Transcriptionally active heterotrophic diazotrophs are widespread in the upper water column of the Arabian Sea

RESEARCH ARTICLE Transcriptionally active heterotrophic diazotrophs are widespread in the upper water column of the Arabian Sea Clare Bird & Michael Wyman Biological and Environmental Sciences, School of Natural Sciences, University of Stirling, Stirling, UK Present address: Clare Bird, School of Geosciences, Grant Institute, University of Edinburgh, The King’s Buildings, West Mains Road, Edinburgh, EH9 3JW, UK Received 5 June 2012; revised 22 November 2012; accepted 25 November 2012. Final version published online 27 December 2012. MICROBIOLOGY ECOLOGY DOI: 10.1111/1574-6941.12049 Editor: Gary King Keywords nifH; nitrogenase; nitrogen-fixation; reverse transcriptase-polymerase chain reaction. Abstract Pelagic nitrogen fixation makes an important contribution to the fixed nitrogen budget of the world’s oceans. Filamentous and unicellular cyanobacteria are significant players in this process but less is known of the potential activity of heterotrophic diazotrophs, although they are present and can be quite numerous in the nitrogen-deplete surface waters of the tropical and sub-tropical oceans. In this study we focused on the potential activity of several clades of heterotrophic nitrogen-fixers identified by phylogenetic analysis of 44 nonTrichodesmium-related, nifH (encoding the Fe-subunit of nitrogenase) clones from the Arabian Sea. Specific Northern slot blot protocols were developed to quantify nifH mRNAs from each clade and showed that two groups of Gammaproteobacteria, including the previously characterized UMB clade, and a third, novel phylotype affiliated with cluster III anaerobes, were actively expressing nitrogenase in the equatorial waters of this region. Transcripts (nifH mRNAs) from the latter clade were particularly abundant and were also detected in the suboxic waters of the oxygen minimum zone further north. Like the gammaproteobacterial groups, nifH expression by these organisms appeared to be insensitive to combined nitrogen concentrations and was readily detected in the nutrient-replete waters below the upper mixed layer as well as at shallower depths. Introduction For much of the world’s oceans, the limited availability of fixed nitrogen sets an upper boundary on the net rate of photosynthetic carbon incorporation into the planktonic biomass, thereby constraining the biogenic flux of atmospheric CO2 into the ocean interior (Falkowski, 1997; Gruber & Galloway, 2008). Once considered to be of little consequence, diazotrophic (N2-fixing) microorganisms are now known to play a pivotal role in the nitrogen cycle of the oceans (Gruber & Sarmiento, 1997; Karl et al., 2002). They are an important source of ‘new nitrogen’ that fuels biological production over vast swathes of the tropical and sub-tropical oceans (Michaels et al., 1996; Karl et al., 1997) and help to rebalance the losses of fixed nitrogen from the oceans via anammox and denitrification (Codispoti, 1995; Gruber & Sarmiento, 1997; Canfield et al., 2010). The most well known marine diazotrophs are all members of the Cyanobacteria. The most conspicuous FEMS Microbiol Ecol 84 (2013) 189–200 and best studied of these are the colonial forms that belong to the genus Trichodesmiuim (Capone et al., 1997) and the non-colonial heterocyst-forming Richelia spp., which enter into symbioses with various diatoms such as Rhizosolenia spp. (Carpenter et al., 1999). Unicellular diazotrophic cyanobacteria belonging to three distinct lineages are also present in the surface waters of the tropical and subtropical oceans (Zehr et al., 2001; Montoya et al., 2004). The distribution of one of these, the uncultivated UCYN-A group that lacks the oxygen-evolving photosystem II and CO2-fixing enzyme RubisCO (Zehr et al., 2008) and enters into symbioses with picoeukaryotic prymnesiophytes (Thompson et al., 2012), extends also to higher latitudes and to deeper waters (Moisander et al., 2010). Like the other unicellular cyanobacteria, the overall importance of these unusual diazotrophs in the oceanic nitrogen budget remains to be assessed but some estimates of N2-fixation by the UCYN-B lineage, which includes the cultivated representative, Crocosphaera watsonii WH8501 (Webb et al., 2009), suggest that it may be ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Correspondence: Dr Michael Wyman, Biological and Environmental Sciences, School of Natural Sciences, University of Stirling, Stirling FK9 4LA, UK. Tel.: +44 (0) 1786 467784; fax: +44 (0)1786 467843; e-mail: 190 ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Materials and methods Study site and sample collection Observations were made in September 2001 aboard RRS Charles Darwin (cruise CD 132) during the NERC AMBITION (Assessing Microbial Biodiversity In The Indian OceaN) cruise in the Arabian Sea. Eleven stations were occupied along a 5150-km northerly transect from the Seychelles Islands to Muscat, Oman (Bird et al., 2005). Hydrographical data and seawater samples were collected at each station with a Sea-Bird 911plus CTD profiler (SeaBird Electronics, Inc., Bellevue, WA) equipped with a rosette of 24, 30-L volume Niskin bottles. Plankton samples were obtained from discrete depths and concentrated by filtering 5–10 L seawater under gentle vacuum (10–20 mmHg) through 90-mm diameter, 0.2 pore-size polycarbonate membranes (Osmonics Inc., Fileder Filter Systems, Maidstone, UK). The retained cell material was resuspended in DNA lysis buffer [250 mM NaCl, 100 mM EGTA, 100 mM Tris pH 8.0, 1% (w/v) lithium dodecyl sulphate] and stored frozen at –70 °C or, for RNA samples, in the preservative, RNALater (Ambion, Applied Biosystems, Warrington, UK) and kept refrigerated at 4 °C. All samples were shipped by air to the UK at the end of the cruise on dry ice and stored at –80 °C prior to the extraction of nucleic acids as described by Bird et al. (2005). Continuous depth profiles of chlorophyll concentrations, dissolved oxygen and photosynthetically active radiation were determined with a Chelsea Aquatracker III fluorometer (Chelsea Instruments, West Molesey, UK), Sea-Bird 43B oxygen detector, and Chelsea 2Π irradiance sensor deployed with the CTD profiler during the sampling hydrocasts at each station. Combined nitrogen (nitrate and nitrite) concentrations were determined using a Technicon AAII segmented flow colorimetric autoanalyser configured with high sensitivity, liquid waveguide capillary cells and detectors as described (Fuller et al., 2006). PCR amplification and TA cloning A ~ 360-bp fragment of nifH (encoding dinitrogenase reductase) was amplified from DNA samples obtained from the upper water column (
300 m depth) at six of the stations occupied during the cruise (see Table 1 for the locations of the stations and the depths sampled) using a nested PCR protocol. Reactions were performed with the external primer pairs nifH4 (...truncated)