Comparative Transcriptional Profiling of Bacillus cereus Sensu Lato Strains during Growth in CO2-Bicarbonate and Aerobic Atmospheres

Bacillus species are spore-forming bacteria that are ubiquitous in the environment and display a range of virulent and avirulent phenotypes. This range is particularly evident in the Bacillus cereus sensu lato group; where closely related strains cause anthrax, food-borne illnesses, and pneumonia, but can also be non-pathogenic. Although much of this phenotypic range can be attributed to the presence or absence of a few key virulence factors, there are other virulence-associated loci that are conserved throughout the B. cereus group, and we hypothesized that these genes may be regulated differently in pathogenic and non-pathogenic strains.Here we report transcriptional profiles of three closely related but phenotypically unique members of the Bacillus cereus group--a pneumonia-causing B. cereus strain (G9241), an attenuated strain of B. anthracis (Sterne 34F(2)), and an avirulent B. cereus strain (10987)--during exponential growth in two distinct atmospheric environments: 14% CO(2)/bicarbonate and ambient air. We show that the disease-causing Bacillus strains undergo more distinctive transcriptional changes between the two environments, and that the expression of plasmid-encoded virulence genes was increased exclusively in the CO(2) environment. We observed a core of conserved metabolic genes that were differentially expressed in all three strains in both conditions. Additionally, the expression profiles of putative virulence genes in G9241 suggest that this strain, unlike Bacillus anthracis, may regulate gene expression with both PlcR and AtxA transcriptional regulators, each acting in a different environment.We have shown that homologous and even identical genes within the genomes of three closely related members of the B. cereus sensu lato group are in some instances regulated very differently, and that these differences can have important implications for virulence. This study provides insights into the evolution of the B. cereus group, and highlights the importance of looking beyond differences in gene content in comparative genomics studies

Structure and Complexity of a Bacterial Transcriptome▿ †

Although gene expression has been studied in bacteria for decades, many aspects of the bacterial transcriptome remain poorly understood. Transcript structure, operon linkages, and information on absolute abundance all provide valuable insights into gene function and regulation, but none has ever been determined on a genome-wide scale for any bacterium. Indeed, these aspects of the prokaryotic transcriptome have been explored on a large scale in only a few instances, and consequently little is known about the absolute composition of the mRNA population within a bacterial cell. Here we report the use of a high-throughput sequencing-based approach in assembling the first comprehensive, single-nucleotide resolution view of a bacterial transcriptome. We sampled the Bacillus anthracis transcriptome under a variety of growth conditions and showed that the data provide an accurate and high-resolution map of transcript start sites and operon structure throughout the genome. Further, the sequence data identified previously nonannotated regions with significant transcriptional activity and enhanced the accuracy of existing genome annotations. Finally, our data provide estimates of absolute transcript abundance and suggest that there is significant transcriptional heterogeneity within a clonal, synchronized bacterial population. Overall, our results offer an unprecedented view of gene expression and regulation in a bacterial cell

The <i>B. anthracis</i> chromosome, Leading and Lagging strand gene score distributions, frequency distributions and median gene expression scores for Sense and Antisense expression by leading and lagging strands.

<p>(A) Basic outline of the ∼5.2 Mb chromosome of <i>B. anthracis</i>, showing approximate origin and terminus of replication, and the division scheme of genes on leading and lagging strands. Note that red is the plus strand and green is the minus strand, but “leading” and “lagging” directionality switches at the terminus, with “leading” strand genes going in the same 5′-3′ direction of replication, and “lagging” going in opposite direction. (B-C) Frequency distributions of percentage of genes per leading and lagging strands for sense (B) and antisense (C) transcripts in Control sample. (0* means score of 0.00). (D-E) Median gene expression and interquartile range per quadrant divisions outlined in (A) for sense (D) and antisense (E) gene expression.</p

Bar charts illustrating proportions of Antisense percentage scores (AS%) per leading and lagging strands.

<p>(A) Percentages of genes on leading and lagging strands with greater than 10% AS scores. Percentages calculated per all annotated genes on strands (Leading = 4,049 genes; Lagging = 1458 genes). (B) Same as A, except only considering genes with sense-directed transcription of>2.5, representing those genes present at approximately 0.001 copy per cell.</p

Strand-specific expression measurements of 5 genes by both ssRNAseq and nanoString Technology¹ (5 out of 17 represented – all nanoString data in Tables S4 and S5).

1<p>Only data for Control sample listed here – all data included in Supplemental Materials.</p>2<p>ssRNAseq measurements are “gene scores” (average hits per nucleotide).</p>3<p>nanoString data listed in arbitrary fluorescent units (background subtracted – average of 3 biological replicates).</p>4<p>Spearman Rank Correlations (17 df): rho = 0.946 (Control); 0.949 (Cold); 0.947 (EtOH); and 0.949 (NaCl). All p<1E-09.</p>*<p>Note that gene GBAA4499 had AS signals of 0.00, and so the ratio is listed as the Sense score only (i.e., a denominator of 1, as in nanoString assay – thus, these ratios are an underestimate).</p

Examples of expression coverage illustrating single nucleotide scores with transcriptional activity of surrounding genomic regions.

<p>(A) <i>B. anthracis</i> chromosome, from coordinates 1–5.2 million. Red = plus strand signal; Green = minus strand signal. <i>For all plots, y-axis = log10 of the average coverage of each nucleotide</i>. <i>(i.e., how many times each nucleotide was counted per RNA-seq sequencing run – normalized by total coverage)</i> for 4 biological replicates of Control bacteria (grown in rich broth). Detail inset for (A) is locus tag GBAA1047, coordinates 1033577 to 1034011. (B-D) Expression coverage plots illustrating 3 categories of sense plus AS transcription for 6 genomic regions. Genome Coordinates from <i>B. anthracis</i> Ames Ancestor genome: (B) left = 1605215-1608898 (GBAA1703-07); right = 4092484-4095786 (GBAA4498-4500) (C) left = 4105611-4111707 (GBAA4512-18); right = 4397107-4402721 (GBAA4833-39) (D) left = 141515-144006 (GBAA0147-79); right = 646019-655528 (GBAA0630-37). NOTE: See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043350#pone.0043350.s006" target="_blank">Table S3</a> for average gene scores with Standard Deviations and 95% Confidence Intervals for 4 biological replicates. <i>e.g.,</i> flagellin in panel (B) has a square root transformed average and SD of: Sense = 85.62±0.25 and AS = 0.00; <i>sigA</i> in panel (C) is Sense = 14.06±2.38 and AS = 6.59±1.75; and, <i>gerD</i> in panel (D) is Sense = 0.46±0.12 and AS = 5.10±1.58.</p

Descriptive statistics of Sense and Antisense gene expression per Leading and Lagging strands.

*<p>Square root transformed scores from 4 biological replicates - S_R and AS_R are Leading to Lagging Strand ratios for Sense and Antisense data. Leading strand (n = 4049); Lagging strand (n = 1458).</p