Project description:To establish the SinR regulon in B. anthracis, gene expression of the fully virulent Ames strain and the isogenic sinR-null strains were compared using expression microarray analysis.
Project description:Purpose: Small regulatory RNAs (sRNAs) are short transcripts that base-pair to mRNA targets or interact with regulatory proteins. sRNA function has been extensively studied in Gram-negative bacteria; comparatively less is known about sRNAs in Firmicutes. Here we investigate two sRNAs encoded by the virulence plasmid pXO1 of Bacillus anthracis, the causative agent of anthrax. We designated the sRNAs as as “XrrA” and “XrrB” (for pXO1-encoded regulatory RNA). We constructed deletion mutants ∆xrrA, ∆xrrB, and ∆xrrA∆xrrB in an Ames strain background and compared gene expression of these mutants to that of the Ames-derived parent strain using RNA-seq. Methods: Total RNA from Ames strains UTA37 (Parent), UTA38 (∆xrrB), UTA39 (∆xrrA), UTA41 (∆xrrA∆xrrB) was isolated from cultures grown in CA-CO2 in triplicate, using saturated phenol: chloroform extraction. One microgram of pure and quality-checked RNA from each sample was subjected to Illumina sequencing by synthesis using a NextSeq550 sequencer. Sequencing by synthesis was performed to generate 75bp paired-end reads. Two 130M-read sequencing runs (Run1 and Run2) were performed and an average of 31M reads per sample were obtained. Results: All bioinformatic analysis was performed using the publicly available Galaxy web resource (https://usegalaxy.org/). Comparison of the transcriptomes of a virulent Ames-derived strain to isogenic sRNA-null mutants revealed multiple 4.0- to >100-fold differences in gene expression. Deletion of xrrA resulted in 50 transcripts showing a ≥ 4.0-fold change in expression compared to the parent strain. In contrast, deletion of xrrB led to one transcript having a ≥ 4.0-fold change in expression compared to the parent strain. In the ∆xrrA∆xrrB mutant, levels of 116 transcripts were affected with a fold-change of ≥ 4.0. Many sRNA-regulated targets were chromosome genes associated with branched-chain amino acid metabolism, proteolysis, and transmembrane transport. Conclusions: In this work, we provide experimental evidence for sRNA-mediated regulation in the mammalian pathogen B. anthracis. XrrA and XrrB are the first regulatory RNAs examined in B. anthracis and are shown here to have multiple effects on gene expression in this pathogen. The data suggest distinct regulatory roles for XrrA, as well as some functional overlap between XrrA and XrrB.
Project description:Investigation of whole genome expression level changes in Bacillus anthracis Sterne deltaClpX mutant compared to the wild-type strain after growth in nutrient rich media. The deltaClpX mutant used in this study is described in McGillivray et al. 2009. ClpX Protease Contributes to Antimicrobial Peptide Resistance and Virulence Phenotypes of Bacillus anthracis. Journal of Innate Immunity 1(5): 494-506.
Project description:AtxA, the master virulence regulator of Bacillus anthracis, regulates the expression of three toxins that are required for the pathogenicity of Bacillus anthracis. Recent transcriptome analyses also showed that AtxA affects a large number of genes on both chromosome and plasmid, suggesting its role as a global regulator. Its mechanism of gene regulation nor binding target in vivo was, however, not well understood. In this work, we conducted ChIP-seq for cataloging binding sites of AtxA in vivo and Cappable-seq for catalogging the transcription start sites on the B. anthracis genome. For detected regulons, single knockout strains were constructed and RNA-seq was conducted for each strain.
Project description:To establish the SinR regulon in B. anthracis, gene expression of the fully virulent Ames strain and the isogenic sinR-null strains were compared using expression microarray analysis. Cultures of the two B. anthracis strains tested were grown in triplicate in LB broth to mid-exponential phase (O.D.600 = 0.5-0.6) or early stationary phase of growth (O.D.600 = 3.5-3.9). Cells were pelleted through centrifugation and frozen at -80ºC. Total RNA was isolated using a hot phenol method, converted to cDNA, fragmented, labeled, and hybridized to our Affymetrix microarray. Estimates of gene expression were calculated using GCOS software v1.4.
Project description:In this study we focused on understanding the effect of over expression of recombinant protective antigen (rPA) on the Bacillus anthracis metabolism and genes expression with the purpose of improving the production of this recombinant toxin in particular and recombinant proteins in general. To achieve this goal controlled growth of the B. anthracis ames strain transformed with pYS5 (PA producer) plasmid encoding protective antigen and the corresponding empty vector pSW4 (non-producer) were performed and protein production, bacterial growth characteristics and gene transcription pattern were analyzed. Controlled condition batch runs were performed for both strains and samples from early log (OD 3), log (OD 10) and late log (OD 16) phase were used for gene expression profiling. We found that most of the genes belonging to transport, TCA, glycolysis, PPP and amino acid biosynthesis were up-regulated in the lag phase samples which help the cell coping with the increased requirements of nutrient and energy for rPA expression. These pathways are down-regulated in the log and latelog phase as cellular stress response onsets, which is reflected in the decreased specific growth rate.