RNAseq analysis of Campylobacter jejuni's response to glutamine addition in growth media
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ABSTRACT: Campylobacter jejuni is a human pathogen which causes campylobacteriosis, one of the most widespread zoonotic enteric diseases worldwide. Growth of Campylobacter can be improved through the addition of glutamine to media which serves as the nitrogen source. RNAseq was used to identify the transcriptomic response of Campylobacter jejuni when the nitrogen source was switched from serine (poor growth) to glutamine (good growth) in chemostat cultures.
Project description:Campylobacter jejuni is a human pathogen which causes campylobacteriosis, one of the most widespread zoonotic enteric diseases worldwide. Most cases of sporadic C. jejuni infection occur through the handling or consumption of undercooked chicken meat, or cross-contamination of other foods with raw poultry fluid. A common practice to combat Campylobacter infection is to treat chickens with chlorine which kills the microbe. This analysis aimed to elucidate the transcriptomic response of Campylobacter jejuni treated with hypochlorite through Illumina sequencing. C. jejuni was grown and treated with hypochlorite. Samples were taken 5, 20 and 45 min after treatment for RNAseq analysis.The data generated were compared to the transcriptome pre-exposure to determine C. jejuni's response to hypochlorite.
Project description:Campylobacter jejuni is a human pathogen which causes Campylobacteriosis, one of the most widespread zoonotic enteric diseases worldwide. ModE is a transcriptional regulator that controls molybdenum uptake in many bacteria. modE (cj1507c) was deleted from C. jejuni NCTC11168 and grown alongside the parental wild type to mid-log phase in defined medium containing replete Mo, W and Se. Samples were taken for RNAseq analysis and used to compare gene expression.
Project description:Campylobacter jejuni is susceptible to killing through exposure to blue light (405 nm) due to its poor ability to detoxify reactive oxygen species. This analysis aimed to elucidate the transcriptomic response of Campylobacter jejuni exposed to 405 nm light through illumina sequencing. C. jejuni was grown and exposed to 405nm light. Samples were taken at 15 min (7 J cm-1) and 30 min (14 J cm-1) after exposure. The data generated were compared to the transcriptome pre-exposure to determine the changes associated with blue light exposure
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.
Project description:Campylobacter jejuni is a major zoonotic pathogen transmitted to humans via the food chain. C. jejuni is prevalent in chickens, a natural reservoir for this pathogenic organism. Due to the importance of macrolide antibiotics in clinical therapy of human campylobacteriosis, development of macrolide resistance in Campylobacter has become a concern for public health.To facilitate understanding the molecular basis associated with the fitness difference between Erys and Eryr Campylobacter, we compared the transcriptomes between ATCC 700819 and its isogenic Eryr transformant T.L.101 using DNA microarray. The design utilized an available two color microarray slide for the entire transcriptome of Campylobacter jejuni. Four hybridizations were performed each with independently extracted samples of either macrolide susceptible ATCC 700819 cDNA samples or its isogenic Eryr transformant T.L.101 cDNA samples. A dye swap was utilized to help minimize dye dependent bias. Thus, there were four biological replicates of each sample.
Project description:Expression arrays comparing Campylobacter jejuni NCTC11168 during growth in the cecum of germ-free C57 BL/6 IL-10 knockout mice to C. jejuni NCTC11168 during growth in Bolton broth. Four biological replicates comparing C. jejuni NCTC11168 growth in vivo to in vitro. Two biological replicates were dye swaps.
Project description:We performed transcriptome (RNA-seq) analyses for Campylobacter jejuni 11168 wild-type (WT) and Cj1608 deletion mutant (ΔCj1608::aphA-3) under oxidative stress and optimal microaerobic growth conditions. The expression of 231 genes was affected by oxidative stress in stressed wild-type cells (WTS) compared to non-stressed cells (WT). A comparison of genes expressed in the ΔCj1608 and WT strains under optimal growth conditions revealed 380 differently expressed genes. Moreover, transcriptional changes and overall final protein levels correlated across multiple genes. The data were validated through RT-qPCR and phenotype experiments for selected processes.
Project description:The pathogenic bacterium Campylobacter jejuni is the leading cause of bacterial foodborne gastroenteritis worldwide yet it does not grow in the aerobic environment. The paralogues RrpA and RrpB which are members of MarR family of DNA binding proteins have been shown to be important for the survival of C. jejuni under aerobic and redox stress. We report that RrpA is a positive regulator of mdaB, encoding a flavin-dependent quinone reductase. MdaB confers protection to the cell from redox stress mediated by structurally diverse quinones. RrpB negatively regulates the expression of nfrA (Cj1555c), a flavin reductase. NfrA reduces riboflavin at a much higher rate than flavin mononucleotide (FMN),suggesting exogenous free flavins are the natural substrate. Enzymatic activity of MdaB and NfrA towards their substrates revealed both reductases preferred NADPH as an electron donor. DNA-binding and post translational modification analyses showed that the mechanism of RrpA and RrpB DNA binding is likely a cysteine-based redox switch. Complete genome sequences analysis indicated that MdaB is predominant in Campylobacter spp. and the related Helicobacter spp., whilst NfrA is more often found in C. jejuni strains. Quinones and flavins are antimicrobial redox cycling agents secreted by a wide range of cell-types that can form damaging superoxide by one-electron reactions. We propose that MdaB and NfrA production allows a two-electron reduction mechanism to the less toxic quinol forms. These enzymes thus aid the survival and persistence of C. jejuni in the face of toxic compounds from competing microbes.
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.
Project description:Campylobacter spp. cause food-borne illnesses worldwide due to contaminated food and cross-contamination. This is at least partly the result of Campylobacter resistance in the food production chain, as modern food production facilitates the emergence and spread of resistance through intensive use of antimicrobials and international trade in raw materials and food products. The biofilm 'lifestyle' of Campylobacter contributes to this spread as it enables them to withstand stress in the environment both outside and inside the host. Campylobacter adhesion and biofilm formation has major implications for the food industry, where biofilms can be persistent sources of contamination. In our study, we described how the proteome of C. jejuni is affected by the deletion of the luxS gene on the planktonic cell type of C. jejuni, which is the first step of biofilm formation. In C. jejuni, the presence of the luxS gene has been associated with several phenotypes, including intercellular signalling, motility, biofilm formation, host colonisation, virulence, autoagglutination, cellular adherence and invasion, oxidative stress and chemotaxis. Deletion of the luxS gene is associated with a reduction or absence of the above properties compared to wild type (Elvers and Park, 2002; Guerry et al., 2006; He et al., 2008; Jeon et al., 2003; Quiñones et al., 2009; Plummer et al., 2011; Plummer, 2012; Reeser et al., 2007).