Analysis of the Microbial Community Structure by Monitoring an Hg Methylation Gene (hgcA) in Paddy Soils along an Hg Gradient

Knowledge of the diversity of mercury (Hg)-methylating microbes in the environment is limited due to a lack of available molecular biomarkers. Here, we developed novel degenerate PCR primers for a key Hg-methylating gene (hgcA) and amplified successfully the targeted genes from 48 paddy soil samples along an Hg concentration gradient in the Wanshan Hg mining area of China. A significant positive correlation was observed between hgcA gene abundance and methylmercury (MeHg) concentrations, suggesting that microbes containing the genes contribute to Hg methylation in the sampled soils. Canonical correspondence analysis (CCA) showed that the hgcA gene diversity in microbial community structures from paddy soils was high and was influenced by the contents of total Hg, SO₄²⁻, NH₄⁺, and organic matter. Phylogenetic analysis showed that hgcA microbes in the sampled soils likely were related to Deltaproteobacteria, Firmicutes, Chloroflexi, Euryarchaeota, and two unclassified groups. This is a novel report of hgcA diversity in paddy habitats, and results here suggest a link between Hg-methylating microbes and MeHg contamination in situ, which would be useful for monitoring and mediating MeHg synthesis in soils.     

Impacts of Mercury on Freshwater Fish-Eating Wildlife and Humans

This paper reviews the current state of knowledge of the toxic effects of mercury on fish-eating birds, mammals, and humans associated with freshwater ecosystems, including new information on the relative risk of elevated methyl Hg exposure for fish-eating birds inhabiting aquatic ecosystems impacted by mining/smelting activities and areas characterized by high geological sources of Hg. The influence of various environmental conditions such as lake pH, DOC, and chemical speciation of Hg, on fish-Hg concentrations and Hg exposure in fish-eating wildlife, are discussed. Although a continuing global effort to decrease the release of this nonessential metal into the environment is warranted, Hg methylation and biomagnification may be limited in some environments due to chemical speciation of mercury in soils and sediments (e.g., HgS) and water quality conditions (e.g., high alkalinity and pH) that do not facilitate high methylation rates. We have shown such limitations for a lake where historic Hg mining greatly increased sediment-Hg loadings, yet Hg increases in small fish of various species are currently lower than expected, and top predators (bald eagles), despite having elevated concentrations of Hg in their blood compared with individuals from nearby lakes, exhibit no Hg-related reproductive impairment or other signs of MeHg intoxication. Recent epidemiological studies have shown that fish-eating human populations may be exposed to Hg sufficient to cause significant developmental effects. However, for humans, we conclude that the current USEPA reference dose for MeHg may be too restrictive, particularly for the less sensitive adult. The health status of indigenous peoples relying on the subsistence harvest of wild foods may be negatively affected by such restrictions.     

Targeted metagenomics: a high-resolution metagenomics approach for specific gene clusters in complex microbial communities

A major research goal in microbial ecology is to understand the relationship between gene organization and function involved in environmental processes of potential interest. Given that more than an estimated 99% of microorganisms in most environments are not amenable to culturing, methods for culture-independent studies of genes of interest have been developed. The wealth of metagenomic approaches allows environmental microbiologists to directly explore the enormous genetic diversity of microbial communities. However, it is extremely difficult to obtain the appropriate sequencing depth of any particular gene that can entirely represent the complexity of microbial metagenomes and be able to draw meaningful conclusions about these communities. This review presents a summary of the metagenomic approaches that have been useful for collecting more information about specific genes. Specific subsets of metagenomes that focus on sequence analysis were selected in each metagenomic studies. This 'targeted metagenomics' approach will provide extensive insight into the functional, ecological and evolutionary patterns of important genes found in microorganisms from various ecosystems.     

Oxidation of organoarsenicals and antimonite by a novel flavin monooxygenase widely present in soil bacteria

Arsenic can be biomethylated to form a variety of organic arsenicals differing in toxicity and environmental mobility. Trivalent methylarsenite (MAs(III)) produced in the methylation process is more toxic than inorganic arsenite (As(III)). MAs(III) also serves as a primitive antibiotic and, consequently, some environmental microorganisms have evolved mechanisms to detoxify MAs(III). However, the mechanisms of MAs(III) detoxification are not well understood. In this study, we identified an arsenic resistance (ars) operon consisting of three genes, arsRVK, that contribute to MAs(III) resistance in Ensifer adhaerens ST2. ArsV is annotated as an NADPH-dependent flavin monooxygenase with unknown function. Expression of arsV in the arsenic hypersensitive Escherichia coli strain AW3110Δars conferred resistance to MAs(III) and the ability to oxidize MAs(III) to MAs(V). In the presence of NADPH and either FAD or FMN, purified ArsV protein was able to oxidize both MAs(III) to MAs(V) and Sb(III) to Sb(V). Genes with arsV-like sequences are widely present in soils and environmental bacteria. Metagenomic analysis of five paddy soils showed the abundance of arsV-like sequences of 0.12–0.25 ppm. These results demonstrate that ArsV is a novel enzyme for the detoxification of MAs(III) and Sb(III) and the genes encoding ArsV are widely present in soil bacteria.     

Unveiling metabolic characteristics of an uncultured Gammaproteobacterium responsible for in situ PAH biodegradation in petroleum polluted soil

Exploring the metabolic characteristics of indigenous PAH degraders is critical to understanding the PAH bioremediation mechanism in the natural environment. While stable-isotopic probing (SIP) is a viable method to identify functional microorganisms in complex environments, the metabolic characteristics of uncultured degraders are still elusive. Here, we investigated the naphthalene (NAP) biodegradation of petroleum polluted soils by combining SIP, amplicon sequencing and metagenome binning. Based on the SIP and amplicon sequencing results, an uncultured Gammaproteobacterium sp. was identified as the key NAP degrader. Additionally, the assembled genome of this uncultured degrader was successfully obtained from the ¹³C-DNA metagenomes by matching its 16S rRNA gene with the SIP identified OTU sequence. Meanwhile, a number of NAP degrading genes encoding naphthalene/PAH dioxygenases were identified in this genome, further confirming the direct involvement of this indigenous degrader in the NAP degradation. The degrader contained genes related to the metabolisms of several carbon sources, energy substances and vitamins, illuminating potential reasons for why microorganisms cannot be cultivated and finally realize their cultivation. Our findings provide novel information on the mechanisms of in situ PAH biodegradation and add to our current knowledge on the cultivation of non-culturable microorganisms by combining both SIP and metagenome binning.     

Arsenic biotransformation by Streptomyces sp. isolated from rice rhizosphere

Isolation and functional analysis of microbes mediating the methylation of arsenic (As) in paddy soils is important for understanding the origin of dimethylarsinic acid (DMA) in rice grains. Here, we isolated from the rice rhizosphere a unique bacterium responsible for As methylation. Strain GSRB54, which was isolated from the roots of rice plants grown in As‐contaminated paddy soil under anaerobic conditions, was classified into the genus Streptomyces by 16S ribosomal RNA sequencing. Sequence analysis of the arsenite S‐adenosylmethionine methyltransferase (arsM) gene revealed that GSRB54 arsM was phylogenetically different from known arsM genes in other bacteria. This strain produced DMA and monomethylarsonic acid when cultured in liquid medium containing arsenite [As(III)]. Heterologous expression of GSRB54 arsM in Escherichia coli promoted methylation of As(III) by converting it into DMA and trimethylarsine oxide. These results demonstrate that strain GSRB54 has a strong ability to methylate As. In addition, DMA was detected in the shoots of rice grown in liquid medium inoculated with GSRB54 and containing As(III). Since Streptomyces are generally aerobic bacteria, we speculate that strain GSRB54 inhabits the oxidative zone around roots of paddy rice and is associated with DMA accumulation in rice grains through As methylation in the rice rhizosphere.     

Aquatic metagenomes implicate Thaumarchaeota in global cobalamin production

Cobalamin (vitamin B₁₂) is a complex metabolite and essential cofactor required by many branches of life, including most eukaryotic phytoplankton. Algae and other cobalamin auxotrophs rely on environmental cobalamin supplied from a relatively small set of cobalamin-producing prokaryotic taxa. Although several Bacteria have been implicated in cobalamin biosynthesis and associated with algal symbiosis, the involvement of Archaea in cobalamin production is poorly understood, especially with respect to the Thaumarchaeota. Based on the detection of cobalamin synthesis genes in available thaumarchaeotal genomes, we hypothesized that Thaumarchaeota, which are ubiquitous and abundant in aquatic environments, have an important role in cobalamin biosynthesis within global aquatic ecosystems. To test this hypothesis, we examined cobalamin synthesis genes across sequenced thaumarchaeotal genomes and 430 metagenomes from a diverse range of marine, freshwater and hypersaline environments. Our analysis demonstrates that all available thaumarchaeotal genomes possess cobalamin synthesis genes, predominantly from the anaerobic pathway, suggesting widespread genetic capacity for cobalamin synthesis. Furthermore, although bacterial cobalamin genes dominated most surface marine metagenomes, thaumarchaeotal cobalamin genes dominated metagenomes from polar marine environments, increased with depth in marine water columns, and displayed seasonality, with increased winter abundance observed in time-series datasets (e.g., L4 surface water in the English Channel). Our results also suggest niche partitioning between thaumarchaeotal and cyanobacterial ribosomal and cobalamin synthesis genes across all metagenomic datasets analyzed. These results provide strong evidence for specific biogeographical distributions of thaumarchaeotal cobalamin genes, expanding our understanding of the global biogeochemical roles played by Thaumarchaeota in aquatic environments.     

Single Cells within the Puerto Rico Trench Suggest Hadal Adaptation of Microbial Lineages

Hadal ecosystems are found at a depth of 6,000 m below sea level and below, occupying less than 1% of the total area of the ocean. The microbial communities and metabolic potential in these ecosystems are largely uncharacterized. Here, we present four single amplified genomes (SAGs) obtained from 8,219 m below the sea surface within the hadal ecosystem of the Puerto Rico Trench (PRT). These SAGs are derived from members of deep-sea clades, including the Thaumarchaeota and SAR11 clade, and two are related to previously isolated piezophilic (high-pressure-adapted) microorganisms. In order to identify genes that might play a role in adaptation to deep-sea environments, comparative analyses were performed with genomes from closely related shallow-water microbes. The archaeal SAG possesses genes associated with mixotrophy, including lipoylation and the glycine cleavage pathway. The SAR11 SAG encodes glycolytic enzymes previously reported to be missing from this abundant and cosmopolitan group. The other SAGs, which are related to piezophilic isolates, possess genes that may supplement energy demands through the oxidation of hydrogen or the reduction of nitrous oxide. We found evidence for potential trench-specific gene distributions, as several SAG genes were observed only in a PRT metagenome and not in shallower deep-sea metagenomes. These results illustrate new ecotype features that might perform important roles in the adaptation of microorganisms to life in hadal environments.     

Local root status: a neglected bio-factor that regulates the home-field advantage of leaf litter decomposition

Background and aims

Bacterial Non-Specific Acid Phosphatase (NSAP) enzymes are capable of dephosphorylating diverse organic phosphoesters but are rarely studied: their distribution in natural and managed environments is poorly understood. The aim of this study was to generate new insight into the environmental distribution of NSAPs and establish their potential global relevance to cycling of organic phosphorus.

Methods

We employed bioinformatic tools to determine NSAP diversity and subcellular localization in microbial genomes; used the corresponding NSAP gene sequences to census metagenomes from diverse ecosystems; studied the effect of long-term land management upon NSAP diversity and abundance.

Results

Periplasmic class B NSAPs are poorly represented in marine and terrestrial environments, reflecting their association with enteric and pathogenic bacteria. Periplasmic class A and outer membrane-associated class C NSAPs are cosmopolitan. NSAPs are more abundant in marine than terrestrial ecosystems and class C more abundant than class A genes, except in an acidic peat where class A genes dominate. A clear effect of land management upon gene abundance was identified.

Conclusions

NSAP genes are cosmopolitan. Class C genes are more widely distributed: their association with the outer-membrane of cells gives them a clear role in the cycling of organic phosphorus, particularly in soils.

     

Metagenomic Analysis of 0.2-μm-Passable Microorganisms in Deep-Sea Hydrothermal Fluid

We pyrosequenced the bulk DNA extracted from microorganisms that passed through 0.2-μm-pore-size filters and trapped by 0.1-μm-pore-size filters in the hydrothermal fluid of the Mariana Trough. Using the 454-FLX sequencer, we generated 202,648 sequences with an average length of 173.8 bases. Functional profiles were assigned by the SEED Annotation Engine. In the metagenome of the 0.2-μm-passable microorganisms, genes related to membrane function, including potassium homeostasis classified as membrane transport, and multidrug-resistance efflux pumps classified as virulence, were dominant. There was a higher proportion of genes pertinent to the subsystem of membrane transport in our metagenomic library than in other oceanic and hydrothermal vent metagenomes. Genes associated with a RND-type efflux transporter for exogenous substances were specifically identified in the present study. After a comparative analysis with the genome of the known ultramicrobacterium Sphingopyxis alaskensis RB2256, we discovered 1,542 cases of significant hits (E < 1 × 10⁻²) in our metagenome, and 1,172 of those were related to the DNA repair protein RadA. In this way, the microbial functional profile of 0.2-μm-passable fraction in the present study differs from oceanic metagenomes in the 0.2-μm-trapped fractions and hydrothermal vent metagenomes reported in previous research.     

东北湿地沉积物中T4型噬菌体g23基因的多样性

【目的】揭示我国东北典型湿地沉积物中T4型噬菌体g23基因的多样性,明确湿地环境T4型噬菌体群落分布特征,为噬菌体生态学研究提供数据支撑。【方法】采用简并性引物MZIA1bis和MZIA6对采自东北6个地点不同类型湿地沉积物土壤DNA进行PCR扩增,采用克隆测序方法,解析沉积物中T4型噬菌体g23基因组成,通过UniFrac分析T4型噬菌体群落结构在湿地沉积物中与其他环境中的差异。【结果】在东北湿地沉积物中共得到262条不同的g23基因序列,构建的系统进化树分析表明,我国东北湿地沉积物T4型噬菌体g23基因分布与海洋、湖泊及稻田生态系统中g23基因亲缘关系较近,而与东北旱地黑土g23基因分布较远;以g23基因群集表征的T4型噬菌体群落在不同地点湿地中分异明显。【结论】东北湿地生态系统T4型噬菌体群落结构复杂多样,存在着一些未知的噬菌体类群。     

Environmental Conditions Constrain the Distribution and Diversity of Archaeal <Emphasis Type="Italic">merA</Emphasis> in Yellowstone National Park,Wyoming, U.S.A.

The distribution and phylogeny of extant protein-encoding genes recovered from geochemically diverse environments can provide insight into the physical and chemical parameters that led to the origin and which constrained the evolution of a functional process. Mercuric reductase (MerA) plays an integral role in mercury (Hg) biogeochemistry by catalyzing the transformation of Hg(II) to Hg(0). Putative merA sequences were amplified from DNA extracts of microbial communities associated with mats and sulfur precipitates from physicochemically diverse Hg-containing springs in Yellowstone National Park, Wyoming, using four PCR primer sets that were designed to capture the known diversity of merA. The recovery of novel and deeply rooted MerA lineages from these habitats supports previous evidence that indicates merA originated in a thermophilic environment. Generalized linear models indicate that the distribution of putative archaeal merA lineages was constrained by a combination of pH, dissolved organic carbon, dissolved total mercury and sulfide. The models failed to identify statistically well supported trends for the distribution of putative bacterial merA lineages as a function of these or other measured environmental variables, suggesting that these lineages were either influenced by environmental parameters not considered in the present study, or the bacterial primer sets were designed to target too broad of a class of genes which may have responded differently to environmental stimuli. The widespread occurrence of merA in the geothermal environments implies a prominent role for Hg detoxification in these environments. Moreover, the differences in the distribution of the merA genes amplified with the four merA primer sets suggests that the organisms putatively engaged in this activity have evolved to occupy different ecological niches within the geothermal gradient.     

Mercury Methylation by the Methanogen Methanospirillum hungatei

Methylmercury (MeHg), a neurotoxic substance that accumulates in aquatic food chains and poses a risk to human health, is synthesized by anaerobic microorganisms in the environment. To date, mercury (Hg) methylation has been attributed to sulfate- and iron-reducing bacteria (SRB and IRB, respectively). Here we report that a methanogen, Methanospirillum hungatei JF-1, methylated Hg in a sulfide-free medium at comparable rates, but with higher yields, than those observed for some SRB and IRB. Phylogenetic analyses showed that the concatenated orthologs of the Hg methylation proteins HgcA and HgcB from M. hungatei are closely related to those from known SRB and IRB methylators and that they cluster together with proteins from eight other methanogens, suggesting that these methanogens may also methylate Hg. Because all nine methanogens with HgcA and HgcB orthologs belong to the class Methanomicrobia, constituting the late-evolving methanogenic lineage, methanogenic Hg methylation could not be considered an ancient metabolic trait. Our results identify methanogens as a new guild of Hg-methylating microbes with a potentially important role in mineral-poor (sulfate- and iron-limited) anoxic freshwater environments.     

Tree phyllospheres are a habitat for diverse populations of CO-oxidizing bacteria

Carbon monoxide (CO) is both a ubiquitous atmospheric trace gas and an air pollutant. While aerobic CO-degrading microorganisms in soils and oceans are estimated to remove ~370 Tg of CO per year, the presence of CO-degrading microorganisms in above-ground habitats, such as the phyllosphere, and their potential role in CO cycling remains unknown. CO-degradation by leaf washes of two common British trees, Ilex aquifolium and Crataegus monogyna, demonstrated CO uptake in all samples investigated. Based on the analyses of taxonomic and functional genes, diverse communities of candidate CO-oxidizing taxa were identified, including members of Rhizobiales and Burkholderiales which were abundant in the phyllosphere at the time of sampling. Based on predicted genomes of phyllosphere community members, an estimated 21% of phyllosphere bacteria contained CoxL, the large subunit of CO-dehydrogenase. In support of this, data mining of publicly available phyllosphere metagenomes for genes encoding CO-dehydrogenase subunits demonstrated that, on average, 25% of phyllosphere bacteria contained CO-dehydrogenase gene homologues. A CO-oxidizing Phyllobacteriaceae strain was also isolated from phyllosphere samples which contains genes encoding both CO-dehydrogenase as well as a ribulose-1,5-bisphosphate carboxylase-oxygenase. These results suggest that the phyllosphere supports diverse and potentially abundant CO-oxidizing bacteria, which are a potential sink for atmospheric CO.     

Reduced sulphur sources favour HgII reduction during anoxygenic photosynthesis by Heliobacteria

The consumption of rice has become a global food safety issue because rice paddies support the production of high levels of the potent neurotoxin, methylmercury. The production of methylmercury is carried out by chemotrophic anaerobes that rely on a diversity of terminal electron acceptors, namely sulphate. Sulphur can be a limiting nutrient in rice paddies, and sulphate amendments are often used to stimulate crop production, which can increase methylmercury production. Mercury (Hg) redox cycling can affect Hg methylation by controlling the delivery of inorganic Hg substrates to methylators in anoxic habitats. Whereas sulphur is recognized as a key substrate controlling methylmercury production, the controls sulphur exerts on other microbe‐mediated Hg transformations remain poorly understood. To explore the potential coupling between sulphur assimilation and anaerobic Hg^II reduction to Hg⁰, we studied Heliobacillus mobilis, a mesophilic anoxygenic phototroph representative from the Heliobacteriacea family originally isolated from a rice paddy. Here, we tested whether the redox state of the sulphur sources available to H. mobilis would affect its ability to reduce Hg^II. By comparing Hg⁰ production over a redox gradient of sulphur sources, we demonstrate that phototrophic Hg^II reduction is favoured in the presence of reduced sulphur sources such as thiosulphate and cysteine. We also show that cysteine exerts dynamic control on Hg cycling by affecting not only Hg's bioavailability but also its abiotic photoreduction under low light conditions. Specifically, in the absence of cells we show that organic matter (as yeast extract) and cysteine are both required for photoreduction to occur. This study offers insights into how one of the most primitive forms of photosynthesis affects Hg redox transformations and frames Heliobacteria as key players in Hg cycling within paddy soils, forming a basis for management strategies to mitigate Hg accumulation in rice.     

Unravelling the Identity,Metabolic Potential and Global Biogeography of the Atmospheric Methane‐Oxidizing Upland Soil Cluster α

Understanding of global methane sources and sinks is a prerequisite for the design of strategies to counteract global warming. Microbial methane oxidation in soils represents the largest biological sink for atmospheric methane. However, still very little is known about the identity, metabolic properties and distribution of the microbial group proposed to be responsible for most of this uptake, the uncultivated upland soil cluster α (USCα). Here, we reconstructed a draft genome of USCα from a combination of targeted cell sorting and metagenomes from forest soil, providing the first insights into its metabolic potential and environmental adaptation strategies. The 16S rRNA gene sequence recovered was distinctive and suggests this crucial group as a new genus within the Beijerinckiaceae, close to Methylocapsa. Application of a fluorescently labelled suicide substrate for the particulate methane monooxygenase enzyme (pMMO) coupled to 16S rRNA fluorescence in situ hybridisation (FISH) allowed for the first time a direct link of the high‐affinity activity of methane oxidation to USCα cells in situ. Analysis of the global biogeography of this group further revealed its presence in previously unrecognized habitats, such as subterranean and volcanic biofilm environments, indicating a potential role of these environments in the biological sink for atmospheric methane.     

Mercury transport and fate in municipal solid waste landfills and its implications

Mercury (Hg) release and migration from municipal solid waste landfills has been an important issue to nearby ecosystems and human health. To completely understand the Hg biogeochemical cycle in landfills, this review presents the Hg emission processes via different pathways, related controlling mechanisms, and critical Hg transport and transformation processes involving the diffusion and advection of Hg, Hg volatilization, Hg adsorption and desorption, Hg redox reactions, and Hg methylation and demethylation. These critical physical, chemical, and biological processes result in the phase transfer of Hg and the distribution of different Hg species in landfill gas (LFG), leachates, and cover soils. In addition, key factors (e.g., LFG, meteorological conditions, cover soils, and vegetation) affecting Hg emission processes and their impacts are discussed here. This work provides a comprehensive picture of Hg behavior in landfills, and has positive implications for the development of a process-based model and the control of Hg emissions from landfills.

     

Analysis of Hydrocarbon-Contaminated Groundwater Metagenomes as Revealed by High-Throughput Sequencing

The tendency for chlorinated aliphatics and aromatic hydrocarbons to accumulate in environments such as groundwater and sediments poses a serious environmental threat. In this study, the metabolic capacity of hydrocarbon (aromatics and chlorinated aliphatics)-contaminated groundwater in the KwaZulu-Natal province of South Africa has been elucidated for the first time by analysis of pyrosequencing data. The taxonomic data revealed that the metagenomes were dominated by the phylum Proteobacteria (mainly Betaproteobacteria). In addition, Flavobacteriales, Sphingobacteria, Burkholderiales, and Rhodocyclales were the predominant orders present in the individual metagenomes. These orders included microorganisms (Flavobacteria, Dechloromonas aromatica RCB, and Azoarcus) involved in the degradation of aromatic compounds and various other hydrocarbons that were present in the groundwater. Although the metabolic reconstruction of the metagenome represented composite cell networks, the information obtained was sufficient to address questions regarding the metabolic potential of the microbial communities and to correlate the data to the contamination profile of the groundwater. Genes involved in the degradation of benzene and benzoate, heavy metal-resistance mechanisms appeared to provide a survival strategy used by the microbial communities. Analysis of the pyrosequencing-derived data revealed that the metagenomes represent complex microbial communities that have adapted to the geochemical conditions of the groundwater as evidenced by the presence of key enzymes/genes conferring resistance to specific contaminants. Thus, pyrosequencing analysis of the metagenomes provided insights into the microbial activities in hydrocarbon-contaminated habitats.