Transcriptomic studies of the bacterium Rhodobacter capsulatus

Grüll, Marc (2019) Transcriptomic studies of the bacterium Rhodobacter capsulatus. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Rhodobacter capsulatus is a purple, non-sulfur alpha-proteobacterium that is studied for different aspects of physiology and its ability to produce the bacteriophage like particle called gene transfer agent (RcGTA). These particles are capable of transferring ~4 kb of random host DNA to other R. capsulatus through a process analogous to transduction. The genes that encode the RcGTA particles are located in a region of ~15 kb called the RcGTA structural gene cluster. The exact regulatory mechanisms involved in the production of RcGTA remain elusive. I investigated the regulation of GTA regulatory genes on a global scale using next generation sequencing methods. I performed investigations to analyse small RNA (sRNA) sequences genome-wide using RNA sequencing. I compared an R. capsulatus wild-type strain to a ctrA null mutant due to previous studies that suggested the global response regulator CtrA might controll a sRNA that regulates RcGTA gene expression. Using the latest bioinformatic approach, 422 putative sRNAs were detected in R. capsulatus during early stationary phase. To get a more detailed insight into the expression of the RcGTA structural gene cluster I performed total RNA sequencing on a RcGTA overproducer mutant strain of R. capsulatus. The previously developed differential RNA sequencing (dRNA-seq) protocol, which can distinguish between primary and processed transcripts, has been modified to identify transcription start sites (TSS) and to predict transcription termination sites (TTS). The combination of total RNA and 5’ -3’ specific sequencing data allowed for the prediction of transcriptional units and to analyze operon complexity in R. capsulatus. The analysis revealed a complex operon architecture, similar to other bacterial species, with operons having multiple TSS and TTS, genomic regions of high transcriptional activity and novel transcripts. Finally, to gain further insight into how RcGTA-associated genes affect each other, I performed qPCR experiments on eight RcGTA-associated genes within and outside of the RcGTA structural gene cluster and within five different R. capsulatus mutant strains. The results indicate that the absence of any one of the known and putative GTA regulators investigated can have effects on the transcription of GTA loci.

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