The ␤-proteobacterial strain ULPAs1, isolated from an arsenic-contaminated environment, is able to efficiently oxidize arsenite [As(III)] to arsenate [As(V)]. Mutagenesis with a lacZ-based reporter transposon yielded two knockout derivatives deficient in arsenite oxidation. Sequence analysis of the DNA flanking the transposon insertions in the two mutants identified two adjacent open reading frames, named aoxA and aoxB, as well as a putative promoter upstream of the aoxA gene. Reverse transcription-PCR data indicated that these genes are organized in an operonic structure. The proteins encoded by aoxA and aoxB share 64 and 72% identity with the small Rieske subunit and the large subunit of the purified and crystallized arsenite oxidase of Alcaligenes faecalis, respectively (P. J. Ellis, T. Conrads, R. Hille, and P. Kuhn, Structure [Cambridge] 9:125-132, 2001). Importantly, almost all amino acids involved in cofactor interactions in both subunits of the A. faecalis enzyme were conserved in the corresponding sequences of strain ULPAs1. An additional Tat (twin-arginine translocation) signal peptide sequence was detected at the N terminus of the protein encoded by aoxA, strongly suggesting that the Tat pathway is involved in the translocation of the arsenite oxidase to its known periplasmic location.Arsenic is present in various environments, is released either by natural weathering of rocks or by anthropogenic sources (e.g., mining industries and agricultural practices), and is found in the oxidation states ϩ5 (arsenate), ϩ3 (arsenite), 0 (elemental arsenic), and Ϫ3 (arsine). Contamination of drinkingwater supplies with the inorganic soluble forms arsenite and arsenate has often been reported, and arsenic has been identified as a major risk for human health in different parts of the world (northeast India, Bangladesh, northwest United States) (31). The biogeochemical cycle of this element strongly depends on microbial transformation, which affects the mobility and the distribution of arsenic species in the environment (33, 41). Several bacteria involved in transformation processes comprising reduction, oxidation, and methylation of arsenic species have been described (8,11,26,36,40).The toxicological effects of arsenic are related to its chemical form and oxidation state; the organic forms are the less toxic. Among inorganic forms, As(III) is reported to be on average 100 times more toxic than the less mobile As(V) (25). Several remediation processes have been described for arsenic removal (19) based on chemical oxidation of arsenite to arsenate followed by alkaline precipitation (5, 15-17, 24). The major drawbacks of these processes are that they generate additional pollution and are expensive. This has led to the exploration of alternative methods for arsenic remediation based on its biological oxidation. Several arsenite-oxidizing bacteria have been isolated, starting with an Achromobacter strain in 1918 (14). Since then, different arsenite-oxidizing bacteria, including several Pseudomonas strains (18, 42-44), A...

Microbial biotransformations have a major impact on contamination by toxic elements, which threatens public health in developing and industrial countries. Finding a means of preserving natural environments—including ground and surface waters—from arsenic constitutes a major challenge facing modern society. Although this metalloid is ubiquitous on Earth, thus far no bacterium thriving in arsenic-contaminated environments has been fully characterized. In-depth exploration of the genome of the β-proteobacterium Herminiimonas arsenicoxydans with regard to physiology, genetics, and proteomics, revealed that it possesses heretofore unsuspected mechanisms for coping with arsenic. Aside from multiple biochemical processes such as arsenic oxidation, reduction, and efflux, H. arsenicoxydans also exhibits positive chemotaxis and motility towards arsenic and metalloid scavenging by exopolysaccharides. These observations demonstrate the existence of a novel strategy to efficiently colonize arsenic-rich environments, which extends beyond oxidoreduction reactions. Such a microbial mechanism of detoxification, which is possibly exploitable for bioremediation applications of contaminated sites, may have played a crucial role in the occupation of ancient ecological niches on earth.

An arsenite-oxidizing bacterium, designated strain ULPAs1 T , was isolated from industrial sludge heavily contaminated with arsenic. Cells of this isolate were Gram-negative, curved rods, motile by means of a polar flagellum. The strain was positive for oxidase and catalase activities, was able to reduce nitrate to nitrite, used acetate, lactate and peptone as organic carbon sources under aerobic conditions and was able to oxidize arsenite (As[III]) to arsenate (As [V]

An efficient, inexpensive microplate colorimetric assay for screening of bacteria which can be used in bioremediation of arsenic was developed. The assay is based on the colorimetric analysis of the precipitates formed upon reaction of silver nitrate with arsenic. The method proved reliable and sensitive for the detection of As[III] oxidizers and As[V] reducers and can be used over a large pH range (5.8-8.4). Seventy-eight bacterial strains isolated from different environments were tested by this method. It also showed agreement with results obtained by high-performance liquid chromatography coupled with inductively coupled plasma atomic emission spectrometry.

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BackgroundThe Delta-Proteobacterium Desulfotignum phosphitoxidans is a type strain of the genus Desulfotignum, which comprises to date only three species together with D. balticum and D. toluenicum. D. phosphitoxidans oxidizes phosphite to phosphate as its only source of electrons, with either sulfate or CO2 as electron acceptor to gain its metabolic energy, which is of exclusive interest. Sequencing of the genome of this bacterium was undertaken to elucidate the genomic basis of this so far unique type of energy metabolism.ResultsThe genome contains 4,998,761 base pairs and 4646 genes of which 3609 were assigned to a function, and 1037 are without function prediction. Metabolic reconstruction revealed that most biosynthetic pathways of Gram negative, autotrophic sulfate reducers were present. Autotrophic CO2 assimilation proceeds through the Wood-Ljungdahl pathway. Additionally, we have found and confirmed the ability of the strain to couple phosphite oxidation to dissimilatory nitrate reduction to ammonia, which in itself is a new type of energy metabolism. Surprisingly, only two pathways for uptake, assimilation and utilization of inorganic and organic phosphonates were found in the genome. The unique for D. phosphitoxidans Ptx-Ptd cluster is involved in inorganic phosphite oxidation and an atypical C-P lyase-coding cluster (Phn) is involved in utilization of organophosphonates.ConclusionsWe present the whole genome sequence of the first bacterium able to gain metabolic energy via phosphite oxidation. The data obtained provide initial information on the composition and architecture of the phosphite–utilizing and energy-transducing systems needed to live with phosphite as an unusual electron donor.

Desulfotignum phosphitoxidans is a strictly anaerobic, Gram-negative bacterium that utilizes phosphite as the sole electron source for homoacetogenic CO 2 reduction or sulfate reduction. A genomic library of D. phosphitoxidans, constructed using the fosmid vector pJK050, was screened for clones harboring the genes involved in phosphite oxidation via PCR using primers developed based on the amino acid sequences of phosphite-induced proteins. Sequence analysis of two positive clones revealed a putative operon of seven genes predicted to be involved in phosphite oxidation. Four of these genes (ptxD-ptdFCG) were cloned and heterologously expressed in Desulfotignum balticum, a related strain that cannot use phosphite as either an electron donor or as a phosphorus source. The ptxD-ptdFCG gene cluster was sufficient to confer phosphite uptake and oxidation ability to the D. balticum host strain but did not allow use of phosphite as an electron donor for chemolithotrophic growth. Phosphite oxidation activity was measured in cell extracts of D. balticum transconjugants, suggesting that all genes required for phosphite oxidation were cloned. Genes of the phosphite gene cluster were assigned putative functions on the basis of sequence analysis and enzyme assays.Phosphorus (P) is an important nutrient for all living organisms. The predominant forms of phosphorus in biological systems are inorganic phosphate and its organic esters and acid anhydrides in which P is at its highest oxidation state (ϩV). The P requirements of living cells can be fulfilled with phosphate in various forms, including reduced organic and inorganic phosphorus compounds (23). Several aerobic bacteria were shown to be able to oxidize hypophosphite (ϩI) and phosphite (ϩIII) to phosphate (ϩV) and to incorporate the last into their biomass (5, 15-17, 31, 34). Phosphite can also be oxidized under anaerobic conditions, as shown for an anaerobic Bacillus strain (7) and for Pseudomonas stutzeri which can use phosphite under denitrifying conditions (17, 21). The only bacterium known to oxidize phosphite as the sole source of electrons in lithoautotrophic energy metabolism is Desulfotignum phosphitoxidans (24,25).Three different metabolic pathways for the use of phosphite as a single P source have been characterized so far. Two of them were discovered and characterized with Escherichia coli and one with Pseudomonas stutzeri. The first pathway in E. coli is mediated by the enzyme carbon phosphorus lyase (C-P lyase), and the second one by the alkaline phosphatase encoded by phoA (16,34). This alkaline phosphatase not only hydrolyzes phosphate esters but also hydrolyzes phosphite to phosphate and molecular hydrogen (32). This is a particular property only of the E. coli alkaline phosphatase and is not observed with alkaline phosphatases of other bacteria. The third pathway is encoded by the ptxABCDE gene cluster in P. stutzeri (17). In this system, phosphite is transported into the cell by a binding protein-dependent phosphite transporter at the expense of ATP (Pt...

Arsenic is one of the major groundwater contaminants worldwide. It was previously demonstrated that the beta-proteobacterium Cenibacterium arsenoxidans has an efficient As[III] oxidation ability. The present study was conducted to evaluate the performance of alginate-immobilized ULPAs1 in the oxidation of As[III] to As[V] in batch reactors. A two-level full factorial experimental design was applied to investigate the influence of main parameters involved in the oxidation process, i.e., pH (7-8), temperature (4 degrees C-25 degrees C), kind of nutrient media (2%-20% sauerkraut brine), and arsenic concentration (10-100 mg/L). One hundred milligram per liter of As[III] was fully oxidized by calcium-alginate immobilized cells in 1 h. It was found that the temperature as well as the kind of nutrient media used were significant parameters at a 95% confidence interval whereas only temperature was a significant parameter at a 99% confidence interval. The immobilization of the As[III] oxidizing strain in alginate beads offers a promising way to implement new treatment processes in the remediation of arsenic contaminated waters.