Genome-Wide TSS Distribution in Three Related Clostridia with Normalized Capp-Switch Sequencing

Transcription initiation is a tightly regulated process that is crucial for many aspects of prokaryotic physiology. High-throughput transcription start site (TSS) mapping can shed light on global and local regulation of transcription initiation, which in turn may help us understand and predict microbial behavior. In this study, we used Capp-Switch sequencing to determine the TSS positions in the genomes of three model solventogenic clostridia: Clostridium acetobutylicum ATCC 824, C. beijerinckii DSM 6423, and C. beijerinckii NCIMB 8052. We first refined the approach by implementing a normalization pipeline accounting for gene expression, yielding a total of 12,114 mapped TSSs across the species. We further compared the distributions of these sites in the three strains. Results indicated similar distribution patterns at the genome scale, but also some sharp differences, such as for the butyryl-CoA synthesis operon, particularly when comparing C. acetobutylicum to the C. beijerinckii strains. Lastly, we found that promoter structure is generally poorly conserved between C. acetobutylicum and C. beijerinckii. A few conserved promoters across species are discussed, showing interesting examples of how TSS determination and comparison can improve our understanding of gene expression regulation at the transcript level. IMPORTANCE Solventogenic clostridia have been employed in industry for more than a century, initially being used in the acetone-butanol-ethanol (ABE) fermentation process for acetone and butanol production. Interest in these bacteria has recently increased in the context of green chemistry and sustainable development. However, our current understanding of their genomes and physiology limits their optimal use as industrial solvent production platforms. The gene regulatory mechanisms of solventogenesis are still only partly understood, impeding efforts to increase rates and yields. Genome-wide mapping of transcription start sites (TSSs) for three model solventogenic Clostridium strains is an important step toward understanding mechanisms of gene regulation in these industrially important bacteria.