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A biochemical characterization of the DNA binding activity of the response regulator VicR from Streptococcus mutans.

ABSTRACT: Two-component systems (TCSs) are ubiquitous among bacteria and are among the most elegant and effective sensing systems in nature. They allow for efficient adaptive responses to rapidly changing environmental conditions. In this study, we investigated the biochemical characteristics of the Streptococcus mutans protein VicR, an essential response regulator that is part of the VicRK TCS. We dissected the DNA binding requirements of the recognition sequences for VicR in its phosphorylated and unphosphorylated forms. In doing so, we were able to make predictions for the expansion of the VicR regulon within S. mutans. With the ever increasing number of bacteria that are rapidly becoming resistant to even the antibiotics of last resort, TCSs such as the VicRK provide promising targets for a new class of antimicrobials.

SUBMITTER: Ayala E

PROVIDER: S-EPMC4168254 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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A biochemical characterization of the DNA binding activity of the response regulator VicR from Streptococcus mutans.

Ayala Eduardo E Downey Jennifer S JS Mashburn-Warren Lauren L Senadheera Dilani B DB Cvitkovitch Dennis G DG Goodman Steven D SD

PloS one 20140917 9

Two-component systems (TCSs) are ubiquitous among bacteria and are among the most elegant and effective sensing systems in nature. They allow for efficient adaptive responses to rapidly changing environmental conditions. In this study, we investigated the biochemical characteristics of the Streptococcus mutans protein VicR, an essential response regulator that is part of the VicRK TCS. We dissected the DNA binding requirements of the recognition sequences for VicR in its phosphorylated and unpho ...[more]

PMID: 25229632

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Project description:The VicRK 2-component system of Streptococcus mutans regulates genes associated with cell wall biogenesis and biofilm formation. A putative RNase III-encoding gene ( rnc) is located downstream from the vicRKX operon. The goals of this study were to investigate the potential role of VicR in the regulation of adjacent downstream genes and evaluate transcription levels of vicR during planktonic and biofilm growth. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to investigate whether vicRKX and adjacent downstream genes were cotranscribed. Binding of purified recombinant VicR protein to promoter regions of vicR, rnc, and syfA genes was confirmed by electrophoretic mobility shift assay and by chromatin immunoprecipitation analyses. VicR antisense (AS vicR) RNA was detected by Northern blotting and qRT-PCR assays. AS vicR overexpression mutants were constructed, and the biofilm biomass was determined by crystal violet microtiter assay. Adjacent downstream genes rnc, smc, syfA, smu.1511, and syfB were cotranscribed with vicRKX. The predicted promoter regions of vicR, rnc, and syfA genes were directly regulated by VicR. An AS vicR RNA transcript was detected upstream of the rnc gene. Expression of the AS vicR RNA transcript was elevated in planktonic cultures and repressed during biofilm growth. In addition, Western blot data showed that expression of the VicR protein decreased by 35% in planktonic as compared with biofilm cultures. Furthermore, we show that overexpression of AS vicR led to a reduction in biofilm formation. The downstream genes rnc, smc, syfA, smu.1511, and syfB are cotranscribed with vicRKX. VicR is autophosphorylated, and rnc and syfA are directly regulated by VicR. Expression of VicR protein correlated inversely with different levels of AS vicR RNA transcript and growth conditions. The biofilm biomass decreased in the AS vicR overexpression mutant. These data suggest a role for the AS vicR RNA transcript in posttranscriptional regulation of VicR protein production in S. mutans.

Project description:BackgroundGlutamate racemase (MurI) is a cofactor-independent enzyme that is essential to the bacterial peptidoglycan biosynthesis pathway and has therefore been considered an attractive target for the development of antimicrobial drugs. While in our previous study the essentiality of the murI gene was shown in Streptococcus mutans, the primary aetiologic agent of human dental caries, studies on S. mutans MurI have not yet provided definitive results. This study aimed to produce and characterize the biochemical properties of the MurI from the S. mutans UA159 genome.MethodsStructure characterization prediction and multiple sequence alignment were performed by bioinformatic analysis. Recombinant His6-tagged S. mutans MurI was overexpressed in the expression vector pColdII and further purified using a Ni2+ affinity chromatography method. Protein solubility, purity and aggregation state were analyzed by SDS-PAGE, Western blotting, native PAGE and SEC-HPLC. Kinetic parameters were assessed by a circular dichroism (CD) assay. Kinetic constants were calculated based on the curve fit for the Michaelis-Menten equation. The effects of temperature and pH on enzymatic activity were determined by a series of coupled enzyme reaction mixtures.ResultsThe glutamate racemase gene from S. mutans UA159 was amplified by PCR, cloned and expressed in Escherichia coli BL21 (DE3). The 264-amino-acid protein, as a mixture of dimeric and monomeric enzymes, was purified to electrophoretic homogeneity. In the CD assay, S. mutans MurI displayed unique kinetic parameters (K m, d-Glu?l-Glu = 0.3631 ± 0.3205 mM, V max, d-Glu?l-Glu = 0.1963 ± 0.0361 mM min-1, k cat, d-Glu?l-Glu = 0.0306 ± 0.0065 s-1, k cat/K m, d-Glu?l-Glu = 0.0844 ± 0.0128 s-1 mM-1, with d-glutamate as substrate; K m, l-Glu?d-Glu = 0.8077 ± 0.5081 mM, V max, l-Glu?d-Glu = 0.2421 ± 0.0418 mM min-1, k cat , l - Glu?d-Glu = 0.0378 ± 0.0056 s-1, k cat/K m, l-Glu?d-Glu = 0.0468 ± 0.0176 s-1 mM-1, with l-glutamate as substrate). S. mutans MurI possessed an assay temperature optimum of 37.5 °C and its optimum pH was 8.0.ConclusionThe findings of this study provide insight into the structure and biochemical traits of the glutamate racemase in S. mutans and supply a conceivable guideline for employing glutamate racemase in anti-caries drug design.

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A biochemical characterization of the DNA binding activity of the response regulator VicR from Streptococcus mutans.

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A biochemical characterization of the DNA binding activity of the response regulator VicR from Streptococcus mutans.

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