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Regulation of fatty acid biosynthesis by the global regulator CcpA and the local regulator FabT in Streptococcus mutans.

ABSTRACT: SMU.1745c, encoding a putative transcriptional regulator of the MarR family, maps to a location proximal to the fab gene cluster in Streptococcus mutans. Deletion of the SMU.1745c (fabTS m ) coding region resulted in a membrane fatty acid composition comprised of longer-chained, unsaturated fatty acids (UFA), compared with the parent strain. Previous reports have indicated a role for FabT in regulation of genes in the fab gene cluster in other organisms, through binding to a palindromic DNA sequence. Consensus FabT motif sequences were identified in S. mutans in the intergenic regions preceding fabM, fabTSm and fabK in the fab gene cluster. Chloramphenicol acetyltransferase (cat) reporter fusions, using the fabM promoter, revealed elevated transcription in a ?fabTS m background. Transcription of fabTS m was dramatically elevated in cells grown at pH values of 5 and 7 in the ? fabTS m background. Transcription of fabTS m was also elevated in a strain carrying a deletion for the carbon catabolite repressor CcpA. Purified FabTS m and CcpA bound to the promoter regions of fabTS m and fabM. Hence, the data indicate that FabTS m acts as a repressor of fabM and fabTS m itself and the global regulator CcpA acts as a repressor for fabTS m .

SUBMITTER: Faustoferri RC

PROVIDER: S-EPMC4415630 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Regulation of fatty acid biosynthesis by the global regulator CcpA and the local regulator FabT in Streptococcus mutans.

Faustoferri R C RC Hubbard C J CJ Santiago B B Buckley A A AA Seifert T B TB Quivey R G RG

Molecular oral microbiology 20141027 2

SMU.1745c, encoding a putative transcriptional regulator of the MarR family, maps to a location proximal to the fab gene cluster in Streptococcus mutans. Deletion of the SMU.1745c (fabTS m ) coding region resulted in a membrane fatty acid composition comprised of longer-chained, unsaturated fatty acids (UFA), compared with the parent strain. Previous reports have indicated a role for FabT in regulation of genes in the fab gene cluster in other organisms, through binding to a palindromic DNA sequ ...[more]

PMID: 25131436

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Project description:In the dental caries pathogen Streptococcus 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 in S. mutans had not been investigated. Here, we show that inactivation in S. mutans of ccpA or codY reduced the activity of the ackA promoter, whereas a ccpA mutant displayed elevated pta promoter 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 the ackA promoter 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 (cre1 and cre2) that can be bound by CcpA. Notably, the cre2 site of ackA overlaps 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 by S. mutans CodY were noted. Transcription of the pta and ackA genes in the ccpA mutant strain was markedly different at low pH relative to transcription at neutral pH. Thus, CcpA and CodY are direct regulators of transcription of ackA and pta in S. mutans that optimize acetate metabolism in response to carbohydrate, amino acid availability, and environmental pH.IMPORTANCE The human dental caries pathogen Streptococcus mutans is remarkably adept at coping with extended periods of carbohydrate limitation during fasting periods. The phosphotransacetylase-acetate kinase (Pta-Ack) pathway in S. mutans modulates 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 the pta and ackA genes 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 the ackA gene. A working model is proposed to explain how regulation of pta and ackA genes 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.

Project description:CcpA is a global regulator of transcription in Gram-positive bacteria that controls gene expression in response to carbohydrate availability. Using the fructan hydrolase (fruA) gene of S. mutans as a model, we demonstrate that CcpA does indeed play a major role in carbohydrate catabolite repression. Using DNA microarrays, the expression of at least 170 genes differed in CcpA-deficient cells compared to the parental strains when cells are grown in the presence of glucose, but only 90 genes showed altered expression when cells were cultivated in the poorly-repressing substrate galactose. Interestingly, 90 genes were differently expressed in wild-type S. mutans in response to cultivation in glucose versus galactose, but the expression of over 500 genes was altered when growth in glucose and galactose were compared in the CcpA-deficient strain. The results support a major role for CcpA in regulation of gene expression, but reveal that a substantial CcpA-independent network exists in S. mutans to regulate gene expression in response to carbohydrate source. A reference RNA that had been isolated from 2 liters of UA159 cells grown in BHI broth to OD600 = 0.5 was used in every experiment. Our experimental conditions consisted of S. mutans UA159 and TW1 grown in TV supplemented with 0.5% glucose or 0.5% galactose and collected at the mid-exponential phase of growth (OD600 = 0.5). All RNAs were purified as described above and used to generate cDNA according to the protocol provided by TIGR with the following minor modifications. The amount of RNA in each reaction was increased to 10 ug and the molar ratio of dTTP:aa-dUTP was increased to 1:2. SuperscriptIII Reverse transcriptase (Invitrogen, Gaithersburg, MD) was used to increase cDNA yields. Purified cDNAs were coupled with indocarbocyanine (Cy3)-dUTP, while reference cDNA was coupled with indodicarbocyanine (Cy5)-dUTP (Amersham Biosciences, Piscataway, NJ). Four individual Cy3-labeled cDNA samples originating from four different cultures of UA159 or TW1, grown in each experimental condition, were hybridized to the arrays along with Cy5-labeled reference cDNA, generating a total of 16 slides. Hybridizations were carried out in a Maui 4-chamber hybridization system (BioMicro Systems, Salt Lake City, Utah) for 17 h at 42ºC. The slides were then washed according to TIGR protocols and scanned using a GenePix Scanner (Axon Instruments Inc., Union City, CA). After the slides were scanned, single-channel images were loaded into TIGR Spotfinder software and overlayed. A spot grid was created according to TIGR specifications and manually adjusted to fit all spots within the grid, then the intensity values of each spot were measured. Data was normalized using TIGR Microarray data analysis software (MIDAS), by using LOWESS and Iterative Log Mean Centering with default settings, followed by In-Slide Replicate Analysis. Statistical analysis was carried out using BRB Array Tools with a cutoff P value of 0.001 for class prediction and class comparison.

Dataset Information

Regulation of fatty acid biosynthesis by the global regulator CcpA and the local regulator FabT in Streptococcus mutans.

Publications

Regulation of fatty acid biosynthesis by the global regulator CcpA and the local regulator FabT in Streptococcus mutans.

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