Interference of PTS components with PrfA of L.monocytogenes
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ABSTRACT:
ORGANISM(S): Listeria monocytogenes
PROVIDER: GSE6028 | GEO | 2007/02/01
SECONDARY ACCESSION(S): PRJNA97841
REPOSITORIES: GEO
Similar Datasets
Project description:PrfA activity was studied in L. monocytogenes strain EGD and in an isogenic prfA deletion mutant (EGDΔprfA) carrying multiple copies of the wild-type prfA or the mutant prfA* gene (strains EGDΔprfApPrfA and EGDΔprfApPrfA*) after growth in brain heart infusion (BHI), Luria-Bertani broth (LB) or a defined minimal medium (MM) supplemented either with one of the three PTS-carbohydrates, glucose, mannose and cellobiose, or the non-PTS carbon source glycerol. Low PrfA activity was observed in the wild-type EGD strain in BHI and LB with either of these carbon sources, while PrfA activity was high in minimal medium in presence of glycerol but significantly reduced in presence of cellobiose. The strains expressing the prfA and prfA* gene under the prfA promoters, P1 and P2, produced equally large amounts of PrfA protein and high PrfA activity was observed in strain EGDΔprfApPrfA* under all growth conditions. In contrast, high PrfA activity in strain EGDΔprfApPrfA was only observed when this strain was cultured in BHI but not in LB or MM (in presence of either carbon source). A ptsH mutant (lacking a functional HPr) was able to grow in BHI suggesting that growth of L. monocytogenes in this culture medium is supported by carbon sources whose uptake and metabolism are independent of the PTS pathway. However, this mutant was unable to grow in LB and MM regardless which of the four carbon sources was added, suggesting that uptake of the used carbohydrates and the catabolism of glycerol depend fully on the functional common PTS pathway. Furthermore, the growth rates of L. monocytogenes are strongly reduced in presence of large amounts of PrfA protein when growing MM but less in LB and only slightly in BHI. The expression profiles of the genes encoding PTS permeases were determined in the three strains under various growth conditions. The data suggest that PrfA activity correlates with the expression level and the phosphorylation state of specific PTS permeases. This SuperSeries is composed of the following subset Series: GSE12143: Listeria monocytogenes EGD after growth BHI vs. LB vs. MM GSE12145: Listeria monocytogenes EGDΔprfApPrfA and EGDΔprfApPrfA* compared to the wild type strain EGD GSE12146: Listeria monocytogenes EGD and EGD-e
2008-11-30 | E-GEOD-12151 | biostudies-arrayexpress
Project description:Listeria monocytogenes is able to efficiently utilize glycerol as carbon source. In a defined minimal medium the growth rate is similar (during balanced growth) in presence of glycerol as in presence of glucose or cellobiose. Comparative transcriptome analyses of L. monocytogenes showed in the presence of glycerol (compared to glucose and/or cellobiose) high transcriptional upregulation of the known genes involved in glycerol uptake and metabolism (glpFK, glpD). Expression of the genes encoding a second putative glycerol uptake facilitator (GlpF-2) and a second putative glycerol kinase (GlpK-2) was less enhanced under these conditions. GlpK-1 but not GlpK-2 was essential for glycerol catabolism in L. monocytogenes under extracellular conditions, while loss of GlpK-1 affected replication in Caco-2 cells less than loss of GlpK-2 and GlpD. Additional genes whose transcription was higher in presence of glycerol than in presence of glucose and cellobiose included those for two dihydroxyacetone (Dha) kinases and many genes that are under carbon catabolite repression (CCR) control. Transcriptional down-regulation in the presence of glycerol (compared to glucose and cellobiose) was observed for several genes and operons that are positively regulated by glucose, including genes involved in glycolysis, N-metabolism and biosynthesis of branched chain amino acids. The highest transcriptional up-regulation was observed for all PrfA-dependent genes during early and late logarithmic growth in glycerol. Under these conditions a low level of HPr-Ser-P and a high level of HPr-His-P was present in the cells, suggesting that all EIIA (B) components of the PTS permeases expressed will be phosphorylated. These and other data reported suggest that the phosphorylation state of PTS permeases correlates with PrfA activity. Keywords: Response of Listeria monocytogenes to different carbon sources
2008-05-30 | GSE11459 | GEO
Project description:PrfA activity was studied in L. monocytogenes strain EGD and in an isogenic prfA deletion mutant (EGDΔprfA) carrying multiple copies of the wild-type prfA or the mutant prfA* gene (strains EGDΔprfApPrfA and EGDΔprfApPrfA*) after growth in brain heart infusion (BHI), Luria-Bertani broth (LB) or a defined minimal medium (MM) supplemented either with one of the three PTS-carbohydrates, glucose, mannose and cellobiose, or the non-PTS carbon source glycerol. Low PrfA activity was observed in the wild-type EGD strain in BHI and LB with either of these carbon sources, while PrfA activity was high in minimal medium in presence of glycerol but significantly reduced in presence of cellobiose. The strains expressing the prfA and prfA* gene under the prfA promoters, P1 and P2, produced equally large amounts of PrfA protein and high PrfA activity was observed in strain EGDΔprfApPrfA* under all growth conditions. In contrast, high PrfA activity in strain EGDΔprfApPrfA was only observed when this strain was cultured in BHI but not in LB or MM (in presence of either carbon source). A ptsH mutant (lacking a functional HPr) was able to grow in BHI suggesting that growth of L. monocytogenes in this culture medium is supported by carbon sources whose uptake and metabolism are independent of the PTS pathway. However, this mutant was unable to grow in LB and MM regardless which of the four carbon sources was added, suggesting that uptake of the used carbohydrates and the catabolism of glycerol depend fully on the functional common PTS pathway. Furthermore, the growth rates of L. monocytogenes are strongly reduced in presence of large amounts of PrfA protein when growing MM but less in LB and only slightly in BHI. The expression profiles of the genes encoding PTS permeases were determined in the three strains under various growth conditions. The data suggest that PrfA activity correlates with the expression level and the phosphorylation state of specific PTS permeases. This SuperSeries is composed of the SubSeries listed below.
2008-12-01 | GSE12151 | GEO
Project description:Listeria monocytogenes is able to efficiently utilize glycerol as carbon source. In a defined minimal medium the growth rate is similar (during balanced growth) in presence of glycerol as in presence of glucose or cellobiose. Comparative transcriptome analyses of L. monocytogenes showed in the presence of glycerol (compared to glucose and/or cellobiose) high transcriptional upregulation of the known genes involved in glycerol uptake and metabolism (glpFK, glpD). Expression of the genes encoding a second putative glycerol uptake facilitator (GlpF-2) and a second putative glycerol kinase (GlpK-2) was less enhanced under these conditions. GlpK-1 but not GlpK-2 was essential for glycerol catabolism in L. monocytogenes under extracellular conditions, while loss of GlpK-1 affected replication in Caco-2 cells less than loss of GlpK-2 and GlpD. Additional genes whose transcription was higher in presence of glycerol than in presence of glucose and cellobiose included those for two dihydroxyacetone (Dha) kinases and many genes that are under carbon catabolite repression (CCR) control. Transcriptional down-regulation in the presence of glycerol (compared to glucose and cellobiose) was observed for several genes and operons that are positively regulated by glucose, including genes involved in glycolysis, N-metabolism and biosynthesis of branched chain amino acids. The highest transcriptional up-regulation was observed for all PrfA-dependent genes during early and late logarithmic growth in glycerol. Under these conditions a low level of HPr-Ser-P and a high level of HPr-His-P was present in the cells, suggesting that all EIIA (B) components of the PTS permeases expressed will be phosphorylated. These and other data reported suggest that the phosphorylation state of PTS permeases correlates with PrfA activity. Keywords: Response of Listeria monocytogenes to different carbon sources A total of four independently isolated RNA samples from each condition at each growth phase were used for the analysis. RNA from two isolations were pooled and hybridized onto two microarray slides with dye swap. Another two microarray slides were hybridized using the same principle. In total, we used four RNAs and four microarray slides to generate 16 replicate expression values for each combination except for the comparison between glucose and cellobiose, phase B where data generated from three microarray slides were used for further analysis
2008-05-30 | E-GEOD-11459 | biostudies-arrayexpress
Project description:Identification of the major PEP-phosphotransferase systems (PTS) for glucose, mannose and cellobiose of Listeria monocytogenes, and their significance for extra- and intracellular growth. Transcriptional profile of prfA, the dependent genes and the PTS genes after growth in MM (minimal medium (Premaratne et al. 2001)) (supplemented with 10 mM Glucose). Listeria monocytogenes EGD-e possesses 86 pts genes encoding 29 complete and several incomplete PEP-dependent phosphotransferase systems (PTS) for the transport of carbohydrates and sugar alcohols. By a systematic deletion analysis we identified the major PTS involved in glucose, mannose and cellobiose transport, when L. monocytogenes grows in a defined minimal medium in the presence of either of these carbohydrates. Under these conditions two of the four PTSMan and at least one of the five PTSGlc function as glucose transporter with different affinities. Cellobiose is transported (with different efficiencies) mainly by two of the six PTSLac and by a PTSGlc that also transports glucose. One of the PTSMan and both PTSLac are regulated by LevR-homologous PRD containing transcriptional activators, detected with PRD deletion mutants. The growth rates of mutants that are lacking these PTS are drastically reduced compared to the wild-type strain upon growth in a defined medium with low concentrations of glucose and cellobiose, respectively. In contrast, replication of these PTS mutants within epithelial cells or macrophages is as efficient as that of the wild-type strain. Keywords: pts and prd deletion mutants A total of three independently isolated RNA samples from each condition were used for the analysis. PTS Samples: GSM458191-GSM458208 PRD Samples: GSM458209-GSM458214
2010-05-19 | E-GEOD-18363 | biostudies-arrayexpress
Project description:Identification of the major PEP-phosphotransferase systems (PTS) for glucose, mannose and cellobiose of Listeria monocytogenes, and their significance for extra- and intracellular growth. Transcriptional profile of prfA, the dependent genes and the PTS genes after growth in MM (minimal medium (Premaratne et al. 2001)) (supplemented with 10 mM Glucose). Listeria monocytogenes EGD-e possesses 86 pts genes encoding 29 complete and several incomplete PEP-dependent phosphotransferase systems (PTS) for the transport of carbohydrates and sugar alcohols. By a systematic deletion analysis we identified the major PTS involved in glucose, mannose and cellobiose transport, when L. monocytogenes grows in a defined minimal medium in the presence of either of these carbohydrates. Under these conditions two of the four PTSMan and at least one of the five PTSGlc function as glucose transporter with different affinities. Cellobiose is transported (with different efficiencies) mainly by two of the six PTSLac and by a PTSGlc that also transports glucose. One of the PTSMan and both PTSLac are regulated by LevR-homologous PRD containing transcriptional activators, detected with PRD deletion mutants. The growth rates of mutants that are lacking these PTS are drastically reduced compared to the wild-type strain upon growth in a defined medium with low concentrations of glucose and cellobiose, respectively. In contrast, replication of these PTS mutants within epithelial cells or macrophages is as efficient as that of the wild-type strain. Keywords: pts and prd deletion mutants
2009-12-01 | GSE18363 | GEO
Project description:In Bacillus subtilis and its relatives carbon catabolite control, a mechanism enabling to reach maximal efficiency of carbon and energy sources metabolism, is achieved by the global regulator CcpA (carbon catabolite protein A). CcpA in a complex with HPr-Ser-P (seryl-phosphorylated form of histidine-containing protein, HPr) binds to operator sites called catabolite responsive elements, cre. Depending on the cre box position relative to the promoter, the CcpA/HPr-Ser-P complex can either act as a positive or a negative regulator. The cre boxes are highly degenerate semi-palindromes with a lowly conserved consensus sequence. So far, studies aimed at revealing how CcpA can bind such diverse sites were focused on the analysis of single cre boxes. In this study, a genome-wide analysis of cre sites was performed in order to identify differences in cre sequence and position, which determine their binding affinity. The transcriptomes of B. subtilis cultures with three different CcpA expression levels were compared. The higher the amount of CcpA in the cells, the more operons possessing cre sites were differentially regulated. The cre boxes that mediated regulation at low CcpA levels were designated as strong (high affinity) and those which responded only to high amounts of CcpA, as weak (low affinity). Differences in the sequence and position in relation to the transcription start site between strong and weak cre boxes were revealed. Certain residues at specific positions in the cre box as well as, to a certain extent, a more palindromic nature of cre sequences and the location of cre in close vicinity to the transcription start site contribute to the strength of CcpA-dependent regulation. The main factors contributing to cre regulatory efficiencies, enabling subtle differential control of various subregulons of the CcpA regulon, are identified.
2012-08-14 | GSE35154 | GEO
Project description:In Bacillus subtilis and its relatives carbon catabolite control, a mechanism enabling to reach maximal efficiency of carbon and energy sources metabolism, is achieved by the global regulator CcpA (carbon catabolite protein A). CcpA in a complex with HPr-Ser-P (seryl-phosphorylated form of histidine-containing protein, HPr) binds to operator sites called catabolite responsive elements, cre. Depending on the cre box position relative to the promoter, the CcpA/HPr-Ser-P complex can either act as a positive or a negative regulator. The cre boxes are highly degenerate semi-palindromes with a lowly conserved consensus sequence. So far, studies aimed at revealing how CcpA can bind such diverse sites were focused on the analysis of single cre boxes. In this study, a genome-wide analysis of cre sites was performed in order to identify differences in cre sequence and position, which determine their binding affinity. The transcriptomes of B. subtilis cultures with three different CcpA expression levels were compared. The higher the amount of CcpA in the cells, the more operons possessing cre sites were differentially regulated. The cre boxes that mediated regulation at low CcpA levels were designated as strong (high affinity) and those which responded only to high amounts of CcpA, as weak (low affinity). Differences in the sequence and position in relation to the transcription start site between strong and weak cre boxes were revealed. Certain residues at specific positions in the cre box as well as, to a certain extent, a more palindromic nature of cre sequences and the location of cre in close vicinity to the transcription start site contribute to the strength of CcpA-dependent regulation. The main factors contributing to cre regulatory efficiencies, enabling subtle differential control of various subregulons of the CcpA regulon, are identified. Bacillus subtilis strain MP902 [strain MP901 (Ptet-ccpA, KmR) carrying pWH119 (Pxyl-tetR, EmR)] was grown in presence of three different concentrations of Ptet inducer, anhydrotetracycline (ATc), leading to three levels of ccpA expression induction. The control culture was grown in absence of ATc. The total RNA for transcriptome analyses was isolated at OD600 = 0.8 from 16 ml culture. Three independent cultures of each strain (target strains and controls) were used, and cells were sampled for microarray experiment.
2012-08-13 | E-GEOD-35154 | biostudies-arrayexpress
Project description:Listeria monocytogenes SigB and PrfA are pleiotropic regulators of stress response and virulence gene expression, which have been shown to co-regulate genes in L. monocytogenes. We performed whole genome transcriptional profiling in the presence of PrfA* and active SigB, to identify the overlaps between the PrfA virulence regulon and the SigB stress response regulon. In L. monocytogenes, the PrfA* allele contributes to the activation of virulence genes to a level comparable to that of intracellular growing L. monocytogenes. Our results showed that the core PrfA regulon consists of 12 genes previously described as PrfA regulated. Furthermore, we found that the role of SigB during virulence gene regulation changes, dependent on the presence or absence of PrfA*. In the absence of PrfA*, SigB activated the transcription of virulence genes such as inlA and inlB. In the presence of PrfA*, SigB negatively influenced the transcription of genes in the PrfA core regulon. The observed effect of SigB on the transcript level of PrfA regulated genes was shown to reduce the cytotoxic effect of the PrfA* allele in HepG-2 cells. Our results indicate that the SigB-PrfA regulatory network is important for the adjustment of virulence gene transcription to ensure L. monocytogenes success as an intracellular pathogen. Keywords: comparison of gene expression of regulatory mutants
2008-10-01 | GSE11347 | GEO
Project description:Listeria monocytogenes SigB and PrfA are pleiotropic regulators of stress response and virulence gene expression, which have been shown to co-regulate genes in L. monocytogenes. We performed whole genome transcriptional profiling in the presence of PrfA* and active SigB, to identify the overlaps between the PrfA virulence regulon and the SigB stress response regulon. In L. monocytogenes, the PrfA* allele contributes to the activation of virulence genes to a level comparable to that of intracellular growing L. monocytogenes. Our results showed that the core PrfA regulon consists of 12 genes previously described as PrfA regulated. Furthermore, we found that the role of SigB during virulence gene regulation changes, dependent on the presence or absence of PrfA*. In the absence of PrfA*, SigB activated the transcription of virulence genes such as inlA and inlB. In the presence of PrfA*, SigB negatively influenced the transcription of genes in the PrfA core regulon. The observed effect of SigB on the transcript level of PrfA regulated genes was shown to reduce the cytotoxic effect of the PrfA* allele in HepG-2 cells. Our results indicate that the SigB-PrfA regulatory network is important for the adjustment of virulence gene transcription to ensure L. monocytogenes success as an intracellular pathogen. Keywords: comparison of gene expression of regulatory mutants The experimental design included 4 mutant strains of L. monocytogenes 10403S (PrfA*, delta prfA, delta prfA delta sigB, and PrfA* delta sigB), of which cDNA generated from 4 biological replicates were hybridized in all possible pairwise comparisons. Data were analyzed using a one way ANOVA in R/MAANOVA to determine significant differences in gene expression among the different strains. A two way ANOVA implemented in R/MAANOVA determined significant differences in gene expression due to the presence or absence of SigB and PrfA.
2010-05-19 | E-GEOD-11347 | biostudies-arrayexpress