A Na ⁺ :H ⁺ Antiporter and a Molybdate Transporter Are Essential for Arsenite Oxidation in Agrobacterium tumefaciens

Abstract

Transposon Tn5-B22 mutagenesis was used to identify genetic determinants required for arsenite [As(III)] oxidation in an Agrobacterium tumefaciens soil isolate, strain 5A. In one mutant, the transposon interrupted modB, which codes for the permease component of a high-affinity molybdate transporter. In a second mutant, the transposon insertion occurred in mrpB, which is part of a seven-gene operon encoding an Mrp-type Na ؉ :H ؉ antiporter complex. Complementation experiments with mod and mrp operons PCR cloned from the genomesequenced A. tumefaciens strain C58 resulted in complementation back to an As(III)-oxidizing phenotype, confirming that these genes encode activities essential for As(III) oxidation in this strain of A. tumefaciens. As expected, the mrp mutant was extremely sensitive to NaCl and LiCl, indicating that the Mrp complex in A. tumefaciens is involved in Na ؉ circulation across the membrane. Gene expression studies (lacZ reporter and reverse transcriptase PCR experiments) failed to show evidence of transcriptional regulation of the mrp operon in response to As(III) exposure, whereas expression of the mod operon was found to be up-regulated by As(III) exposure. In each mutant, the loss of As(III)-oxidizing capacity resulted in conversion to an arsenate [As(V)]-reducing phenotype. Neither mutant was more sensitive to As(III) than the parental strain.Microbe-arsenic interactions are viewed as a major driver of arsenic (As) chemical speciation in nature, which in turn is a critical factor in determining As fate and transport in the environment (reviewed in references 19 and 38). Many types of As transformations have been documented in various microorganisms (6,14,18,34,41,47), although those currently seen to dominate As speciation in the environment involve either arsenite [H 3 AsO 3 , As(III)] oxidation or arsenate [HAsO 4 2Ϫ , As(V)] reduction. These redox transformation reactions are generally thought to be used by microorganisms either for detoxification or for generating cellular energy to support growth (see reviews in references 19, 38, 49 and 50).Detoxification-based As(V) reduction has been documented to occur in microorganisms throughout the domains Bacteria and Archaea (38, 49), and involves As(V) reduction to As(III) via an As(V) reductase, with the As(III) then extruded by the ArsB efflux pump that is efficient at removing As(III) and antimonite [Sb(III)]. This process, as well as the genes encoding the enzymes and regulatory proteins involved, has been extensively studied and recently reviewed by Silver and Phung (49). Dissimilatory As(V) reduction has also been documented to occur in numerous prokaryotes in both prokaryotic domains (38), although its original discovery was more recent (2), and as a consequence, far less is known about the genetic determinants required for anaerobic As(V) respiration. Dissimilatory As(V) reductases from Chrysiogenes arsenatis (25) and Bacillus selenitireducens (1) have been purified and characterized, and the arr genes have been identified in Shewanella...

New Insights into Microbial Oxidation of Antimony and Arsenic

“…1A). As expected for these culturing conditions and genotypes (12,13), expression of the As(III) oxidase structural genes, aoxAB, was observed in the wild-type strain but not in the aoxR regulatory mutant (Fig. 1B).…”

“…These observations suggest the possibility that the same enzymatic pathways used for As(III) oxidation may also be used for Sb(III). In recent studies regarding the genetics underlying As(III) oxidation in Agrobacterium tumefaciens, we discovered regulatory (aoxR) and Na ϩ :H ϩ antiporter (mrpB) mutants which are defective in As(III) oxidation (12,13). In subsequent investigations, we found the wild-type A. tumefaciens strain to also be capable of oxidizing Sb(III).…”

Catalytic Properties of Staphylococcus aureus and Bacillus Members of the Secondary Cation/Proton Antiporter-3 (Mrp) Family Are Revealed by an Optimized Assay in an Escherichia coli Host

“…The putative Mrp complex is also expected to include one or more anion transporters whose particular substrate(s) may be species specific (56,58). This is suggested by roles of Mrp in bile salt and arsenite resistance in B. subtilis and Agrobacterium tumefaciens, respectively (19,20,27). A multifunctional complex could provide synergies such as the presentation of a large protein surface area on the external membrane surface.…”

Single Site Mutations in the Hetero-oligomeric Mrp Antiporter from Alkaliphilic Bacillus pseudofirmus OF4 That Affect Na+/H+ Antiport Activity, Sodium Exclusion, Individual Mrp Protein Levels, or Mrp Complex Formation

“…Mrp antiporters are also broadly distributed among non-alkaliphilic Gram-positive and Gram-negative bacteria and play roles in diverse physiological processes that may depend upon pH and Na ϩ homeostasis (2). In addition to Na ϩ /H ϩ antiport, several of these Mrp antiporters support resistance to anions, such as arsenite and bile salts (13)(14)(15). We hypothesize that Mrp complexes are consortiums of more than one transporter type (2).…”