title: CcpA and CodY Coordinate Acetate Metabolism in Streptococcus mutans.
abstract: In the dental caries pathogenStreptococcus mutans, phosphotransacetylase (Pta) and acetate kinase (Ack) convert pyruvate into acetate with the concomitant generation of ATP. The genes for this pathway are tightly regulated by multiple environmental and intracellular inputs, but the basis for differential expression of the genes for Pta and Ack inS. mutanshad not been investigated. Here, we show that inactivation inS. mutansofccpAorcodYreduced the activity of theackApromoter, whereas accpAmutant displayed elevatedptapromoter activity. The interactions of CcpA with the promoter regions of both genes were observed using electrophoretic mobility shift and DNase protection assays. CodY bound to theackApromoter region but only in the presence of branched-chain amino acids (BCAAs). DNase footprinting revealed that the upstream region of both genes contains two catabolite-responsive elements (cre1andcre2) that can be bound by CcpA. Notably, thecre2site ofackAoverlaps with a CodY-binding site. The CcpA- and CodY-binding sites in the promoter region of both genes were further defined by site-directed mutagenesis. Some differences between the reported consensus CodY binding site and the region protected byS. mutansCodY were noted. Transcription of theptaandackAgenes in theccpAmutant strain was markedly different at low pH relative to transcription at neutral pH. Thus, CcpA and CodY are direct regulators of transcription ofackAandptainS. mutansthat optimize acetate metabolism in response to carbohydrate, amino acid availability, and environmental pH.IMPORTANCEThe human dental caries pathogenStreptococcus mutansis remarkably adept at coping with extended periods of carbohydrate limitation during fasting periods. The phosphotransacetylase-acetate kinase (Pta-Ack) pathway inS. mutansmodulates carbohydrate flux and fine-tunes the ability of the organisms to cope with stressors that are commonly encountered in the oral cavity. Here, we show that CcpA controls transcription of theptaandackAgenes via direct interaction with the promoter regions of both genes and that branched-chain amino acids (BCAAs), particularly isoleucine, enhance the ability of CodY to bind to the promoter region of theackAgene. A working model is proposed to explain how regulation ofptaandackAgenes by these allosterically controlled regulatory proteins facilitates proper carbon flow and energy production, which are essential functions during infection and pathogenesis as carbohydrate and amino acid availability continually fluctuate.
authors.affiliation: Department of Microbiology, College of Natural Sciences, Pusan National University, Busan, Republic of Korea.
authors.forename: Jeong Nam
authors.initials: JN
authors.lastname: Kim
authors.affiliation: Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, Florida, USA rburne@dental.ufl.edu.
authors.forename: Robert A
authors.identifier: http://orcid.org/0000-0002-4234-0316
authors.initials: RA
authors.lastname: Burne
mesh_terms: D000084:Acetate Kinase / Q000235:genetics; D000085:Acetates / Q000378:metabolism*; D000597:Amino Acids, Branched-Chain / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D003731:Dental Caries / Q000382:microbiology; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D010709:Phosphate Acetyltransferase / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D019289:Pyruvic Acid / Q000378:metabolism; D013295:Streptococcus mutans / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D000085:Acetates; D000597:Amino Acids, Branched-Chain; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D019289:Pyruvic Acid; D010709:Phosphate Acetyltransferase; D000084:Acetate Kinase
keywords: acid tolerance; carbohydrate metabolism; dental caries; gene regulation; pyruvate