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      Aerobic proteobacterial methylotrophs in Movile Cave: genomic and metagenomic analyses

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          Abstract

          Background

          Movile Cave (Mangalia, Romania) is a unique ecosystem where the food web is sustained by microbial primary production, analogous to deep-sea hydrothermal vents. Specifically, chemoautotrophic microbes deriving energy from the oxidation of hydrogen sulphide and methane form the basis of the food web.

          Results

          Here, we report the isolation of the first methane-oxidizing bacterium from the Movile Cave ecosystem, Candidatus Methylomonas sp. LWB, a new species and representative of Movile Cave microbial mat samples. While previous research has suggested a prevalence of anoxic conditions in deeper lake water and sediment, using small-scale shotgun metagenome sequencing, we show that metabolic genes encoding enzymes for aerobic methylotrophy are prevalent in sediment metagenomes possibly indicating the presence of microoxic conditions. Moreover, this study also indicates that members within the family Gallionellaceae ( Sideroxydans and Gallionella) were the dominant taxa within the sediment microbial community, thus suggesting a major role for microaerophilic iron-oxidising bacteria in nutrient cycling within the Movile Cave sediments.

          Conclusions

          In this study, based on phylogenetic and metabolic gene surveys of metagenome sequences, the possibility of aerobic microbial processes (i.e., methylotrophy and iron oxidation) within the sediment is indicated. We also highlight significant gaps in our knowledge on biogeochemical cycles within the Movile Cave ecosystem, and the need to further investigate potential feedback mechanisms between microbial communities in both lake sediment and lake water.

          Electronic supplementary material

          The online version of this article (10.1186/s40168-017-0383-2) contains supplementary material, which is available to authorized users.

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          Most cited references23

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          FunGene: the functional gene pipeline and repository

          Ribosomal RNA genes have become the standard molecular markers for microbial community analysis for good reasons, including universal occurrence in cellular organisms, availability of large databases, and ease of rRNA gene region amplification and analysis. As markers, however, rRNA genes have some significant limitations. The rRNA genes are often present in multiple copies, unlike most protein-coding genes. The slow rate of change in rRNA genes means that multiple species sometimes share identical 16S rRNA gene sequences, while many more species share identical sequences in the short 16S rRNA regions commonly analyzed. In addition, the genes involved in many important processes are not distributed in a phylogenetically coherent manner, potentially due to gene loss or horizontal gene transfer. While rRNA genes remain the most commonly used markers, key genes in ecologically important pathways, e.g., those involved in carbon and nitrogen cycling, can provide important insights into community composition and function not obtainable through rRNA analysis. However, working with ecofunctional gene data requires some tools beyond those required for rRNA analysis. To address this, our Functional Gene Pipeline and Repository (FunGene; http://fungene.cme.msu.edu/) offers databases of many common ecofunctional genes and proteins, as well as integrated tools that allow researchers to browse these collections and choose subsets for further analysis, build phylogenetic trees, test primers and probes for coverage, and download aligned sequences. Additional FunGene tools are specialized to process coding gene amplicon data. For example, FrameBot produces frameshift-corrected protein and DNA sequences from raw reads while finding the most closely related protein reference sequence. These tools can help provide better insight into microbial communities by directly studying key genes involved in important ecological processes.
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            Life without light: microbial diversity and evidence of sulfur- and ammonium-based chemolithotrophy in Movile Cave.

            Microbial diversity in Movile Cave (Romania) was studied using bacterial and archaeal 16S rRNA gene sequence and functional gene analyses, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), soxB (sulfate thioesterase/thiohydrolase) and amoA (ammonia monooxygenase). Sulfur oxidizers from both Gammaproteobacteria and Betaproteobacteria were detected in 16S rRNA, soxB and RuBisCO gene libraries. DNA-based stable-isotope probing analyses using 13C-bicarbonate showed that Thiobacillus spp. were most active in assimilating CO2 and also implied that ammonia and nitrite oxidizers were active during incubations. Nitrosomonas spp. were detected in both 16S rRNA and amoA gene libraries from the 'heavy' DNA and sequences related to nitrite-oxidizing bacteria Nitrospira and Candidatus 'Nitrotoga' were also detected in the 'heavy' DNA, which suggests that ammonia/nitrite oxidation may be another major primary production process in this unique ecosystem. A significant number of sequences associated with known methylotrophs from the Betaproteobacteria were obtained, including Methylotenera, Methylophilus and Methylovorus, supporting the view that cycling of one-carbon compounds may be an important process within Movile Cave. Other sequences detected in the bacterial 16S rRNA clone library included Verrucomicrobia, Firmicutes, Bacteroidetes, alphaproteobacterial Rhodobacterales and gammaproteobacterial Xanthomonadales. Archaeal 16S rRNA sequences retrieved were restricted within two groups, namely the Deep-sea Hydrothermal Vent Euryarchaeota group and the Miscellaneous Crenarchaeotic group. No sequences related to known sulfur-oxidizing archaea, ammonia-oxidizing archaea, methanogens or anaerobic methane-oxidizing archaea were detected in this clone library. The results provided molecular biological evidence to support the hypothesis that Movile Cave is driven by chemolithoautotrophy, mainly through sulfur oxidation by sulfur-oxidizing bacteria and reveal that ammonia- and nitrite-oxidizing bacteria may also be major primary producers in Movile Cave.
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              A novel family of functional operons encoding methane/ammonia monooxygenase-related proteins in gammaproteobacterial methanotrophs.

              Genomes of alphaproteobacterial and verrucomicrobial methane-oxidizing bacteria (MOB) encode sequence-divergent copies of particulate methane monooxygenase [pMMO = (PmoABC); pmoCAB]. In contrast, sequenced gammaproteobacterial MOB (Gamma-MOB) genomes contain single or multiple near-identical copies of pmoCAB operons. In betaproteobacterial ammonia-oxidizing bacteria (Beta-AOB), near-identical amoCAB operons encode ammonia monooxygenase (AMO), a homologue of pMMO. Here, we report that Gamma-MOB in the genera Methylomonas, Methylobacter and Methylomicrobium also encode a sequence-divergent particulate monooxygenase (pXMO). Whereas all known genes encoding pMMO or AMO cluster in the order 'CAB', the genes encoding pXMO are uniquely organized in the non-canonical form 'pxmABC.' Steady state pxm mRNA was detected in cultures of Methylomonas sp. as well as in freshwater creek sediment samples, demonstrating that pxm genes are expressed in culture and in situ. Inclusion of PxmA and PxmB proteins in phylogenetic analyses of the Pmo/Amo protein superfamilies created trifurcated trees with three major clades: (i) Pmo of Alpha- and Gamma-MOB and Amo of Gamma-AOB; (ii) Amo of Beta-AOB, Pmo of putative ethane-oxidizing Gamma-MOB and Pxm of Gamma-MOB; and (iii) verrucomicrobial Pmo and Amo of ammonia-oxidizing Archaea. These data support but do not prove the hypothesis that oxygen-dependent methane and ammonia monooxygenases evolved from a substrate-promiscuous ancestor after horizontal transfer while being integrated into the catabolic contexts of their extant hosts. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.
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                Author and article information

                Contributors
                +44 28 9097 2181 , d.kumaresan@qub.ac.uk
                jason.stephenson@warwick.ac.uk
                acdoxey@uwaterloo.ca
                hbandukwala@waterloo.ca
                e.brooks@uea.ac.uk
                alexandra.hillebrand@gmail.com
                andy.whiteley@uwa.edu.au
                j.c.murrell@uea.ac.uk
                Journal
                Microbiome
                Microbiome
                Microbiome
                BioMed Central (London )
                2049-2618
                2 January 2018
                2 January 2018
                2018
                : 6
                : 1
                Affiliations
                [1 ]ISNI 0000 0001 1092 7967, GRID grid.8273.e, School of Environmental Sciences, , University of East Anglia, ; Norwich, UK
                [2 ]ISNI 0000 0004 0374 7521, GRID grid.4777.3, School of Biological Sciences and Institute for Global Food Security, , Queen’s University Belfast, ; 97 Lisburn Road, Belfast, BT9 7BL UK
                [3 ]ISNI 0000 0000 8809 1613, GRID grid.7372.1, School of Life Sciences, , University of Warwick, ; Coventry, UK
                [4 ]ISNI 0000 0000 8644 1405, GRID grid.46078.3d, Department of Biology, , University of Waterloo, ; Waterloo, Canada
                [5 ]Department of Biospeleology and Karst Edaphobiology, Emil Racovita Institute of Speleology, Bucharest, Romania
                [6 ]ISNI 0000 0004 1936 7910, GRID grid.1012.2, UWA School of Agriculture and Environment, , University of Western Australia, ; Perth, Australia
                Author information
                http://orcid.org/0000-0001-9072-3058
                Article
                383
                10.1186/s40168-017-0383-2
                5748958
                29291746
                10787207-c338-40de-ac69-55d47bfc0550
                © The Author(s). 2017

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 19 May 2017
                : 14 December 2017
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100000270, Natural Environment Research Council;
                Award ID: NE/G017956
                Award Recipient :
                Funded by: University of East Anglia (GB)
                Funded by: FundRef http://dx.doi.org/10.13039/501100000873, Queen's University Belfast;
                Categories
                Research
                Custom metadata
                © The Author(s) 2018

                movile cave,methylotrophic bacteria,one-carbon metabolism,methane,methanotrophs,extreme ecosystem

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