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.

Identification of Campylobacter jejuni Genes Contributing to Acid Adaptation by Transcriptional Profiling and Genome-Wide Mutagenesis

“…RNA was extracted from cells grown to early midlogarithmic phase (OD 600 ϭ 0.9) at 37°C under microaerophilic conditions as described previously (65). Briefly, RNA turnover was prevented by mixing each sample (25 ml) with a cold RNA stabilization solution (2.5 ml of 10% [vol/vol] buffer-saturated phenol in ethanol) on ice.…”

“…Bacteria were maintained and grown at 37°C on Mueller-Hinton (MH) agar plates or in biphasic MH cultures under microaerophilic conditions (83% N 2 , 4% H 2 , 8% O 2 , and 5% CO 2 ) in a MACS-VA500 microaerophilic workstation (Don Whitley, West Yorkshire, England). The C. jejuni NCTC 11168 fur deletion mutant (65) and the kpsM insertion mutant (77) used in this study were described previously.…”

“…Data were collected and analyzed as described previously (65). Briefly, microarray slides were scanned at 532-nm (Cy3) and 635-nm (Cy5) wavelengths using a laser-activated confocal scanner (ScanArray Gx; Perkin Elmer) at a 10-m resolution.…”

“…Aminoallyl-dUTP was included in the reverse transcription reaction to permit the labeling of the cDNA with the monoreactive fluors indocarbocyanine (Cy3 dye; used to label pH 7.0 control samples) and indodicarbocyanine (Cy5 dye; used to label test samples) (Amersham Biosciences). Fluorescent labeling of the probes was described previously (65). The labeled cDNA probes from each acid stress condition (e.g., pH 6.5) were individually cohybridized with labeledcDNA probes from cells grown at pH 7.0 on microarray slides.…”

CmeR Functions as a Pleiotropic Regulator and Is Required for Optimal Colonization of Campylobacter jejuni In Vivo

“…Fur functions as a transcriptional repressor and controls iron homeostasis in C. jejuni (31,45). Mutation of Fur affected the expression of 53 genes and significantly reduced the colonization of chickens by Campylobacter (31).…”