Project description:Global transcriptional analysis of acid-inducible genes in Streptococcus mutans: multiple two-component systems involved in acid adaptation pH is a major environmental factor that regulates gene expression in many bacteria. Streptococcus mutans in dental biofilms is regularly exposed to cycles of acidic pH during the ingestion of fermentable dietary carbohydrates. The ability of S. mutans to tolerate low pH is crucial for its virulence and the pathogenesis in dental caries. To better understand its acid tolerance mechanisms, we used DNA microarray to perform genome-wide transcriptional analysis of S. mutans in response to acidic pH. The results showed that adaptation of S. mutans to pH 5.5 for 2 hrs induced differential expression of nearly 14% of genes in the genome, including 169 up-regulated genes and 108 down-regulated genes, largely categorized into six groups. Especially, we found that the genes encoding multiple two-component systems, including CiaHR, LevSR, LiaSR, ScnKR, HK/RR07 and ComDE, were up-regulated during acid adaptation. These findings were further confirmed by real time qRT-PCR and phenotypic assays of the gene deletion mutants. The results support that the multiple two-component systems are required for S. mutans to orchestrate its signal transduction networks for optimal adaptation to acidic pH. Total RNAs were isolated from S. mutans UA159 cells (0.6 at OD600) grown in a TYG broth (3% tryptone, 0.3% yeast extract and 20 mM glucose) at either pH 5.5 or pH 7.5. The RNAs were treated with RNase-free DNase 1 and purified by Qiagen RNeasy mini columns. The purified RNAs were used to generate cDNA probes by an indirect labeling method based on the protocol from TIGR. The cDNAs were coupled with AlexaFluor 555 or AlexaFluor 647 (Invitrogen). The labeled cDNA probes from three different cultures of UA159 were hybridized to the S. mutans microarray slides obtained from PFGRC (http://pfgrc.tigr.org). Array hybridization was conducted using a protocol from the PFGRC with minor modification. After hybridization, washes and dried, the array slides were scanned by ScanArray 5000XL Reader (Perkin Elmer, Boston, MA). After the array slides were scanned, the resulting images were loaded into TIGR Spotfinder software (http://www.tigr.org/software/) and overlaid. A spot grid was created according to TIGR specifications and manually adjusted to fit all spots within the grid, and the intensity values of each spot were determined. Signal intensities of individual channels from an array slide were averaged and normalized using an array data analysis software (MIDAS) by using LOWESS and iterative log mean centering with default settings, followed by in-slide replicate analysis. A t-test was used to determine the consistency of ratios across replicate hybridizations. Only genes whose ratios were â¥ 2-fold changes (either increase or decrease) with 99% confidence interval (P â¤ 0.01) were considered statistically significant.

Project description:Dental caries is closely associated with the virulence of Streptococcus mutans (S. mutans). The stress adaptation of S. mutans in the fluctuating oral environment is critical to the virulence expression of this bacterium. Here we used whole-genome microarrays to profile the dynamic transcriptomic responses of S. mutans during physiological heat stress. We also evaluated the phenotypic changes, including initial biofilm formation, acid production and ATP turnover of S. mutans during heat stress. We found that S. mutans responded to heat stress in a distinct pattern, which featured a differential transcription of 885 genes in total and 114 “core” transcripts throughout the six post-exposure time points investigated (5 min, 10 min, 15 min, 30 min, 45 min and 60 min). Further gene ontology analysis showed that transcriptional changes of genes involved in transcriptional regulation and cellular homeostasis were critical for heat stress responses of S. mutans. In addition to those highly conserved heat-shock proteins, we observed differential expression of multiple transcriptional regulators. The expression of genes involved in sugar transport, soluble glucans biosynthesis (gtfC) and binding (gbpC) were upregulated, whereas genes involved in ABC transporters, insoluble glucans biosynthesis (gtfB) and binding (gbpB), which are critical for biofilm skeleton, were either down-regulated or unchanged. These results together with enhanced glycolytic activity, attenuated sucrose-dependent initial attachment and impaired biofilm architecture indicate metabolic adaptations by this bacterium to compensate the extra energy demand for a better fitness under adverse conditions. We used time series microarrays to detect the dynamic changes of S. mutans under heat stress. Mid-logarithmic phase cell cultures of S. mutans (OD600nm = 0.5) were incubated at 42℃ for 5 min, 10 min, 15 min, 30 min, 45 min and 60 min, which were compared with the S. mutans cultured at 37℃. We applied three biological replicates at each time point.

Project description:Transcriptional profiling of S. mutans rpoE knockout mutant comparing to the wild type. The mutation of rpoE cause dramatic changes in phenotypes. The goal is to determine the genes affected by rpoE. The delta subunit of RNA polymerase, RpoE, is wide spread in low G+C Gram-positive bacteria and is thought to play a role in enhancing transcriptional specificity by blocking RNA polymerase binding at weak-promoter sites and stimulating RNA synthesis by accelerating core enzyme recycling. Despite the well-studied biochemical properties of RpoE, a role for this protein in vivo has not been defined in depth. In this study, we show that inactivation of rpoE in the human dental caries pathogen Streptococcus mutans causes impaired growth, and loss of important virulence traits, including biofilm formation, resistance to antibiotics, and tolerance to environmental stresses. Complementation of the mutant with rpoE expressed in trans restored its phenotype to wild type. The luciferase fusion reporter showed that rpoE was highly transcribed throughout growth, and that acid and hydrogen peroxide stresses repressed rpoE expression. Transcriptome profiling of wild type and ΔrpoE cells in the exponential and early stationary phase of growth, under acid and hydrogen peroxide stress and under both stresses combined revealed that genes involved in histidine synthesis, malolactic fermentation, biofilm formation, and antibiotic resistance were down-regulated in the ΔrpoE mutant in all conditions. Moreover, the loss of RpoE resulted in dramatic changes in transport and metabolism of carbohydrates and amino acids. Interestingly, differential expression, mostly up-regulation, of 330 non-coding regions was found. In conclusion, this study demonstrates that RpoE is an important global modulator of gene expression in S. mutans, which is required for optimal growth and environmental adaptation. 5 conditions, mutant vs wild type, biological replicates: 2, technical replicates: 2

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Streptococcus mutans

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