Functional Analyses and Application of Bacterial Non-coding RNAs

Bacterial non-coding RNAs, including ribozymes and riboswitches, have diverse roles in gene regulation and many other processes. My dissertation research has focused on three bacterial non-coding RNAs: OLE RNA, a putative ribozyme that protects extremophilic bacteria from alcohol toxicity and other membrane stress; NiCo riboswitch, a riboswitch class that senses heavy metal ions and controls related transporter genes; fluoride riboswitch, a widespread riboswitch that class senses F- ion. In the study of OLE RNA, its consensus sequence, secondary structure and gene association was updated. OLE was found to be one of the most stable and abundant RNAs and its production is increased in the presence of short-chain alcohols such as ethanol. OLE/OAP (OLE associated protein) knockout Bacillus halodurans cells were shown to have growth defects under ethanol or cold stress. I developed and optimized several growth assays as tools to study these phenotypes quantitatively. I demonstrated that these phenotypes can be rescued by reintroducing the genes for OLE and OAP into B. halodurans, either on a plasmid or somewhere else on the chromosome. I identified an OAP mutant that confers lethality, an even stronger phenotype, in B. halodurans in the presence of short chain alcohols or at low temperature. I showed this lethal phenotype is dominant negative and is dependent both on the presence of a structurally intact OLE RNA and the interaction between OLE and OAP. Several general hypotheses about the function of OLE and OAP were proposed and explored. I performed genetic selection for mutations that could suppress the lethality of the OAP mutant and isolated one surviving variant. Whole genome sequencing to map its mutations is in progress. In the study of NiCo riboswitches, ligand specificity was established as Ni2+ and Co2+ by in-line probing analysis, and ligand binding was shown to be cooperative. Transcription assays showed this riboswitch could control gene expression in vitro. I demonstrated that high external concentration of Ni2+ triggers the expression of a gene under control of this riboswitch in Clostridium scindens, thereby confirming in vivo gene regulation. A molecular structure model was established from X-ray crystallography data, revealing the details of ligand binding. I performed in-line probing analysis on several aptamer mutants to further study the nucleotides and functional groups that are responsible for ligand binding and the observed ligand binding cooperativity. In the study of antibiotic activity of gramicidin D, a fluoride riboswitch was used to show gramicidin D increases the internal concentration of fluoride Bacillus subtilis. I performed growth assay to show gramicidin D enhances the antibiotic activity of fluoride in B. subtilis and B. halodurans, which helped to elucidate the mechanism of this antibiotics. Overall, my dissertation research expands our current knowledge about the roles of bacterial non-coding RNA in nature, especially in coping with various environmental stresses.