Tracking the elusive function of the regulatory RNA SolB in solventogenesis and its relation to the RNA-binding protein Hfq in Clostridium acetobutylicum
Auch gedruckt in der BibliothekW: W-H 15.192
FakultätFakultät für Naturwissenschaften
InstitutionInstitut für Mikrobiologie und Biotechnologie
Ressourcen- / MedientypDissertation, Text
Datum der Erstveröffentlichung2017-07-19
Here, we focus on the small regulatory RNA (sRNA) SolB from the Gram-positive Clostridium acetobutylicum. This solvent-producing anaerobic bacterium is of importance as it naturally produces butanol, an important bulk chemical and biofuel. SolB is able to repress the production of acetone and butanol as well as the sporulation process in a C. acetobutylicum solB overexpression strain. The aim of this study was to identify targets and the role of SolB in solventogenesis. The transcriptome analysis and quantitative real-time PCR revealed down-regulation of all genes encoded on the megaplasmid pSOL1 of the C. acetobutylicum solB overexpression strain compared to the C. acetobutylicum wild type strain. DNA sequencing data and quantitative real-time PCR demonstrated a decreased copy number of the megaplasmid pSOL1 in the C. acetobutylicum solB overexpression strain compared to the control strain. Concluding, SolB regulates the copy number of pSOL1, and the reduced level of pSOL1 in the C. acetobutylicum solB overexpression strain seems to be the bottleneck for the low expression of the genes encoded on the megaplasmid pSOL1. Simultaneous overexpression of solB and adhE2 in C. acetobutylicum restored solvent production and increased the copy number of pSOL1 supporting the interaction of SolB with the transcript of adhE2. Further growth experiments with genetically modified C. acetobutylicum strains indicated that the overexpression of adhE2 as well as the overexpression of spo0A lead to a titration effect of SolB. Moreover, the function of the RNA-binding protein Hfq was elucidated in C. acetobutylicum. Hfq of C. acetobutylicum was able to compensate cell changes and growth deficiency under different conditions in an E. coli ∆hfq strain. However, C. acetobutylicum hfq knockdown and knockout strains did not show phenotypic changes supporting a minor important role as displayed for Hfq in E. coli. As most trans-encoded sRNAs require the RNA-binding protein Hfq for stability or functionality, the importance of Hfq for SolB was investigated. Pulldown and electrophoretic mobility shift assays proved that SolB is bound to Hfq. Moreover, faster degradation of SolB was observed in presence of Hfq. Furthermore, the effects of solB overexpression in an hfq-deficient C. acetobutylicum strain were diminished confirming the dependency of SolB on Hfq.
LCSHSmall interfering RNA