Comparative genomics reveals surprising divergence of two closely related strains of uncultivated UCYN-A cyanobacteria

The ISME Journal (2014) 8, 2530–2542 & 2014 International Society for Microbial Ecology All rights reserved 1751-7362/14 www.nature.com/ismej ORIGINAL ARTICLE Comparative genomics reveals surprising divergence of two closely related strains of uncultivated UCYN-A cyanobacteria Deniz Bombar1,4,5, Philip Heller2,4, Patricia Sanchez-Baracaldo3, Brandon J Carter1 and Jonathan P Zehr1 1 Ocean Sciences Department, University of California, Santa Cruz, CA, USA; 2Biomolecular Engineering Department, University of California, Santa Cruz, CA, USA and 3Schools of Biological and Geographical Sciences, University of Bristol, Bristol, UK Marine planktonic cyanobacteria capable of fixing molecular nitrogen (termed ‘diazotrophs’) are key in biogeochemical cycling, and the nitrogen fixed is one of the major external sources of nitrogen to the open ocean. Candidatus Atelocyanobacterium thalassa (UCYN-A) is a diazotrophic cyanobacterium known for its widespread geographic distribution in tropical and subtropical oligotrophic oceans, unusually reduced genome and symbiosis with a single-celled prymnesiophyte alga. Recently a novel strain of this organism was also detected in coastal waters sampled from the Scripps Institute of Oceanography pier. We analyzed the metagenome of this UCYN-A2 population by concentrating cells by flow cytometry. Phylogenomic analysis provided strong bootstrap support for the monophyly of UCYN-A (here called UCYN-A1) and UCYN-A2 within the marine Crocosphaera sp. and Cyanothece sp. clade. UCYN-A2 shares 1159 of the 1200 UCYN-A1 protein-coding genes (96.6%) with high synteny, yet the average amino-acid sequence identity between these orthologs is only 86%. UCYN-A2 lacks the same major pathways and proteins that are absent in UCYN-A1, suggesting that both strains can be grouped at the same functional and ecological level. Our results suggest that UCYN-A1 and UCYN-A2 had a common ancestor and diverged after genome reduction. These two variants may reflect adaptation of the host to different niches, which could be coastal and open ocean habitats. The ISME Journal (2014) 8, 2530–2542; doi:10.1038/ismej.2014.167; published online 16 September 2014 Introduction Marine pelagic cyanobacteria play a major role in biogeochemical cycling of carbon and nitrogen in the ocean. Prochlorococcus and Synechococcus together are the most abundant phototrophic prokaryotes on Earth, and are responsible for a major fraction of oceanic carbon fixation (Partensky et al., 1999; Scanlan and West, 2002; Scanlan, 2003; Johnson et al., 2006). Likewise, cyanobacteria capable of fixing molecular nitrogen (‘diazotrophs’) dominate global oceanic N2 fixation although they are typically orders of magnitude less abundant than Prochlorococcus or Synechococcus (Zehr and Paerl, 2008; Zehr and Kudela, 2011; Voss et al., 2013). Together with upward fluxes of deep-water NO3 to the surface ocean, diazotrophs supply the Correspondence: JP Zehr, Ocean Sciences Department, University of California, 1156 High Street, Santa Cruz, CA 95064, USA. E-mail: 4 These authors contributed equally to this work. 5 Current address: Marine Biological Section, Department of Biology, University of Copenhagen, Helsingør, Denmark. Received 3 June 2014; revised 5 August 2014; accepted 8 August 2014; published online 16 September 2014 nitrogen requirement of primary productivity and quantitatively balance losses by sinking of organic material, which can sequester CO2 from the atmosphere to deep waters (Karl et al., 1997; Sohm et al., 2011). There are several groups of quantitatively significant diazotrophic cyanobacteria in the open ocean, all of which thrive mainly in tropical and subtropical latitudes (Stal, 2009). Traditionally, the filamentous, aggregate-forming cyanobacterium Trichodesmium sp. was viewed as the most important oceanic N2 fixer, based on its wide distribution and direct measurements of its N2 fixation capacity (Dugdale et al., 1961; Capone et al., 1997; Bergman et al., 2013). Other diazotrophic cyanobacteria discovered in early microscopic studies are the filamentous heterocyst-forming types of the Richelia and Calothrix lineages, which live in symbioses with several different diatom species (Villareal, 1992; Janson et al., 1999; Foster and Zehr, 2006). More recently, the application of molecular approaches resulted in the discovery of unexpected and unusual cyanobacteria involved in oceanic N2 fixation (Zehr et al., 1998, 2001). These have usually been grouped as ‘unicellular’ diazotrophic cyanobacteria, but, UCYN-A genome comparison D Bombar et al 2531 among them, different types have very different lifestyles, with Crocosphaera watsonii being photosynthetic and mostly free-living cells (but see Foster et al., 2011), whereas UCYN-A (Candidatus Atelocyanobacterium thalassa) is a photoheterotroph that is symbiotic with prymnesiophyte algae (Thompson et al., 2012). While the major biogeochemical role of all diazotrophic cyanobacteria is to provide new nitrogen to the system, their different lifestyles suggest important differences regarding their distribution in the ocean, and the fate of the fixed nitrogen and carbon (Glibert and Bronk, 1994; Scharek et al., 1999; Mulholland, 2007). As a diazotrophic cyanobacterium, UCYN-A (termed UCYN-A1 from here on) is remarkable in several ways. Although somewhat closely related to Cyanothece sp. strain ATCC 51142, the UCYN-A1 genome is only 1.44 Mb and lacks many genes including whole metabolic pathways and proteins, such as the oxygen-evolving photosystem II and RuBisCO, that is, features that normally define cyanobacteria (Tripp et al., 2010). The recent identification of a symbiotic eukaryotic prymnesiophyte partner, to which UCYN-A1 provides fixed nitrogen while receiving carbon in return, is the first known example of a symbiosis between a cyanobacterium and a prymnesiophyte alga (Thompson et al., 2012). Further, UCYN-A1 can be detected in colder and deeper waters compared with other major N2 fixers like Trichodesmium sp. and C. watsonii (Needoba et al., 2007; Langlois et al., 2008; Rees et al., 2009; Moisander et al., 2010; Diez et al., 2012), and is also abundant in some coastal waters (Mulholland et al., 2012). There is now evidence that there are at least three nifH lineages of UCYN-A in the ocean (Thompson et al., 2014). These different clades were previously unrecognized because their nifH amino-acid sequences are nearly identical, with sequence variation primarily only occurring in the third base pair of each codon (Thompson et al., 2014). It is unknown whether these strains are different metabolic variants of UCYN-A, analogous to observations in free-living cyanobacteria like Prochlorococcus and Synechococcus, which have extensive heterogeneity in their genome contents that enable them to occupy different niches along gradients of nutrients and light (Moore et al., 1998; Ahlgren et al., 2006; Kettler et al., 2007). Phylotype ‘UCYN-A2’ shares only 95 (...truncated)