SUPPLEMENTARY INFORMATION

Transcription

1 Supplementary Table 1. Quantity of phosphorus spared via phospholipid substitutions by picocyanobacteria. (1 6 P atoms cell -1 )* (% of P in DNA) Synechococcus WH812.7 ±.1 15 ± 2 Synechococcus WH ±.5 57 ± 11 Synechococcus WH ±.3 86 ± 5 Prochlorococcus MED4.3 ± <.1 1 ± <1 Data are mean ± s.d. * Difference in the per cell concentrations of phosphorus in phosphatidylglycerol (PG) between cultures grown under phosphorus-replete and phosphorus-limited conditions. Difference expressed as a percentage of the phosphorus in one copy of double-stranded genomic DNA as calculated from the genome sizes for these organisms; genome size data were obtained from either the DOE Joint Genome Institute website or published papers 31,

2 Supplementary Table 2. Media used in phosphorus-replete and -limited cultures. Medium Phosphate concentration in replete media* Phosphate concentration in depleted media Refs. Synechococcus SN 91 µmol L -1 1 µmol L Prochlorococcus AMP1 5 µmol L µmol L Crocosphaera SN 45 µmol L Trichodesmium RMP 8 µmol L Thalassiosira & Chaetoceros f/2 + Si 36 µmol L -1 1 µmol L Emiliania f/2 36 µmol L -1 1 µmol L Pelagibacter SAR11 1 nmol L -1 N.A. 37 * The nitrogen to phosphorus ratio (N:P) of the standard media used in the phosphorusreplete cultures was approximately 16:1; with the exception of phosphate, the concentrations of all salts and nutrients were identical in the phosphorus-replete and - depleted media. Added to Sargasso seawater base. Added to Vineyard Sound seawater base. Added to North Pacific subtropical gyre seawater base. N.A. not applicable. 2

3 Supplementary Figure 1. Structures of phosphatidylglycerol (PG), sulfoquinovosyldiacylglycerol (SQDG), phosphatidylcholine (PC), and the betaine lipids (BLs) diacylglyceryl hydroxymethyl-trimethyl-β-alanine (DGTA), diacylglyceryl trimethylhomoserine (DGTS) and diacylglyceryl carboxyhydroxymethylcholine (DGCC). R indicates the positions of acyl (i.e. fatty acid) groups. Arrows indicate direction of substitution under conditions of phosphorus starvation. In planktonic communities of the Sargasso Sea, DGTS and DGTA were approximately equally abundant and together composed 8-9% of the total BLs with DGCC composing the remaining 1 2%. In the South Pacific, DGTS and DGTA were also approximately equally abundant but DGCC composed <1%. E. huxleyi contained DGTS but was dominated by DGCC. DGTA was the exclusive BL in C. affinis and DGCC the exclusive BL in T. pseudonana. 3

4 Supplementary Figure 2. Base peak HPLC/MS chromatograms of lipid extracts from phosphorus-replete (top) and phosphorus-limited (middle) cultures of Roseobacter sp. strain COL2P 38. Aside from the SAR11 clade, the Roseobacter genus is one of the most abundant groups of heterotrophic bacteria in the open ocean 39. Like many Roseobacter spp. this particular strain is capable of aerobic anoxygenic photosynthesis, but is still obligately heterotrophic. Phosphatidylglycerol (PG) was the primary phospholipid in this organism, but when limited by phosphorus ornithine lipids (OLs) became abundant. However, unlike all of the phytoplankton we examined, there was no concomitant decrease in the per cell abundance of phospholipids. Instead the OLs appeared to 4

5 contribute to a general increase in the size of the cells as indicated by the inset photomicrographs (scale bar 1 µm). So in this particular example, OLs did not act as a substitute lipid, which agrees with previous results suggesting that OLs may not be as effective substitute lipids as other non-phosphorus lipids. For example, Geiger et al 4 showed in Sinorhizobium meliloti that betaine lipid (BL) synthesis and not OL synthesis was controlled by the pho genes that regulate the response to phosphorus-limitation. Note that unlike all of the other membrane lipids discussed in this paper, OLs are not glycerol lipids (bottom). We emphasize that we did not detect OLs in either the Sargasso Sea water column or regrowth incubations even though we were clearly able to detect them with our HPLC/MS method (top and middle). 5

6 1 PO 4 3- concentration (nmol L -1 ) Incubation A Incubation B Incubation C Phospholipid concentration (ng L -1 ) PG Incubation A PE Incubation A PG Incubation B PE Incubation B PG Incubation C PE Incubation C Time (d) Supplementary Figure 3. Plot of phosphate (top) and phospholipid (bottom) concentrations as a function of time in replicate (A,B, and C) regrowth incubations. These data show that phospholipid concentrations increased even though phosphate was becoming scarcer during the course of the incubations. PG = phosphatidylglycerol; PE = phosphatidylethanolamine. 6

7 3 Substitute lipid concentration (ng L -1 ) SQDG Incubation A SQDG Incubation B SQDG Incubation C Substitute lipid concentration (ng L -1 ) DGTS Incubation A DGTA Incubation A DGCC Incubation A DGTS Incubation B DGTA Incubation B DGCC Incubation B DGTS Incubation C DGTA Incubation C DGCC Incubation C Time (d) Supplementary Figure 4. Plot of sulfoquinovosyldiacylglycerol (SQDG; top) and betaine lipid (BL, bottom) concentrations as a function of time in replicate (A,B, and C) regrowth incubations. These data show that concentrations of SQDG and BLs from phytoplankton in the inoculums remained constant or decreased with time, unlike phospholipids from heterotrophic bacteria (Suppl. Fig. 3). Ornithine lipids (OLs) remained undetectable in all of the regrowth incubations. For BL abbreviations and structures, see Suppl. Fig

8 Cell concentration (x 1 3 cells ml -1 ) Prochlorococcus Synechococcus Picoeukaryotes Nanoeukaryotes Heterotrophic bacteria 2.5 PG + PE concentration (atoms P cell -1 ) Time (d) Supplementary Figure 5. Plot of cell concentrations (top) and cellular phospholipid contents (bottom) as a function of time in regrowth incubation A. These data show that only heterotrophic bacterial cells grew during the incubations, and that the cellular phospholipid contents of these cells remained relatively stable even though phosphate concentrations were decreasing (Suppl. Fig. 3). The dashed line represents the cellular phospholipid contents of cultured P. ubique (see main text). 8

9 References for Supplementary Information. 31. Palenik, B. & others, a. The genome of a motile marine Synechococcus. Nature 424, (23). 32. Rocap, G. et al. Genome divergence in two Prochlorococcus ecotypes reflects oceanic niche differentiation. Nature 424, (23). 33. Waterbury, J. B., Watson, S. W., Valois, F. W. & Franks, D. G. in Photosynthetic Picoplankton (eds. Platt, T. & Li, W. K. W.) (Canadian Department of Fisheries and Oceans, Ottawa, 1986). 34. Moore, L. R. et al. Culturing the marine cyanobacterium Prochlorococcus. Limnol. Oceanogr. Meth. 5, (27). 35. Webb, E. A., Moffett, J. W. & Waterbury, J. B. Iron Stress in Open Ocean Cyanobacteria (Synechococcus, Trichodesmium, and Crocosphaera): Identification of the IdiA protein. Appl. Environ. Microbiol. 67, (21). 36. Guillard, R. R. L. in Culture of marine invertebrate animals (eds. Smith, W. C. & Chanley, M. H.) 29-6 (Plenum, New York, 1975). 37. Rappé, M. S., Connon, S. A., Vergin, K. L. & Giovannoni, J. Cultivation of ubiquitous SAR11 marine bacterioplankton clade. Nature 418, (22). 38. Rontani, J.-F., Christodoulou, S. & Koblizek, M. GC-MS stuctural characterization of fatty acids from marine aerobic anoxygenic phototrophic bacteria. Lipids 4, (25). 39. Moran, M. A. et al. Genome sequence of Silicibacter pomeroyi reveals adaptations to the marine environment. Nature 432, (24). 4. Geiger, O., Röhrs, V., Weissenmayer, B., Finan, T. M. & Thomas-Oates, J. E. The regulator gene phob mediates phosphate stress-controlled synthesis of the membrane lipid dacylglycerol-n,n,n-trimethylhomoserine in Rhozobium (Sinorhizobium) meliloti. Molec. Microbiol. 32, (1999). 9