The role of planktonic Flavobacteria in processing algal organicFor matter Peer in coastal Review East Antarctica Only revealed using metagenomics and metaproteomics Journal: Environmental Microbiology and Environmental Microbiology Reports Manuscript ID: EMI-2012-0714.R1 Manuscript Type: EMI - Research article Journal: Environmental Microbiology Date Submitted by the Author: n/a Complete List of Authors: Cavicchioli, Ricardo ecophysiology, environmental genomics, functional diversity, Keywords: genomics/functional genomics/comparative genomics, bacteria, metagenomics/community genomics, microbial ecology, archaea Wiley-Blackwell and Society for Applied Microbiology Page 1 of 165 The role of planktonic Flavobacteria in processing algal organic matter in coastal East Antarctica revealed using metagenomics and metaproteomics For Peer Review Only Timothy J. Williams 1, David Wilkins 1, Emilie Long 1,2 , Flavia Evans 1, Mathew Z. DeMaere 1, Mark J. Raftery 3, and Ricardo Cavicchioli 1† 1 School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, New South Wales, 2052, Australia. 2 UFR 927, Université Pierre et Marie Curie (UPMC) Paris VI, 4 place Jussieu 75532, Paris, France 3 Bioanalytical Mass Spectrometry Facility, The University of New South Wales, Sydney, New South Wales, 2052, Australia. † To whom correspondence should be addressed. E-mail: [email protected] Running head: Metaproteomics of marine Antarctic Flavobacteria 1 Wiley-Blackwell and Society for Applied Microbiology Page 2 of 165 1 Summary 2 3 Heterotrophic marine bacteria play key roles in remineralizing organic matter 4 generated from primary production. However, far more is known about which 5 groups areFor dominant Peerthan about the Review cellular processes they Only perform in order to 6 become dominant. In the Southern Ocean, eukaryotic phytoplankton are the 7 dominant primary producers. In this study we used metagenomics and 8 metaproteomics to determine how the dominant bacterial and archaeal plankton 9 processed bloom material. We examined the microbial community composition in 10 fourteen metagenomes and found that the relative abundance of Flavobacteria 11 (dominated by Polaribacter ) was positively correlated with chlorophyll a 12 fluorescence, and the relative abundance of SAR11 was inversely correlated with 13 both fluorescence and Flavobacteria abundance. By performing metaproteomics on 14 the sample with the highest relative abundance of Flavobacteria (Newcomb Bay, 15 East Antarctica) we defined how Flavobacteria attach to and degrade diverse 16 complex organic material, how they make labile compounds available to 17 Alphaproteobacteria (especially SAR11) and Gammaproteobacteria , and how these 18 heterotrophic Proteobacteria target and utilize these nutrients. The presence of 19 methylotrophic proteins for archaea and bacteria also indicated the importance of 20 metabolic specialists. Overall, the study provides functional data for the microbial 21 mechanisms of nutrient cycling at the surface of the coastal Southern Ocean. 22 2 Wiley-Blackwell and Society for Applied Microbiology Page 3 of 165 23 Introduction 24 25 Bacterioplankton populations in the world’s oceans are typically dominated by three 26 bacterial clades: Alphaproteobacteria , Gammaproteobacteria , and Bacteroidetes 27 (Glöckner Foret al. , 1999; Peer Kirchman, 2002; Review Kirchman et al. , 2003; Only O’Sullivan et al. , 2004; 28 Abell and Bowman, 2005a). They also dominate Southern Ocean bacterioplankton 29 populations in the austral summer (Abell and Bowman, 2005a,b; Murray and Grzymski, 30 2007; Jamieson et al. , 2012; Grzymski et al., 2012). 31 Flavobacteria , the major clade of Bacteroidetes in the marine environment, are 32 heterotrophs that target complex organic matter, and specialize in the degradation of 33 biopolymers (Pinhassi et al. , 1999; Cottrell and Kirchman. 2000; Kirchman, 2002; Abell 34 and Bowman, 2005a,b; González et al. , 2008; Teeling et al. , 2012). Flavobacteria target 35 high molecular weight compounds and tend to be abundant during phytoplankton blooms 36 (DeLong et al., 1993; Glöckner et al. , 1999; Pinhassi, 2004; West et al. , 2008; Teeling et 37 al. , 2012). Increased Flavobacteria abundance has been linked to enhanced primary 38 production (Brown and Bowman, 2001; Kirchman, 2002; Horner-Devine et al. , 2003; 39 Abell and Bowman, 2005a; Murray and Grzymski, 2007), consistent with their role as 40 major mineralizers of organic matter (Cottrell and Kirchman, 2000). Flavobacteria are 41 especially important as the “first responders” to phytoplankton blooms, and by breaking 42 down complex organic matter by direct attachment and exoenzymatic attack of algal cells 43 and algal-derived detrital particles (Kirchman, 2002; Gómez-Pereira et al. , 2012; Teeling 44 et al. , 2012). Consistent with this, Flavobacteria tend to be abundant in the Southern 3 Wiley-Blackwell and Society for Applied Microbiology Page 4 of 165 45 Ocean where the waters are enriched in nutrients and phytoplankton (Abell and Bowman 46 2005a), particularly in summer (Grzymski et al., 2012; Williams et al., 2012). 47 Marine Alphaproteobacteria populations are dominated by the SAR11 and 48 Roseobacter clades, which favor labile substrates, including byproducts from the growth 49 of algae andFor Flavobacteria Peer (Mou et al.Review, 2008; Teeling et al. , 2012).Only Members of the 50 SAR11 clade are obligately planktonic, scavenge low concentrations of nutrients from 51 seawater (Morris et al. , 2002; Giovannoni et al. , 2005), and are most abundant where 52 phytoplankton biomass and primary production are low (Morris et al. , 2002; Sowell et 53 al. , 2009). Members of the Roseobacter clade of the Rhodobacterales are metabolically 54 diverse, targeting a wider range of substrates than SAR11 (Wagner-Döbler and Biebl, 55 2006; Moran et al. , 2007), and are often found in close association with phytoplankton 56 (Pinhassi et al. , 2004; West et al. , 2008). SAR116 is a clade of Alphaproteobacteria that 57 has also been detected in a range of marine environments, and has been regarded as a 58 metabolic generalist (Oh et al. , 2010). Gammaproteobacteria also comprise a major 59 component of marine bacterioplankton, and are represented by phylogenetically diverse 60 clades. Marine members of Oceanospirillales and Alteromonadales include heterotrophs 61 with broad substrate preferences, with cold-adapted genera such as Colwellia , 62 Pseudoalteromonas , Marinobacter and Psychromonas recorded in Southern Ocean 63 seawater and sea-ice off Antarctica (Bowman et al. , 1997; Piquet et al. , 2011). The 64 Oligotrophic Marine Gammaproteobacteria (OMG) group are physiologically diverse 65 marine heterotrophs that have been shown to be obligately oligotrophic when grown in 66 culture (Cho and Giovannoni, 2004). 4 Wiley-Blackwell and Society for Applied Microbiology Page 5 of 165 67 In addition to these groups, Antarctic surface waters include putative 68 chemolithoautotrophs, notably sulfur-oxidizing Gammaproteobacteria and ammonia- 69 oxidizing Marine Group I Crenarchaeota (MGI), as major components of the 70 picoplankton (Murray and Grzymski, 2007; Gzymski et al. , 2012; Williams et al. , 2012). 71 Bacteria ofFor the gammaproteobacterial Peer Reviewsulfur oxidizer EOSA-1 Only (GSO-EOSA-1) complex 72 have been reported in global mesopelagic waters (Swan et al., 2011) and oxygen 73 minimum zones (Walsh et al., 2009; Canfield et al., 2010), and their ecological role in 74 surface waters is yet to be determined (Grzymski et al. , 2012; Williams et al. , 2012). 75 MGI (also called Thaumarchaeota ) appear to play an important role performing ‘dark’ 76 chemolithotrophic carbon fixation and nitrification in the bulk ocean (Konneke et al. , 77 2005; Wuchter et al., 2006; Berg et al. , 2007), as well as in Antarctic surface waters in 78 winter (Grzymski et al. , 2012; Williams et al. , 2012). 79 In the North Sea, specific populations of Alphaproteobacteria , 80 Gammaproteobacteria and Bacteroidetes have been shown to successively exploit 81 organic matter in response to diatom blooms (Teeling et al. , 2012). During the decline in 82 chlorophyll a levels that followed a bloom (marked by a rapid increase in chlorophyll a 83 levels from essentially zero to ~25 µg L -1), the relative abundances of specific 84 Flavobacteria (Ulvibacter spp., Formosa spp., and Polaribacter spp.) and 85 Gammaproteobacteria (Reinekea spp. and SAR92 clade) were found to peak, while 86 specific groups of Alphaproteobacteria (SAR11 and Roseobacter clade) remained 87 comparatively constant (Teeling et al. , 2012). 88 The Southern Ocean is dominated by eukaryotic phytoplankton (rather than 89 Cyanobacteria ), and consists mainly of diatoms, dinoflagellates, and haptophytes (Wright 5 Wiley-Blackwell and Society for Applied Microbiology Page 6 of 165 90 et al. , 2010). Antarctic surface blooms occur as a result of release of nutrients 91 (particularly iron) upwelled over winter or through other nutrient incursions (Boyd and 92 Ellwood, 2010). For much of the Southern Ocean, surface concentrations of <0.3 – 0.4 µg 93 L-1 are typical, while higher chlorophyll concentrations (>1 µg L -1) are associated with 94 the major SouthernFor Ocean Peer fronts, ice edgeReview blooms during sea-ice Only retreat in summer, and 95 coastal/shelf waters (Moore and Abbott, 2000). However to date, metagenome- or 96 metaproteome-based studies of the bacterial and archaeal communities associated with 97 high chlorophyll