Microbial oxidation of arsenite in a subarctic environment: diversity of arsenite oxidase genes and identification of a psychrotolerant arsenite oxidiser

Background  

Arsenic is toxic to most living cells. The two soluble inorganic forms of arsenic are arsenite (+3) and arsenate (+5), with arsenite the more toxic. Prokaryotic metabolism of arsenic has been reported in both thermal and moderate environments and has been shown to be involved in the redox cycling of arsenic. No arsenic metabolism (either dissimilatory arsenate reduction or arsenite oxidation) has ever been reported in cold environments (i.e. < 10°C).     

Selective phylogenetic analysis targeted at 16S rRNA genes of thermophiles and hyperthermophiles in deep-subsurface geothermal environments

Deep-subsurface samples obtained by deep drilling are likely to be contaminated with mesophilic microorganisms in the drilling fluid, and this could affect determination of the community structure of the geothermal microflora using 16S rRNA gene clone library analysis. To eliminate possible contamination by PCR-amplified 16S rRNA genes from mesophiles, a combined thermal denaturation and enzyme digestion method, based on a strong correlation between the G+C content of the 16S rRNA gene and the optimum growth temperatures of most known prokaryotic cultures, was used prior to clone library construction. To validate this technique, hot spring fluid (76 degrees C) and river water (14 degrees C) were used to mimic a deep-subsurface sample contaminated with drilling fluid. After DNA extraction and PCR amplification of the 16S rRNA genes from individual samples separately, the amplified products from river water were observed to be denatured at 82 degrees C and completely digested by exonuclease I (Exo I), while the amplified products from hot spring fluid remained intact after denaturation at 84 degrees C and enzyme digestion with Exo I. DNAs extracted from the two samples were mixed and used as a template for amplification of the 16S rRNA genes. The amplified rRNA genes were denatured at 84 degrees C and digested with Exo I before clone library construction. The results indicated that the 16S rRNA gene sequences from the river water were almost completely eliminated, whereas those from the hot spring fluid remained.     

Metagenomics of microbial and viral life in terrestrial geothermal environments

Geothermally heated regions of Earth, such as terrestrial volcanic areas (fumaroles, hot springs, and geysers) and deep-sea hydrothermal vents, represent a variety of different environments populated by extremophilic archaeal and bacterial microorganisms. Since most of these microbes thriving in such harsh biotopes, they are often recalcitrant to cultivation; therefore, ecological, physiological and phylogenetic studies of these microbial populations have been hampered for a long time. More recently, culture-independent methodologies coupled with the fast development of next generation sequencing technologies as well as with the continuous advances in computational biology, have allowed the production of large amounts of metagenomic data. Specifically, these approaches have assessed the phylogenetic composition and functional potential of microbial consortia thriving within these habitats, shedding light on how extreme physico-chemical conditions and biological interactions have shaped such microbial communities. Metagenomics allowed to better understand that the exposure to an extreme range of selective pressures in such severe environments, accounts for genomic flexibility and metabolic versatility of microbial and viral communities, and makes extreme- and hyper-thermophiles suitable for bioprospecting purposes, representing an interesting source for novel thermostable proteins that can be potentially used in several industrial processes.     

Linking geochemical processes with microbial community analysis: successional dynamics in an arsenic-rich, acid-sulphate-chloride geothermal spring

The source waters of acid‐sulphate‐chloride (ASC) geothermal springs located in Norris Geyser Basin, Yellowstone National Park contain several reduced chemical species, including H₂, H₂S, As(III), and Fe(II), which may serve as electron donors driving chemolithotrophic metabolism. Microorganisms thriving in these environments must also cope with high temperatures, low pH (～3), and high concentrations of sulphide, As(III), and boron. The goal of the current study was to correlate the temporal and spatial distribution of bacterial and archaeal populations with changes in temperature and geochemical energy gradients occurring throughout a newly formed (redirected) outflow channel of an ASC spring. A suite of complimentary analyses including aqueous geochemistry, microscopy, solid phase identification, and 16S rDNA sequence distribution were used to correlate the appearance of specific microbial populations with biogeochemical processes mediating S, Fe, and As cycling and subsequent biomineralization of As(V)‐rich hydrous ferric oxide (HFO) mats. Rapid As(III) oxidation (maximum first order rate constants ranged from 4 to 5 min^?1, t_1/2 = 0.17 ? 0.14 min) was correlated with the appearance of Hydrogenobaculum and Thiomonas–like populations, whereas the biogenesis of As(V)‐rich HFO microbial mats (mole ratios of As:Fe ～0.7) was correlated with the appearance of Metallosphaera, Acidimicrobium, and Thiomonas–like populations. Several 16S sequences detected near the source were closely related to sequences of chemolithotrophic hyperthermophilic populations including Stygiolobus and Hydrogenobaculum organisms that are known H₂ oxidizers. The use of H₂, reduced S(–II,0), Fe(II) and perhaps As(III) by different organisms represented throughout the outflow channel was supported by thermodynamic calculations, confirming highly exergonic redox couples with these electron donors. Results from this work demonstrated that chemical energy gradients play an important role in establishing distinct community structure as a function of distance from geothermal spring discharge.     

Detection, diversity and expression of aerobic bacterial arsenite oxidase genes

The arsenic (As) drinking water crisis in south and south-east Asia has stimulated intense study of the microbial processes controlling the redox cycling of As in soil-water systems. Microbial oxidation of arsenite is a critical link in the global As cycle, and phylogenetically diverse arsenite-oxidizing microorganisms have been isolated from various aquatic and soil environments. However, despite progress characterizing the metabolism of As in various pure cultures, no functional gene approaches have been developed to determine the importance and distribution of arsenite-oxidizing genes in soil-water-sediment systems. Here we report for the first time the successful amplification of arsenite oxidase-like genes (aroA/asoA/aoxB) from a variety of soil-sediment and geothermal environments where arsenite is known to be oxidized. Prior to the current work, only 16 aroA/asoA/aoxB-like gene sequences were available in GenBank, most of these being putative assignments from homology searches of whole genomes. Although aroA/asoA/aoxB gene sequences are not highly conserved across disparate phyla, degenerate primers were used successfully to characterize over 160 diverse aroA-like sequences from 10 geographically isolated, arsenic-contaminated sites and from 13 arsenite-oxidizing organisms. The primer sets were also useful for confirming the expression of aroA-like genes in an arsenite-oxidizing organism and in geothermal environments where arsenite is oxidized to arsenate. The phylogenetic and ecological diversity of aroA-like sequences obtained from this study suggests that genes for aerobic arsenite oxidation are widely distributed in the bacterial domain, are widespread in soil-water systems containing As, and play a critical role in the biogeochemical cycling of As.     

Cultural and phylogenetic analysis of mixed microbial populations found in natural and commercial bioleaching environments.

A range of autotrophic and heterotrophic enrichment cultures were established to determine the cultural bacterial diversity present in samples obtained from the acidic runoff of a chalcocite overburden heap and from laboratory-scale (1- to 4-liter) batch and continuous bioreactors which were being used for the commercial assessment of the bioleachability of zinc sulfide ore concentrates. Strains identified as Thiobacillus ferrooxidans, Thiobacillus thiooxidans, "Leptospirillum ferrooxidans," and Acidiphilium cryptum were isolated from both the natural site and the batch bioreactor, but only "L. ferrooxidans," a moderately thermophilic strain of T. thiooxidans, and a moderately thermophilic iron-oxidizing bacterium could be recovered from the continuous bioreactor running under steady-state conditions. Sequence analysis of the 16S rRNA genes of 33 representative strains revealed that all of the strains were closely related to strains which have been sequenced previously and also confirmed the phylogenetic diversity of bacteria present in bioleaching environments.     

New insights into microbial oxidation of antimony and arsenic

Sb(III) oxidation was documented in an Agrobacterium tumefaciens isolate that can also oxidize As(III). Equivalent Sb(III) oxidation rates were observed in the parental wild-type organism and in two well-characterized mutants that cannot oxidize As(III) for fundamentally different reasons. Therefore, despite the literature suggesting that Sb(III) and As(III) may be biochemical analogs, Sb(III) oxidation is catalyzed by a pathway different than that used for As(III). Sb(III) and As(III) oxidation was also observed for an eukaryotic acidothermophilic alga belonging to the order Cyanidiales, implying that the ability to oxidize metalloids may be phylogenetically widespread.     

Recent advances in molecular techniques for the detection of phylogenetic markers and functional genes in microbial communities

The detection and analysis of nucleic acids extracted from microbial communities are the ultimate ways to determine the diversity and functional capability of microbial communities in the environments. However, it remains a challenge to use molecular techniques for unequivocal determination and quantification of microbial species composition and functional activities. Considerable efforts have been made to enhance the capability of molecular techniques. Here an update of the recent developments in molecular techniques for environmental microbiology is provided.     

Diversity and abundance of the arsenite oxidase gene aioA in geothermal areas of Tengchong, Yunnan, China

A total of 12 samples were collected from the Tengchong geothermal areas of Yunnan, China, with the goal to assess the arsenite (AsIII) oxidation potential of the extant microbial communities as inferred by the abundance and diversity of the AsIII oxidase large subunit gene aioA relative to geochemical context. Arsenic concentrations were higher (on average 251.68 μg/L) in neutral or alkaline springs than in acidic springs (on average 30.88 μg/L). aioA abundance ranged from 1.63 × 10¹ to 7.08 × 10³ per ng of DNA and positively correlated with sulfide and the ratios of arsenate (AsV):total dissolved arsenic (As_Tot). Based on qPCR estimates of bacterial and archaeal 16S rRNA gene abundance, aioA-harboring organisms comprised as much as ~15 % of the total community. Phylogenetically, the major aioA sequences (270 total) in the acidic hot springs (pH 3.3–4.4) were affiliated with Aquificales and Rhizobiales, while those in neutral or alkaline springs (pH 6.6–9.1) were inferred to be primarily bacteria related to Thermales and Burkholderiales. Interestingly, aioA abundance at one site greatly exceeded bacterial 16S rRNA gene abundance, suggesting these aioA genes were archaeal even though phylogenetically these aioA sequences were most similar to the Aquificales. In summary, this study described novel aioA sequences in geothermal features geographically far removed from those in the heavily studied Yellowstone geothermal complex.     

The role of denitrification on arsenite oxidation and arsenic mobility in an anoxic sediment column model with activated alumina

Arsenite (As(III)) is the predominant arsenic (As) species in reducing environments. As(III) is less strongly adsorbed than As(V) at circumneutral pH conditions by common non‐iron metal oxides in sediments such as those of aluminum. Therefore, oxidation of As(III) to As(V) could contribute to an improved immobilization of As and thus help mitigate As contamination in groundwater. Microbial oxidation of As(III) is known to readily under aerobic conditions, however, the dissolved oxygen (O₂) concentration in groundwater may be limited due to the poor solubility of O₂ and its high chemical reactivity with reduced compounds. Nitrate (${\rm NO}_{3}^{{-} } $ ), can be considered as an alternative electron acceptor, which can support oxidation of As(III) to As(V) by denitrifying bacteria. In this study, two up‐flow sediment columns packed with activated alumina (AA) were utilized to demonstrate the role of denitrification on the oxidation of As(III) to As(V) and its contribution to improved As adsorption onto AA. One column was supplied with ${\rm NO}_{3}^{{-} } $ (C1) and its performance was compared with a control column lacking ${\rm NO}_{3}^{{-} } $ (C2). During most of the operation when the pH was in the circumneutral range (days 50–250), the release of arsenic was greater from C2 compared to C1. The effluent As concentrations started increasing on days 60 and 100 in C2 and C1, respectively. Complete breakthrough started on day 200 in C2; whereas in C1, complete breakthrough was never achieved. The effluent and solid phase As speciation was dominated by As(V) in C1, indicating the occurrence of As(III) oxidation due to ${\rm NO}_{3}^{{-} } $ ; whereas in C2, only As(III) was dominant. This study illustrates a bioremediation or natural attenuation process based on anoxic microbial ${\rm NO}_{3}^{{-} } $ ‐dependent oxidation of As(III) to more readily adsorbed As(V) as a means to enhance the immobilization of As on alumina oxide particles in subsurface environments. Biotechnol. Bioeng. 2010;107: 786–794. © 2010 Wiley Periodicals, Inc.     

Molecular phylogenetic analysis of archaeal intron-containing genes coding for rRNA obtained from a deep-subsurface geothermal water pool

Molecular phylogenetic analysis of a naturally occurring microbial community in a deep-subsurface geothermal environment indicated that the phylogenetic diversity of the microbial population in the environment was extremely limited and that only hyperthermophilic archaeal members closely related to Pyrobaculum were present. All archaeal ribosomal DNA sequences contained intron-like sequences, some of which had open reading frames with repeated homing-endonuclease motifs. The sequence similarity analysis and the phylogenetic analysis of these homing endonucleases suggested the possible phylogenetic relationship among archaeal rRNA-encoded homing endonucleases.     

Comparative and phylogenetic analysis of alpha-L-fucosidase genes

Fucosylated glycoconjugates play a role in a wide variety of biological processes, including immune responses, signal transduction, ontogenic events and pathogenesis of several human diseases. Alpha-L-fucosidases, which are responsible for their processing, have been demonstrated to be involved in lysosomal storage disease, inflammation, cystic fibrosis, cancer development and in the interactions between gametes in vertebrates as well as invertebrates. The sequence and comparative genomic analysis of these glycosyl hydrolases and the study of their evolutionary relationships appear therefore to be of considerable interest. In this work we carried out extensive similarity searches and comparative analyses to identify sequences encoding alpha-L-fucosidases. We have identified novel alpha-L-fucosidase coding sequences in worms, insects, sea urchin, ascidians, fish, chicken, amphibians, mammals and various bacteria resulting in a total of 39 alpha-L-fucosidase sequences. Two alpha-L-fucosidases that are present in all vertebrates likely reflect a distinct biological role for paralogous genes. Comparative sequence analysis of all metazoan alpha-L-fucosidases reveals a broad conservation of features, including the aspartate residue that constitutes the catalytic nucleophile. However, a cysteine which is thought to be part of the active site is also conserved in metazoa but not in arthropods, where it is replaced by an alanine. Phylogenetic analysis suggests a gene duplication event very early in metazoan evolution with the subsequent differential loss of isoforms in various metazoan lineages.     

Biogeochemistry and microbial ecology of methane oxidation in anoxic environments: a review

Evidence supporting a key role for anaerobic methane oxidation in the global methane cycle is reviewed. Emphasis is on recent microbiological advances. The driving force for research on this process continues to be the fact that microbial communities intercept and consume methane from anoxic environments, methane that would otherwise enter the atmosphere. Anaerobic methane oxidation is biogeochemically important because methane is a potent greenhouse gas in the atmosphere and is abundant in anoxic environments. Geochemical evidence for this process has been observed in numerous marine sediments along the continental margins, in methane seeps and vents, around methane hydrate deposits, and in anoxic waters. The anaerobic oxidation of methane is performed by at least two phylogenetically distinct groups of archaea, the ANME-1 and ANME-2. These archaea are frequently observed as consortia with sulfate-reducing bacteria, and the metabolism of these consortia presumably involves a syntrophic association based on interspecies electron transfer. The archaeal member of a consortium apparently oxidizes methane and shuttles reduced compounds to the sulfate-reducing bacteria. Despite recent advances in understanding anaerobic methane oxidation, uncertainties still remain regarding the nature and necessity of the syntrophic association, the biochemical pathway of methane oxidation, and the interaction of the process with the local chemical and physical environment. This review will consider the microbial ecology and biogeochemistry of anaerobic methane oxidation with a special emphasis on the interactions between the responsible organisms and their environment. This revised version was published online in August 2006 with corrections to the Cover Date.     

Photosynthetic and phylogenetic primers for detection of anoxygenic phototrophs in natural environments.

Primer sets were designed to target specific 16S ribosomal DNA (rDNA) sequences of photosynthetic bacteria, including the green sulfur bacteria, the green nonsulfur bacteria, and the members of the Heliobacteriaceae (a gram-positive phylum). Due to the phylogenetic diversity of purple sulfur and purple nonsulfur phototrophs, the 16S rDNA gene was not an appropriate target for phylogenetic rDNA primers. Thus, a primer set was designed that targets the pufM gene, encoding the M subunit of the photosynthetic reaction center, which is universally distributed among purple phototrophic bacteria. The pufM primer set amplified DNAs not only from purple sulfur and purple nonsulfur phototrophs but also from Chloroflexus species, which also produce a reaction center like that of the purple bacteria. Although the purple bacterial reaction center structurally resembles green plant photosystem II, the pufM primers did not amplify cyanobacterial DNA, further indicating their specificity for purple anoxyphototrophs. This combination of phylogenetic- and photosynthesis-specific primers covers all groups of known anoxygenic phototrophs and as such shows promise as a molecular tool for the rapid assessment of natural samples in ecological studies of these organisms.     

Patterns of ecological specialization among microbial populations in the Red Sea and diverse oligotrophic marine environments

Large swaths of the nutrient‐poor surface ocean are dominated numerically by cyanobacteria (Prochlorococcus), cyanobacterial viruses (cyanophage), and alphaproteobacteria (SAR11). How these groups thrive in the diverse physicochemical environments of different oceanic regions remains poorly understood. Comparative metagenomics can reveal adaptive responses linked to ecosystem‐specific selective pressures. The Red Sea is well‐suited for studying adaptation of pelagic‐microbes, with salinities, temperatures, and light levels at the extreme end for the surface ocean, and low nutrient concentrations, yet no metagenomic studies have been done there. The Red Sea (high salinity, high light, low N and P) compares favorably with the Mediterranean Sea (high salinity, low P), Sargasso Sea (low P), and North Pacific Subtropical Gyre (high light, low N). We quantified the relative abundance of genetic functions among Prochlorococcus, cyanophage, and SAR11 from these four regions. Gene frequencies indicate selection for phosphorus acquisition (Mediterranean/Sargasso), DNA repair and high‐light responses (Red Sea/Pacific Prochlorococcus), and osmolyte C1 oxidation (Red Sea/Mediterranean SAR11). The unexpected connection between salinity‐dependent osmolyte production and SAR11 C1 metabolism represents a potentially major coevolutionary adaptation and biogeochemical flux. Among Prochlorococcus and cyanophage, genes enriched in specific environments had ecotype distributions similar to nonenriched genes, suggesting that inter‐ecotype gene transfer is not a major source of environment‐specific adaptation. Clustering of metagenomes using gene frequencies shows similarities in populations (Red Sea with Pacific, Mediterranean with Sargasso) that belie their geographic distances. Taken together, the genetic functions enriched in specific environments indicate competitive strategies for maintaining carrying capacity in the face of physical stressors and low nutrient availability.     

Molecular phylogenetic analysis of dominant microbial populations in aged refuse

The phylogenetic analysis of dominant microbial populations in 8-year-old refuse samples was done in terms of the whole Bacterial and Archaeal domains. The results indicated that the Bacterial 16S rRNA genes sequences from the aged refuse were largely affiliated with the genus Bacillus, and that more than 60 % of the Archaeal sequences were closely related to the methanogenic archaeon. Some inferentially identified extremophilic organisms, particularly alkaliphiles and/or halophiles, were noted to be present in the aged refuse. Moreover, molecular evidence for the occurrence of ammonia-oxidizing Archaea in aged refuse was reported, which opens up avenues for elucidating its role in ammonia transformation in landfill systems. It seems reasonable to assume that the highly complex environment within the landfill systems may select for microbial populations with versatile metabolism and strong adaptation. These findings underline the need for further biochemical and ecological study of these organisms in aged refuse.     

Genomic insights into microbial iron oxidation and iron uptake strategies in extremely acidic environments

This minireview presents recent advances in our understanding of iron oxidation and homeostasis in acidophilic Bacteria and Archaea. These processes influence the flux of metals and nutrients in pristine and man-made acidic environments such as acid mine drainage and industrial bioleaching operations. Acidophiles are also being studied to understand life in extreme conditions and their role in the generation of biomarkers used in the search for evidence of existing or past extra-terrestrial life. Iron oxidation in acidophiles is best understood in the model organism Acidithiobacillus ferrooxidans. However, recent functional genomic analysis of acidophiles is leading to a deeper appreciation of the diversity of acidophilic iron-oxidizing pathways. Although it is too early to paint a detailed picture of the role played by lateral gene transfer in the evolution of iron oxidation, emerging evidence tends to support the view that iron oxidation arose independently more than once in evolution. Acidic environments are generally rich in soluble iron and extreme acidophiles (e.g. the Leptospirillum genus) have considerably fewer iron uptake systems compared with neutrophiles. However, some acidophiles have been shown to grow as high as pH 6 and, in the case of the Acidithiobacillus genus, to have multiple iron uptake systems. This could be an adaption allowing them to respond to different iron concentrations via the use of a multiplicity of different siderophores. Both Leptospirillum spp. and Acidithiobacillus spp. are predicted to synthesize the acid stable citrate siderophore for Fe(III) uptake. In addition, both groups have predicted receptors for siderophores produced by other microorganisms, suggesting that competition for iron occurs influencing the ecophysiology of acidic environments. Little is known about the genetic regulation of iron oxidation and iron uptake in acidophiles, especially how the use of iron as an energy source is balanced with its need to take up iron for metabolism. It is anticipated that integrated and complex regulatory networks sensing different environmental signals, such as the energy source and/or the redox state of the cell as well as the oxygen availability, are involved.     

Comparative phylogenetic analysis of microbial communities in pristine and hydrocarbon-contaminated Alpine soils

A molecular characterization of pristine and petroleum hydrocarbon-contaminated Alpine soils sampled in Tyrol (Austria) was performed. To identify predominant bacteria, PCR-amplified 16S rRNA gene fragments from five pristine and nine contaminated soils were analysed using denaturing gradient gel electrophoresis (DGGE). Sequencing and phylogenetic analyses demonstrated that the majority of the DGGE bands represented bacteria in the Actinobacteria and Proteobacteria phyla: 18 and 73%, respectively, in pristine soils, compared with 20 and 76%, respectively, in contaminated soils. A different distribution pattern of bacterial classes in the Proteobacteria was observed between pristine and contaminated soils. The relative proportion of microorganisms belonging to the Alphaproteobacteria was larger in pristine (46%) than in contaminated (24%) soils, while Betaproteobacteria and Gammaproteobacteria were detected only in the hydrocarbon-contaminated soils. This result compared favourably with earlier work in which hydrocarbon-degradation genotypes, largely pseudomonads and Acinetobacter, belonging to the Gammaproteobacteria, were enriched following oil hydrocarbon contamination. In contrast, members of the Actinobacteria phylum, represented by Rhodococcus and Mycobacterium, were found in pristine soils where contamination events had not occurred. The results demonstrate a significant shift in the microbial community structure in Alpine soils following contamination. Furthermore, more potentially novel phylotypes were found in the pristine soils than in the contaminated soils.     

Ecophysiology and geochemistry of microbial arsenic oxidation within a high arsenic, circumneutral hot spring system of the Alvord Desert

Microbial metabolism of arsenic has gained considerable interest, due to the potential of microorganisms to drive arsenic cycling and significantly influence the geochemistry of naturally arsenic-rich or anthropogenically arsenic-polluted environments. Alvord Hot Spring in southeastern Oregon is a circumneutral hot spring with an average arsenic concentration of 4.5 mg L(-1) (60 microM). Hydrogeochemical analyses indicated significant arsenite oxidation, increased pH and decreased temperature along the stream channels flowing into Alvord Hot Spring. The dynamic range of pH and temperature over the length of three stream channels were 6.76-7.06 and 69.5-78.2 degrees C, respectively. Biofilm samples showed As(III) oxidation ex situ. 16S rRNA gene studies of sparse upstream biofilm indicated a dominance of bacteria related to Sulfurihydrogenibium, Thermus, and Thermocrinis. The lush downstream biofilm community included these same three groups but was more diverse with sequences related to uncultured OP10 bacterial phylum, uncultured Bacteroidetes, and an uncultured clade. Isolation of an arsenite oxidizer was conducted with artificial hot spring medium and yielded the isolate A03C, which is closely related to Thermus aquaticus based on 16S rRNA gene analysis. Thus, this study demonstrated the bacterial diversity along geochemical gradients of temperature, pH and As(III): As(V), and provided evidence of microbial arsenite oxidation within the Alvord Hot Spring system.