The fdh genes are divided into two operons that are transcribed selleck kinase inhibitor in the same orientation and separated by ~ 67 nucleotides. The operon downstream of fdhA contains fdhD and Cj1507c (encodes the DNA binding protein ModE) . However, the introduction of the individual native genes into the ΔfdhA as well as the other RPs mutants
resulted in the complementation of the impacted phenotypes (motility, H2O2 resistance and biofilm formation) (Additional file 1: Table S1). Conclusions In this study, we showed that RPs contribute differentially to key C. jejuni phenotypes in a manner that depends on the temperature and/or oxygen content of the environment (Table 1). Consequently, we conclude that these proteins partially bestow C. jejuni with its remarkable ability to adapt and survive in a variety of niches, a characteristic that is crucial for understanding this bacterium’s prevalence, persistence and success as a pathogen. Methods Bacterial strains and growth see more conditions RPs mutants were previously generated in the C. jejuni NCTC-11168 background and included ΔnapA (encoding a subunit of the nitrate reductase), ΔnrfA (encoding a subunit of the nitrite reductase), ΔfdhA (encoding a subunit of the formate dehydrogenase), ΔhydB (encoding a subunit of the hydrogenase), and ΔmfrA (encoding a subunit of the methylmenaquinol:fumarate reductase) [8–10]. All strains were cultured
on MH agar under microaerobic conditions (85% N2, 10% CO2, 5% O2). Incubation at 37°C or 42°C was performed for comparison between temperatures, while oxygen-limited conditions were generated using the BD GasPak Sachets system, which constitutes an atmosphere of less than 1% oxygen and greater than or equal to 13% carbon dioxide (BD diagnostics, NJ, USA). In this paper, oxygen-limited atmosphere was designated as anaerobic to make a clear distinction with microaerobic conditions. Leaked horse blood (5%, Oxoid, KS, USA), antibiotics (chloramphenicol: 20 μg.ml-1), and the Campylobacter selective supplement (SR155E, Oxoid, KS, USA) were added to the MH medium when necessary. For growth curve analysis, the mutants and wildtype strain were inoculated
into MH broth and incubated shaking (200 rpm) at different temperature and oxygen PAK5 conditions. Growth was monitored by measuring optical density (λ = 600 nm) at different time points. Construction of complementation strains To construct complementation strains, individual native RPs genes (napA, nrfA, mfrA, hydB, and fdhA) along with their potential promoter sequences were amplified from the genomic DNA of C. jejuni NCTC-11168 using specific primers (Additional file 2: Table S2). The primers were designed to include restriction sites that facilitate directional cloning. The PCR products were digested, purified and ligated into a similarly digested pRY108 plasmid using a Fast-Link DNA ligation kit (Epicentre). The ligated product was then cloned into Library Efficiency DH5α E.