Molecular genetics and physiology of arsenic transforming bacteria

Arsenic is found ubiquitously in the earths crust and when ingested can cause cancer in humans. Despite its toxicity, microorganisms have evolved biotransformation pathways that function to couple the oxidation/reduction of arsenicals to energy conservation and growth. Bacteria have been isolated that can couple the reduction of arsenate (As(V)) to the oxidation of organic carbon using the arsenate respiratory reductase enzyme complex (ArrAB). Similarly, bacteria have been isolated that can oxidize arsenite (As(III)). Most of these microbes oxidize arsenite for detoxification purposes using the AoxAB enzyme complex while others can couple the oxidation of arsenite to chemoautotrophic growth using a novel arsenite oxidase complex. Though they catalyze similar reactions, ArrAB and AoxAB form distinct clades with in the DMSO reductase family of enzymes (5).;In Shewanella sp. ANA-3, three proteins have been identified that are required for arsenate respiratory reduction. These proteins give ANA-3 the capability to couple the oxidation of lactate to the reduction of arsenate leading to energy conservation and growth. Two of these proteins make up the arsenate respiratory reductase complex, ArrAB. The larger subunit (ArrA) is a molybdenum containing subunit predicted to catalyze the reduction of arsenate, and the smaller subunit (ArrB) is an Fe-S containing protein. It is predicted that ArrB accepts electrons from a third protein CymA, a tetraheme c-type cytochrome anchored to the periplasmic face of the cytoplasmic membrane (3, 4). The first project (Chapter 2) of this dissertation, explores the interaction of CymA and menaquinols. The results from this chapter demonstrate that during arsenate reduction, CymA interact with and is reduced by menaquinol analogs (HQNO and DMNQH2) and that lysine 91 is crucial for that interaction (6).;Up until now, it was thought the AoxAB complex solely catalyzed the oxidation of arsenite. Project 2 (Chapter 3 and 4) describes the identification of a novel arsenite oxidase complex, ArxB'ABCDE, which couples the oxidation of arsenite to chemoautotrophic growth. This novel system was identified in the bacterium, Alkalilimincoli ehrlichii strain MLHE-1, isolated from the anoxic bottom water of Mono Lake (CA) (1). The results from this project show that ArxA, a molybdenum containing oxidoreductase, is required for chemoautotrophic arsenite oxidation Furthermore, transcription analysis shows that arxA is only expressed under anaerobic conditions with arsenite. These results and past observations support the position that ArxA is a distinct clade with in the DMSO reductase family of enzymes.;The identification of the Arx system in MLHE-1 lead to the search of this enzyme complex in other microorganisms. In 2008 Kulp et al. (2) identified Ectothiorhodospira str. PHS-1 from Paoha Island in Mono Lake. PHS-1 was shown to couple arsenite oxidation to anoxygenic photosynthesis. Chapter 4 describes; (i) the arx operon in PHS-1 and (ii) design of molecular tools to detect arxA-like arsenite oxidase gene in pure culture and environmental DNA and (iii) the molecular ecology of arxA with in Mono Lake beach sediment and Hot Creek riverbed sediments (CA). Results from inverse PCR reactions revealed that the genome of PHS-1 contains an arx operon similar to that found in MLHE-1. So far the arx operon in PHS-1 is shown to be composed of arxABCD with ∼40%--70% amino acid similarity to those found in MLHE-1. Further work will elucidate if the arx operon of PHS-1 also contain arxB' and arxE homologues as seen in MLHE-1. Based on the sequences of arxA from MLHE-1 and PHS-1, and data obtained from bioinformatics analysis, molecular detection tools were designed for the detection of arxA-like genes in environmental samples. Implementation of these tools revealed that arxA-like genes, different from those found in PHS-1 and MLHE-1, were present in Mono Lake sediment. Furthermore, arxA-like genes were also identified in environmental DNA isolated from the arsenic rich riverbed sediments of Hot Creek. This creek, located about 25 miles south of Mono Lake, originates in the Eastern Sierra Nevada mountain range and runs through a series of geothermal system mixing with arsenic rich geothermal fluids. The AoxAB system has been found in various environments ranging from fresh water streams to geothermal systems and mine tailing sites. The initial results from the molecular ecology studies of arxA-like genes indicate that the Arx system might also be widespread beyond organisms found in Mono Lake. Implementation of the designed molecular detection tools on diverse environmental samples will reveal more information about the ecology of the Arx system in the environment.