Transcriptome Sequencing Data of Bacillus anthracis Vollum ΔhtrA and Its Parental Strain, Isolated under Oxidative Stress

ANNOUNCEMENT

The Gram-positive spore-forming obligate pathogen Bacillus anthracis is the etiological cause of anthrax. The lethality of B. anthracis is attributed to its exotoxins and its optimal adaptation to tolerate stress constraints encountered in the course of infection (1). Proteomic/serologic surveys of B. anthracis (1–3) showed that the secreted protease/chaperone high-temperature requirement (HtrA) (involved in protein synthesis quality control and necessary for tolerance to heat, oxidative, and other stress regimens) belongs to a class of immunogenic vaccine and disease biomarker candidates (4). Virulence-attenuating htrA gene disruption was implemented for the development of a live spore vaccine (5–7). Recently, we suggested that HtrA acts as both a protease/chaperone and a pleiotropic factor of gene expression (8). Here, we present transcriptome sequencing (RNA-seq) data sets describing the effect of htrA gene disruption on the global gene expression of B. anthracis in the presence/absence of H₂O₂.

B. anthracis parental strain ΔVollum (acapsular and nontoxinogenic, referred to in this report as wild type [WT]) and an htrA-disrupted strain, in biological triplicate or duplicate cultures (14 sets of data, as detailed in Table 1), were grown in brain heart infusion broth at 37°C to mid-log phase and split into twin cultures in the presence or absence of 3 mM H₂O₂. Cells were collected from the initial culture before treatment (Table 1, samples 1 and 2) and 10 min after treatment (Table 1, without H₂O₂, samples 3 and 5; with H₂O₂, samples 4 and 6). Total RNA was extracted using the RNeasy kit (Qiagen), and residual DNA was digested using RNase-free DNase (Qiagen). RNA-seq was performed in-house (IIBR, Ness Zionna, Israel). Libraries were generated using the TruSeq RNA library prep kit version 2 (Illumina), assessed for correct sizing using a high-sensitivity Bioanalyzer DNA chip (Agilent), quantified by quantitative PCR (qPCR), and normalized to 2 nM. Pooling and clustering of libraries were performed using the Illumina cBot system; 35-bp single-end sequencing was performed on the Illumina Genome Analyzer IIx system with TruSeq sequencing-by-synthesis (SBS) kit version 2 reagents. FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc) was used for quality control of the data. Reads were mapped to the B. anthracis Ames Ancestor reference genome (GenBank accession number NC_007530) using Novoalign, version 3.02.07. The HTSeq software (9), version 0.6, was used to quantify the number of reads mapped to each gene. Sequencing yielded 4.3 million to 11.9 million reads (Table 1) with a mapping percentage that ranged from 89.3% to 99.5%. An analysis of differentially expressed genes under various conditions was performed using the R package DESeq, version 1.16.0 (10). All analytical software programs were used at their respective default settings.

The analysis revealed the following categories of H₂O₂-modulated genes: (i) induced upon treatment in both strains (792 genes), (ii) repressed in both strains (868 genes), (iii) uniquely upregulated in the WT strain (271 genes), (iv) uniquely downregulated in the WT strain (221), (v) uniquely upregulated in the mutant (330 genes), and (vi) uniquely downregulated in the mutant (648 genes). Further inspection of these classes of genes will enable a better understanding of the response of B. anthracis to oxidative stress in general and the regulatory role of the protein HtrA in particular. Furthermore, this database may facilitate identification of proteins for the future development of countermeasures against B. anthracis.