Identification of a novel arsenite oxidase gene, arxA, in the haloalkaliphilic, arsenite-oxidizing bacterium alkalilimnicola ehrlichii strain MLHE-1

Arsenic is toxic to most organisms and is known to cause cancer in humans. However, bacteria have adapted several biotransformation pathways that function to either couple the reduction or oxidation of arsenicals to energy conservation and growth (1). The enzymologies of these two pathways have several features in common. The arsenate respiratory reductase (ArrAB) and arsenite oxidase (AoxAB) enzymes are usually composed of at least two subunits, a small iron-sulfur cluster-containing subunit (ArrB and AoxA) and a larger molybdopterin-containing catalytic subunit (ArrA and AoxB). Although they catalyze arsenic redox chemistry, ArrA and AoxB form distinct phylogenetic clades within the dimethyl sulfoxide (DMSO) reductase family of molybdenum-containing enzymes (16, 24).

Culture-dependent approaches have resulted in the isolation of a variety of diverse bacteria that metabolize arsenic (reviewed in reference 26). Many of these isolates have had their genomes sequenced, which has been insightful for understanding the composition and diversity of arrand aox gene clusters. In the arsenite-oxidizing nitrate reducer Alkalilimnicola ehrlichii strain MLHE-1 (a haloalkaliphile isolated from Mono Lake [CA]) (10, 15), bioinformatic analysis of its genome revealed the absence of genes homologous to the arsenite oxidase genes of the aoxBtype. Instead, two genes (mlg_0216 and mlg_2426) were identified that better resembled the catalytic subunit of the arsenate respiratory reductase (20); however, MLHE-1 has not been shown to respire (or reduce) arsenate (15). Recent work by Richey et al. (20) showed that the Mlg_0216 protein (and not Mlg_2426) was expressed under chemolithoautotrophic (10 mM arsenite and 10 mM nitrate) growth conditions. Moreover, it was shown that Mlg_0216 exhibits both arsenate reductase and arsenite oxidase activities in vitro. These observations raised the question, is the mlg_0216 gene required for arsenite oxidation in vivo? In this report, we addressed this question by developing a genetic system in MLHE-1, generating strains with mutations in mlg_0216 and mlg_2426, and physiologically characterizing the resulting strains. Our results implicate mlg_0216 in chemolithoautotrophic arsenite oxidation coupled to nitrate respiration.