@phdthesis{Pernitzsch2021,
  author    = {Pernitzsch, Sandy Ramona},
  title     = {Functional Characterization of the abundant and conserved small regulatory RNA RepG in Helicobacter pylori},
  doi       = {10.25972/OPUS-12268},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-122686},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Bacterial small non-coding RNAs (sRNAs) play fundamental roles in controlling and finetuning gene expression in a wide variety of cellular processes, including stress responses, environmental signaling and virulence in pathogens. Despite the identification of hundreds of sRNA candidates in diverse bacteria by genomics approaches, the mechanisms and regulatory capabilities of these posttranscriptional regulators have most intensively been studied in Gram-negative Gammaproteobacteria such as Escherichia coli and Salmonella. So far, almost nothing is known about sRNA-mediated regulation (riboregulation) in Epsilonproteobacteria, including the major human pathogen Helicobacter pylori. H. pylori was even thought to be deficient for riboregulation as none of the sRNAs known from enterobacteria are conserved in Helicobacter and since it lacks the major RNA chaperone Hfq, which is crucial for sRNA function as well as stability in many bacteria. Nonetheless, more than 60 cis- and trans-acting sRNA candidates were recently identified in H. pylori by a global RNA sequencing approach, indicating that this pathogen, in principle, has the capability to use riboregulation for its gene expression control. However, the functions and underlying mechanisms of H. pylori sRNAs remained unclear. This thesis focused on the first functional characterization and target gene identification of a trans-acting sRNA, RepG (Regulator of polymeric G-repeats), in H. pylori. Using in-vitro and in-vivo approaches, RepG was shown to directly base-pair with its C/Urich terminator loop to a variable homopolymeric G-repeat in the 5' untranslated region (UTR) of the tlpB mRNA, thereby regulating expression of the chemotaxis receptor TlpB. While the RepG sRNA is highly conserved, the length of the G-repeat in the tlpB mRNA leader varies among different H. pylori isolates, resulting in a strain-specific tlpB regulation. The modification of the number of guanines within the G-stretch in H. pylori strain 26695 demonstrated that the length of the homopolymeric G-repeat determines the outcome of posttranscriptional control (repression or activation) of tlpB by RepG. This lengthdependent targeting of a simple sequence repeat by a trans-acting sRNA represents a new twist in sRNA-mediated regulation and a novel mechanism of gene expression control, since it uniquely links phase variation by simple sequence repeats to posttranscriptional regulation. In almost all sequenced H. pylori strains, tlpB is encoded in a two gene operon upstream of HP0102, a gene of previously unknown function. This study provided evidence that HP0102 encodes a glycosyltransferase involved in LPS O-chain and Lewis x antigen production. Accordingly, this glycosyltransferase was shown to be essential for mice colonization by H. pylori. The coordinated posttranscriptional regulation of the tlpB-HP0102 operon by antisense base-pairing of RepG to the phase-variable G-repeat in the 5' UTR of the tlpB mRNA allows for a gradual, rather than ON/OFF, control of HP0102 expression, thereby affecting LPS biosynthesis in H. pylori. This fine-tuning of O-chain and Lewis x antigen expression modulates H. pylori antibiotics sensitivity and thus, might be advantageous for Helicobacter colonization and persistence. Whole transcriptome analysis based on microarray and RNA sequencing was used to identify additional RepG target mRNAs and uncover the physiological role of this riboregulator in H. pylori. Altogether, repG deletion affected expression of more than 40 target gene candidates involved various cellular processes, including membrane transport and adhesion, LPS modification, amino acid metabolism, oxidative and nitrosative stress, and nucleic acid modification. The presence of homopolymeric G-repeats/G-rich sequences in almost all target mRNA candidates indicated that RepG hijacks a conserved motif to recognize and regulate multiple target mRNAs in H. pylori. Overall, this study demonstrates that H. pylori employs riboregulation in stress response and virulence control. In addition, this thesis has successfully established Helicobacter as a new model organism for investigating general concepts of gene expression control by Hfq-independent sRNAs and sRNAs in bacterial pathogens.},
  subject      = {Small RNA},
  language  = {en}
}