The contribution of ArsB to arsenic resistance in Campylobacter jejuni

Abstract

Arsenic, a toxic metalloid, exists in the natural environment and its organic form is approved for use as a feed additive for animal production. As a major foodborne pathogen of animal origin, Campylobacter is exposed to arsenic selection pressure in the food animal production environments. Previous studies showed that Campylobacter isolates from poultry were highly resistant to arsenic compounds and a 4-gene operon (containing arsP, arsR, arsC, and acr3) was associated with arsenic resistance in Campylobacter. However, this 4-gene operon is only present in some Campylobacter isolates and other arsenic resistance mechanisms in C. jejuni have not been characterized. In this study, we determined the role of several putative arsenic resistance genes including arsB, arsC2, and arsR3 in arsenic resistance in C. jejuni and found that arsB, but not the other two genes, contributes to the resistance to arsenite and arsenate. Inactivation of arsB in C. jejuni resulted in 8- and 4-fold reduction in the MICs of arsenite and arsenate, respectively, and complementation of the arsB mutant restored the MIC of arsenite. Additionally, overexpression of arsB in C. jejuni 11168 resulted in a 16-fold increase in the MIC of arsenite. PCR analysis of C. jejuni isolates from different animals hosts indicated that arsB and acr3 (the 4-gene operon) are widely distributed in various C. jejuni strains, suggesting that Campylobacter requires at least one of the two genes for adaptation to arsenic-containing environments. These results identify ArsB as an alternative mechanism for arsenic resistance in C. jejuni and provide new insights into the adaptive mechanisms of Campylobacter in animal food production environments.

Figure 1. The membrane topologies of ArsB predicted by TMHMM.

The transmembrane domains are shaded in red. The blue line indicates loops facing inside (cytoplasma), while the pink line depicts loops facing outside (periplasmic space). The numbers at the bottom indicate the amino acid numbers in ArsB.

Figure 2. Diagrams showing the genomic localizations and mutant generation of various ars genes.

(A) Genomic organization of arsB and inactivation of arsB by insertion of a choramphenicol resistance cassette. (B) Genomic localization of arsC2 and inactivation of this gene by insertion of a kanamycin resistance cassette. (C) arsR3 and its flanking gene. Inactivation of arsR3 was accomplished by insertion of a kanamycin resistance cassette. (D) Complementation of the arsB mutant by insertion of an extra copy of the arsB gene downstream of 16S rRNA. (E) The ars operon identified in C. jejuni CB5-28 and inactivation of arsC by insertion of a kanamycin resistance cassette.

Figure 3. Dose-dependent induction of arsB in 11168 by arsenite and arsenate.

The concentrations of the arsenic compounds supplemented into the culture media are labeled at the bottom of the panel.