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

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

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 ( MBp)

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

nutrients light thermocline upwelling euphotic zone

nutrients light thermocline upwelling euphotic zone

Dot-blot hybridisation with Prochlorococcus genotype-specific oligonucleotides 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

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 HLIHLIILLSS120EUB m 30 m 40 m 50 m 60 m 70 m 90 m 110 m HLIHLIILLSS120EUB338 Eastern North AtlanticSargasso Sea

Depth profile 1 37°N Med Natl2A Sarg Tatl m M+P+ S+T2 Denaturing Gradient Gel Electrophoresis (DGGE) 36% constant denaturant Thermocline Depth m Temperature °C Chlorophyll mg/m 3

Med Natl2A SargTatl2 Tatl1 Natl m Depth profile 2 37°N 36% constant denaturant Thermocline Depth m Temperature °C Chlorophyll mg/m 3

Flow cytometry data at 37°N, 20°W Cells ml Depth (m) Total picoplanktonSynechococcusProchlorococcusPicoeukaryotes

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 (%) 645HLI181LLCYA < < Red Sea –positive signals with HLII

MED4 SS120 chlb 2 /a 2 ratio low ( ) high ( ) optimal growth irradiance  mol photons m -2 s  mol photons m -2 s -1** major antenna apoproteins ~ 32.5 kDa 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)

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

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?

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