Genome-Scale Analyses of Escherichia coli and Salmonella enterica AraC Reveal Noncanonical Targets and an Expanded Core Regulon

Abstract

c 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.

Self-regulating genomic island encoding tandem regulators confers chromatic acclimation to marine Synechococcus

“…3D) appears to be quite different from other AraC-class family members. Recent RNA-Seq studies of araC mutants in Escherichia coli and Salmonella demonstrate that AraC has a much broader regulon than previously known (27), and other AraC family members also control large regulons in a diverse set of bacteria (28,29). The small and highly specific FciA/FciB regulon is also different from the widespread responses in gene expression that have been observed for other forms of chromatic acclimation (13,30).…”

Defining bacterial regulons using ChIP-seq

“…Many successful ChIP-seq studies have used affinity-tagged versions of the TF of interest, often using triple FLAG affinity tags [4,5,7,8,12,15,18,21-25] or myc tags [14]. Using an affinity-tagged protein allows the use of a commercial antibody that likely has little cross-reactivity with other proteins from the bacterium under study and negates the requirement of an antibody specific to the TF of interest.…”

“…Chromatin immunoprecipitation (ChIP) followed by microarray analysis (ChIP-chip) or, more recently, high-throughput sequencing (ChIP-seq), which has a much higher resolution and signal-to-noise ratio than ChIP-chip, has been used to map genome-wide binding of many bacterial TFs [2-25]. While many of the studies have focused on TFs in Escherichia coli , recent studies have examined transcription factors in many diverse bacterial species, such as Vibrio cholerae, Vibrio harveyi, Rhodobacter sphaeroides, Salmonella enterica, Mycobacterium tuberculosis, and Caulobacter crescentus [2-25].…”

“…While many of the studies have focused on TFs in Escherichia coli , recent studies have examined transcription factors in many diverse bacterial species, such as Vibrio cholerae, Vibrio harveyi, Rhodobacter sphaeroides, Salmonella enterica, Mycobacterium tuberculosis, and Caulobacter crescentus [2-25]. A subset of these studies also correlated occupancy data with expression data to investigate the regulons of certain TFs [2,3,5,7,8,13,16,21,22,25], and we predict the desire to use these assays to study other TFs will increase as the costs of the performing ChIP-seq decrease. Here, we provide an overview to using ChIP-seq to identify bacterial regulons, from sample preparation, data generation, data visualization, data analysis, and downstream bioinformatic and computational analyses.…”

“…ChIP-exo has shown promise in the few bacterial studies where it has been used [26-29] but the analysis of ChIP-exo data has its own unique issues including sample preparation and data analysis. We encourage those interested in performing ChIP-seq experiments or variations on ChIP-seq to review other studies [2-25] if they want a broader sense of the experimental and analytical variables.…”