Identification of a novel arsenite oxidase gene, arxA, in the haloalkaliphilic, arsenite-oxidizing bacterium Alkalilimnicola ehrlichii strain MLHE-1

Abstract

Although arsenic is highly toxic to most organisms, certain prokaryotes are known to grow on and respire toxic metalloids of arsenic (i.e., arsenate and arsenite). Two enzymes are known to be required for this arsenic-based metabolism: (i) the arsenate respiratory reductase (ArrA) and (ii) arsenite oxidase (AoxB). Both catalytic enzymes contain molybdopterin cofactors and form distinct phylogenetic clades (ArrA and AoxB) within the dimethyl sulfoxide (DMSO) reductase family of enzymes. Here we report on the genetic identification of a "new" type of arsenite oxidase that fills a phylogenetic gap between the ArrA and AoxB clades of arsenic metabolic enzymes. This "new" arsenite oxidase is referred to as ArxA and was identified in the genome sequence of the Mono Lake isolate Alkalilimnicola ehrlichii MLHE-1, a chemolithoautotroph that can couple arsenite oxidation to nitrate reduction. A genetic system was developed for MLHE-1 and used to show that arxA (gene locus ID mlg_0216) was required for chemoautotrophic arsenite oxidation. Transcription analysis also showed that mlg_0216 was only expressed under anaerobic conditions in the presence of arsenite. The mlg_0216 gene is referred to as arxA because of its greater homology to arrA relative to aoxB and previous reports that implicated Mlg_0216 (ArxA) of MLHE-1 in reversible arsenite oxidation and arsenate reduction in vitro. Our results and past observations support the position that ArxA is a distinct clade within the DMSO reductase family of proteins. These results raise further questions about the evolutionary relationships between arsenite oxidases (AoxB) and arsenate respiratory reductases (ArrA).

FIG. 1.

MLHE-1 arx operon. Genes are shown as arrows to scale, and each arrow indicates the orientation of the coding sequence. The arx operon is predicted to contain six genes (shaded in black); arxA (mlg_0216) encodes the molybdopterin oxidoreductase, arxB′ (mlg_0217) encodes a [4Fe-4S]-containing protein, arxB (mlg_0215) encodes another [4Fe-4S]-containing protein, arxC (mlg_0214) encodes a membrane protein, arxD (mlg_0213) encodes a TorD like protein, and arxE (mlg_0212) encodes a peptidylprolyl isomerase. The genes depicted in white are predicted to be a separate operon transcribed in the direction opposite to that of the arx operon and predicted to comprise the regulatory system for the arx operon. The genes in this operon include mlg_0218 (encoding an oxyanion transport protein), mlg_0219 (encoding a histidine kinase resembling AoxS), and mlg_0220 (thought to encode a response regulator resembling AoxR). Plasmid pSMV20 is shown to indicate a single homologous recombination event leading to the creation of insertion mutant strains.

FIG. 2.

Confirmation of insertion mutations in the MLHE-1 wild-type genome. Shown are an mlg_0216 (arxA) and mlg_2426 insertion mutation cat PCR (panel A), an mlg_0216 insertion PCR with M13 F/mlg_0216 R (panel B), and an mlg_2426 insertion PCR with M13 F/mlg_2426 R (panel C). Samples: MLHE_0216 DNA (lanes 1 and 5), MLHE_2426 DNA (lanes 2 and 7), MLHE-1 wild-type DNA (lanes 3, 6, and 9), and a water control (lanes 4, 7, and 10).

FIG. 3.

Cell counts of MLHE-1 cultures grown anaerobically with 5 mM arsenite and 5 mM nitrate. Symbols: ▪, wild type; ▴, MLHE1_0216; •, MLHE1_2426. Data points and error bars represent averages and standard deviations of triplicate cultures, respectively.

FIG. 4.

Anaerobic growth analysis of MLHE-1 variants grown on 5 mM arsenite and 5 mM nitrate. Panels: A, mM nitrate; B, mM nitrite; C, mM arsenite; D, mM arsenate. Symbols: ▪, wild type; ▴, MLHE1_0216; •, MLHE1_2426. Data points and error bars represent averages and standard deviations of triplicate cultures, respectively.

FIG. 5.

Growth analysis of MLHE-1 variants on 40 mM acetate and (A) oxygen or (B) 20 mM nitrate and (C) nitrite analysis. Symbols: ▪, wild type; ▴, MLHE1_0216; •, MLHE1_2426; ⧫, no cells. Data points and error bars represent averages and standard deviations of triplicate cultures, respectively.

FIG. 6.

(A) RT-PCR analysis of 16S rRNA gene (top) and mlg_0216 (bottom) in MLHE-1 after addition of 5 mM arsenate or 5 mM arsenite. Triplicate cultures of MLHE-1 were grown in MLC medium under aerobic or anaerobic conditions with 40 mM acetate or with 40 mM acetate and 20 mM nitrate, respectively. Once the cultures reached mid-log phase, cells were collected, followed by addition of 5 mM arsenate or 5 mM arsenite. After 6 h, the cultures were sampled once again. One representative sample from each triplicate condition is presented in the gel. Lanes 1 and 2 are aerobic samples before and after addition of arsenate. Lanes 3 and 4 are aerobic samples before and after addition of arsenite. Lanes 5 and 6 are anaerobic samples before and after addition of arsenate. Lanes 7 and 8 are anaerobic samples before and after addition of arsenite. Lane 9 is MLHE-1 DNA, lane 10 is Shewanella sp. strain ANA-3 DNA, and lane 11 is the water control. (B) Semiquantitative analysis of arxA gene expression done by subtracting the gel background from the band intensities of the arxA RT-PCR products (bottom) and from the 16S rRNA gene RT-PCR product (top). The black bars indicate relative arxA gene expression levels before addition of arsenate or arsenite, and the white bars indicate arxA gene expression levels 6 h after addition of arsenate or arsenite. Bars and error bars represent averages and standard deviations of triplicate cultures, respectively.

FIG. 7.

Phylogenetic analysis of arsenate respiratory reductases (ArrA), AoxB-type arsenite oxidases, and the ArxA-type arsenite oxidase of MLHE-1. The unrooted tree was constructed using the neighbor-joining method; gaps were ignored in the final phylogeny. The numbering refers to representative amino acid sequences of ArrA and AoxB as follows, where * or ** indicates that the organism is known to respire arsenate or oxidize arsenite, respectively. Arsenate respiratory reductase group (ArrA): 1, Chrysiogenes arsenatis AAU11839*; 2, Geobacter lovleyi ZP_01593421; 3, Geobacter uraniireducens Rf4 ZP_01140714; 4, Bacillus selenitireducens AAQ19491*; 5, Bacillus arsenicoselenatis AAU11841*; 6, Sulfurospirillum barnesii AAU11840*; 7, Wolinella succinogenes NP_906980*; 8, Desulfosporosinus ABB02056*; 9 and 10, Desulfitobacterium YP_520364 and ZP_01372404*, respectively; 11, unidentified deltaproteobacterium MLMS-1 ZP_01288668*; 12, Natranaerobius thermophilus YP_001916826; 13, Halarsenatibacter silvermanii SLAS-1 ACF74513*; 14, Desulfonatronospira thiodismutans ASO3-1 ZP_03737819; 15, “Alkaliphilus metalliredigens” ZP_00800578; 16, Alkaliphilus oremlandii OhILAs ZP_01360543*; 17, Shewanella piezotolerans WP3 YP_002311519; 18 to 20, Shewanella group AAQ01672*, ZP_01704274, and YP_964317. Arsenite oxidase AoxB group: 21, Rhizobium sp. NT-26 AAR05656**; 22, Agrobacterium tumefaciens ABB51928**; 23, Ochrobactrum tritici ACK38267**; 24, Xanthobacter autotrophicus Py2 ZP_01198801; 25, Nitrobacter hamburgensis YP_571843; 26, Roseovarius sp. strain 217 ZP_01034989; 27, Ralstonia sp. strain 22 ACX69823; 28, Alcaligenes faecalis AAQ19838**; 29, Herminiimonas arsenoxydans AAN05581**; 30, Burkholderia multivorans ZP_0157266830; 31, Rhodoferax ferrireducens YP_524325; 32, Thiomonas sp. strain 3As CAM58792**; 33, Pseudomonas sp. strain TS44 ACB05943; 34, Halomonas sp. strain HAL1 ACF77048; 35, Chloroflexus aurantiacus ZP_00356; 36, Thermus thermophilus YP_145366**.