Arsenate-reducing bacteria
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Arsenate-reducing bacteria are bacteria which reduce arsenates. Arsenate-reducing bacteria are ubiquitous in arsenic-contaminated groundwater (aqueous environment). Arsenates are salts or esters of arsenic acid (H3AsO4), consisting of the ion AsO43−. They are moderate oxidizers that can be reduced to arsenites and to arsine. Arsenate can serve as a respiratory electron acceptor for oxidation of organic substrates and H2S or H2. Arsenates occur naturally in minerals such as adamite, alarsite, legrandite, and erythrite, and as hydrated or anhydrous arsenates. Arsenates are similar to phosphates since arsenic (As) and phosphorus (P) occur in group 15 (or VA) of the periodic table. Unlike phosphates, arsenates are not readily lost from minerals due to weathering. They are the predominant form of inorganic arsenic in aqueous aerobic environments. On the other hand, arsenite is more common in anaerobic environments, more mobile, and more toxic than arsenate. Arsenite is 25–60 times more toxic and more mobile than arsenate under most environmental conditions. Arsenate can lead to poisoning, since it can replace inorganic phosphate in the glyceraldehyde-3-phosphate --> 1,3-biphosphoglycerate step of
glycolysis Glycolysis is the metabolic pathway that converts glucose () into pyruvate (). The free energy released in this process is used to form the high-energy molecules adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH ...
, producing 1-arseno-3-phosphoglycerate instead. Although glycolysis continues, 1 ATP molecule is lost. Thus, arsenate is toxic due to its ability to uncouple glycolysis. Arsenate can also inhibit pyruvate conversion into acetyl-CoA, thereby blocking the TCA cycle, resulting in additional loss of ATP.


Niche

Arsenate is the major arsenic form in oxidizing environments; however, in one study, bacteria from arsenic-contaminated soil at a smelter site was able to reduce As(+5) to As(+3) under anaerobic conditions at arsenic concentration as high as 75 mg/L. Arsenate-respiring bacteria and
Archaea Archaea ( ; singular archaeon ) is a domain of single-celled organisms. These microorganisms lack cell nuclei and are therefore prokaryotes. Archaea were initially classified as bacteria, receiving the name archaebacteria (in the Archaebac ...
have also recently been isolated from a diversity of natural environments, including freshwater streams and sediments, alkaline and saline lakes, and hot springs. However, arsenate-reducing bacteria may exist in low numbers until provided with new sources of carbon especially and possibly the selective pressure of higher concentrations of arsenic. Some common locations with arsenate reducing bacteria that is causing major contamination problems by releasing arsenic into drinking water in shallow wells include Bangladesh, the American Midwest, and the Canadian Maritime Provinces.


Ecological importance & markers

Arsenic(+3) fuels anoxygenic photosynthesis, such as in hot spring biofilms of Mono Lake, California. Anoxygenic photosynthesis, or photosynthesis that does not produce oxygen and is common with photosynthetic bacteria and certain
cyanobacteria Cyanobacteria (), also known as Cyanophyta, are a phylum of gram-negative bacteria that obtain energy via photosynthesis. The name ''cyanobacteria'' refers to their color (), which similarly forms the basis of cyanobacteria's common name, blu ...
, uses electron donors other than water to reduce CO2 for incorporation into biomass. This mechanism of photoautotrophy usually uses
hydrogen sulfide Hydrogen sulfide is a chemical compound with the formula . It is a colorless chalcogen-hydride gas, and is poisonous, corrosive, and flammable, with trace amounts in ambient atmosphere having a characteristic foul odor of rotten eggs. The unde ...
(H2S) as an electron donor and a single photosystem as a catalyst, oxidizing sulfide to sulfur and sulfate to support the growth of phototrophs in anoxic sulfidic environments exposed to light, such as laminated microbial mats and pycnoclines of stratified lakes.


Identification & characteristics

Arsenate-reducing bacteria derive their energy from reducing arsenate (As(+5)) to arsenite (As(+3)) via arsenate reductase enzymes. As(+5) can be directly reduced to As(+3) by dissimilatory arsenate reducing prokaryotes (DARPs), yielding energy to support their growth. They synthesize organic molecules by using the energy from arsenic
redox reactions Redox (reduction–oxidation, , ) is a type of chemical reaction in which the oxidation states of substrate change. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is the gain of electrons or a d ...
. The complete reduction process takes about 21 hours. Dissimilatory As(+5)-respiring prokaryotes consist of a diverse phylogenetic group, including ''Chrysiogenes'', ''Bacillus'', ''Desulfomicrobium'', ''Sulfurospirillum'', ''Shewanella'', ''Citrobacter'', and ''Sulfurihydrogenibium'' species. Some specific species include ''Klebsiella oxytoca'', ''Citrobacter freundii'', and ''Bacillus anthracis''. Although the ability to respire As(+5) is spread across several phylogenetic groups, the As(+5) reduction mechanism in these organisms seems to be conserved.


Genome/Molecular composition

Living cells (microbial or human) are generally exposed to arsenic as arsenate or arsenite. Arsenate (As(+5)) has a pKa of 7.0, with HAsO42− and H2AsO41− being equally abundant at pH 7.0. Although arsenate is regarded as highly soluble, in many environments with calcium or insoluble iron compounds, arsenate is precipitated just like phosphates are. Arsenite (As(+3)), has a pKa of 9.3, and occurs at a neutral or acidic pH as As(OH)3. Arsenite in water can be thought of as an inorganic equivalent of non-ionized glycerol and is transported across cell membranes from bacterial cells to human cells by glyceroporin membrane channel proteins. Two
enzymes Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different molecule ...
are involved in two separate processes for reducing arsenate: a membrane-bound or periplasmic respiratory arsenate reductase and a cytoplasmic arsenate reductase. The anaerobic respiratory arsenate reductase reduces AsO43− to As(OH)3. It is a heterodimer periplasmic or membrane-associated protein consisting of a larger molybdopterin subunit (''ArrA''), which contains an iron-sulfur (FeS) center. This includes the FeS cofactors involved in 2 e transfer pathways and amino acids cysteine or histidine linking the FeS cofactors to the ArrA, or HIPIP (''high potential iron protein'') polypeptides. It is also composed of a smaller FeS center protein ArrB. This enzyme in Gram-positive '' Bacillus'' differs from that of Gram-negative bacteria since it is anchored to the membrane of the Gram-positive cell, which lacks a periplasmic compartment. The cytoplasmic arsenate reductase, found widely in microbes, is for intracellular defense and also reduces AsO43− to As(OH)3 with part of the process taking place in the cytoplasm. The ''arsC'' gene occurs in ''ars'' operons for arsenic resistance in most bacteria and some archaeal genomes. It is a monomeric protein of about 135 amino acids containing 3 essential cysteine residues involved in a cascade sequence of enzyme activity. There are no
cofactors Cofactor may also refer to: * Cofactor (biochemistry), a substance that needs to be present in addition to an enzyme for a certain reaction to be catalysed * A domain parameter in elliptic curve cryptography, defined as the ratio between the order ...
in the ArsC enzyme. The first recognized cytoplasmic arsenate reductase was found on a Gram-positive ''
Staphylococcus ''Staphylococcus'' is a genus of Gram-positive bacteria in the family Staphylococcaceae from the order Bacillales. Under the microscope, they appear spherical (cocci), and form in grape-like clusters. ''Staphylococcus'' species are facultative ...
'' plasmid. The thioredoxin-coupled clade of arsenate reductases is found widely among plasmids and genomes of Gram-positive bacteria and also in some Gram-negative bacteria. The '' Pseudomonas aeruginosa'' genome has separate genes for glutaredoxin- and thioredoxin-coupled ArsC reductases. In contrast, those for
cyanobacteria Cyanobacteria (), also known as Cyanophyta, are a phylum of gram-negative bacteria that obtain energy via photosynthesis. The name ''cyanobacteria'' refers to their color (), which similarly forms the basis of cyanobacteria's common name, blu ...
seem to be an unusual hybrid with strong sequence similarity to thioredoxin-dependent reductase, but functioning with glutaredoxin and glutathione instead. The cyanobacteria arsenate reductase is also a homodimer, different from other known bacterial enzymes but similar to the yeast enzyme.


Isolation techniques

One study used for enrichment a sample of mud from an arsenic-contaminated gold mine in Bendigo, Victoria, Australia (pH 7.6, 2.5 mg l−1 arsenic). The mud was placed in anoxic minimal medium containing arsenate (5 mM) and acetate (10 mM) and the enrichment was incubated for five days. The enrichment was subcultured twice and the third transfer was serially diluted and inoculated into minimal medium containing 1.5% (w/v) Oxoid agar (Oxoid, Hants, England), arsenate (5 mM) and acetate (10 mM) in Hungate roll tubes or onto agar plates in an anaerobic chamber. Several colonies were selected, purified, and tested for their ability to respire with arsenate (5 mM) using acetate (10 mM) as the electron donor. A motile, rod-shaped bacterium was isolated and designated JMM-4.


Growth media & conditions

The anoxic minimal medium contained 20 mM NaCl, 4 mM KCl, 2.8 mM NH4Cl, 1.5 mM KH2PO4, 0.2 mM Na2SO4, 2mM MgCl2, 1mM CaCl2, 0.05% NaHCO3, 1 ml l−1 SL10 trace elements, and vitamins (Macy et al. 1989). At no time was a reducing agent added to the medium. The initial pH of the medium was 7.8. The standard anaerobic culture technique of Hungate was employed (Macy et al. 1989). All incubations were carried out at 28 °C. -->


Phylogeny & diversity

Phylogenetic analysis shows that microbial arsenic metabolism probably extends back to the anoxic primordial Earth. As(+5) produced by anoxygenic photosynthesis might have created niches for primordial Earth's first As(+5)-respiring prokaryotes. In microbial biofilms growing on the rock surfaces of anoxic brine pools fed by hot springs containing arsenite and sulfide at high concentrations, light-dependent oxidation of arsenite (+3) to arsenate (+5) was discovered occurring under anoxic conditions. A pure culture of a photosynthetic bacterium grew as a photoautotroph when As(+3) was used as the sole photosynthetic electron donor. The strain contained genes supposedly encoding a As(+5) reductase. However, no detectable homologs of the As(+3) oxidase genes of aerobic chemolithotrophs, suggesting a reverse functionality for the reductase.


Notable species

In a study, a total of 9 arsenate-respiring prokaryotes have been described, 6 of which use the non-respiratory substrate lactate as the electron donor. These organisms group together phylogenetically as follows:


Bacteria

• '' Sulfurospirillum arsenophilum'' • '' Sulfurospirillum barnesii'' • '' Bacillus arsenicoselenatis'' str. E1H • '' B. selenitireducens'' str.MLS10 • '' Desulfotomaculum auripigmentum'' • '' Desulfomicrobium sp''. str. Ben-RB • ''
Chrysiogenes arsenatis ''Chrysiogenes arsenatis'' is a species of bacterium in the family Chrysiogenaceae. It has a unique biochemistry. Instead of respiring with oxygen, it respires using the most oxidized form of arsenic, arsenate. It uses arsenate as its terminal el ...
''


Archaea

• ''
Pyrobaculum arsenaticum ''Pyrobaculum'' is a genus of the Thermoproteaceae. Description and significance As its Latin name ''Pyrobaculum'' (the "fire stick") suggests, the archaeon is rod-shaped and isolated from locations with high temperatures. It is Gram-negative ...
'' • ''
Pyrobaculum aerophilum ''Pyrobaculum aerophilum'' is a single-celled microorganism in the genus ''Pyrobaculum''. The first ''Pyrobaculum'' species to be sequenced was ''P. aerophilum''. It is a rod-shaped hyperthermophilic archaeum first isolated from a boiling marin ...
''Santini, Joanne M., John F. Stolz, and Joan M. Macy. "Isolation of a New Arsenate-Respiring Bacterium--Physiological and Phylogenetic Studies." Geomicrobiology Journal 19.1 (2002): 41-52. Print.


Biochemistry


Reducing process

Arsenic occurs in nature in three oxidation states: As(+5) (arsenate), As(+3) (arsenite), and As(-3) (arsine). Although transfers between these states can be achieved by purely chemical means, microorganisms can also mediate a diversity of reactions including reduction, oxidation, and methylation.Bacterial Dissimilatory Reduction of Arsenic(V) to Arsenic(III) in Anoxic Sediments. PHILIP R. DOWDLE,1 ANNIET M. LAVERMAN,2 AND RONALD S. OREMLAND1* Some bacteria obtain energy by oxidizing various reduced substrates while reducing arsenates to form arsenites. The enzymes involved are known as arsenate reductases. In 2008, bacteria were discovered that employ a version of photosynthesis with arsenites as electron donors, producing arsenates (analogous to PSII in oxygenic photosynthesis uses water as electron donor, producing molecular oxygen). The researchers conjectured that historically these photosynthesizing organisms produced the arsenates that allowed the arsenate-reducing bacteria to thrive.Arsenic-loving bacteria rewrite photosynthesis rules
Chemistry World, 15 August 2008


Mechanism

In ''Desulfomicrobium'' strain Ben-RB arsenate is reduced by a membrane-bound enzyme that is either a c-type cytochrome or is associated with such a cytochrome; benzyl-viologen-dependent arsenate reductase activity was greater in cells grown with arsenate/sulfate than in cells grown with sulfate only. It appears that arsenate reduction by the ''Desulfovibrio'' strain Ben-RA is catalyzed by an arsenate reductase that is encoded by a chromosomally-borne gene shown to be homologous to the ''arsC'' gene of the '' Escherichia coli'' plasmid, R773 ars system.Macy, J. M., J. M. Santini, B. V. Pauling, A. H. O'Neill, and L. I. Sly. "Two New Arsenate/sulfate-reducing Bacteria: Mechanisms of Arsenate Reduction." Archives of Microbiology 173.1 (2000): 49-57. Print.


Contamination

Arsenic poisoning of groundwater used for drinking and irrigation is a global issue, with the risk of harmful human exposure occurring at numerous locations across the Americas, Asia, and also central Europe. Many recent studies have reported arsenic-enriched groundwater within the Ganges-Brahmaputra-Meghna Delta, with more than 35 million people at risk of arsenic poisoning in Bangladesh alone. The weathering of arsenic-rich minerals prevalent in the Himalayas and their gradual transport and deposition in the alluvial deltas below, followed by microbially mediated arsenic solubilization, are thought to be major mechanisms of arsenic mobilization into aquifers within the region. Conditions similarly conducive to the development of arsenic-enriched groundwater are thought to be present within the Red River and Mekong River deltas of Southeast Asia, where elevated concentrations of arsenic have also been reported.


Uses

Microbial metabolism undoubtedly worsens environmental arsenic problems by releasing arsenite into drinking water, including shallow wells. Understanding the mechanisms can help minimize the impact. It is proposed that microbial anaerobic respiratory arsenate reductase releases previously immobilized sub-surface As(+5) into water in newly drilled wells. It is possible that microbial metabolism (arsenite oxidase coupled with precipitation in mineral deposits) can be harnessed for practical bioremediation of wastewater or drinking water contaminated with arsenic. However, this prospect is just beginning to be recognized and no sustained efforts in this direction have been made. Microbial batch reactors to remove arsenic by oxidation of As(+3) to As(+5) and the use of bacterial arsenate reductase genes in transgenic plants for potential phytoremediation by intracellular sequestration after reduction from As(+5) to As(+3) have been recently reported.


References

{{reflist Arsenates Extremophiles