@article{SharmaDugarHerbigetal.2013,
  author    = {Sharma, Cynthia M. and Dugar, Gaurav and Herbig, Alexander and F{\"o}rstner, Konrad U. and Heidrich, Nadja and Reinhardt, Richard and Nieselt, Kay},
  title     = {High-Resolution Transcriptome Maps Reveal Strain-Specific Regulatory Features of Multiple Campylobacter jejuni Isolates},
  series = {PLoS Genetics},
  journal   = {PLoS Genetics},
  doi       = {10.1371/journal.pgen.1003495},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-96610},
  year      = {2013},
  abstract  = {Campylobacter jejuni is currently the leading cause of bacterial gastroenteritis in humans. Comparison of multiple Campylobacter strains revealed a high genetic and phenotypic diversity. However, little is known about differences in transcriptome organization, gene expression, and small RNA (sRNA) repertoires. Here we present the first comparative primary transcriptome analysis based on the differential RNA-seq (dRNA-seq) of four C. jejuni isolates. Our approach includes a novel, generic method for the automated annotation of transcriptional start sites (TSS), which allowed us to provide genome-wide promoter maps in the analyzed strains. These global TSS maps are refined through the integration of a SuperGenome approach that allows for a comparative TSS annotation by mapping RNA-seq data of multiple strains into a common coordinate system derived from a whole-genome alignment. Considering the steadily increasing amount of RNA-seq studies, our automated TSS annotation will not only facilitate transcriptome annotation for a wider range of pro- and eukaryotes but can also be adapted for the analysis among different growth or stress conditions. Our comparative dRNA-seq analysis revealed conservation of most TSS, but also single-nucleotide-polymorphisms (SNP) in promoter regions, which lead to strain-specific transcriptional output. Furthermore, we identified strain-specific sRNA repertoires that could contribute to differential gene regulation among strains. In addition, we identified a novel minimal CRISPR-system in Campylobacter of the type-II CRISPR subtype, which relies on the host factor RNase III and a trans-encoded sRNA for maturation of crRNAs. This minimal system of Campylobacter, which seems active in only some strains, employs a unique maturation pathway, since the crRNAs are transcribed from individual promoters in the upstream repeats and thereby minimize the requirements for the maturation machinery. Overall, our study provides new insights into strain-specific transcriptome organization and sRNAs, and reveals genes that could modulate phenotypic variation among strains despite high conservation at the DNA level.},
  language  = {en}
}
@phdthesis{Dugar2016,
  author    = {Dugar, Gaurav},
  title     = {Comparative transcriptomics and post-transcriptional regulation in \(Campylobacter\) \(jejuni\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146180},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {The transcriptome is defined as the set of all RNA molecules transcribed in a cell. These include protein-coding messenger RNAs (mRNAs) as well as non-coding RNAs, such as ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and small non-coding RNAs (sRNAs). sRNAs are known to play an important role in regulating gene expression and virulence in pathogens. In this thesis, the transcriptome of the food-borne pathogen Campylobacter jejuni was characterized at single nucleotide resolution by use of next-generation sequencing approaches. The first genome of a C. jejuni strain was published in the year 2000. However, its transcriptome remained uncharacterized at large. C. jejuni can survive in a variety of ecological niches and hosts. However, how strain-specific transcriptional changes contribute to such adaptation is not known. In this study, the global transcriptome maps of four closely related C. jejuni strains were defined using a differential RNA-seq (dRNA-seq) approach. This analysis also included a novel automated method to annotate the transcriptional start sites (TSS) at a genome-wide scale. Next, the transcriptomes of four strains were simultaneously mapped and compared by the use of a common coordinate system derived from whole-genome alignment, termed as SuperGenome. This approach helped to refine the promoter maps by comparison of TSS within strains. Most of the TSS were found to be conserved among all four strains, but some single-nucleotide-polymorphisms (SNPs) around promoter regions led to strain-specific transcriptional output. Most of these SNPs altered transcription only slightly, but some others led to a complete abrogation of transcription leading to differential molecular phenotypes. These in turn might help the strains to adapt to their specific host or microniche. The transcriptome also unveiled a plethora of sRNAs, some of which were conserved among the four strains while others were strain specific. Furthermore, a Cas9-dependent minimal type-II CRISPR-Cas system with only three Cas genes and multiple promoters to drive the transcription of the CRISPR locus was also characterized in C. jejuni using the dRNA-seq dataset. Apart from sRNAs, the role of global RNA binding proteins (RBPs) is also unclear in C. jejuni. Aided by the global transcriptome data, the role of RBPs in post-transcriptional regulation of C. jejuni was studied at a global scale. Two of the most widely studied RNA binding proteins in bacteria are Hfq and CsrA. The RNA interactome of the translational regulator CsrA was defined using another global deep-sequencing technique that combines co-immunoprecipitation (coIP) with RNA sequencing (RIP-seq). Using this interactome dataset, the direct targets of this widespread global post-transcriptional regulator were defined, revealing a significant enrichment for mRNAs encoding genes involved in flagella biosynthesis. Unlike Gammaproteobacteria, where sRNAs such as CsrB/C, antagonize CsrA activity, no sRNAs were enriched in the CsrA-coIP in C. jejuni, indicating absence of any sRNA antagonists and novel modes of CsrA activity regulation. Instead, the CsrA regulatory pathway revealed flaA mRNA, encoding the major flagellin, as a dual-function mRNA. flaA mRNA was the main target of CsrA but it also served to antagonize CsrA activity along with the protein antagonist FliW previously identified in the Gram-positive bacterium Bacillus subtilis. Furthermore, this regulatory mRNA was also shown in this thesis to localize to the poles of elongating C. jejuni cells in a translation-dependent manner. It was also shown that this localization is dependent on the CsrA-FliW regulon, which controls the translation of flaA mRNA. The role and mechanism of flaA mRNA localization or mRNA localization in general is not yet clear in bacteria when compared to their eukaryotic counterparts. Overall, this study provides first insights into riboregulation of the bacterial pathogen C. jejuni. The work presented in this thesis unveils several novel modes of riboregulation in C. jejuni, which could be applicable more generally. Moreover, this study also lays out several unsolved intriguing questions, which may pave the way for interesting studies to come.},
  subject      = {Campylobacter jejuni},
  language  = {en}
}
@article{DugarSvenssonBischleretal.2016,
  author    = {Dugar, Gaurav and Svensson, Sarah L. and Bischler, Thorsten and Waldchen, Sina and Reinhardt, Richard and Sauer, Markus and Sharma, Cynthia M.},
  title     = {The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni},
  series = {Nature Communications},
  volume    = {7},
  journal   = {Nature Communications},
  doi       = {10.1038/ncomms11667},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-173201},
  year      = {2016},
  abstract  = {The widespread CsrA/RsmA protein regulators repress translation by binding GGA motifs in bacterial mRNAs. CsrA activity is primarily controlled through sequestration by multiple small regulatory RNAs. Here we investigate CsrA activity control in the absence of antagonizing small RNAs by examining the CsrA regulon in the human pathogen Campylobacter jejuni. We use genome-wide co-immunoprecipitation combined with RNA sequencing to show that CsrA primarily binds flagellar mRNAs and identify the major flagellin mRNA (flaA) as the main CsrA target. The flaA mRNA is translationally repressed by CsrA, but it can also titrate CsrA activity. Together with the main C. jejuni CsrA antagonist, the FliW protein, flaA mRNA controls CsrA-mediated post-transcriptional regulation of other flagellar genes. RNA-FISH reveals that flaA mRNA is expressed and localized at the poles of elongating cells. Polar flaA mRNA localization is translation dependent and is post-transcriptionally regulated by the CsrA-FliW network. Overall, our results suggest a role for CsrA-FliW in spatiotemporal control of flagella assembly and localization of a dual-function mRNA.},
  language  = {en}
}