Project description:Macrolide resistance, increasingly identified in Streptococcus pneumoniae and a wide range of other Gram-positive bacteria, is often due to efflux pumps encoded by the mef/mel(msr) operon found on discrete mobile genetic elements. The regulation of mef/mel(msr) in these elements is not well understood. We defined the promoter controlling the mef(E)/mel operon in S. pneumoniae, identified cis-acting 5′ regulatory elements and determined the mechanism of macrolide-inducible expression of the efflux pump. The mef(E)/mel transcriptional start site was a guanine 327 bp upstream of the mef(E) start codon. Consensus pneumococcal promoter -10 (5′-TATACT-3′) and -35 (5′-TTGAAC-3′) boxes separated by a 17 bp spacer were identified 7 bp upstream of the start site. Analysis of the predicted secondary structure of the 327 bp 5’ region identified four pairs of inverted repeats R1-R8 predicted to fold into stem-loops, a small leader peptide (MTASMRLR) required for macrolide induction and a Rho-independent transcription terminator involving the R5/R6 stem loop. Mutational analyses of the regulatory region identified transcriptional attenuation as the model for the inducible expression of macrolide efflux, which was confirmed by RNA-Seq expression data. The 327 bp region 5’ of mef(E) was highly conserved in other mef/mel(msr)-containing genetic elements complexes including Tn1207.1 and the 5612IQ complex in pneumococci and Tn1207.3 in Group A streptococci. Induction of the mef(E)/mel operon and macrolide efflux occurs by anti-attenuation in the presence of inducing macrolides and appears to be a mechanism RNA-seq was performed on wild type and mutant cultures of four strains of Streptococcus pneumoniae untreated, or treated with spiramycin, LL-37 or erythromycin
Project description:Macrolide resistance, increasingly identified in Streptococcus pneumoniae and a wide range of other Gram-positive bacteria, is often due to efflux pumps encoded by the mef/mel(msr) operon found on discrete mobile genetic elements. The regulation of mef/mel(msr) in these elements is not well understood. We defined the promoter controlling the mef(E)/mel operon in S. pneumoniae, identified cis-acting 5′ regulatory elements and determined the mechanism of macrolide-inducible expression of the efflux pump. The mef(E)/mel transcriptional start site was a guanine 327 bp upstream of the mef(E) start codon. Consensus pneumococcal promoter -10 (5′-TATACT-3′) and -35 (5′-TTGAAC-3′) boxes separated by a 17 bp spacer were identified 7 bp upstream of the start site. Analysis of the predicted secondary structure of the 327 bp 5’ region identified four pairs of inverted repeats R1-R8 predicted to fold into stem-loops, a small leader peptide (MTASMRLR) required for macrolide induction and a Rho-independent transcription terminator involving the R5/R6 stem loop. Mutational analyses of the regulatory region identified transcriptional attenuation as the model for the inducible expression of macrolide efflux, which was confirmed by RNA-Seq expression data. The 327 bp region 5’ of mef(E) was highly conserved in other mef/mel(msr)-containing genetic elements complexes including Tn1207.1 and the 5612IQ complex in pneumococci and Tn1207.3 in Group A streptococci. Induction of the mef(E)/mel operon and macrolide efflux occurs by anti-attenuation in the presence of inducing macrolides and appears to be a mechanism
Project description:Microarray analysis of Streptococcus pneumoniae TIGR4 transcriptome in response to manganese as the transcriptome changes in response to intracellular manganese accumulation via a mutation in sp1552/mntE a manganese efflux protein. Investigating role of manganese efflux and accumulation in S. pneumoniae: 3 TIGR4 in ThyB vs. TIGR4 in Mn and 3 TIGR4 in Mn vs mntE1 in Mn replicate 3
Project description:Microarray analysis of Streptococcus pneumoniae TIGR4 transcriptome in response to manganese as the transcriptome changes in response to intracellular manganese accumulation via a mutation in sp1552/mntE a manganese efflux protein.
Project description:Comparison of the Streptococcus pneumoniae D39 wild type in M17 medium+ 0.5 % (w/v) Cellobiose (CM17) compared to M17 medium+ 0.5 % (w/v) Glucose (GM17) hervasted at time point T2 The human pathogen Streptococcus pneumoniae has the ability to use the carbon- and energy source cellobiose due to the presence of a cellobiose-utilizing gene cluster (cel locus) in its genome. This system is regulated by the cellobiose-dependent transcriptional activator CelR, which has been previously shown to contribute to pneumococcal virulence. To get a broader understanding of the response of S. pneumoniae to cellobiose, we compared the pneumococcal transcriptome during growth on glucose as the main carbon source to that with cellobiose as the main carbon source. The expression of various carbon metabolic genes was altered, including a PTS operon (which we here denote as the bgu operon) that has high similarity with the cel locus. In contrast to the cel locus, the bgu operon is conserved in all sequenced strains of S. pneumoniae, indicating an important physiological function in the lifestyle of pneumococci. We next characterized the transcriptional regulation of the bgu operon in more detail. Its expression was increased in the presence of cellobiose, and decreased in the presence of glucose. A novel GntR-type transcriptional regulator (which we here denote as BguR) was shown to act as a transcriptional repressor of the bgu operon and its repressive effect was relieved in the presence of cellobiose. BguR-dependent repression was demonstrated to be mediated by a 20-bp DNA operator site (5M-bM-^@M-^Y-AAAAATGTCTAGACAAATTT-3M-bM-^@M-^Y) present in PbguA as verified by promoter truncation experiments. In conclusion, we have identified a new cellobiose-responsive PTS operon, together with its transcriptional regulator in S. pneumoniae. Two condition design, comparison of one strain including a dye swap
Project description:Comparison of the Streptococcus pneumoniae D39 wild type in M17 medium+ 0.5 % (w/v) Cellobiose (CM17) compared to M17 medium+ 0.5 % (w/v) Glucose (GM17) hervasted at time point T1 The human pathogen Streptococcus pneumoniae has the ability to use the carbon- and energy source cellobiose due to the presence of a cellobiose-utilizing gene cluster (cel locus) in its genome. This system is regulated by the cellobiose-dependent transcriptional activator CelR, which has been previously shown to contribute to pneumococcal virulence. To get a broader understanding of the response of S. pneumoniae to cellobiose, we compared the pneumococcal transcriptome during growth on glucose as the main carbon source to that with cellobiose as the main carbon source. The expression of various carbon metabolic genes was altered, including a PTS operon (which we here denote as the bgu operon) that has high similarity with the cel locus. In contrast to the cel locus, the bgu operon is conserved in all sequenced strains of S. pneumoniae, indicating an important physiological function in the lifestyle of pneumococci. We next characterized the transcriptional regulation of the bgu operon in more detail. Its expression was increased in the presence of cellobiose, and decreased in the presence of glucose. A novel GntR-type transcriptional regulator (which we here denote as BguR) was shown to act as a transcriptional repressor of the bgu operon and its repressive effect was relieved in the presence of cellobiose. BguR-dependent repression was demonstrated to be mediated by a 20-bp DNA operator site (5M-bM-^@M-^Y-AAAAATGTCTAGACAAATTT-3M-bM-^@M-^Y) present in PbguA as verified by promoter truncation experiments. In conclusion, we have identified a new cellobiose-responsive PTS operon, together with its transcriptional regulator in S. pneumoniae. Two condition design, comparison of one strain including a dye swap
Project description:Transcriptome comparison of bguR mutant to wild-type in the Streptococcus pneumoniae D39 grown in GM17 The human pathogen Streptococcus pneumoniae has the ability to use the carbon- and energy source cellobiose due to the presence of a cellobiose-utilizing gene cluster (cel locus) in its genome. This system is regulated by the cellobiose-dependent transcriptional activator CelR, which has been previously shown to contribute to pneumococcal virulence. To get a broader understanding of the response of S. pneumoniae to cellobiose, we compared the pneumococcal transcriptome during growth on glucose as the main carbon source to that with cellobiose as the main carbon source. The expression of various carbon metabolic genes was altered, including a PTS operon (which we here denote as the bgu operon) that has high similarity with the cel locus. In contrast to the cel locus, the bgu operon is conserved in all sequenced strains of S. pneumoniae, indicating an important physiological function in the lifestyle of pneumococci. We next characterized the transcriptional regulation of the bgu operon in more detail. Its expression was increased in the presence of cellobiose, and decreased in the presence of glucose. A novel GntR-type transcriptional regulator (which we here denote as BguR) was shown to act as a transcriptional repressor of the bgu operon and its repressive effect was relieved in the presence of cellobiose. BguR-dependent repression was demonstrated to be mediated by a 20-bp DNA operator site (5M-bM-^@M-^Y-AAAAATGTCTAGACAAATTT-3M-bM-^@M-^Y) present in PbguA as verified by promoter truncation experiments. In conclusion, we have identified a new cellobiose-responsive PTS operon, together with its transcriptional regulator in S. pneumoniae. One condition design, comparison of two strains including a dye swap
Project description:In this study, we explore the impact of fucose on the transcriptome of S. pneumoniae D39. The expression of various genes and operons, including the fucose uptake PTS and utilization operon (fcs operon), was altered in the presence of fucose. By means of quantitative RT-PCR and β-galactosidase analysis, we demonstrate the role of the transcriptional regulator FcsR, present upstream of the fcs operon, as a transcriptional activator of the fcs operon. We also predict a 19-bp putative FcsR regulatory site (5’-ATTTGAACATTATTCAAGT-3’) in the promoter region of the fcs operon. The functionality of this predicted FcsR regulatory site was further confirmed by promoter-truncation experiments, where deletion of half of the FscR regulatory site or full deletion led to the abolition of expression of the fcs operon. Comparison of the Streptococcus pneumoniae D39 wild-type in M17 plus 0.5% glucose (GM17) and M17 plus 0.5% fucose (FM17) Two condition design comparison of Wild-type strain including a dye swap
Project description:The polyamine transport operon in Streptococcus pneumoniae TIGR4 is necessary for survival in murine models of pneumococcal pneumonia. To date, there is no description of polyamine transport dependent pneumococcal gene expression. In this study, we compared gene expression between the wild-type and transport deficient (potABCD) TIGR4 by RNA-Seq analysis.
Project description:In this study, we have explored the impact of ascorbic acid on the transcriptome of Streptococcus pneumoniae D39. The expression of several genes and operons, including the ula operon (which has been previously shown to be involved in ascorbic acid utilization), the AdcR regulon (which has been previously shown to be involved in zinc transport and virulence) and a PTS operon (which we denote here as ula2 operon) were altered in the presence of ascorbic acid. The ula2 operon consists of five genes, including the transcriptional activator ulaR2. Our M-NM-2-galactosidase assay data and transcriptome comparison of the ulaR2 mutant with the wild-type demonstrated that the transcriptional activator UlaR2 in the presence of ascorbic acid activates the expression of the ula2 operon. We further predict a 16-bp regulatory site (5M-bM-^@M-^Y-ATATTGTGCTCAAATA-3M-bM-^@M-^Y) for UlaR2 binding in the Pula2. Furthermore, we have explored the effect of ascorbic acid on the expression of the AdcR regulon. Our ICP-MS analysis showed that addition of ascorbic acid to the medium causes zinc starvation in the cell that leads to the activation of the AdcR regulon. Comparison of the Streptococcus pneumoniae D39 compared to D39 wild type in M17 medium+ 10mM ascorbic acid (AM17) Two conditions design comparision of one strain including a dye swap