1 Ecology and Evolution of Viruses Infecting Uncultivated SUP05 Bacteria As Revealed 2 by Single-Cell- and Meta- Genomics 3 1 2 2 2 4 Simon Roux , Alyse K
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1 Ecology and evolution of viruses infecting uncultivated SUP05 bacteria as revealed 2 by single-cell- and meta- genomics 3 1 2 2 2 4 Simon Roux , Alyse K. Hawley , Monica Torres Beltran , Melanie Scofield , 5 Patrick Schwientek3, Ramunas Stepanauskas4, Tanja Woyke3, Steven J. Hallam2,5*, 1* 6 and Matthew B. Sullivan 7 8 Affiliations: 9 1Ecology and Evolutionary Biology, University of Arizona, USA 10 2 Department of Microbiology and Immunology, University of British Columbia, Canada 11 3 DOE Joint Genome Institute, Walnut Creek, California, USA 12 4 Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, USA 13 5 Graduate Program in Bioinformatics, University of British Columbia, Canada 14 15 *To whom correspondence should be addressed: 16 Ecology and Evolutionary Biology Department and Molecular and Cellular Biology 17 Department, Life Sciences South (Office LSS246, Lab LSS203 + LSS207). 1007 East 18 Lowell Street, Tucson AZ 85721, USA. Office: (520) 626-6297 e-mail: 19 [email protected] 20 21 University of British Columbia, Life Sciences Institute for Personalized Medicine, 22 Department of Microbiology & Immunology. 2552-2350 Health Sciences Mall, 23 Vancouver, British Columbia, V6T1Z3, Canada. Office: (604) 827-3420 FAX: (604) 822- 24 6041 e-mail: [email protected] 25 26 The authors declare no competing interests. 27 Summary: Single-cell amplified genome sequencing uncovers virus-host interactions in 28 uncultivated SUP05 bacteria with relevance to coupled biogeochemical cycling in marine 29 oxygen minimum zones. 30 Keywords: SUP05, bacteriophages, viruses, single cell genomics, oxygen minimum 31 zones, viral dark matter 1 32 Abstract: 33 Viruses modulate microbial communities and alter ecosystem functions. However, due to 34 cultivation bottlenecks specific virus-host interaction dynamics remain cryptic. Here we 35 examined 127 single-cell amplified genomes (SAGs) from uncultivated SUP05 bacteria 36 isolated from a marine oxygen minimum zone (OMZ) to identify 69 viral contigs 37 representing five new genera within dsDNA Caudovirales and ssDNA Microviridae. 38 Infection frequencies suggest that ~1/3 of SUP05 bacteria are viral-infected, with higher 39 infection frequency where oxygen-deficiency was most severe. Observed Microviridae 40 clonality suggests recovery of bloom-terminating viruses, while systematic co-infection 41 between dsDNA and ssDNA viruses posits previously unrecognized cooperation modes. 42 Analyses of 186 microbial and viral metagenomes revealed that SUP05 viruses persisted 43 for years, but remained endemic to the OMZ. Finally, identification of virus-encoded 44 dissimilatory sulfite reductase suggests SUP05 viruses reprogram their host’s energy 45 metabolism. Together these results demonstrate closely coupled SUP05 virus-host co- 46 evolutionary dynamics with potential to modulate biogeochemical cycling in climate- 47 critical and expanding OMZs. 48 49 2 50 Main text: 51 Introduction 52 Microbial communities are critical drivers of the nutrient and energy flow in natural and 53 engineered ecosystems (Falkowski, Fenchel, and Delong 2008). In the last two decades, it 54 has progressively become clear that viral mediated predation, gene transfer and metabolic 55 reprogramming modulate the structure, function and evolutionary trajectory of these 56 microbial communities (Suttle 2007; Abedon 2009; Rodriguez-Valera et al. 2009; 57 Hurwitz, Hallam, and Sullivan 2013). At the same time, the vast majority of microbes and 58 viruses remain uncultivated and their diversity is extensive, so that model system-based 59 measurements rarely reflect the network properties of natural microbial communities. 60 While culture-independent methods, such as metagenomics and metatranscriptomics, can 61 illuminate latent and expressed metabolic potential of microbial (Frias-Lopez et al. 2008; 62 Venter et al. 2004; Stewart, Ulloa, and DeLong 2012; DeLong et al. 2006) or viral 63 communities (Angly et al. 2006; Hurwitz, Hallam, and Sullivan 2013; Mizuno et al. 64 2013), interactions between community members remain difficult to resolve. 65 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) containing 66 short stretches of viral or plasmid DNA separated between repeat sequences can provide 67 a record of past infections in uncultivated microbial communities. Together with 68 associated Cas (CRISPR-associated) genes, CRISPRs function as an adaptive immune 69 system in prokaryotes with the potential to suppress viral replication or horizontal gene 70 transfer (Sorek, Kunin, and Hugenholtz 2008). However, an application of CRISPR- 71 based virus-host association to both uncultivated hosts and viruses require the assembly 72 of complete or near-complete genomes of both entities, limiting their utility to lower 73 diversity ecosystems (Andersson and Banfield 2008; Anderson et al. 2011). Alternatively, 74 single-cell amplified genome (SAG) sequencing is emerging as a more direct method to 75 chart metabolic potential of individual cells within microbial communities with special 76 emphasis on candidate phyla that have no cultured representatives, (Rinke et al. 2013; 77 Swan et al. 2013; Yoon et al. 2011; Martinez-Garcia et al. 2012). Here we combine 78 metagenomic and single-cell genomic sequencing to explore virus-host interactions 79 within uncultivated bacteria inhabiting a marine oxygen minimum zone (OMZ). 80 Marine OMZs, defined by dissolved oxygen concentrations <20 μmol kg-1, are 3 81 oceanographic features that arise from elevated demand for respiratory oxygen in poorly 82 ventilated, highly stratified waters. OMZs are crucial for the biogeochemical cycles in 83 global ocean, as they represent hotspots for microbial-driven carbon, nitrogen and sulfur 84 transformations (Ulloa et al. 2012; Wright, Konwar, and Hallam 2012), and play a 85 disproportionate role in nitrogen loss processes and greenhouse gas cycling (Lam et al. 86 2009; Ward et al. 2009). Moreover, these zones are expanding due to changing ocean 87 water temperatures and circulation patterns (Stramma et al. 2008; Whitney, Freeland, and 88 Robert 2007). Given these changing physical and chemical conditions and the importance 89 of OMZs to ocean-atmosphere functioning, a clearer understanding of biological 90 responses is critical to develop a much-needed predictive modeling capacity for OMZs. 91 In OMZs, microbial communities drive matter and energy transformations, and 92 are typically dominated by sulfur-oxidizing Gammaproteobacteria related to the 93 chemoautotrophic gill symbionts of deep-sea clams and mussels (Stewart, Ulloa, and 94 DeLong 2012; Wright, Konwar, and Hallam 2012). Phylogenetic analysis indicates that 95 these bacteria are comprised of two primary lineages; one consisting of sequences 96 affiliated with SUP05 and clam and mussel symbionts, and the other consisting of 97 sequences affiliated with Arctic96BD-19 (Wright, Konwar, and Hallam 2012; Walsh et al. 98 2009). Both groups partition along gradients of oxygen and sulfide, with Arctic96BD-19 99 most prevalent in oxygenated waters and SUP05 most prevalent in anoxic or 100 anoxic/sulfidic waters (Wright, Konwar, and Hallam 2012). Niche partitioning between 101 SUP05 and Arctic96BD-19 is driven by complementary modes of carbon and energy 102 metabolism that harness alternative terminal electron acceptors. While both Arctic96BD- 103 19 and SUP05 use reduced sulfur compounds as electron donors to drive inorganic 104 carbon fixation, SUP05 manifests a more versatile energy metabolism linking carbon, 105 nitrogen and sulfur cycling within OMZ and hydrothermal vent waters (Stewart, Ulloa, 106 and DeLong 2012; Anantharaman et al. 2013; Canfield et al. 2010; Swan et al. 2011; 107 Mattes et al. 2013; Zaikova et al. 2010; Hawley et al. 2014; Anantharaman et al. 2014) . 108 Ocean viruses, predominantly investigated in the sunlit or photic zone, are 109 abundant, dynamic, and diverse (Suttle 2005), with growing evidence for direct roles in 110 metabolic reprogramming of microbial photosynthesis, central carbon metabolism and 111 sulfur cycling (Mann et al. 2003; Lindell et al. 2005; Clokie et al. 2006; Breitbart et al. 4 112 2007; Dammeyer et al 2008; Sharon et al. 2009; Sharon et al. 2011; Thompson et al. 113 2011; Hurwitz, Hallam, and Sullivan 2013). Preliminary studies suggest that similar 114 patterns are emerging in OMZ waters. In the Eastern Tropical South Pacific, a 115 metagenomic survey revealed specific viral populations endemic to OMZ waters 116 (Cassman et al. 2012). Consistent with most viral metagenome surveys, approximately 117 3% of sequences were affiliated with functionally annotated genes in public databases. 118 From a nitrogen and sulfur cycling perspective, viromes from the oxycline contained 119 genes encoding components of nitric oxide synthase, nitrate and nitrite ammonification 120 and ammonia assimilation pathways as well as inorganic sulfur assimilation (Cassman et 121 al. 2012). In anoxic waters, viromes contained genes encoding components of 122 denitrification, nitrate and nitrite ammonification and ammonia assimilation pathways as 123 well as sulfate reduction, thioredoxin-disulfide reductase and inorganic sulfur 124 assimilation (Cassman et al. 2012). More recently, metagenomic analyses of 125 hydrothermal vent plume microbial communities dominated by SUP05 bacteria enabled 126 phage genome assemblies presumed to infect SUP05 (Anantharaman et al. 2014). 127 Consistent with viruses encoding auxiliary metabolic genes (AMGs, Breitbart et al. 2007)