Campylobacter jejuni gene expression in response to iron limitation and the role of Fur

Abstract

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.

Oxygen- and NssR-dependent Globin Expression and Enhanced Iron Acquisition in the Response of Campylobacter to Nitrosative Stress

“…However, some of the differences observed between the two-dimensional gel and the microarray results are not explained by membrane protein issues. P19 (Cj1959) is readily visible on a protein gel and changes in this and other iron associated proteins occur under iron-limiting conditions (41). The changes in transcript for P19 were much lower (7.94-fold) in this study with no observed difference in protein compared with the iron depletion study (110-fold) where an 11-fold increase in protein was also found.…”

“…For each microarray slide, one sample was labeled with Cy3-dCTP, whereas another sample was labeled with Cy5-dCTP. Methods for reverse transcription and hybridization, scanning, and data analysis were as before (41). A control experiment (comparison of two mRNA samples from replicated independent cultures) was used to estimate the boundaries between genes that were equally and differentially expressed in the two samples.…”

“…The primers used are listed in supplemental . Extracts were kept on ice for 20 min before centrifuging at 18,500 ϫ g for 20 min; supernatants were analyzed by two-dimensional proteomic analysis (41). Protein concentrations were determined using the 2D Quant Kit (GE Healthcare).…”

“…Indeed, 18 up-regulated genes (9 of which are in Table 1) were previously reported (41) to undergo significant transcriptional changes under low iron conditions. Furthermore, katA, ahpC, and the genes encoding the thioredoxin and thioredoxin reductase (see below) are all iron-regulated by the two C. jejuni Fur homologues, Fur and PerR (41,48). In C. jejuni, perR adopts the role of oxyR of E. coli, having a role in peroxide stress (48,49).…”

The hrcA and hspR regulons of Campylobacter jejuni

“…The third upregulated gene, Cj0168c, encodes a putative integral membrane protein that is conserved among Campylobacter spp., and shows 34 % amino acid identity to a MotA/TolQ/ ExbB proton channel protein from Shewanella frigidimarina, but otherwise has no obvious homologues. Interestingly, this protein is also upregulated by iron limitation (Holmes et al, 2005). As HrcA binds to the CIRCE consensus sequence, we searched for CIRCE sequences in the C. jejuni NCTC 11168 genome using fuzznuc, part of the EMBOSS software suite.…”