Project description:Transcriptional regulation mediates adaptation of pathogens to environmental stimuli and is important for host colonisation. The Campylobacter jejuni genome sequence reveals a surprisingly small set of regulators, mostly of unknown function, suggesting an intricate regulatory network. Interestingly, C. jejuni lacks the homologues of ubiquitous regulators involved in stress response found in many other Gram-negative bacteria. Nonetheless, cj1000 is predicted to code for the sole LysR-type regulator in the C. jejuni genome, and thus may be involved in major adaptation pathways. A cj1000 mutant strain was constructed and found to be attenuated in its ability to colonise 1-day old chicks. Complementation of cj1000 mutation restored the colonisation ability to that of wild type levels. The mutant strain was also outcompeted in a competitive colonisation assay of the piglet intestine. High resolution oxygraphy was carried out for the first time on C. jejuni and revealed a role for Cj1000 in controlling O2 consumption. Furthermore, microarray analysis of the cj1000 mutant revealed both direct and indirect regulatory targets, including genes involved in energy metabolism and oxidative stress defences. These results highlight the importance of Cj1000 regulation in host colonisation and in major physiological pathways.
Project description:Batch cultures of Wild-type C. jejuni NCTC 11168 were grown in 150 ml volumes of Mueller-Hinton broth in 250 ml baffled flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (10 % Oxygen, 10 % Carbon dioxide, 80 % Nitrogen) from Don Whitley Scientific, Ltd which also maintained the growth temperature at 42 ºC. When mid-exponential phase was reached 0.25 mM GSNO was added to half of the cultures. After a 5/10/15/45 minute exposure samples of both treated and untreated cells were harvested into phenol/ethanol to stabilize the RNA and total RNA was purified using Qiagen’s RNeasy Mini kit (as recommended by the suppliers) prior to use in microarray analysis. Keywords: Time Course
Project description:Transcriptional regulation mediates adaptation of pathogens to environmental stimuli and is important for host colonisation. The Campylobacter jejuni genome sequence reveals a surprisingly small set of regulators, mostly of unknown function, suggesting an intricate regulatory network. Interestingly, C. jejuni lacks the homologues of ubiquitous regulators involved in stress response found in many other Gram-negative bacteria. Nonetheless, cj1000 is predicted to code for the sole LysR-type regulator in the C. jejuni genome, and thus may be involved in major adaptation pathways. A cj1000 mutant strain was constructed and found to be attenuated in its ability to colonise 1-day old chicks. Complementation of cj1000 mutation restored the colonisation ability to that of wild type levels. The mutant strain was also outcompeted in a competitive colonisation assay of the piglet intestine. High resolution oxygraphy was carried out for the first time on C. jejuni and revealed a role for Cj1000 in controlling O2 consumption. Furthermore, microarray analysis of the cj1000 mutant revealed both direct and indirect regulatory targets, including genes involved in energy metabolism and oxidative stress defences. These results highlight the importance of Cj1000 regulation in host colonisation and in major physiological pathways. Microarray data was collected from three independent biological replicates and 3-9 technical replicates for each biological replicate.
Project description:The Cj1223c gene was cloned downstream of a strong promoter into the replicating plasmid pRY108, and was highly expressed in the wild type Campylobacter jejuni 81-176 strain (overexpressed). The Cj1223c gene was knocked out by allelic replacement in the 81-176 strain background (mutant). This mutant also carried an empty pRY108 vector. The Cj1223c overexpressing strain and the mutant were grown overnight in liquid broth, supplemented with kanamycin. The following morning, both cultures were diluted back to O.D.600=0.100, and allowed to continue to grow, shaking. At various time intervals (2, 6, 10, and 24 hours), samples of each culture were removed, combined with 1/10 stop solution, and the bacteria pelleted for RNA preparation.
Project description:In Campylobacter jejuni CmeR functions as a transcriptional repressor modulating the expression of the multidrug efflux pump CmeABC, which plays an important role in the resistance to antimicrobial agents and bile compounds. Using DNA microarray, we identified multiple genes that are either activated or repressed by CmeR in C. jejuni. The DNA microarray data was independently confirmed by quantitative real-time RT-PCR. The CmeR-regulated genes encode products of diverse functions including membrane proteins, drug efflux transporters, the C4-dicarboxylate transport/utilization system, and enzymes involved in the biosynthesis of capsular polysaccharide (CPS). Immunoblotting and Alcian blue staining further showed that CPS production is reduced in the cmeR mutant, confirming the regulation of CPS production by CmeR. Electrophoretic mobility shift assay revealed that recombinant CmeR bound specifically to several intergenic regions in the CPS gene cluster, suggesting that CmeR directly regulates this gene cluster. In the chicken host, the mutant carrying a null mutation in cmeR was severely outcompeted by the isogenic wild-type strain. Together these data indicate that CmeR functions as a global regulator in C. jejuni, modulates the expression of genes encoding diverse functions, and is important for the fitness of Campylobacter in the intestinal tract. Keywords: cell type comparison
Project description:Campylobacter jejuni causes food- and water-borne gastroenteritis, and as such must survive passage through the stomach in order to reach the gastrointestinal tract. While little is known about how C. jejuni survives transit through the stomach, its low infectious dose suggests it is well equipped to sense and respond to acid shock. In this study, the transcriptional profile of C. jejuni NCTC 11168 was obtained after exposure to in vitro acid shock. Keywords: acid shock; in vitro study; time course