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Expression of an Extracellular Protein (SMU.63) Is Regulated by SprV in Streptococcus mutans.

ABSTRACT: 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.

SUBMITTER: Pandey SD

PROVIDER: S-EPMC7688222 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Streptococcus mutans utilizes numerous metabolite transporters to obtain essential nutrients in the "feast or famine" environment of the human mouth. S. mutans and most other streptococci are considered auxotrophic for several essential vitamins including riboflavin (vitamin B2), which is used to generate key cofactors and to perform numerous cellular redox reactions. Despite the well-known contributions of this vitamin to central metabolism, little is known about how S. mutans obtains and metabolizes B2 The uncharacterized protein SMU.1703c displays high sequence homology to the riboflavin transporter RibU. Deletion of SMU.1703c hindered S. mutans growth in complex and defined medium in the absence of saturating levels of exogenous riboflavin, whereas deletion of cotranscribed SMU.1702c alone had no apparent effect on growth. Expression of SMU.1703c in a Bacillus subtilis riboflavin auxotroph functionally complemented growth in nonsaturating riboflavin conditions. S. mutans was also able to grow on flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) in an SMU.1703c-dependent manner. Deletion of SMU.1703c and/or SMU.1702c impacted S. mutans acid stress tolerance, as all mutants showed improved growth at pH 5.5 compared to that of the wild type when medium was supplemented with saturating riboflavin. Cooccurrence of SMU.1703c and SMU.1702c, a hypothetical PAP2 family acid phosphatase gene, appears unique to the streptococci and may suggest a connection of SMU.1702c to the acquisition or metabolism of flavins within this genus. Identification of SMU.1703c as a RibU-like riboflavin transporter furthers our understanding of how S. mutans acquires essential micronutrients within the oral cavity and how this pathogen successfully competes within nutrient-starved oral biofilms.IMPORTANCE Dental caries form when acid produced by oral bacteria erodes tooth enamel. This process is driven by the fermentative metabolism of cariogenic bacteria, most notably Streptococcus mutans Nutrient acquisition is key in the competitive oral cavity, and many organisms have evolved various strategies to procure carbon sources or necessary biomolecules. B vitamins, such as riboflavin, which many oral streptococci must scavenge from the oral environment, are necessary for survival within the competitive oral cavity. However, the primary mechanism and proteins involved in this process remain uncharacterized. This study is important because it identifies a key step in S. mutans riboflavin acquisition and cofactor generation, which may enable the development of novel anticaries treatment strategies via selective targeting of metabolite transporters.

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.

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Expression of an Extracellular Protein (SMU.63) Is Regulated by SprV in Streptococcus mutans.

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