Genetic identification of a respiratory arsenate reductase

Abstract

For more than a decade, it has been recognized that arsenate [H2AsO4 1-; As(V)] can be used by microorganisms as a terminal electron acceptor in anaerobic respiration. Given the toxicity of arsenic, the mechanistic basis of this process is intriguing, as is its evolutionary origin. Here we show that a two-gene cluster (arrAB; arsenate respiratory reduction) in the bacterium Shewanella sp. strain ANA-3 specifically confers respiratory As(V) reductase activity. Mutants with in-frame deletions of either arrA or arrB are incapable of growing on As(V), yet both are able to grow on a wide variety of other electron acceptors as efficiently as the wild-type. Complementation by the wild-type sequence rescues the mutants' ability to respire As(V). arrA is predicted to encode a 95.2-kDa protein with sequence motifs similar to the molybdenum containing enzymes of the dimethyl sulfoxide reductase family. arrB is predicted to encode a 25.7-kDa iron-sulfur protein. arrA and arrB comprise an operon that contains a twin arginine translocation (Tat) motif in ArrA (but not in ArrB) as well as a putative anaerobic transcription factor binding site upstream of arrA, suggesting that the respiratory As(V) reductase is exported to the periplasm via the Tat pathway and under anaerobic transcriptional control. These genes appear to define a new class of reductases that are specific for respiratory As(V) reduction.T he consumption of arsenic (As)-tainted surface waters and ground waters has created a public health crisis in many countries (1, 2). Although much of the As contamination derives from natural weathering and dissolution of As-bearing minerals, recognition that microorganisms can alter the mobility of As in natural waters through redox transformations (3) drove the discovery of the first arsenate [As(V)]-respiring bacterium nearly a decade ago (4). Since then, many more microorganisms that can reduce As(V) to arsenite [H 3 AsO 3 , As(III)] have been discovered (5-8), but a mechanistic understanding of this metabolism has lagged. To date, only three studies have described the biochemistry of arsenate respiration (5, 9, 10), and detailed biochemical analyses have not been performed. In part, the limitations of these studies can be attributed to the fact that the As(V)-respiring organisms being studied were not genetically tractable. To quantify the geochemical impact of As(V)-respiring microorganisms in a given locale, we must be able to predict when these organisms will be active, and how rapidly they will transform As(V). Identification of the gene(s) that control this process, elucidation of their regulation, and determination of the kinetics of their protein products, are necessary steps toward understanding the specific contribution of As(V)-respiring bacteria to As-cycling in the environment.In response to this need, a new As(V)-respiring species, Shewanella strain ANA-3 that is amenable to genetic analysis, was recently isolated (8). This organism contains two systems for reducing As(V). One is similar to the well conserved...

Extracellular respiration of dimethyl sulfoxide by Shewanella oneidensis strain MR-1

“…Directed mutagenesis was carried out as described in ref. 43. The construction of the SO1427::Km strain was described in ref.…”

Rate enhancement of bacterial extracellular electron transport involves bound flavin semiquinones

“…The suspension was centrifuged and the cells were then washed with DM before use in electrochemical experiments. Mutant strains of MR-1 lacking complete genes for MtrC and SO3177 were previously constructed by allele replacement using a two-step homologous recombination method (28,37). The growth procedures for the two mutant strains were the same as described for the WT MR-1 strain.…”

Solution Structures and Backbone Dynamics of Arsenate Reductase from Bacillus subtilis

“…Briefly, the B. subtilis arsC gene was cloned into the pET28a expression vector and expressed in Escherichia coli strain BL21 pLysS(DE3). The culture was grown in LB medium; centrifuged; and resuspended in M9 minimal medium with antibiotics and 15 NH 4 Cl in the presence and absence of [ 13 C 6 ]glucose for preparation of 13 C/ 15 Nlabeled and 15 N-labeled samples, respectively (25). B. subtilis ArsC was purified by ion exchange chromatography (Mono Q) and gel filtration (Superdex 75) using an Ä KTA FPLC system (Amersham Biosciences).…”

“…A set of two-dimensional 15 N-and 13 C-edited HSQC spectra and three-dimensional HNCA, HNCO, HNCACB, HBHA(CO)NH, CBCA(CO)NH, and (H)CC(CO)NH total correlation spectroscopy and (H)CCH COSY spectra were collected to obtain the backbone and side chain assignments (26 -30). The three-dimensional 15 N-edited nuclear Overhauser effect (NOE) spectroscopy (NOESY)-HSQC spectra (mixing times of 50 and 100 ms) were collected to confirm the backbone assignments and in combination with the threedimensional 13 C-edited NOESY-HSQC spectra (mixing times of 50 and 100 ms) to obtain distance restraints for structure calculations (31). The three-dimensional HNHA experiment was performed to obtain dihedral angle restraints (32).…”