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      Archaeal and bacterial communities in deep-sea hydrogenetic ferromanganese crusts on old seamounts of the northwestern Pacific

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          Abstract

          Deep-sea ferromanganese crusts are found ubiquitously on the surface of seamounts of the world’s oceans. Considering the wide distribution of the crusts, archaeal and bacterial communities on these crusts potentially play a significant role in biogeochemical cycling between oceans and seamounts; however little is known about phylogenetic diversity, abundance and function of the crust communities. To this end, we collected the crusts from the northwest Pacific basin and the Philippine Sea. We performed comprehensive analysis of the archaeal and bacterial communities of the collected crust samples by culture-independent molecular techniques. The distance between the sampling points was up to approximately 2,000 km. Surrounding sediments and bottom seawater were also collected as references near the sampling points of the crusts, and analyzed together. 16S rRNA gene analyses showed that the community structure of the crusts was significantly different from that of the seawater. Several members related to ammonia-oxidizers of Thaumarchaeota and Betaproteobacteria were detected in the crusts at most of all regions and depths by analyses of 16S rRNA and amoA genes, suggesting that the ammonia-oxidizing members are commonly present in the crusts. Although members related to the ammonia-oxidizers were also detected in the seawater, they differed from those in the crusts phylogenetically. In addition, members of uncultured groups of Alpha-, Delta- and Gammaproteobacteria were commonly detected in the crusts but not in the seawater. Comparison with previous studies of ferromanganese crusts and nodules suggests that the common members determined in the present study are widely distributed in the crusts and nodules on the vast seafloor. They may be key microbes for sustaining microbial ecosystems there.

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          Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean.

          Nitrification, the microbial oxidation of ammonia to nitrite and nitrate, occurs in a wide variety of environments and plays a central role in the global nitrogen cycle. Catalyzed by the enzyme ammonia monooxygenase, the ability to oxidize ammonia was previously thought to be restricted to a few groups within the beta- and gamma-Proteobacteria. However, recent metagenomic studies have revealed the existence of unique ammonia monooxygenase alpha-subunit (amoA) genes derived from uncultivated, nonextremophilic Crenarchaeota. Here, we report molecular evidence for the widespread presence of ammonia-oxidizing archaea (AOA) in marine water columns and sediments. Using PCR primers designed to specifically target archaeal amoA, we find AOA to be pervasive in areas of the ocean that are critical for the global nitrogen cycle, including the base of the euphotic zone, suboxic water columns, and estuarine and coastal sediments. Diverse and distinct AOA communities are associated with each of these habitats, with little overlap between water columns and sediments. Within marine sediments, most AOA sequences are unique to individual sampling locations, whereas a small number of sequences are evidently cosmopolitan in distribution. Considering the abundance of nonextremophilic archaea in the ocean, our results suggest that AOA may play a significant, but previously unrecognized, role in the global nitrogen cycle.
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            The influence of soil pH on the diversity, abundance and transcriptional activity of ammonia oxidizing archaea and bacteria.

            Autotrophic ammonia oxidation occurs in acid soils, even though laboratory cultures of isolated ammonia oxidizing bacteria fail to grow below neutral pH. To investigate whether archaea possessing ammonia monooxygenase genes were responsible for autotrophic nitrification in acid soils, the community structure and phylogeny of ammonia oxidizing bacteria and archaea were determined across a soil pH gradient (4.9-7.5) by amplifying 16S rRNA and amoA genes followed by denaturing gradient gel electrophoresis (DGGE) and sequence analysis. The structure of both communities changed with soil pH, with distinct populations in acid and neutral soils. Phylogenetic reconstructions of crenarchaeal 16S rRNA and amoA genes confirmed selection of distinct lineages within the pH gradient and high similarity in phylogenies indicated a high level of congruence between 16S rRNA and amoA genes. The abundance of archaeal and bacterial amoA gene copies and mRNA transcripts contrasted across the pH gradient. Archaeal amoA gene and transcript abundance decreased with increasing soil pH, while bacterial amoA gene abundance was generally lower and transcripts increased with increasing pH. Short-term activity was investigated by DGGE analysis of gene transcripts in microcosms containing acidic or neutral soil or mixed soil with pH readjusted to that of native soils. Although mixed soil microcosms contained identical archaeal ammonia oxidizer communities, those adapted to acidic or neutral pH ranges showed greater relative activity at their native soil pH. Findings indicate that different bacterial and archaeal ammonia oxidizer phylotypes are selected in soils of different pH and that these differences in community structure and abundances are reflected in different contributions to ammonia oxidizer activity. They also suggest that both groups of ammonia oxidizers have distinct physiological characteristics and ecological niches, with consequences for nitrification in acid soils.
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              Quantitative analysis of small-subunit rRNA genes in mixed microbial populations via 5'-nuclease assays.

              Few techniques are currently available for quantifying specific prokaryotic taxa in environmental samples. Quantification of specific genotypes has relied mainly on oligonucleotide hybridization to extracted rRNA or intact rRNA in whole cells. However, low abundance and cellular rRNA content limit the application of these techniques in aquatic environments. In this study, we applied a newly developed quantitative PCR assay (5'-nuclease assay, also known as TaqMan) to quantify specific small-subunit (SSU) rRNA genes (rDNAs) from uncultivated planktonic prokaryotes in Monterey Bay. Primer and probe combinations for quantification of SSU rDNAs at the domain and group levels were developed and tested for specificity and quantitative reliability. We examined the spatial and temporal variations of SSU rDNAs from Synechococcus plus Prochlorococcus and marine Archaea and compared the results of the quantitative PCR assays to those obtained by alternative methods. The 5'-nuclease assays reliably quantified rDNAs over at least 4 orders of magnitude and accurately measured the proportions of genes in artificial mixtures. The spatial and temporal distributions of planktonic microbial groups measured by the 5'-nuclease assays were similar to the distributions estimated by quantitative oligonucleotide probe hybridization, whole-cell hybridization assays, and flow cytometry.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                24 February 2017
                2017
                : 12
                : 2
                : e0173071
                Affiliations
                [1 ]Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo, Japan
                [2 ]Ore Genesis Research Unit, Project Team for Development of New-generation Research Protocol for Submarine Resources, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Kanagawa, Japan
                [3 ]Center for Advanced Marine Core Research, Kochi University, Kochi-shi, Kochi, Japan
                [4 ]Department of Earth and Planetary Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan
                Wageningen University, NETHERLANDS
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: SK AK.

                • Data curation: SN SK AU TU AK.

                • Formal analysis: SK.

                • Funding acquisition: AU TU KS AY.

                • Investigation: SN SK AU TU.

                • Project administration: AU TU KS AY.

                • Writing – original draft: SN SK AY.

                • Writing – review & editing: SN SK AU TU KS AK.

                Article
                PONE-D-16-43537
                10.1371/journal.pone.0173071
                5325594
                28235095
                6be6e099-390b-4ac8-88fa-92596d98944f
                © 2017 Nitahara et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 1 November 2016
                : 14 February 2017
                Page count
                Figures: 4, Tables: 2, Pages: 21
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100001700, Ministry of Education, Culture, Sports, Science, and Technology;
                Award ID: Project TAIGA
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100002770, Cabinet Office, Government of Japan;
                Award ID: SIP Zipangu-in-the-ocean project
                Award Recipient :
                This work was supported by the Ministry of Education, Culture, Science and Technology (MEXT), Japan, through a special coordination fund (Project TAIGA: Trans-crustal Advection and In-situ biogeochemical processes of Global sub-seafloor Aquifer)( http://www.mext.go.jp/a_menu/shinkou/hojyo/chukan-jigohyouka/1301256.htm), and partially by the Cabinet Office, Government of Japan, though the Next-generation technology for ocean resources exploration (called as Zipangu-in-the-ocean project) in the Cross-ministerial Strategic Innovation Promotion Program (SIP)( https://www.jamstec.go.jp/sip/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Earth Sciences
                Geology
                Petrology
                Sediment
                Earth Sciences
                Geology
                Sedimentary Geology
                Sediment
                Earth Sciences
                Hydrology
                Sea Water
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Non-coding RNA
                Ribosomal RNA
                Biology and life sciences
                Biochemistry
                Ribosomes
                Ribosomal RNA
                Biology and life sciences
                Cell biology
                Cellular structures and organelles
                Ribosomes
                Ribosomal RNA
                Ecology and Environmental Sciences
                Terrestrial Environments
                Seamounts
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Cloning
                Research and Analysis Methods
                Molecular Biology Techniques
                Cloning
                Physical Sciences
                Chemistry
                Chemical Compounds
                Ammonia
                Biology and Life Sciences
                Evolutionary Biology
                Evolutionary Systematics
                Phylogenetics
                Biology and Life Sciences
                Taxonomy
                Evolutionary Systematics
                Phylogenetics
                Computer and Information Sciences
                Data Management
                Taxonomy
                Evolutionary Systematics
                Phylogenetics
                Physical Sciences
                Chemistry
                Chemical Elements
                Manganese
                Custom metadata
                The nucleotide sequences of 16S rRNA and amoA genes determined in the present study have been deposited in the DDBJ database under the following accession numbers: LC138362–LC139974 for 16S rRNA gene clones and LC139975–LC140541 for amoA gene clones.

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