Isolation and characterization of a novel As(V)-reducing bacterium: implications for arsenic mobilization and the genus Desulfitobacterium

Abstract

Dissimilatory arsenate-reducing bacteria have been implicated in the mobilization of arsenic from arsenic-enriched sediments. An As(V)-reducing bacterium, designated strain GBFH, was isolated from arsenic-contaminated sediments of Lake Coeur d'Alene, Idaho. Strain GBFH couples the oxidation of formate to the reduction of As(V) when formate is supplied as the sole carbon source and electron donor. Additionally, strain GBFH is capable of reducing As(V), Fe(III), Se(VI), Mn(IV) and a variety of oxidized sulfur species. 16S ribosomal DNA sequence comparisons reveal that strain GBFH is closely related to Desulfitobacterium hafniense DCB-2(T) and Desulfitobacterium frappieri PCP-1(T). Comparative physiology demonstrates that D. hafniense and D. frappieri, known for reductively dechlorinating chlorophenols, are also capable of toxic metal or metalloid respiration. DNA-DNA hybridization and comparative physiological studies suggest that D. hafniense, D. frappieri, and strain GBFH should be united into one species. The isolation of an Fe(III)- and As(V)-reducing bacterium from Lake Coeur d'Alene suggests a mechanism for arsenic mobilization in these contaminated sediments while the discovery of metal or metalloid respiration in the genus Desulfitobacterium has implications for environments cocontaminated with arsenious and chlorophenolic compounds.

FIG. 1

(A) Scanning electron micrograph illustrating the cells of strain GBFH are curved rods measuring 2 to 4 μm in length and 0.3 to 0.5 μm in width. (B) Transmission electron micrograph of strain GBFH illustrating cell morphology and two laterally attached flagella. The sample was negatively stained with 2% uranyl acetate. (C) Transmission electron micrograph of endospore formation by strain GBFH. Note the five lateral flagella. The sample was negatively stained with 2% phosphotungstic acid.

FIG. 2

Growth curve of strain GBFH (A) and D. frappieri (B) at pH 7.5 illustrating an increase in cell number (●) and As(III) (▴) and a decrease in As(V) (▵) and formate (○). Experiments were performed in triplicate, and error bars represent standard deviations.

FIG. 3

(A) Effect of temperature on specific growth rate (h⁻¹) of strain GBFH grown on 10 mM formate and 10 mM As(V) at pH 7.0. (B) Effect of pH on specific growth rate of strain GBFH grown on 10 mM formate and 10 mM As(V) at 37°C. Experiments were performed in triplicate; each error bar represents one standard deviation.

FIG. 4

Growth of D. dehalogenans (A) and D. hafniense (B) in the presence of As(V) and other electron acceptors. Cells grown fermentatively on pyruvate were inoculated into one of the following eight treatments: ▪, pyruvate only; □, pyruvate plus As(V); ●, lactate plus 3-Cl-4-OHPA; ○, lactate plus 3-Cl-4-OHPA plus As(V); ▴, lactate plus nitrate; ▵, lactate plus nitrate plus As(V); ▾, lactate alone; ▿, lactate plus As(V). Experiments were performed in duplicate; symbols represent average values.

FIG. 5

(A) Unrooted phylogenetic tree showing the positions of currently recognized As(V)-reducing microorganisms (in bold) among the major lines of prokaryotes. The tree is based on an optimized global tree reconstructed from almost full-length SSU rRNA gene sequences belonging to all three domains using various treeing programs included in the ARB package. The bar indicates 10% estimated sequence divergence. (B) Phylogenetic tree based on partial 16S rRNA gene sequences (nucleotides 98 to 1542, E. coli numbering) showing the placement of strain GBFH (in bold) in the low-GC gram-positive clade among members of the genus Desulfitobacterium. A maximum-likelihood method was used for tree reconstruction. Reference sequences (not shown) belonging to major lineages of the Bacillus-Clostridium group of bacteria were used as the out-group. The bar indicates 1% estimated sequence divergence.

FIG. 5

(A) Unrooted phylogenetic tree showing the positions of currently recognized As(V)-reducing microorganisms (in bold) among the major lines of prokaryotes. The tree is based on an optimized global tree reconstructed from almost full-length SSU rRNA gene sequences belonging to all three domains using various treeing programs included in the ARB package. The bar indicates 10% estimated sequence divergence. (B) Phylogenetic tree based on partial 16S rRNA gene sequences (nucleotides 98 to 1542, E. coli numbering) showing the placement of strain GBFH (in bold) in the low-GC gram-positive clade among members of the genus Desulfitobacterium. A maximum-likelihood method was used for tree reconstruction. Reference sequences (not shown) belonging to major lineages of the Bacillus-Clostridium group of bacteria were used as the out-group. The bar indicates 1% estimated sequence divergence.