ABSTRACT: We mapped the genome-wide binding of C-terminally FLAG-tagged AraC in S. enterica subsp. enterica serovar Typhimurium strain 14028s using ChIP coupled with deep sequencing (ChIP-seq). We identified five putative target loci for AraC: upstream of araB/araC, araE, araJ, STM14_0178, and within sseD.
Project description:We mapped the genome-wide binding of C-terminally FLAG-tagged AraC in S. enterica subsp. enterica serovar Typhimurium strain 14028s using ChIP coupled with deep sequencing (ChIP-seq). We identified five putative target loci for AraC: upstream of araB/araC, araE, araJ, STM14_0178, and within sseD.
Project description:Escherichia coli AraC is a well-described transcription activator of genes involved in arabinose metabolism. Using complementary genomic approaches, chromatin immunoprecipitation (ChIP)-chip, and transcription profiling, we identify direct regulatory targets of AraC, including five novel target genes: ytfQ, ydeN, ydeM, ygeA, and polB. Strikingly, only ytfQ has an established connection to arabinose metabolism, suggesting that AraC has a broader function than previously described. We demonstrate arabinose-dependent repression of ydeNM by AraC, in contrast to the well-described arabinose-dependent activation of other target genes. We also demonstrate unexpected read-through of transcription at the Rho-independent terminators downstream of araD and araE, leading to significant increases in the expression of polB and ygeA, respectively. AraC is highly conserved in the related species Salmonella enterica. We use ChIP sequencing (ChIP-seq) and RNA sequencing (RNA-seq) to map the AraC regulon in S. enterica. A comparison of the E. coli and S. enterica AraC regulons, coupled with a bioinformatic analysis of other related species, reveals a conserved regulatory network across the family Enterobacteriaceae comprised of 10 genes associated with arabinose transport and metabolism.
Project description:We used RNA-seq to determine the effects of AraC and arabinose on RNA levels genome-wide in S. enterica. Wild-type or delta araC mutant cells were grown in both the presence and absence of 0.2% L-arabinose. RNA-Seq libraries from two independent biological replicate samples were sequenced for (i) wild-type cells with no treatment, (ii) wild-type cells treated with arabinose, (iii) delta araC cells with no treatment, (iv) delta araC cells treated with arabinose. Comparisons were made between (i) and (ii) [analysis file name: Salmonella RNA-seq processed WT without arabinose vs WT with arabinose.xls], between (i) and (iii) [analysis file name: Salmonella RNA-Seq proessed no arabinose WT vs no arabinose delta araC.xls], and between (ii) and (iv) [analysis file name: Salmonella RNA-Seq processed arabinose WT vs arabinose delta araC.xls]. All analysis files are available from https://www.ebi.ac.uk/arrayexpress/files/E-MTAB-1901/E-MTAB-1901.additional.1.zip
Project description:HilD is a regulator of Salmonella pathogenicity island 1 (SPI-1) virulence genes in Salmonella enterica serovar Typhimurium. To identify novel HilD-regulated genes, we mapped the genome-wide association of HilD in S. Typhimurium under SPI-1-inducing conditions (high salt, low aeration) using ChIP-seq. HilD was C-terminally tagged with 3 FLAG tags in strain 14028s.
Project description:The Bacillus subtilis araC locus, mapped at about 294 degrees on the genetic map, was defined by mutations conferring an Ara- phenotype to strains bearing the metabolic araA, araB, and araD wild-type alleles (located at about 256 degrees on the genetic map) and by mutants showing constitutive expression of the three genes. In previous work, it has been postulated that the gene in which these mutations lie exerts its effect on the ara metabolic operon in trans, and this locus was named araC by analogy to the Escherichia coli regulatory gene. Here, we report the cloning and sequencing of the araC locus. This region comprises two open reading frames with divergently arranged promoters, the regulatory gene, araC, encoding a 41-kDa polypeptide, and a partially cloned gene, termed araE, which most probably codes for a permease involved in the transport of L-arabinose. The DNA sequence of araC revealed that its putative product is very similar to a number of bacterial negative regulators (the GalR-LacI family). However, a helix-turn-helix motif was identified in the N-terminal region by its identity to the consensus signature sequence of another group of repressors, the GntR family. The lack of similarity between the predicted primary structure of the product encoded by the B. subtilis regulatory gene and the AraC regulator from E. coli and the apparently different modes of action of these two proteins lead us to propose a new name, araR, for this gene. The araR gene is monocistronic, and the promoter region contains -10 and -35 regions (as determined by primer extension analysis) similar to those recognized by RNA polymerase containing the major vegetative cell sigma factor sigmaA. An insertion-deletion mutation in the araR gene leads to constitutive expression of the L-arabinose metabolic operon. We demonstrate that the araR gene codes for a negative regulator of the ara operon and that the expression of araR is repressed by its own product.
Project description:Salmonellosis outbreaks associated with sprouted legumes have been a food safety concern for over two decades. Despite evidence that Salmonella enterica triggers biotic plant defense pathways, it has remained unclear how plant defenses impact Salmonella growth on sprouted legumes. We used Medicago truncatula mutants in which the gene for the flagellin receptor FLS2 was disrupted to demonstrate that plant defenses triggered by FLS2 elicitation do not impact the growth of Salmonella enterica serovar Typhimurium ATCC 14028S. As a control, we tested the growth of Salmonella enterica serovar Typhimurium LT2, which has a defect in rpoS that increases its sensitivity to reactive oxygen species. LT2 displayed enhanced growth on M. truncatula FLS2 mutants in comparison to wild-type M. truncatula. We hypothesize that these growth differences are primarily due to differences in 14028S and LT2 reactive oxygen species sensitivity. Results from this study show that FLS2-mediated plant defenses are ineffective in inhibiting growth of Salmonella entrica 14028S.