Involvement of the Acr3 and DctA anti‐porters in arsenite oxidation in Agrobacterium tumefaciens 5A

Microbial arsenite (AsIII) oxidation forms a critical piece of the arsenic cycle in nature, though our understanding of how and why microorganisms oxidize AsIII remains rudimentary. Our model organism Agrobacterium tumefaciens 5A contains two distinct ars operons (ars1 and ars2) that are similar in their coding region content. The ars1 operon is located nearby the aio operon that is essential for AsIII oxidation. The AsIII/H⁺ anti‐porters encoded by acr3‐1 and acr3‐2 are required for maximal AsIII and antimonite (SbIII) resistance, but acr3‐1 (negatively regulated by ArsR‐1) appears more active in this regard and also required for AsIII oxidation and expression of aioBA. A malate‐phosphate anti‐porter DctA is regulated by RpoN and AsIII, and is required for normal growth with malate as a sole carbon source. Qualitatively, a ΔdctA mutant was normal for AsIII oxidation and AsIII/SbIII resistance at metalloid concentrations inhibitory to the Δacr3‐1 mutant; however, aioBA induction kinetics was significantly phase‐shift delayed. Acr3 involvement in AsIII/SbIII resistance is reasonably well understood, but the role of Acr3 and DctA anti‐porters in AsIII oxidation and its regulation is unexpected, and suggests that controlled AsIII trafficking across the cytoplasmic membrane is important to a process understood to occur in the periplasm.     

A periplasmic arsenite-binding protein involved in regulating arsenite oxidation

Arsenic (As) is the most common toxic element in the environment, ranking first on the Superfund List of Hazardous Substances. Microbial redox transformations are the principal drivers of As chemical speciation, which in turn dictates As mobility and toxicity. Consequently, in order to manage or remediate environmental As, land managers need to understand how and why microorganisms react to As. Studies have demonstrated a two-component signal transduction system comprised of AioS (sensor kinase) and AioR (response regulator) is involved in regulating microbial AsIII oxidation, with the AsIII oxidase structural genes aioB and aioA being upregulated by AsIII. However, it is not known whether AsIII is first detected directly by AioS or by an intermediate. Herein we demonstrate the essential role of a periplasmic AsIII-binding protein encoded by aioX, which is upregulated by AsIII. An ΔaioX mutant is defective for upregulation of the aioBA genes and consequently AsIII oxidation. Purified AioX expressed without its TAT-type signal peptide behaves as a monomer (MW 32?kDa), and Western blots show AioX to be exclusively associated with the cytoplasmic membrane. AioX binds AsIII with a K(D) of 2.4?μM AsIII; however, mutating a conserved Cys108 to either alanine or serine resulted in lack of AsIII binding, lack of aioBA induction, and correlated with a negative AsIII oxidation phenotype. The discovery and characterization of AioX illustrates a novel AsIII sensing mechanism that appears to be used in a range of bacteria and also provides one of the first examples of a bacterial signal anchor protein.     

An Arabidopsis mutant of inositol pentakisphosphate 2‐kinase AtIPK1 displays reduced arsenate tolerance

Arsenate [As(V)] toxicity is considered to be derived from similarities in the chemical properties of As(V) and phosphate (Pi). An Arabidopsis thaliana mutant of inositol pentakisphosphate 2‐kinase (AtIPK1), atipk1‐1, has previously exhibited lower level of phytate and higher level of Pi, relative to wild‐type (WT). Here, atipk1‐1 displayed hypersensitivity to As(V) stress and less As(V) uptake when compared to WT. Overexpression of AtIPK1 controlled by the CaMV 35S promoter partially rescued the As(V)‐sensitive phenotype of atipk1‐1. When compared to control Pi status, addition of Pi enhanced As(V) tolerance of both WT and atipk1‐1 plants, while the arsenic concentration was less reduced in the latter genotype. Despite the higher Pi level in atipk1‐1 than did WT plants, the mutant suffered more severe Pi starvation under Pi limitation stress, indicating that Pi homeostasis was altered in the mutant. Gene expression analysis of WT and atipk1‐1 plants showed the diverse effect of As(V) stress on Pi starvation‐dependent regulation of Pi‐responsive genes. Our study suggested that a particular mechanism of As(V) toxicity existed in atipk1‐1 mutant, and may offer new insights into the interactions between Pi homeostasis and As(V) detoxification in plants.     

Fate of arsenate following arsenite oxidation in Agrobacterium tumefaciens GW4

The fate of arsenate (As^V) generated by microbial arsenite (As^III) oxidation is poorly understood. Agrobacterium tumefaciens wild‐type strain (GW4) was studied to determine how the cell copes with As^V generated in batch culture. GW4 grown heterotrophically with mannitol used As^III as a supplemental energy supply as reflected by enhanced growth and increased cellular levels of NADH and ATP. Under low phosphate (Pi) conditions and presence of As^III oxidation, up to ～ 50% of the resulting As^V was taken up and found associated with the periplasm, membrane or cytoplasm fractions of the cells. Arsenic was found associated with proteins and polar lipids, but not in nucleic acids or sugars. Thin‐layer chromatography and gas chromatography–mass spectrometry analysis suggested the presence of arsenolipids in membranes, presumably as part of the bilayer structure of the cell membrane and replacing Pi under Pi‐limiting conditions. The potential role of a Pi‐binding protein (PstS) for As^V uptake was assessed with the His‐tag purified protein. Intrinsic tryptophan fluorescence spectra analysis suggests that PstS can bind As^V, but with lower affinity as compared with Pi. In early stationary phase cells, the As^V : Pi ratio was approximately 4.3 and accompanied by an altered cell ultrastructure.     

Linking microbial oxidation of arsenic with detection and phylogenetic analysis of arsenite oxidase genes in diverse geothermal environments

The identification and characterization of genes involved in the microbial oxidation of arsenite will contribute to our understanding of factors controlling As cycling in natural systems. Towards this goal, we recently characterized the widespread occurrence of aerobic arsenite oxidase genes (aroA‐like) from pure‐culture bacterial isolates, soils, sediments and geothermal mats, but were unable to detect these genes in all geothermal systems where we have observed microbial arsenite oxidation. Consequently, the objectives of the current study were to measure arsenite‐oxidation rates in geochemically diverse thermal habitats in Yellowstone National Park (YNP) ranging in pH from 2.6 to 8, and to identify corresponding 16S rRNA and aroA genotypes associated with these arsenite‐oxidizing environments. Geochemical analyses, including measurement of arsenite‐oxidation rates within geothermal outflow channels, were combined with 16S rRNA gene and aroA functional gene analysis using newly designed primers to capture previously undescribed aroA‐like arsenite oxidase gene diversity. The majority of bacterial 16S rRNA gene sequences found in acidic (pH 2.6–3.6) Fe‐oxyhydroxide microbial mats were closely related to Hydrogenobaculum spp. (members of the bacterial order Aquificales), while the predominant sequences from near‐neutral (pH 6.2–8) springs were affiliated with other Aquificales including Sulfurihydrogenibium spp., Thermocrinis spp. and Hydrogenobacter spp., as well as members of the Deinococci, Thermodesulfobacteria and β‐Proteobacteria. Modified primers designed around previously characterized and newly identified aroA‐like genes successfully amplified new lineages of aroA‐like genes associated with members of the Aquificales across all geothermal systems examined. The expression of Aquificales aroA‐like genes was also confirmed in situ, and the resultant cDNA sequences were consistent with aroA genotypes identified in the same environments. The aroA sequences identified in the current study expand the phylogenetic distribution of known Mo‐pterin arsenite oxidase genes, and suggest the importance of three prominent genera of the order Aquificales in arsenite oxidation across geochemically distinct geothermal habitats ranging in pH from 2.6 to 8.     

Urease gene‐containing Archaea dominate autotrophic ammonia oxidation in two acid soils

The metabolic traits of ammonia‐oxidizing archaea (AOA) and bacteria (AOB) interacting with their environment determine the nitrogen cycle at the global scale. Ureolytic metabolism has long been proposed as a mechanism for AOB to cope with substrate paucity in acid soil, but it remains unclear whether urea hydrolysis could afford AOA greater ecological advantages. By combining DNA‐based stable isotope probing (SIP) and high‐throughput pyrosequencing, here we show that autotrophic ammonia oxidation in two acid soils was predominately driven by AOA that contain ureC genes encoding the alpha subunit of a putative archaeal urease. In urea‐amended SIP microcosms of forest soil (pH 5.40) and tea orchard soil (pH 3.75), nitrification activity was stimulated significantly by urea fertilization when compared with water‐amended soils in which nitrification resulted solely from the oxidation of ammonia generated through mineralization of soil organic nitrogen. The stimulated activity was paralleled by changes in abundance and composition of archaeal amoA genes. Time‐course incubations indicated that archaeal amoA genes were increasingly labelled by ¹³CO₂ in both microcosms amended with water and urea. Pyrosequencing revealed that archaeal populations were labelled to a much greater extent in soils amended with urea than water. Furthermore, archaeal ureC genes were successfully amplified in the ¹³C‐DNA, and acetylene inhibition suggests that autotrophic growth of urease‐containing AOA depended on energy generation through ammonia oxidation. The sequences of AOB were not detected, and active AOA were affiliated with the marine Group 1.1a‐associated lineage. The results suggest that ureolytic N metabolism could afford AOA greater advantages for autotrophic ammonia oxidation in acid soil, but the mechanism of how urea activates AOA cells remains unclear.     

Comparative genetic analysis of,Arabidopsis purple acid phosphatases AtPAP10, AtPAP12, and AtPAP26 provides new insights into their roles in plant adaptation to phosphate deprivation

Induction and secretion of acid phosphatases （APases） is thought to be an adaptive mechanism that helps plants survive and grow under phosphate （Pi） deprivation, in Arabidopsis, there are 29 purple acid phosphatase （AtPAP） genes. To systematically investigate the roles of different AtPAPs, we first identified knockout or knock-down T-DNA lines for all 29 AtPAP genes. Using these atpap mutants combined with in-gel and quantitative APase enzyme assays, we demonstrated that AtPAP12 and AtPAP26 are two major intracellular and secreted APases in Arabidopsis while AtPAPlo is mainly a secreted APase. On Pi-deficient （P-） medium or P- medium supplemented with the organophosphates ADP and fructose-6-phosphate （Fru-6-P）, growth of atpaplo was significantly reduced whereas growth of atpap12 was only moderately reduced, and growth of atpap26 was nearly equal to that of the wild type （WT）. Overexpression of the AtPAP12 or AtPAP26 gene, however, caused plants to grow better on P- or P- medium supplemented with ADP or Fru-6-P. Interest-ingly, Pi levels are essentially the same for the WT and overexpressing lines, although these two types of plants have significantly different growth phenotypes. These results suggest that the APases may have other roles besides enhancing internal Pi recycling or releasing Pi from external organophosphates for plant uptake.     

Down‐regulation of OsSPX1 caused semi‐male sterility,resulting in reduction of grain yield in rice

OsSPX1, a rice SPX domain gene, involved in the phosphate (Pi)‐sensing mechanism plays an essential role in the Pi‐signalling network through interaction with OsPHR2. In this study, we focused on the potential function of OsSPX1 during rice reproductive phase. Based on investigation of OsSPX1 antisense and sense transgenic rice lines in the paddy fields, we discovered that the down‐regulation of OsSPX1 caused reduction of seed‐setting rate and filled grain number. Through examination of anthers and pollens of the transgenic and wild‐type plants by microscopy, we found that the antisense of OsSPX1 gene led to semi‐male sterility, with lacking of mature pollen grains and phenotypes with a disordered surface of anthers and pollens. We further conducted rice whole‐genome GeneChip analysis to elucidate the possible molecular mechanism underlying why the down‐regulation of OsSPX1 caused deficiencies in anthers and pollens and lower seed‐setting rate in rice. The down‐regulation of OsSPX1 significantly affected expression of genes involved in carbohydrate metabolism and sugar transport, anther development, cell cycle, etc. These genes may be related to pollen fertility and male gametophyte development. Our study demonstrated that down‐regulation of OsSPX1 disrupted rice normal anther and pollen development by affecting carbohydrate metabolism and sugar transport, leading to semi‐male sterility, and ultimately resulted in low seed‐setting rate and grain yield.     

The secretion of the bacterial phytase PHY‐US417 by Arabidopsis roots reveals its potential for increasing phosphate acquisition and biomass production during co‐growth

Phytic acid (PA) is a major source of inorganic phosphate (Pi) in the soil; however, the plant lacks the capacity to utilize it for Pi nutrition and growth. Microbial phytases constitute a group of enzymes that are able to remobilize Pi from PA. Thus, the use of these phytases to increase the capacity of higher plants to remobilize Pi from PA is of agronomical interest. In the current study, we generate transgenic Arabidopsis lines (ePHY) overexpressing an extracellular form of the phytase PHY‐US417 of Bacillus subtilis, which are characterized by high levels of secreted phytase activity. In the presence of PA as sole source of Pi, while the wild‐type plants show hallmark of Pi deficiency phenotypes, including the induction of the expression of Pi starvation‐induced genes (PSI, e.g. PHT1;4) and the inhibition of growth capacity, the ePHY overexpressing lines show a higher biomass production and no PSI induction. Interestingly, when co‐cultured with ePHY overexpressors, wild‐type Arabidopsis plants (or tobacco) show repression of the PSI genes, improvement of Pi content and increases in biomass production. In line with these results, mutants in the high‐affinity Pi transporters, namely pht1;1 and pht1;1‐1;4, both fail to accumulate Pi and to grow when co‐cultured with ePHY overexpressors. Taken together, these data demonstrate the potential of secreted phytases in improving the Pi content and enhancing growth of not only the transgenic lines but also the neighbouring plants.     

The Arabidopsis thaliana aquaglyceroporin AtNIP7;1 is a pathway for arsenite uptake

We studied the effect of loss of function in the NIP subfamily II in Arabidopsis thaliana to assess their potential role(s) in arsenite (AsIII) uptake. Loss of function in AtNIP7;1 led to increased plant tolerance to AsIII and reduced total As in planta. AtNIP7;1 expression in various yeast backgrounds increased AsIII sensitivity. In the acr3Δ yeast genotype, AtNIP7;1 caused a moderate increase in AsV tolerance. Short-term As uptake in fsp1Δ expressing AtNIP7;1 was significantly larger than that in the empty vector control.

The data suggest that AtNIP7;1 can mediate AsIII transport and contributes to AsIII uptake in plants.