Rototorque biofilm reactor in a tryptone-yeast extract-sucrose medium with or without pH control. Both CAT and urease activities in biofilms were measured at 'quasi-steady state' and after a 25mM glucose pulse. The results showed that CAT expression in PurelCAT was repressed at relatively neutral pH values, and that expression could be induced by acidic pH after carbohydrate challenge. Biofilms of PurelCAT grown at low pH, without buffering, had about 20-fold higher CAT levels, and only a modest further induction could be elicited with carbohydrate pulsing. The levels of CAT in biofilms of PurelCAT grown in buffered medium were slightly higher than those reported for planktonic cells cultured at pH 7.0, and the levels of CAT in Purel-CAT growing at low pH or after induction were similar to those reported for fully induced planktonic cells. CAT activity in Pureldelta100CAT was constitutively high, regardless of growth conditions. Interestingly, urease activity detected in biofilms of the parent strain, S. salivarius 57.1, could be as much as 130-fold higher than that reported for fluid chemostat cultures grown under similar conditions. The higher level of urease activity in biofilms was probably caused by the accumulation of the stable urease enzyme within biofilm cells, low pH microenvironments and the growth phase of populations of cells in the biofilm. The ability of S. salivarius biofilm cells to upregulate urease expression in response to pH gradients and to accumulate greater quantities of urease enzyme when growing in biofilms may have a significant impact on oral biofilm pH homeostasis and microbial ecology in vivo. Additionally, S. salivarius carrying the pH-sensitive urease gene promoter fused to an appropriate reporter gene may be a useful biological probe for sensing biofilm pH in situ.     

Extent of horizontal gene transfer in evolution of Streptococci of the salivarius group

The phylogenetically closely related species Streptococcus salivarius and Streptococcus vestibularis are oral bacteria that are considered commensals, although they can also be found in human infections. The relationship between these two species and the relationship between strains isolated from carriers and strains responsible for invasive infections were investigated by multilocus sequence typing and additional sequence analysis. The clustering of several S. vestibularis alleles and the extent of genomic divergence at certain loci support the conclusion that S. salivarius and S. vestibularis are separate species. The level of sequence diversity in S. salivarius alleles is generally high, whereas that in S. vestibularis alleles is low at certain loci, indicating that the latter species might have evolved recently. Cluster analysis indicated that there has been genetic exchange between S. salivarius and S. vestibularis at three of the nine loci investigated. Horizontal gene transfer between streptococci belonging to the S. salivarius group and other oral streptococci was also detected at several loci. A high level of recombination in S. salivarius was revealed by allele index association and split decomposition sequence analyses. Commensal and infection-associated S. salivarius strains could not be distinguished by cluster analysis, suggesting that the pathogen isolates are opportunistic. Taken together, our results indicate that there is a high level of gene exchange that contributes to the evolution of two streptococcal species from the human oral cavity.     

Identification of Streptococcus salivarius by PCR and DNA probe

AIMS: To establish species-specific PCR and DNA probe methods for Streptococcus salivarius and to clarify the distribution of dextranase in oral isolates of Strep. salivarius. METHODS AND RESULTS: A pair of PCR primers and a DNA probe were designed based on the nucleotide sequence of the dextranase gene of Strep. salivarius JCM5707. Both the PCR primer and the DNA probe specifically detected Strep. salivarius but none of the other oral streptococci (23 strains of 13 species). The primer and the probe were capable of detecting 1 pg and 1 ng of the genomic DNA, respectively, purified from Strep. salivarius JCM5707. All oral isolates (130 strains from 12 subjects) of Strep. salivarius from human saliva were positive by both methods. CONCLUSION: The present PCR and DNA probe methods are highly specific to Strep. salivarius and are useful for the its detection and identification of this bacterium. The dextranase widely distributes among oral isolates of Strep. salivarius. Significance and Impact of the Study: The DNA sequence of a dextranase gene present in the genome of Strep. salivarius is useful as the target DNA of the species-specific PCR and DNA probe.     

Characterization of the Actinomyces naeslundii ureolysis and its role in bacterial aciduricity and capacity to modulate pH homeostasis

Ammonia production from urea by ureolytic oral bacteria is believed to have a significant impact on oral health and ecological balance of oral microbial populations. Actinomyces naeslundii is an important ureolytic organism in the oral cavity. In this study, we aimed to investigate the substrate affinity and pH optimum for ureolysis of A. naeslundii (ATCC12104), and expression of urease under different environmental factors. In addition, in vitro acid killing and pH drop experiments were used to detect the role of ureolysis in bacterial aciduricity and capacity to modulate pH homeostasis. We observed the K_s value of the ureolytic activity was 7.5 mM and a pH optimum near 6.5. Urease expression by A. naeslundii (ATCC12104) was affected by multiple factors, including environmental pH, glucose and nitrogen availability. The cells could be protected against acid killing through hydrolysis of physiologically relevant concentrations of urea. A. naeslundii (ATCC12104) demonstrated a significant capacity to temper glycolytic acidification in vitro at urea concentrations normally found in the oral cavity.     

Influence of pH,temperature and culture media on the growth and bacteriocin production by vaginal Lactobacillus salivarius CRL 1328

AIMS: To study the influence of pH, temperature and culture medium on the growth and bacteriocin production by vaginal Lactobacillus salivarius subsp. salivarius CRL 1328. METHODS AND RESULTS: The study was performed using a complete factorial experimental design. Lactobacillus salivarius was cultivated in LAPTg and MRS broths, adjusted to specific initial pH, and at different temperatures of incubation. The growth, which was evaluated by the Gompertz model, was higher in MRS broth than in LAPTg broth. The initial pH of the culture medium and the temperature had a dramatic effect on the production of bacteriocin. The optimal conditions for bacteriocin production were different to those for optimal growth. The decrease in the pH of the culture medium was parallel to the growth; pH had similar final values in both the MRS and the LAPTg broths. CONCLUSIONS: The optimal growth conditions were recorded in MRS broth, with an initial pH of 6.5 and a temperature of 37 degrees C. The maximum bacteriocin activity was obtained in LAPTg after 6 h at 37 degrees C, and at an initial pH of 6.5 or 8.0. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of a complete factorial design, and the evaluation of the growth parameters through the Gompertz model, enabled a rapid and simultaneous exploration of the influence of pH, temperature and growth medium on both growth and bacteriocin production by vaginal Lact. salivarius CRL 1328.     

Urease-positive, acid-sensitive mutants of Helicobacter pylori: urease-independent acid resistance involved in growth at low pH

Acid resistance is considered an important virulence factor of the human pathogen Helicobacter pylori. The enzyme urease plays an important role in this acid resistance, but there are indications that other systems are present. We set out to establish the relevance of these urease-independent acid-resistance systems for growth at low pH. Four mutants out of a total of 1000 UV-mutants were urease positive, grew identical to wild-type on pH 7 plates, but did not grow on pH 5 plates. Whereas transformation of a mutant with its own chromosomal DNA did not restore growth at pH 5, transformation with wild-type DNA or DNA of one of the other mutants did restore the growth. From these complementation studies, we conclude that in H. pylori a urease-independent acid-resistance system, probably depending on the expression of more than one gene, is involved in the growth at low pH.     

Isolation and characterization of Lactobacillus salivarius MTC 1026 as a potential probiotic

Researchers are becoming more interested in studying probiotics at present due to their benefit to human and animal health. In this study, newly isolated strains from human feces were evaluated for probiotic properties. A total of sixty isolated strains were collected from feces and six out of sixty isolated strains could inhibit the growth of Salmonella typhi and Salmonella typhimurium. The strain which gave the best inhibitory effect was selected for further characterization as a probiotic strain. The identification of this strain was analyzed on the basis of morphological and biochemical characteristics and 16S rDNA gene sequence. This strain was Gram-positive, rod shaped, and catalase and oxidase-negative, and produced acids from D-glucose, D-fructose, lactose, mannitol, sorbitol, inulin and starch. It could not hydrolyze esculin or red blood cells. Based on its 16S rDNA gene sequence, it was Lactobacillus salivarius, and so was called L. salivarius MTC 1026 in this study, and was closely related with L. salivarius DSPV 344T isolated from the calf gut. It was able to survive in gastric and small intestinal juices at pH 2.0 and 1.0% bile salt for several hours and also could grow at 45°C. Moreover, this strain showed inhibitory activity against a variety of food-borne pathogens. It was highly sensitive to piperacillin, chloramphenicl, ampicillin, erythromycin and ceftazidime. After plasmid curing, this strain was susceptible to gentamicin, amikacin, norfloxacin and ciprofloxacin. L. salivarius MTC 1026 could significantly inhibit the adhesion process of E. coli ATCC 25922 and S. typhimurium ATCC 13311 on Caco-2 monolayers in a competition assay and also reduced invasion of both pathogens (4-log cfu/ml) in an exclusion and displacement assay. Therefore, it was clearly demonstrated in this study that L. salivarius MTC 1026 has shown promising properties as a candidate for a potential probiotic for applications in humans and animals.     

Effect of Streptococcus salivarius K12 on the in vitro growth of Candida albicans and its protective effect in an oral candidiasis model

Oral candidiasis is often accompanied by severe inflammation, resulting in a decline in the quality of life of immunosuppressed individuals and elderly people. To develop a new oral therapeutic option for candidiasis, a nonpathogenic commensal oral probiotic microorganism, Streptococcus salivarius K12, was evaluated for its ability to modulate Candida albicans growth in vitro, and its therapeutic activity in an experimental oral candidiasis model was tested. In vitro inhibition of mycelial growth of C. albicans was determined by plate assay and fluorescence microscopy. Addition of S. salivarius K12 to modified RPMI 1640 culture medium inhibited the adherence of C. albicans to the plastic petri dish in a dose-dependent manner. Preculture of S. salivarius K12 potentiated its inhibitory activity for adherence of C. albicans. Interestingly, S. salivarius K12 was not directly fungicidal but appeared to inhibit Candida adhesion to the substratum by preferentially binding to hyphae rather than yeast. To determine the potentially anti-infective attributes of S. salivarius K12 in oral candidiasis, the probiotic was administered to mice with orally induced candidiasis. Oral treatment with S. salivarius K12 significantly protected the mice from severe candidiasis. These findings suggest that S. salivarius K12 may inhibit the process of invasion of C. albicans into mucous surfaces or its adhesion to denture acrylic resins by mechanisms not associated with the antimicrobial activity of the bacteriocin. S. salivarius K12 may be useful as a probiotic as a protective tool for oral care, especially with regard to candidiasis.     

天然药物对唾液链球菌生长与产酸影响的体外研究

目的：通过研究不同天然药物对唾液链球菌生长及产酸的影响,为今后筛选出能有效调理口腔菌群生态平衡的药物奠定基础,方法：选用唾液链球菌SS196作为实验菌株,测定川芎,血藤,五倍子等11种天然药物的最低抑菌浓度MIC,再以低于MIC的4个浓度梯度配制含药的TPY液体培养基,调定其初始pH为7．4,接种唾液链球菌,厌氧培养后测定其终末pH,结果：当药物浓度低于或等于8．00mg/ml时,除槟榔,蜂房,三七外,其他药物对唾液链球菌的生长都有一定的抑制作用,且以大黄,五倍子和黄芩较强,槟榔,茶多酚,大黄,蜂房,黄芩,三七,五倍子和儿茶对唾液链球菌的产酸具有一定的抑制能力,而白芷,川芎和血藤没有明显的抑制能力,结论：茶多酚,大黄,黄芩,五倍子和儿茶对唾液链球菌的生长和产酸都有一定的抑制作用。     

幽门螺旋杆菌重组尿素酶B亚基的纯化及其免疫学性质的研究

目的:幽门螺旋杆菌(Hp)尿素酶是Hp重要的定制因子和致病因子,Hp尿素酶活性位点位于Hp尿素酶B亚基(UreB),研发基于UreB的Hp疫苗是一种很有前景的防治Hp感染的策略。方法:主要利用基因克隆技术从幽门螺旋杆菌标准菌株SS1(Hp SS1)获得Hp尿素酶B亚基基因,并构建含有重组Hp尿素酶B亚基(rUreB)基因的重组表达载体pET-rUreB及其重组菌株;重组菌株经蛋白表达和优化后,利用Ni-NTP镍离子亲和层析和DEAE Sepharose FF阴离子交换层析纯化重组尿素酶B亚基(rUreB),并进一步通过腹腔注射免疫BALB/c小鼠,研究rUreB的免疫学性质。结果:通过基因克隆技术成功获得了Hp尿素酶B亚基基因,并成功构建了重组表达载体pET-rUreB及其重组菌株BL21(DE3)/pET-rUreB,经蛋白表达优化及纯化,可获得高纯度(96.5%)的重组蛋白rUreB。重组蛋白rUreB辅以弗氏佐剂腹腔注射免疫BALB/c小鼠,经间接ELISA鉴定小鼠能够产生针对天然Hp尿素酶和UreB的高滴度特异性抗体,且能够显著性抑制Hp尿素酶的活性。结论:重组Hp尿素酶B亚基能够在大肠杆菌表达系统中获得较高水平的表达,具有较高的免疫学特异性,其抗体能够有效抑制Hp尿素酶活性。为研究基于尿素酶的防治Hp感染的Hp疫苗奠定了一定的实验基础。     

Complete genome sequence of the ureolytic Streptococcus salivarius strain 57.I

Streptococcus salivarius 57.I is one of the most abundant and highly ureolytic bacteria in the human mouth. It can utilize urea as the sole nitrogen source via the activity of urease. Complete genome sequencing of S. salivarius 57.I revealed a chromosome and a phage which are absent in strain SK126.     

The global gene expression response of Escherichia coli to L-phenylalanine

We investigated the global gene expression changes of Escherichia coli due to the presence of different concentrations of phenylalanine or shikimate in the growth medium. The response to 0.5 g l(-1) phenylalanine primarily reflected a perturbed aromatic amino acid metabolism, in particular due to TyrR-mediated regulation. The addition of 5g l(-1) phenylalanine reduced the growth rate by half and elicited a great number of likely indirect effects on genes regulated in response to changed pH, nitrogen or carbon availability. Consistent with the observed gene expression changes, supplementation with shikimate, tyrosine and tryptophan relieved growth inhibition by phenylalanine. In contrast to the wild-type, a tyrR disruption strain showed increased expression of pckA and of tktB in the presence of phenylalanine, but its growth was not affected by phenylalanine at the concentrations tested. The absence of growth inhibition by phenylalanine suggested that at high phenylalanine concentrations TyrR-defective strains might perform better in phenylalanine production.     

Energetics of Helicobacter pylori and its implications for the mechanism of urease-dependent acid tolerance at pH 1

In the presence of urea the neutrophilic human pathogen Helicobacter pylori survives for several hours at pH 1 with concomitant cytoplasmic pH homeostasis. To study this effect in detail, the transmembrane proton motive force and cytoplasmic urease activity of H. pylori were determined at various pH values. In the absence of urea, the organism maintained a close-to-neutral cytoplasm and an internally negative membrane potential at external pH values greater than 4 to 5. In the presence of urea, H. pylori accomplished cytoplasmic pH homeostasis down to an external pH of 1.2. At this external pH, the cytoplasmic pH was 4.9 and the membrane potential was slightly negative inside. The latter finding is in contrast to the situation in acidophiles, which develop inside-positive membrane potentials under similar conditions. Measurements of the time course of the membrane potential confirmed that addition of urea to the cells led to hyperpolarization. Most likely, this effect was due to electrogenic export of ammonium cations from the cytoplasm. The urease activity of intact cells increased nearly exponentially with decreasing external pH. This activation was not due to enhanced gene expression at low external pH values. In cell extracts the pH optimum of urease activity was dependent on the buffer system and was about pH 5 in sodium citrate buffer. Since this is the cytoplasmic pH of the cells at pH 1 to 2, we propose that cytoplasmic pH is a factor in the in vivo activation of the urease at low external pH values. The mechanism by which urease activity leads to cytoplasmic pH homeostasis in H. pylori is discussed.     

Molecular characterization of a cluster of at least two glucosyltransferase genes in Streptococcus salivarius ATCC 25975.

The oral micro-organism Streptococcus salivarius ATCC 25975 synthesizes extracellular glucosyltransferases (GTFs) which polymerize the glucose moiety of sucrose into glucan polymers. Two separate genes encoding the activities of a GTF-I (a GTF that synthesizes an insoluble product) and a GTF-S (a GTF that synthesizes soluble product) were cloned into bacteriophage lambda L47.1. The inserts in the lambda-clones were characterized by restriction mapping and Southern hybridization and were found to overlap, implying that the two genes lay very close to one another on the S. salivarius chromosome. Both genes were subcloned into phagemid vector pIBI30 where they were expressed at a high level. The GTF-I-encoding gene was named gtfJ and the GTF-S-encoding gene, gtfK. Nucleotide sequencing showed that gtfJ and most probably gtfK were closely related to the gtf genes of the mutans streptococci. Sequence alignment also indicated that gtfK lay very close to and downstream from gtfJ, and that both were transcribed in the same direction.     

基于不同氮源培养条件的巴氏芽孢杆菌脲酶功能转录组分析

巴氏芽孢杆菌是源于土壤的革兰氏阳性菌,人们利用其高效的脲酶活性诱导产生碳酸钙的现象开发了多种应用场景.然而,巴氏芽孢杆菌的生物矿化相关代谢机制还不够明确,尤其是对在矿化作用中发挥核心作用的脲酶基因结构、表达调控机制及关联代谢等方面的研究相对较少.当前,巴氏芽孢杆菌应用研究中面临的矿化反应不可控性及不稳定性等问题都源于脲酶代谢机制的研究匮乏.因此,进一步揭示巴氏芽孢杆菌脲酶的基因信息、表达调控机制及相关代谢机理迫在眉睫.本文通过转录组测序,对比了4种培养条件下巴氏芽孢杆菌的生长情况和基因表达情况,解析了脲酶的代谢机制,结果进一步证明ATP合成与脲酶表达及尿素水解相关联,最终预测了脲酶的双操纵子结构.