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Expression of BrpA in Streptococcus mutans is regulated by FNR-box mediated repression.

ABSTRACT: Our previous studies showed that brpA in Streptococcus mutans, which encodes a member of the LytR-CpsA-Psr family of proteins, can be co-transcribed with brpB upstream as a bicistronic operon, and the intergenic region also has strong promoter activity. To elucidate how brpA expression is regulated, the promoter regions were analyzed using polymerase chain reaction-based deletions and site-directed mutagenesis and a promoterless luciferase gene as a reporter. Allelic exchange mutagenesis was also used to examine genes encoding putative trans-acting factors, and the impact of such mutations on brpA expression was analyzed by reporter assays. Multiple elements in the short brpA promoter (nucleotide -1 to -344 relative to start cordon ATG) were shown to have a major impact on brpA expression, including an FNR-box, for a putative binding site of an FNR-type of transcriptional regulator. When compared with the intact brpA promoter, mutations of the highly conserved nucleotides in FNR-box from TTGATgtttAcCtt to TTACAgaaaGtTac resulted in 1362-fold increases of luciferase activity (P < .001), indicative of the FNR-box-mediated repression as a major mechanism in regulation of brpA expression. When luciferase reporter was fused to the upstream brpBA promoter (nucleotides -784 to -1144), luciferase activity was decreased by 4.5-fold (P < .001) in the brpA mutant, TW14D, and by 67.7-fold (P < .001) in the brpB mutant, JB409, compared with the wild-type, UA159. However, no such effects were observed when the reporter gene was fused to the short brpA promoter and its derivatives. These results also suggest that brpA expression in S. mutans is auto-regulated through the upstream brpBA promoter.

SUBMITTER: Liao S

PROVIDER: S-EPMC5785582 | biostudies-other | 2017 Dec

REPOSITORIES: biostudies-other

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Expression of BrpA in Streptococcus mutans is regulated by FNR-box mediated repression.

Liao S S De A A Thompson T T Chapman L L Bitoun J P JP Yao X X Yu Q Q Ma F F Wen Z T ZT

Molecular oral microbiology 20170824 6

Our previous studies showed that brpA in Streptococcus mutans, which encodes a member of the LytR-CpsA-Psr family of proteins, can be co-transcribed with brpB upstream as a bicistronic operon, and the intergenic region also has strong promoter activity. To elucidate how brpA expression is regulated, the promoter regions were analyzed using polymerase chain reaction-based deletions and site-directed mutagenesis and a promoterless luciferase gene as a reporter. Allelic exchange mutagenesis was als ...[more]

PMID: 28744965

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Project description:Intracellular polysaccharides (IPS) are glycogen-like storage polymers which contribute significantly to Streptococcus mutans-induced cariogenesis. We previously identified and cloned a locus from the S. mutans chromosome which is required for the accumulation of IPS. Sequencing of this locus revealed at least four contiguous open reading frames, all of which are preceded by a common promoter region and are transcribed in the same direction. Analysis of the amino acid sequence deduced from the first of these open reading frames (ORF1) revealed domains which are highly conserved among D-alanine-activating enzymes (DltA) in Lactobacillus rhamnosus (formerly Lactobacillus casei) and Bacillus subtilis. The deduced amino acid sequences derived from ORF2, -3, and -4 also exhibit extensive similarity to DltB, -C, and -D, respectively, in these microorganisms. However, Southern hybridization experiments indicate that this operon maps to a locus on the S. mutans chromosome which is separate from the glgP, glgA, and glgD genes, whose products are known mediators of bacterial IPS accumulation. We therefore assigned a new dlt designation to the locus which we had formerly called glg. We maintain that the dlt genes are involved in S. mutans IPS accumulation, however, since they complement a mutation in trans which otherwise renders S. mutans IPS deficient. In this study, we found that expression of the S. mutans dlt genes is growth phase dependent and is modulated by carbohydrates internalized via the phosphoenolpyruvate phosphotransferase system (PTS). We demonstrated that the S. mutans dlt genes are expressed constitutively when non-PTS sugars are provided as the sole source of carbohydrate. Consistent with a role for the PTS in dlt expression is a similar constitutive expression of the dlt genes in an S. mutans PTS mutant grown in a chemically defined medium supplemented with glucose. In summary, these findings support a novel role for the dlt gene products in S. mutans IPS accumulation and suggest that dlt expression in this oral pathogen is subject to complex mechanisms of control imposed by growth phase, dietary carbohydrate, and other factors present in the plaque environment.

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

Expression of BrpA in Streptococcus mutans is regulated by FNR-box mediated repression.

Publications

Expression of BrpA in Streptococcus mutans is regulated by FNR-box mediated repression.

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