Response of Prochlorococcus ecotypes to co-culture with diverse marine bacteria The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Sher, Daniel et al. “Response of Prochlorococcus ecotypes to co-culture with diverse marine bacteria.” ISME J (2011). As Published http://dx.doi.org/10.1038/ismej.2011.1 Publisher Nature Publishing Group Version Author's final manuscript Accessed Thu May 26 18:27:49 EDT 2016 Citable Link http://hdl.handle.net/1721.1/61703 Terms of Use Creative Commons Attribution-Noncommercial-Share Alike 3.0 Detailed Terms http://creativecommons.org/licenses/by-nc-sa/3.0/ RESPONSE OF PROCHLOROCOCCUS ECOTYPES TO CO-CULTURE WITH DIVERSE MARINE BACTERIA 5 Daniel Sher*, Jessie W. Thompson, Nadav Kashtan, Laura Croal, and Sallie W. Chisholm Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA 10 * Current address: Department of Marine Biology, Leon H. Charney School of Marine Sciences, University of Haifa, Mt. Carmel, 31905, Israel Running title: Prochlorococcus-heterotroph interactions 15 Subject category: Microbe-microbe and microbe-host interactions Abstract Interactions between microorganisms shape microbial ecosystems. Systematic studies of mixed microbes in co-culture have revealed widespread potential for growth inhibition 20 among marine heterotrophic bacteria, but similar synoptic studies have not been done with autotroph/heterotroph pairs, nor have precise descriptions of the temporal evolution of interactions been attempted in a high throughput system. Here, we describe patterns in the outcome of pair-wise co-cultures between two ecologically distinct, yet closely related, strains of the marine cyanobacterium Prochlorococcus and hundreds of 25 heterotrophic marine bacteria. Co-culture with the collection of heterotrophic strains influenced the growth of Prochlorococcus strain MIT9313 much more than that of strain MED4, reflected both in the number of different types of interactions and in the magnitude of the effect of co-culture on various culture parameters. Enhancing interactions, where the presence of heterotrophic bacteria caused Prochlorococcus to 30 grow faster and reach a higher final culture chlorophyll fluorescence , were much more common than antagonistic ones, and for a selected number of cases were shown to be mediated by diffusible compounds. In contrast, for one case at least, temporary inhibition of Prochlorococcus MIT9313 appeared to require close cellular proximity. Bacterial strains whose 16S gene sequences differed by 1-2% tended to have similar effects on 35 MIT9313, suggesting that the patterns of inhibition and enhancement in co-culture observed here are due to phylogenetically-cohesive traits of these heterotrophs. Keywords: heterotrophic bacteria/interactions/phylogeny/Prochlorococcus 2 40 Introduction Interactions such as symbiosis, competition and allelopathy are a central feature of microbial communities (Azam and Malfatti, 2007; Bassler and Losick, 2006; Hibbing et al.). Even in dilute oceanic environments, microbial interactions abound: Antagonistic 45 interactions can promote biodiversity (Bidle and Falkowski, 2004; Czaran et al., 2002; Pernthaler, 2005), and synergistic interactions can provide sources of sustenance in complex communities (Amin et al., 2009; Azam et al., 1983; Azam and Malfatti, 2007; Boetius et al., 2000; Croft et al., 2005; Tripp et al., 2010). While marine microbial interactions often occur on scales of nanometers or microns (Blackburn et al., 1998; 50 Malfatti and Azam, 2009; Seymour et al., 2010; Stocker et al., 2008), they ultimately affect entire ecosystems and global biogeochemical cycles (Azam and Malfatti, 2007). Heterotrophic bacteria have been shown to both enhance and inhibit the growth of marine and freshwater algae (Grossart et al., 2006; Grossart and Simon, 2007; Mayali et al., 55 2008) and cyanobacteria (Bratbak and Thingstad, 1985; Manage et al., 2000; Morris et al., 2008) in liquid culture and on solid media. Through these and similar studies we have come to recognize specific mechanisms of interaction which can occur in the marine environment, such as facilitation of iron uptake (Amin et al., 2009; D'Onofrio et al., 2010), transfer of essential vitamins (Croft et al., 2005), inter- and intra-specific 60 communication (Bassler and Losick, 2006; Vardi et al., 2006) and allelopathy (Hibbing et al., 2009; Mayali et al., 2008). Hypothesizing that bacterium-bacterium antagonistic interactions shape microbial community structure at the microscale, Long and Azam 3 (Long and Azam, 2001) analyzed interactions among 86 pairs of co-isolated marine bacteria on solid media, revealing the widespread distribution of the potential for growth 65 inhibition among these bacterial strains (Grossart et al., 2004; Long and Azam, 2001; Rypien et al.). More recently, several strains of heterotrophic bacteria have been shown to enhance the growth of a number of ecotypes of Prochlorococcus – the dominant phototroph in temperate and tropical oceans (Coleman and Chisholm, 2007; Partensky and Garczarek, 2010) - at low cell concentrations on solid and liquid media (Morris et al., 70 2008). It was shown that the mechanism of enhancement in this case was the reduction of oxidative stress, explaining in part long-standing anecdotal observations that culturing Prochlorococcus is usually more robust when indigenous bacterial contaminants are present. 75 While Prochlorococcus have been extensively studied vis-à-vis the role of environmental factors such as light, temperature and nutrient availability in shaping their ecology (Bouman et al., 2006; Coleman and Chisholm, 2007; Johnson et al., 2006; Moore et al., 2002; Moore et al., 1998), and ‘top down’ processes such as predation and viral lysis have also been studied to some degree (Frias-Lopez et al., 2009; Lindell et al., 2007; 80 Lindell et al., 2005; Sullivan et al., 2005), systematic studies of their interaction with heterotrophic bacteria are limited to that of Morris and Zinser (Morris et al., 2008) described above, who focused on the growth-enhancing role of bacteria in low density cultures of Prochlorococcus. Inspired by this work, and by systemic analyses of Long and Azam (27), we undertook a broad- based and quantitative analysis of co-cultures of 85 two axenic Prochlorococcus ecotypes (Moore et al., 2005; Saito et al., 2002) with 4 hundreds of diverse heterotrophic bacteria, examining the response of the Prochlorococcus cells to the presence of bacteria over the entire growth curve of the cultures. 90 We chose two strains of Prochlorococcus, one adapted to low light (MIT9313) and one adapted to high light (MED4), for these studies because they are ecologically and phylogenetically distinct. Additionally, MIT9313 is known to produce a diverse array of secondary metabolites of unknown function, whereas the genes encoding this system are absent in MED4 (Li et al., 2010). We paired each strain with each of 344 strains of 95 heterotrophic bacteria isolated from an oligotrophic marine environment. We asked: 1) How does the presence of added heterotrophic bacteria influence the growth of each Prochlorococcus strain over the course of its growth curve? 2) Do the two ecotypes respond differently to the presence of the same heterotroph? 3) Do different strains of heterotrophs have different effects, and are they related to the phylogeny of the 100 heterotrophs? 4) Are the observed interactions mediated by soluble compounds or do they require close cellular proximity or contact? While the experimental system does not mimic the natural environment in many ways (Sup Info), it reveals some fundamental differences between the responses of two 105 Prochlorococcus ecotypes to co-culture with hundreds of bacteria - differences that may hold clues to factors governing their realized niches in the ocean. It further highlights a strong correlation of the outcome of co-culture with the phylogeny of the heterotrophic 5 bacteria, yielding hypotheses for further study on the mechanisms of these interactions and their potential role in marine microbial communities. 110 Results and Discussion Differences between Prochlorococcus MIT9313 and MED4 in outcome of co-culture To determine what kinds of interactions occur when Prochlorococcus is grown in co115 culture with many different strains of bacteria, we constructed a “library” of 344 heterotrophic bacterial isolates from seawater collected in the open ocean, at the Hawaii Ocean Time Series (HOT) station ALOHA (22º45’ N, 158º W) (Sup Fig. 1). The heterotrophic strains were isolated on solid media (see Sup. Info) and consist of at least 65 unique ribotypes (based on partial 16S rDNA sequences) clustering into 23, 13, 8 and 120 6 distinct OTUs at 1%, 3%, 5% and 7% rDNA sequence divergence, respectively (Sup Fig 1). The strains belong to the gamma-proteobacteria (primarily Alteromonas, Marinobacter and Alcanivorax) and alpha-proteobacteria (Rhodobacter) classes. Each of the 344 heterotrophic strains was inoculated into co-culture with axenic Prochlorococcus strains MED4 and MIT9313 in 96 well plates (under our conditions the outcome of co- 125 culture does not depend on the initial number of heterotrophs inoculated – see Sup Info, Sup Fig 2). We measured the bulk in-vivo chlorophyll fluorescence (FL) of the cultures, which is widely used (Grossart, 1999; Malmstrom et al., 2010; Mayali et al., 2008) to follow the dynamics of phytoplankton cultures in a non-invasive manner. While FL is only proportional to cell number when the cultures are in balanced growth (Log phase, 6 130 see Sup. Info) the shape of the FL curve can reveal differences between the bulk behavior of the cultures throughout the culture period. From the hundreds of co-cultures analyzed, only a few general types of co-culture outcomes emerged, as defined by the shape of the FL curves (Fig 1). Fifty seven percent 135 of the MIT9313 co-cultures fell into the group described as “early” (green, Fig. 1B) as these cultures entered exponential growth earlier, and reached higher maximal fluorescence than the heterotroph-free MIT9313 cultures (Fig. 1C). A small fraction of the co-cultures (3%) displayed the same initial timing as the “early” group, but fluorescence stopped increasing at an early stage and then declined rapidly (“early 140 arrested”, purple Fig. 1B). Thirty four percent of the cultures stopped increasing in fluorescence after 2-3 days, declined to undetectable levels, and then increased again much later (the “late” group, red Fig. 1B). Finally, only 6 % of the co-cultures with MIT9313 behaved similarly to the heterotroph-free cultures (“intermediate” black Fig. 1B). 145 The synoptic response of MED4 to co-culture with the same library of bacterial strains was dramatically different from that of MIT9313. Ninety-eight percent of the heterotroph culture collection revealed no clear effect on the growth of MED4 – as evidenced by their “intermediate” growth patterns which are very similar to the 150 heterotroph-free cultures. The growth of Prochlorococcus MED4 in the remaining 2% of the co-cultures was arrested early, displaying strong inhibition by the presence of these 7 heterotrophs (Fig 1B). The heterotrophic bacterial strains that inhibited MED4 were the same strains that defined the “early arrested” group in the MIT9313 cultures. 155 Quantifying the parameter space of the MED4 and MIT9313 co-culture outcomes To provide a quantitative estimate of the effect microbial interactions can have on Prochlorococcus culture dynamics, we extracted from the FL curves shown in Fig. 1 biologically-relevant descriptive parameters (similar to those used by Warringer et al (Warringer et al., 2008)): the maximum growth rate (, the time it took the cultures to 160 reach half of the maximal fluorescence (T50), and the maximum FL (Fmax). As was clear in the qualitative analysis, the parameter space is not homogenously covered (Fig 2; Suppl. Figs 3,4). Rather, parts of the parameter space are densely populated, whereas others regions are empty or sparse, representing parameter combinations which are not observed in our co-culture curves (e.g. co-cultures in which the log phase growth rate was 165 significantly reduced compared to heterotroph-free cultures). While the growth rate in log phase was influenced by the presence of bacteria in most of the MIT9313 co-cultures, the median of this parameter actually increased in most of the types of co-culture outcomes compared to the heterotroph-free cultures (Sup Fig. 3) even 170 when the overall effect was clearly one of much later onset of growth. Therefore, in agreement with other studies (Warringer et al., 2008), our results suggest that a combination of different growth parameters is necessary in order to fully describe the complex effect of microbial interactions. 8 175 As described above, the most striking is the difference between the large parameter space inhabited by MIT9313 co-cultures and the much more limited space inhabited by MED4 co-cultures (Fig. 2). The suite of heterotrophic bacteria that strongly influences the growth of MIT9313, decreasing some parameters up to ten-fold or increasing them up to four-fold has minimal, if any, impact on MED4. 180 Heterotroph phylogeny and co-culture outcome. We next asked whether closely related bacteria, as defined by their partial 16S rDNA sequence (ribotype), affect the growth of Prochlorococcus cultures similarly. As shown in Fig. 3, the heterotroph ribotypes which induced “early”, “early arrested” and “late 185 growth” phenotypes were significantly different for MIT9313 (UniFrac test with Bonferroni correction, P≤0.06 [Lozupone and Knight, 2005]), as were the groups that induced “intermediate” and “early inhibited” for MED4 (P≤0.01). For example, all but two of the heterotrophic strains which induced a “late” outcome of MIT9313 belong to two well-defined clades of Alteromonads (Fig. 3, Sup Fig. 1). Similarly, the same strains 190 induced the “early arrested” outcome in both MED4 and MIT9313, and all of these strains belong to a well-defined clade of Rhodobacters, similar to Marinovum algicola and Ruegeria sp. In most of these cases, the differentiation between strains which inhibit Prochlorococcus in co-culture and strains which do not is relatively deep-rooted, within the resolution afforded by our cultured collection of heterotrophs. For example, two 195 Alteromonad clades differing by 1-2% in their partial 16S sequence both inhibit MIT9313, whereas a third clade which differs by 4-5% from these two clades enhances MIT9313. Similarly, the clade of Rhodobacters inducing “early arrested” phenotype 9 differs from the most closely related strains in our collection that do not induce this phenotype by about 4% in their 16S. This level of divergence corresponds to one 200 commonly used to delineate species or genus level differentiation (Schloss and Handelsman, 2005). Co-culture outcome and proximity of cells While many interactions between microorganisms are mediated by diffusible soluble 205 compounds, some have also been observed to occur when cells live in close proximity or even necessitate direct cell-cell contact (Croft et al., 2005; Mayali and Azam, 2004). To test whether close cell-cell proximity is necessary for the different co-culture outcomes observed with MIT9313, we selected five heterotroph strains representing different phylogenetic clades and co-culture outcomes, and co-cultured them with MIT9313 either 210 separated by a membrane permeable to small molecules or mixed together as in the experiment presented above. As shown in Fig. 4, when the FL of the co-cultures increased earlier than that of the axenic cultures this happened regardless of whether or not the heterotrophic bacteria were separated from MIT9313 by a membrane. Thus, the “early” outcome of Prochlorococcus cultures is likely mediated in these cases by soluble, 215 diffusible compounds, although we cannot preclude the possibility that the small number of heterotrophic bacteria than can cross the membrane during these 19-day long experiments (see Sup Info) may also directly impact the growth of MIT9313. In contrast, the late co-culture outcome occurred only when MIT9313 and Alteromonas strain HOTo1A3 were grown in close proximity and not when they were separated by a 220 membrane. 10 Potential mechanisms underlying different co-culture outcomes MIT9313 and MED4 represent two taxonomic extremes within the Prochlorococcus 225 lineage, differing by ~3% in their 16SrRNA sequence. MED4 is a small cell with a highly streamlined genome, and is a member of the high-light adapted clade of Prochlorococcus. MIT9313, in contrast, is a slightly larger cell with a larger genome, and is better adapted for growth at the low light levels found deeper in the water column (Bouman et al., 2006; Coleman and Chisholm, 2007; Johnson et al., 2006; Moore et al., 230 2002; Moore et al., 1998; Rocap et al., 2003). Both strains are growing in these experiments below their respective temperature and light optima (although closer to those of MIT9313, (Rocap et al., 2003; Zinser et al., 2007)), but have been pre-acclimated to the experimental conditions for >7 months (~120 generations) and thus the difference in co-culture outcome is likely not caused by a general stress response in one strain due to 235 culture conditions. The “early” culture outcome is the one most commonly observed with MIT9313, is widely distributed among the different phylogenetic groups, and in all cases tested is caused by soluble, diffusible molecules. This is consistent with a “helper” effect where 240 the growth of Prochlorococcus increases as a result of basic attributes common to many lineages of heterotrophic cells, as suggested by Morris et al (Morris et al., 2008). Such attributes may include scavenging of reactive oxygen species, (Morris et al., 2008), increasing carbon dioxide concentration (Moore et al., 2007) or cycling waste products. 11 MED4 as a high-light adapted strain, may be better adapted to deal with oxidative stress 245 (often generated during photosynthesis) than MIT9313, thus the latter strain may benefit more from interacting with heterotrophs. Notably, however, MED4 can readily form colonies on solid media only with the help of heterotrophs, and thus this strain is not immune to the effect of co-occurring bacteria (Morris et al., 2008). 250 In contrast, inhibition of MIT9313 (early arrested or late outcomes) was observed mainly in co-cultures with two-well defined groups of bacteria belonging to the Alteromonads and Rhodobacters, with the latter group being the only one to clearly affect the growth of MED4 under our conditions. Related bacteria have previously been shown to inhibit other microbes through the production of secreted allelochemicals (e.g. (Gram et al.; Mayali 255 and Azam, 2004)). An intriguing observation is that inhibition of MIT9313 by an Alteromonas strain required proximity between the heterotrophic bacteria and MIT9313 – i.e. the effect could not be mimicked when the cells were kept apart by a semi-permeable membrane. Recently, close physical association (cell-cell contact) has been observed in natural seawater samples between Synechococcus cells, which are closely related to 260 Prochlorococcus, and heterotrophic bacteria of unknown taxonomy (Malfatti and Azam, 2009; Malfatti et al.). These observations suggest the potential for close or contactmediated interactions even in tiny picoplankton cells. 265 Conclusions 12 While some features of our experimental system limit extrapolation of our results to the experience of wild Prochlorococcus – e.g. the co-cultured strains were not co-isolated and the cell densities were higher than found in the wild (see also Sup Info) – our study has revealed some properties of these microbial interaction that likely have ecological 270 relevance. First, the two Prochlorococcus ecotypes display fundamentally different responses to the presence of bacteria, both in terms of general patterns, and in terms of specific responses to specific bacterial strains. These differences could influence the connectivity of these two strains within the microbial network in the wild. If so, MIT9313 may be more susceptible to changes in the microbial community than MED4. Similar 275 trends have been suggested for other marine bacterioplankton based on network analysis of patterns of co-occurrence in the oceans (Fuhrman and Steele, 2008). Second, both the antagonistic and enhancing interactions in our system revealed a clear phylogenetic signature, with closely related bacteria causing similar responses in the co280 cultured Prochlorococcus. Furthermore, only a handful of different interaction types, as measured through their effect on Prochlorococcus growth curves, were observed. The heterotroph culture collection we used represents only a fraction of diversity found in the oceans, and does not include many of the most common lineages. Future work with a wider diversity of bacteria may either reveal additional types of interactions or highlight 285 unknown constrains on the types of interactions which can affect cells in the aquatic environment. 13 Considering the high levels of microheterogeneity in both marine microbial populations (Hunt et al., 2008; Thompson et al., 2005) and their environment (Azam and Malfatti, 290 2007; Blackburn et al., 1998; Seymour et al., 2010; Stocker et al., 2008), the task of understanding how complex microbial populations interact in the oceans is a daunting one. Although it is encouraging, as we seek general patterns, that the co-culture outcomes we observe are not random with respect to the phylogeny of the heterortophs, the opposite has been observed in cultures of interacting heterotrophc bacteria (Long and 295 Azam, 2001). Clearly expanded and in depth study of the network of possible interactions between microbial groups is essential, if we ever wish to incorporate microbial interactions into our understanding of marine microbial communities. Materials and Methods 300 We isolated heterotrophic bacteria from the Hawaii Ocean Time Series (HOT) station ALOHA (22º45’ N, 158º W), one of the most comprehensively studied sites in the ocean, with a microbial community dominated by Prochlorococcus and characterized in some detail (DeLong et al., 2006). The heterotrophs were re-streaked for purity three times, and 305 the final library was preserved at -80°C in 25% glycerol. Prochlorococcus strains MIT9313 and MED4 were isolated from the Gulf Stream and the Mediterranean Sea, respectively (Rocap et al., 2003), and were maintained in the lab at 20°C and 27E constant cold white illumination. Co-culture was initiated by adding 2L of an overnight culture of each heterotroph from the library to 200L of Prochlorococcus culture (106 310 cells/mL) in 96 well plates. The culture media was Pro99 (Moore et al., 2007) with the 14 addition of 0.01% w/v Pyruvate, Acetate, Lactate and Glycerol as well as a vitamin mix (Morris et al., 2008). The co-culture plates were maintained for 42 days at 20°C and 27E constant cold white illumination, and the bulk chlorophyll fluorescence (ex440 em680) measured almost daily using a Bio-Tek Synergy HT plate reader. The resulting 315 curves were filtered to retain consistent curves, defined as those in which the Euclidian Distance between normalized curves fell within the range defined by 95% of the between-plate replicates of axenic curves. The growth parameters were extracted from the growth curves using macros written in Excel VBA, which are available from the authors upon request. Hierarchical Clustering was performed in Matlab. 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A clearly different pattern can be seen between four major clusters in MIT9313 but only two in MED4. B and C) FL curves of the 250 co-cultures (B) and 88 axenic Prochlorococcus cultures (C). The curves are 540 colored as shown in the legend based on the clustering results in panel A. Four different types of curves which differ in their growth timing and maximal FL, can be observed for MIT9313, whereas only two clusters are observed for MED4. Note the similarity in the shape of the “early arrested” outcome between MIT9313 and MED4. 545 Figure 2: The quantitative three-dimensional parameter spaces defining the effect co-culture on Prochlorococcus MIT9313 and MED4. A three-dimensional parameter space is shown, with the axes being the maximum growth rate (, the time it took the cultures to reach half of the maximal fluorescence (T50), and the maximum FL (Fmax). The parameter spaces shown includes both the co-cultures and the control axenic 550 cultures, and are normalized to axenic wells on the same plates (i.e. values larger than one represent an increase in the relevant parameter compared to axenic culture, smaller than one represent a decrease). The data points are colored based on the clustering shown in Fig 1. Large circles represent the median coordinates of each co-culture outcome. 25 555 Figure 3: The relationship between patterns observed in co-cultures and the ribotype of the heterotroph. A maximum likelihood (ML) tree (partial 16S rDNA sequences) is shown, with the co-culture outcome (as defined in Fig. 1) shown for MIT9313 (middle ring) and MED4 (outer ring). Spheres on the tree branches denote >80% aLRT confidence (Anisimova and Gascuel, 2006). Different shading of the 560 branches of the tree denotes Operational Taxonomic Units (OTUs) at 0.01 resolution (see also Sup Fig. 1 and Sup Table 1). 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Nature 464: 90-4. 755 Vardi A, Formiggini F, Casotti R, De Martino A, Ribalet F, Miralto A et al (2006). A stress surveillance system based on calcium and nitric oxide in marine diatoms. PLoS Biol 4: e60. Warringer J, Anevski D, Liu B, Blomberg A (2008). Chemogenetic fingerprinting by analysis of cellular growth dynamics. BMC Chem Biol 8: 3. 760 Zinser ER, Johnson ZI, Coe A, Karaca E, Veneziano D, Chisholm SW (2007). Influence of light and temperature on Prochlorococcus ecotype distributions in the Atlantic Ocean. Limnology and Oceanography 52: 2205-2220. 765 31 Supplementary Information 770 775 780 785 790 795 Detailed Materials and Methods General considerations and caveats: The experimental system described here was designed in order to enable the quantitative analysis of hundreds of cultures in parallel. It does not mimic the natural oceanic environment in many ways: the concentrations of nutrients and cell densities were significantly higher than in the oligotrophic ocean (see below for media composition); the heterotrophs were supplemented with organic compounds in the media, and not dependent on the phototrophs for sustenance; the light intensity was constant, and the diffusion of gasses was likely limited in the 96 well plates. The heterotrophic strains used are those which are relatively easily isolated on solid medium, and do not represent the majority of bacteria in the oceans (although Alteromonads and Rhodobacters are an important part – up to 10% - of the total marine microbial population, as measured using metagenomic methods (DeLong et al., 2006; Ivars-Martinez et al., 2008)). Finally, batch cultures were used by necessity, whereas the ocean environment is more like a chemostat. It is important to recognize these caveats (and others, described below) when interpreting the results of the described experiments. We isolated heterotrophic bacteria from the Hawaii Ocean Time Series (HOT) station ALOHA (22º45’ N, 158º W), one of the most comprehensively studied sites in the ocean, with a microbial community dominated by Prochlorococcus and characterized in some detail (DeLong et al., 2006). The bacteria were isolated on a relatively nutrient-poor seawater-based media, ProMM, which is similar to PLAG (Morris et al., 2008) but using 100% rather than 75% seawater. This media is based on the Pro99 media used to culture Prochlorococcus (Moore et al., 2007), with the addition of a set of defined organic compounds (0.05% w/v each of Lactate, Pyruvate, Acetate and Glycerol) and vitamins (Waterbury and Willey, 1988)). We chose to use this media because growth of Prochlorococcus from low cell numbers in 96 well plates is much more robust with the addition of low concentrations (1:20 v/v) of ProMM in Pro99 (manuscript in preparation). Thus, we could perform the co-culture in diluted ProMM, providing for robust growth of both Prochlorococcus and the co-cultured heterotroph. 800 805 810 Several methods are used traditionally to measure growth rates of phytoplankton cultures, including bulk fluorescence (Grossart, 1999; Malmstrom et al., 2010; Mayali et al., 2008; Osburne et al., 2010), optical density at 600nm (OD600, (Bruckner et al., 2008)), chlorophyll concentration (Bruckner et al., 2008) and cell number (Grossart and Simon, 2007). We chose to measure bulk chlorophyll fluorescence (ex440 em680) (FL) since it is widely used and the easiest to measure in a non-invasive, high throughput manner. During steady-state, balanced growth, bulk fluorescence is linearly correlated with Prochlorococcus cell numbers (R2 = 0.82 for MIT9313 and 0.97 for MED4, see also (Malmstrom et al., 2010; Osburne et al., 2010)). During lag, stationary and decline phases, however, FL is no longer correlated with cell number and thus can only be used to compare the state of one culture with respect to another. We extracted three biologically-intuitive parameters of the fluorescence curve: growth rate, growth time and 32 815 820 825 maximal fluorescence. Briefly, the growth rate, , was defined as the maximal slope of a linear regression, performed on Log10-transformed fluorescence data, in a sliding window of four consecutive data points within the exponential growth phase. The growth time, T50, was defined as the time it took the bulk culture fluorescence to reach 50% of the maximal fluorescence, Fmax, value for that curve. In order to simplify the experimental setup, the same volume of heterotrophic bacteria was added into each co-culture well. Thus, the number of heterotrophs initially inoculated into co-culture differs between different heterotroph strains. However, the outcome of coculture in our experimental system does not depend on the initial density of heterotrophic bacteria added, even when this density varied over six orders of magnitude (sup Fig. 2). This was observed for 8 out of 9 strains tested in co-culture with MIT9313 and for all 8 strains tested in co-culture with MED4. The lack of dependence on initial heterotroph density is likely due to the significantly higher growth rates of the heterotrophs in this media, which reach stationary growth (“quasi steady state”) typically after 1-2 days compared to 7-10 days for Prochlorococcus (Sup Fig. 2B, D, F). 830 835 840 845 While heterotrophs were inoculated into each well of the co-culture plates, occasionally wells were observed in which the inoculated heterotrophs did not visibly grow. If indeed no heterotrophs grew in these wells this could lead to the Prochlorococcus revealing a similar pattern as axenic cultures (e.g. “intermediate” response, Fig. 1). To bound how important this issue was, we monitored in parallel to the bulk culture fluorescence the turbidity (OD600) for all co-cultures. While these turbidity measurements cannot allow quantitative estimation of the heterotroph numbers, they do enable us to roughly determine whether heterotroph growth occurred in a specific well, based on a sharp increase in turbidity after on a different timescale from that caused by Prochlorococcus. In 94% of the co-cultures with MIT9313 and 91% of the co-cultures with MED4, clear heterotroph growth was observed after 1.5 days, defined as an OD600 value of >0.086, and these included co-cultures revealing all of the different effects on Prochlorococcus (Early, Early arrested, Intermediate and Late growth). Of the remaining cultures, in the case of MIT9313 more than 2/3 revealed an “Early” phenotype. Thus, while we cannot rule out that some of “indifferent” co-cultures were due to a failed heterotroph inoculation, this possibility affects at most 2% of the MIT9313 and 9% of the MED4 cocultures, and does not affect the overall conclusions of this study. 850 855 Isolation of heterotrophic bacteria from ALOHA: Seawater was collected during the C-MORE BLOOMER cruise on August 18, 2007, transported back to the lab and maintained for one month in clear polycarbonate bottles at 21ºC under 18 E constant cold white illumination. 1-100L of seawater were spread on ProMM agar plates, and colonies picked as they appeared over a period of 16 days into liquid ProMM. Colonies were further re-streaked three times for purity on ProMM plates, and the final library was maintained at -80°C in 25% glycerol. 33 860 865 870 875 880 885 PCR amplification and sequence analysis of the 16S gene: PCR amplification was performed from 2l of the bacteria in ProMM using the universal bacterial primers 8F and 1492R and sequenced using Sanger sequencing. Several samples which did not amplify in several attempts using these primers were amplified using the ReaX kit (Q Chip, Cardiff, UK, utilizing 27F and 905R primers). Sequence traces were analyzed in CodonCode Aligner (CodonCode Corporation, Dedham, MA, http://www.codoncode.com/aligner/download.htm), with the poor quality segments of the sequences removed using clip parameters set to “maximize region with error rate below 0.01”. Reads with fewer than 200 consecutive high quality base pairs were also removed from the dataset. 257 sequences (Sup Fig. 1) which passed the quality threshold were analyzed using GreenGenes ((DeSantis et al., 2006a; DeSantis et al., 2006b), http://greengenes.lbl.gov). The sequences were aligned to the Core Set of aligned sequences using NAST (minimum length set to 250) to produce a set of aligned 7682character sequences (DeSantis et al., 2006b), and were checked for chimeric sequences using Bellerophon 3.0 (600bp Core set threshold and 200bp window size) (DeSantis et al., 2006a). The nearest neighbors and nearest isolates were determined using Simrank, and sequence divergence from near-neighbors calculated using the DNAML option of DNADIST (PHYLIP package, all performed within GreenGenes) with Lane Mask restricting calculation to 1,287 conserved columns, the A, C, G, T 16S rRNA base frequency being 0.2537, 0.2317, 0.3167, 0.1979, respectively, and the Transition:Transversion Ratio assumed to be 2.0. The phylogenetic classification is shown using the Hugenholz Genus classifier. Operational Taxonomic Unites (OTUs) were estimated using DOTUR (Schloss and Handelsman, 2005) using the furthest neighbor algorithm and a precision value of 1000. The phylogenetic trees in Fig 3 and Sup Fig. 1 were created using PhyML (model=HKY, estimated t, v and a, (Guindon and Gascuel, 2003)) with aLRT test for branches (Anisimova and Gascuel, 2006) and visualized using iTOL ((Letunic and Bork, 2007), http://itol.embl.de/). Percent divergence in the 16S gene sequences reported throughout this manuscript were based on BLAST2seq comparisons performed at the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using the default parameters. The sequences have been submitted to the GenBank under accession numbers HQ537134-HQ537390. 890 895 900 Co-culture setup and parameter extraction: Prochlorococcus strains MIT9313 and MED4 were maintained at 20°C and 27E constant cold white illumination. Under these conditions the two strains grow at comparable rates (=0.32±0.04 for MIT9313, 0.43±0.01 for MED4, growth rate is described using base e). Duplicate mid-exponential cultures were counted by using an Influx flow cytometer (BD biosciences), diluted to a final cell density of 106 cells/ml in 5% ProMM in Pro99 and distributed into the wells of 96 microtitre plates. Duplicates of the heterotroph library were inoculated into 5% ProMM in Pro99 24 hours prior to the initiation of the experiment, and 2L of the heterotroph plates were used to inoculate the co-culture plates. The outer wells of the 96 well plates were not used for the experiments as growth in these wells is less robust, possibly due to a higher evaporation rate. Each of the 96 well plates used in this experiment had at least 12 control wells (columns 2 and 11), in which Prochlorococcus was grown axenically, and 48 co-culture wells. The co-culture plates were maintained for 42 days at 20°C and 27E constant cold white illumination, and the fluorescence 34 905 (ex440 em680) read almost daily using a Bio-Tek Synergy HT plate reader. The culture parameters described above were extracted from the FL curves using several macros written in Excel VBA, which are available from the authors upon request. 910 Selecting consistent curves and hierarchical clustering: The 96 well plates used for the experiment contained 344 heterotroph strains, of which 257 had their 16S sequences determined (Sup Fig 1B). We next removed from the study co-cultures in which the two biological duplicates were not consistent. We defined a consistent pair of fluorescencebased growth curves as one in which the Euclidian distance between the normalized curves is within the range defined by 95% of the between-plate pairs of axenic curves (Sup Fig 5). Using this definition, 94.4% of the MED4 and 77.9% of the MIT 9313 duplicate curves were consistent, and we from the 344 original strains retained for further analysis 250 strains which were consistent in co-culture with both Prochlorococcus strains. These include 180 strains for which partial 16S sequences was determines, and from which the phylogenetic tree in Fig. 3 was constructed. Duplicate curves deemed to be consistent and the parameters extracted from these curves were averaged. For Hierarchical Clustering, each averaged fluorescence curve was first normalized to a vector of length one. We then applied standard Hierarchical Clustering using Matlab (UPGMA linkage (Sokal and Michener, 1958)) using the default Euclidian distances and a threshold for determining the clusters of 1 for the large experiment and 0.7 for the smaller one described below. Similar results were observed in both the clustering of fluorescence curves and in the phylogenetic analysis of the outcomes both with and without the filtered “inconsistent” curves. 915 920 925 930 935 940 945 950 Testing the robustness of the observed patterns across different experiments: To test the consistency of the general pattern of interactions observed, we randomly selected a subset of 96 strains from the heterotroph library and performed a second co-culture experiment under similar conditions several months later. Similar patterns emerged in the second experiment, including the main different co-culture outcomes for MIT9313, the striking lack of effect of co-culture on MED4, and the dependence of the effect on the phylogeny of the heterotrophs (sup Fig. 6). These results show that the pattern of interactions observed across the collection of bacteria sampled, as well as the specific effect of each heterotroph strain, is highly robust. Co-cultures separated by membranes: To test whether diffusible compounds mediated the interactions we observed, or whether close cellular proximity was needed, we performed co-culture experiments in 6-well plates containing “ThinCert” tissue culture inserts containing a transparent 0.4m PET membrane (Greiner Bio-one). These inserts allow each well to be partitioned into two compartments separated by a porous membrane, which enables the rapid exchange of small molecules between the compartments (~95% exchange of the dye phenol red within 27 hours) but blocks almost completely the exchange of bacteria (<0.1% exchange of either Prochlorococcus or Alteromonas HOTo1A3 (Fig 4) in 1:20 ProMM after 24 hours, as counted by flow cytometry). The 6-well plates and tissue-culture inserts were acid washed for 15 minutes, washed extensively with sterile mili-Q water and sterilized by UV irradiation (5 minutes 35 955 960 using a Stratagene UV cross-linker). Each compartment contained 3.5mL of 5% ProMM in Pro99. Prochlorococcus MIT9313 was inoculated at 2×106 cells/mL into the interior compartment, and 35L of the appropriate heterotroph strain (from an overnight culture) were inoculated either together with MIT9313 in the interior compartment or in the exterior compartment, separated by the membrane from MIT9313. The 6-well plates were maintained for 19 days at 20°C and 27E constant cold white illumination. The fluorescence (ex440 em680) was read almost every two days a Bio-Tek Synergy HT plate reader using a 9×9 matrix per well, the middle 32 cells were averaged, and the reading from a cell not containing any Prochlorococcus (blank cell) subtracted to produce the final fluorescence value. 36 Titles and legends to supplementary figures: 965 970 975 980 985 Sup Fig 1: Composition and diversity of the heterotroph library used for co-culture experiments with Prochlorococcus. A) Phylogeny of the heterotroph library and the outcome of co-culture with Prochlorococcus. A maximum likelihood (ML) tree is shown built from the partial 16S rDNA sequences of 257 library strains and 13 of cultured reference strains. Spheres on the tree branches denote >80% aLRT confidence (Anisimova and Gascuel, 2006). The leaves are colored according to the genus level identity, and with black boxes showing Operational Taxonomic Units at 0.01 resolution calculated using DOTUR (Schloss and Handelsman, 2005). The results of the co-culture experiments are shown as in Figure 3. Strains which did not produce consistent curves in replicate experiments are shown in white. For the Alteromonad reference strains, the location where they were isolated is shown in parentheses to stress the global distribution of these strains. The insert shows the bacterial classes represented in the library (highlighted in red) in the context of a global phylogenetic tree (“Tree of life” downloaded from iTOL (Letunic and Bork, 2007), http://itol.embl.de/)). Cyanobacteria (the phylum including Prochlorococcus) are highlighted in green. B) Order level composition of the heterotroph library, determined using the Hugenholz Genus classifier in GreenGenes (DeSantis et al., 2006a; DeSantis et al., 2006b). Unknown means that either no PCR amplicon, or good quality sequence, was obtained in several reactions using the16S primers described in the materials and methods. C) Number of Operational Taxonomic Units (OTUs) as a function of 16S rDNA gene sequence divergence. There are 257 sequenced heterotroph strains in the library, corresponding to 65 distinct partial 16S sequences. These cluster into 23, 13, 8 and 6 distinct OTUs at 1%, 3%, 5% and 7% rDNA sequence divergence, respectively. 990 995 1000 1005 Sup Fig 2: The outcome of a co-culture as a function of initial density of heterotrophs. Ten-fold serial dilutions of three selected heterotroph strains (each representing one of the co-culture outcomes shown in Fig. 1), spanning 5-6 orders of magnitude, were co-inoculated with duplicate Prochlorococcus MIT9313 cultures. The left panels (A, C, E) show fluorescence curves, the right panels (B, D, F) show OD600 curves. The purpose of the OD600 curves is to assess the growth of the heterotrophs: during the first few days of co-culture the increase in OD600 is caused only by heterotroph growth, as Prochlorococcus cells numbers remain low and no such increase in OD600 is seen in axenic Prochlorococcus cultures. A second increase in OD600 mirrors the increase in culture fluorescence and is likely due to the increase in Prochlorococcus cell numbers. Green curves are axenic controls, colored curves show co-cultures at different serial dilutions (dark-more heterotrophic cells). Results show mean and range of duplicates. No clear difference could be seen between the outcomes of the co-cultures at different initial heterotroph densities. The growth rates of the heterotrophic strains, as calculated from the OD600 curves during the initial four days of co-culture, were 1.44±0.1 for Alteromonas HOTo3B7, 1.19±0.1 for Alteromonas HOTo1A3 and 1.01±0.1 for Roseobacter HOTo5H2, significantly higher than the growth rate of MIT9313 as determined using culture fluorescence (0.28-0.40 calculated during the MIT9313 exponential stage and depending on the heterotroph strain). These data support our interpretation that upon 37 1010 initiation of co-culture the heterotrophic bacteria grow much faster than Prochlorococcus and reach a quasi-steady-state which does not depend on the initial number of cells. A, B) Early Growth (Alteromonas HOTo3B7,dilution range 2-2x105 cells/mL), C, D) Late Growth (Alteromonas HOTo1A3, dilution range 5-5x105 cells/mL), E, F) Intermediate Growth (Roseobacter HOTo5H2, dilution range 0.24-2.4x104 cells/mL). 1015 1020 1025 1030 1035 1040 1045 1050 Sup Fig. 3: Details of the co-culture parameter space for MIT9313. For each culture (shown by a single curve in Fig. 1) three parameters were measured: max growth rate , growth time T50 and maximal fluorescence Fmax. Histograms are shown of these three parameters in all of the cultures belonging to a specific co-culture outcome (Early growth, intermediate growth, late growth, as defined in Fig. 1) as well as in the MIT9313only cultures. X-axis = relative parameter value, Y-axis = number of cultures. Within each histogram the median +/- one standard deviation are written. Sup Fig. 4: Details of the co-culture parameter space for MED4. For each culture (shown by a single curve in Fig. 1) three parameters were measured: max growth rate , growth time T50 and maximal fluorescence Fmax. Histograms are shown of these three parameters in all of the cultures belonging to a specific co-culture outcome (Early growth, intermediate growth, late growth, as defined in Fig. 1) as well as in the MED4only cultures. X-axis = relative parameter value, Y-axis = number of cultures. Within each histogram the median +/- one standard deviation are written. Sup Fig. 5: Estimating the consistency of duplicate co-cultures. Histograms are shown of the Euclidian distance between pairs of axenic cultures (upper panels) and co-cultures (lower panels). Pairs of control cultures were defined as those controls found in corresponding wells from duplicate plates. The dashed line represents the 95% cutoff used to determine which co-cultures were consistent. 94.4% of the MED4 cultures and 77.9% of the MIT9313 co-cultures from the large experiment were deemed to be consistent using these criteria. Sup Fig. 6: Reproducibility of general patterns observed using a smaller, randomly selected group of heterotrophs in co-cultures. A second set of co-culture experiments was performed with 96 randomly-selected strains from the heterotroph library, several months after the experiments shown in Figs 1-3. A) Heat maps of the normalized bulk chlorophyll fluorescence of 85 co-cultures and 20 controls clustered using Hierarchical Clustering (HC). B and C) Fluorescence curves of the co-cultures (B) and axenic cultures (C). Six main co-culture outcomes can be differentiated using HC, three of which are similar to the “Late Growth” observed in Fig. 1 but differing in the timing of the final growth. Only two co-culture outcomes can be seen for MED4. Note the similarity in the shape of the “early arrested growth” outcome between MIT9313 and MED4. D) Similarity in the phylogenetic pattern of co-culture outcomes between the two experiments. The maximum likelihood tree from Fig. 3 is shown, with the coculture outcomes (see legend) of the second experiment added. Middle rings are MIT9313, outer rings are MED4. Spheres on the tree branches denote >80% aLRT confidence (Anisimova and Gascuel, 2006). 1055 38 OTU Order number Rhodobacterales 1 Rhodobacterales 2 Rhodobacterales 3 4 Oceanospirillales OM60 5 6 Alcanivoracaceae 7 Alcanivoracaceae 8 Alcanivoracaceae 9 Alcanivoracaceae Marinobacter 10 Marinobacter 11 Marinobacter 12 Marinobacter 13 Marinobacter 14 Marinobacter 15 Alteromonadales 17 Alteromonadales 18 Alteromonadales 19 Alteromonadales 20 Alteromonadales 21 1060 Effect on MIT9313 Early Early Intermearrested diate 0 4 1 1 0 0 7 0 0 0 0 1 0 0 0 0 Effect on MED4 Early Intermearrested diate 4 1 0 1 0 7 0 1 3 3 0 0 0 0 0 0 0 0 3 3 1 0 0 0 0 1 1 0 0 0 0 1 2 0 0 0 0 2 0 3 19 10 1 37 4 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 1 0 0 0 0 0 0 1 1 0 0 0 0 1 0 0 32 1 41 0 0 0 0 0 0 0 0 0 0 0 1 3 21 11 1 38 4 1 32 1 42 Late Supplementary Table 1: Operational Taxonomic Units (OTUs) at 0.01 resolution and their effect on MIT9313 and MED4 in co-culture. The OTUs are shown in the same order as in Figure 3 and Supplemental Figure 6D, ordered clockwise. The number of strains revealing each effect is shown. References for Supplementary Information 1065 1070 Anisimova M, Gascuel O (2006). Approximate likelihood-ratio test for branches: A fast, accurate, and powerful alternative. Syst Biol 55: 539-52. Bruckner CG, Bahulikar R, Rahalkar M, Schink B, Kroth PG (2008). Bacteria associated with benthic diatoms from Lake Constance: phylogeny and influences on diatom growth and secretion of extracellular polymeric substances. Appl Environ Microbiol 74: 7740-9. 39 DeLong EF, Preston CM, Mincer T, Rich V, Hallam SJ, Frigaard NU et al (2006). Community genomics among stratified microbial assemblages in the ocean's interior. Science 311: 496-503. 1075 DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K et al (2006a). Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB. Appl. Environ. Microbiol. 72: 5069-5072. 1080 1085 DeSantis TZ, Jr, Hugenholtz P, Keller K, Brodie EL, Larsen N, Piceno YM et al (2006b). NAST: a multiple sequence alignment server for comparative analysis of 16S rRNA genes. Nucl. Acids Res. 34: W394-399. Grossart H-P (1999). Interactions between marine bacteria and axenic diatoms (Cylindrotheca fusiformis, Nitzschia laevis, and Thalassiosira weissflogii) incubated under various conditions in the lab. Aquatic Microbial Ecology 19: 1-11. Grossart HP, Simon M (2007). Interactions of planktonic algae and bacteria: effects on algal growth and organic matter dynamics. Aquatic Microbial Ecology 47: 163-176. 1090 Guindon S, Gascuel O (2003). A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52: 696-704. 1095 1100 Ivars-Martinez E, Martin-Cuadrado A-B, D'Auria G, Mira A, Ferriera S, Johnson J et al (2008). Comparative genomics of two ecotypes of the marine planktonic copiotroph Alteromonas macleodii suggests alternative lifestyles associated with different kinds of particulate organic matter. Isme J 2: 1194-1212. Letunic I, Bork P (2007). Interactive Tree Of Life (iTOL): an online tool for phylogenetic tree display and annotation. Bioinformatics 23: 127-8. Malmstrom RR, Coe A, Kettler GC, Martiny AC, Frias-Lopez J, Zinser ER et al (2010). Temporal dynamics of Prochlorococcus ecotypes in the Atlantic and Pacific oceans. Isme J. 1105 Mayali X, Franks PJ, Azam F (2008). Cultivation and ecosystem role of a marine roseobacter clade-affiliated cluster bacterium. Appl Environ Microbiol 74: 2595-603. 1110 1115 Moore LR, Coe A, Zinser ER, Saito MA, Sullivan MB, Lindell D et al (2007). Culturing the marine cyanobacterium Prochlorococcus. Limnology and Oceanography-Methods 5: 353-362. Morris JJ, Kirkegaard R, Szul MJ, Johnson ZI, Zinser ER (2008). Facilitation of robust growth of Prochlorococcus colonies and dilute liquid cultures by "helper" heterotrophic bacteria. Appl Environ Microbiol 74: 4530-4. 40 1120 Osburne MS, Holmbeck BM, Frias-Lopez J, Steen R, Huang K, Kelly L et al (2010). UV hyper-resistance in Prochlorococcus MED4 results from a single base pair deletion just upstream of an operon encoding nudix hydrolase and photolyase. Environmental Microbiology. Schloss PD, Handelsman J (2005). Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Appl Environ Microbiol 71: 1501-6. 1125 Sokal RR, Michener CD (1958). A statistical method for evaluating systematic relationships. University of Kansas Scientific Bulletin 38: 1409-1438. 1130 Waterbury JB, Willey JM (1988). Isolation and Growth of Marine Planktonic Cyanobacteria. Methods in Enzymology 167: 100-105. 41 9313 A 0.8 0.6 0.4 0.2 0 Fluorescence 0.1 -0.1 sample Color legend for co-culture outcomes: Prochlorococcus + heterotrophs 15000 15000 10000 10000 5000 5000 0 0 10 20 30 C Fluorescence 0.3 -0.2 sample B MED4 0.5 40 0 Early 0 10 20 30 40 Prochlorococcus alone 15000 15000 10000 10000 5000 5000 0 0 10 20 30 40 0 In te rm e d ia te E a rly arrested 0 time (days) 10 20 30 40 Late 3 2 1 0 0 0 ax 1 1 T50 2 2 Fm Growth Rate µ MIT9313 Early 3 3 MED4 Interm ediate 3 E arly arrested 2 Late 1 0 0 0 ax 1 1 T50 2 2 3 3 Fm Growth Rate µ Color legend for co-culture outcomes: Outer ring: Middle ring: MED4 931 3 Inner ring: phylogeny: Scale: 0.05 * 5C4 HOT4G8 HOT4C7 HOT HOT5G 7 HOT5D 8 HO T5F4 HO T5D1 0 HO T4G9 HO T9 C6 HO T4 G5 HO T4 E4 HO T4 D4 HO T4 F1 HO 0 T4 E10 HO T4C HO 3 T7G HO 7 T5E HO 8 T4 H E9 O T4 E6 HOT5G5 HOT4D10 HOT4E8 HOT2G5 HOT4F6 HOT1D9 HOT4F8 HOT4F4 HOT4C5 HOT5C7 HO HO F6 T5 HO B3 T4 HO E7 T4 HO B9 T9 10 0F T1 10 HO T5G 3 HO T8D 8 HO T8G 3 C HO T5 F4 HO T8 O H 4B5 HOT 8 HOT9B 10 HOT9B G9 HOT7 T9C7 HO 7 T10F HO T3F6 HOT4F7 * * HO HO HO T5 HO HO T5 HO HO HO T8 T5B 8 T9B B10 T7 B8 F3 T5 G T5 HO 6 E8 E5 9 HO 5 T4E 10 T4C HO F8 T5 3 HO 0G T1 HO G6 T5 HO C4 T4 T5 HO E6 G8 T2 HO HO * HOT2E HOT2D 8 HOT 1C10 T5 C9 T9C5 HO T4B7 HO T2G6 HO 0D7 HOT1 C5 HOT10 7 HOT5E HOT HOT1E6 HOT8F3 HOT3F9 HOT5D3 HOT3E7 HOT1G8 HOT3D9 HOT1G9 HOT3C8 HOT2G7 HOT1E5 HOT3D6 HOT3G6 HOT1G3 HOT4C8 HOT1E9 HOT1D10 HOT10B5 HOT1E4 HOT8D9 HOT1E7 HOT3E1 HOT5C10 0 HOT4D5 4B6 HOT 3 HOT4F 10 HOT3F HOT 9C3 HOT3C HOT9B E6 T3 E5 T3 0 F1 T2 F9 10 9 T4B C5 HO T3 HO 6 0D T1 10 O G H T4 6 HO T4G 9 HO T4F 5 HO T4F HO B8 T2 HO G8 T3 HO F3 T3 HO D8 T3 HO D6 T1 HO T3E8 HO T3E9 HO T4G4 HO 6 7 HOT3D5 1B10 HOT T2E9 HOT2B HOT2C HO HOT2B9 HOT1B5 HOT10C8 Rhodobacterales Oceanospirillales OM60 Alcanivoracaceae Marinobacter Alteromonadales HOT2E8 α proteobacteria HOT2D9 Inner ring: P hylog eny (Clockwise) HOT2D7 HOT2C8 T4B H O T3 HO D 3 T2 HO C T2B 7 HO 6 T1D HO 7 T3C HO 9 T1 D8 HO T3 G7 HO T2 E5 HO T3 B4 HO T3 B1 0 HO T10F 8 HO T2D6 HO T3D7 HO T4D7 HOT3B 9 HOT4C 6 HOT 3C4 HO HOT2B4 T2 HO HOT1E3 HO HOT3B5 HO HOT1B3 HO HOT3F5 T3E3 HO T3B6 HO 0 T3D1 HO G5 T3 HO C6 T3 HO 7 3 HO T8G1 0 HO T5E4 HO T4D6 HO T8D7 HO T3 B8 HO T5 F9 HO T5 G4 HO T5 D4 HO T4 D9 HO T3 F4 HO T1G HO 10 T1G HO 5 T1 0F 6 T3E4 HO γ proteobacteria 10 1G6 HOT1G7 HOT3G10 * O uter rings: co culture outcom e Early Interm ediate E aarly arrested Late Fluorescence 250 200 Marinobacter HOT4G9 Alcanivorax HOT7G9 Rhodobacter HOT5F3 Alteromonas HOTs2G3 150 100 50 0 250 200 150 100 50 0 250 200 0 Alteromonas HOTo1A3 15 co-culture together 100 50 0 10 9313 alone 150 0 5 5 10 Days 15 20 Co-culture separated by membrane 20 Co-culture outcome MIT9313 MED4 α-Proteobacteria A Prochlorococcus MIT9313 Pelagibacter ubique Dinoroseobacter shibae Roseobacter denitrificans Marinovum algicola Pro MIT931 Pelagibac Dinoroseo HOT10F8 HOT10C7 Roseobact HOT9B6 HOT10B9 HOT5E6 HOT7E8 HOT5E5 HOT5F3 Marinovum HOT5B10 HOT8G6 HOT8G8 HOT8C6 HOT8F5 HOT8C5 HOT8D3 HOT8G5 HOT8B6 HOT8F10 HOT5C3 HOT8F4 HOT8F6 Rhodobacter HOT5G10 HOT8B4 HOT9C10 HOT8F9 HOT8B9 HOT9B5 HOT8C10 HOT8C8 HOT8B5 HOT8C9 HOT5B8 HOT8B8 HOT8D8 HOT8B3 HOT8G9 HOT7G10 HOT8F7 HOT8C7 HOT8D5 HOT8D6 HOT7C10 HOT9C7 HOT7G9 HOT10F9 HOT10B10 HOT10G9 Alcanivorax borkumensis HOT9B8 HOT9B10 Alcanivor HOT10G3 HOT10C4 HOT10F7 Alcanivorax HOT5F6 HOT10G4 HOT10D3 HOT10F10 HOT10F3 HOT10G8 Marinobacter AP10 HOT3F6 HOT10C3 Marinobact HOT4E5 HOT4C10 HOT10B3 HOT5F8 HOT5G9 HOT4E6 HOT4B5 Scale 0.01 HOT4F8 HOT4F4 HOT4C5 HOT5C7 HOT1D9 HOT5G5 HOT4F6 HOT4D10 HOT5F4 HOT5D10 HOT4G9 HOT9C6 HOT4G5 HOT4E4 HOT4D4 HOT4F10 HOT4E10 HOT4C3 HOT7G7 HOT4E9 HOT5E8 HOT4E8 HOT2G5 HOT4F7 HOT4G8 HOT4C7 HOT5C4 HOT5G7 HOT5D8 HOT5G6 HOT10C9 HOT5C9 Marinobacter HOT2G8 HOT4C4 HOT10C5 HOT10F5 HOT9C5 HOT4B7 HOT2G6 HOT10F4 HOT10G5 HOT10D8 HOT5E10 HOT1G7 HOT1G6 HOT8F3 HOT5D3 HOT1G8 HOT1G9 HOT2G7 HOT1G3 HOT10C8 HOT1E9 HOT10D5 HOT8D9 HOT9C3 HOT3C7 HOT5C10 HOT4D5 HOT9B3 HOT8G10 HOT5E4 HOT4D6 HOT8D7 ia cter ba yano C HOT3B8 HOT5F9 HOT5G4 HOT4D9 HOT3F4 HOT1G10 HOT1G5 HOT10G10 Spongiibacter tropicus HOT9C4 HOT2G9 Spongiibac HOT9B9 HOT9C8 HOT4E7 Vibrio splendidus HOT4B3 Vibrio spl HOT10B6 OM60 Alt MED HOT10B6 Alteromonas MED102 (Mediterranean) HOT4G10 HOT4G6 HOT10G6 HOT10D6 HOT10F6 HOT10D7 HOT11C6 Alteromonas macleodii Deep Ecotype (Adriatic Sea) HOT4F9 Alt DE γ-proteobacteria HOT3D8 HOT3E8 HOT2F5 HOT3C3 HOT1E8 HOT2B8 HOT1D6 HOT4G7 HOT3G8 HOT4F5 HOT3E9 HOT2E9 β-p rot bac eoteri a Firmicutes Oceanospirillales HOT5D4 pylo s m a C e eral bact γ-Proteobacteria HOT3D6 HOT2C6 HOT3B9 HOT1D5 HOT2G10 HOT1C6 HOT4D3 HOT3B5 HOT1F3 Alteromonas CF12-6 (South China Sea) HOT2F6 HOT2D7 Alt SCS HOT3D7 HOT4B4 pr α- HOT4D7 HOT2D6 HOT4C6 HOT2E8 HOT1B9 HOT2D9 HOT1B5 HOT3D5 HOT2B9 HOT2F8 HOT3F5 HOT1B10 Alteromonadales b eo HOT1B7 HOT2B4 HOT1E3 HOT3F3 HOT2C8 HOT1B3 HOT4G4 Alteromonas L188s.610 (Mid-Atlantic Ridge sediment) HOT2B7 ac HOT2F7 Alt MAR HOT4C8 HOT3G6 te HOT3C8 HOT1E5 HOT1C10 HOT2G4 ria HOT1C4 HOT3D9 HOT3B6 HOT3E7 HOT1D7 HOT3G10 HOT1E6 HOT1B6 HOT2D8 HOT2D10 eae ttsiac Ricke ot HOT3C4 HOT2E7 HOT1D4 HOT2B6 HOT3F10 HOT2F10 HOT3E4 HOT3C10 HOT3D10 HOT2F9 HOT3E3 HOT2C4 HOT3E5 HOT3G3 HOT3E6 HOT3C6 HOT3G5 HOT4F3 HOT4B6 HOT1E4 Color legend for co-culture outcomes: HOT2D5 HOT3E10 HOT2C7 HOT2E4 HOT2C10 HOT4B10 HOT4B9 Archaea HOT3B10 HOT3B4 HOT2E6 HOT1B8 HOT1D3 HOT3C5 Eukarya HOT1B4 HOT2E5 HOT3G7 HOT1F4 HOT2B3 HOT1D10 HOT1E7 HOT3F9 HOT1D8 HOT3C9 HOT2C9 HOT3D3 C 1% unkno wn 26% 1% 26% 33% Rho d o b a cte ra le s 12% A lca nivo ra ca ce a e 3% M a rino b a cte r 24% 12% 3% 24% A lte ro m o na d a le s 33% O M 6 0 1% O ce a no sp irilla le s 1% Number of OTUs B Early Interm ediate E arly arrested Late 70 60 50 40 30 20 10 0 0 0 .1 0 .2 Sequence divergence 0 .3 A B C ulture fluorescence 100000 C ulture turbidity 1 10000 0.1 1000 100 Alteromonas sp H O To3B 7 10 C 0 20 30 0.01 D 40 O D600nm F lu o re s c e n c e 10000 1000 100 0 10 20 30 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 1 0.1 Alteromonas sp H O To1A 3 10 E 10 0.01 40 F 10000 1 1000 0.1 100 Roseobacter sp H O To5H 2 10 0 10 20 30 40 0.01 D ays Heterotroph density (cells/mL) Alteromonas HOTo3B7 2x10^ 5 2x10^ 4 2x10^ 3 200 20 2 Alteromonas HOTo1A3 5x10^ 5 5x10^ 4 5x10^ 3 500 50 5 M ean all con trols Roseobacter HOTo5H2 2.4x10^ 4 2.4x10^ 3 240 24 2.4 0.24 Relative Growth Time T50 Relative Growth Rate µ 40 20 0 Early 0 0.5 1 Interm ediate 1.5 2 2.5 1.49 (+0.28 −0.31) 20 0 0 0.5 1 1.5 2 2.5 1.16 (+0.42 −0.27) 2 0 0 0.5 1 100 0 0 0.5 1 1.5 2 2.5 1.5 2 2.5 0 0.5 1 1.5 2 2.5 1.01 (+0.16 −0.24) 0 0 0.5 1 40 2 2.5 1.25 (+0.11 −0.05) 20 0 1.5 0 0.5 1 1.5 2 Relative growth rate 2.5 0 0 0.5 1 1.5 2 2.5 0.42 (+0.03 −0.05) 1 2 3 4 1.71 (+0.49 −0.83) 0 0 1 2 4 3 4 1.11 (+0.71 −0.40) 0 0 1 4 2 3 4 0.41 (+0.05 −0.14) 2 0 0.5 1 40 1.5 2 2.5 1.94 (+0.21 −0.26) 20 0 0 2 5 E arly arrested 0 20 10 1.58 (+0.03 −0.09) 1.01 (+0.16 −0.16) 40 0.74 (+0.10 −0.07) 4 0 40 20 2 5 Late 0.99 (+0.11 −0.09) 50 4 0 40 20 40 Number of cultures Co-cultures 9313 alone 1.00 (+0.10 −0.10) Relative Maximal Fluorescence Fmax 0 0 1 2 40 3 4 0.32 (+0.17 −0.42) 20 0 0.5 1 1.5 Relative growth time 2 2.5 0 0 1 2 3 Relative maximal fluorescence 4 Relative Maximal Fluorescence Fmax Relative Growth Rate µ Relative Growth Time T50 60 60 60 1.00 (+0.03 −0.03) 1.00 (+0.03 −0.03) MED4 alone 40 40 40 20 20 20 Co-cultures Number of cultures 0 Interm ediate 0 0.5 1 200 1.5 2 2.5 0 0 0.5 1 150 1.03 (+0.06 −0.05) 150 1.5 2 2.5 1 2 3 4 1.10 (+0.23 −0.23) 40 50 50 0 0.5 1 1.5 2 2.5 0 20 0 0.5 1 3 1.5 2 2.5 0 0 1 2 3 4 4 0.62 (+0.05 −0.05) 0.98 (+0.17 −0.17) 1.5 0.13 (+0.05 −0.12) 3 2 1 2 1 0.5 0 0 60 100 0 0 80 1.01 (+0.05 −0.03) 100 2 E arly arrested 1.00 (+0.08 −0.10) 0 0.5 1 1.5 2 Relative growth rate 2.5 0 1 0 0.5 1 1.5 2 Relative growth time 2.5 0 0 1 2 3 Relative maximal fluorescence 4 MIT9313 alone 15 MED4 alone 15 mean=0.18 std=0.07 Number of curves mean=0.38 std=0.18 10 10 5 5 0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 0.1 0.2 0.3 MIT9313 Co-cultures 30 40 10 20 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 0.4 0.5 0.6 0.7 0.8 0.6 0.7 0.8 MED4 Co-cultures 60 20 0 0 0 0.1 0.2 0.3 0.4 Euclidian distance between duplicate curves 0.5 MIT9313 A MED4 0. 7 0. 5 0. 6 0. 5 0. 4 0. 4 0. 3 0. 3 0. 2 0. 2 0. 1 0. 1 0 0 −0. 1 B D sample Prochlorococcus + heterotrophs Fluorescence C 15000 15000 10000 10000 5000 5000 0 0 10 20 30 0 10 20 30 40 20 30 40 Prochlorococcus alone 15000 15000 10000 10000 5000 5000 0 0 10 20 30 40 0 time (days) 0 10 HOT5G5 HOT4D10 HOT4E8 HO HO T8 T5 B8 H HO OT9 B8 B T HO 5B10 6 HO T7E 8 HO T5F3 HO T5E5 T5 E6 HOT1G9 HOT2G7 HOT3D6 HOT1G3 HOT1E9 HOT10B5 HOT8D9 HOT5C10 HOT4D5 HOT9C HOT3C 3 HOT9 7 B3 HO T8G1 HO T5E4 0 HO T4D6 HO T8D7 HO HO T3B8 HO T5F9 HO T5G HO T5D 4 HO T4D 4 HO T3F 9 H T1 4 HO OT1 G1 T1 G5 0 0F 6 HOT10C8 HOT2B9 HOT1B5 H O HO T3 HO T2 D3 HO T2 C7 B HO T1D 6 HO T3C 7 HO T1D 9 T3 8 HO G7 HO T2E5 HO T3B4 T3 B1 HO 0 T1 HO 0F8 T2D6 HO T3D7 HO T4 HOT3 D7 B9 HOT4C 6 HOT3C 4 HOT2B4 HOT1E3 HOT3B5 HOT1B3 HOT3F5 HOT2C8 9 G T5 E5 HO T4 C10 HO T4 8 HO T5F G3 HO T10 6 HO T5G 4 HO T4C 8 HO T2G HO T5C9 HO T9C5 HO T4B7 HO T2G6 HO 0D7 HOT1 0C5 HOT1 10 HOT5E 6 HOT1G HOT1G7 HOT8F3 HOT5D3 HOT1G8 6 0D 0 T1 G1 O H OT4 G6 H T4 F9 HO T4 5 HO T4F 8 HO T2B 8 HO T3G HO T3F3 HO T3D8 D6 HO T1 HO T3E8 HO T3E9 HO G4 T4 HO T2E9 HO C6 HOT2 7 HOT2B 10 HOT1B HOT3D5 HOT2D7 HOT2D9 HOT2E8 HOT2 E7 HOT2D HOT1C 8 10 HOT1E6 HOT3G10 HOT3F9 HOT3E7 HOT3D9 HOT3C8 HOT1E5 HOT3G6 HOT4C8 HOT1D10 HOT1E4 HOT1E7 0 HOT3E1 6 HOT4B 3 HOT4F 0 F1 HOT3 E4 T3 HO T3E3 HO T3B6 HO D10 T3 5 HO T3G C6 HO T3 HO T3E6 5 HO T3E HO 2F10 9 T HO OT2F 10 H 4B 9 T 4B HO OT 3C5 H T HO HOT2G5 HOT4F7 HOT4G8 HOT4C7 HOT5C HOT5G 4 HOT5 7 D8 HO T5F4 HO T5D1 HO T4G9 0 HO HO T9C6 HO T4G5 HO T4E4 HO T4D HO T4F1 4 HO T4E 0 HO T4C 10 HO T7 3 H T5 G7 H OT E8 O 4E T4 9 E6 Scale: 0.05 HOT4F6 HOT1D9 HOT5C7 HOT4C5 HOT4F4 HOT4F8 5 HOT4B 8 HOT9B 0 B1 HOT9 G9 HOT7 C7 T9 HO 0F7 T1 HO T3F6 HO F6 T5 HO B3 T4 HO T4E7 9 HO T9B HO F10 T10 10 HO T5G 8D3 HO OT G8 H T8 C3 HO T5 F4 8 HO OT H MED4 experiment 2 0 40 MIT9313 experiment 1 MIT9313 experiment 2 MED4 experiment 1 −0. 1 sample Color legend for co-culture outcomes: Early Interm ediate E arly arrested Late (1) Late (2) Late (3)