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      Nitrogenase Activity in Thermophilic Chemolithoautotrophic Bacteria in the Phylum Aquificae Isolated under Nitrogen-Fixing Conditions from Nakabusa Hot Springs

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          Abstract

          The phylum Aquificae comprises chemolithoautotrophic thermophilic to hyperthermophilic bacteria, in which the nitrogenase reductase gene ( nifH) has been reported. However, nitrogen-fixing activity has not yet been demonstrated in members of this deeply branching bacterial phylum. We isolated two thermophilic diazotrophic strains from chemosynthetic microbial communities in slightly alkaline hot springs (≥70°C) in Nakabusa, Nagano Prefecture, Japan. A phylogenetic analysis based on 16S rRNA genes identified these strains as members of the genus Hydrogenobacter within Aquificae. Their NifH sequences showed 96.5 and 97.4% amino acid sequence identities to that from Hydrogenobacter thermophilus TK-6. Nitrogenase activity, measured by acetylene reduction, was confirmed in both strains at 70°C. These novel strains grew under semi-aerobic conditions by using CO 2 as the sole carbon source and N 2 as the sole nitrogen source in media containing hydrogen and/or thiosulfate. To the best of our knowledge, this is the first demonstration of active nitrogen fixation in thermophilic bacteria at 70°C and in the phylum Aquificae.

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          Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses.

          D. J. Lane, B. Pace, G. J. Olsen … (1985)
          Although the applicability of small subunit ribosomal RNA (16S rRNA) sequences for bacterial classification is now well accepted, the general use of these molecules has been hindered by the technical difficulty of obtaining their sequences. A protocol is described for rapidly generating large blocks of 16S rRNA sequence data without isolation of the 16S rRNA or cloning of its gene. The 16S rRNA in bulk cellular RNA preparations is selectively targeted for dideoxynucleotide-terminated sequencing by using reverse transcriptase and synthetic oligodeoxynucleotide primers complementary to universally conserved 16S rRNA sequences. Three particularly useful priming sites, which provide access to the three major 16S rRNA structural domains, routinely yield 800-1000 nucleotides of 16S rRNA sequence. The method is evaluated with respect to accuracy, sensitivity to modified nucleotides in the template RNA, and phylogenetic usefulness, by examination of several 16S rRNAs whose gene sequences are known. The relative simplicity of this approach should facilitate a rapid expansion of the 16S rRNA sequence collection available for phylogenetic analyses.
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            Nitrogenase gene diversity and microbial community structure: a cross-system comparison.

            Jonathan P. Zehr, Bethany D Jenkins, Steven Short … (2003)
            Biological nitrogen fixation is an important source of fixed nitrogen for the biosphere. Microorganisms catalyse biological nitrogen fixation with the enzyme nitrogenase, which has been highly conserved through evolution. Cloning and sequencing of one of the nitrogenase structural genes, nifH, has provided a large, rapidly expanding database of sequences from diverse terrestrial and aquatic environments. Comparison of nifH phylogenies to ribosomal RNA phylogenies from cultivated microorganisms shows little conclusive evidence of lateral gene transfer. Sequence diversity far outstrips representation by cultivated representatives. The phylogeny of nitrogenase includes branches that represent phylotypic groupings based on ribosomal RNA phylogeny, but also includes paralogous clades including the alternative, non-molybdenum, non-vanadium containing nitrogenases. Only a few alternative or archaeal nitrogenase sequences have as yet been obtained from the environment. Extensive analysis of the distribution of nifH phylotypes among habitats indicates that there are characteristic patterns of nitrogen fixing microorganisms in termite guts, sediment and soil environments, estuaries and salt marshes, and oligotrophic oceans. The distribution of nitrogen-fixing microorganisms, although not entirely dictated by the nitrogen availability in the environment, is non-random and can be predicted on the basis of habitat characteristics. The ability to assay for gene expression and investigate genome arrangements provides the promise of new tools for interrogating natural populations of diazotrophs. The broad analysis of nitrogenase genes provides a basis for developing molecular assays and bioinformatics approaches for the study of nitrogen fixation in the environment.
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              Improvement in the RFLP procedure for studying the diversity of nifH genes in communities of nitrogen fixers in soil.

              F Poly, L Monrozier, R Bally (2015)
              Several specific primers for the nifH gene were tested with different pure telluric N2-fixing strains. A PolF/PolR primer set provided successful amplification of 19 representative N2-fixing strains. Three restriction enzymes, HaeIII, NdeII and MnlI, chosen for restriction fragment length polymorphism (RFLP) analyses, were the most discriminating for the study of nifH gene diversity as they resulted in differences between strains at the species level. Amplification by selected primers and RFLP were applied to assess the genetic diversity of the nifH gene pool in soil. Pair soils, one under cultivation, the second under permanent pasture, were found to harbor a contrasting diversity of nifH genes. Pure strain profiles could not be recognized in the nifH soil patterns. Using the simple procedure described, it was shown that the structure of nitrogen fixers in soil was influenced by soil functioning.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Microbes Environ
                Journal ID (iso-abbrev): Microbes Environ
                Title: Microbes and Environments
                Publisher: the Japanese Society of Microbial Ecology (JSME)/the Japanese Society of Soil Microbiology (JSSM)/the Taiwan Society of Microbial Ecology (TSME)/the Japanese Society of Plant Microbe Interactions (JSPMI)
                ISSN (Print): 1342-6311
                ISSN (Electronic): 1347-4405
                Publication date (Print): December 2018
                Publication date (Electronic): 23 November 2018
                Volume: 33
                Issue: 4
                Pages: 394-401
                Affiliations
                [1 ] Department of Biological Sciences, Tokyo Metropolitan University Minami-Osawa, Hachioji, Tokyo 192–0397 Japan
                [2 ] Earth-Life Science Institute, Tokyo Institute of Technology Ookayama, Meguro-ku, Tokyo 152–8551 Japan
                [3 ] Biofunctional Catalyst Research Team, RIKEN Center for Sustainable Resource Science Wako-shi 351–0198 Japan
                [4 ] Blue Marble Space Institute of Science Seattle, WA 98145–1561 USA
                Author notes
                [* ]Corresponding author. E-mail: arisa.nishihara@ 123456gmail.com ; Tel: +81–42–677–2581; Fax: +81–42–677–2559.
                Article
                Publisher ID: 33_394
                DOI: 10.1264/jsme2.ME18041
                PMC ID: 6307999
                PubMed ID: 30473565
                SO-VID: 6a399360-1b48-4411-a9b3-652a8fb02790
                Copyright © Copyright © 2018 by Japanese Society of Microbial Ecology / Japanese Society of Soil Microbiology / Taiwan Society of Microbial Ecology / Japanese Society of Plant Microbe Interactions.
                License:

                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 work is properly cited.

                History
                Date received : 16 March 2018
                Date accepted : 12 August 2018
                Categories
                Subject: Articles

                Keywords: nitrogen fixation,thermophile,aquificales,nifh gene,hydrogen-oxidizing bacteria
                Data availability:
                Keywords: nitrogen fixation, thermophile, aquificales, nifh gene, hydrogen-oxidizing bacteria

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