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Global oceanographic monitoring initiatives originally measured abiotic essential ocean variables but are currently incorporating biological and metagenomic sampling programs. There is, however, a large knowledge gap on how to infer bacterial functions, the information sought by biogeochemists, ecologists, and modelers, from the bacterial taxonomic information (produced by bacterial marker gene surveys). Here, we provide a correlative understanding of how a bacterial marker gene (16S rRNA) can be used to infer latitudinal trends for metabolic pathways in global monitoring campaigns. From a transect spanning 7000 km in the South Pacific Ocean we infer ten metabolic pathways from 16S rRNA gene sequences and 11 corresponding metagenome samples, which relate to metabolic processes of primary productivity, temperature-regulated thermodynamic effects, coping strategies for nutrient limitation, energy metabolism, and organic matter degradation. This study demonstrates that low-cost, high-throughput bacterial marker gene data, can be used to infer shifts in the metabolic strategies at the community scale.

Exploratory drilling for deep-sea oil and gas resources is planned for the Great Australian Bight (GAB). There is scant knowledge of the region's benthic ecosystems and no baseline information of the region's indigenous oil degrading bacteria. To address this knowledge gap, we used next generation sequencing (NGS) of three marker genes (alkB, c23o and pmoA) to detect and characterize the microbial communities capable of aerobic hydrocarbon degradation. Unique, highly novel microbial communities capable of degrading hydrocarbons occur in surface sediments at depths between 200 and 2800 m. Clustering at 97% demonstrated differences in community structure with depth, changing most markedly between 400 and 1000 m depth on the continental slope, and identified putative functional 'ecotypes' related to depth. Observed differences in community structure showed strong correlations with temperature, other physicochemical properties of the overlying water column and are further modulated by differences in sediment grain size. This study provides important baseline data on hydrocarbon degrading microbial communities prior to the start of petroleum resource extraction. Our data will inform future ecological monitoring of the GAB deep-sea ecosystem., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Marine microbes along with microeukaryotes are key regulators of oceanic biogeochemical pathways. Here we present a high-resolution (every 0.5° of latitude) dataset describing microbial pro- and eukaryotic richness in the surface and just below the thermocline along a 7,000-km transect from 66°S at the Antarctic ice edge to the equator in the South Pacific Ocean. The transect, conducted in austral winter, covered key oceanographic features including crossing of the polar front (PF), the subtropical front (STF), and the equatorial upwelling region. Our data indicate that temperature does not determine patterns of marine microbial richness, complementing the global model data from Ladau et al. [Ladau J, et al. (2013) ISME J 7:1669-1677]. Rather, NH 4 , nanophytoplankton, and primary productivity were the main drivers for archaeal and bacterial richness. Eukaryote richness was highest in the least-productive ocean region, the tropical oligotrophic province. We also observed a unique diversity pattern in the South Pacific Ocean: a regional increase in archaeal and bacterial diversity between 10°S and the equator. Rapoport's rule describes the tendency for the latitudinal ranges of species to increase with latitude. Our data showed that the mean latitudinal ranges of archaea and bacteria decreased with latitude. We show that permanent oceanographic features, such as the STF and the equatorial upwelling, can have a significant influence on both alpha-diversity and beta-diversity of pro- and eukaryotes.+ , nanophytoplankton, and primary productivity were the main drivers for archaeal and bacterial richness. Eukaryote richness was highest in the least-productive ocean region, the tropical oligotrophic province. We also observed a unique diversity pattern in the South Pacific Ocean: a regional increase in archaeal and bacterial diversity between 10°S and the equator. Rapoport's rule describes the tendency for the latitudinal ranges of species to increase with latitude. Our data showed that the mean latitudinal ranges of archaea and bacteria decreased with latitude. We show that permanent oceanographic features, such as the STF and the equatorial upwelling, can have a significant influence on both alpha-diversity and beta-diversity of pro- and eukaryotes., Competing Interests: The authors declare no conflict of interest.

Sustained observations of microbial dynamics are rare, especially in southern hemisphere waters. The Australian Marine Microbial Biodiversity Initiative (AMMBI) provides methodologically standardized, continental scale, temporal phylogenetic amplicon sequencing data describing Bacteria, Archaea and microbial Eukarya assemblages. Sequence data is linked to extensive physical, biological and chemical oceanographic contextual information. Samples are collected monthly to seasonally from multiple depths at seven sites: Darwin Harbour (Northern Territory), Yongala (Queensland), North Stradbroke Island (Queensland), Port Hacking (New South Wales), Maria Island (Tasmania), Kangaroo Island (South Australia), Rottnest Island (Western Australia). These sites span ~30° of latitude and ~38° longitude, range from tropical to cold temperate zones, and are influenced by both local and globally significant oceanographic and climatic features. All sequence datasets are provided in both raw and processed fashion. Currently 952 samples are publically available for bacteria and archaea which include 88,951,761 bacterial (72,435 unique) and 70,463,079 archaeal (24,205 unique) 16 S rRNA v1-3 gene sequences, and 388 samples are available for eukaryotes which include 39,801,050 (78,463 unique) 18 S rRNA v4 gene sequences.

Sphagnum-associated methanotrophs (SAM) are an important sink for the methane (CH4) formed in boreal peatlands. We aimed to reveal how peatland succession, which entails a directional change in several environmental variables, affects SAM and their activity. Based on the pmoA microarray results, SAM community structure changes when a peatland develops from a minerotrophic fen to an ombrotrophic bog. Methanotroph subtypes Ia, Ib, and II showed slightly contrasting patterns during succession, suggesting differences in their ecological niche adaptation. Although the direct DNA-based analysis revealed a high diversity of type Ib and II methanotrophs throughout the studied peatland chronosequence, stable isotope probing (SIP) of the pmoA gene indicated they were active mainly during the later stages of succession. In contrast, type Ia methanotrophs showed active CH4 consumption in all analyzed samples. SIP-derived (13)C-labeled 16S rRNA gene clone libraries revealed a high diversity of SAM in every succession stage including some putative Methylocella/Methyloferula methanotrophs that are not detectable with the pmoA-based approach. In addition, a high diversity of 16S rRNA gene sequences likely representing cross-labeled nonmethanotrophs was discovered, including a significant proportion of Verrucomicrobia-related sequences. These results help to predict the effects of changing environmental conditions on SAM communities and activity., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Microbes catalyze all major geochemical cycles on earth. However, the role of microbial traits and community composition in biogeochemical cycles is still poorly understood mainly due to the inability to assess the community members that are actually performing biogeochemical conversions in complex environmental samples. Here we applied a polyphasic approach to assess the role of microbial community composition in modulating methane emission from a riparian floodplain. We show that the dynamics and intensity of methane consumption in riparian wetlands coincide with relative abundance and activity of specific subgroups of methane-oxidizing bacteria (MOB), which can be considered as a minor component of the microbial community in this ecosystem. Microarray-based community composition analyses demonstrated linear relationships of MOB diversity parameters and in vitro methane consumption. Incubations using intact cores in combination with stable isotope labeling of lipids and proteins corroborated the correlative evidence from in vitro incubations demonstrating γ-proteobacterial MOB subgroups to be responsible for methane oxidation. The results obtained within the riparian flooding gradient collectively demonstrate that niche partitioning of MOB within a community comprised of a very limited amount of active species modulates methane consumption and emission from this wetland. The implications of the results obtained for biodiversity-ecosystem functioning are discussed with special reference to the role of spatial and temporal heterogeneity and functional redundancy.

We investigated methanotrophic bacteria in slightly alkaline surface water (pH 7.4-8.7) of oilsands tailings ponds in Fort McMurray, Canada. These large lakes (up to 10 km(2)) contain water, silt, clay and residual hydrocarbons that are not recovered in oilsands mining. They are primarily anoxic and produce methane but have an aerobic surface layer. Aerobic methane oxidation was measured in the surface water at rates up to 152 nmol CH4 ml(-1) water d(-1). Microbial diversity was investigated via pyrotag sequencing of amplified 16S rRNA genes, as well as by analysis of methanotroph-specific pmoA genes using both pyrosequencing and microarray analysis. The predominantly detected methanotroph in surface waters at all sampling times was an uncultured species related to the gammaproteobacterial genus Methylocaldum, although a few other methanotrophs were also detected, including Methylomonas spp. Active species were identified via (13)CH4 stable isotope probing (SIP) of DNA, combined with pyrotag sequencing and shotgun metagenomic sequencing of heavy (13)C-DNA. The SIP-PCR results demonstrated that the Methylocaldum and Methylomonas spp. actively consumed methane in fresh tailings pond water. Metagenomic analysis of DNA from the heavy SIP fraction verified the PCR-based results and identified additional pmoA genes not detected via PCR. The metagenome indicated that the overall methylotrophic community possessed known pathways for formaldehyde oxidation, carbon fixation and detoxification of nitrogenous compounds but appeared to possess only particulate methane monooxygenase not soluble methane monooxygenase.

Advances in microbial ecology research are more often than not limited by the capabilities of available methodologies. Aerobic autotrophic nitrification is one of the most important and well studied microbiological processes in terrestrial and aquatic ecosystems. We have developed and validated a microbial diagnostic microarray based on the ammonia-monooxygenase subunit A (amoA) gene, enabling the in-depth analysis of the community structure of bacterial and archaeal ammonia oxidisers. The amoA microarray has been successfully applied to analyse nitrifier diversity in marine, estuarine, soil and wastewater treatment plant environments. The microarray has moderate costs for labour and consumables and enables the analysis of hundreds of environmental DNA or RNA samples per week per person. The array has been thoroughly validated with a range of individual and complex targets (amoA clones and environmental samples, respectively), combined with parallel analysis using traditional sequencing methods. The moderate cost and high throughput of the microarray makes it possible to adequately address broader questions of the ecology of microbial ammonia oxidation requiring high sample numbers and high resolution of the community composition.

Roots are the primary site of interaction between plants and microorganisms. To meet food demands in changing climates, improved yields and stress resistance are increasingly important, stimulating efforts to identify factors that affect plant productivity. The role of bacterial endophytes that reside inside plants remains largely unexplored, because analysis of their specific functions is impeded by difficulties in cultivating most prokaryotes. Here, we present the first metagenomic approach to analyze an endophytic bacterial community resident inside roots of rice, one of the most important staple foods. Metagenome sequences were obtained from endophyte cells extracted from roots of field-grown plants. Putative functions were deduced from protein domains or similarity analyses of protein-encoding gene fragments, and allowed insights into the capacities of endophyte cells. This allowed us to predict traits and metabolic processes important for the endophytic lifestyle, suggesting that the endorhizosphere is an exclusive microhabitat requiring numerous adaptations. Prominent features included flagella, plant-polymer-degrading enzymes, protein secretion systems, iron acquisition and storage, quorum sensing, and detoxification of reactive oxygen species. Surprisingly, endophytes might be involved in the entire nitrogen cycle, as protein domains involved in N(2)-fixation, denitrification, and nitrification were detected and selected genes expressed. Our data suggest a high potential of the endophyte community for plant-growth promotion, improvement of plant stress resistance, biocontrol against pathogens, and bioremediation, regardless of their culturability.

In lake ecosystems a major proportion of methane (CH(4) ) emissions originate from the littoral zone, which can have a great spatial variability in hydrology, soil quality and vegetation. Hitherto, spatial heterogeneity and the effects it has on functioning and diversity of methanotrophs in littoral wetlands have been poorly understood. A diagnostic microarray based on the particulate methane monooxygenase gene coupled with geostatistics was used to analyse spatial patterns of methanotrophs in the littoral wetland of a eutrophic boreal lake (Lake Kevätön, Eastern Finland). The wetland had a hydrology gradient with a mean water table varying from -8 to -25 cm. The wettest area, comprising the highest CH(4) oxidation, had the highest abundance and species richness of methanotrophs. A high water table favoured the occurrence of type Ib methanotrophs, whereas types Ia and II were found under all moisture conditions. Thus the spatial heterogeneity in functioning and diversity of methanotrophs in littoral wetlands is highly dependent on the water table, which in turn varies spatially in relation to the geomorphology of the wetland. We suggest that changes in water levels resulting from regulation of lakes and/or global change will affect the abundance, activity and diversity of methanotrophs, and consequently CH(4) emissions from such systems., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Landfills are large sources of CH(4), but a considerable amount of CH(4) can be removed in situ by methanotrophs if their activity can be stimulated through the addition of nitrogen. Nitrogen can, however, lead to increased N(2)O production. To examine the effects of nitrogen and a selective inhibitor on CH(4) oxidation and N(2)O production in situ, 0.5 M of NH(4)Cl and 0.25 M of KNO(3), with and without 0.01% (w/v) phenylacetylene, were applied to test plots at a landfill in Kalamazoo, MI from 2007 November to 2009 July. Nitrogen amendments stimulated N(2)O production but had no effect on CH(4) oxidation. The addition of phenylacetylene stimulated CH(4) oxidation while reducing N(2)O production. Methanotrophs possessing particulate methane monooxygenase and archaeal ammonia-oxidizers (AOAs) were abundant. The addition of nitrogen reduced methanotrophic diversity, particularly for type I methanotrophs. The simultaneous addition of phenylacetylene increased methanotrophic diversity and the presence of type I methanotrophs. Clone libraries of the archaeal amoA gene showed that the addition of nitrogen increased AOAs affiliated with Crenarchaeal group 1.1b, while they decreased with the simultaneous addition of phenylacetylene. These results suggest that the addition of phenylacetylene with nitrogen reduces N(2)O production by selectively inhibiting AOAs and/or type II methanotrophs.

A microbial diagnostic microarray for the detection of the most relevant bacterial food- and water-borne pathogens and indicator organisms was developed and thoroughly validated. The microarray platform based on sequence-specific end labelling of oligonucleotides and the pyhylogenetically robust gyrB marker gene allowed a highly specific (resolution on genus/species level) and sensitive (0.1% relative and 10(4) cfu absolute detection sensitivity) detection of the target pathogens. Validation was performed using a set of reference strains and a set of spiked environmental samples. Reliability of the obtained data was additionally verified by independent analysis of the samples via fluorescence in situ hybridization (FISH) and conventional microbiological reference methods. The applicability of this diagnostic system for food analysis was demonstrated through extensive validation using artificially and naturally contaminated spiked food samples. The microarray-based pathogen detection was compared with the corresponding microbiological reference methods (performed according to the ISO norm). Microarray results revealed high consistency with the reference microbiological data., (© 2010 The Authors. Journal compilation © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

The impact of substrates for probe immobilization was studied using a microbial diagnostic microarray consisting of probes designed against the pmoA genes of methanotrophs and functionally related bacteria. The proprietary ARChip Epoxy was compared to seven epoxy-modified competitor slides, and one porous 3D, furthermore two substrates recommended for oligoprobes without description of the surface chemistry. The diagnostic microarray on Cel silylated aldehyde (CSS) slide was used as a reference for this comparison as it was actually used for assay development and validation. The suitability of binding chemistries for microarrays was evaluated by specificity, signal, and inter- and intra-slide precision and ranked accordingly. The performance of four epoxy substrates (ARChip Epoxy, Cel Epoxy, Corning Epoxy, sciChip) and Spot On slides has proved satisfactory and comparable to the reference Cel CSS in that inter-slide precision was between 8% and 18% CV, intra-slide precision below 30%, respectively. The four parameter ranking shows great promise of providing deeper insight in the performance of materials and protocols tested.

Background: Pathogen identification in clinical routine is based on the cultivation of microbes with subsequent morphological and physiological characterisation lasting at least 24 hours. However, early and accurate identification is a crucial requisite for fast and optimally targeted antimicrobial treatment. Molecular biology based techniques allow fast identification, however discrimination of very closely related species remains still difficult., Results: A molecular approach is presented for the rapid identification of pathogens combining PCR amplification with microarray detection. The DNA chip comprises oligonucleotide capture probes for 25 different pathogens including Gram positive cocci, the most frequently encountered genera of Enterobacteriaceae, non-fermenter and clinical relevant Candida species. The observed detection limits varied from 10 cells (e.g. E. coli) to 10(5) cells (S. aureus) per mL artificially spiked blood. Thus the current low sensitivity for some species still represents a barrier for clinical application. Successful discrimination of closely related species was achieved by a signal pattern recognition approach based on the k-nearest-neighbour method. A prototype software providing this statistical evaluation was developed, allowing correct identification in 100 % of the cases at the genus and in 96.7 % at the species level (n = 241)., Conclusion: The newly developed molecular assay can be carried out within 6 hours in a research laboratory from pathogen isolation to species identification. From our results we conclude that DNA microarrays can be a useful tool for rapid identification of closely related pathogens particularly when the protocols are adapted to the special clinical scenarios.

A major challenge in microbial diagnostics is the parallel detection and identification of low-bundance pathogens within a complex microbial community. In addition, a high specificity providing robust, reliable identification at least at the species level is required. A microbial diagnostic microarray approach, using single nucleotide extension labeling with gyrB as the marker gene, was developed. We present a novel concept applying competitive oligonucleotide probes to improve the specificity of the assay. Our approach enabled the sensitive and specific detection of a broad range of pathogenic bacteria. The approach was tested with a set of 35 oligonucleotide probes targeting Escherichia coli, Shigella spp., Salmonella spp., Aeromonas hydrophila, Vibrio cholerae, Mycobacterium avium, Mycobacterium tuberculosis, Helicobacter pylori, Proteus mirabilis, Yersinia enterocolitica, and Campylobacter jejuni. The introduction of competitive oligonucleotides in the labeling reaction successfully suppressed cross-reaction by closely related sequences, significantly improving the performance of the assay. Environmental applicability was tested with environmental and veterinary samples harboring complex microbial communities. Detection sensitivity in the range of 0.1% has been demonstrated, far below the 5% detection limit of traditional microbial diagnostic microarrays.

Soil microbial communities are responsible for important physiological and metabolic processes. In the last decade soil microorganisms have been frequently analysed by cultivation-independent techniques because only a minority of the natural microbial communities are accessible by cultivation. Cultivation-independent community analyses have revolutionized our understanding of soil microbial diversity and population dynamics. Nevertheless, many methods are still laborious and time-consuming, and high-throughput methods have to be applied in order to understand population shifts at a finer level and to be better able to link microbial diversity with ecosystems functioning. Microbial diagnostic microarrays (MDMs) represent a powerful tool for the parallel, high-throughput identification of many microorganisms. Three categories of MDMs have been defined based on the nature of the probe and target molecules used: phylogenetic oligonucleotide microarrays with short oligonucleotides against a phylogenetic marker gene; functional gene arrays containing probes targeting genes encoding specific functions; and community genome arrays employing whole genomes as probes. In this review, important methodological developments relevant to the application of the different types of diagnostic microarrays in soil ecology will be addressed and new approaches, needs and future directions will be identified, which might lead to a better insight into the functional activities of soil microbial communities.

Oligonucleotide microarrays are highly parallel hybridization platforms, allowing rapid and simultaneous identification of many different microorganisms and viruses in a single assay. In the past few years, researchers have been confronted with a dramatic increase in the number of studies reporting development and/or improvement of oligonucleotide microarrays for microbial diagnostics, but use of the technology in routine diagnostics is still constrained by a variety of factors. Careful development of microarray essentials (such as oligonucleotide probes, protocols for target preparation and hybridization, etc.) combined with extensive performance testing are thus mandatory requirements for the maturation of diagnostic microarrays from fancy technological gimmicks to robust and routinely applicable tools.

The diversity and composition of soil bacterial communities were compared among six Austrian natural forests, including oak-hornbeam, spruce-fir-beech, and Austrian pine forests, using terminal restriction fragment length polymorphism (T-RFLP, or TRF) analysis and sequence analysis of 16S rRNA genes. The forests studied differ greatly in soil chemical characteristics, microbial biomass, and nutrient turnover rates. The aim of this study was to relate these differences to the composition of the bacterial communities inhabiting the individual forest soils. Both TRF profiling and clone sequence analysis revealed that the bacterial communities in soils under Austrian pine forests, representing azonal forest types, were distinct from those in soils under zonal oak-hornbeam and spruce-fir-beech forests, which were more similar in community composition. Clones derived from an Austrian pine forest soil were mostly affiliated with high-G+C gram-positive bacteria (49%), followed by members of the alpha-Proteobacteria (20%) and the Holophaga/Acidobacterium group (12%). Clones in libraries from oak-hornbeam and spruce-fir-beech forest soils were mainly related to the Holophaga/Acidobacterium group (28 and 35%), followed by members of the Verrucomicrobia (24%) and the alpha-Proteobacteria (27%), respectively. The soil bacterial communities in forests with distinct vegetational and soil chemical properties appeared to be well differentiated based on 16S rRNA gene phylogeny. In particular, the outstanding position of the Austrian pine forests, which are determined by specific soil conditions, was reflected in the bacterial community composition.

ARChip Epoxy and ARChip UV are presented as novel chip platforms for oligonucleotide immobilization. ARChip Epoxy is made of reactive epoxy resin available commercially. ARChip UV consists of photoactivatable poly(styrene-co-4-vinylbenzylthiocyanate). Both ARChip surfaces are tested in a model assay based on oligonucleotide probes from a real-life genotyping project and are evaluated in comparison with five commercial chip surfaces based on nitrocellulose, epoxy, and aldehyde polymer, and two different aminosilanes. Optimum print buffer, spotter compatibility, and data normalization are discussed.

The potential of DNA microarray technology in high-throughput detection of bacteria and quantitative assessment of their community structures is widely acknowledged but has not been fully realised yet. A generally applicable set of techniques, based on readily available technologies and materials, was developed for the design, production and application of diagnostic microbial microarrays. A microarray targeting the particulate methane monooxygenase (pmoA) gene was developed for the detection and quantification of methanotrophs and functionally related bacteria. A microarray consisting of a set of 59 probes that covers the whole known diversity of these bacteria was validated with a representative set of extant strains and environmental clones. The potential of the pmoA microarray was tested with environmental samples. The results were in good agreement with those of clone library sequence analyses. The approach can currently detect less dominant bacteria down to 5% of the total community targeted. Initial tests assessing the quantification potential of this system with artificial PCR mixtures showed very good correlation with the expected results with standard deviations in the range of 0.4-17.2%. Quantification of environmental samples with this method requires the design of a reference mixture consisting of very close relatives of the strains within the sample and is currently limited by biases inherent in environmental DNA extraction and universal PCR amplification.