Selenate-dependent anaerobic arsenite oxidation by a bacterium from Mono Lake, California

Abstract

Arsenate was produced when anoxic Mono Lake water samples were amended with arsenite and either selenate or nitrate. Arsenite oxidation did not occur in killed control samples or live samples with no added terminal electron acceptor. Potential rates of anaerobic arsenite oxidation with selenate were comparable to those with nitrate ( approximately 12 to 15 mumol.liter(-1) h(-1)). A pure culture capable of selenate-dependent anaerobic arsenite oxidation (strain ML-SRAO) was isolated from Mono Lake water into a defined salts medium with selenate, arsenite, and yeast extract. This strain does not grow chemoautotrophically, but it catalyzes the oxidation of arsenite during growth on an organic carbon source with selenate. No arsenate was produced in pure cultures amended with arsenite and nitrate or oxygen, indicating that the process is selenate dependent. Experiments with washed cells in mineral medium demonstrated that the oxidation of arsenite is tightly coupled to the reduction of selenate. Strain ML-SRAO grows optimally on lactate with selenate or arsenate as the electron acceptor. The amino acid sequences deduced from the respiratory arsenate reductase gene (arrA) from strain ML-SRAO are highly similar (89 to 94%) to those from two previously isolated Mono Lake arsenate reducers. The 16S rRNA gene sequence of strain ML-SRAO places it within the Bacillus RNA group 6 of gram-positive bacteria having low G+C content.

FIG. 1.

Arsenate production in water collected from Mono Lake amended with 2 mM arsenite and selenate ○, 5 mM arsenite and nitrate ▾, and filter-sterilized controls •.

FIG. 2.

Arsenic and selenium speciation in Mono Lake water diluted 1:25 with mineral medium and amended with 1 mM arsenite and selenate. Symbols: •, arsenite; ○, arsenate; ▾, selenite; ▵, selenate.

FIG. 3.

Scanning electron micrograph of strain ML-SRAO. Scale bar, 5 μm.

FIG. 4.

(A) Growth of strain ML-SRAO on selenate and lactate. Symbols: ○, selenite; • selenate; ▴, cell density. (B) Growth of strain ML-SRAO on arsenate and lactate. Symbols: • arsenate; ○, arsenite; ▴, cell density. Symbols represent the mean of three replicates; error bars represent ±1 standard deviation.

FIG. 5.

(A) Arsenate (•) and selenite (○) production and arsenite (▾) and selenate (▵) consumption by strain ML-SRAO in AMLW amended with 5 mM selenate and arsenite. (B) Selenite produced by strain ML-SRAO in AMLW amended with 5 mM arsenite and selenate (○); 5 mM selenate, 2.5 mM lactate, and 2.5 mM arsenite (▾); or 5 mM selenate and lactate (•). Symbols represent the mean of three replicates; error bars represent ±1 standard deviation.

FIG. 6.

Arsenate production versus selenite production (•) in washed-cell experiments. The solid line (slope, 0.93; r² = 0.9998, n = 18, and P < 0.0001) shows the relationship of these two variables. The dashed line represents a 1:1 slope.

FIG. 7.

Phylogenetic relationships among cultured members of the genus Bacillus, environmental clones from Mono Lake (*), and strain ML-SRAO based on nearly full-length (>1,400 bp) 16S rRNA gene sequences. The neighbor-joining tree is rooted with the sequence from Paenibacillus polymyxa. Bootstrap values (numbers at nodes) were calculated from 1,000 iterations; values less than 50% are not shown. Scale bar indicates 0.01 nucleotide substitutions per position. GenBank accession numbers for the sequences are given in parentheses.

FIG. 8.

Neighbor-joining tree based ∼200 deduced amino acid sequences based on the respiratory arsenate reductase gene (arrA) of strain ML-SRAO and other cultured arsenate reducing bacteria. GenBank accession numbers are given in parentheses. Evolutionary distances were computed using the maximum composite likelihood method. The tree is rooted with the putative deduced ArrA sequence from A. ehrlichii MLHE-1 (NC_008340). Bootstrap values (>50% only; based on 1,000 iterations) are shown at the nodes of the tree. The scale bar indicates 0.05 nucleotide substitutions per position.