The CsrA pathway and the mechanism of regulation have Selleckchem MAPK inhibitor been studied extensively in the γ-proteobacteria and further studies of the

role of CsrA in various pathogens have extended its importance to the expression of virulence factors and the regulation of pathogenesis [22–26]. Despite these advances, very little is known about the mechanism of action of CsrA in the ε-proteobacteria. Examination of the C. jejuni genome [7, 27–29] suggests that this bacterium lacks several genes in the CsrA pathway, including apparent orthologs of the small RNA molecules csrB and csrC[30], the barA/uvrY two-component signal transduction system, and csrD which is responsible for csrB and csrC turnover [31]. One report describing the role of CsrA in the gastric pathogen Helicobacter pylori indicated that CsrA was required for motility, survival under oxidative stress, and host colonization, and plays a role in the expression of several virulence and oxidative stress related proteins [23]. It was also suggested that the H. pylori ortholog was unable to function when exogenously expressed in E. coli because it failed to complement the glycogen accumulation phenotype of an E. coli csrA mutant [23]. Considering these observations in H. pylori, the phenotypes of a C. jejuni csrA mutant, and the lack of knowledge concerning the functions of CsrA within the ε-proteobacteria, we examined the ability

of C. jejuni CsrA to complement the phenotypes of an E. coli csrA mutant with the hope of gaining further insight into the molecular mechanism of C. jejuni CsrA. Phylogenetic comparison revealed that C. jejuni CsrA exhibits Alisertib solubility dmso variability in amino acids that constitute the published RNA binding domains, as well as in other residues that are important for CsrA-mediated regulation in E. coli. Surprisingly, although the C. jejuni ortholog was unable to complement the glycogen accumulation phenotype of E. coli, successful rescue of several other E. coli mutant phenotypes was achieved, demonstrating both similarities and

differences in the C. jejuni and E. coli Csr systems. Methods Bacterial strains and routine growth conditions All bacterial strains used in this Janus kinase (JAK) study are listed in Table 1. Overnight cultures of E. coli strains were routinely carried out at 37°C on LB agar or in LB broth with shaking. One Shot® TOP10 chemically competent E. coli (Invitrogen, Carlsbad, CA) was used as a cloning host for TA-cloning procedures. E. coli MG1655 and TRMG1655 (csrA::Kan) were obtained from T. Romeo (University of Florida). When appropriate, E. coli strains were selected in LB medium using ampicillin (100 μg/ml) or kanamycin (50 μg/ml). Cloned genes were induced by the addition of 0.002% L-arabinose to the growth media. C. jejuni strain 81–176 was grown on MH agar at 42°C under microaerophilic contitions (10% CO2, 10% O2, and 80% N2) supplemented with 5% sheep’s blood (Remel, Lenexa, KS).