Regulation of fatty acid biosynthesis by the global regulator CcpA and the local regulator FabT in Streptococcus mutans.
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ABSTRACT:
SUBMITTER: Faustoferri RC
PROVIDER: S-EPMC4415630 | biostudies-literature | 2015 Apr
REPOSITORIES: biostudies-literature
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Project description:The Streptococcus mutans Cid/Lrg system represents an ideal model for studying this organism's ability to withstand various stressors encountered in the oral cavity. The lrg and cid operons display distinct and opposite patterns of expression in response to growth phase and glucose levels, suggesting that the activity and regulation of these proteins must be tightly coordinated in the cell and closely associated with metabolic pathways of the organism. Here, we demonstrate that expression of the cid and lrg operons is directly mediated by a global transcriptional regulator CcpA in response to glucose levels. Comparison of the cid and lrg promoter regions with the conserved CcpA binding motif revealed the presence of two potential cre sites (for CcpA binding) in the cid promoter (designated cid-cre1 and cid-cre2), which were arranged in a similar manner to those previously identified in the lrg promoter region (designated lrg-cre1 and lrg-cre2). We demonstrated that CcpA binds to both the cid and lrg promoters with a high affinity, but has an opposing glucose-dependent effect on the regulation of cid (positive) and lrg (negative) expression. DNase I footprinting analyses revealed potential binding sequences for CcpA in both cid and lrg promoter regions. Collectively, these data suggest that CcpA is a direct regulator of cid and lrg expression, and are suggestive of a potential mechanism by which Cid/Lrg-mediated virulence and cellular homeostasis is integrated with signals associated with both the environment and cellular metabolic status.
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Project description:A bacterial transcriptome of the primary etiological agent of human dental caries, Streptococcus mutans, is described here using deep RNA sequencing. Differential expression profiles of the transcriptome in the context of carbohydrate source, and of the presence or absence of the catabolite control protein CcpA, revealed good agreement with previously-published DNA microarrays. In addition, RNA-seq considerably expanded the repertoire of DNA sequences that showed statistically-significant changes in expression as a function of the presence of CcpA and growth carbohydrate. Novel mRNAs and small RNAs were identified, some of which were differentially expressed in conditions tested in this study, suggesting that the function of the S. mutans CcpA protein and the influence of carbohydrate sources has a more substantial impact on gene regulation than previously appreciated. Likewise, the data reveal that the mechanisms underlying prioritization of carbohydrate utilization are more diverse than what is currently understood. Collectively, this study demonstrates the validity of RNA-seq as a potentially more-powerful alternative to DNA microarrays in studying gene regulation in S. mutans because of the capacity of this approach to yield a more precise landscape of transcriptomic changes in response to specific mutations and growth conditions.
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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.
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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. Keywords: Catabolite repression, sugar transport, biofilm, virulence, stress response
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Project description:CcpA globally regulates transcription in response to carbohydrate availability in many gram-positive bacteria, but its role in Streptococcus mutans remains enigmatic. Using the fructan hydrolase (fruA) gene of S. mutans as a model, we demonstrated that CcpA plays a direct role in carbon catabolite repression (CCR). Subsequently, the expression of 170 genes was shown to be differently expressed (> or = 2-fold) in glucose-grown wild-type (UA159) and CcpA-deficient (TW1) strains (P < or = 0.001). However, there were differences in expression of only 96 genes between UA159 and TW1 when cells were cultivated with the poorly repressing substrate galactose. Interestingly, 90 genes were expressed differently in wild-type S. mutans when glucose- and galactose-grown cells were compared, but the expression of 515 genes was altered in the CcpA-deficient strain in a similar comparison. Overall, our results supported the hypothesis that CcpA has a major role in CCR and regulation of gene expression but revealed that in S. mutans there is a substantial CcpA-independent network that regulates gene expression in response to the carbohydrate source. Based on the genetic studies, biochemical and physiological experiments demonstrated that loss of CcpA impacts the ability of S. mutans to transport and grow on selected sugars. Also, the CcpA-deficient strain displayed an enhanced capacity to produce acid from intracellular stores of polysaccharides, could grow faster at pH 5.5, and could acidify the environment more rapidly and to a greater extent than the parental strain. Thus, CcpA directly modulates the pathogenic potential of S. mutans through global control of gene expression.
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Project description:Transcriptional analysis of the regulator FabT in S. mutans
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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.
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