Identification of an arsenic resistance and arsenic-sensing system in Campylobacter jejuni

Abstract

Arsenic is commonly present in the natural environment and is also used as a feed additive for animal production. Poultry is a major reservoir for Campylobacter jejuni, a major food-borne human pathogen causing gastroenteritis. It has been shown that Campylobacter isolates from poultry are highly resistant to arsenic compounds, but the molecular mechanisms responsible for the resistance have not been determined, and it is unclear if the acquired arsenic resistance affects the susceptibility of Campylobacter spp. to other antimicrobials. In this study, we identified a four-gene operon that contributes to arsenic resistance in Campylobacter. This operon encodes a putative membrane permease (ArsP), a transcriptional repressor (ArsR), an arsenate reductase (ArsC), and an efflux protein (Acr3). PCR analysis of various clinical C. jejuni isolates indicated a significant association of this operon with elevated resistance to arsenite and arsenate. Gene-specific mutagenesis confirmed the role of the ars operon in conferring arsenic resistance. It was further shown that this operon is subject to regulation by ArsR, which directly binds to the ars promoter and inhibits the transcription of the operon. Arsenite inhibits the binding of ArsR to the ars promoter DNA and induces the expression of the ars genes. Mutation of the ars genes did not affect the susceptibility of C. jejuni to commonly used antibiotics. These results identify the ars operon as an important mechanism for arsenic resistance and sensing in Campylobacter.

FIG. 1.

Genomic organization of the ars operon and confirmation of the generated ars mutants. (A) Diagram of the ars genes in C. jejuni strains RM1221, CB5-28, NCTC 11168, and 81-176. ORFs are indicated by boxed arrows. The single promoter in front of arsP is depicted by a curved arrow. In strains NCTC 11168 and 81-176, arsC and acr3 are absent and arsP and arsR are degenerate due to frameshift mutations (indicated by black strips in the genes). An additional arsR homologue in NCTC 11168 and 81-176 (named arsR2) is indicated with a black arrow. (B) PCR confirmation of the arsC::Kan^r insertion in LP001. Lane 1, 1-kb DNA ladder; lane 2, LP001; and lane 3, wild-type CB5-28. (C) PCR confirmation of the acr3::cat insertion in LP002. Lane 1, 1-kb DNA ladder; lane 2, wild-type CB5-28; and lane 3, LP002. (D) PCR confirmation of the in-frame deletion in arsR in LP004. Lane 1, 100-bp DNA ladder; lane 2, wild-type CB5-28; and lane 3, LP004.

FIG. 2.

Expression levels of the ars genes as determined by qRT-PCR. (A) Elevated expression of arsP, arsC, and acr3 in the arsR deletion mutant (LP004) compared to the wild-type CB5-28. (B) Induction of arsP, arsC, and acr3 in CB5-28 by arsenite [As(III)] and arsenate [As(V)] at a subinhibitory concentration. In both panels, each bar represents the mean ± standard deviation of three independent experiments.

FIG. 3.

Regulation of the ars operon. (A) Sequence features of the ars promoter. The start codon (ATG) of arsP is bold. The transcript start (base T) is marked as +1, and a curved arrow indicates the direction of transcription. The predicated ribosomal binding site (RBS) is underlined. The putative −10, −16, and −35 boxes are underlined. The inverted repeat is overlined with inverted arrows. (B) Alignment of the ArsR binding sites among C. jejuni, B. subtilis, and Synechocystis. The conserved sequences are bold and underlined. (C) An electropherogram showing the result of primer extension analysis of the ars promoter. The extension product is indicated by an arrowhead. The 5-6-carboxyfluorescein-labeled size standards are marked with asterisks. (D) Binding of ArsR to the ars promoter as determined by EMSA. A 164-bp DIG-labeled DNA fragment containing the ars promoter was incubated with 0 ng (lane 1), 5 ng (lane 2), 10 ng (lane 3), and 20 ng (lane 4) of rArsR. (E) Competition assay to confirm the binding specificity of ArsR. Each binding reaction was conducted with the DIG-labeled promoter DNA and 20 ng of ArsR in the presence of the competing DNA (a 50-bp probe containing the IR). The competing DNA was added at a zerofold (lane 2), 50-fold (lane 3), 100-fold (lane 4), and 200-fold (lane 5) molar excess of the DIG-labeled promoter DNA. Lane 1 contains the DIG-labeled promoter DNA only (no rArsR). (F) Competition assay using a DNA fragment amplified from the coding sequence of arsP, which was added with no (lane 2), 50-fold (lane 3), 100-fold (lane 4), and 200-fold (lane 5) excess of the DIG-labeled promoter DNA. Lane 1 contains the DIG-labeled promoter DNA only.

FIG. 4.

Effect of arsenite (A) and arsenate (B) on the formation of ArsR-DNA complexes as determined by EMSA. The concentrations of arsenic used in the reaction are indicated at top of each panel. Each reaction was performed with 5 ng of DIG-labeled ars promoter DNA. +, 20 ng of rArsR was added in the reaction; −, rArsR was absent in the reaction.