Genetic identification of a respiratory arsenate reductase

Abstract

For more than a decade, it has been recognized that arsenate [H2AsO41-; 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.

Fig. 1.

Molecular organization of the arrAB gene cluster. The arrA gene is located upstream in the opposite orientation to an ars operon. CDS1 and CDS2 are similar to glutathione synthetases and a S. oneidensis MR-1 conserved hypothetical protein, respectively. The arrow between arsD and arrA indicates the location of potential Fnr-like binding sites. The loop after arrB is a putative transcriptional terminator region. Analysis of the putative Tat signal sequence within the first 42 amino acid residues of ArrA reveals a potential cleavage site (arrow).

Fig. 2.

As(V) respiration by wild-type ANA-3 (circles) and mutant strains ARRA3 (ΔarrA) (squares) and ARRB1 (ΔarrB) (triangles). (A) The time course for growth inferred by optical density at 600 nm. (B) The As(V) concentration in the medium at the various time points. (C) Anaerobic growth on As(V) by strain ARRA3 (ΔarrA) and strain ARRB1 (ΔarrB) is restored by providing a wild-type copy of the arrA or arrB gene on a complementation vector (filled symbols). Open symbols in C represent strains containing the complementation vector alone. Points and error bars in each panel represent the averages and standard deviations of triplicate samples.

Fig. 3.

Unrooted neighbor-joining trees for representative sequences from the DMSO reductase family of molybdoenzymes (19) (A) and iron–sulfur proteins (B). Aso, arsenite oxidase; Fdh, formate dehydrogenase; Nap, periplasmic nitrate reductase; Dor/Dms, DMSO reductase; Bis, biotinsulfoxide reductase; Tor, trimethylamineoxide reductase; Ser, selenate reductase; Nar, membrane-associated nitrate reductase; Arr, arsenate respiratory reductase; Psr/Phs, polysulfide reductase; Sdh, succinate dehydrogenase; Frd, fumarate reductase; Nrf, nitrite reductase. GenBank accession numbers are included next to the corresponding species. Scale bar represents the number of amino acid changes per site.