1. Comparison of Synechococcus and Prochlorococcus photosynthetic pigments and cell size Characteristic Prochlorococcus Synechococcus Primary photosynthetic pigment divinyl chl-a, divinyl chl-b chl-a Phycobilisomesnoyes Accessory pigmentsphycoerythrin (+/-) chl-c-like pigment  -carotene zeaxanthin phycoerythrin phycourobilin/ phycoerythrobilin  -carotene zeaxanthin Cell diameter (µm)~ 0.6 ~ 1

3. Prochlorococcus : a model system for studying marine microbial ecology I Responsible for ~ 50% of total chlorophyll over a significant fraction of the world’s oceans It inhabits a relatively simple, well mixed environment that covers 70% of the earth It is relatively easy to isolate into culture and has minimal growth requirements It is widespread and abundant in the oceans, and is easily identified and studied in situ using flow cytometry

4. Prochlorococcus : a model system for studying marine microbial ecology II Its unique form of chlorophyll a allows measurement of its proportional contribution to photosynthetic biomass Its cell division is highly synchronised, simplifying measurements of in situ growth rates There is a rapidly growing molecular database for the genus, which facilitates the development of probes to study the distribution of different ecotypes in situ It has an extremely small genome size (1.8 -2.0 MBp)

5. Prochlorococcus specific primers TOTAL DNA PCR of 16S rRNA Oxygenic phototroph-biased primers Clone librariesDot-blot hybridisation DGGE SequencesRFLPQuantification of genotypes SINGLE CELLS Fluorescent In-Situ Hybridisation (FISH) Prochlorococcus genotype-specific probes GENETIC DIVERSITY P & N STATUS TOTAL PROTEINS SDS PAGE Western-blotting Interrogation with PstS/Amt antibodies Physiological status with respect to P & N for: Sequence diversity CELLS POPULATIONS Single-cell Immunofluorescence

6. nutrients light thermocline upwelling euphotic zone

7. nutrients light thermocline upwelling euphotic zone

8. Dot-blot hybridisation with Prochlorococcus genotype-specific oligonucleotides 10 30 40 50 60 70 90 110 Surface1 Surface2 Deep Mit9303 Sarg Eub338 E. coli Med Natl1 Tatl2 Mit9303 Sarg WH8103 DeepSurface1Surface2 Mit9303 Sarg Eub338 Depth m Control DNA Depth profile 2 37°N

9. Geographical and vertical distribution of Prochlorococcus 5 m 20 m 40 m 60 m 80 m 90 m 100 m 120 m 150 m 300 m HLIHLIILLSS120EUB338 10 m 30 m 40 m 50 m 60 m 70 m 90 m 110 m HLIHLIILLSS120EUB338 Eastern North AtlanticSargasso Sea

10. Depth profile 1 37°N Med Natl2A Sarg Tatl2 10 20 30 40 50 60 70 m M+P+ S+T2 Denaturing Gradient Gel Electrophoresis (DGGE) 36% constant denaturant Thermocline 0 5 10 15 20 25 0102030405060708090 Depth m Temperature °C 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Chlorophyll mg/m 3

11. Med Natl2A SargTatl2 Tatl1 Natl1 10 30 40 50 60 70 90 110 m Depth profile 2 37°N 36% constant denaturant Thermocline 0 5 10 15 20 25 0102030405060708090100110 Depth m Temperature °C 0 0.05 0.1 0.15 0.2 0.25 0.3 Chlorophyll mg/m 3

12. Flow cytometry data at 37°N, 20°W 10304050607090110 0 50000 100000 150000 200000 250000 Cells ml 10304050607090110 Depth (m) Total picoplanktonSynechococcusProchlorococcusPicoeukaryotes

13. FISH analysis of natural Prochlorococcus populations North Atlantic –positive signals with HLI and LL Depth (m)Proportion of DAPI stained cells giving a signal with each probe (%) 645HLI181LLCYA664 322<123 4020<123 8001314 Red Sea –positive signals with HLII

14. MED4 SS120 chlb 2 /a 2 ratio low (0.05 -0.15) high (0.4 -2.4) optimal growth irradiance 15-80  mol photons m -2 s -1 8-30  mol photons m -2 s -1** major antenna apoproteins ~ 32.5 kDa 34-28 kDa copies of pcb gene single multiple (7) phycoerythrin absent present P inducible proteinpresent absent growth on nitrate no yes(?) * photoinhibited only around 450  mol photons m -2 s -1 ** photinhibited at light intensities greater than 37  mol photons m -2 s -1 N.B. MED and SS120 genomes appear to be co-linear; 16S rDNA identity = 98.3% Comparison of physiological properties of Prochlorococcus strains MED4 (HLI) and SS120 (LL)

15. Conclusions Distribution of Prochlorococcus genotypes is dependent on hydrological conditions and oceanic region Molecular techniques e.g. DGGE, dot-blots, or FISH in combination with TSA, allows the community structure of natural populations to be rapidly evaluated Niche adaptation of specific strains (species?) potentially involves a response to both gradients of light and nutrients

16. Future perspectives Determination of carbon fixation potential of distinct Prochlorococcus genotypes in situ Correlation of genotype and phenotype with hydrological properties and nutrients - optimisation of single-cell IF assay - analysis of FISH and single-cell IF assays with flow cytometry Comparative genome analysis of HL and LL strains : what are the specific adaptations of these strains to their niche?

17. Acknowledgments Nyree West FISHWilli Schönhuber Rudi Amann, Rosi Rippka N.Atlantic samplesMike Zubkov Red Sea samplesAnton Post, Nick Fuller Sargasso samplesJames Ammerman Royal Society