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

Background: 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. Methodology/Principal Findings: Single-stranded RNA-seq results revealed a widespread and non-random pattern of antisense transcription covering more than two thirds of the B. anthracis 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 B. anthracis 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. Conclusions/Significance: 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 Bacillus species. This study illustrates the surprising complexity of prokaryotic RNA abundance for both strands of a bacterial chromosome.