An ArsRC fusion protein enhances arsenate sensing and detoxification

Arsenical resistance (ars) operons encode genes for arsenic resistance and biotransformation. The majority are composed of individual genes, but fusion of ars genes is not uncommon, although it is not clear if the fused gene products are functional. Here we report identification of a four-gene ars operon from Paracoccus sp. SY that has two arsR-arsC gene fusions. ArsRC1 and ArsRC2 are related proteins that consist of an N-terminal ArsR arsenite (As(III))-responsive repressor with a C-terminal ArsC arsenate reductase. The other two genes in the operon are gapdh and arsJ. GAPDH, glyceraldehyde 3-phosphate dehydrogenase, forms 1-arseno-3-phosphoglycerate (1As3PGA) from 3-phosphoglyceraldehyde and arsenate (As(V)), ArsJ is an efflux permease for 1As3PGA that dissociates into extracellular As(V) and 3-phosphoglycerate. The net effect is As(V) extrusion and resistance. ArsRs are usually selective for As(III) and do not respond to As(V). However, the substrates and products of this operon are pentavalent, which would not be inducers of the operon. We propose that ArsRC fusions overcome this limitation by channelling the ArsC product into the ArsR binding site without diffusion through the cytosol, a de facto mechanism for As(V) induction. This novel mechanism for arsenate sensing can confer an evolutionary advantage for detoxification of inorganic arsenate.     

A novel pathway of arsenate detoxification

Microorganisms have evolved various mechanisms to detoxify arsenic, an ubiquitous environmental toxin. Known mechanisms include arsenite efflux, arsenate reduction followed by arsenite efflux and arsenite methylation. In this issue, Chen et al. describe a novel mechanism for arsenate detoxification via synergistic interaction of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and a major facilitator superfamily protein (ArsJ). They propose that GAPDH catalyzes the formation of 1‐arseno‐3‐phosphoglycerate, which is then extruded out of the cell by ArsJ. The significance of this pathway and questions for further research are discussed.     

ArsV and ArsW provide synergistic resistance to the antibiotic methylarsenite

Toxic organoarsenicals enter the environment from biogenic and anthropogenic activities such as microbial methylation of inorganic arsenic and pentavalent herbicides such as monosodium methylarsenate (MSMA or MAs(V)). Trivalent MAs(III) is considerably more toxic than arsenite or arsenate. Microbes have evolved mechanisms to detoxify organoarsenicals. We previously identified ArsV, a flavin-linked monooxygenase and demonstrated that it confers resistance to methylarsenite by oxidation to methylarsenate. The arsV gene is usually in an arsenic resistance (ars) operon controlled by an ArsR repressor and adjacent to a methylarsenite efflux gene, either arsK or a gene for a putative transporter. Here we show that Paracoccus sp. SY oxidizes methylarsenite. It has an ars operon with three genes, arsR, arsV and a transport gene termed arsW. Heterologous expression of arsV in Escherichia coli conferred resistance to MAs(III), while arsW did not. Co-expression of arsV and arsW increased resistance compared with either alone. The cells oxidized methylarsenite and accumulated less methylarsenate. Everted membrane vesicles from E. coli cells expressing arsW-accumulated methylarsenate. We propose that ArsV is a monooxygenase that oxidizes methylarsenite to methylarsenate, which is extruded by ArsW, one of only a few known pentavalent organoarsenical efflux permeases, a novel pathway of organoarsenical resistance.     

Mutational and gene expression analysis of mtrDEF,omcA and mtrCAB during arsenate and iron reduction in Shewanella sp. ANA‐3

Arsenate respiration and Fe(III) reduction are important processes that influence the fate and transport of arsenic in the environment. The goal of this study was to investigate the impact of arsenate on Fe(III) reduction using arsenate and Fe(III) reduction deficient mutants of Shewanella sp. strain ANA‐3. Ferrihydrite reduction in the absence of arsenate was similar for an arsenate reduction mutant (arrA and arsC deletion strain of ANA‐3) compared with wild‐type ANA‐3. However, the presence of arsenate adsorbed onto ferrihydrite impeded Fe(III) reduction for the arsenate reduction mutant but not in the wild‐type. In an Fe(III) reduction mutant (mtrDEF, omcA, mtrCAB null mutant of ANA‐3), arsenate was reduced similarly to wild‐type ANA‐3 indicating the Fe(III) reduction pathway is not required for ferrihydrite‐associated arsenate reduction. Expression analysis of the mtr/omc gene cluster of ANA‐3 showed that omcA and mtrCAB were expressed under soluble Fe(III), ferrihydrite and arsenate growth conditions and not in aerobically grown cells. Expression of arrA was greater with ferrihydrite pre‐adsorbed with arsenate relative to ferrihydrite only. Lastly, arrA and mtrA were simultaneously induced in cells shifted to anaerobic conditions and exposed to soluble Fe(III) and arsenate. These observations suggest that, unlike Fe(III), arsenate can co‐induce operons (arr and mtr) implicated in arsenic mobilization.     

Characterization of As efflux from the roots of As hyperaccumulator <Emphasis Type="Italic">Pteris vittata</Emphasis> L.

In some plant species, various arsenic (As) species have been reported to efflux from the roots. However, the details of As efflux by the As hyperaccumulator Pteris vittata remain unknown. In this study, root As efflux was investigated for different phosphorus (P) supply conditions during or after a 24-h arsenate uptake experiment under hydroponic growth conditions. During an 8-h arsenate uptake experiment, P-supplied (P+) P. vittata exhibited much greater arsenite efflux relative to arsenate uptake when compared with P-deprived (P–) P. vittata, indicating that arsenite efflux was not proportional to arsenate uptake. In the As efflux experiment following 24 h of arsenate uptake, arsenate efflux was also observed with arsenite efflux in the external solution. All the results showed relatively low rates of arsenate efflux, ranging from 5.4 to 16.1% of the previously absorbed As, indicating that a low rate of arsenate efflux to the external solution is also a characteristic of P. vittata, as was reported with arsenite efflux. In conclusion, after 24 h of arsenate uptake, both P+ and P– P. vittata loaded/effluxed similar amounts of arsenite to the fronds and the external solution, indicating a similar process of xylem loading and efflux for arsenite, with the order of the arsenite concentrations being solution ≪ roots ≪ fronds.     

Genomic potential for arsenic efflux and methylation varies among global Prochlorococcus populations

The globally significant picocyanobacterium Prochlorococcus is the main primary producer in oligotrophic subtropical gyres. When phosphate concentrations are very low in the marine environment, the mol:mol availability of phosphate relative to the chemically similar arsenate molecule is reduced, potentially resulting in increased cellular arsenic exposure. To mediate accidental arsenate uptake, some Prochlorococcus isolates contain genes encoding a full or partial efflux detoxification pathway, consisting of an arsenate reductase (arsC), an arsenite-specific efflux pump (acr3) and an arsenic-related repressive regulator (arsR). This efflux pathway was the only previously known arsenic detox pathway in Prochlorococcus. We have identified an additional putative arsenic mediation strategy in Prochlorococcus driven by the enzyme arsenite S-adenosylmethionine methyltransferase (ArsM) which can convert inorganic arsenic into more innocuous organic forms and appears to be a more widespread mode of detoxification. We used a phylogenetically informed approach to identify Prochlorococcus linked arsenic genes from both pathways in the Global Ocean Sampling survey. The putative arsenic methylation pathway is nearly ubiquitously present in global Prochlorococcus populations. In contrast, the complete efflux pathway is only maintained in populations which experience extremely low PO₄:AsO₄, such as regions in the tropical and subtropical Atlantic. Thus, environmental exposure to arsenic appears to select for maintenance of the efflux detoxification pathway in Prochlorococcus. The differential distribution of these two pathways has implications for global arsenic cycling, as their associated end products, arsenite or organoarsenicals, have differing biochemical activities and residence times.     

Mutation of the rice TCM12 gene encoding 2,3‐bisphosphoglycerate‐independent phosphoglycerate mutase affects chlorophyll synthesis,photosynthesis and chloroplast development at seedling stage at low temperatures

• Glycolysis is a central metabolic pathway that provides energy and products of primary metabolites. 2,3‐Biphosphoglycerate‐independent phosphoglycerate mutase (iPGAM) is a key enzyme that catalyses the reversible interconversion of 3‐phosphoglycerate (3‐PGA) to 2‐phosphoglycerate (2‐PGA) in glycolysis. Low temperature is a common abiotic stress in rice production. However, the mechanism for rice iPGAM genes is not fully understood at low temperature.
• In this study, the rice mutant tcm12, with chlorosis, malformed chloroplasts and impaired photosynthesis, was grown at a low temperature (<20 °C) to the three‐leaf stage, while the normal phenotype at 32 °C was used. Chlorophyll fluorescence analysis and transmission electron microscopy were used to examine features of the tcm12 mutant. The inheritance behaviour and function of TCM12 were then analysed thorough map‐based cloning, transgenic complementation and subcellular localisation.
• The thermo‐sensitive chlorosis phenotype was caused by a single nucleotide mutation (T→C) on the fifth exon of TCM12 (LOC_Os12g35040) encoding iPGAM, localised to both nucleus and membranes. In addition, TCM12 was constitutively expressed, and its disruption resulted in down‐regulation of some genes associated with chlorophyll biosynthesis and photosynthesis at low temperatures (20 °C).
• This is the first report of the involvement of rice iPGAM gene in chlorophyll synthesis, photosynthesis and chloroplast development, providing new insights into the mechanisms underlying early growth of rice at low temperatures.

     

Overexpression of the triose phosphate translocator (TPT) complements the abnormal metabolism and development of plastidial glycolytic glyceraldehyde‐3‐phosphate dehydrogenase mutants

The presence of two glycolytic pathways working in parallel in plastids and cytosol has complicated the understanding of this essential process in plant cells, especially the integration of the plastidial pathway into the metabolism of heterotrophic and autotrophic organs. It is assumed that this integration is achieved by transport systems, which exchange glycolytic intermediates across plastidial membranes. However, it is unknown whether plastidial and cytosolic pools of 3‐phosphoglycerate (3‐PGA) can equilibrate in non‐photosynthetic tissues. To resolve this question, we employed Arabidopsis mutants of the plastidial glycolytic isoforms of glyceraldehyde‐3‐phosphate dehydrogenase (GAPCp) that express the triose phosphate translocator (TPT) under the control of the 35S (35S:TPT) or the native GAPCp1 (GAPCp1:TPT) promoters. TPT expression under the control of both promoters complemented the vegetative developmental defects and metabolic disorders of the GAPCp double mutants (gapcp1gapcp2). However, as the 35S is poorly expressed in the tapetum, full vegetative and reproductive complementation of gapcp1gapcp2 was achieved only by transforming this mutant with the GAPCp1:TPT construct. Our results indicate that the main function of GAPCp is to supply 3‐PGA for anabolic pathways in plastids of heterotrophic cells and suggest that the plastidial glycolysis may contribute to fatty acid biosynthesis in seeds. They also suggest a 3‐PGA deficiency in the plastids of gapcp1gapcp2, and that 3‐PGA pools between cytosol and plastid do not equilibrate in heterotrophic cells.     

Precipitation of alacranite (As8S9) by a novel As(V)‐respiring anaerobe strain MPA‐C3

Strain MPA‐C3 was isolated by incubating arsenic‐bearing sediments under anaerobic, mesophilic conditions in minimal media with acetate as the sole source of energy and carbon, and As(V) as the sole electron acceptor. Following growth and the respiratory reduction of As(V) to As(III), a yellow precipitate formed in active cultures, while no precipitate was observed in autoclaved controls, or in uninoculated media supplemented with As(III). The precipitate was identified by X‐ray diffraction as alacranite, As₈S₉, a mineral previously only identified in hydrothermal environments. Sequencing of the 16S rRNA gene indicated that strain MPA‐C3 is a member of the Deferribacteres family, with relatively low (90%) identity to Denitrovibrio acetiphilus DSM 12809. The arsenate respiratory reductase gene, arrA, was sequenced, showing high homology to the arrA gene of Desulfitobacterium halfniense. In addition to As(V), strain MPA‐C3 utilizes NO₃^?, Se(VI), Se(IV), fumarate and Fe(III) as electron acceptors, and acetate, pyruvate, fructose and benzoate as sources of carbon and energy. Analysis of a draft genome sequence revealed multiple pathways for respiration and carbon utilization. The results of this work demonstrate that alacranite, a mineral previously thought to be formed only chemically under hydrothermal conditions, is precipitated under mesophilic conditions by the metabolically versatile strain MPA‐C3.     

Validation of Arsenic Resistance in <Emphasis Type="Italic">Bacillus cereus</Emphasis> Strain AG27 by Comparative Protein Modeling of <Emphasis Type="Italic">ars</Emphasis>C Gene Product

The ars gene system provides arsenic resistance to a variety of microorganisms and can be chromosomal or plasmid-borne. The arsC gene, which codes for an arsenate reductase is essential for arsenate resistance and transforms arsenate into arsenite, which is extruded from the cell. Therefore, arsC gene from Bacillus cereus strain AG27 isolated from soil was amplified, cloned and sequenced. The strain exhibited a minimum inhibitory concentration of 40 and 35 mM to sodium arsenate and sodium arsenite, respectively. Homology of the sequence, when compared with available database using BLASTn search showed that 300 bp amplicons obtained possess partial arsC gene sequence which codes for arsenate reductase, an enzyme involved in the reduction of arsenate to arsenite which is then effluxed out of the cell, thereby indicating the presence of efflux mechanism of resistance in strain. The efflux mechanism was further confirmed by atomic absorption spectroscopy and scanning electron microscopy studies. Moreover, three dimensional structure of modeled arsC from Bacillus cereus strain shares significant structural similarity with arsenate reductase protein of B.subtilis, consisting of, highly similar overall fold with single α/β domain containing a central four stranded, parallel, open-twisted β-sheet flanked by α-helices on both sides. The structure harbors the arsenic binding motif AB loop or P-loop that is highly conserved in arsenate reductase family.     

Microbial ecology of arsenic‐mobilizing Cambodian sediments: lithological controls uncovered by stable‐isotope probing

Microbially mediated arsenic release from Holocene and Pleistocene Cambodian aquifer sediments was investigated using microcosm experiments and substrate amendments. In the Holocene sediment, the metabolically active bacteria, including arsenate‐respiring bacteria, were determined by DNA stable‐isotope probing. After incubation with ¹³C‐acetate and ¹³C‐lactate, active bacterial community in the Holocene sediment was dominated by different Geobacter spp.‐related 16S rRNA sequences. Substrate addition also resulted in the enrichment of sequences related to the arsenate‐respiring Sulfurospirillum spp. ¹³C‐acetate selected for ArrA related to Geobacter spp. whereas ¹³C‐lactate selected for ArrA which were not closely related to any cultivated organism. Incubation of the Pleistocene sediment with lactate favoured a 16S rRNA‐phylotype related to the sulphate‐reducing Desulfovibrio oxamicus DSM1925, whereas the ArrA sequences clustered with environmental sequences distinct from those identified in the Holocene sediment. Whereas limited As(III) release was observed in Pleistocene sediment after lactate addition, no arsenic mobilization occurred from Holocene sediments, probably because of the initial reduced state of As, as determined by X‐ray Absorption Near Edge Structure. Our findings demonstrate that in the presence of reactive organic carbon, As(III) mobilization can occur in Pleistocene sediments, having implications for future strategies that aim to reduce arsenic contamination in drinking waters by using aquifers containing Pleistocene sediments.     

Conserved cysteine residues determine substrate specificity in a novel As(III) S‐adenosylmethionine methyltransferase from Aspergillus fumigatus

Methylation of inorganic arsenic is a central process in the organoarsenical biogeochemical cycle. Members of every kingdom have ArsM As(III) S‐adenosylmethionine (SAM) methyltransferases that methylates inorganic As(III) into mono‐ (MAs(III)), di‐ (DMAs(III)) and tri‐ (TMAs(III)) methylarsenicals. Every characterized ArsM to date has four conserved cysteine residues. All four cysteines are required for methylation of As(III) to MAs(III), but methylation of MAs(III) to DMAs(III) requires only the two cysteines closest to the C‐terminus. Fungi produce volatile and toxic arsines, but the physiological roles of arsenic methylation and the biochemical basis is unknown. Here they demonstrate that most fungal species have ArsM orthologs with only three conserved cysteine residues. The genome of Aspergillus fumigatus has four arsM genes encoding ArsMs with only the second, third and fourth conserved cysteine residues. AfArsM1 methylates MAs(III) but not As(III). Heterologous expression of AfarsM1 in an Escherichia coli conferred resistance to MAs(III) but not As(III). The existence of ArsMs with only three conserved cysteine residues suggest that the ability to methylate MAs(III) may be an evolutionary step toward enzymes capable of methylating As(III), the result of a loss of function mutation in organisms with infrequent exposure to inorganic As(III) or as a resistance mechanism for MAs(III).     

Indigenous arsenic(V)‐reducing microbial communities in redox‐fluctuating near‐surface sediments of the Mekong Delta

Arsenic (As) cycling within soils and sediments of the Mekong Delta of Cambodia is affected by drastic redox fluctuations caused by seasonal monsoons. Extensive flooding during monsoon seasons creates anoxic soil conditions that favor anaerobic microbial processes, including arsenate [As(V)] respiration—a process contributing to the mobilization of As. Repeated oxidation and reduction in near‐surface sediments, which contain 10–40 mg kg^?1 As, lead to the eventual downward movement of As to the underlying aquifer. Amplification of a highly conserved functional gene encoding dissimilatory As(V) reductase, arrA, can be used as a molecular marker to detect the genetic potential for As(V) respiration in environmental samples. However, few studies have successfully amplified arrA from sediments without prior enrichment, which can drastically shift community structure. In the present study, we examine the distribution and diversity of arrA genes amplified from multiple sites within the Cambodian Mekong Delta as a function of near‐surface depth (10, 50, 100, 200, and 400 cm), where sediments undergo seasonal redox fluctuations. We report successful amplification of 302 arrA gene sequences (72 OTUs) from near‐surface Cambodian soils (without prior enrichment or stimulation with carbon amendments), where a large majority (>70%) formed a well‐supported clade that is phylogenetically distinct from previously reported sequences from Cambodia and other South and Southeast Asian sediments, with highest sequence similarity to known Geobacter species capable of As(V) respiration, further supporting the potentially important role of Geobacter sp. in arsenic mobilization in these regions.     

Phylogenetic analysis of bacterial and archaeal <Emphasis Type="Italic">arsC</Emphasis> gene sequences suggests an ancient,common origin for arsenate reductase

Background  

The ars gene system provides arsenic resistance for a variety of microorganisms and can be chromosomal or plasmid-borne. The arsC gene, which codes for an arsenate reductase is essential for arsenate resistance and transforms arsenate into arsenite, which is extruded from the cell. A survey of GenBank shows that arsC appears to be phylogenetically widespread both in organisms with known arsenic resistance and those organisms that have been sequenced as part of whole genome projects.     

Arsenate‐induced apoptosis in murine embryonic maxillary mesenchymal cells via mitochondrial‐mediated oxidative injury

BACKGROUND : Arsenic is a ubiquitous element that is a potential carcinogen and teratogen and can cause adverse developmental outcomes. Arsenic exerts its toxic effects through the generation of reactive oxygen species (ROS) that include hydrogen peroxide (H₂O₂), superoxide‐derived hydroxyl ion, and peroxyl radicals. However, the molecular mechanisms by which arsenic induces cytotoxicity in murine embryonic maxillary mesenchymal (MEMM) cells are undefined. METHODS : MEMM cells in culture were treated with different concentrations of pentavalent sodium arsenate [As (V)] for 24 or 48 hr and various end points measured. RESULTS : Treatment of MEMM cells with the pentavalent form of inorganic arsenic resulted in caspase‐mediated apoptosis, accompanied by generation of ROS and disruption of mitochondrial membrane potential. Treatment with caspase inhibitors markedly blocked apoptosis. In addition, the free radical scavenger N‐acetylcysteine dramatically attenuated arsenic‐mediated ROS production and apoptosis, and exposure to arsenate increased Bax and decreased Bcl protein levels in MEMM cells. CONCLUSIONS : Taken together, these findings suggest that in MEMM cells arsenate‐mediated oxidative injury acts as an early and upstream initiator of the cell death cascade, triggering cytotoxicity, mitochondrial dysfunction, altered Bcl/Bax protein ratios, and activation of caspase‐9. Birth Defects Research (Part A), 2010. © 2009 Wiley‐Liss, Inc.     

Structures of glyceraldehyde 3‐phosphate dehydrogenase in Neisseria gonorrhoeae and Chlamydia trachomatis

Neisseria gonorrhoeae (Ng) and Chlamydia trachomatis (Ct) are the most commonly reported sexually transmitted bacteria worldwide and usually present as co‐infections. Increasing resistance of Ng to currently recommended dual therapy of azithromycin and ceftriaxone presents therapeutic challenges for syndromic management of Ng‐Ct co‐infections. Development of a safe, effective, and inexpensive dual therapy for Ng‐Ct co‐infections is an effective strategy for the global control and prevention of these two most prevalent bacterial sexually transmitted infections. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) is a validated drug target with two approved drugs for indications other than antibacterials. Nonetheless, any new drugs targeting GAPDH in Ng and Ct must be specific inhibitors of bacterial GAPDH that do not inhibit human GAPDH, and structural information of Ng and Ct GAPDH will aid in finding such selective inhibitors. Here, we report the X‐ray crystal structures of Ng and Ct GAPDH. Analysis of the structures demonstrates significant differences in amino acid residues in the active sites of human GAPDH from those of the two bacterial enzymes suggesting design of compounds to selectively inhibit Ng and Ct is possible. We also describe an efficient in vitro assay of recombinant GAPDH enzyme activity amenable to high‐throughput drug screening to aid in identifying inhibitory compounds and begin to address selectivity.     

Orphan enzyme or patriarch of a new tribe: the arsenic resistance ATPase of bacterial plasmids

The plasmid-determined arsenite and antimonite efflux ATPase of bacteria differs from other membrane transport ATPases, which are classified into several families (such as the F₀F₁-type H⁺-translocating ATP synthases, the related vacuolar H⁺-translocating ATPases, the P-type cation-translocating ATPases, and the superfamily which includes the periplasmic binding-protein-dependent systems in Gram-negative bacteria, the human multidrug resistance P-glycoprotein, and the cystic fibrosis transport regulator). The amino acid sequences of the components of the arsenic resistance system are not similar to known ATPase proteins. New findings with the arsenic resistance operons of bacterial plasmids suggest that instead of being an orphan the Ars system will now be the first recognized member of a new class of ATPases. Furthermore, fundamental questions of energy-coupling (ATP-driven or chemiosmotic) have recently been raised and the finding that the arsC gene product is a soluble enzyme that reduces arsenate to arsenite changes the previous picture of the functioning of this widespread bacterial system.