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.     

A cDNA from pea petals with sequence similarity to pollen allergen, cytokinin-induced and genetic tumour-specific genes: identification of a new family of related sequences

A cDNA isolated from pea petals exhibits extensive similarity to pollen allergen genes, a cytokinin-regulated cDNA from soybean suspension cultures, a partial cDNA preferentially expressed in tobacco genetic tumours, four Arabidopsis expressed sequence tags (ESTs) and fifteen rice ESTs. This diverse family of pollen allergen-likes genes may have a common ancestor or at least share common functional domains. Possession of a putative signal peptide and a presumed extracellular location is a common aspect of this family of sequences.     

滨海湿地生态系统微生物驱动的氮循环研究进展

滨海湿地生态系统介于陆地生态系统和海洋生态系统之间,其类型多种多样,环境差异极大,微生物种类丰富。近年来,随着人为氮源的大量输入,造成滨海湿地生态系统富营养化污染问题日趋严重。本文主要总结了滨海湿地生态系统微生物驱动的固氮、硝化、反硝化、厌氧氨氧化、NO_3~-还原成铵等主要氮循环过程,并综述了通过功能基因(如nifH、amoA、hzo、nirS、nirK、nrfA)检测微生物群落多样性及其环境影响因素的相关研究,旨在更好理解微生物驱动氮循环过程以去除氮,以期为减轻富营养化和危害性藻类爆发提供科学依据。     

Solution Hybrid Selection Capture for the Recovery of Functional Full-Length Eukaryotic cDNAs From Complex Environmental Samples

Eukaryotic microbial communities play key functional roles in soil biology and potentially represent a rich source of natural products including biocatalysts. Culture-independent molecular methods are powerful tools to isolate functional genes from uncultured microorganisms. However, none of the methods used in environmental genomics allow for a rapid isolation of numerous functional genes from eukaryotic microbial communities. We developed an original adaptation of the solution hybrid selection (SHS) for an efficient recovery of functional complementary DNAs (cDNAs) synthesized from soil-extracted polyadenylated mRNAs. This protocol was tested on the Glycoside Hydrolase 11 gene family encoding endo-xylanases for which we designed 35 explorative 31-mers capture probes. SHS was implemented on four soil eukaryotic cDNA pools. After two successive rounds of capture, >90% of the resulting cDNAs were GH11 sequences, of which 70% (38 among 53 sequenced genes) were full length. Between 1.5 and 25% of the cloned captured sequences were expressed in Saccharomyces cerevisiae. Sequencing of polymerase chain reaction-amplified GH11 gene fragments from the captured sequences highlighted hundreds of phylogenetically diverse sequences that were not yet described, in public databases. This protocol offers the possibility of performing exhaustive exploration of eukaryotic gene families within microbial communities thriving in any type of environment.     

Changes in Bacterial and Archaeal Community Structure and Functional Diversity along a Geochemically Variable Soil Profile

Spatial heterogeneity in physical, chemical, and biological properties of soils allows for the proliferation of diverse microbial communities. Factors influencing the structuring of microbial communities, including availability of nutrients and water, pH, and soil texture, can vary considerably with soil depth and within soil aggregates. Here we investigated changes in the microbial and functional communities within soil aggregates obtained along a soil profile spanning the surface, vadose zone, and saturated soil environments. The composition and diversity of microbial communities and specific functional groups involved in key pathways in the geochemical cycling of nitrogen, Fe, and sulfur were characterized using a coupled approach involving cultivation-independent analysis of both 16S rRNA (bacterial and archaeal) and functional genes (amoA and dsrAB) as well as cultivation-based analysis of Fe(III)-reducing organisms. Here we found that the microbial communities and putative ammonia-oxidizing and Fe(III)-reducing communities varied greatly along the soil profile, likely reflecting differences in carbon availability, water content, and pH. In particular, the Crenarchaeota 16S rRNA sequences are largely unique to each horizon, sharing a distribution and diversity similar to those of the putative (amoA-based) ammonia-oxidizing archaeal community. Anaerobic microenvironments within soil aggregates also appear to allow for both anaerobic- and aerobic-based metabolisms, further highlighting the complexity and spatial heterogeneity impacting microbial community structure and metabolic potential within soils.     

The Sphagnum microbiome supports bog ecosystem functioning under extreme conditions

Sphagnum‐dominated bogs represent a unique yet widely distributed type of terrestrial ecosystem and strongly contribute to global biosphere functioning. Sphagnum is colonized by highly diverse microbial communities, but less is known about their function. We identified a high functional diversity within the Sphagnum microbiome applying an Illumina‐based metagenomic approach followed by de novo assembly and MG‐RAST annotation. An interenvironmental comparison revealed that the Sphagnum microbiome harbours specific genetic features that distinguish it significantly from microbiomes of higher plants and peat soils. The differential traits especially support ecosystem functioning by a symbiotic lifestyle under poikilohydric and ombrotrophic conditions. To realise a plasticity–stability balance, we found abundant subsystems responsible to cope with oxidative and drought stresses, to exchange (mobile) genetic elements, and genes that encode for resistance to detrimental environmental factors, repair and self‐controlling mechanisms. Multiple microbe–microbe and plant–microbe interactions were also found to play a crucial role as indicated by diverse genes necessary for biofilm formation, interaction via quorum sensing and nutrient exchange. A high proportion of genes involved in nitrogen cycle and recycling of organic material supported the role of bacteria for nutrient supply. 16S rDNA analysis indicated a higher structural diversity than that which had been previously detected using PCR‐dependent techniques. Altogether, the diverse Sphagnum microbiome has the ability to support the life of the host plant and the entire ecosystem under changing environmental conditions. Beyond this, the moss microbiome presents a promising bio‐resource for environmental biotechnology – with respect to novel enzymes or stress‐protecting bacteria.     

Expression profile of calcium-dependent protein kinase (CDPKs) genes during the whole lifespan and under phytohormone treatment conditions in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L. ssp. <Emphasis Type="Italic">indica</Emphasis>)

Calcium-dependent protein kinases (CDPKs) control plant development and response to various stress environments through the important roles in the regulation of Ca²⁺ signaling. Thirty-one CDPK genes have been identified in the rice genome by a complete search of the genome based upon HMM profiles. In this study, the expression of this gene family was analyzed using the Affymetrix rice genome array in three rice cultivars: Minghui 63, Zhenshan 97, and their hybrid Shanyou 63 independently. Twenty-seven tissues sampled throughout the entire rice life-span were studied, along with three hormone treatments (GA3, NAA and KT), applied to the seedling at the trefoil stage. All 31 genes were found to be expressed in at least one of the experimental stages studied and revealed diverse expression patterns. We identified differential expression of the OsCPK genes in the stamen (1 day before flowering), the panicle (at the heading stage), the endosperm (days after pollination) and also in callus, in all three cultivars. Eight genes, OsCPK2, OsCPK11, OsCPK14, OsCPK22, OsCPK25, OsCPK26, OsCPK27 and OsCPK29 were found dominantly expressed in the panicle and the stamen, and five genes, OsCPK6, OsCPK7, OsCPK12, OsCPK23 and OsCPK31 were up-regulated in the endosperm stage. The OsCPK genes were also found to be regulated in rice seedlings subjected to different hormone treatment conditions, however their expression were not the same for all varieties. These diverse expression profiles trigger the functional analysis of the CDPK family in rice. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Distribution,diversity and functional dissociation of the mac genes in marine biofilms

Bacteria produce metamorphosis-associated contractile (MAC) structures to induce larval metamorphosis in Hydroides elegans. The distribution and diversity of mac gene homologs in marine environments are largely unexplored. In the present study mac genes were examined in marine environments by analyzing 101 biofilm and 91 seawater metagenomes. There were more mac genes in biofilms than in seawater, and substratum type, location, or sampling time did not affect the mac genes in biofilms. The mac gene clusters were highly diverse and often incomplete while the three MAC components co-occurred with other genes of different functions. Genomic analysis of four Pseudoalteromonas and two Streptomyces strains revealed the mac genes transfers among different microbial taxa. It is proposed that mac genes are more specific to biofilms; gene transfer among different microbial taxa has led to highly diverse mac gene clusters; and in most cases, the three MAC components function individually rather than forming a complex.     

Cloning the Soil Metagenome: a Strategy for Accessing the Genetic and Functional Diversity of Uncultured Microorganisms

Recent progress in molecular microbial ecology has revealed that traditional culturing methods fail to represent the scope of microbial diversity in nature, since only a small proportion of viable microorganisms in a sample are recovered by culturing techniques. To develop methods to investigate the full extent of microbial diversity, we used a bacterial artificial chromosome (BAC) vector to construct libraries of genomic DNA isolated directly from soil (termed metagenomic libraries). To date, we have constructed two such libraries, which contain more than 1 Gbp of DNA. Phylogenetic analysis of 16S rRNA gene sequences recovered from one of the libraries indicates that the BAC libraries contain DNA from a wide diversity of microbial phyla, including sequences from diverse taxa such as the low-G+C, gram-positive Acidobacterium, Cytophagales, and Proteobacteria. Initial screening of the libraries in Escherichia coli identified several clones that express heterologous genes from the inserts, confirming that the BAC vector can be used to maintain, express, and analyze environmental DNA. The phenotypes expressed by these clones include antibacterial, lipase, amylase, nuclease, and hemolytic activities. Metagenomic libraries are a powerful tool for exploring soil microbial diversity, providing access to the genetic information of uncultured soil microorganisms. Such libraries will be the basis of new initiatives to conduct genomic studies that link phylogenetic and functional information about the microbiota of environments dominated by microorganisms that are refractory to cultivation.     

Isolation and sequence analysis of <Emphasis Type="Italic">Sox</Emphasis> genes from lizard <Emphasis Type="Italic">Eremias</Emphasis> multiocellata

The Sox (SRY-related high-mobility-group box) family of genes shares a conserved HMG box and is involved in a diverse range of developmental processes and sex determination in vertebrates. Twenty Sox genes are present in the genomes of humans and mice, but far less is known about the Sox gene family in reptiles. Using two pairs of highly degenerate primers designed from a multiple alignment of Sox amino acid sequences in several species, different positive clones were obtained from male and female Eremias multiocellata, a viviparous lizard which is subject to TSD (temperature-dependent sex determination). These clones were sequenced and identified. They are members of the SoxB (Sox2, Sox14), SoxC (Sox11, Sox12) and SoxE (Sox9a, Sox9b, Sox10) groups. No sex-specific differences were observed. Based on the amino acid sequence similarities, the phylogenetic analysis was carried out and these genes clustered with their orthologues. In addition, we found the gene duplication in E. multiocellata, it may be a mechanism to produce new functional genes.     

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.     

Molecular Characterization of the Total Bacteria and Dissimilatory Arsenate-Reducing Bacteria in Core Sediments of the Jianghan Plain,Central China

Arsenic contamination in groundwater has been reported in the Jianghan Plain of China since 2005, yet little is known about the microbial communities involved in As mobilization in this area, especially the dissimilatory arsenate-reducing bacteria (DARB) communities. Here, we conducted a cultivation-independent investigation on core sediments collected from a region with arsenic-contaminated groundwater in the Jianghan Plain to reveal the total bacteria and DARB community structures. Highly diverse As-resistant bacteria communities were found from sediment samples via high-throughput sequencing of 16S rRNA genes. Notably, we identified 27 unique arrA gene (encoding the alpha subunit of dissimilatory arsenate reductase) phylotypes, none of which was related to any previously described arrA gene sequence. This suggests a novel and unique DARB community in the sediments of the Jianghan Plain and expands our knowledge about the distribution and diversity of this group of bacteria in natural environments. Moreover, RDA and CCA demonstrated that total bacterial communities and specific functional groups are controlled by different environmental factors. Specifically, sediment pH, NH₄⁺, total nitrogen, total Fe, total organic carbon and total phosphorus were the key factors driving total bacterial community compositions, while As significantly shaped DARB community structures. This report is the first to describe DARB communities and their correlation with environmental factors in Jianghan Plain sediments, which could give us clues about the origin of the arsenic contamination of groundwater in this region.     

Differentiation strategies of soil rare and abundant microbial taxa in response to changing climatic regimes

Despite the important roles of soil microbes, especially the most diverse rare taxa in maintaining community diversity and multifunctionality, how different climate regimes alter the stability and functions of the rare microbial biosphere remains unknown. We reciprocally transplanted field soils across a latitudinal gradient to simulate climate change and sampled the soils annually after harvesting the maize over the following 6 years (from 2005 to 2011). By sequencing microbial 16S ribosomal RNA gene amplicons, we found that changing climate regimes significantly altered the composition and dynamics of soil microbial communities. A continuous succession of the rare and abundant communities was observed. Rare microbial communities were more stable under changing climatic regimes, with lower variations in temporal dynamics, and higher stability and constancy of diversity. More nitrogen cycling genes were detected in the rare members than in the abundant members, including amoA, napA, nifH, nirK, nirS, norB and nrfA. Random forest analysis and receiver operating characteristics analysis showed that rare taxa may act as potential contributors to maize yield under changing climatics. The study indicates that the taxonomically and functionally diverse rare biosphere has the potential to increase functional redundancy and enhance the ability of soil communities to counteract environmental disturbances. With ongoing global climate change, exploring the succession process and functional changes of rare taxa may be important in elucidating the ecosystem stability and multifunctionality that are mediated by microbial communities.