Single-nucleotide resolution analysis of the transcriptome structure of Clostridium beijerinckii NCIMB 8052 using...

Clostridium beijerinckii is an important solvent producing microorganism. The genome of C. beijerinckii NCIMB 8052 has recently been sequenced. Although transcriptome structure is important in order to reveal the functional and regulatory architecture of the genome, the physical structure of transcriptome for this strain, such as the operon linkages and transcript boundaries are not well understood.

In this study, we conducted a single-nucleotide resolution analysis of the C. beijerinckii NCIMB 8052 transcriptome using high-throughput RNA-Seq technology. We identified the transcription start sites and operon structure throughout the genome. We confirmed the structure of important gene operons involved in metabolic pathways for acid and solvent production in C. beijerinckii 8052, including pta-ack, ptb-buk, hbd-etfA-etfB-crt (bcs) and ald-ctfA-ctfB-adc (sol) operons; we also defined important operons related to chemotaxis/motility, transcriptional regulation, stress response and fatty acids biosynthesis along with others. We discovered 20 previously non-annotated regions with significant transcriptional activities and 15 genes whose translation start codons were likely mis-annotated. As a consequence, the accuracy of existing genome annotation was significantly enhanced. Furthermore, we identified 78 putative silent genes and 177 putative housekeeping genes based on normalized transcription measurement with the sequence data. We also observed that more than 30% of pseudogenes had significant transcriptional activities during the fermentation process. Strong correlations exist between the expression values derived from RNA-Seq analysis and microarray data or qRT-PCR results.

Transcriptome structural profiling in this research provided important supplemental information on the accuracy of genome annotation, and revealed additional gene functions and regulation in C. beijerinckii.