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Preliminary X-ray crystallographic analysis of SMU.2055 protein from the caries pathogen Streptococcus mutans.

ABSTRACT: The SMU.2055 gene from the major caries pathogen Streptococcus mutans is annotated as a putative acetyltransferase with 163 amino-acid residues. In order to identify its function via structural studies, the SMU.2055 gene was cloned into the expression vector pET28a. Native and SeMet-labelled SMU.2055 proteins with a His(6) tag at the N-terminus were expressed at a high level in Escherichia coli strain BL21 (DE3) and purified to homogeneity by Ni(2+)-chelating affinity chromatography. Diffraction-quality crystals of SeMet-labelled SMU.2055 were obtained using the sitting-drop vapour-diffusion method and diffracted to a resolution of 2.5 A on beamline BL17A at the Photon Factory, Tsukuba, Japan. The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 92.0, b = 95.0, c = 192.2 A. The asymmetric unit contained four molecules, with a solvent content of 57.1%.

SUBMITTER: Zhao WH

PROVIDER: S-EPMC2864685 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Preliminary X-ray crystallographic analysis of SMU.2055 protein from the caries pathogen Streptococcus mutans.

Zhao Wang Hong WH Zhan Xiu Rong XR Gao Xiong Zhuo XZ Liu Xiang X Zhang Yi Fei YF Lin Jiuxiang J Li Lan Fen LF Wei Shi Cheng SC Su Xio Dong XD

Acta crystallographica. Section F, Structural biology and crystallization communications 20100429 Pt 5

The SMU.2055 gene from the major caries pathogen Streptococcus mutans is annotated as a putative acetyltransferase with 163 amino-acid residues. In order to identify its function via structural studies, the SMU.2055 gene was cloned into the expression vector pET28a. Native and SeMet-labelled SMU.2055 proteins with a His(6) tag at the N-terminus were expressed at a high level in Escherichia coli strain BL21 (DE3) and purified to homogeneity by Ni(2+)-chelating affinity chromatography. Diffraction ...[more]

PMID: 20445252

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Project description:In Streptococcus mutans, SprV (SMU.2137) is a pleiotropic regulator that differentially regulates genes related to competence, mutacin production, biofilm formation, and the stress tolerance response, along with some other pathways. In this study, we established a link between SprV and an ∼67-kDa protein in the culture supernatant of strain UA159 that was later confirmed as SMU.63 by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. We discovered that SprV downregulates the transcription and translation of SMU.63. We found that the seven amino acids from the C-terminal region of SprV were also crucial for the expression of SMU.63. Deletion of smu.63 led to increased sucrose-independent biofilm formation and competence. The sprV deletion also increased biofilm formation although this could be partially attributed to the downregulation of smu.63 In an smu.63 sprV double mutant, a synergistic effect was observed in biofilm formation in contrast to effects on competence development. We found that low or excess magnesium ion repressed sprV transcription that, in turn, affected the expression of smu.63 As expected, a magnesium ion-dependent effect of competence and biofilm formation was observed in the UA159 strain. We also replicated the results of SMU.63 expression and competence in S. mutans GS5 that encodes both SprV and SMU.63 homologs and found that the GS5 strain behaves similarly to the UA159 strain, indicating that SprV's effect is strain independent.IMPORTANCE We previously identified a pleiotropic regulator, SprV, in Streptococcus mutans This regulator appears to be highly conserved among streptococci. Here, we showed that SprV regulates the expression of a secreted protein encoded by SMU.63 in S. mutans SMU.63 has been known to impact biofilm formation and genetic competence, two important characteristics that help in colonization of the organism. SMU.63 is also unique since it is known to form amyloid fiber. We found that SprV regulates the expression of SMU.63 at both the transcriptional and translational levels. We also found that the expression of SprV is regulated by magnesium ion concentration. Interestingly, both low and high magnesium ion concentrations affected biofilm formation and genetic competence. Since SMU.63 is also highly conserved among streptococci, we hypothesized that SprV will have a similar effect on its expression.

Dataset Information

Preliminary X-ray crystallographic analysis of SMU.2055 protein from the caries pathogen Streptococcus mutans.

Publications

Preliminary X-ray crystallographic analysis of SMU.2055 protein from the caries pathogen Streptococcus mutans.

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