SUMMARY The minute photosynthetic prokaryote Prochlorococcus, which was discovered about 10 years ago, has proven exceptional from several standpoints. Its tiny size (0.5 to 0.7 μm in diameter) makes it the smallest known photosynthetic organism. Its ubiquity within the 40°S to 40°N latitudinal band of oceans and its occurrence at high density from the surface down to depths of 200 m make it presumably the most abundant photosynthetic organism on Earth. Prochlorococcus typically divides once a day in the subsurface layer of oligotrophic areas, where it dominates the photosynthetic biomass. It also possesses a remarkable pigment complement which includes divinyl derivatives of chlorophyll a (Chl a) and Chl b, the so-called Chl a2 and Chl b2, and, in some strains, small amounts of a new type of phycoerythrin. Phylogenetically, Prochlorococcus has also proven fascinating. Recent studies suggest that it evolved from an ancestral cyanobacterium by reducing its cell and genome sizes and by recruiting a protein originally synthesized under conditions of iron depletion to build a reduced antenna system as a replacement for large phycobilisomes. Environmental constraints clearly played a predominant role in Prochlorococcus evolution. Its tiny size is an advantage for its adaptation to nutrient-deprived environments. Furthermore, genetically distinct ecotypes, with different antenna systems and ecophysiological characteristics, are present at depth and in surface waters. This vertical species variation has allowed Prochlorococcus to adapt to the natural light gradient occurring in the upper layer of oceans. The present review critically assesses the basic knowledge acquired about Prochlorococcus both in the ocean and in the laboratory.

The novel dye SYBR Green I binds specifically to nucleic acids and can be excited by blue light (488-nm wavelength). Cell concentrations of prokaryotes measured in marine samples with this dye on a low-cost compact flow cytometer are comparable to those obtained with the UV-excited stain Hoechst 33342 (bisbenzimide) on an expensive flow cytometer with a water-cooled laser. In contrast to TOTO-1 and TO-PRO-1, SYBR Green I has the advantage of clearly discriminating both heterotrophic bacteria and autotrophic Prochlorococcus cells, even in oligotrophic waters. As with TOTO-1 and TO-PRO-1, two groups of heterotrophic bacteria (B-I and B-II-like types) can be distinguished. Moreover, the resolution of DNA distribution obtained with SYBR Green I is similar to that obtained with Hoechst 33342 and permits the analysis of the cell cycle of photosynthetic prokaryotes over the whole water column.

For many years, a small but dedicated group of scientists have been using flow cytometry for the evaluation of marine microorganisms. One of these scientists now provides us with a detailed series of protocols in this area, spelling out the variations in method and instrument operation that are crucial to the successful extraction of quality flow data from marine organisms. In addition, the use of a number of less frequently employed fluorescent probes gives some insight into alternative staining procedures. As our collection of microbiologically oriented techniques increases, this knowledge database becomes invaluable.

Prochlorococcus marinus. The purification and properties of the axenic strain PCC 9511, derived from the same primary culture (SARG) as the type species, are reported here. Prochlorococcus PCC 9511 differs from the latter in possessing horseshoeshaped thylakoids, exhibiting a low chlorophyll b 2 content and lacking phycoerythrin, but shares these phenotypic properties with Prochlorococcus strain CCMP 1378. This relationship was confirmed by 16S rRNA sequence analyses, which clearly demonstrated that the axenic isolate is not co-identic with the nomenclatural type. Strain PCC 9511 has a low mean DNA base composition (32 mol % GMC) and harbours the smallest genome of all known oxyphotobacteria (genome complexity 13 GDa l 2 Mbp). Urea and ammonia are the preferred sources of nitrogen for growth, whereas nitrate is not utilized. Several different organic phosphorus compounds efficiently replace phosphate in the culture medium, indicative of ecto-phosphohydrolase activity. In order to distinguish strain PCC 9511 from the nomenclatural type, a new subspecies is proposed, Prochlorococcus marinus Chisholm et al. 1992 subsp. pastoris subsp. nov. This paper is dedicated in gratitude to Professor Germaine Cohen-Bazire on the occasion of her 80th birthday. Together with her late husband, Professor R. Y. Stanier, Germaine gave the members of the Physiologie Microbienne (Institute Pasteur, Paris) generous scientific guidance and spiritual support over many years (1971)(1972)(1973)(1974)(1975)(1976)(1977)(1978)(1979)(1980)(1981)(1982)(1983)(1984)(1985)(1986)(1987)(1988) Abbreviations : chl, chlorophyll ; HL, high light ; LL, low light ; PPFD, photosynthetic photon flux density ; PE, phycoerythrin ; T m , temperature mid-point of denaturation. Keywords :The GenBank accession numbers for the 16S rRNA sequences of PCC 9511, CCMP 1426 and NATL1 are AF180967, AF133833 and AF133834, respectively. INTRODUCTIONIn the last edition of Bergey's Manual of Systematic Bacteriology, the class Oxyphotobacteria was subdivided into the Cyanobacteria, whose ordinal recognition still awaits validation, and the order Prochlorales Lewin 1977(Castenholz & Waterbury, 1989. In the light, like algae and plants, these photosynthetic prokaryotes use H # O for the generation of chemical energy and reducing power and liberate molecular O # as a by-product. The demand for cellular carbon is met by CO # fixation. Cyanobacteria synthesize monovinyl chlorophyll a (chl a " ) and harvest light by aid of watersoluble multimeric complexes, the phycobilisomes, composed of biliproteins and linker polypeptides R. Rippka and others (Glazer, 1987(Glazer, , 1989Sidler, 1994). In contrast, oxyphotobacteria of the order Prochlorales Lewin 1977 either lack phycobiliproteins entirely, or synthesize only trace amounts (Lewin, 1977(Lewin, , 1989 BurgerWiersma et al., 1986 ;Chisholm et al., 1992 ;Hess et al., 1996). Their light-harvesting complexes are membrane-associated proteins containing chlorophyll a (a " or a # ) and chlorophyll b (b " or b # ) as the major photosyntheti...