Iron acquisition and regulation in Campylobacter jejuni

Abstract

Iron affects the physiology of bacteria in two different ways: as a micronutrient for bacterial growth and as a catalyst for the formation of hydroxyl radicals. In this study, we used DNA microarrays to identify the C. jejuni genes that have their transcript abundance affected by iron availability. The transcript levels of 647 genes were affected after the addition of iron to iron-limited C. jejuni cells. Several classes of affected genes were revealed within 15 min, including immediate-early response genes as well as those specific to iron acquisition and metabolism. In contrast, only 208 genes were differentially expressed during steady-state experiments comparing iron-rich and iron-limited growth conditions. As expected, genes annotated as being involved in either iron acquisition or oxidative stress defense were downregulated during both time course and steady-state experiments, while genes encoding proteins involved in energy metabolism were upregulated. Because the level of protein glycosylation increased with iron limitation, iron may modulate the level of C. jejuni virulence by affecting the degree of protein glycosylation. Since iron homeostasis has been shown to be Fur regulated in C. jejuni, an isogenic fur mutant was used to define the Fur regulon by transcriptome profiling. A total of 53 genes were Fur regulated, including many genes not previously associated with Fur regulation. A putative Fur binding consensus sequence was identified in the promoter region of most iron-repressed and Fur-regulated genes. Interestingly, a fur mutant was found to be significantly affected in its ability to colonize the gastrointestinal tract of chicks, highlighting the importance of iron homeostasis in vivo. Directed mutagenesis of other genes identified by the microarray analyses allowed the characterization of the ferric enterobactin receptor, previously named CfrA. Chick colonization assays indicated that mutants defective in enterobactin-mediated iron acquisition were unable to colonize the gastrointestinal tract. In addition, a mutation in a receptor (Cj0178) for an uncharacterized iron source also resulted in reduced colonization potential. Overall, this work documents the complex response of C. jejuni to iron availability, describes the genetic network between the Fur and iron regulons, and provides insight regarding the role of iron in C. jejuni colonization in vivo.

FIG.1.

Hierarchical cluster analysis of genes found to be significantly up- or downregulated at mid-log phase. Going from left to right, the columns represent the transcriptome change at 1, 3, 5, 7, 9, and 15 min after the addition of ferrous sulfate and at mid-log phase. The intensity of the color is proportional to the change, as represented by the scale at the bottom. Detailed gene names are shown for each cluster.

FIG. 2.

Operon mapping by RT-PCR analysis of iron- and Fur-regulated genes. The template RNA was purified from mid-log-phase bacteria grown in iron-rich and iron-limited MEM-α for iron-induced (exbB3, exbD3, and Cj0111) and iron-repressed genes, respectively. Predicted RT-PCR fragments with gene names are shown at the bottom. The gel lanes match the RT-PCR fragment labels. Lanes M1 and M2 show the 1-kb and 100-bp DNA ladders, respectively.

FIG. 3.

Whole-cell lysates of C. jejuni proteins analyzed on SDS-12.5% PAGE. (A) Silver staining; (B) lectin blotting. Total proteins were prepared from C. jejuni grown to mid-log phase in iron-limited medium (MEM-α; lanes labeled 0 min and mid-log) or iron-rich medium (MEM-α with 40 μM FeSO₄). Lanes labeled 5 min, 9 min, and 15 min correspond to the protein profiles of C. jejuni grown in iron-limited medium at 5, 9, and 15 min after the addition of ferrous sulfate, respectively. The lane labeled mid-log (+Fe) corresponds to the protein profile of C. jejuni grown to mid-log phase in the iron-rich medium.

FIG. 4.

Hierarchical cluster analysis of Fur-regulated genes. Columns 1 to 7 correspond to C. jejuni gene expression changes in response to the addition of iron to an iron-limited medium at 1, 3, 5, 7, 9, and 15 min and at mid-log phase, respectively. Columns 8 and 9 represent the change in transcript level of the wild-type C. jejuni strain compared to the fur mutant grown to mid-log phase in iron-limited medium and 15 min after the addition of FeSO₄, respectively. The shade of red and green indicates the level of change. Genes are subgrouped into four clusters, named A, B, C, and D.

FIG. 5.

Colonization properties of the C. jejuni mutant strains in the chick model. Groups of five chicks were inoculated with the C. jejuni wild-type (wt) strain NCTC 11168 or with the fur, cfrA, ceuE, and Cj0178 mutants (as indicated) at a dose of 1 × 10⁵ to 3 × 10⁵ CFU. The columns represent the means, and the error bars indicate the standard deviations.

FIG. 6.

Sequence logo of the potential Fur binding site. The height of each letter indicates the relative frequency of that base at that position. The height of each stack of letters corresponds to the sequence conservation at that position.

FIG. 7.

Enterobactin growth promotion tests of the wild-type strain C. jejuni NCTC 11168 and the mutants ΔCj0178, ΔceuE, and ΔcfrA. A halo of growth around the filter paper disk containing 10 μl of enterobactin (10 mM) indicates utilization of the siderophore by the tested strain. The diameters of growth promotion zones ± standard deviation are shown in parentheses.

FIG. 8.

Diagram of the genetic organization of the cfrA, ceuE, and Cj0178 mutants described in this study. Each mutant was constructed by site-directed deletion and insertional mutagenesis with the chloramphenicol resistance marker (Cm^r). The length of each deletion is shown in parentheses (in base pairs). The solid black arrow represents the position and orientation of the inserted chloramphenicol antibiotic resistance cassette in each gene. Given the absence of a transcriptional terminator downstream of the chloramphenicol resistance gene and the orientation of this gene with respect to the mutated gene, the constructed mutations are likely nonpolar. All genes are drawn approximately to scale.