Abstract
BackgroundSmall non-coding RNAs (sRNA) are emerging as major components of the cell’s regulatory network, several possessing their own regulons. A few sRNAs have been reported as being involved in general or toxic-metabolite stress, mostly in Gram- prokaryotes, but hardly any in Gram+ prokaryotes. Significantly, the role of sRNAs in the stress response remains poorly understood at the genome-scale level. It was previously shown that toxic-metabolite stress is one of the most comprehensive and encompassing stress responses in the cell, engaging both the general stress (or heat-shock protein, HSP) response as well as specialized metabolic programs.ResultsUsing RNA deep sequencing (RNA-seq) we examined the sRNome of C. acetobutylicum in response to the native but toxic metabolites, butanol and butyrate. 7.5% of the RNA-seq reads mapped to genome outside annotated ORFs, thus demonstrating the richness and importance of the small RNome. We used comparative expression analysis of 113 sRNAs we had previously computationally predicted, and of annotated mRNAs to set metrics for reliably identifying sRNAs from RNA-seq data, thus discovering 46 additional sRNAs. Under metabolite stress, these 159 sRNAs displayed distinct expression patterns, a select number of which was verified by Northern analysis. We identified stress-related expression of sRNAs affecting transcriptional (6S, S-box & solB) and translational (tmRNA & SRP-RNA) processes, and 65 likely targets of the RNA chaperone Hfq.ConclusionsOur results support an important role for sRNAs for understanding the complexity of the regulatory network that underlies the stress response in Clostridium organisms, whether related to normophysiology, pathogenesis or biotechnological applications.
Highlights
Small non-coding RNAs are emerging as major components of the cell’s regulatory network, several possessing their own regulons
The goal of this study is to identify Small non-coding RNAs (sRNA), at the genome scale, that respond to butanol and/or butyrate stress and possibly start assigning mechanistic roles for these sRNAs. sRNAs that modulate the stress response can be engaged to engineer strains tolerant to these toxic metabolites, as we and others have recently reported for both C. acetobutylicum [34] and Escherichia coli [36,41]
The goal of this study was to identify sRNAs that respond to butanol and/or butyrate stress, and general stress, since, as we discussed, it was previously shown that toxic-chemical stress in Clostridium organisms engages both the general heatshock protein (HSP) systems as well as specialized systems
Summary
Small non-coding RNAs (sRNA) are emerging as major components of the cell’s regulatory network, several possessing their own regulons. Small non-coding regulatory-RNAs (sRNAs), discovered on the genome of all bacteria so far examined, have been established as an integral component of the regulatory system of the cell [1,2,3]. Unlike their counterparts in eukaryotes, which are about 20 nucleotides long, sRNAs in bacteria span a wider size range between 50 to 500 nts [4]. Regulation of gene expression at post-transcriptional level by sRNAs has been established in both Gram-, such as Vibrio fischeri [5], Pseudomonas aeruginosa [6], and Escherichia coli [1,7], and Gram+ bacteria,. Clostridium organisms are Gram+, endospore-forming firmicutes capable of fermenting a very broad set of substrates and are of great importance in human and animal pathogenesis and health, cellulose degradation, non-photosynthetic CO2 fixation, bioremediation and biotechnology, such as for the production of solvents and other chemicals in the context of biofuel and biorefinery applications [27,28]
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