Categories of Sense + AS expression – numbers of genes per category (n = 5,507 genes).

*<p>Numbers represent counts of genes in each category per the entire annotated genome. Top number is total. Numbers in parentheses are leading (left) plus lagging (right) strand counts.</p

Strand-Specific RNA-Seq Reveals Ordered Patterns of Sense and Antisense Transcription in <em>Bacillus anthracis</em>

<div><h3>Background</h3><p>Although genome-wide transcriptional analysis has been used for many years to study bacterial gene expression, many aspects of the bacterial transcriptome remain undefined. One example is antisense transcription, which has been observed in a number of bacteria, though the function of antisense transcripts, and their distribution across the bacterial genome, is still unclear.</p> <h3>Methodology/Principal Findings</h3><p>Single-stranded RNA-seq results revealed a widespread and non-random pattern of antisense transcription covering more than two thirds of the <em>B. anthracis</em> genome. Our analysis revealed a variety of antisense structural patterns, suggesting multiple mechanisms of antisense transcription. The data revealed several instances of sense and antisense expression changes in different growth conditions, suggesting that antisense transcription may play a role in the ways in which <em>B. anthracis</em> responds to its environment. Significantly, genome-wide antisense expression occurred at consistently higher levels on the lagging strand, while the leading strand showed very little antisense activity. Intrasample gene expression comparisons revealed a gene dosage effect in all growth conditions, where genes farthest from the origin showed the lowest overall range of expression for both sense and antisense directed transcription. Additionally, transcription from both strands was verified using a novel strand-specific assay. The variety of structural patterns we observed in antisense transcription suggests multiple mechanisms for this phenomenon, suggesting that some antisense transcription may play a role in regulating the expression of key genes, while some may be due to chromosome replication dynamics and transcriptional noise.</p> <h3>Conclusions/Significance</h3><p>Although the variety of structural patterns we observed in antisense transcription suggest multiple mechanisms for antisense expression, our data also clearly indicate that antisense transcription may play a genome-wide role in regulating the expression of key genes in <em>Bacillus</em> species. This study illustrates the surprising complexity of prokaryotic RNA abundance for both strands of a bacterial chromosome.</p> </div

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

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

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

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

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