bioRxiv preprint doi: https://doi.org/10.1101/460428; this version posted November 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 2 Co-culture and biogeography of Prochlorococcus and SAR11 3 4 Jamie W. Becker1*, Shane L. Hogle1, Kali Rosendo1, and Sallie W. Chisholm1,2 5 6 7 1Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, 8 Cambridge, MA, USA 9 10 2Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA 11 12 Correspondence: JW Becker* or SW Chisholm, Departments of Civil and Environmental 13 Engineering & Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Bldg 14 48-419, Cambridge, MA 02139, USA 15 E-mail: [email protected] or [email protected] 16 17 *Current address: Department of Biology, Haverford College, Haverford, PA, USA 18 19 Running title: Prochlorococcus/SAR11 interactions 20 21 Conflict of interest: The authors declare that they have no conflict of interest. 1 bioRxiv preprint doi: https://doi.org/10.1101/460428; this version posted November 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 22 Abstract 23 Prochlorococcus and SAR11 are among the smallest and most abundant organisms on 24 Earth. With a combined global population of about 2.7 x 1028 cells, they numerically dominate 25 bacterioplankton communities in oligotrophic ocean gyres and yet they have never been grown 26 together in vitro. Here we describe co-cultures of Prochlorococcus and SAR11 isolates 27 representing both high- and low-light adapted clades. We examined: (1) the influence of 28 Prochlorococcus on the growth of SAR11 and vice-versa, (2) whether Prochlorococcus can 29 meet specific nutrient requirements of SAR11, and (3) how co-culture dynamics vary when 30 Prochlorococcus is grown with SAR11 compared with sympatric copiotrophic bacteria. SAR11 31 grew as much as 70% faster in the presence of Prochlorococcus, while the growth of the latter 32 was unaffected. When Prochlorococcus populations entered stationary phase, SAR11 33 abundances decreased dramatically. In parallel experiments with copiotrophic bacteria however, 34 the heterotrophic partner increased in abundance as Prochlorococcus densities leveled off. The 35 presence of Prochlorococcus was able to meet SAR11’s central requirement for organic carbon, 36 but not reduced sulfur. Prochlorococcus strain MIT9313, but not MED4, could meet the unique 37 glycine requirement of SAR11, likely due to production and release of glycine betaine by 38 MIT9313. Evidence suggests that Prochlorococcus MIT9313 may also compete with SAR11 for 39 the uptake of 3-dimethylsulfoniopropionate (DMSP). To place our results in context, we assessed 40 the relative contribution of Prochlorococcus and SAR11 genome equivalents to those of 41 identifiable bacteria and archaea in over 800 marine metagenomes. At many locations, more than 42 half of the identifiable genome equivalents in the euphotic zone belonged to Prochlorococcus 43 and SAR11 – highlighting the biogeochemical potential of these two groups. 2 bioRxiv preprint doi: https://doi.org/10.1101/460428; this version posted November 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 44 Introduction 45 The global ocean is numerically dominated by Prochlorococcus and SAR11 (Pelagibacterales). 46 Prochlorococcus, a cyanobacterium, is the most abundant primary producer in tropical and 47 subtropical waters, where its estimated 2.9 x 1027 cells produce ca. 4 Gt of organic carbon 48 annually [1]. As such, they support a notable fraction of the secondary production in these 49 nutrient-poor waters [2]. Members of the alphaproteobacteria known as SAR11 are found 50 throughout the marine environment with an estimated global abundance of 2.4 x 1028 cells, about 51 half of which are in the euphotic zone [3]. Prochlorococcus and SAR11 together have been 52 estimated to comprise roughly 40-60% of the total bacteria in oligotrophic surface waters at 53 Station ALOHA in the North Pacific [4]. 54 Since its discovery three decades ago, Prochlorococcus has emerged as a powerful model 55 organism for microbial ecology [5]. Its ecotype diversity is well-characterized [6-9] and there is 56 an increasing wealth of genomic, transcriptomic and proteomic information applied to this group 57 [10-12]. The discovery of SAR11 a few years later further advanced our understanding of 58 microbial ecology and evolution in the oligotrophic ocean [13,14]. SAR11 possesses many traits 59 that highlight adaptations to the oligotrophic marine environment, including a small genome size 60 [15,16] and unique metabolic dependencies [17-20] partitioned among diverse ecotypes with 61 distinct biogeography [21-24]. 62 In addition to having large population sizes consisting of genetically distinct ecotypes, or 63 clades, Prochlorococcus and SAR11 have adapted to their oligotrophic habitat by minimizing 64 their genome content and metabolic versatility. SAR11 and high-light adapted Prochlorococcus 65 have small cell sizes between 0.2 and 0.8 microns in diameter, small genomes (1.2 to 1.8 Mb) 66 with low guanine-cytosine content (29 to 32 %GC) [10,15], and fewer regulatory s-factors than 3 bioRxiv preprint doi: https://doi.org/10.1101/460428; this version posted November 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 67 would be predicted from the size of their genomes [25]. Low-light adapted Prochlorococcus 68 genomes are larger (ca. 2.5 Mb) with higher guanine-cytosine content (33-50 %GC), but are still 69 relatively streamlined compared to copiotrophic bacteria. Genome reduction has resulted in the 70 loss of metabolic capabilities in both Prochlorococcus and SAR11, some of which, such as 71 reduction of oxidative stress [26] or a requirement for exogenous reduced sulfur [17] 72 respectively, are provided by neighboring microbes. Likely as a result of these types of 73 interdependencies, both taxa are difficult to isolate and maintain in-vitro compared to faster- 74 growing (r-selected) microbes; many of the same traits that confer a competitive advantage in 75 their native habitat likely render them difficult to maintain in a laboratory. 76 Prochlorococcus and SAR11 cells can interact with each other in the surface ocean either 77 directly or indirectly through the exchange of metabolites. Like all heterotrophs, SAR11 cells 78 rely on organic matter derived from primary producers and recent experiments revealed that 79 some members of the SAR11 clade likely rely heavily on organic compounds as an important 80 source of phosphorus in addition to carbon [27]. SAR11 also has several unique metabolic 81 requirements that implicate potential mutualism with Prochlorococcus. For example, SAR11 82 exhibits auxotrophy for a thiamin precursor molecule found in spent medium from 83 Prochlorococcus [20] and some strains have a glycine requirement that can be partially met by 84 glycolate, a byproduct of Prochlorococcus photorespiration [2,28]. Furthermore, evidence from 85 comparative genomics and metabolic modeling suggests that Prochlorococcus and SAR11 may 86 exchange glycolate, pyruvate, and malate through complementary and/or co-dependent 87 metabolic pathways, and it is likely that the evolution of these two organisms has been tightly 88 interwoven [29]. 4 bioRxiv preprint doi: https://doi.org/10.1101/460428; this version posted November 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 89 Despite the high abundances and frequently observed habitat overlap of Prochlorococcus 90 and SAR11 in the tropical and subtropical oligotrophic ocean, a system for co-culturing and 91 exploring interactions between members of these two model groups has remained elusive, 92 largely due to their cryptic growth requirements and inherent challenges in maintaining 93 laboratory isolates. Here we report the development of stable (over two year) co-cultures of 94 Prochlorococcus and SAR11, and explorations into the interrelated growth dynamics of these 95 groups. Specifically, we sought to determine if Prochlorococcus can provide SAR11 with 96 individual growth requirements and how SAR11/Prochlorococcus co-culture dynamics compare 97 with the co-culture of Prochlorococcus and a suite of sympatric copiotrophic bacteria. Given the 98 high abundance of these two groups throughout the oligotrophic surface ocean, these co-cultures 99 establish a novel and ecologically significant model system for the study of marine microbial 100 ecology. 101 102 Materials and methods 103 Biogeography analysis 104 The computational steps for determining the