Analysis of an agmatine deiminase gene cluster in Streptococcus mutans UA159

An operon encoding enzymes of the agmatine deiminase system (AgDS) has been identified in the cariogenic bacterium Streptococcus mutans UA159. The AgDS is regulated by agmatine induction and carbohydrate catabolite repression. Ammonia is produced from agmatine at low pH, suggesting that the AgDS could augment acid tolerance.     

Role of HtrA in growth and competence of Streptococcus mutans UA159

We report here that HtrA plays a role in controlling growth and competence development for genetic transformation in Streptococcus mutans. Disruption of the gene for HtrA resulted in slow growth at 37 degrees C, reduced thermal tolerance at 42 degrees C, and altered sucrose-dependent biofilm formation on polystyrene surfaces. The htrA mutant also displayed a significantly reduced ability to undergo genetic transformation. A direct association between HtrA and genetic competence was demonstrated by the increased expression of the htrA gene upon exposure to competence-stimulating peptide. The induction of htrA gradually reached a maximum at around 20 min, suggesting that HtrA may be involved in a late competence response. Complementation of the htrA mutation in a single copy on the chromosome of the mutant could rescue the defective growth phenotypes but not transformability, apparently because a second gene, spo0J, immediately downstream of htrA, also affects transformation. The htrA and spo0J genes were shown to be both individually transcribed and cotranscribed and probably have a functional connection in competence development. HtrA regulation appears to be finely tuned in S. mutans, since strains containing multiple copies of htrA exhibited abnormal growth phenotypes. Collectively, the results reveal HtrA to be an integral component of the regulatory network connecting cellular growth, stress tolerance, biofilm formation, and competence development and reveal a novel role for the spo0J gene in genetic transformation.     

Mutacins from Streptococcus mutans UA159 are active against multiple streptococcal species

Streptococcus mutans UA159, whose genome is completely sequenced, produces two nonlantibiotic mutacins, mutacin IV (encoded by nlmAB) and mutacin V (encoded by nlmC). In this study, we investigated the contribution of nlmA and nlmB to mutacin IV activity and demonstrated by performing genetic studies as well as by using semipurified molecules that, in contrast to a previous report, both of these genes are required for optimum mutacin IV activity. We also showed that mutacin IV is active against multiple Streptococcus species. In contrast, mutacin V displayed a narrower inhibitory range than mutacin IV. Our results suggest that mutacin IV and mutacin V may act synergistically to inhibit various organisms.     

Effects of mutating putative two-component systems on biofilm formation by Streptococcus mutans UA159

Streptococcus mutans has at least six pairs of open reading frames that are homologous to bacterial two-component regulatory systems. Putative response regulators from five out of six of these pairs were successfully mutated by insertion of a kanamycin resistance marker and the effects of inactivation of the genes on the ability of the cells to form biofilms in an in vitro model were assessed. Disruption of the response regulators of four systems had no effect on biofilm formation, whereas disruption of one response regulator caused a substantial decrease in biofilm formation as compared to the wild-type S. mutans.     

An Extracelluar Protease,SepM, Generates Functional Competence-Stimulating Peptide in Streptococcus mutans UA159

Cell-cell communication in Gram-positive bacteria often depends on the production of extracellular peptides. The cariogenic bacterium Streptococcus mutans employs so-called competence-stimulating peptide (CSP) to stimulate mutacin (bacteriocin) production and competence development through the activation of the ComDE two-component pathway. In S. mutans, CSP is secreted as a 21-residue peptide; however, mass spectrometric analysis of culture supernatant indicates the presence of an 18-residue proteolytically cleaved species. In this study, using a transposon mutagenesis screening, we identified a cell surface protease that is involved in the processing of 21-residue CSP to generate the 18-residue CSP. We named this protease SepM for streptococcal extracellular protease required for mutacin production. We showed that the truncated 18-residue peptide is the biologically active form and that the specific postexport cleavage is a prerequisite to activate the ComDE two-component signal transduction pathway. We also showed that the CSP and the mutacins are exported outside the cell by the same ABC transporter, NlmTE. Our study further confirmed that the ComDE two-component system is absolutely necessary for mutacin production in S. mutans.     

Acid adaptation in Streptococcus mutans UA159 alleviates sensitization to environmental stress due to RecA deficiency

Abstract A RecA-deficient stain of Streptococcus mutans , isolated previously, was found to be more susceptible than the prototroph organism to acid killing and also showed reduced colony-forming ability on sucrose-containing medium. The deficient strain was able to grow in chemostat culture at a low pH value of 5 and did not show reduced capacity to produce acid in standard pH-drop experiments with excess glucose. Moreover, it was able to undergo an adaptive response when grown at a low pH to become more resistant to acid killing and also to killing by ultraviolet radiation or hydrogen peroxide. In fact, after adaptation, it was nearly as resistant as the prototroph strain. These findings were interpreted, in part, in terms of an acid-inducible DNA repair system which functions independently of RecA.     

The antimicrobial effects of deglycyrrhizinated licorice root extract on Streptococcus mutans UA159 in both planktonic and biofilm cultures

The objective of the study was to investigate the antimicrobial effects of deglycyrrhizinated licorice root extracts (DG-LRE) against Streptococcus mutans UA159 in both the planktonic and biofilm phases by determining the minimum inhibitory concentration and minimum bactericidal concentration, and by performing time-kill kinetic, growth, adhesion, and biofilm assays. The cell toxicity of DG-LRE on normal human gingival fibroblast (NHGF) cells was tested using a methyl thiazolyl tetrazolium assay. This study showed that DG-LRE had strong antimicrobial activity against S. mutans in the planktonic phase with little cytotoxic effect on NHGF cells. In addition, DG-LRE significantly inhibited biofilm formation by S. mutans UA159 at concentrations over 4 μg/ml for glucose or 16 μg/ml for sucrose, respectively, regardless of the presence of saliva-coating. To the best of our knowledge, this is the first report to provide evidence that DG-LRE demonstrates antimicrobial activity against S. mutans. These results suggest that DG-LRE can be used in developing oral hygiene products, such as gargling solution and dentifrice to prevent human dental caries.     

Effects of combined oleic acid and fluoride at sub-MIC levels on EPS formation and viability of Streptococcus mutans UA159 biofilms

Despite the widespread use of fluoride, dental caries, a biofilm-related disease, remains an important health problem. This study investigated whether oleic acid, a monounsaturated fatty acid, can enhance the effect of fluoride on extracellular polysaccharide (EPS) formation by Streptococcus mutans UA159 biofilms at sub-minimum inhibitory concentration levels, via microbiological and biochemical methods, confocal fluorescence microscopy, and real-time PCR. The combination of oleic acid with fluoride inhibited EPS formation more strongly than did fluoride or oleic acid alone. The superior inhibition of EPS formation was due to the combination of the inhibitory effects of oleic acid and fluoride against glucosyltransferases (GTFs) and GTF-related gene (gtfB, gtfC, and gtfD) expression, respectively. In addition, the combination of oleic acid with fluoride altered the bacterial biovolume of the biofilms without bactericidal activity. These results suggest that oleic acid may be useful for enhancing fluoride inhibition of EPS formation by S. mutans biofilms, without killing the bacterium.     

iTRAQ-based quantitative analysis reveals the mechanism underlying the changes in physiological activity in a glutamate racemase mutant strain of Streptococcus mutans UA159

Molecular Biology Reports - Streptococcus mutans UA159 is responsible for human dental caries with robust cariogenic potential. Our previous study noted that a glutamate racemase (MurI) mutant...     

Bacteriocin (mutacin) production by Streptococcus mutans genome sequence reference strain UA159: elucidation of the antimicrobial repertoire by genetic dissection

Streptococcus mutans UA159, the genome sequence reference strain, exhibits nonlantibiotic mutacin activity. In this study, bioinformatic and mutational analyses were employed to demonstrate that the antimicrobial repertoire of strain UA159 includes mutacin IV (specified by the nlm locus) and a newly identified bacteriocin, mutacin V (encoded by SMU.1914c).     

Bacteriocin (Mutacin) Production by Streptococcus mutans Genome Sequence Reference Strain UA159: Elucidation of the Antimicrobial Repertoire by Genetic Dissection

Streptococcus mutans UA159, the genome sequence reference strain, exhibits nonlantibiotic mutacin activity. In this study, bioinformatic and mutational analyses were employed to demonstrate that the antimicrobial repertoire of strain UA159 includes mutacin IV (specified by the nlm locus) and a newly identified bacteriocin, mutacin V (encoded by SMU.1914c).     

SELEX技术筛选变形链球菌UA159适配子可行性的研究

目的:研究SELEX技术用于筛选口腔致龋菌适配子的可行性。方法:化学合成长度为35mer的随机ssDNA文库,利用SE-LEX技术,分别以变形链球菌UA159(以下简称变链UA159)、乳杆菌和离心管作为靶物质,筛选适配子,不对称PCR扩增筛选产物,所得适配子进行克隆、测序,分析其二级结构,并对其二级结构进行了初步分析。结果:显示各个靶物质的筛选产物在第二轮筛选时就已经表现出具有特征性的二级结构。结论:SELEX技术可以用于口腔致龋菌适配子的筛选。     

Two‐dimensional gel‐based proteomic of the caries causative bacterium Streptococcus mutans UA159 and insight into the inhibitory effect of carolacton

Streptococcus mutans is considered to be the most cariogenic organism. Carolacton, isolated from the myxobacterium Sorangium cellulosum, shows the ability to disturb S. mutans biofilm viability that makes it a potential anti‐biofilm drug. However, the molecular mechanism of carolacton remains to be elucidated. In order to use proteomics to characterize the effect of carolacton, we constructed a 2DE‐based proteome reference map of the cytoplasmic and extracellular proteins for S. mutans in the present study. In total, 239 protein spots representing 192 different cytoplasmic proteins were identified by MALDI‐TOF MS and PMF. This represents the highest number of identified proteins so far for S. mutans UA159 in the pI range of 4–7 and would benefit further research on the physiology and pathogenicity of this strain. Based on the constructed reference map, the inhibitory effects of carolacton on S. mutans biofilm and planktonic‐growing cells were investigated. The results of the comparative proteome analysis indicate that carolacton exerts its inhibitory effects by disturbing the peptidoglycan biosynthesis and degradation and thereby causes damages to the integrity of the cell envelope, leading ultimately to cell death.