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The foodborne bacterial pathogen Campylobacter jejuni is an obligate microaerophile that is exposed to atmospheric oxygen during transmission through the food chain. Survival under aerobic conditions requires the concerted control of oxidative stress systems, which in C. jejuni are intimately connected with iron metabolism via the PerR and Fur regulatory proteins. Here, we have characterized the roles of C. jejuni PerR in oxidative stress and motility phenotypes, and its regulon at the level of transcription, protein expression and promoter interactions. Insertional inactivation of perR in the C. jejuni reference strains NCTC 11168, 81-176 and 81116 did not result in any growth deficiencies, but strongly increased survival in atmospheric oxygen conditions, and allowed growth around filter discs infused with up to 30 % H2O2 (8.8 M). Expression of catalase, alkyl hydroperoxide reductase, thioredoxin reductase and the Rrc desulforubrerythrin was increased in the perR mutant, and this was mediated at the transcriptional level as shown by electrophoretic mobility shift assays of the katA, ahpC and trxB promoters using purified PerR. Differential RNA-sequencing analysis of a fur perR mutant allowed the identification of eight previously unknown transcription start sites of genes controlled by Fur and/or PerR. Finally, inactivation of perR in C. jejuni did not result in reduced motility, and did not reduce killing of Galleria melonella wax moth larvae. In conclusion, PerR plays an important role in controlling oxidative stress resistance and aerobic survival of C. jejuni, but this role does not extend into control of motility and associated phenotypes.

The food-borne bacterial pathogen Campylobacter jejuni efficiently utilizes organic acids such as lactate and formate for energy production. Formate is rapidly metabolized via the activity of the multisubunit formate dehydrogenase (FDH) enzyme, of which the FdhA subunit is predicted to contain a selenocysteine (SeC) amino acid. In this study we investigated the function of the cj1500 and cj1501 genes of C. jejuni, demonstrate that they are involved in selenium-controlled production of FDH, and propose the names fdhT and fdhU, respectively. Insertional inactivation of fdhT or fdhU in C. jejuni resulted in the absence of FdhA and FdhB protein expression, reduced fdhABC RNA levels, the absence of FDH enzyme activity, and the lack of formate utilization, as assessed by (1)H nuclear magnetic resonance. The fdhABC genes are transcribed from a single promoter located two genes upstream of fdhA, and the decrease in fdhABC RNA levels in the fdhU mutant is mediated at the posttranscriptional level. FDH activity and the ability to utilize formate were restored by genetic complementation with fdhU and by supplementation of the growth media with selenium dioxide. Disruption of SeC synthesis by inactivation of the selA and selB genes also resulted in the absence of FDH activity, which could not be restored by selenium supplementation. Comparative genomic analysis suggests a link between the presence of selA and fdhTU orthologs and the predicted presence of SeC in FdhA. The fdhTU genes encode accessory proteins required for FDH expression and activity in C. jejuni, possibly by contributing to acquisition or utilization of selenium.

Campylobacter jejuni is a leading cause of bacterial gastroenteritis in the developed world. The role of a homologue of the negative transcriptional regulatory protein HspR, which in other organisms participates in the control of the heat-shock response, was investigated. Following inactivation of hspR in C. jejuni, members of the HspR regulon were identified by DNA microarray transcript profiling. In agreement with the predicted role of HspR as a negative regulator of genes involved in the heat-shock response, it was observed that the transcript amounts of 13 genes were increased in the hspR mutant, including the chaperone genes dnaK, grpE and clpB, and a gene encoding the heat-shock regulator HrcA. Proteomic analysis also revealed increased synthesis of the heat-shock proteins DnaK, GrpE, GroEL and GroES in the absence of HspR. The altered expression of chaperones was accompanied by heat sensitivity, as the hspR mutant was unable to form colonies at 44 degrees C. Surprisingly, transcriptome analysis also revealed a group of 17 genes with lower transcript levels in the hspR mutant. Of these, eight were predicted to be involved in the formation of the flagella apparatus, and the decreased expression is likely to be responsible for the reduced motility and ability to autoagglutinate that was observed for hspR mutant cells. Electron micrographs showed that mutant cells were spiral-shaped and carried intact flagella, but were elongated compared to wild-type cells. The inactivation of hspR also reduced the ability of Campylobacter to adhere to and invade human epithelial INT-407 cells in vitro, possibly as a consequence of the reduced motility or lower expression of the flagellar export apparatus in hspR mutant cells. It was concluded that, in C. jejuni, HspR influences the expression of several genes that are likely to have an impact on the ability of the bacterium to successfully survive in food products and subsequently infect the consumer.

Campylobacter jejuni is a zoonotic pathogen and the most common cause of bacterial foodborne diarrhoeal illness worldwide. To establish intestinal colonization prior to either a commensal or pathogenic interaction with the host, C. jejuni will encounter iron-limited niches where there is likely to be intense competition from the host and normal microbiota for iron. To gain a better understanding of iron homeostasis and the role of ferric uptake regulator (Fur) in iron acquisition in C. jejuni, a proteomic and transcriptome analysis of wild-type and fur mutant strains in iron-rich and iron-limited growth conditions was carried out. All of the proposed iron-transport systems for haemin, ferric iron and enterochelin, as well as the putative iron-transport genes p19, Cj1658, Cj0177, Cj0178 and cfrA, were expressed at higher levels in the wild-type strain under iron limitation and in the fur mutant in iron-rich conditions, suggesting that they were regulated by Fur. Genes encoding a previously uncharacterized ABC transport system (Cj1660-Cj1663) also appeared to be Fur regulated, supporting a role for these genes in iron uptake. Several promoters containing consensus Fur boxes that were identified in a previous bioinformatics search appeared not to be regulated by iron or Fur, indicating that the Fur box consensus needs experimental refinement. Binding of purified Fur to the promoters upstream of the p19, CfrA and CeuB operons was verified using an electrophoretic mobility shift assay (EMSA). These results also implicated Fur as having a role in the regulation of several genes, including fumarate hydratase, that showed decreased expression in response to iron limitation. The known PerR promoters were also derepressed in the C. jejuni Fur mutant, suggesting that they might be co-regulated in response to iron and peroxide stress. These results provide new insights into the effects of iron on metabolism and oxidative stress response as well as the regulatory role of Fur.