Properties of Arsenite Efflux Permeases (Acr3) from Alkaliphilus metalliredigens and Corynebacterium glutamicum

Hseuh-Liang Fu,Yuling Meng,Efrén Ordóñez,Almudena F Villadangos,Hiranmoy Bhattacharjee,José A Gil,Luís M Mateos,Barry P Rosen

doi:10.1074/jbc.m109.011882

Abstract

Members of the Acr3 family of arsenite permeases confer resistance to trivalent arsenic by extrusion from cells, with members in every phylogenetic domain. In this study bacterial Acr3 homologues from Alkaliphilus metalliredigens and Corynebacterium glutamicum were cloned and expressed in Escherichia coli. Modification of a single cysteine residue that is conserved in all analyzed Acr3 homologues resulted in loss of transport activity, indicating that it plays a role in Acr3 function. The results of treatment with thiol reagents suggested that the conserved cysteine is located in a hydrophobic region of the permease. A scanning cysteine accessibility method was used to show that Acr3 has 10 transmembrane segments, and the conserved cysteine would be predicted to be in the fourth transmembrane segment.

Highlights

Arsenic is a carcinogen that ranks first on the Superfund List of Hazardous Substances
The third arsenic resistance transporter is Acr3, which is a member of the BART superfamily and includes members found in bacteria, archaea, and fungi and is more widely distributed than members of the ArsB family [10]
We examined the properties of Acr3 orthologues from Alkaliphilus metalliredigens and Corynebacterium glutamicum

Summary

EXPERIMENTAL PROCEDURES

Plasmids, Media, and Reagents—Strains and plasmids used are given in supplemental Tables 1 and 2. The DNA fragment was ligated into vector plasmid pTrcHis2A digested, generating plasmid pTrcHis2A-AmAcr3-His. A similar strategy was used to clone the Cgacr gene using chromosomal DNA from C. glutamicum strain ATCC 13032 [19] and the corresponding primers indicated in supplemental Table 3. The Cgacr gene was PCR-amplified using the primer pair Cgacr3-NcoI forward/HindIII reverse, and the isolated 1.1-kb band was NcoI and HindIII digested and further ligated into pTrcHis2A generating plasmid pTrcHis2A-CgAcr3-His. Mutants in acr genes from both organisms were generated using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using the primers listed in supplemental Table 4. For assays in which the cells were treated with thiol-modifying reagents, 50 ml of freshly grown cells were centrifuged and washed twice at A600 nm ϭ 1 with buffer C lacking MgSO4 and suspended in 25 ml of the same buffer. 1), indicating that there might be a fundamental mechanistic difference between ArsB and Acr

Conserved Cysteine Plays a Role in

RESULTS

DISCUSSION