Prochlorococcus and Synechococcus are the two most abundant and widespread phytoplankton in the global ocean. To better understand the factors controlling their biogeography, a reference database of the high-resolution taxonomic marker petB, encoding cytochrome b 6 , was used to recruit reads out of 109 metagenomes from the Tara Oceans expedition. An unsuspected novel genetic diversity was unveiled within both genera, even for the most abundant and well-characterized clades, and 136 divergent petB sequences were successfully assembled from metagenomic reads, significantly enriching the reference database. We then defined Ecologically Significant Taxonomic Units (ESTUs)-that is, organisms belonging to the same clade and occupying a common oceanic niche. Three major ESTU assemblages were identified along the cruise transect for Prochlorococcus and eight for Synechococcus. Although Prochlorococcus HLIIIA and HLIVA ESTUs codominated in irondepleted areas of the Pacific Ocean, CRD1 and the yet-to-be cultured EnvB were the prevalent Synechococcus clades in this area, with three different CRD1 and EnvB ESTUs occupying distinct ecological niches with regard to iron availability and temperature. Sharp community shifts were also observed over short geographic distances-for example, around the Marquesas Islands or between southern Indian and Atlantic Oceans-pointing to a tight correlation between ESTU assemblages and specific physico-chemical parameters. Together, this study demonstrates that there is a previously overlooked, ecologically meaningful, fine-scale diversity within some currently defined picocyanobacterial ecotypes, bringing novel insights into the ecology, diversity, and biology of the two most abundant phototrophs on Earth. molecular ecology | metagenomics | Tara Oceans | Synechococcus | Prochlorococcus

Marine photosynthetic picoeukaryotes (PPEs), representing organisms < 3 µm in size, are major contributors to global carbon cycling. However, the key members of the PPE community and hence the major routes of carbon fixation, particularly in the open ocean environment, are poorly described. Here, we have accessed PPE community structure using the plastid encoded 16S rRNA gene. Plastid 16S rRNA genes were sequenced from 65 algal cultures, about half being PPEs, representing 14 algal classes. These included sequences from 5 classes where previously no such sequences from cultured representatives had been available (Bolidophyceae, Dictyochophyceae, Eustigmatophyceae, Pelagophyceae and Pinguiophyceae). Sequences were also obtained for 6 of the 7 (according to 18S rRNA gene sequence) prasinophyte clades. Phylogenetic analysis revealed plastids from the same class as clustering together. Using all the obtained sequences, as well as plastid sequences currently in public databases, a non-degenerate marine algal plastid-biased PCR primer, PLA491F, was developed to minimize amplification of picocyanobacteria, which often dominate numerically environmental samples. Clone libraries subsequently constructed from the pico-sized fraction from 2 open ocean sites in the Arabian Sea, revealed an abundance of 16S rRNA gene clones phylogenetically related to chrysophytes, whilst prymnesiophyte, clade II prasinophyte (Ostreococcus-like) and pelagophyte clones were also well represented. The finding of a wealth of novel clones related to the Chrysophyceae highlights the utility of a PCR biased towards marine algal plastids as a valuable complement to 18S rDNA based studies of PPE diversity. KEY WORDS: Photosynthetic picoeukaryotes · Plastid 16S rRNA · Arabian Sea · PCR Resale or republication not permitted without written consent of the publisherAquat Microb Ecol 43: [79][80][81][82][83][84][85][86][87][88][89][90][91][92][93] 2006 guiophyceae (Kawachi et al. 2002). Yet despite the evident ecological significance of PPEs, relatively little is known of their diversity in the marine environment, particularly in the open ocean. This has been attributed mainly to difficulties in identification by light microscopy. Only recently, with the advent of molecular techniques, has picoeukaryote diversity begun to be revealed (for a recent review see Moreira & LopezGarcia 2002). Thus, phylogenetic studies based on 18S rRNA gene sequence analysis are now beginning to show the extent of taxonomic diversity within this group of organisms (Diez et al. 2001, Lopez-Garcia et al. 2001, Moon-van der Staay et al. 2001, Stoeck & Epstein 2003. Such studies, using 'universal' 18S rRNA gene primers (which target both [photo]autotrophs and heterotrophs) and clone library construction from environmental samples have demonstrated the presence of PPEs affiliated with many different algal classes including the Bacillariophyceae, Bolidophyceae, Chrysophyceae, Cryptophyceae, Dictyochophyceae, Dinophyceae, Eustigmatophyceae, Glaucocystophyceae, Pelagoph...

Synechocystis sp. strain PCC 6803 possesses two putative ABC-type inorganic phosphate (P i ) transporters with three associated P i -binding proteins (PBPs), SphX (encoded by sll0679), PstS1 (encoded by sll0680), and PstS2 (encoded by slr1247), organized in two spatially discrete gene clusters, pst1 and pst2. We used a combination of mutagenesis, gene expression, and radiotracer uptake analyses to functionally characterize the role of these PBPs and associated gene clusters. Quantitative PCR (qPCR) demonstrated that pstS1 was expressed at a high level in P i -replete conditions compared to sphX or pstS2. However, a P i stress shift increased expression of pstS2 318-fold after 48 h, compared to 43-fold for pstS1 and 37-fold for sphX. A shift to high-light conditions caused a transient increase of all PBPs, whereas N stress primarily increased expression of sphX.Interposon mutagenesis of each PBP demonstrated that disruption of pstS1 alone caused constitutive expression of pho regulon genes, implicating PstS1 as a major component of the P i sensing machinery. The pstS1 mutant was also transformation incompetent.32 P i radiotracer uptake experiments using pst1 and pst2 deletion mutants showed that Pst1 acts as a low-affinity, high-velocity transporter (K s , 3.7 ؎ 0.7 M; V max , 31.18 ؎ 3.96 fmol cell ؊1 min ؊1 ) and Pst2 acts as a high-affinity, low-velocity system (K s , 0.07 ؎ 0.01 M; V max , 0.88 ؎ 0.11 fmol cell ؊1 min ؊1 ). These P i ABC transporters thus exhibit differences in both kinetic and regulatory properties, the former trait potentially dramatically increasing the dynamic range of P i transport into the cell, which has potential implications for our understanding of the ecological success of this key microbial group.Phosphorus input into aquatic systems is largely in the form of poorly soluble, eroded mineral phosphate, which enters these systems via runoff from land, making P i a key growthlimiting nutrient, particularly in freshwater environments (13,23,41). A recent survey of 34 inland lakes from three (physiographic) regions of Canada (25) revealed total P i concentrations ranging between 0.058 and 7.64 M. Thus, organisms occupying such environments invariably need to make key biochemical and regulatory adaptations to their P i uptake system in order to sustain growth. One such group is the cyanobacteria, one of the largest, most diverse, and most widely distributed prokaryotic lineages (42). Their ability to acclimate to a varying-light environment as well as their ability to acquire nutrients present at low ambient concentrations has led to their present-day dominance in vast tracts of oligotrophic open ocean waters (40) and in freshwater systems (14).Studies of bacterial P i acquisition have largely focused on model organisms such as Escherichia coli (52) and Bacillus subtilis (26). In E. coli, uptake utilizes both a low-affinity permease, the Pit system (54) [with uptake of P i being reliant on cotransport with divalent metal cations such as Mg(II) or Ca(II) through the formation of a soluble...