The genus Clostridium includes major human pathogens and species important to cellulose degradation, the carbon cycle, and biotechnology. Small RNAs (sRNAs) are emerging as crucial regulatory molecules in all organisms, but they have not been investigated in clostridia. Research on sRNAs in clostridia is hindered by the absence of a systematic method to identify sRNA candidates, thus delegating clostridial sRNA research to a hit-and-miss process. Thus, we wanted to develop a method to identify potential sRNAs in the Clostridium genus to open up the field of sRNA research in clostridia. Using comparative genomics analyses combined with predictions of rho-independent terminators and promoters, we predicted sRNAs in 21 clostridial genomes: Clostridium acetobutylicum, C. beijerinckii, C. botulinum (eight strains), C. cellulolyticum, C. difficile, C. kluyveri (two strains), C. novyi, C. perfringens (three strains), C. phytofermentans, C. tetani, and C. thermocellum. Although more than one-third of predicted sRNAs have Shine-Dalgarno (SD) sequences, only one-sixth have a start codon downstream of SD sequences; thus, most of the predicted sRNAs are noncoding RNAs. Quantitative reverse transcription-PCR (Q-RT-PCR) and Northern analysis were employed to test the presence of a randomly chosen set of sRNAs in C. acetobutylicum and several C. botulinum strains, leading to the confirmation of a large fraction of the tested sRNAs. We identified a conserved, novel sRNA which, together with the downstream gene coding for an ATP-binding cassette (ABC) transporter gene, responds to the antibiotic clindamycin. The number of predicted sRNAs correlated with the physiological function of the species (high for pathogens, low for cellulolytic, and intermediate for solventogenic), but not with 16S rRNA-based phylogeny. IMPORTANCE Clostridia include major human pathogens and species important to human physiology, cellulose degradation, the carbon cycle, and biotechnology. Small RNAs (sRNAs) are increasingly recognized as crucial regulatory molecules in all organisms, but they remain virtually unexplored in clostridia. We provide the first comprehensive list of computationally identified and experimentally verified small RNAs in the genus Clostridium aiming to accelerate interest in and studies of small RNA molecules in a very important genus. The higher number of sRNAs found in clostridial pathogens suggests a good correlation between the physiological function or niche of the species and the number of predicted and conserved sRNAs. Our list of predicted sRNAs displays a strong enrichment of sRNAs upstream or downstream of ATP-binding cassette (ABC) transporter genes. This, combined with the identification of a conserved sRNA apparently involved in clindamycin resistance, provides a new perspective for future studies of possible regulation of antibiotic resistance genes by sRNAs in bacteria.
