(378b) The Small Rnome of Clostridium Acetobutylicum That Responds to Butanol and Butyrate Stress

In the genus Clostridium, which is of major importance to human and animal pathogenesis, human physiology, the carbon cycle, biorefinery and biotechnological application, very few regulatory non-coding small RNA (sRNA) have been so far identified. Significantly, no sRNAs have been reported as being involved in toxic metabolite stress or any stress.  In C. acetobutylicum, we have previously predicted 113 sRNAs and have experimentally validated a subset of 31 of them. Here, using deep RNA sequencing (RNA-seq), we investigated the expression of the small RNome of C. acetobutylicum in response to two important toxic metabolites, butanol and butyrate. Butanol is the major metabolite of the solventogenic stationary phase while butyrate is the major product of the acidogenic growth phase. The goal of this study is to identify sRNAs that respond to butanol and/or butyrate stress, aiming to identify sRNAs that can be engaged to engineer strains tolerant to these toxic metabolites, but significantly also discover if and how sRNAs respond to metabolic stresses.

We collected a large set of temporal RNA-seq data from multiple experiments with biological replicates employing various levels of metabolite stress. We mined the RNA-seq data for robustly identifying the presence and differential expression of sRNAs by relying on the analysis, based on the RNA-seq data, of the previously predicted 113 and the validated subset of 31 sRNAS. This analysis enabled us to set metrics (read counts) for reliably identifying the presence of new sRNAs from RNA-seq data. Based on these metrics, we identified an additional set of 46 sRNAs not previously known or predicted for this organism. We then examined the differential expression under metabolite stress of all identified sRNAs and verified the presence of a select subset by Northern analysis. Based on the conservation of the putative Hfq binding module, we identified the subset of these sRNAs that are likely to be regulated by the well-conserved RNA chaperone Hfq. Finally, we examined the likely role of a few of these stress-related sRNAs by relating their expression pattern to identifiable physiological characteristics.

This is the first comprehensive study of the stress-related sRNome in a Clostridium organism. Elucidation of the role of sRNA in clostridial response to metabolite stress will be an essential component for understanding the complexity of the regulatory network that underlies these stress responses.