Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 May;191(10):3203-11.
doi: 10.1128/JB.00122-09. Epub 2009 Mar 20.

Structure and complexity of a bacterial transcriptome

Karla D Passalacqua  1 , Anjana Varadarajan, Brian D Ondov, David T Okou, Michael E Zwick, Nicholas H Bergman
Affiliations

Structure and complexity of a bacterial transcriptome

Karla D Passalacqua et al. J Bacteriol. 2009 May.

Abstract

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.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Sample collection for and representative global structure of the B. anthracis transcriptome. (A) Representative growth curve of B. anthracis in MGM, with approximate RNA collection points shown by arrows. The atmosphere under which each sample was collected is indicated; note that growth rates in air and 15% CO2 environments were similar, with slightly slower growth in CO2. OD600, optical density at 600 nm. (B) Sequence coverage across the entire B. anthracis genome (5.2 Mb). Data shown are from sample 5. (C) Magnified portion of the plot from panel B, showing sequence coverage over an ∼10-kb region of the B. anthracis chromosome, with genes GBAA1980-7 indicated below by arrows.
FIG. 2.
FIG. 2.
Single-nucleotide resolution-sequencing data allow multiple mapping strategies to reveal global transcriptome composition. (A) Sequence coverage near the 5′ terminus of the asbA (GBAA1981) gene. The green line indicates the start codon, with the arrow beneath showing the direction of transcription. The blue and red lines indicate the transcript start site determined by sequence coverage and template-switching extension (TSx) data, respectively. (B) Sequence coverage in the GBAA5506-8 region of the B. anthracis chromosome, showing transcription across a region not included in the current genome annotation. Arrows beneath show the positions of the GBAA5506 and GBAA5508 loci. (C) Sequence coverage near the GBAA0688 locus, with a clear boundary inside the annotated gene (gap between annotated start codon and beginning of sequence coverage noted by red bracket).
FIG. 3.
FIG. 3.
Sequence coverage depth is quantitative and accurately reflects B. anthracis transcript abundance. (A) Comparison of sequence coverage depth and absolute Affymetrix GeneChip intensities. Shown is a plot of sequence coverage depth for sample 5 and raw microarray intensities for an equivalent sample (i.e., collected at the same point in the life cycle under the same growth conditions) analyzed previously and reported in reference . (B) Comparison of sequence coverage depth with SYBR green qRT-PCR data using RNA from sample 6.
FIG. 4.
FIG. 4.
Transcriptional profiling in B. anthracis using RNA-Seq. (A) Sequence coverage plots for samples 5 (mid-log phase; in black) and 7 (late sporulation; in green), normalized to the genome-wide total number of reads mapped unambiguously. The representative region shown is an ∼11-kb genome segment surrounding the asb operon (GBAA1981-6), and specific genes are indicated. Below each gene or operon is the change in expression level that was measured by comparing microarray data from equivalent samples (previously described in reference 1) (ArrayExpress accession number E-MEXP-788). (B) Coverage plots for the ∼12-kb region surrounding the ilvE-1-leuD operon (GBAA1416-23) from samples 5 (mid-log phase in air; in black) and 6 (late-log phase in CO2; in gray). The T-box structural element predicted by Griffiths-Jones et al. (7) is indicated in red.
FIG. 5.
FIG. 5.
The distribution of mRNA abundance in B. anthracis. The histogram shows the distribution of transcription levels (sequence coverage depth) for all genes in the B. anthracis genome in RNA sample 5. Arrows indicate the coverage depth expected (95% probability) for an mRNA molecule of average length present at 1 copy per cell (28.2-8.3), 0.1 copies per cell (24.7-5.0), and 0.01 copies per cell (21.3-1.9), based on the model described in the text in the supplemental material.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bergman, N. H., E. C. Anderson, E. E. Swenson, M. M. Niemeyer, A. D. Miyoshi, and P. C. Hanna. 2006. Transcriptional profiling of the Bacillus anthracis life cycle in vitro and an implied model for regulation of spore formation. J. Bacteriol. 1886092-6100. - PMC - PubMed
    1. Bourgogne, A., M. Drysdale, S. G. Hilsenbeck, S. N. Peterson, and T. M. Koehler. 2003. Global effects of virulence gene regulators in a Bacillus anthracis strain with both virulence plasmids. Infect. Immun. 712736-2743. - PMC - PubMed
    1. Cendrowski, S. R. 2004. Role of the asb operon in Bacillus anthracis pathogenesis. Ph.D. dissertation. University of Michigan, Ann Arbor.
    1. Coker, P. R., K. L. Smith, P. F. Fellows, G. Rybachuck, K. G. Kousoulas, and M. E. Hugh-Jones. 2003. Bacillus anthracis virulence in guinea pigs vaccinated with Anthrax Vaccine Adsorbed is linked to plasmid quantities and clonality. J. Clin. Microbiol. 411212-1218. - PMC - PubMed
    1. Elowitz, M. B., A. J. Levine, E. D. Siggia, and P. S. Swain. 2002. Stochastic gene expression in a single cell. Science 2971183-1186. - PubMed

Publication types

MeSH terms

Associated data