Chlorophyll b-containing oxygenic photosynthetic prokaryotes: oxychlorobacteria (prochlorophytes).
Abstract Introduction Gross Morphology and Genome Size Cellular Ultrastructure Location and Stacking of Thylakoids The Cell Wall Vacuoles Carboxysomes and Other Cellular Inclusions Localization of DNA Pigments and Other Cellular Constituents Chlorophylls Chlorophyll-Protein Complexes Carotenoids Phycobilins Lipids Ecology Worldwide Distribution Photosynthetic Productivity In Situ and In Vitro Growth Rates Contribution to Food Chains Taxonomic and Evolutionary Considerations Relatedness of the Three Prochlorophyte Genera Species, Strain, or Ecotype Differentiation Relatedness to Cyanobacteria Relatedness to Chloroplasts and Cyanelles Summary and Conclusions Acknowledgment Literature Cited
Before reports in the mid-1970s of the existence of some photosynthetic prokaryotes having chlorophylls a and b but lacking phycobilin pigments (Lewin, 1975; Lewin & Withers, 1975; Newcomb & Pugh, 1975; Schulz-Baldes & Lewin, 1976), the only known oxygenic photosynthetic prokaryotes were the blue-green algae (cyanobacteria), which have chlorophyll (chl a) and phycobilins but no chl b. Because of its unusual pigment composition, the chl b-containing photosynthetic prokaryote first described growing in association with certain colonial ascidians (Didemnidae) and named Prochloron didemni, could not, according to Lewin (1976), be logically assigned to either the Cyanophyta (cyanobacteria) or the Chlorophyta, nor indeed to any other existing algal division. He therefore placed it in a new division, the Prochlorophyta (Lewin, 1976). Two other genera, Prochlorococcus, a unicellular marine phytoplankter (Chisholm et al., 1988) and Prochlorothrix, a filamentous freshwater form (Burger-Wiersma et al., 1986), sharing the prokaryotic ultrastructure and pigment characteristics of Prochloron were later added to the Prochlorophyta. The fact that Prochlorococcus and Prochlorothrix were both free-living and, unlike Prochloron, could be isolated and grown in culture, allowed a much more detailed investigation of the group. In particular, the possible relevance of these chl b-containing prokaryotes to considerations of the evolution of chloroplasts could be investigated. This relevance was suggested by their superficial resemblance to the presumed free-living green prokaryote, which in accordance with the symbiotic theory for the evolutionary origin of chloroplasts (Margulis, 1970) has been proposed as the precursor of the chloroplasts of green algae and plants.
Application of techniques such as nucleotide sequence analysis, spectral and chemical analyses of pigments and pigment-protein complexes, and the powerful tool of flow cytometry (the latter specifically for studies of Prochlorococcus) have allowed a reassessment of the status and significance of this interesting group of prokaryotes. Some of the findings have confirmed strong similarities between these chl b-containing prokaryotes and cyanobacteria, despite the differences in pigment composition; others have revealed differences among the three known genera sufficient to raise questions as to whether they really compose a natural taxonomic grouping.
A number of different taxonomic schemes have been proposed that allow the convenience of maintaining the distinction between these three genera and the cyanobacteria while at the same time acknowledging their relatedness. Their prokaryotic nature suggests that preference should be given to schemes that comply with the code of nomenclature applied to bacteria, although the type genus Prochloron, when it was first described by Lewin (1977), was validly published under the Botanical Code in the family Prochloraceae, order Prochlorales, division Prochlorophyta. Florenzano et al. (1986) proposed that the order Prochlorales be placed in the class Photobacteria, while Lewin (1989) placed the Prochlorales in a group later named the Oxychlorobacteria (Matthijs et al., 1994) in the class Oxygenic Photosynthetic Bacteria. Burger-Wiersma et al. (1989) in formally describing Prochlorothrix hollandica proposed that it be placed in the order Prochlorales, but in the new family Prochlorotrichaceae (on the grounds of its filamentous morphology, which contrasted with that of the other two coccoid, unicellular genera). The discovery of Prochlorococcus and, in particular, the observation that its major membrane-bound chlorophylls occur in the divinyl (DV) form, namely 8-desethyl, 8-vinyl chlorophyll a (chl [a.sub.2]) and 8-desethyl, 8-vinyl chlorophyll b (chl [b.sub.2]) (Chisholm et al., 1988, 1992) further weakened the case for regarding the Prochlorales as a natural grouping. This led Urbach et al. (1992) to suggest abandonment of the order Prochlorales, and later Pinevich et al. (1997) recommended that the three prochlorophyte genera be allocated to already existing orders of the Oxygenic Photosynthetic Bacteria, namely the Chroococcales (for Prochloron and Prochlorococcus) and the Oscillatoriales (for Prochlorothrix).
Discussion of the taxonomic status of the prochlorophytes, and their relationship to the cyanobacteria on the one hand and green chloroplasts on the other has been greatly advanced by analyses of nucleotide sequences and the phylogenetic trees derived from the comparisons involved. Such studies have tended to dominate more recent considerations of the prochlorophytes as a group, with comparisons of physiology, biochemistry, cellular ultrastructure, and ecology of the three genera concerned receiving less attention. This review attempts to bring together these other major features of the three known prochlorophyte genera, recognizing that they are organisms of considerable interest in their own right, quite apart from their possible significance to considerations of the evolution of photosynthetic systems.
In this review, the term "prochlorophytes" as used in the title and elsewhere is simply a convenient way of referring collectively to the three genera under consideration but without thereby necessarily implying the taxonomic validity of the group as a separate division (Prochlorophyta) or order (Prochlorales).
Gross Morphology and Genome Size
The range of cell sizes represented by the three genera extends from the minute bacteria-sized cells of the marine picophytoplankter Prochlorococcus marinus (diameter, 0.5-0.8 [micro]m) (Chisholm et al., 1992) to the large spherical cells (diameter, > 25 [micro]m) of a strain of Prochloron symbiotic with the tropical marine ascidian Didemnum molle (Newcomb and Pugh, 1975). The freshwater Prochlorothrix, on the other hand, occurs in the form of trichomes (diameter, 1.3-1.5 [micro]m; cell length, 10-18 [micro]m) (Burger-Wiersma et al., 1989; Pinevich et al., 1999), resembling filamentous cyanobacteria belonging to the Order Oscillatoriales.
The large size (diameter up to 30 [micro]m) of the spherical unicells of Prochloron, compared with the cells of the other two prochlorophytes and indeed compared with most other prokaryote cells, probably reflects their specialized ecological niche as symbionts embedded in the test or contained within the cloacal cavity of the host, where they are clearly not subject to the same size constraints that are generally considered to apply to free-living prokaryotes. Prochlorococcus, on the other hand, is one of the smallest of all oxygenic autotrophic organisms. It has a high surface-to-volume ratio, probably important for maximizing nutrient and gaseous exchange and light capture in its low-nutrient and, for deepwater ecotypes, low-light environment in oceanic water columns.
The genome size of Prochloron has been estimated as 3.59 x [10.sup.9] Da, comprising two fractions of 3.51 x [10.sup.9] and 7.61 x [10.sup.7] Da (Herdman, 1981), close to the upper limit for cyanobacteria and about 30 times the size of the residual genome of higher plant chloroplasts. The Prochlorothrix genome is fairly similar in size; that of Prochlorothrix hollandica is 5.5 Mbp (i.e., 5.5 x [10.sup.9] Da) (Schyns et al., 1997). The genome of Prochlorococcus is much smaller than that of the other two prochlorophyte genera, and smaller than that of all known cyanobacteria (Herdman et al., 1979). The CCMP 1375 strain of P. marinus, for example, has a genome size of 1.81 [+ or -] 0.04 Mbp, about one-half the size of the Synechocystis genome and considerably smaller than that of Synechococcus (Strehl et al., 1999), from which ancestral group, according to Urbach et al. (1998), it is thought to have evolved by reduction of cell size and genome size. The absence from P. marinus of certain genes widespread among the cyanobacteria generally and the fact that its ribosomal operon exists as a single copy (Strehl et al., 1999) have been interpreted as indicating that deletion of nonessential DNA sequences may have played a major role in the evolution of this prochlorophyte. Many of the known genetic characteristics of P. marinus as described by Partensky et al. (1999) have been interpreted as being consistent with a relatively compact genome, although there is considerable variation in genome size among different strains. One of the highlight-adapted strains has a very small genome (1,657,990 base pairs; 1716 genes)--the smallest of any known oxygenic phototroph--compared with the much larger genome (2,410,873 base pairs; 2275 genes) in one of the low-light-adapted strains (Rocap et al., 2003).
LOCATION AND STACKING OF THYLAKOIDS
Apart from the presence of chl b and the absence of phycobilins, another feature of the photosynthetic machinery of the prochlorophytes that separates them from the cyanobacteria is the stacking of thylakoids. In the cyanobacteria, the thylakoids occur singly, as they do in the Rhodophyta, another group having phycobilin pigments (complexed, in that group, with protein molecules and arranged as phycobilisomes distributed over the thylakoid surface). In Prochloron, as was first described by Schulz-Baldes and Lewin (1976) and confirmed by Whatley (1977), Thinh (1978, 1979), and Giddings et al. (1980), adjacent thylakoids are in contact, forming pairs or further aggregated into stacks, which may comprise as many as 20 or so thylakoids but usually far fewer than this. Stacking of thylakoids into parallel arrays of membranes has also been described for Prochlorothrix (Burger-Wiersma et al., 1986; Miller et al., 1988) and for Prochlorococcus (Chisholm et al., 1988, 1992; Bullerjahn & Post, 1993).
Sometimes, at least in Prochloron (Whatley & Whatley, 1981), stacking is accompanied by invagination or evagination and folding over of the thylakoids, a feature known to be common in the chloroplasts of green algae and euglenoids (Gibbs, 1978). The significance of this, and indeed of thylakoid stacking generally, as an indicator of affinities between prochlorophytes and green chloroplasts, has been the subject of considerable debate. Cavalier-Smith (1982) and Walsby (1986), among others, have pointed out that since thylakoid stacking is common in all plastid types lacking phycobilisomes, it may be no more than a quasimechanical consequence of the absence of phycobilisomes (and the presence of chl b). On the latter point, it should be noted, however, that higher plant chloroplasts depleted of chl b, such as barley mutants (Miller et al., 1976) and clover mutants (Nakatani and Baliga, 1985), nevertheless have stacked thylakoids.
In green algae and in higher plant chloroplasts, stacking of the thylakoids is associated with the recognition in freeze-fracture preparations of four fracture faces distinguishable by the distribution and size of intramembrane particles. Such lateral heterogeneity of particle distribution has also been reported for Prochloron (Giddings et al., 1980) and Prochlorothrix (Miller et al., 1988). For both these prochlorophyte genera, the largest intramembrane particles were found in stacked regions of the exoplasmic fracture face, and were presumed to be photosystem II (PSII) reaction centers surrounded by light-harvesting chi a/b complexes (Golecki & Jurgens, 1989), like those in Chlamydomonas reinhardtii (Goodenough & Staehelin, 1971) and in higher plant chloroplasts (Armond et al., 1977). The greater number of the smaller particles than is common in other chl b-containing organisms prompted the suggestion (Giddings et al., 1980) that the PSII unit size of prochlorophytes is smaller than that of higher plants. This would be consistent with the hypothesis (Thornber et al., 1976; Withers et al., 1978) that the smaller photosynthetic unit size in Prochloron (approximately 240 chlorophyll molecules per P700) is due primarily to a reduced amount of the chl a/b light-harvesting complex. In Prochlorococcus, however, immunolocalization of thylakoid proteins showed that the proteins of light-harvesting complexes and those of the photosystem I (PSI) and PSII reaction centers were equally distributed within the thylakoid membranes and not as segregated as they are in the thylakoids of green algae and higher plants (Lichtle et al., 1995).
THE CELL WALL
In all three prochlorophyte genera, the cell wall is multilayered, as in bacteria and cyanobacteria, but without the external sheath common in the latter group (Burger-Wiersma et al., 1986, Golecki & Jurgens, 1989, Rippka et al., 2000). The main component is peptidoglycan material (Moriarty, 1979; Jurgens, 1989), as in the wall of cyanobacteria and Gram-positive bacteria (Jurgens & Burger-Wiersma, 1989).
The vacuoles of prochlorophytes, defined here as membrane-bound, cytoplasm-free areas of the cell, occur in Prochlorothrix as gas vacuoles (Golecki & Jurgens, 1989) and in Prochloron either as a single, large, central area of the cell devoid of any electrondense material (Thinh, 1978) or as what appear to be the enormously inflated lumens of thylakoids (Cox, 1986). The latter feature sets Prochloron apart from most of the cyanobacteria, in which the membranes of the thylakoids are almost invariably close together, leaving only a narrow lumen.
The gas vesicles of Prochlorothrix are similar in size and shape to those of certain buoyant cyanobacteria (Walsby, 1972) and are aggregated together to form gas vacuoles located at the poles of the constituent cells of the trichome (Burger-Wiersma et al., 1989). They have been reported to have a mean critical pressure (0.9 MPa) toward the upper end of the pressure range of freshwater cyanobacterial gas vacuoles (Walsby, 1980).
CARBOXYSOMES AND OTHER CELLULAR INCLUSIONS
The carboxysomes of Prochloron have a clearly polygonal shape and are either bounded by or closely associated with a tripartite membrane thought to be a modified thylakoid membrane (Cox & Dwarte, 1981, Griffiths et al., 1984). Those of Prochlorothrix and Prochlorococcus are smaller (Burger-Wiersma et al., 1989; Lichtle et al., 1995), but are also closely associated with thylakoid membranes. In this latter respect, the prochlorophyte carboxysomes differ from those of cyanobacteria, which are free of the thylakoids and either have no bounding membrane (Cox et al., 1985) or are bounded by a membrane that is much thinner than a typical thylakoid membrane (Shively, 1974). Prochlorophyte carboxysomes react to immunogold staining in a way consistent with the presence of the photosynthetic enzyme protein ribulose-bisphosphate carboxylase (RuBisCO) (Pinevich et al., 1999). Antibodies raised against the large subunit of RuBisCO have been shown to react specifically with the carboxysomes of all three prochlorophyte genera (Swift & Leser, 1989; Lichtle et al., 1995). The carboxysomal shell of Prochlorococcus has been shown to contain carbonic anhydrase protein, which is presumed to play a role in supplying the active sites of RuBisCO with the high concentration of C[O.sub.2] necessary for optimal carbon fixation (So et al., 2004).
The observation that the carboxysomes of Prochloron are generally larger and more numerous than those of the other two prochlorophyte genera is not unexpected given their presumed major function. A close metabolic integration between symbiont and host, essential in any successful and stable symbiotic relationship, might pose particular problems in an association as morphologically loose as that between Prochloron and its invertebrate host. The greater carboxysome development in Prochloron is probably a reflection of the extent to which synthesis of RuBisCO outstrips the immediate requirement for photosynthetic C[O.sub.2] fixation. It is interesting to note, in this connection, that in some cyanobacteria, carboxysome numbers increase significantly under conditions of a limiting inorganic carbon supply (Turpin et al., 1984).
LOCALIZATION OF DNA
The three prochlorophyte genera differ with respect to the location of DNA-containing sites within the cell. In the filamentous cells of Prochlorothrix, DNA is located primarily in the central region of the stroma (Burger-Wiersma et al., 1986; Swift & Leser, 1989), resembling the central "nucleoid" region in most cyanobacteria. The same is true for Prochlorococcus (Lichtle et al., 1995). Prochloron cells, on the other hand, whatever their type of ascidian host, have their DNA filaments distributed within the stromal region lying between the thylakoid lamellae (Schulz-Baldes & Lewin, 1976; Coleman & Lewin, 1983), a location confirmed by anti-DNA antibodies and by specific reaction to uranyl ions and susceptibility to removal by treatments known to extract DNA (Swift & Leser, 1989).
Pigments and Other Cellular Constituents
It was the presence in Prochloron of chl b as the major accessory pigment, and the apparent absence of any phycobilin pigments, both features previously regarded as diagnostic of green algae and higher plant chloroplasts, that first triggered speculation on the possible significance of Prochloron (and later the other two prochlorophyte genera) in the evolution of chloroplasts. Whether these pigment characteristics (and prokaryote ultrastructure) shared by the three prochlorophyte genera are sufficient, in the face of their other major morphological and ecological differences, to warrant their inclusion in a separate division remained an open question, as also was the related question of whether they (the prochlorophytes) shared a common ancestry with the cyanobacteria or whether they were derived from that group through loss of phycobilins and gain of chl b (and their associated proteins), perhaps independently in each genus. The later discovery, first in Prochlorococcus (Chisholm et al., 1988; Goericke & Repeta, 1992), then in Prochlorothrix (Goericke & Repeta, 1992) and Prochloron (Larkum et al., 1994), of traces of a chl c-like pigment further distinguished the prochlorophytes from the cyanobacteria, at least with respect to their photosynthetic pigment characteristics. The chl c-like pigment in all three prochlorophyte genera has been identified as magnesium 3,8-divinylphaeoporphyrin [a.sub.5], monomethyl ester (Mg [DVPa.sub.5]). Whether the chl c-like pigment is an integral component of the prochlorophyte photosystem or whether it occurs only as an intermediate of chlorophyll synthesis (Rudiger & Schoch, 1988) has not been resolved, but its presence in the group suggests an affinity, at least with regard to chlorophyll composition, with the presumed prokaryotic ancestor from which the chloroplast of a range of eukaryotic algal groups may have evolved (Larkum & Barrett, 1983).
The divinyl (DV) form of the chlorophyll molecule (chl [a.sub.2]), a unique feature of the pigments of Prochlorococcus, has the methyl group on the side chain attached to ring 2 replaced by a formyl (CHO) group. This structural modification allows absorption by the DV form of longer wavelengths in the Soret region (red shift), perhaps contributing to the ability of Prochlorococcus to adapt so successfully to conditions toward the bottom of the euphotic zone (Chisholm et al., 1988; Goericke & Repeta, 1992; Morel et al., 1993). The fact that DV chlorophyllide a is known to be a biosynthetic intermediate of chlorophyll synthesis in higher plants (Rudiger & Schoch, 1988) raises the intriguing possibility that the chl [a.sub.2] of Prochlorococcus may be a vestige of an earlier form of photosynthetic apparatus.
The presence of the DV form of chlorophyll in Prochlorococcus implies absence of the enzyme 3,8-divinyl protochlorophyllide or 8-vinyl reductase (DVR), which in other organisms catalyzes synthesis of monovinyl chlorophyll from the divinyl form. Nagata et al. (2005), in reporting the absence of a DVR homolog from three Prochlorococcus marinus genomes (and its presence in, for example, a Synechococcus genome), suggested that the presence of the DV form of chlorophyll in the prochlorophyte is the result of evolutionary loss of the DVR gene. It was further speculated that loss of DVR in the Prochlorococcus lineage probably occurred initially in strains adapted to low-light conditions, where survival without the monovinyl form of chlorophyll would be possible. The presence of the monovinyl form of chlorophyll in some high-light-adapted ecotypes (Moore et al., 1995) might be the result of later adaptation involving expression of the appropriate enzyme (Partensky et al., 1993).
Compared with the green algae (with the exception of the Euglenophyta) and higher plant chloroplasts, Prochloron and Prochlorothrix (but not Prochlorococcus) have quite high chl a:b ratios. Values ranging from 2.6 to 12.0 have been reported for Prochloron (Thorne et al., 1977; Withers et al., 1978; Schuster et al., 1984; Hiller & Larkum 1985), compared with corresponding values of 2-3 for eukaryotic green algae and higher plant chloroplasts. In Prochlorothrix, the values for the chl a:b ratio can be even higher. Interestingly, uniformly high values for this ratio were recorded in batch cultures of Prochlorothrix maintained at photon flux densities ranging from 4 to 150 [micro]ol [m.sup.-2] [s.sup.-1] (Burger-Wiersma et al., 1986).
All known isolates of Prochlorococcus have chl a:b ratios much lower than those of the other two prochlorophytes. Values of 1.0 or even lower for this ratio (Goericke 1990; Goericke & Repeta 1992; Moore et al., 1995) indicate that Prochlorococcus may either have pigment-protein complexes consisting predominantly of chl [b.sub.2] or reaction centers with a high concentration of chl [b.sub.2]. There is generally a consistent difference in their DV chl a/b ratios between the so-called low-light-adapted (low a/b ratio) and the highlight-adapted (high a/b ratio) forms, reflecting adaptations to their different irradiance environments (Moore et al., 1998; Moore & Chisholm, 1999). Equally low values for this ratio were, however, recorded in axenic cultures of strain PCC 5511 under all light conditions tested (van der Staay et al., 2000). In all isolates of Prochlorococcus that have thus far been studied, most of the chl [b.sub.2] is located in the antennae systems serving PSII, but a significant quantity of this pigment is also found in the PSI complex (Garczarek et al., 1998).
The chlorophylls of prochlorophytes are structurally bound to specific proteins (pcb proteins) to form chl a/b-protein complexes. These, as in the chloroplasts of green algae and plants, are further integrated into the larger reaction center complexes, PSI and PSII, common to all oxygenic photosynthetic organisms.
In Prochloron, the PSI complex has been described as containing four polypeptide units (Schuster et al., 1985), while the PSII complex has as many as ten pcb subunits around a dimeric reaction center (Bibby et al., 2003). It has been estimated that the presence of the pcb proteins increases the PSII antenna size by about 200%, allowing, according to fluorescence emission studies, an efficient transfer of energy to the PSII reaction center (Bibby et al., 2003). Also involved in the PSII energy transfer processes in this and other prochlorophytes, are the chlorophyll-binding polypeptides CP47 and CP43, also known to occur in cyanobacteria, algae, and green plants (Ikeuchi et al., 1991). In Prochlorothrix, five chlorophyll-protein complexes (CP1-CP5) have been resolved by electrophoresis (Bullerjahn et al., 1987). One of them (CP1) was shown to belong to PSI, with at least some of its chl b functionally coupled to the PSI reaction center. Another (CP4) was identified as the major chl a-binding protein of PSII. In Prochlorococcus, the pigment-protein subunits of PSI are arranged as a central PSI trimer core surrounded by 18 copies of one or more of the seven chl a/b-binding (pcb) proteins known for this organism (Garczarek et al., 2000), with the divinyl chl b linked directly to the photosystem core itself (Garczarek et al., 1998). According to this model, and assuming that each pcb binds to 15 chlorophyll molecules, the ring of pcb proteins would contribute 270 light-collecting chlorophyll molecules in addition to the 300 presumed to be associated with the PSI reaction-center trimer, giving the pcb-PSI supercomplex a 90% increase in light-collecting capacity over that of the PSI alone. Such an apparently efficient light-collecting antenna system would be particularly advantageous to low-light-adapted strains of this prochlorophyte in their deepwater locations, bringing the light-trapping efficiencies of their PSI close to that of the PSII, consistent with the PSI:PSII ratio of 1 reported for Prochlorococcus (Bibby et al., 2001b) (compared with the value of about 3 for this ratio in other cyanobacteria).
The chl a/b-(pcb) protein complexes of the prochlorophytes differ in a number of ways from equivalent complexes in green plants, algae, and cyanobacteria. In Prochloron, for example, only 26% of the total chlorophyll is associated with chl a/b-protein complexes (Hiller & Larkum, 1985) compared with the 50% generally found in green algae and higher plants. The P700 chl-protein complex of Prochloron, estimated to contain 240 chlorophyll molecules compared with the equivalent 180 for cyanobacteria and 400 for green plants (Withers et al., 1978) accounts for 17% of the total chlorophyll, intermediate between the 30% for this component in cyanobacteria and the 10% in eukaryotic chloroplasts (Thornber et al., 1976). In Prochlorothrix, the three complexes (CP 2, 3, and 5) functionally bound to chl a and b contain polypeptides that Bullerjahn et al. (1987) showed were immunologically distinct from those of light-harvesting complexes of higher plant thylakoids. The pigment-protein subunits (PsaF and PsaL) of the PSI of Prochlorococcus have been reported to be larger and less conserved than those of other cyanobacteria and higher plant chloroplasts (van der Staay et al., 1998; van der Staay & Partensky, 1999) with the divinyl chl b being linked directly to the photosystem core itself (Garczarek et al., 1998).
Attempts have been made to relate expression of genes encoding for the pigmentprotein complexes of prochlorophytes to ambient light conditions. Thus, Partensky et al. (1997) have shown that in the SS120 strain of Prochlorococcus (a low-light-adapted strain), the abundance of antennae proteins relative to other photosynthetic proteins increases with decreasing irradiance. In a high-light-adapted strain of Prochlorococcus (PCC 9511), grown under a modulated light:dark cycle, there were reported strong changes in the mRNA transcription level of its one pcb gene during the day, with two maxima, one at the end of the light period, the other at the end of the dark period (Garczarek et al., 2001a).
The carotenoids of Prochloron and Prochlorothrix are dominated by [beta]-carotene (Lewin & Withers, 1975; Burger-Wiersma et al., 1986), which accounts for well over half of the total carotenoid pool, with zeaxanthin as the next most abundant. Prochlorococcus differs from the other two prochlorophytes in having zeaxanthin and [alpha]-carotene as the major carotenoids. The ability to synthesize [epsilon]-cyclic carotenoids (such as [alpha]-carotene), previously thought to occur only in eukaryotes (Goodwin & Britton, 1988) sets Prochlorococcus (P. marinus) apart from the other prokaryotes and from all of the cyanobacteria. Carotenoid biosynthesis involves as a key step cyclization of lycopene, which in cyanobacteria is catalyzed by a single enzyme, [beta]-cyclase. Higher plants have two cyclases, [epsilon]- and [beta]-lycopene cyclases. Two genes encoding lycopene cyclases have also been functionally identified in Prochlorococcus (MED 4 strain) (Stickforth et al., 2003). One, both functionally and in terms of its base sequences, is very similar to the [beta]-cyclase of cyanobacteria, while the other catalyzes the formation of [epsilon]- as well as [beta]-ionene end groups. Uniquely among the prokaryotes, therefore, Prochlorococcus has two enzymes catalyzing the formation of cyclic carotenoids with either [beta]- or [epsilon]- end groups, a feature that Stickforth et al. (2003) suggest may have originated through duplication of a single gene.
The presence in prochlorophytes of abundant carotenes, and the consequent strong absorption at shorter wavelengths, together with subsequent efficient transfer of captured energy to chlorophyll, suggests a combined light-harvesting/photo-oxidation protection role for these pigments, consistent with the finding that they are largely associated with thylakoid membranes (Omata et al., 1985). Zeaxanthin, by contrast, has been found to be associated mostly with cellular membranes other than thylakoids suggesting a predominantly light-shielding function.
In the relative proportions of their constituent carotenoids, and in the ratio of [beta]-carotene to chl a, Prochloron and Prochlorothrix more closely resemble the cyanobacteria than the Chlorophyta (Paerl et al., 1984). Prochlorococcus, in having [alpha]-carotene as a major yellow pigment, resembles the marine prasinophytes (Omata et al., 1985). It should be noted, however, that the ratio of carotenoid to chlorophyll can fluctuate markedly, even on a diurnal basis. The ratio of zeaxanthin to DV chl a in the PCC511 clone of Prochlorococcus, for example, increases by as much as 52% between sunrise and sunset (Claustre et al., 2002).
Some strains of Prochlorococcus differ from the prochlorophytes generally in having traces of phycobilin pigments in addition to the universally present chl b. Strain CCMP 1375, for example, has been reported to contain a phycoerythrin (PE) pigment (PE-III) as well as the (divinyl) chl a/b-binding antennae complexes, although apparently not aggregated into phycobilin-like structures (Chisholm et al., 1992; Lichtle et al., 1995; Hess et al., 1999; Partensky et al., 1999). In the low-light-adapted strain SS120, the major chromophore is phycourobilin (Steglich et al., 2005). Other strains, notably strain PCC 9511, the first to be cultured axenically, have no phycoerythrin (van der Staay et al., 2000). Functional, but only weakly expressed, genes coding for the [alpha] and [beta] subunits of phycoerythrin have been shown to be present in a number of low-light-adapted strains of Prochlorococcus marinus isolated from deep waters of the tropical Pacific Ocean (Hess et al., 1996; Ting et al., 1999; Penno et al., 2000). Analysis of nucleotide sequences of the relevant genes has shown that PE-III is distinct from both the common cyanobacterial phycoerythrin C-PE-I, and from PE-II, which occurs mainly in marine cyanobacteria (Ong & Glazer, 1991; Swanson et al., 1991).
The genes encoding for the [alpha] (cpeA) and [beta] (cpeB) subunits of the phycobilin pigment in strain SS120, when subjected to phylogenetic analysis of their nucleotide sequences, fall into a cluster that also contains genes encoding other phycobiliproteins and bilin biosynthesis proteins (Hess et al., 1999). But the low levels of expression of these genes, and similar genes in strains MIT 9303 and MIT 9313 (Ting et al., 2001), casts some doubt on their functional significance. The green-light-absorbing phycoerythrin of the high-light strain MED 4 is coded for by a single gene (cpeB) and is considered to have the potential to serve as a photoreceptor (Steglich et al., 2005).
Evidence has been provided by Lokstein et al. (1999) that the PE of Prochlorococcus strain CCMP 1375 is indeed capable of transferring excitation energy to the divinyl chlorophylls. Thus, it was shown that at room temperature, excitation of suspensions at 495 nm (i.e., the absorption and fluorescence excitation maximum of PE) caused low PE fluorescence emission, but strong divinyl chl a fluorescence at 675 nm. Glycerol treatment of the suspensions caused a pronounced increase in PE fluorescence, paralleled by a considerable decline in chlorophyll fluorescence intensity. This was interpreted as a glycerol-induced detachment of the originally coupled PE from the photosynthetic apparatus. Following such detachment, the PE excitation, no longer quenched by energy transfer, is reemitted as fluorescence. This apparent capacity of the PE of CCMP 1375 to transfer excitation energy to divinyl chlorophyll, in the absence of phycocyanin, ailophycocyanin, or any phycobilisome-like structures involves, according to Lokstein et al. (1999), an initial transfer of PE excitation to the [S.sub.3] energy level of divinyl chl b, which rapidly relaxes by internal conversion to the [S.sub.1] state, from where it is finally transferred to the [S.sub.1] state of divinyi chl a.
The presence of phycobilin pigments in some strains of Prochlorococcus, whether or not they are functionally important in the transfer of excitation energy, gives that prochlorophyte genus a unique status as the only living photosynthetic prokaryote to have both chl a/b antenna genes and phycobiliprotein genes.
The overall distribution of lipid classes in the prochlorophytes has been described as similar to that of many bacteria and cyanobacteria and unlike that of eukaryotic algae. Thus, in Prochloron (Johns et al., 1981) fatty acids compose the major lipid component (5.4% of dry weight), with the carotenoids (2.5% of dry weight) as the next most abundant lipid component. Similarly for Prochlorothrix, the high abundance of free fatty acids, the low amount of triglycerols, and the absence of sterols (Volkman et al., 1988) confirm the alignment of prochlorophytes in their lipid composition with other prokaryotes and separates them from the eukaryotic algae.
Prochloron also lines up with the cyanobacteria in the nature of its constituent fatty acids, with [C.sub.14] and [C.sub.16] acid components dominant (Perry et al., 1978; Johns et al., 1981), and as unlike the eukaryotic algae, where [C.sub.18] and [C.sub.20] acids are more common. The fatty acid composition of Prochlorothrix has been found to be generally similar to that in Prochloron (Volkman et al., 1988), with both prochlorophyte genera lacking the longer chain polyunsaturated fatty acids typical of many eukaryotic algae. It is interesting to note, however, that in terms of the positioning of the double bonds, often regarded as useful to chemotaxonomic considerations (Gillan & Johns, 1986), Prochlorothrix has some fatty acids found neither in Prochloron nor in the cyanobacteria (Gombos & Murata, 1991). The presence of monogalactosyl diacylglycerol (MGDG) as a major lipid component in the prochlorophytes (Murata & Sato, 1983; Gombos & Murata, 1991) sets them apart from the eukaryotic algae and from chloroplasts.
The ecology of the three known prochlorophyte genera reflects the high degree of divergence presumed to have occurred within the group (e.g., Palenik & Haselkorn, 1992). Thus, while Prochloron has been found only in association with a small group of tropical marine invertebrates and Prochlorothrix only in shallow freshwater lakes, Prochlorococcus is a common constituent of the picoplankton across a wide range of oceanic waters.
Ascidian Species harboring Prochloron as a symbiont have a wide distribution between latitudes 30[degrees]N and 30[degrees]S in the Indo-West Pacific region (Kott, 1980). They have been recorded from localities as distant from each other as the Red Sea and the east coast of Africa, and Baja California (Mexico) on the west coast of North America (Lewin, 1975; Lewin & Cheng, 1975; Kott, 1980). Some have also been reported from the Caribbean Sea (Lafargue & Duclaux, 1979; Lewin et al., 1980). Prochlorothrix, on the other hand, has been reported from only a few sites in western Europe (BurgerWiersma et al., 1986; Pinevich et al., 1999). This may simply reflect gaps in the record, particularly since it requires sequence analysis based on ribosomal RNA genes (Zwart et al., 2005) or specific amplification of pcb genes (Geiss et al., 2003) to differentiate clearly between Prochlorothrix and some other morphologically indistinguishable species such as Pseudanabaena and Planktothrix.
The widespread distribution of Prochlorococcus across the warmer, mostly tropical and subtropical waters of the world is well documented, as also is its clear preference for open oceanic rather than coastal environments (Wood et al., 2001). It has been studied in both the Atlantic (Veldhuis & Kraay, 1990; Partensky et al., 1993, 1996; McManus & Dawson, 1994; Claustre & Marty, 1995) and Pacific (Olson et al., 1990; Chavez et al., 1991; Shimada et al., 1993; Campbell et al., 1994; Vaulot et al., 1995; Blanchot & Rodier, 1996) oceans. It has also been extensively studied in the Mediterranean (Vaulot et al., 1990; Partensky et al., 1993; Bustillos-Guzman et al., 1995) and Sargasso (Li et al., 1992; Goericke & Welschmeyer, 1993; Urbach & Chisholm, 1998) seas as well as in the Red Sea (Lindell & Post, 1995), the Arabian Sea (Shalapyonok et al., 1998), shelf waters of the Great Barrier Reef (GBR), Australia (Crosbie & Furnas, 2001), and in the South Pacific and Antarctic oceans (Shimada et al., 1999). The first indication that Prochlorococcus might also occur in nonmarine waters was provided by Vaulot et al. (1990), who reported its presence in the low-salinity dilution zone of the Rhone River at salinities as low as 1.2 ppt. Corzo et al. (1999) have reported the presence of small coccoid cells with red autofluorescence and matching the flow cytometric signature of P. marinus in a eutrophic reservoir (La Concepcion) in southern Spain.
That autotrophic C[O.sub.2] fixation in the prochlorophytes involves operation of the Calvin-Benson cycle is evident from studies showing operation of the two key enzymes unique to that process, RuBisCO and phosphoribulokinase (Akazawa et al., 1978; Kremet et al., 1982; Swift & Leser, 1989; Shimada et al., 1995), and demonstrating that these enzymes are present at levels comparable to those in other C[O.sub.2]-fixing microorganisms (Berhow & McFadden, 1983). Prochlorophyte RuBisCO has a large (ca. 55 kDa) and small (ca. 15 kDa) subunit and sediments in sucrose density gradients similarly to all other sources of this enzyme (e.g., Berhow & McFadden, 1983).
Prochlorococcus is the only prochlorophyte known to make a significant contribution to global primary productivity. Recognition by Chisholm et al. (1988) and Goericke & Repeta (1992) that all isolates of Prochlorococcus uniquely have as major photosynthetic pigments the divinyl forms of chlorophyll (chl a2 and be), provided a marker upon which could be based more accurate biomass estimations than had hitherto been possible. In their studies of winter phytoplankton in the Mediterranean Sea, Vaulot et al. (1990) were able to show that prochlorophytes were present at all stations, and they were estimated to contribute between 3.7 x [10.sup.-4] and 1.3 x [10.sup.-1] [micro]g biomass [L.sup.-1]. Prochlorococcus was estimated to account on average for about 60% of total prokaryotic chlorophyll (largely Prochlorococcus and the cyanobacterium Synechococcus) and about 40% of prokaryotic photosynthetically fixed carbon.
Studies by Campbell et al. (1994), which are among the earliest to distinguish clearly between populations of Prochlorococcus and heterotrophic bacteria, showed that in sites in the central north Pacific Ocean, the prochiorophyte contributes an estimated 31% of the total bacterial-sized biomass in the upper 100 m. Of the total microbial community, approximately 80% was prokaryotic biomass, and a half of that was photosynthetic biomass contributed by Prochlorococcus.
Detailed analysis of the phytoplankton composition in surface waters in the northeast Atlantic showed that while in the northern region (62-52.5[degrees]N) the most abundant phytoplankton classes were the prymnesiophytes (38%), cryptophytes (20%), chlorophytes (14%), and diatoms (15%), further south (50-37[degrees]N), the population was dominated by cyanobacteria and prochlorophytes, which together accounted for a mean of 53% of the total chl a at 37[degrees]N (Gibbs et al., 2001). At this most southerly region sampled, the contribution of cyanobacteria to total chl a declined with depth, the deep chlorophyll maximum being dominated by prochlorophytes but with significant contributions from prymnesiophytes and cryptophytes.
In the equatorial Pacific, Partensky et al. (1999) observed no drastic change in the abundance of Prochlorococcus between the oligotrophic warm pool and the more nutrient-rich areas. Of special note was its presence in the water column down to depths of 150-200 m, where ambient irradiance was less than 0.1% of that at the surface. In waters of the GBR, Australia, Prochlorococcus was at some sites a significant contributor to the total picophytoprokaryote biomass, accounting (e.g., in the central GBR region) for as much as 80% of the total phytoplankton biomass (Crosbie & Furnas, 2001). The prochlorophyte was most abundant (up to 1.3 x [10.sup.5] cells [mL.sup.-1]) in mid- or outer-shelf sites, where the waters are of a more oceanic character. But it was also observed at some inshore sites, especially in the central GBR region, where, in the absence of clear evidence of significant intrusions from nonshelf waters, it was presumed to be the result of active in situ growth.
Li (1994) used a combination of [sup.14]C labeling and flow cytometric sorting to assess the primary production of prochlorophytes relative to that of other groups in a range of North Atlantic sites and concluded that although eukaryotes were generally the dominant primary producers, there were some stations where the prochlorophytes were clearly dominant (e.g., 57% of aggregate productivity at station 32[degrees]39.5'N, 26[degrees]39.2'W). Partensky et al. (1999) have concluded that Prochlorococcus, despite its relatively narrow geographical distribution, nevertheless makes a considerably greater contribution than, for example, Synechococcus to phytoplankton carbon biomass on a global scale. This view is based on its greater abundance (estimated as x100 or x22 in terms of carbon) in warm oligotrophic waters, which are typical of the greater part of the world's oceans.
The contribution of Prochlorococcus to oceanic productivity is strongly influenced by the apparent ability of certain strains to undergo photoadaptation to ambient light intensity and quality at different levels in the water column (Chisholm et al., 1988; Velduis & Kraay, 1990; Partensky et al., 1993). The bimodal nature of red fluorescence peaks in depth profiles around the deep chlorophyll maximum of oligotrophic water columns (McManus & Dawson, 1994; Binder et al., 1996; Blanchot & Rodier, 1996; Partensky et al., 1996) suggests that different populations of the prochlorophyte may exist at different depths. Two distinct populations, low-light and high-light ecotypes, have been identified (Partensky et al., 1999), with the former ecotype having a higher red fluorescence and a higher (2- to 10-fold) chl [b.sub.2]:chl [a.sub.2] ratio than the latter.
Other differences between the two ecotypes relate to their respective abilities to utilize different available sources of nitrogen. Isolates of high-light-adapted ecotypes were shown to grow well in culture on N[H.sub.4], but could use neither N[O.sub.3] nor N[O.sub.2] (Moore et al., 2002). It was further shown that while isolates of low-light-adapted ecotypes could also grow on N[H.sub.4], four of the six strains tested could also grow on N[O.sub.2] (but not on N[O.sub.3]). These findings were interpreted to indicate that in nutrient-depleted surface waters, the high-light-adapted ecotypes that grow there do so largely through utilization of recycled sources of nitrogen, such as N[H.sub.4] and urea, while in the deep euphotic zone of stratified water columns, the resident low-light-adapted ecotypes utilize the N[O.sub.2] that is available there.
A combination of cytometry and tracer methods using [sup.35]S-methionine has shown that, of the considerable uptake of organic nitrogenous compounds by the bacterioplankton of oligotrophic waters of the Arabian Sea, approximately one-third of the total turnover of amino acids can be attributed to Prochlorococcus spp. (Zubkov et al., 2003). Optimal nitrogen utilization by Prochlorococcus under different environments is thus ensured by a range of regulatory mechanisms, including gene loss (Garcia-Fernandez et al., 2004), accounting for the competitive dominance of the prochlorophyte over other photoautotrophs in oligotrophic oceanic waters. Increases over the past decade of Prochlorococcus populations in the North Pacific subtropical gyre have been attributed to the acquisition by some strains of more efficient mechanisms of phosphorus assimilation under conditions of greater P limitation (Moore et al., 2005).
IN SITU AND IN VITRO GROWTH RATES
Since Prochloron has never been successfully grown in culture away from its ascidian host, estimations of its growth rate have necessarily been based on indirect observations. Thus, Thinh et al. (1981), in their observation of the growth and spread of the ascidian Diplosoma virens on an artificial substrate, noted that the colonies increased exponentially in number (0.136 [d.sup.-1]) over a period of 30 days. There was, over this period, no evidence of any significant diluting out of the symbiont, indicating that, at a minimum, growth and division of the prochlorophyte kept pace with that of the host colonies. Lewin et al. (1984), from their observations of cell division frequencies, estimated a doubling time of 6 days (i.e., 0.167 [d.sup.-1]) for Prochloron in association with the same host. These low rates of growth and division, compared with those of cyanobacteria generally, probably reflect the much larger size of the prochlorophyte and the constraints imposed by the requirement that it balance its growth rate with that of its host.
Maximum growth rate of Prochlorothrix hollandica in culture (0.02 [h.sup.-1] i.e., 0.48 [d.sup.-1]) was reported by Burger-Wiersma et al. (1989) to occur at 20 [degrees]C and 40 [micro]mol [m.sup.-2] [s.sup.-1]. There was no growth in the absence of combined forms of nitrogen, even under anaerobic conditions. Growth ceased completely in media containing NaCl at concentrations equivalent to approximately 20% that of seawater. Blooms of P. hollandica occur in summer, and the lakes where it has been found (some originating from past peat excavations) are generally shallow and thus well mixed throughout the year. Such vertical mixing, it has been suggested, ensures reinoculation from the sediment layers. In situ growth rates of this prochlorophyte have been shown to be 30-40% lower than those recorded for co-occurring oscillatorioid populations (Pel et al., 2004), but under conditions of phosphorus limitation in continuous culture, Prochlorothrix was a very good competitor for P, competitively displacing Planktothrix agardhii whether P was supplied under a constant or pulsed regime (Ducobu et al., 1998).
Some of the earliest estimations of in situ growth rates of Prochlorococcus were conducted by Vaulot et al. (1995), who by measuring cell cycle fractions in samples from the equatorial Pacific Ocean were able to record maximum rates of approximately one doubling per day at 30 m depth, with decreasing rates above and below that point. These rates are slightly higher than those recorded in cultures of isolates from the Mediterranean and Sargasso seas (Moore et al., 1995).
More accurate measurement of in situ growth rates of Prochlorococcus, as with other picophytoplankton groups, requires that due allowance be made for losses due to grazing. This is usually accomplished through the use of dilution techniques (Landry, 1993), which are based on the assumption that progressively diluted samples suffer progressively less grazing impact. This technique has yielded in situ growth rates for Prochlorococcus in the central equatorial Pacific Ocean of 0.8 [d.sup.-1] near the surface, 0.34 [d.sup.-1] in the mid-euphotic zone, and 0.22 [d.sup.-1] lower down (Landry et al., 1995). When applied to samples from the lower euphotic zone in the subtropical North Atlantic, the dilution technique has yielded procblorophyte growth rates of only 0.3 [d.sup.-1], just above the deep chlorophyll maximum. The various assumptions upon which the method rests, as they apply to Prochlorococcus, have been revised by Worden and Binder (2003), who use an alternative, more conservative approach that does not, for example, assume a finear relationship between dilution and net growth rate. In their estimations based on experiments conducted in the Sargasso Sea and the California Current, they obtained growth rates ranging from 0.32 to 0.76 [d.sup.-1], consistent with rates reported by others from cell cycle-based estimations.
The observation that the cell cycle of Prochlorococcus is tightly phased to the light:dark cycle (Vaulot et al., 1995; Partensky et al., 1996; Liu et al., 1997) suggests a maximum daily division rate of 1.0. However, some in situ growth rate estimations have yielded values significantly higher than this (Reckermann & Veldhuis, 1997; Veldhuis et al., 1997). Studies monitoring DNA frequency distributions over diel cycles have confirmed the potential for growth rates exceeding one doubling per day in Prochlorococcus samples from the northwest Arabian Sea (Shalapyonok et al., 1998). Cultures of this prochlorophyte isolated from different sources and maintained under a range of light intensities and different light:dark regimes also yielded growth rates well in excess of one division per day. Thus, at light intensities of 40 [micro]mol [m.sup.-2] [s.sup.-1] and higher, the DNA frequency distribution indicates that some of the daughter cells released toward the end of the light period undergo a second division during the subsequent dark period. At lower light intensities, this second wave of cell division is absent. This feature of Prochlorococcus, with strict timing of both DNA replication and cell division and the very short period of growth needed before the second division, distinguishes it from some other phytoplankton, such as Synechococcus, which can also undergo more than one division per day but has continuous DNA replication and cell divisions that can take place throughout the day and into the night (Binder & Chisholm, 1995; Mori et al., 1996). The pattern of growth shown by Prochlorococcus (phased ultradian growth) has been described as one that combines the efficient use of daylight hours for photosynthesis with the potential for maximum cell division during the night, so that cells that have fixed enough carbon during the day are able to divide more than once (Shalapyonok et al., 1998).
Jacquet et al. (2001) have confirmed that in Prochlorococcus, the key parameter for synchronized cell cycling is the "light on" signal, which initiates the S (i.e., DNA synthesis) phase. Moving Prochlorococcus cultures from a low- to a high-light light:dark regime produced an increase in the number of cells in both the S phase and the [G.sub.2] (i.e., the post-DNA replication) growth phase, with a consequent increase in growth. The re verse shift (from high- to low-light regime) reduced the growth rate of the population, confirming close coupling between irradiance levels and cell cycling.
The close synchronization of the cell cycle with a light:dark regimen simulating that in the upper layers of the ocean was studied by Holtzendorff et al. (2001), using turbidostat cultures of the PCC 9511 strain of Prochlorococcus. They cloned and sequenced two genes (dnaA and fisZ) coding for enzymes known to be involved in cell cycle-related processes and observed that both genes exhibited clear diel expression patterns with mRNA maxima during the replication (S) phase. Peak of FtsZ concentration occurred at night (at the time of cell division).
CONTRIBUTION TO FOOD CHAINS
Prochloron contributes to organic production through transfer of its photosynthetic products to the colonial ascidian (or other invertebrate) host. Significant transfer of photosynthetically fixed carbon from Prochloron to host has been demonstrated for associations of the prochlorophyte with Diplosoma sp. and Lissoclinum patella (Pardy & Lewin 1981) and with Diplosoma similis, Lissoclinum bistratum, and Trididemnum cyclops (Griffiths & Thinh, 1983). Prochloron associated with Didemnum molle has been estimated to have the potential to contribute between 12% and 31% of the respiratory carbon requirement of the host (Olson & Porter, 1985). In the Prochloron-Lissoclinum patella association, prochlorophyte photosynthesis may contribute as much as 30% to 56% of the reduced carbon requirement for host respiration (Alberte et al., 1987).
Prochloron may be directly consumed when the ascidian host is subject to predation by a wide range of grazers, including molluscs, echinoderms, and fishes (Parry, 1984). Earlier suggestions (Millar, 1971; Stoecker, 1980; Bak et al., 1981) that ascidians may be resistant to predation, either because of their high vanadium content or because of the presence of acid-containing cells in the test, have not been borne out by direct observation of a range of encrusting ascidian species at Heron Island (GBR) (Parry, 1984). Predation was observed to occur irrespective of the presence or absence of vanadium or of acid-producing cells.
The contribution of Prochlorothrix to the food webs of the eutrophic Loosdrecht lakes (The Netherlands) where it occurs has been demonstrated by noting ingestion and assimilation of the prochlorophyte by one of the major primary consumers in those water bodies, the rotifer Euchlanis (Gulati et al., 1993). Euchlanis utilizes the prochlorophyte with assimilation efficiencies comparable to those obtained with the illamentous cyanobacterium Aphanizomenon flos-aquae. Smaller-sized Euchlanis were particularly efficient consumers of the prochlorophyte, consistent with the observation that in late summer and early autumn, when the phytoplankton community is dominated by Prochlorothrix, the rotifer also occurs in high concentrations.
Prochlorococcus, because of its small size, is more likely to be consumed by small grazers, chiefly flagellates, rather than by mesoplankton grazers such as small copepods or cladocerans (Sherr et al., 1986; Hagstrtm et al., 1988). This was confirmed in laboratory feeding experiments in which Prochlorococcus was supplied to two small flagellates, Symbiomonas scintillans and Picophagus flagellatus. The latter species, which is less than 2 [micro]m in size, was shown to be a substantial feeder of the prochlorophyte, displaying a growth rate of the order of two doublings per day. In a study comparing the consumption of Prochlorococcus and the similarly sized cyanobacterium Synechococcus by two ciliate species, Strombidium sulcatum (normally algivorous) and Uronema sp. (normally bactivorous), Christaki et al. (1999) found that both ciliates showed a clear preference for Synechococcus. The differences in clearance rates for the two prey species was tentatively attributed to differences in surface characteristics. The relatively low growth rates recorded for both consumers, under prey abundances similar to those known to occur in nature, cast doubt on the ability of either species to provide high quality food for these ciliates. The growth rate of Uronema sp. on Prochlorococcus was particularly poor, despite the quite significant ingestion of the prochlorophyte. The extent to which the results obtained with Uronema apply also to other ciliates is not known; it may be relevant that this flagellate is generally more abundant in shallow waters (where Synechococcus is often more numerous than the prochlorophyte). The results tend to confirm that the major in situ consumers of Prochlorococcus are probably smaller nanoflagellates, which provide an additional link between the picoplankton primary producers and higher trophic levels.
Christaki et al. (2001) estimated the relative grazing impact of heterotrophic nanoflagellates on bacteria and cyanobacteria across a range of sampling stations in the Mediterranean Sea. They reported that of the total organic carbon ingested from prokaryotic sources, about 27% was provided by the photosynthetic prokaryotes Synechococcus and Prochlorococcus. By extrapolation from the data relating to the consumption of S vnechococcus, and assuming no selection on the part of the flagellates for or against Prochlorococcus, it was estimated that from 1.4% to 21% (mean, 6%) of the prochlorophyte stock was ingested per day compared with corresponding values of 0.5-45% (mean, 13%) for Synechococcus.
The observed stability of populations of Prochlorococcus in equatorial Pacific waters (Kirchman et al., 1995; Vaulot et al., 1995) was shown by Landry et al. (1995) to be due to grazing control by zooplankton. Using incubation techniques, both on deck and in situ, with serial dilution and prescreening, Kuipers and Witte (2000) estimated the rates of clearance by grazing at different depths (i.e., at, above, and below 100 m) of the deep chlorophyll maximum (DCM) at five stations between 10[degrees] and 35[degrees]N in the Atlantic Ocean and compared them with calculated growth rates of Prochlorococcus. They confirmed that cell division of the prochlorophyte in the upper region of the DCM occurred during the day, and further showed that losses by grazing were also restricted to daylight hours.
The prefiltration trials indicated that the predominant grazers of Prochlorococcus are small heterotrophic nanoflagellates (diameter, 1-3 [micro]m), although these grazers showed a distinct preference for heterotrophic bacteria, which, unlike Prochlorococcus, are motile, thus affording higher rates of encounter. It was concluded that the intensity of grazing on Prochlorococcus is regulated, not by the abundance of the prochlorophyte, but by the predator-prey relationship between the nanoflagellates and heterotrophic bacteria. The observed periodicity and intensity of grazing upon Prochlorococcus was explained as being regulated by the diel cycle in availability, above a certain critical threshold, of the much more abundant primary food source of the nanoflagellates, the heterotrophic bacteria.
Christaki et al. (2002) studied the rates of consumption of picoautotrophs by the marine heterotrophic nanoflagellate Pseudobodo sp. and by a mixed nanoflagellate culture obtained from an oligotrophic open-sea area. Extrapolations from their data indicate that in an oceanic environment containing [10.sup.3] flagellates [mL.sup.-1] and 5 x [10.sup.4] Prochlorococcus cells [mL.sup.-1] (within the range usually encountered in natural waters), the flagellates would consume <5% of the Prochlorococcus stock per day. This finding agrees with other estimations of rates of consumption of Prochlorococcus by nanoflagellates in Mediterranean Sea studies (Christaki et al., 2001), confirming other field studies that indicate that picoautotrophs generally may be no more that a secondary food source for flagellates (Kuipers & Witte, 2000). Studies with another flagellate, Picophagus flagellatus, have yielded ingestion rates for Prochlorococcus of only one individual per flagellate per hour (Guillou et al., 2001) from a single prey concentration of 7 x [10.sup.5] [mL.sup.-1], agreeing with the comparable values reported by Christaki et al. (2002). When fed to ciliates, Prochlorococcus was shown to inhibit or interfere with cell division of Uronema sp. and to permit only moderate growth of another ciliate, Strombidium sulcatum (Christaki et al., 1999).
Prochlorococcus may also contribute to oceanic food webs via the microbial loop that utilizes the prochlorophyte's excreted organic compounds (9-24% of the assimilated carbon) (Bertilsson et al., 2005). In oligotrophic oceanic waters, where input of terrigenous organic matter is negligible, this process may be a significant contributor to the pool of organic substrates available to microbial heterotrophs.
Taxonomic and Evolutionary Considerations
Classification of algae into divisions has traditionally relied heavily on the nature and composition of the accessory photosynthetic pigments, with the cyanobacteria (blue-green algae), despite their prokaryotic ultrastructure, being in some earlier textbooks designated as a division of the algae. It was inevitable, therefore, that discovery of oxygenic photosynthetic prokaryotes having a different pattern of photosynthetic pigments from the cyanobacteria should have led to the proposal for a new division, the Prochlorophyta or, perhaps less controversially, a new order, Prochlorales, within the cyanobacteria. But, as far as the prochlorophytes are concerned, pigment-based systems of classification have become somewhat devalued by the multiplicity of chlorophyll types, chlorophyll-phycobiliprotein combinations, and carotenoids that have come to light within the group. In this section, evidence from phylogenetic analysis of molecular data is summarized to show how they have been interpreted to evaluate the status of the prochlorophytes as a valid taxonomic group and to assess their affinity with cyanobacteria and green chloroplasts.
RELATEDNESS OF THE THREE PROCHLOROPHYTE GENERA
One of the main features shared by the three prochlorophyte genera, distinguishing them from cyanobacteria, is their possession of light-harvesting complexes involving chl b (rather than phycobilins). In any consideration of their relatedness, therefore, the acquisition of the capacity for chl b synthesis assumes critical importance. It is significant that the gene coding for the enzyme chlorophyll a oxygenase, CAO (which catalyses conversion of chl a to chl b), has been shown to be very similar in the two prochlorophytes Prochloron didemni and Prochlorothrix hollandica (Tomitani et al., 1999). In a comparison that included CAO amino acid sequences from green algae, a bryophyte, and some angiosperms, the two prochlorophytes were shown to cluster together with high bootstrap probabilities of 100%, and they shared 69% identical amino acid sequences. The high similarity between the two prochlorophytes was confirmed by analysis of gap positions in the sequences. These results were interpreted by Tomitani et al. (1999) to indicate that the ability to synthesize chl b did not arise independently in these two genera but more probably in a common ancestor of all oxygenic photosynthetic prokaryotes presumed to have both chl b and phycobilins.
Similarly, Litvaitis (2002) has also argued that data based on 16S rRNA sequence analysis are more parsimoniously interpreted in terms of a common cyanobacterial/ prochlorophyte ancestor rather than by invoking de novo acquisition of chl b in each prochlorophyte lineage. Loss from this ancestor of the ability to synthesize phycobilins would, according to Tomitani et al. (1999), have led to the prochlorophytes, and loss of chl b, to the cyanobacteria. Prochlorococcus marinus, with its divinyl forms of chlorophyll and, at least in some strains (Hess et al., 1996), traces of phycoerythrin c, might, with only minor pigment alterations, be regarded as a living model of this hypothetical ancestral form.
These findings, however, contradict earlier evidence based on 16S rRNA sequence data, which yielded phylogenetic trees with branching patterns, suggesting that the development of chl b complexes occurred separately in the three prochlorophyte genera (Palenik & Haselkorn, 1992; Urbach et al., 1992). Further evidence for heterogeneity among the prochlorophytes has come from analysis of the psbA gene, of which there is only one copy in P. marinus (CCMD 1375) compared with two (each encoding proteins of identical amino acid sequence) in Prochlorothrix hollandica (Morden & Golden 1989; Hess et al., 1995). Moreover, the derived amino acid sequence for the P. marinus gene, and that of Prochloron didemni, has been shown to have seven amino acids near the C-terminus that are missing from that location in P. hollandica (Hess et al., 1995; Lockhardt et al., 1993).
The extent to which these differences can be interpreted as evidence for or against a multiple origin for the three prochlorophyte genera depends on the weight given to the seven amino acid deletion. The suspected important role of the C-terminus in some key photosynthetic reactions (Nixon et al., 1992) suggests, according to Hess et al. (1995), some degree of conservation of this part of the [D.sub.1] polypeptide.
SPECIES, STRAIN, OR ECOTYPE-DIFFERENTIATION
There have been many attempts to establish whether all known strains of Prochloron can be accommodated within the one species so far proposed, namely, P. didemni (Lewin,1977). Thus, despite the fact that on the basis of their morphology, different Prochloron strains appear to fall into three groups (Cox, 1986), nucleotide sequence analysis of the DNA-dependent RNA polymerase gene (rpoC) of isolates from different ascidian hosts has suggested that they are more likely to be conspecific (Palenik & Swift, 1996). Stackebrandt et al. (1982), in their comparative analysis of the oligonucleotide sequences of 16S rRNA of Prochloron strains from a range of different ascidian hosts, calculated similarity coefficients indicating that the strains were as closely related as the most closely related species of bacteria subjected to the same analysis, or even as different strains of one bacterial species. DNA-DNA reassociation techniques applied by Stam et al. (1985) to seven Prochloron samples from different localities and hosts yielded substantially similar results. The genetic relationship among them was found to be very close compared, for example, with the relatively distant relationship between these samples and a strain of the cyanobacterium Synechococcus.
The genus Prochlorothrix, originally with only one named species, P. hollandica (Burger-Wiersma et al., 1989), now has a second species, P. scandica, proposed by Pinevich et al. (1999) for the isolate from Lake Malaren, Sweden, which differs from P. hollandica in having filaments composed of shorter cells of larger diameter. The two species also differ in the size, shape, and positioning of certain cytoplasmic inclusions, and in the presence in P. scandica of a starch-like polyglycan.
Within the Prochlorococcus genus, two major ecotypes have been recognized on the basis of photosynthetic pigment ratios and associated physiological differences, namely the low chl b/chl a (high-light, HL) and high chl b/chl a (low-light, LL) ecotypes (see the section "Photosynthetic Productivity"). Analysis of the clustering of various strains in phylogenetic trees, based on nucleotide sequences in DNA fragments, has indicated clear differences between the two ecotypes. Thus, trees based on 16S rRNA sequences clearly separate the two ecotypes, suggesting that the variations in their photosynthetic characteristics are genetically determined (Moore et al., 1998; Urbach & Chisholm, 1998). They also suggest that the low chl b/chl a ecotypes are the most recently evolved lineage (Urbach et al., 1998; Rocap et al., 2002), one in which there has been evolutionary loss of the ability to utilize [NO.sub.3.sup.-] and [NO.sub.2.sup.-] (Moore et al., 2002). Those strains able to utilize [NO.sub.2.sup.-] (a number of high chl b/chl a ecotypes) are distributed between at least two phylogenetic clusters, suggesting that loss of the ability to utilize [NO.sub.2.sup.-] may have occurred a number of times in the Prochlorococcus lineage (Moore et al., 2002). The ability to use [NO.sub.3.sup.-], it was concluded, was lost early in the evolution of Prochlorococcus from its closely related ancestor.
A study of various aspects of the photophysiology of 10 isolates of Prochlorococcus from diverse oceanographic regions (Moore & Chisholm, 1999) showed that they could be grouped into two loose clusters, based on their growth responses to varying light intensity and their chl [b/a.sub.2] ratios. Compared with most other phytoplankton groups, all of the Prochlorococcus isolates were able to grow and photosynthesize very efficiently under low light conditions, but one group (comprising previously recognized high-light-adapted ecotypes) grew optimally at light intensities that totally photoinhibited the remaining isolates (previously designated low-light-adapted ecotypes). The former group, which displayed very uniform physiological characteristics, formed a distinct cluster in phylogenetic analyses based on 16S rDNA sequences. The second group did not, however, form such a well-supported phylogenetic group, consistent with their more diverse physiological characteristics.
These differences between (and to an extent within) the groups were interpreted as accounting for the ecological success of Prochlorococcus over a broad range of oceanographic environments (Moore & Chisholm, 1999). The low-light-adapted strain of Prochlorococcus (SS 120) has been shown to have a gene family encoding seven different DV chl a/b-binding proteins, while the high-light-adapted strain (MED 4) has only a single pcb gene (Garczarek et al., 2000). The two PSI pigment protein subunits of the two Prochlorococcus ecotypes had sequence similarities of only 80.2% (for PsaA) and 88.9% (for PsaB). Such a rapid evolutionary divergence as this implies was interpreted by van der Staay et al. (2000) as pointing to light availability as a major driving force in the evolution of those key photosynthetic proteins (and perhaps for the PSI core as well).
The first strain of Prochlorococcus to be brought into axenic culture, P. marinus Chisholm et al., 1992 subsp, pastoris subsp, nov. (van der Staay et al., 2000) (type strain isolate PCC 9511), on the basis of its 16S rRNA sequences, clusters with members of the HL clade (van der Staay et al., 2000). But it differs from another member of this clade, CCMP 1375, in many respects, including in having a lower chl [b/a.sub.2] ratio (<0.1) and a slightly different arrangement of the thylakoids, differences that van der Staay et al. (2000) suggest may be due to its having been a minor component of the parental culture from which both strains are derived. Van der Staay et al. (2000) caution that primary nonclonal cultures established from natural populations may change both phenotypically and genotypically over prolonged periods of culture through selection driven by the conditions to which they are exposed.
Improved probe hybridization and quantitative polymerase chain reaction (PCR) methods, matching data previously obtained by flow cytometry, have confirmed the identification of six evolutionary lineages of Prochlorococcus as ecotypes with distinct oceanic distributions (Zinser et al., 2006).
RELATEDNESS TO CYANOBACTERIA
There has been considerable speculation as to whether the presumed common ancestor of the prochlorophytes, containing both chl a and b and phycobilins, might also be regarded as the ancestor of the lineage leading to cyanobacteria (through loss of chl b) and green chloroplasts (through loss of phycobilins--to be discussed in a later section).
For Prochloron, special attention has been directed toward its possible relatedness to Synechocystis didemni, a phycoerythrin-containing cyanobacterium (Neveux et al., 1988) that, like Prochloron, also grows in association with an ascidian, namely Trididemnum solidum (Lafargue & Duclaux, 1979). On a range of criteria such as DNA-DNA reassociation characteristics (Holton et al., 1990), nucleotide sequences of the 16S rRNA gene, and the gene coding for the large subunit of RuBisCO (rbcL) (Shimada et al., 2003), as well as the DNA-dependent RNA polymerase gene (rpoC) (Palenik & Swift, 1996), the similarity between the prochlorophyte and the cyanobacterium, although considerable, was shown to be much lower than that among the various Prochloron isolates tested. This led to the conclusion (Shimada et al., 2003) that Prochloron, although probably specifically distinct from S. didemni, is nevertheless closely related to it. Shimada et al. (2003) further suggested that both species may have evolved from a Synechocystis-like ancestor, leaving unanswered the intriguing question as to whether Prochloron evolved from its cyanobacterial ancestor before establishing the symbiotic relationship with ascidians, and, if so, whether there may be some other free-living Prochloron-like prokaryotes yet to be discovered.
Analysis of 16S rRNA sequences of Prochlorothrix hollandica have shown that this prochlorophyte is more closely related to the cyanobacterium Synechococcus than to any other of the range of organisms tested (Turner et al., 1989). The same is also true for Prochlorococcus, all strains of which so far tested for their 16S rRNA sequences have been shown to group most closely with a range of marine and freshwater strains of Synechococcus (Urbach et al., 1992; van der Staay et al., 2000). An affinity of this prochlorophyte with Synechococcus was also apparent from analysis of sequences in the rpoC1 gene (Urbach et al., 1992). All strains of Synechococcus have a mean DNA base composition of 59-70 mol% G+C (Herdman et al., 1979) compared with the much lower 32 mol% value reported for the PCC 9511 strain of Prochlorococcus (van der Staay et al., 2000) and confirmed for a number of other strains (maximum value 38 mol%). Only one strain of Prochlorococcus, MIT 4303, has so far been shown to stand apart from the others in this respect, having a G+C content of 52-58 mol% in the third codon position of its psbB and petB/D genes (Urbach et al., 1998).
Another feature that separates the known Prochlorococcus strains from cyanobacteria such as Synechococcus is the size of the genome. Thus, Prochlorococcus strain PCC 9511 has a genome of about 2 Mbp (van der Staay et al., 2000), compared with the genome of Synechococcus, which has about 4 Mbp (Herdman et al., 1979). The small size (1.81 Mbp) of the genome of P. marinus CCMP1375 has been interpreted as indicating that Prochlorococcus may have evolved from an ancestral cyanobacterium, perhaps belonging to the Synechococcus group (Urbach et al., 1998), by a reduction in genome size (and cell size). The fact that some cyanobacteria lose their phycobiliproteins under conditions of iron depletion (Ferreira & Straus, 1994), with a consequent replacement of the phycobilisomes by a reduced antenna system (Partensky et al., 1999), suggests a possible mechanism for such an evolutionary change.
Some key differences between the genome of Prochlorococcus and those of cyanobacteria have been reported by Hess et al. (2001). Strains MED4 (high-light-adapted) and MIT9313 (low-light-adapted) both contain a gene cluster for RuBisCO and carboxysomal proteins, which Hess et al. (2001) describe as being clearly different from that genomic region in cyanobacteria. It has further been reported that the prochlorophyte genomes also contain genes for enzymes involved in the synthesis of [alpha]-carotene, a carotenoid not produced by the cyanobacteria. Several genes and operons that in the cyanobacteria are involved in light harvesting, nitrate utilization, and the generation of circadian rhythms of the cell cycle have been reported by Hess et al. (2001) to be reduced in the prochlorophytes, but to different degrees in the high- and low-light-adapted ecotypes. Thus, MED4 was noted to have more of those genes encoding highlight-inducible proteins and photolyases than does MIT9313. The latter strain, however, has more genes involved in producing more complex light-harvesting structures, including chromophorylated phycoerythrin, which has a structure intermediate between that of the phycobiliproteins of non-chl b-containing cyanobacteria and that of the degenerated phycoerythrin present in MED4.
The presence of phycobiliproteins in some strains of Prochlorococcus, in addition to the chl [a.sub.2]/[b.sub.2] light-harvesting complex, supports the view, based on 16S rRNA phylogenetic analysis (Urbach et al., 1998), that this prochlorophyte shares a common ancestry with the cyanobacteria (e.g., Synechococcus). It has been proposed that in its evolution from this ancestor, Prochlorococcus acquired the chl [a.sub.2]/[b.sub.2] light-harvesting complex while still retaining genes encoding the phycobiliprotein PE (Hess et al., 1996; La Roche et al., 1996; Ting et al., 1999). Although the possibility cannot be discounted that Prochlorococcus may have received its PE genes by lateral transfer from a close relative, it is considered more likely, and more analytically parsimonious, that its PE genes have been retained from a phycobiliprotein-containing ancestor (Hess et al., 1999; Ting et al., 2001).
Ting et al. (2001) sequenced the genes cpeA and cpeB, encoding respectively the et and [beta] subunits of PE, in a number of Prochlorococcus strains and found that they cluster relative to Synechococcus differently from the way they do in trees based on 16S rRNA. Whether this different clustering is due to different selection processes or to elevated mutation rates is yet to be established. The high-light-adapted strain (MED4), which on other criteria is thought to have arisen more recently than its low-light-adapted counterparts from the presumed phycobiliprotein and chl [a.sub.2]/[b.sub.2]-containing ancestor (Urbach et al., 1998), has been shown to lack the gene encoding the [alpha]-PE subunit and to have only a degenerate form of cpeB. The explanation for this is not clear, nor is it known whether PE has a physiological role in Prochlorococcus.
Close similarities between prochloropbytes and certain cyanobacteria have been revealed by comparison of the chl a/b-binding proteins of the former (pcb proteins) and the chl a-binding proteins of the latter. Thus, peptide sequences of chl a/b-binding proteins from a range of prochlorophytes (encompassing the three major genera) have been shown by La Roche et al. (1996) to have a high similarity with the chl a-binding proteins encoded by the cyanobacterial gene isiA. Gene cloning and sequencing confirmed that the prochlorophyte chl a/b protein genes (pcb genes) were related to but distinct from isiA genes, suggesting that the two proteins may have originated by gene duplication in a common ancestor. The amino acid sequences deduced from prochlorophyte pcb genes showed an average 54% correspondence with cyanobacterial isiA sequences.
The antennae proteins of Prochlorothrix hollandica occur as two subunits, pcbA and pcbC (32 and 38 kDa, respectively), and a third minor polypeptide pcbB (33 kDa) (van der Staay et al., 1998). Phylogenetic trees based on sequence analysis cluster the pcbA and pcbB proteins with cyanobacterial isiA proteins, while the pcbC proteins occupy a separate branch, suggesting that they may have originated from a different ancestral gene duplication from the one that led to the cyanobacterial isiA proteins and the rest of the pcbs. It was concluded that the pcb genes of prochlorophytes and the isiA genes of cyanobacteria belong to the same family of related genes encoding proteins that can bind either chl a+b or chl a alone. It has been suggested (e.g., La Roche et al., 1996) that only a relatively small number of changes in the primary sequence of the presumed common ancestor of the pcbs and isiA proteins would be necessary to permit binding of both chl b and chl a (or divinyl chls a and b) as well as a chl c-like pigment. The pcb proteins are therefore seen as originating from an ancestral protein already binding at least one type of chlorophyll (La Roche et al., 1996).
The relationship between prochlorophyte pcb genes and cyanobacterial isiA genes is especially interesting because the latter are induced only when the cyanobacteria are under iron stress (La Roche et al., 1996). In iron-deficient Synechocystis (PCC 6803), for example, expression of the isiA gene results in the accumulation in the cell of its product, the chl a-binding protein CP-43, and an accompanying lowering of the phycobilisome and PSI components (Bibby et al., 2001a).
Although it has been suggested that the characteristic array of prochlorophyte pigment complexes is coded by genes retained from a phycobiliprotein-containing ancestor (Hess et al., 1999; Ting et al., 2001), the possibility of lateral transfer of genes should also be considered. Such lateral transfers have been shown to have occurred, for example, between Prochlorococcus viruses (phages) and their hosts (Lindell et al., 2004), where phage genes encoding for a number of photosynthesis-related proteins were identified as being of host (prochlorophyte) origin, psbA and psbD genes encoding for the [D.sub.1] and [D.sub.2] core components of the PSII reaction center have been identified in a range of such bacteriophage (cyanomyovirus) isolates (Millard et al., 2004). The phage psbA genes were further shown to fall into a clade that includes the psbA genes of the host, including Prochlorococcus, suggesting that they have been acquired through horizontal gene transfer from the host. The fact that the phage psbA genes form a distinct subclade within this lineage was interpreted as indicating that their acquisition was not very recent.
There is evidence that the psbA and psbD genes were acquired by cyanomyoviruses more than once, and that their horizontal transfer between phages via a common phage gene pool may represent a continuing process. Multiple transfers from host to phage have also been suggested for genes encoding for certain high-light-inducible proteins (hli) and for the enzyme transaldolase (Lindell et al., 2004: Millard et al., 2004). Transfer of the genes back to the host after a period of evolution in the phage would mediate expansion of the respective gene families.
An analysis of the genomes of three Prochlorococcus phages by Sullivan et al. (2005) has shown them to contain photosynthetic genes, which, if functional, might be capable of maintaining host photosynthetic activity during infection. Such phage genomes would represent a pool of genes that, through lateral transfer, could play a major role in the evolution of the prochlorophyte photosynthetic apparatus (Zeidner et al., 2005). Genomic islands that have been described for Prochlorococcus populations, resembling the pathogenicity islands of some parasitic bacteria, are thought to have arisen, at least in part, by phage-mediated lateral transfer of genes that are differentially expressed under conditions of light or nutrient stress (Coleman et al., 2006).
RELATEDNESS TO CHLOROPLASTS AND CYANELLES
Analyses of DNA sequences within the 16S rRNA gene (Urbach et al., 1992) and the rpoC1 gene (Palenik & Haselkorn, 1992) have indicated that none of the prochlorophytes that have been examined are positioned in the lineage leading directly to green chloroplasts. Moreover, prochlorophyte pcbs have been reported to be quite different from the chlorophyll-binding proteins CP47 and CP43, which are present in all other oxyphototrophic organisms (Garczarek et al., 2001b). They appear not to belong to the superfamily of light-harvesting chlorophyll-protein complexes, since none of the prochlorophyte pcb sequences had any detectable relatedness to those of eukaryotic chl a/b antennae proteins.
Another form of sequence analysis much used in comparing different photosynthetic organisms is that which focuses on a sequence of seven amino acids near the carboxy terminus of the so-called D1 polypeptides (Morden & Golden, 1989; Maid et al., 1990; Winhauer et al., 1991; Lockhardt et al., 1993). The presence or absence of this fragment has been much used as a phylogenetic marker in studies of cyanobacteria, algae, and plant chloroplasts (e.g., Winhauer et al., 1991). It has been shown to be present in all cyanobacteria and in non-green algal plastids but absent from all green plant chloroplasts. Prochlorothrix hollandica lines up, in this respect, with green plant chloroplasts (Morden & Golden, 1989) in lacking this fragment, while both Prochloron (Lockhardt et al., 1993) and Prochlorococcus marinus (Hess et al., 1995) appear to have it; on this criterion, therefore, the latter might be regarded as being more closely related to the cyanobacteria (and Cyanophora, see below).
Evidence presented by Tomitani et al. (1999), based on analysis of amino acid sequences in CAO, the enzyme that catalyzes conversion of chl a to chl b, reveals strong similarities between prochlorophytes and chlorophytes, indicating that their respective chl b synthesis genes may have a common evolutionary origin. This, together with the presence of phycobilins in Prochlorococcus, cyanobacteria, and some eukaryotic chloroplasts, points to a chl b-containing oxygenic photosynthetic prokaryote (resembling the prochlorophytes) as a likely ancestral form of chloroplasts. Tomitani et al. (1999) further speculated that in the presumed ancestral eukaryotic chloroplast, chl b might have been bound to pcbs (as in prochlorophytes) and then transferred to chlorophyll a/b-binding proteins, which came into being only after the primary symbiotic event.
Interesting information on possible relationships between prochlorophytes and higher plant chloroplasts has come from studies of their respective plastocyanins (Pcs), the redox proteins that function as mobile electron carriers in PSI. The Pc of Prochlorothrix hollandica has been shown to differ from other Pcs in a number of ways, principally in having two unique residues (Tyr-12 and Pro-14) instead of the usual Gly10 and Leu-12 (Babu et al., 1999). Studies with modified forms of Prochlorothrix Pcs, induced by site-directed mutagenesis (Navarro et al., 2001), have shown that when the Pro-14 is replaced by the conserved leucine of higher plant plastocyanins, its reactivity is enhanced. This finding was interpreted by Navarro et al. (2001) as indicating that the Pc of Prochlorothrix is a divergent protein that appeared before evolution of the more effective form having leucine at position 14.
The discovery that Acaryochloris marina, a symbiotic prokaryote capable of high rates of oxygenic photosynthesis, has chlorophyll d (3-desvinyl-3 formyl chl a) as its major light-harvesting pigment (Miyashita et al., 1996), highlighted the wide diversity of pigment composition found among the photo-oxygenic prokaryotes, challenging the former unique status of the prochlorophytes. The major light-harvesting protein complex of A. marina has been isolated by Chen et al. (2002) and shown to contain chl a and chl d. Further characterization of the light-harvesting protein complex by electrophoresis and spectral analysis has confirmed that the main protein component is a polypeptide similar in size (34 kDa) to that of prochlorophyte light-harvesting protein complexes. The evolution of chlorophyll d as a reaction-center pigment in A. marina has been interpreted (Melkonian, 2001) as an adaptation of a presumed primitive photosynthetic prokaryote to maximize photosynthesis under far-red light. The phylogenetic relationships among A. marina, cyanobacteria (including prochlorophytes), and plastids have been examined using sequences in the small subunit (SSU) of rRNA (Miyashita et al., 2003). On the basis of this analysis, A. marina, like the prochlorophytes, was concluded to fall within the cyanobacterial radiation and remote from the plastids.
Cyanophora paradoxa is a flagellated protozoan that contains a plastid (cyanelle) having many cyanobacteria-like features, including the possession of phycobiliproteins (Bryant et al., 1985; Lemaux & Grossman, 1985) and an envelope composed of peptidoglycans outside its bounding membrane (Aitken & Stanier, 1979), suggesting that it may be the result of endosymbiosis between a cyanobacterium and a eukaryotic host. Because of these features and the small size of its genome (similar in size and gene content to plastid genomes), the cyanelle has been regarded by some (e.g., Cavalier-Smith, 1982; Maxwell et al., 1986) as having much in common with the presumed symbiont precursor of plastids. Comparisons of DNA sequence information for a portion of the rpoC1 gene (which encodes subunits of DNA-dependent RNA polymerase) (Palenik & Haselkorn, 1992) and of 16S rRNA data led Bryant (1992) to conclude that although the phycobilin-containing cyanelle may be closely related to the chlorophyll a- and b-containing plastids of algae, liverworts, and higher plants, they have no particular affinity with the prochlorophyte samples tested.
Summary and Conclusions
Among the range of characteristics reported here, some unequivocally align the prochlorophytes with the cyanobacteria, and others can best be accommodated if the prochlorophytes are separated from the other cyanobacteria and kept together as a distinct taxonomic entity. Their prokaryotic ultrastructure, including a bacterial-sized (or smaller) genome, a bacterial-like cell wall, and the universal presence of carboxysomes, all point to affinities with the cyanobacteria. They differ from the other cyanobacteria in having chl b as a major accessory pigment, located in stacked thylakoids and contained in chl a/b-protein complexes, which differ in a number of ways from equivalent complexes in cyanobacteria.
The absence of phycobilin pigments, formerly considered to be another diagnostic feature of the group, ceased to be such following discovery of traces of PE in some strains of Prochlorococcus. Expression of the genes coding for the [alpha] and [beta] subunits of the phycobilin pigment in the SS120 strain of Prochlorococcus has been demonstrated at both the RNA and protein level, but at a low level (Hess et al., 1996, 1999). The Prochlorococcus phycobilin is not, however, identical with that in cyanobacteria, nor is it, like the cyanobacterial pigment, contained within phycobilisomes. Its functional significance is also not clear, and it may, like the presence in that organism of divinyl forms of chlorophyll, represent no more than a special adaptation to low light conditions.
Apart from their gross morphological differences and their widely divergent habitats, the three prochlorophyte genera differ from each other in many other respects. Prochlorococcus, for example, stands apart from the other two genera in having divinyl forms of chlorophyll, presumed to be the result of evolutionary loss of the DVR enzyme, which in the other prochlorophyte genera, as in all other photosynthetic organisms, catalyzes synthesis of the monovinyl form of chlorophyll from the divinyl form. Prochlorococcus is also different from the other two genera in having, generally, much lower chl a:b ratios and in having [alpha]-carotene rather than [beta]-carotene as a major carotenoid.
The evidence from phylogenetic trees based on DNA sequence data is equivocal with respect to both the integrity of the prochlorophytes as a group and affinities between the three prochlorophyte genera and the cyanobacteria. Thus, while some of the data have been interpreted as indicating that chl b and its associated chl a/b-binding proteins probably developed independently in the lineages leading to the three prochlorophyte genera, other data are best interpreted on the basis of a common chl b-containing ancestor for the prochlorophytes, and for the cyanobacteria as well. Molecular data on the latter point has come from a variety of sources, most significantly from data indicating that pcb proteins and the isiA proteins of cyanobacteria are coded by a family of related genes. Prochlorococcus marinus (CCMP 1375) with its chl a/b/c antennae and PE pigment (Hess et al., 1996) might be interpreted as a model of the presumed common ancestor from which, through evolutionary loss of certain antenna systems, lineages of organisms having several types of chlorophyll and biliproteins might have developed.
There is little evidence from DNA sequencing data of any particular affinity between the prochlorophytes and green chloroplasts beyond the presence in both groups of chl b. The DV chl a/b-binding protein of Prochlorococcus, for example, is not directly related to the known chl a/b-binding (CAB) proteins of chloroplasts, but instead appears to be more like an evolutionary derivative of the psbC gene (La Roche et al., 1996).
The limitations inherent in interpreting molecular data have been highlighted by Larkum (1999), who, along with van der Peer et al. (1996), has cautioned against overreliance upon molecular data to the exclusion of structural, chemical, biochemical, and physiological characteristics. When taken together, the molecular and nonmolecular information as presented in this review, favors retention of the three prochlorophyte genera as a subclass (Chloroxybacteria) of the Oxyphotobacteria, sufficiently closely related to the cyanobacteria to indicate that they may share a common origin with them and possibly with eukaryotic chloroplasts. It would appear to be unlikely, however, on the evidence available, that the prochlorophytes and green chloroplasts occupy anything closer than remote divergent branches arising from this common ancestor.
Before discovery of the two free-living genera, the prochlorophytes, as then represented by various strains of the symbiotic Prochloron, were of primary interest only with respect to considerations of the evolution of photosynthetic systems. The inclusion of Prochlorothrix and Prochlorococcus within the group has brought added interest and, in the case of the latter genus especially, considerable ecological significance because of its demonstrated contribution to oceanic productivity. The convenience of retaining three such otherwise dissimilar genera within the one taxonomic grouping (Oxychlorobacteria), as dictated by their unique combination of ultrastructural and pigment characteristics, would appear to outweigh the case for their separate allocation to different orders of the cyanobacteria.
I thank Professor Ralph A. Lewin, Scripps Institution of Oceanography, University of California, San Diego, USA, for reading the manuscript and for his interest.
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DILWYN J. GRIFFITHS
School of Biological Sciences
James Cook University of North Queensland
Townsville, Queensland 4811, Australia
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|Author:||Griffiths, Dilwyn J.|
|Publication:||The Botanical Review|
|Date:||Oct 1, 2006|
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