Veterinary Microbiology and Preventive Medicine Veterinary Microbiology and Preventive Medicine Publications 8-2009 Identification of an Arsenic Resistance and Arsenic- Sensing System in Campylobacter jejuni Liping Wang Iowa State University Byeonghwa Jeon Iowa State University Orhan Sahin Iowa State University, [email protected] Qijing Zhang Iowa State University, [email protected] Follow this and additional works at: http://lib.dr.iastate.edu/vmpm_pubs Part of the Molecular Genetics Commons, Veterinary Microbiology and Immunobiology Commons, and the Veterinary Preventive Medicine, Epidemiology, and Public Health Commons The ompc lete bibliographic information for this item can be found at http://lib.dr.iastate.edu/ vmpm_pubs/165. For information on how to cite this item, please visit http://lib.dr.iastate.edu/ howtocite.html. This Article is brought to you for free and open access by the Veterinary Microbiology and Preventive Medicine at Iowa State University Digital Repository. It has been accepted for inclusion in Veterinary Microbiology and Preventive Medicine Publications by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. 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 utm agenesis 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. Disciplines Genetics and Genomics | Molecular Genetics | Veterinary Microbiology and Immunobiology | Veterinary Preventive Medicine, Epidemiology, and Public Health Comments This article is published as Wang, Liping, Byeonghwa Jeon, Orhan Sahin, and Qijing Zhang. "Identification of an arsenic resistance and arsenic-sensing system in Campylobacter jejuni." Applied and environmental microbiology 75, no. 15 (2009): 5064-5073. doi: 10.1128/AEM.00149-09. Posted with permission. This article is available at Iowa State University Digital Repository: http://lib.dr.iastate.edu/vmpm_pubs/165 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2009, p. 5064–5073 Vol. 75, No. 15 0099-2240/09/$08.00ϩ0 doi:10.1128/AEM.00149-09 Copyright © 2009, American Society for Microbiology. All Rights Reserved. Identification of an Arsenic Resistance and Arsenic-Sensing System in Campylobacter jejuniᰔ† Liping Wang,1,2 Byeonghwa Jeon,1 Orhan Sahin,1 and Qijing Zhang1* Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, Iowa 50011,1 and College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China2 Downloaded from Received 21 January 2009/Accepted 1 June 2009 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 deter- mined, 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 http://aem.asm.org/ 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. on September 29, 2017 by IOWA STATE UNIVERSITY Arsenic is a toxic metalloid present in the natural environ- Sinorhizobium meliloti (31, 59, 60). The newly identified ment. At high levels, arsenic is toxic to most cells, including arsenic resistance gene arsM encodes an arsenite S-adeno- microbial organisms (32, 33). To survive this toxicity, bacterial sylmethionine methyltransferase that methylates arsenite to organisms have evolved multiple mechanisms for arsenic de- volatile trimethylarsine, contributing to arsenic detoxifica- toxification, including extrusion mediated by efflux transport- tion in bacteria (39). In bacteria, the ars genes can be carried ers, reduction of arsenate [As(V)] to arsenite [As(III)], which on either plasmids or chromosomes and are often organized is subsequently extruded by efflux transporters, and methyl- in operons, such as arsRBC, arsRABC, and arsRDABC, but ation of As(III) by S-adenosylmethionine methyltransferase in some cases the ars genes exist singly (3–5, 10, 29, 45, 50). (39, 41). These resistance mechanisms are encoded by various Campylobacter jejuni is a leading cause of bacterial gastro- ars genes, and the best-characterized ones include arsR, arsA, enteritis in humans and is estimated to be responsible for 400 arsB, arsC, arsD, arsH, and arsM. The arsR gene encodes to 500 million cases of diarrhea each year worldwide (43). As an As(III)-responsive transcriptional repressor, which controls a food-borne pathogen, C. jejuni is widely distributed in food- the expression of other ars genes by binding to target promot- producing animals, including both livestock and poultry (18). ers (42, 55). ArsA and ArsB form an oxyanion-translocating In animal reservoirs, C. jejuni colonizes the intestinal tracts of complex, in which ArsA functions as an ATPase, while ArsB animals and usually does not cause clinical diseases. But once is a membrane efflux transporter (41). In addition, ArsB can transmitted to a human host, it can cause severe gastroenteritis function alone (without ArsA) in the extrusion of arsenite (30). Transmission of Campylobacter bacteria from animals to (9). arsC encodes a arsenate reductase, which converts ar- humans is mainly via contaminated poultry, water, and raw senate to arsenite (20). ArsD was previously thought to be milk (18). Among domestic animals, poultry is considered the an inducer-independent regulatory protein, but recent work major reservoir for C. jejuni (61). The high prevalence of C. indicated that it functions as an arsenic metallochaperone jejuni on chicken farms suggests that this organism is well transferring As(III) to ArsA and increasing the rate of ar- senic extrusion (26, 27, 57). arsH encodes an NADPH-flavin adapted in the poultry production system, but the mechanisms mononucleotide oxidoreductase and has been shown to be involved in the adaptation are poorly understood. Understand- involved in arsenic resistance in Yersinia enterocolitica and ing the adaptive mechanisms may facilitate the development of intervention strategies to control the prevalence of this patho- genic organism. * Corresponding author. Mailing address: Department of Veteri- During the past decade, Campylobacter species have become nary Microbiology and Preventive Medicine, 1116 Veterinary Medi- increasingly resistant to clinically important antibiotics such cine Complex, Iowa State University, Ames, IA 50011. Phone: (515) as fluoroquinolones and macrolides (13, 35). Antimicrobials 294-2038. Fax: (515) 294-8500. E-mail: [email protected]. including organoarsenical compounds, such as 3-nitro-4-hy- † Supplemental material for this article may be found at http://aem .asm.org/. droxyphenylarsonate (roxarsone), are commonly used in ᰔ Published ahead of print on 5 June 2009. poultry production in the United States and other countries 5064 VOL. 75, 2009 MECHANISMS OF ARSENIC RESISTANCE IN CAMPYLOBACTER 5065 (6). Roxarsone is given to broiler chickens as a feed