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.Although more than a thousand bacterial genomes have been sequenced, our understanding of bacterial transcriptomes has lagged far behind (see the NIH database at http: //www.ncbi.nlm.nih.gov/genomes/genlist.cgi?taxidϭ2&type ϭ0&nameϭComplete%20Bacteria). The physical structure of prokaryotic transcriptomes-operon linkages and transcript boundaries, for instance-is not defined on a genomewide level for any species. Even though this information represents a critical step in understanding the functional and regulatory architecture of the genome, it has been explored on a large scale (Ͼ10% of the transcriptome) in only a few instances (6, 16). Similarly, although global gene expression in bacteria is routinely studied in a relative sense, where expression patterns occurring in two or more conditions are compared, there have been few attempts to comprehensively profile a bacterial transcriptome from an unbiased perspective. Consequently, little is known about the absolute composition of the mRNA population within a bacterial cell or about how individual cells' mRNA content might differ.Recent advances in high-throughput DNA sequencing have made it possible to define nucleic acid populations at an unprecedented depth and resolution. Here we report the use of a sequencing-based approach (RNA-Seq) (17, 24) in assembling the first comprehensive, single-nucleotide resolution view of a bacterial transcriptome. Sterne (34F 2 ). B. anthracis Sterne (34F 2 ) was grown in modified G medium (MGM) at 37°C (with shaking at 250 rpm) and in MGM plus 0.8% sodium bicarbonate in 14 to 15% CO 2 at 37°C (with shaking at 150...

Summary: Here, we report the development of SOCS (short oligonucleotide color space), a program designed for efficient and flexible mapping of Applied Biosystems SOLiD sequence data onto a reference genome. SOCS performs its mapping within the context of ‘color space’, and it maximizes usable data by allowing a user-specified number of mismatches. Sequence census functions facilitate a variety of functional genomics applications, including transcriptome mapping and profiling, as well as ChIP-Seq.Availability: Executables, source code, and sample data are available at http://socs.biology.gatech.edu/Contact: nickbergman@gatech.eduSupplementary information: Supplementary data are available at Bioinformatics Online.

Background 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.Methodology/Principal FindingsHere 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 34F2), and an avirulent B. cereus strain (10987)—during exponential growth in two distinct atmospheric environments: 14% CO2/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 CO2 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.Conclusions/SignificanceWe 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.

BackgroundAlthough 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 FindingsSingle-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/SignificanceAlthough 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.