RNAseq analysis of wild type Campylobacter jejuni compared to modE deletion strain
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 3000
ORGANISM(S): Campylobacter jejuni subsp. jejuni NCTC 11168
SUBMITTER: Dave Kelly
PROVIDER: E-MTAB-11503 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:In eukaryotes glycosylation plays a role in proteome stability, protein quality control and modulating protein function, however, similar studies in bacteria are lacking. Here, we investigate the role of general protein glycosylation systems in bacteria using the enteropathogen Campylobacter jejuni as a well-defined example. By using a quantitative proteomics strategy, we were able to monitor changes in the C. jejuni proteome when glycosylation is disrupted. We demonstrate that in C. jejuni, N-glycosylation is essential to maintain proteome stability and protein quality control. These findings guided us to investigate the role of N-glycosylation in modulating bacterial cellular activities. In glycosylation deficient C. jejuni, the multidrug efflux pump and electron transport pathways were significantly impaired. In vivo, we demonstrate that fully glycosylation deficient C. jejuni were unable to colonise its natural avian host. These results provide the first evidence of a link between proteome stability and complex functions via a bacterial general glycosylation system.
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Project description: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 RNAM-bM-^@M-^Sseq (dRNAM-bM-^@M-^Sseq) 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 RNAM-bM-^@M-^Sseq data of multiple strains into a common coordinate system derived from a whole-genome alignment. Considering the steadily increasing amount of RNAM-bM-^@M-^Sseq 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 dRNAM-bM-^@M-^Sseq 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. Our dRNA-seq study of multiple C. jejuni strains represents the first comparative analysis of the primary transcriptomes of multiple strains and provides new insights into riboregulation in this bacterial pathogen.
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Project description:Strain specific growth of C. jejuni on fucose has been linked to a plasticity region of the chromosome (PR2) and confers a competitive advantage during intestinal colonization. Growth on fucose induces gene expression of PR2 genes, but the regulatory mechanism of the structural genes involved with fucose utilization is unknown. A mutant was constructed to examine the role of Cj0480c, a putative IclR-type transcriptional regulator, on PR2 gene expression. Transcriptional profiles of wild-type C. jejuni and the Cj0480c mutant strain grown without fucose may provide insight in to the extent of the fucose regulon and genes that are necessary for fucose utilization. The design utilized an available two color microarray slide for the entire transcriptome of Campylobacter jejuni wild type strain NCTC 11168. Each sample represents one competitive hybridization: wild-type NCTC 11168 v.s. Cj0480c isogenic mutant. There were four biological replicates of each sample with a dye swap introduced in alternating replicates. Samples were independently grown, treated and harvested.
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