Major maker of oxygen and eater carbon dioxide.

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Prochlorococcus marinus SS120

One of the smallest photosynthetic organisms known

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The marine cyanobacteria from the Prochlorococcus genus are the smallest photosynthetic organisms known; they are also the most abundant photosynthetic organisms in the oceans, and therefore on the planet. With a size of less than one micrometer (diameter between 0.5 and .07 micrometer), these prokaryotic cells were not noticed until the end of the 1980s, although the existence of photosynthetic organisms of the order of one micron in size (picophytoplankton) has been known since 1979. In fact, the cells of Prochlorococcus were indistinguishable from heterotrophic bacteria with traditional microscopic techniques, and only flow cytometry revealed their presence: in 1988, S.W. Chisholm and her group from the Massachusetts Institute of Technology (MIT) announced that they had discovered a new class of prokaryotic picoplanktonic organisms using a flow cytometer on board a ship. These cells were different from typical cyanobacteria of the genus Synechococcus in their weak red fluorescence signal and almost complete absence of orange fluorescence, which indicated a very unusual composition of photosynthetic pigments for a cyanobacterium. Furthermore, these organisms seemed to be very abundant - more than 100 000 cells/milliliter in open seas, which are very poor in minerals (oligotrophy). We know today that the cyanobacteria of the genus Prochlorococcus are the dominant organisms in terms of numbers in the central zones of all the oceans between 40 degrees north latitude and 40 degrees south. In these oligotrophic waters, they represent 40 to 50% of the phytoplankton biomass. Moreover, they are present down to 200 meters under the surface, a depth at which light energy is a thousand times weaker than at the surface.

The reasons for studying the bacteria from the Prochlorococcus genus are numerous. First of all, these are organisms of great ecological importance; along with other marine cyanobacteria from the Synechococcus genus, these cells are some of the principal primary producers of the phytoplankton, which is responsible of half of the photosynthesis on the planet. These bacteria are therefore prominent actors in global oceanic function, in the carbon cycle, and consequently in the evolution of climate.

Bacteria of the Prochlorococcus genus are also quite remarkable in their adaptations:

• a tiny size which facilitates the acquisition of mineral salts - which are present in very low concentrations — due to the increase in the surface/volume ratio; furthermore, the reduction in cell diameter increases the coefficient of absorption of the photosynthetic pigments by reducing the package effect: the presence of several layers of photosynthetic membranes (or thylakoids) has the effect of reducing the contribution of pigments situated in the interior parts of the cell relative to the more peripheral pigments, and this effect diminishes with the size of the cell.
• a genome with a very small size in some strains, a characteristic which is certainly due to the small size of the cells and the low concentrations of nitrogen and phosphorus which makes nucleotides costly. The great stability of the environment of these strains makes the consequences of gene loss - i.e. loss of potential for adaptation
   less detrimental. With a size of about 1.7 Mb, the genomes of these strains are the smallest known among the cyanobacteria, and more generally among oxyphototrophic organisms. The inventory of the genes retained in such “minimal” genomes is of great interest.
• an unusual pigment composition, with the presence of divinyl derivatives of chlorophyll a and chlorophyll b, unique to the genus Prochlorococcus. These pigments, which are associated with proteins in the thylakoid membranes, constitute the major photosynthetic antenna of Prochlorococcus. In the “typical” cyanobacteria, on the contrary, and notably in Synechococcus, chlorophyll a plays only a secondary role and the solar energy is mainly collected by complexes called phycobilisomes, which contain highly colored pigments, the phycobilins, and other proteins. The phycobilisomes seem to be absent in Prochlorococcus, or present as only vestigial structures in some strains. This replacement of phycobilisomes by a chlorophyll a/b antenna embedded in the thylakoid membranes is also found in two other organisms, Prochloron andProchlorothrix, which are sometimes grouped with Prochlorococcus under the name “prochlorophytes”. The nature of the antenna is similar in the three prochlorophytes and its organization in the thylakoids is reminiscent to that of green algae and higher plants. It has been shown, however, that the prochlorophytes form three well-separated lineages in the radiation of the cyanobacteria, and that none of the three genera is on the lineage which leads to the chloroplasts of plant cells. This therefore represents a remarkable example of convergent evolution. Furthermore,Prochlorococcus also differs from other prochlorophytes in the presence of alpha-carotene instead of beta-carotene, in addition to the presence of the divinyl derivatives of chlorophylls.

An understanding of the role of Prochlorococcus cyanobacteria in the global oceanic ecosystem will require a detailed study of their physiological characteristics. These characteristics seem to be remarkably diverse, with the presence of at least two ecotypes which are genetically and physiologically quite different; between these ecotypes, and even within them, there is a great variation in traits such as pigment ratios, optimal light intensity for growth, efficacy of absorption of the antenna, capacity to use nitrogen, specificity with regard to cyanophages (phages which infect cyanobacteria), etc.

These differences may appear surprising at first for a photoautotrophic organism which requires only light, carbon dioxide and mineral salts for growth and which lives in an environment made uniform by the movement of the water. The water column in the central zones of the oceans at tropical and sub-tropical latitudes is in fact stratified by density and offers several ecological niches: the surface waters receive a large amount of light energy and the concentration in mineral salts is very low; on the other hand, the region between depths of 100 and 200 meters receives very little light with wavelengths shifted toward the blues, but is richer in nutrient salts. The strains of Prochlorococcus which grow in the surface waters form the high-light-adapted ecotype; their antennae contain mainly the divinyl derivative of chlorophyll a. The strains which grow preferentially at depths between 80 and 200 meters form the low-light-adapted ecotype and contain a lot of divinyl-chlorophyll b, which absorbs optimally in the blue area of the spectrum. The genomes of these strains possess several copies of the gene which codes for the antenna protein; this is likely an adaptation to weak luminosity.

In order to elucidate the genetic basis of these adaptations more completely, scientists have undertaken the sequencing of strains of Prochlorococcus from different ecological niches. The Joint Genome Institute (Walnut Creek, California) has sequenced the genomes of two strains which are representative of high-light-adapted(the MED4 strain) and low-light-adapted (the MIT9313 strain) ecotypes. However, the genetic diversity of the low-light-adapted strains is much greater than that of high-light-adapted strains, and the genome sequence of the strain MIT9313 is not sufficient to illustrate evolutionary tendencies in these “deep water” strains. The sequencing of another low-light-adapted strain was therefore undertaken in parallel at Genoscope on the initiative of the "oceanic phytoplankton" team at the Roscoff Biological Station. This strain, named Prochlorococcus marinus SS120, is the type strain of the genus Prochlorococcus, and the best characterized from an ecophysiological point of view. From the phylogenetic point of view, the SS120 strain seems rather distant from the other deep water strain sequenced, MIT9313, which is rooted at the base of the radiation of Prochlorococcus.

The genomic sequences of Prochlorococcus SS120, MED4 and MIT9313 were described in two papers published in August 2003, at the same time as the sequence of a strain of marineSynechococcus, and their comparison have been instructive. The first lesson from these sequences is the confirmation of the heterogeneity of the deep water strains: the genome of SS120, which is 1.75 Mb and contains 1884 genes, is almost as small as the genome of the high-light-adapted strain MED4 (1.66 Mb, 1716 genes) and presents as a quasi-minimal genome; on the contrary, the genome of the second deep-water strain, MIT9313, is much larger (2.4 Mb, 2275 genes). Moreover, the annotation indicates that this latter strain is capable of exploiting a larger range of nutrients. For example, MIT9313 can use ammonium ions, nitrite, nitrate, cyanate, urea, amino acids and peptides as a source of nitrogen, whereas SS120 can only use ammonia and amino acids. SS120 seems to be adapted to a very stable environment at the limit of the zone of penetration of light, whereas MIT9313 seems to be adapted to less deep waters, corresponding to a transition zone in which the available resources are more diverse.

A comparison of the genomic sequence of Prochlorococcus marinus SS120 with that of the three fresh-water cyanobacteria which have already been sequenced reveals the nature of the absent or under-represented genes in the “minimal” genome of SS120: certain photosynthetic genes, and genes implicated in DNA repair, importation of solutes, intermediary metabolism and in numerous other functions. In particular, the genes involved in signal transduction and the response to environmental stress have suffered a drastic reduction in SS120, probably because of the great stability of the environment of this strain. Another factor in the compactness of the genome of SS120: it contains very few paralogous genes. One of the rare examples of gene amplification involves the 8 pcb genes encoding the antenna proteins (compared with 2 in MIT9313).

The ensemble of this genomic data will allow detailed ecophysiological studies. The idea is to gain a better understanding of the function of the picoplanktonic ecosystem, in response to environmental changes such as the increase in carbon dioxide in the atmosphere. Several teams implicated in the annotation of SS120, including the Roscoff term, are current participants in the European project MARGENES, which consists in using post-genomic approaches to identify and characterize genes which are specific to ecological niches or essential functions of marine cyanobacteria and diatoms (photoprotection, nutrition, detection of environmental signals, etc.).

Another subject that is being studied in several European and American laboratories is the population dynamics of Prochlorococcus and Synechococcus, which are assaulted by miniature eukaryotic predators of several micrometers in size, and are infected by cyanophages which seem moreover to be responsible for horizontal transfer. Here also, the idea is to characterize the genotypes which are involved in these interspecific relationships. Finally, the American Department of Energy (DOE), in the framework of its Genomes to Life (GTL) programme, is supporting the modeling of the global metabolism of Prochlorococcus MED4, based on the genomic sequence of this high-light-adapted strain. Totally ignored less than 20 years ago, Prochlorococcus is today the target of an impressive number of genomic, post-genomic, physiologic and ecologic studies befitting its important role in global oceanic function.

The annotation of the genomic sequence of Prochlorococcus marinus SS120 has been performed by a European consortium composed of the following laboratories:
"Oceanic phytoplankton" team (F. Partensky and D. Vaulot) at the Roscoff center for oceanography studies and marine biology (UMR 7127 CNRS and University Paris 6);Cyanobacteria unit (N. Tandeau de Marsac) at the Pasteur Institute (URA 2172 CNRS); Institüt für Biologie und Genetik (W. Hess, now at Ocean Genome Legacy), Humboldt Universität zu Berlin; Department of Biological Sciences (D. Scanlan), Warwick University, Coventry, UK.

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Publications

A. Dufresne et al. (2003), Genome sequence of the cyanobacterium Prochlorococcus marinusSS120, a nearly minimal oxyphototrophic genome, Proc. Natl. Acad. Sci. USA 100, 10020-10025.

G. Rocap et al. (2003), Genome divergence in two Prochlorococcus ecotypes reflects oceanic niche differentiation, Nature 424, 1042-1047.