INTRODUCTION

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The large body of work achieved since the discovery about 10 years ago of the minute and ubiquitous photosynthetic prokaryote Prochlorococcus (20, 21) has changed our view of the community structure of oceanic picoplankton. It also has implications in fields as different as the phylogeny of cyanobacteria and photosynthesis. Prochlorococcus has proven exceptional from several standpoints.

(i) The tiny size of Prochlorococcus (equivalent spherical diameter in culture, 0.5 to 0.7 µm [110]) makes it the smallest known photosynthetic organism, having the lowest predictable size for an O₂ evolver (136). The discovery and first field studies of this organism were made possible only by the use of sensitive flow cytometers onboard research vessels (21, 88, 116). Since then, the development of procedures to fix and preserve picoplanktonic cells has allowed cells to be transported to the laboratory for analysis (104, 173). As a result, the number of studies of picoplankton including Prochlorococcus has steadily increased. The ubiquity of this organism within the 40°S to 40°N latitudinal band, its high density, and its occupation of a 100- to 200-m-deep layer make it the most abundant photosynthetic organism in the ocean and presumably on Earth.

(ii) Prochlorococcus 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 (41), that are unique to this genus. Recent developments of high-performance liquid chromatography (HPLC) (42) and spectrofluorimetric techniques (114) have made it possible to identify these pigments in natural assemblages and therefore to assess precisely the contribution of Prochlorococcus to the total planktonic photosynthetic biomass. Another peculiarity of its photosynthetic apparatus is the presence of light-harvesting complexes which are similar in function but not in structure to those of higher plants or green algae (77). In some strains, there are also small amounts of a particular type of phycoerythrin (55). The combination of Chl a, Chl b, and at least one phycobiliprotein is a unique trait among oxygen-evolving phototrophs.

(iii) In subtropical oligotrophic areas of the Atlantic and Pacific Oceans, the vertical distribution of Prochlorococcus often exceeds the boundaries of the euphotic layer (i.e., the part of the water column extending from the surface to the depth that receives 1% of the surface irradiance). Thus, cells of this genus seem to be able to sustain growth and photosynthesis over an irradiance range extending for more than 3 orders of magnitude. This raises the question whether natural Prochlorococcus populations exhibit an outstanding ability for photoacclimation or whether other processes such as the occurrence of different Prochlorococcus species or pigment types along the vertical light gradient are implicated in this intriguing phenomenon. Recent biochemical studies (132) suggest that different Prochlorococcus strains may have different antenna systems specifically adapted to the light environment from which they have been isolated.

(iv) Prochlorococcus typically divides once a day in the subsurface layer of oligotrophic areas, such as the central oceanic gyres (94), where it dominates the photosynthetic biomass (16). In these environments, where nutrients such as nitrogen are extremely scarce, Prochlorococcus has an obvious advantage with respect to uptake because of its high surface/volume ratio resulting from its very small cell size (19). However, it must also possess either unusually small nutrient requirements (at least compared to other phytoplankton) or an extremely efficient capacity to scavenge elements recycled by heterotrophic bacteria.

(v) The remarkable diversity of Prochlorococcus observed both among laboratory isolates (145, 166) and within field populations (124) raises questions about the role of genetic variability in the success of Prochlorococcus in the field. One may wonder how environmental constraints, such as the absence of iron in oligotrophic areas, conditioned the evolution of Prochlorococcus over geologic timescales from the ancestor it shares with the cyanobacteria.

This review critically assesses the basic knowledge acquired about Prochlorococcus both in the ocean and in the laboratory since its recent discovery. It clearly indicates that some research areas, such as the ecological distribution, photosynthetic capacities, and phylogeny of Prochlorococcus, are well ahead of others, in particular nutrient acquisition, which need to be developed.

CHARACTERIZATION AND CULTIVATION

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Methods of Characterization

The first published records of Prochlorococcus in nature are the electron microscopy sections of "type II" cells from marine samples reported in the paper by Johnson and Sieburth (63). They revealed what is now known as the typical ultrastructure of Prochlorococcus (Fig. 1A). Although these authors clearly suggested that the cells they observed were probably devoid of phycoerythrin (63), Guillard et al. mistakenly identified them as Synechococcus (47), the other coccoid photosynthetic prokaryote discovered in abundance in marine waters around that time (184). The second published historical record came indirectly in 1983 from the discovery of an unknown "red-shifted" Chl a derivative in subtropical Atlantic waters (37) (Fig. 1B). In fact, years later, Gieskes (36) revealed that in 1977 he had found both red-shifted Chl a and Chl b in these waters by using thin layer chromatography. Prochlorococcus was clearly identified only once flow cytometry was used (21). During a cruise out of Barbados in 1985, Rob Olson and Ginger Armbrust were the first to visualize in a deep sample a new population of red-fluorescing particles, dimmer than Synechococcus (117). The finding that these cells possessed an unusual divinyl derivative of Chl a and the visualization of their ultrastructure led to the announcement in 1988 of the discovery of Prochlorococcus by Chisholm et al. (21). By then, other workers had began to record Prochlorococcus either by flow cytometry or by epifluorescence microscopy (88, 116).

View larger version (69K): [in this window] [in a new window]   FIG. 1.   First records of the occurrence of Prochlorococcus. (A) Electron microscope photograph of "type II cells" from deep samples of the North Atlantic ocean. Reprinted from reference 63 with permission of the publisher. ce, cell envelope; pb, polyhedral bodies; th, thylakoids. Scale bar, 0.5 µm. (B) Analysis of a pigment extract obtained by normal-phase HPLC showing the "unknown Chl a derivative," indicated by a star. Reprinted from reference 37 with permission of the publisher.

Since the late 1980s, many observations on Prochlorococcus in natural waters have been made. Most of these observations were obtained by flow cytometry on either live (Fig. 2) or preserved samples (99, 129, 173). One disadvantage of most commercially available flow cytometers is their inability to completely resolve surface populations from background noise in very oligotrophic surface waters, due to the very low Chl fluorescence of surface Prochlorococcus. This remains a serious problem for water column studies in areas where Prochlorococcus dominates the phytoplankton community. Two possible solutions to this problem have been put forward. The first requires the use of very high laser power (above 1 W) and modified optics on old instruments equipped with water-cooled lasers (119). Such bulky instruments are not very convenient for field work. The second possibility involves modifying the optics of the small instruments equipped with air-cooled lasers to focus laser beam more narrowly and increase the excitation power (28).

View larger version (31K): [in this window] [in a new window]   FIG. 2.   Analysis of natural Prochlorococcus populations by flow cytometry. (Top) Side scatter (a function of cell size) plotted against red fluorescence of chlorophyll. (Bottom) Orange fluorescence of phycoerythrin plotted against red fluorescence of chlorophyll. All scales are 4-decades logarithmic from 1 to 10,000 (arbitrary units). (Left) Typical surface-layer sample (45 m, deep Equatorial Pacific, 7°S, 150°W, collected 10 November 94). Synechococcus is easily distinguished from Prochlorococcus by its orange phycoerythrin fluorescence. (Middle) Typical deep sample (105 m deep, Equatorial Pacific, 7°S, 150°W, collected 10 November 94). Synechococcus is virtually absent, and the chlorophyll fluorescence of Prochlorococcus is much higher than near the surface (compare the fluorescence of Prochlorococcus and that of the standard beads). Note also the weak orange fluorescence displayed by Prochlorococcus at this depth. (Right) Example of two Prochlorococcus populations coexisting at the same depth (80 m deep, Mediterranean Sea, 37°5'N, 16°52'E, collected 20 June 1996).

Alternative methods to quantify the Prochlorococcus cell population in the field include epifluorescence microscopy, electron microscopy, and pigment analysis. Although it has been claimed that Prochlorococcus abundance can be recorded by ordinary epifluorescence microscopy (60, 61, 88, 92), sophisticated image recording with, for example, a cooled charge-coupled device camera (158) is necessary to avoid severe underestimates of cell abundance, especially in surface waters, where the fluorescence is very low. For example, concentrations of Prochlorococcus in the Central Pacific estimated by epifluorescence microscopy (60) are twofold lower than those estimated by flow cytometry in the same region. Epifluorescence microscopy can, however, provide direct estimates of cell size, a key characteristic of oceanic biomass budgets (158). Because of its technical difficulty, electron microscopy has very seldom been used with marine samples (1, 21, 63). Finally, pigment analysis can be used to routinely detect Prochlorococcus in the field. Although it has been possible to discriminate Chl a₂ from its monovinyl counterpart by direct-phase HPLC analyses for quite a long time (37), a major step forward came with the ability to separate these pigments by reverse-phase HPLC, the most commonly used oceanographic HPLC technique, which also allows reliable separation of Chl b₁ and b₂ (see, e.g., references 3, 42, 170, and 182). Moreover, HPLC analysis offers the ability to measure the growth rate of Prochlorococcus by monitoring the incorporation of ¹⁴C into Chl a₂ (see "Growth rates and loss processes in the ocean" below). Spectrofluorometry, a technique that is hardly ever used in oceanography, offers a very attractive alternative to detect divinyl Chls, especially because of its sensitivity, speed, and ease of implementation (114).

Available cultures and isolation methods. The first Prochlorococcus strain was isolated by Palenik in May 1988 from the bottom of the euphotic zone in the Sargasso Sea (depth, 120 m). It was initially dubbed LG (125), for "Little Greens," but was then renamed SARG for its geographical origin (131). Since then, several strains have been isolated both at the surface and at depth from various sites in oceans around the world (Table 1 and Fig. 3). Strains are now available from most areas where Prochlorococcus populations have been sampled.

View larger version (37K): [in this window] [in a new window]   FIG. 3.   Locations of some of the available Prochlorococcus strains (see Table 1).

Optimal growth medium. Chisholm et al. (20) showed that among the various media initially tested, seawater enriched with urea, -glycerophosphate, a minimum trace metal mix, and 100 µM CPTC (cis,cis,cis,cis-1,2,3,4-cyclopentanetetracarboxylic acid), a chelator, led to sustained growth of Prochlorococcus (Table 2). However, Prochlorococcus can also be adapted to a seawater-based medium containing only inorganic additions (20). To date, the most widely used culture media (PC, PRO2, and modified K/10-Cu [Table 2]) are derived from the K medium used for marine microalgae (69), with EDTA as the chelator, a 10-fold-diluted trace metal stock solution, and no copper (Table 2). The PRO2 medium proved to be very efficient for isolation purposes (105). Other media, such as PC with trace metals as in f/20 (48) or PCR-S11 (142) (Table 2), which use a modified "Gaffron" metal stock solution (141) have also been successful in tests for culturing. Maximum cell yields with these media are 2 × 10⁸ to 3 × 10⁸ cells ml¹, corresponding to a Chl a₂ yield of ca. 0.2 to 0.4 mg liter¹.

PHYSIOLOGY

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Basic Cellular Features

Ultrastructure. Because of their tiny size, Prochlorococcus cells are very difficult to identify by optical microscopy. They are hardly distinguishable from small heterotrophic bacteria, except for their very weak Chl fluorescence. Transmission electron microscopy reveals typical cyanobacterial architecture (Fig. 4), which is best compared to that of the other abundant oceanic photosynthetic prokaryote, Synechococcus. However, the Prochlorococcus cell is distinctly elongated (Fig. 4A) whereas the Synechococcus cell is much more spherical (see 21, 63, 158). In some cases, the membrane appears fairly electron dense, but it is not known whether this is a consequence of environmental conditions or if it is strain specific. The cytoplasm contains DNA fibrils, carboxysomes that can be labeled with an antibody against ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and glycogen granules, located near or between thylakoids (90). In cross-sections, there are generally between two and four thylakoids, and there are sometimes up to six (157). They run parallel to the cell membrane (Fig. 4) and are much more appressed than in Synechococcus. In Prochlorococcus marinus type strain (SARG) and in most cells from natural populations, they are closed. In contrast, in the surface isolate Prochlorococcus sp. strain MED (90) and in the deep isolate MIT 9313 (Fig. 4A), they are horseshoe shaped. Interestingly, Johnson and Sieburth (63) described a "type III" cell with noncircular thylakoids that looks somewhat like MED. No phycobilisomes are visible, but since phycoerythrin has been found in strains such as CCMP 1375 (55), immunocytochemistry would be useful to investigate its intracellular localization.

View larger version (72K): [in this window] [in a new window]   FIG. 4.   Electron micrographs of longitudinal and cross sections of Prochlorococcus strain MIT9313 showing tightly appressed thylakoids at the periphery of the cell. Scale bar, 0.1 µm. Unpublished photographs courtesy of C. Ting, J. King, and S. W. Chisholm.

Size and carbon content. Most methods used for cell sizing, such as electronic (Coulter) sizing and optical and electronic microscopy, exhibit a number of biases for such tiny objects as Prochlorococcus cells. The best estimates made on cultures give a range of 0.5 to 0.8 µm for length and 0.4 to 0.6 µm for width (90, 110). The cell size appears to vary with environmental conditions. For example, it was shown to increase from 0.45 to 0.75 µm between the surface and a depth of 150 m in the Sargasso Sea (158). Moreover, forward scatter measured by flow cytometry, a function of both size and refractive index, increases from dusk to dawn at the equator, a corollary of synchronized cell division (7, 174).

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Genome size and base composition. Existing data obtained on the axenic strain PCC 9511 either by DNA renaturation kinetics (143) or by flow cytometry (97) suggest a genome size of 1.9 to 2.0 Mbp. This value is within the size range of genomes of free-living eubacteria, which extends from 1.55 Mbp for Aquifex aeolicus (25) to 4.21 Mbp for Bacillus subtilis (71). Genome sizes have also been estimated for a variety of cyanobacteria (mainly freshwater) by renaturation kinetics (49). These range from 2.55 to 13.2 Mbp (1.66 × 10⁹ to 8.58 × 10⁹ Da (49; Catalogue of the Pasteur Culture Collection at http://www.pasteur.fr). For instance, the only sequenced cyanobacterial genome, Synechocystis strain PCC 6803 (67), is about twice as large (3.6 Mbp) as that of Prochlorococcus whereas Prochlorothrix hollandica has a genome size of 5.5 Mbp (149). Although more genome size data are required for marine cyanobacteria, it seems that Prochlorococcus possesses the smallest genome of all prokaryotes evolving oxygen (or oxyphotobacteria; see "Phylogeny" below). Some of the genetic characteristics of P. marinus SS120, such as the presence of only one copy of psbA (57), overlapping genes in the dihydrodipicolinate synthase operon (95), or overlapping promoter regions (53), are consistent with the finding of a relatively compact genome in this microorganism. The genes sequenced to date in Prochlorococcus are listed in Table 3.

Pigment composition. The presence of Chl a₂ and Chl b₂ is a trait common to all Prochlorococcus strains characterized to date (41, 107, 131, 166). Besides Prochlorococcus, Chl a₂ (but not Chl b₂) has been observed only in a mutant of corn (5), although divinyl derivatives of chlorophyll(ide) are probable intermediates in the biosynthesis of Chl in higher plants (126). Both Chl a₂ and Chl b₂ have absorption and fluorescence excitation maxima in the blue part of the visible spectrum red-shifted by 8 to 10 nm compared to their monovinyl counterparts (107, 110). The other pigments of Prochlorococcus, which include zeaxanthin, -carotene, and small amounts of a Chl c-like pigment (Mg,3-8 divinyl phaeoporphyrin a₅), are shared with a limited number of other phytoplanktonic groups, including chlorophytes, cryptomonads, and cyanobacteria (41). It is noteworthy that natural populations of Prochlorococcus from suboxic waters of the Arabian Sea also possess a novel 7,8-dihydro derivative of zeaxanthin, probably parasiloxanthin, which is absent in cultured strains (40). One notable peculiarity of Prochlorococcus is the dramatic difference in pigment ratios among different isolates. For example, several isolates, notably SARG, have a Chl b₂/Chl a₂ ratio equal to or higher than 1 whereas other isolates display much lower ratios, with the MED strain exhibiting the lowest (0.13 [105-107, 131]). At least three isolates (MIT9302, MIT9312, and SARG and its clonal derivative SS120 [Table 1]) also synthesize normal (monovinyl) Chl b (or Chl b₁) when grown under high light conditions (105, 107, 131), suggesting that this light condition may trigger the expression of enzymes which can transform, probably in a single step, Chl b₂ into Chl b₁ (5, 131, 144). Surprisingly, although these enzymes should also be able to allow the transformation of Chl a₂ in Chl a₁, no Chl a₁ has been detected in Prochlorococcus. Another difference between these strains is the presence in SS120 but not in MED4 of a novel type of phycoerythrin (55). Because of its low concentration and the fact that classical pigment HPLC analyses do not detect phycobiliproteins, this pigment remained undetected for a long time. It also was overlooked by researchers making absorption measurements, because the major phycobilin associated with this phycoerythrin is phycourobilin. Its absorption maximum at 495 nm is very close to that of the very abundant Chl b₂ (480 nm), and thus they cannot be discriminated by their absorption spectra. It was only after the discovery of the phycoerythrin gene that Hess et al. (55) examined water-soluble fractions by spectrofluorimetry and found evidence of phycobilins.

Photosynthetic performances. In a study with the MED and SARG isolates (131), the observed ranges of assimilation rates, expressed per Chl unit, at various growth irradiances were similar between these strains (1.5 to 4.8 and 1.4 to 5.6 fg of C fg of Chl¹ h¹ for MED and SARG, respectively), but expressed per cell, they were almost constant for MED (4.9 to 5.8 fg of C cell¹ h¹) and more variable for SARG (2.8 to 6.2 fg of C cell¹ h¹). However, light-saturated carbon fixation rates (P_m^Chl or P_m^cell [m stands for maximum]) were found to vary significantly between strains grown under similar irradiances (131). A more recent study confirmed that differences in pigmentation among isolates correlate with differing photosynthetic efficiencies: when grown at 9 µmol quanta m² s¹, two Prochlorococcus isolates (MIT9303 and MIT9313) with high ratios of Chl b₂ to Chl a₂ (>1.1) had a significantly higher P^Chl_m than did two other isolates (MIT9302 and MIT9312) with Chl b₂/Chl a₂ ratios lower by a factor of 2 (2.4 and 1.8 fg of C fg of Chl¹ h¹) (105). Laboratory values also compare fairly well with data on natural Prochlorococcus populations from the Moroccan upwelling (0.6 to 4 fg of C cell¹ h¹), obtained by labeling a natural seawater sample with ¹⁴C and sorting individual Prochlorococcus cells by flow cytometry (82). However, photosynthetic rates measured by the same method for Prochlorococcus cells at the base of the euphotic zone in the open ocean were significantly lower (0.03 to 0.3 fg of C cell¹ h¹). This method also allowed us to estimate the fraction of the total phytoplanktonic production attributable to Prochlorococcus, which varied from 11 to 57% (82).

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View larger version (27K): [in this window] [in a new window]   FIG. 5.   Vertical distributions from bright and dim Prochlorococcus populations in the subtropical Pacific Ocean off Hawaii. The insert represents the vertical profile of side scatter and red chlorophyll fluorescence of the total Prochlorococcus population measured by flow cytometry. Adapted from reference 17 with permission of the publisher.

Photosynthetic apparatus. One basic question raised by the observation of different Prochlorococcus pigment types both in the field (17, 129) and in culture (107, 110, 131) is whether strains or populations representative of those found near the surface and those found at the bottom of the euphotic zone have not only different pigmentation and ecophysiological features but also structurally different photosynthetic apparatuses (Fig. 6). This question was addressed through the biochemical characterization of the pigment complexes of clones MED4 and SS120 (132). Since these strains differed mainly in their ratios of Chl b₂ to Chl a₂ (even at a given growth irradiance; see "Pigment composition" above) and since the majority of Chl b is located in antenna systems (46), the most striking structural difference between strains was expected to be found at the level of their major light-harvesting complexes. Proteins constituting these antenna complexes have apparent molecular masses on denaturing electrophoresis gels of 32.5 and 34 to 38 kDa in MED4 and SS120, respectively. Moreover, they are more abundant (at low light intensities), their relative amount varies more with irradiance, and they bind ca. 7 times as much Chl b₂ in SS120 as in MED4 (132). Sequencing of the pcb genes, which encode these antenna proteins, confirmed the large differences between strains, since polypeptides deduced from gene sequences have only 76% identity at the amino acid level (77). These polypeptides are, however, similar between MED4 and SS120 in length (352 and 351 amino acids, respectively), mass (44.9 and 44.6 kDa, respectively), and the presence of six putative transmembrane helices (insert in Fig. 6), suggesting that the discrepancy noticed between their apparent molecular masses resulted from posttranslational modifications, e.g., different levels of phosphorylation, affecting their migration properties in denaturing gels (132). The antenna proteins of Prochlorococcus (Pcb), as well as those of the two other "prochlorophytes" (Prochlorothrix and Prochloron; see "Phylogeny" below), are closely related to the IsiA proteins found in iron-stressed freshwater cyanobacteria, such as Synechococcus strain PCC 7942, and are probably derived from them (77). All these proteins belong to the same family of Chl-binding proteins as the psbC and the psbB gene products (CP43 and CP47), which are major constituents of the photosystem II (PS II) internal antenna in all oxygenic photosynthesizing organisms (77). Despite the progress made recently in the identification of "prochlorophyte" antenna proteins, major questions still remain to be answered. The presence in both Prochlorothrix and Prochloron of several pcb genes (77, 169) raises the question whether some or all Prochlorococcus strains might not also possess multiple pcb genes, since these genes could play an important role in the photoacclimation capacity of these organisms. Other important topics are the exact localization of Chl-binding residues within the Pcb amino acid sequences and their total Chl-binding capacity per molecule. Furthermore, state transitions, a characteristic of photosynthesis in many cyanobacteria (see reference 147 and references therein), and the possible role of Pcb-antenna complexes in this phenomenon, if it takes place in Prochlorococcus, might be specifically targeted in future studies.

View larger version (51K): [in this window] [in a new window]   FIG. 6.   Diagram showing the thylakoid proteins in Prochlorococcus and their putative organization by homology to the photosynthetic apparatus of cyanobacteria. The proteins whose genes have been sequenced partially or totally are shown in dark gray (see Table 3), and those which have been characterized only by immunoblotting (35, 90, 132) are shown in light gray. Although phycoerythrin is present in some strains such as SS120, it is not clear whether it is integrated in phycobilisomes, which, if present, would be very scarce. PS I is probably organized in trimers (35), but only one monomer is shown. The insert shows a detail of the Pcb protein which includes six transmembrane hydrophobic domains. For the electron transport chain, only the cytochrome b6-f complex is shown because the existence of other components (such as NADH dehydrogenase and plastoquinone) has not been demonstrated yet. Other probable components of the photosynthetic apparatus such as cytochrome oxidase or ATP synthase are not shown either, since they are still uncharacterized in Prochlorococcus. Abbreviations: CP, chlorophyll-protein complex; Cyt, cytochrome; OEC, oxygen evolving complex.

One of the most intriguing ecological characteristics of Prochlorococcus, besides its capacity to grow over a very wide range of irradiances in nature, is its ability to colonize extremely oligotrophic areas. For example, Prochlorococcus represents on average 73% of the photosynthetic carbon in the surface mixed layer off Hawaii (16). Under these conditions, its very small cell size and the resulting high surface-to-volume ratio are obvious adaptative advantages for nutrient uptake (19). However, very little is known about its basic physiological characteristics with respect to nutrient assimilation, partly because of the unavailability of axenic cultures, a gap only recently filled (143).

Most oceanic areas are assumed to be limited by nitrogen. In tropical areas, Prochlorococcus is present both in the surface layer, where presumably only reduced nitrogen forms are available, and in the deep chlorophyll maximum, where nitrates are present. However, in the Mediterranean Sea in winter, NO₃ addition was able to stimulate Prochlorococcus cell cycling (177), suggesting that some populations might be able to take up oxidized forms of nitrogen. These data raise the possibility that there are different physiological types of Prochlorococcus adapted to grow on different nutrient sources, a hypothesis consistent with the available information for marine Synechococus (185) and with the existence of genetically different Prochlorococcus (see "Genetic diversity" below). Nutrient uptake experiments must be performed with the axenic strain now available (143) and other forthcoming axenic strains more representative of deep ecotypes. It will also be very interesting to investigate whether all Prochlorococcus strains possess the components necessary for NO₃ uptake, such as the ntcA gene, whose product is involved in the activation of transcription of a set of genes required for the use of oxidized nitrogen sources (91).

Although nitrogen is the primary limiting nutrient in many oceanic areas, recent evidence points to phosphorus as limiting, either permanently, in areas such as the Mediterranean Sea, or transiently, in areas such as the Sargasso Sea (23, 162). Prochlorococcus is well represented in both areas, suggesting that it also has the ability to thrive under very low P concentrations. Parpais et al. (127) showed that Prochlorococcus becomes limited only at P-PO₄³ concentrations of the order of 30 nM. Although the initial medium used to isolate Prochlorococcus contained organic instead of mineral phosphorus (see "Optimal growth medium" above), Prochlorococcus can grow very well on the latter form. In fact, organic phosphorus is rapidly mineralized by the heterotrophic bacteria present in the culture (127). It is possible that a similar mechanism occurs in situ and provides natural Prochlorococcus populations with mineral P, which is consumed so quickly that it remains undetectable. It is worth noting that Prochlorococcus possesses a pstS gene encoding a phosphate-binding protein, that is expressed only under P depletion and may play a role in its adaptation to oligotrophy; however, this gene is not unique to this organism (146).

Iron is the third major nutrient recently implicated in the limitation of primary productivity in remote areas of the ocean not subjected to eolian inputs, such as the equatorial Pacific (101), as well as in more coastal areas (76). The low iron requirement of Prochlorococcus could be a key factor in its success in central oceanic areas. This is suggested in particular by the fact that the Prochlorococcus PS II antenna is similar to IsiA, a protein induced under iron stress in Synechococcus sp. strain PCC 7942 (77). However, field experiments suggest that natural populations of Prochlorococcus are somewhat iron limited (although much less so than larger cells such as diatoms), since the addition of iron induces increases in cell size and chlorophyll fluorescence (192). Clearly, future laboratory experiments should aim at a better understanding of the regulation of cellular processes by iron in Prochlorococcus and should search for the eventual presence of siderophores, as evidenced previously in a variety of marine and freshwater cyanobacteria (189).

Although little studied in photosynthetic prokaryotes, cell cycling, a key cellular process that coordinates growth, DNA replication, and cell division, has received some attention in Prochlorococcus because of its application to assessment of growth rate and population status with respect to nutrient limitation in the field (see "Growth rates and loss processes in the ocean" below). The Prochlorococcus cell cycle can be easily studied by flow cytometry after staining with a DNA-binding dye (98, 104). The Prochlorococcus cell cycle resembles that of eukaryotes, with a discrete DNA synthesis phase (S phase) and two well-defined G₁ and G₂ phases, in contrast to some strains of marine Synechococcus, which may have more than two genome copies (6). As in most phytoplankton species (171), light or nutrient deprivation arrests cells in the G₁ phase of the cell cycle. A notable exception occurs when phosphorus is depleted. In this case, cells are blocked in all phases of the cell cycle, including the DNA synthesis S phase. Cells arrested in S are not able to recover upon addition of fresh phosphorus, in contrast to cells arrested in G₁ and G₂ (127). Light-dark entrainment induces a very strong synchronization of the cell cycle both in cultures (150) and in nature (94, 129, 175). The S phase usually takes place in late afternoon, and division occurs during the early part of the night. Natural Prochlorococcus populations from the Arabian Sea, as well as two cultured strains, can divide more than once per photocycle while remaining highly synchronized (150). In such cases, two cohorts of dividing cells occur in rapid succession, the second with a G₁ duration as short as 1 h. It would be interesting to investigate whether Prochlorococcus can display behaviors typical of circadian clock control (e.g., free running in continuous light), as demonstrated for Synechococcus strain PCC 7942 (62). Indeed, S-phase initiation appears to be linked to a light-triggered timer (150).

The only gene related to the cell cycle that has fully been characterized in Prochlorococcus so far is dnaA. In most bacteria, the encoded protein, DnaA, is essential for initiating chromosomal replication recognizing short, asymmetric sequence elements, the DnaA boxes, near the origin of replication, oriC (159). In P. marinus CCMP 1375 (139), DnaA consists of 461 amino acids and has 63% identity in a 315-residue overlap to Synechocystis strain PCC 6803 DnaA (140). Among the more than 35 bacterial DnaA sequences available in data banks, those of Prochlorococcus and Synechocystis form a distinguishable subfamily. The C-terminal DNA-binding domain of the Prochlorococcus recombinant DnaA protein expressed in vitro recognizes and binds specifically the heterologous oriC from Bacillus subtilis and E. coli, suggesting that the primary structure of the DnaA boxes might be evolutionarily conserved in Prochlorococcus. In many bacteria, oriC is physically near the dnaA gene and DnaA boxes are frequently present in its promoter region, providing a basis for binding and autoregulation (159). Surprisingly, no putative DnaA box could be identified in the analyzed genomic region of 5,304 bp containing Prochlorococcus dnaA (139). However, this is consistent with a unique gene organization in Prochlorococcus, which is very different from the highly conserved gene order, rnpA-rpmH-dnaA-recF-gyrB, found in a wide variety of bacteria. It is worth noting that the cyanobacterium Synechocystis strain PCC 6803 also lacks a DnaA-binding box near dnaA and displays an unusual gene arrangement in this region (140). Surprisingly, knockout mutants of Synechocystis strain PCC 6803 grow as well as the wild type, suggesting that DnaA is not essential for this strain, in contrast to all known eubacteria (138).

ECOLOGY

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Oceanic Distribution

Prochlorococcus appears to have a very wide oceanic distribution. This is shown in Fig. 7, which is based on the analysis of more than 8,400 field measurements of Prochlorococcus made throughout the world oceans by flow cytometry (Table 5). Prochlorococcus is virtually ubiquitous in the latitudinal band extending from 40°N to 40°S (Fig. 7). It can still be found beyond 40°, but its concentrations decline fairly rapidly. The highest latitude where it has been recorded is 60°N off Iceland in the North Atlantic (11). This oceanic distribution suggests that low temperatures are lethal to Prochlorococcus, and this is confirmed by culture data (107). In the field, the lowest surface temperature at which Prochlorococcus is recorded is about 10°C (Fig. 8A). This contrasts strongly with that for Synechococcus, the other major marine unicellular cyanobacterium, which can be encountered, albeit at low concentrations, at temperatures as low as 2°C (151). There does not appear to be an upper temperature limit for Prochlorococcus distribution, since it is found in warm equatorial waters that reach 30°C at the surface. Still, the maximum integrated concentrations occur between 26 and 29°C and decrease above that temperature (Fig. 8A).

View larger version (38K): [in this window] [in a new window]   FIG. 7.   Concentrations Prochlorococcus integrated over the water column throughout the world oceans. Based on data from Table 5.

View larger version (32K): [in this window] [in a new window]   FIG. 8.   Relationship between Prochlorococcus integrated concentrations and surface temperature (A) and surface nitrate concentrations (B). Based on data from Table 5.

Although Prochlorococcus is most abundant in oligotrophic waters, both in absolute terms and relative to the other photosynthetic populations, it is by no means restricted to nutrient-depleted waters (Fig. 8B). In particular, it can be found in relatively coastal areas, such as the plume of the Rhone river in the Mediterranean Sea (178) or the Japanese waters of Suruga Bay (157). It has also been observed in the inner lagoons of Pacific atolls (18). Whether Prochlorococcus grows actively in such environments or is simply advected is an open question. In contrast, although it is often present in offshore temperate waters, it has never been observed in some temperate, permanently mixed shallow seas such as the English Channel (175). Very recently, it has been discovered in yet another niche, in secondary deep chlorophyll maxima situated below the oxycline in the Arabian Sea (40, 64). Still, vast areas of the world oceans remain uncharted (Fig. 7), especially in the southern hemisphere, although more data are now available from regions such as the Indian Ocean (Table 5), where large oceanographic surveys have been recently completed as part of the Joint Global Ocean Flux Study.

Vertical plots of the available measurements of Prochlorococcus and Synechococcus concentration by flow cytometry (Fig. 9) reveal the broad features that distinguish these two photosynthetic prokaryotes. On average Synechococcus concentrations are about 1 order of magnitude lower than Prochlorococcus concentrations, although their abundance maxima are very comparable. Maximum Prochlorococcus concentrations on the order of 700,000 cells ml¹ have been recorded in the Arabian Sea (14). Clearly, Prochlorococcus extends much deeper than Synechococcus, since the latter disappears virtually beyond 100 m deep. Three major types of vertical distributions are observed (Fig. 10). The first type is encountered mostly in nearshore waters (Fig. 10A and B). Prochlorococcus is restricted to the surface mixed layer, and its abundance drops abruptly below the thermocline (Fig. 10A), where a sharp and narrow maximum can be localized (Fig. 10B). In such cases, Synechococcus has a vertical distribution that parallels that of Prochlorococcus and its abundance is similar or slightly higher. In the second type of distribution (Fig. 10C and D), Prochlorococcus presents a very sharp maximum concentration on the order of 10⁵ cells ml¹ near the bottom of the euphotic zone, which decreases by at least 1 order of magnitude at the surface. The third type of vertical distribution (Fig. 10E and F), with Prochlorococcus extending from the surface to the bottom of the euphotic zone at nearly constant concentrations on the order of 1 × 10⁵ to 3 × 10⁵ cells ml¹, is the most widespread in oceanic waters. Under these circumstances, the Synechococcus concentration is 1 to 2 orders of magnitude lower. Very often, a slight Prochlorococcus maximum occurs just above the depth at which concentrations begin to drop off (e.g., at 70 m in Fig. 10E). Generally, two different types of populations are found in the surface mixed layer and near the bottom of the euphotic zone (Fig. 5). This type of vertical profile is typical of oligotrophic waters. As oligotrophy increases (e.g., in the Pacific, going from the equator to the south gyre [Fig. 10E and F]), the Prochlorococcus layer deepens and Synechococcus concentrations decrease. Under these circumstances, the chlorophyll fluorescence of Prochlorococcus is very weak near the surface and cells are difficult to detect. This may lead to underestimates of cell concentrations at the surface and to creation of artifactual abundance maxima at depth.

View larger version (44K): [in this window] [in a new window]   FIG. 9.   Vertical distributions of Prochlorococcus (A) and Synechococcus (B). Based on data from Table 5.

View larger version (31K): [in this window] [in a new window]   FIG. 10.   Typical vertical distributions of Prochlorococcus and Synechococcus. (A and B) Surface layer maximum. (C and D) Deep maximum. (E and F) Uniform distribution over the euphotic zone. (A and B) North tropical Atlantic, EUMELI3 cruise (129). (A) EU site, off the coast of Mauritania (20°N, 18°W). (B) MESO site (18°N, 21°W). (C) North Atlantic 30°N, 23°W (11). (D) Eastern Mediterranean Sea, 34°N 18°E, MINOS cruise (176). (E) Equatorial Pacific 150°W, 5°S (174). (F) Tropical Pacific 150°W, 16°S (174). (D and F) Prochlorococcus chlorophyll fluorescence was too weak to be detected at the surface.

In oceanic areas, where the water column is never mixed beyond 100 to 150 m deep (roughly between 30°S and 30°N), Prochlorococcus depth profiles vary only slightly throughout the year and are of the third type, as observed, for example, off Hawaii (15). In contrast, when the water column is mixed seasonally to depths below the euphotic zone, which usually happens in winter in more temperate waters, Prochlorococcus completely disappears during that period of the year (92). In spring, when the water column begins to restratify and abundant nutrients are found at the surface, Synechococcus may start to bloom in the mixed layer, followed by Prochlorococcus, yielding vertical profiles of the first type (119). As the bloom develops and nutrients become depleted, a well-defined Prochlorococcus layer deepens, resulting in a vertical profile of the second type (119). At these sites, profiles of the third type are not observed, at least to our knowledge, suggesting that there is not enough time between the end of the spring bloom and the next winter mixing event for a Prochlorococcus population to recolonize fully the oligotrophic surface layer. However, such recolonization may happen at locations where deep mixing does not occur every winter.

In culture, maximal steady-state division rates reported for Prochlorococcus are in general slightly below (or, more rarely, above [150]) one division per day, which is equivalent to a population growth rate of 0.5 to 0.6 day¹ (107, 131, 156). Besides being under the control of parameters such as light and nutrients, the division rate is under the strict control of temperature. For example, the MED4 and SS120 strains both have optimal growth rates at 24°C and cannot grow at or above 28°C, while their minimum growth temperatures are, respectively, 15 and 12.5°C (107).

In the field, division rates are very difficult to estimate directly from changes in cell concentrations because loss and growth processes balance each other and maintain populations at near constant levels. Initially, average estimates of Prochlorococcus growth rates of 0.2 to 0.3 day¹ in the top, euphotic layer of the Sargasso Sea off Bermuda were provided by the labeling of Chl a₂ with ¹⁴C (43). However, this method yields strong over- or underestimates for populations subjected to photoacclimation (13), a common occurrence in the field. Use of the dilution method (72) to measure division rates in Equatorial Pacific waters gave variable and in some cases unrealistically low estimates for the Prochlorococcus division rate (0 to 0.5 day¹ at 10 m deep in reference 73), while its use to measure rates in the western Arabian Sea off Somalia suggested that Prochlorococcus can undergo more than two divisions per day in the field (137), i.e., higher than ever observed in culture.

Using cell cycle analysis, a method avoiding all artifacts linked to sample incubation, several groups demonstrated that the Prochlorococcus cell cycle is highly syn