Prochlorococcus Isolates Research Articles

Genomes of Diverse Isolates of Prochlorococcus High-Light-Adapted Clade II in the Western Pacific Ocean

Genomes of Diverse Isolates of Prochlorococcus High-Light-Adapted Clade II in the Western Pacific Ocean

Genomic potential for arsenic efflux and methylation varies among global Prochlorococcus populations.

The globally significant picocyanobacterium Prochlorococcus is the main primary producer in oligotrophic subtropical gyres. When phosphate concentrations are very low in the marine environment, the mol:mol availability of phosphate relative to the chemically similar arsenate molecule is reduced, potentially resulting in increased cellular arsenic exposure. To mediate accidental arsenate uptake, some Prochlorococcus isolates contain genes encoding a full or partial efflux detoxification pathway, consisting of an arsenate reductase (arsC), an arsenite-specific efflux pump (acr3) and an arsenic-related repressive regulator (arsR). This efflux pathway was the only previously known arsenic detox pathway in Prochlorococcus. We have identified an additional putative arsenic mediation strategy in Prochlorococcus driven by the enzyme arsenite S-adenosylmethionine methyltransferase (ArsM) which can convert inorganic arsenic into more innocuous organic forms and appears to be a more widespread mode of detoxification. We used a phylogenetically informed approach to identify Prochlorococcus linked arsenic genes from both pathways in the Global Ocean Sampling survey. The putative arsenic methylation pathway is nearly ubiquitously present in global Prochlorococcus populations. In contrast, the complete efflux pathway is only maintained in populations which experience extremely low PO4:AsO4, such as regions in the tropical and subtropical Atlantic. Thus, environmental exposure to arsenic appears to select for maintenance of the efflux detoxification pathway in Prochlorococcus. The differential distribution of these two pathways has implications for global arsenic cycling, as their associated end products, arsenite or organoarsenicals, have differing biochemical activities and residence times.

Prevalence of a calcium‐based alkaline phosphatase associated with the marine cyanobacterium Prochlorococcus and other ocean bacteria

Phosphate plays a key role in regulating primary productivity in several regions of the world's oceans and here dissolved organic phosphate can be an important phosphate source. A key enzyme for utilizing dissolved organic phosphate is alkaline phosphatase and the phoA-type of this enzyme has a zinc cofactor. As the dissolved zinc concentration is low in phosphate depleted environments, this has led to the hypothesis that some phytoplankton may be zinc-P co-limited. Recently, it was shown that many marine bacteria contain an alternative form of alkaline phosphatase called phoX, but it is unclear which marine lineages carry this enzyme. Here, we describe the occurrence in low phosphate environments of phoX that is associated with uncultured Prochlorococcus and SAR11 cells. Through heterologous expression, we demonstrate that phoX encodes an active phosphatase with a calcium cofactor. The enzyme also functions with magnesium and copper, whereas cobalt, manganese, nickel and zinc inhibit enzyme activity to various degrees. We also find that uncultured SAR11 cells and cyanophages contain a different alkaline phosphatase related to a variant present in several Prochlorococcus isolates. Overall, the results suggest that many bacterial lineages including Prochlorococcus and SAR11 may not be subject to zinc-P co-limitation.

Characterization ofProchlorococcusclades from iron-depleted oceanic regions

Prochlorococcus describes a diverse and abundant genus of marine photosynthetic microbes. It is primarily found in oligotrophic waters across the globe and plays a crucial role in energy and nutrient cycling in the ocean ecosystem. The abundance, global distribution, and availability of isolates make Prochlorococcus a model system for understanding marine microbial diversity and biogeochemical cycling. Analysis of 73 metagenomic samples from the Global Ocean Sampling expedition acquired in the Atlantic, Pacific, and Indian Oceans revealed the presence of two uncharacterized Prochlorococcus clades. A phylogenetic analysis using six different genetic markers places the clades close to known lineages adapted to high-light environments. The two uncharacterized clades consistently cooccur and dominate the surface waters of high-temperature, macronutrient-replete, and low-iron regions of the Eastern Equatorial Pacific upwelling and the tropical Indian Ocean. They are genetically distinct from each other and other high-light Prochlorococcus isolates and likely define a previously unrecognized ecotype. Our detailed genomic analysis indicates that these clades comprise organisms that are adapted to iron-depleted environments by reducing their iron quota through the loss of several iron-containing proteins that likely function as electron sinks in the photosynthetic pathway in other Prochlorococcus clades from high-light environments. The presence and inferred physiology of these clades may explain why Prochlorococcus populations from iron-depleted regions do not respond to iron fertilization experiments and further expand our understanding of how phytoplankton adapt to variations in nutrient availability in the ocean.

Minimal genomes, maximal productivity: comparative genomics of the photosystem and light-harvesting complexes in the marine cyanobacterium, Prochlorococcus

Although Prochlorococcus isolates possess the smallest genomes of any extant photosynthetic organism, this genus numerically dominates vast regions of the world's subtropical and tropical open oceans and has evolved to become an important contributor to global biogeochemical cycles. The sequencing of 12 Prochlorococcus genomes provides a glimpse of the extensive genetic heterogeneity and, thus, physiological potential of the lineage. In this study, we present an up-to-date comparative analysis of major proteins of the photosynthetic apparatus in 12 Prochlorococcus genomes. Our analyses reveal a striking diversity within the Prochlorococcus lineage in the major protein complexes of the photosynthetic apparatus. The heterogeneity that has evolved in the photosynthetic apparatus suggests versatility in strategies for optimizing photosynthesis under conditions of environmental variability and stress. This diversity could be particularly important in ensuring the survival of a lineage whose individuals have evolved minimal genomes and, thus, relatively limited repertoires for responding to environmental challenges.

Influence of light and temperature on Prochlorococcus ecotype distributions in the Atlantic Ocean

In a focused analysis of Prochlorococcus population structure in the western North Atlantic, we found that the relative abundances of ecotypes varied significantly with depth and, at seasonally stratified locations, with degree of vertical mixing. More limited regional variation was observed (e.g., Sargasso Sea, Gulf Stream, continental slope, and equatorial current), and local patchiness was minimal. Modeling of a combined North and South Atlantic data set revealed significant, independent effects of light and temperature on ecotype abundances, suggesting that they are key ecological determinants that establish the different habitat ranges of the physiologically and genetically distinct ecotypes. This was in sharp contrast with the genus Synechococcus, whose total abundance was related to light but did not vary in a predictable way with temperature. Comparisons of field abundances with growth characteristics of cultured isolates of Prochlorococcus suggested the presence of ecotypespecific thermal and light adaptations that could be responsible for the distinct distribution patterns of the four dominant ecotypes. Significantly, we discovered that one “low‐light‐adapted” ecotype, eNATL2A, can thrive in deeply mixed surface layers, whereas another, eMIT9313, cannot, even though they have the same growth optimum for (low) light.

Ecotypic variation in phosphorus-acquisition mechanisms within marine picocyanobacteria

Prochlorococcus and Synechococcus are major prokaryotic primary producers in the oligotrophic oceans that may be affected by the climate-related increases in nitrogen fixation and subsequent phosphorus (P) limitation in some parts of the oceans. Evidence that Prochlorococcus populations in the North Pacific subtropical gyre (NPSG) have increased over the past decades, possibly due to having a competitive advantage under conditions of P limitation, suggests aspects of their P physiology that are important for dictating their in situ success. Here, we compared the physiology of P acquisition and response to P stress (indicated by alkaline phosphatase activity, APA) among isolates of Prochlorococcus and Synechococcus representing different genotypic clades within the marine picophytoplankton lineage (sensu Urbach et al. 1998: J Mol Evol 46:188-201). The 2 Synechococcus isolates examined (WH 8102 and WH 7803) can utilize a wide variety of organic P sources. Of the 3 Prochlorococcus isolates examined, only the HLI genotype, MED4, is capable of growth on a variety of organic P sources. Under conditions of P starvation the 2 Synechococcus strains and Prochlorococcus MED4 exhibit significant increases in APA, above their measurable constitutive activities. The genomes of the Synechococcus strains and Prochlorococcus MED4 indicate the presence of many P-uptake and -regulatory genes required under conditions of P stress, including the phoA gene that encodes for an alkaline phosphatase enzyme. The other isolates of Prochloro- coccus, HLII MIT 9312 and LLIV MIT 9313, have distinctly different P-stress responses. MIT 9312, which contains the same P-uptake and -regulatory genes as MED4, except for psip1 and ptrA, has no constitutive APA, but does exhibit measurable, albeit very low, activity when P starved. MIT 9313, which lacks phoA and a functional phosphate-sensing histidine kinase gene, phoR, exhibits low constitutive activity that decreases when the cells become P-starved. These results show variability in P utilization and in P-stress response between the 2 genera of marine unicellular cyanobacteria, and marked differences among Prochlorococcus genotypes, implying that only certain eco/genotypes of these marine cyanobacteria will have an ecological advantage under conditions of P limitation in the oceans. This further points to the critical need to continue developing genotypic- or cell-specific tools to assess the response of the picophytoplankton community to large-scale changes in nutrient conditions in the oceans.

Cyanophages infecting the oceanic cyanobacterium Prochlorococcus.

Prochlorococcus is the numerically dominant phototroph in the tropical and subtropical oceans, accounting for half of the photosynthetic biomass in some areas. Here we report the isolation of cyanophages that infect Prochlorococcus, and show that although some are host-strain-specific, others cross-infect with closely related marine Synechococcus as well as between high-light- and low-light-adapted Prochlorococcus isolates, suggesting a mechanism for horizontal gene transfer. High-light-adapted Prochlorococcus hosts yielded Podoviridae exclusively, which were extremely host-specific, whereas low-light-adapted Prochlorococcus and all strains of Synechococcus yielded primarily Myoviridae, which has a broad host range. Finally, both Prochlorococcus and Synechococcus strain-specific cyanophage titres were low (< 10(3) ml(-1)) in stratified oligotrophic waters even where total cyanobacterial abundances were high (> 10(5) cells x ml(-1)). These low titres in areas of high total host cell abundance seem to be a feature of open ocean ecosystems. We hypothesize that gradients in cyanobacterial population diversity, growth rates, and/or the incidence of lysogeny underlie these trends.

Nitrate is reduced by heterotrophic bacteria but not transferred to Prochlorococcus in non-axenic cultures

The ability to assimilate nitrate in non-axenic isolates of Prochlorococcus spp. was addressed in this work, particularly in three low-irradiance adapted strains originating from ocean depths with measurable nitrate concentrations. None of the studied strains was able to use nitrate as the sole nitrogen source. Nitrate reductase (NR; EC 1.6.6.2) activity was, however, detected using the methyl viologen/dithionite assay in crude extracts from all studied Prochlorococcus strains. Characterization of this activity unambiguously demonstrated its enzymatic origin. We observed that NR activity did not decrease in vivo under darkness. Attempts to detect the narB gene (coding for NR in other cyanobacteria) by PCR with primers designed on the basis of the specific codon usage in Prochlorococcus were unsuccessful. However, when primers were designed considering the codon frequencies typical of other bacteria, we could amplify different fragments of nas genes, coding for bacterial assimilatory NRs. Similar amplification products were obtained using colonies of contaminant bacteria from Prochlorococcus cultures as PCR template. Furthermore, NR activity was found in cultures of these contaminants, demonstrating the non-cyanobacterial origin of the enzyme. These results strongly suggest that the studied strains of Prochlorococcus lack NR, in spite of inhabiting environments with nitrate as the main nitrogen source. In addition, they indicate that the nitrite produced by heterotrophic bacteria is not transferred to Prochlorococcus for growth, thus discarding a trophic nitrogen chain between heterotrophic bacteria and Prochlorococcus in the studied cultures.

Nitrate is reduced by heterotrophic bacteria but not transferred to Prochlorococcus in non-axenic cultures

Abstract The ability to assimilate nitrate in non-axenic isolates of Prochlorococcus spp. was addressed in this work, particularly in three low-irradiance adapted strains originating from ocean depths with measurable nitrate concentrations. None of the studied strains was able to use nitrate as the sole nitrogen source. Nitrate reductase (NR; EC 1.6.6.2) activity was, however, detected using the methyl viologen/dithionite assay in crude extracts from all studied Prochlorococcus strains. Characterization of this activity unambiguously demonstrated its enzymatic origin. We observed that NR activity did not decrease in vivo under darkness. Attempts to detect the narB gene (coding for NR in other cyanobacteria) by PCR with primers designed on the basis of the specific codon usage in Prochlorococcus were unsuccessful. However, when primers were designed considering the codon frequencies typical of other bacteria, we could amplify different fragments of nas genes, coding for bacterial assimilatory NRs. Similar amplification products were obtained using colonies of contaminant bacteria from Prochlorococcus cultures as PCR template. Furthermore, NR activity was found in cultures of these contaminants, demonstrating the non-cyanobacterial origin of the enzyme. These results strongly suggest that the studied strains of Prochlorococcus lack NR, in spite of inhabiting environments with nitrate as the main nitrogen source. In addition, they indicate that the nitrite produced by heterotrophic bacteria is not transferred to Prochlorococcus for growth, thus discarding a trophic nitrogen chain between heterotrophic bacteria and Prochlorococcus in the studied cultures.

Utilization of different nitrogen sources by the marine cyanobacteria Prochlorococcus and Synechococcus

Prochlorococcus is the most abundant phytoplankter throughout the photic zone in stratified marine waters and experiences distinct gradients of light and nitrogen nutrition. Physiologically and genetically distinct Prochlorococcus ecotypes partition the water column: high‐B/A (low‐light adapted) ecotypes are generally restricted to the deep euphotic zone near or at the nitracline. Low‐B/A (high‐light adapted) ecotypes predominate in, but are not limited to, NO3-‐depleted surface waters, where they outnumber coexisting Synechococcus populations. The niche partitioning by different Prochlorococcus ecotypes begs the question of whether they also differ in their nitrogen (N) utilization physiology, especially with respect to NO3- utilization. To explore this possibility, we studied the capabilities of different Prochlorococcus and Synechococcus strains to grow on a variety of N sources. We found that all the isolates grew well on NH4+ and all were capable of urea utilization, occasionally at a lower growth rate. None of the Prochlorococcus isolates were able to grow with NO3-. Four high‐B/A Prochlorococcus isolates grew on NO3-, but all others did not. Whole genome analysis of the low‐B/A Prochlorococcus MED4 revealed that the genes required for NO uptake and reduction were absent. The genome of the high‐B/A Prochlorococcus MIT 9313 also lacked the NO3- utilization genes but has homologs of genes required for NO3- utilization consistent with its physiology and ecology. Thus, the utilization of different N sources in the marine environment is partitioned among closely related ecotypes, each with adaptations optimized for the environment where these sources are available.

Copper toxicity and cyanobacteria ecology in the Sargasso Sea

The closely related cyanobacteria Synechococcus and Prochlorococcus have different distributions in stratified water columns in the northern Sargasso Sea. The abundance of Synechococcus is relatively uniform with depth, but Prochlorococcus cell numbers are low within shallow mixed layers and high in and below the thermocline. Because free cupric ion (free Cu2+) concentrations are high (up to 6 pM) in shallow mixed layers and lower in deeper water, there is an inverse relationship between Prochlorococcus densities and the free Cu2+ concentration. We explored the possibility of a causal underpinning for this relationship by examining the relative copper sensitivities of Prochlorococcus and Synechococcus in cultures and field populations. Prochlorococcus isolates from both the highߚ and lowߚlight adapted ecotypes were inhibited at free Cu2+ concentrations that had no effect on Synechococcus. However, the highߚlight adapted strains were more copper resistant than their lowߚlight adapted counterparts. When copper was added to Prochlorococcus from environments where the in situ free Cu2+ was low (in deeply mixed water columns and below the mixed layer in stratified conditions), net growth rates were substantially reduced and cells arrested in the G1 and early S phases of the cell cycle. Prochlorococcus in shallow mixed layers were less sensitive to copper and were probably members of the copperߚresistant highߚlight adapted ecotype. Synechococcus were relatively copper resistant across a range of environments in the Sargasso Sea. These observations are consistent with our hypothesis that copper plays a role in cyanobacteria ecology in the Sargasso Sea.

Genotyping of axenic and non-axenic isolates of the genus Prochlorococcus and the OMF-‘Synechococcus’ clade by size, sequence analysis or RFLP of the Internal Transcribed Spacer of the ribosomal operon The GenBank accession numbers for the ITS amplicon sequences reported in this paper are AF387607 (PCC 9511T), AF387610 (PCC 6307), AF387608 (PCC 7001) and AF387609 (PCC 7941).

PCR amplicons of the Internal Transcribed Spacer (ITS) of the rrn operon of three axenic OMF (oceanic, marine and freshwater) strains of 'Synechococcus' (WH7803, PCC 7001 and PCC 6307, respectively) differ greatly in length from that of the axenic Prochlorococcus marinus subsp. pastoris PCC 9511(T), although these four cyanobacteria cluster relatively closely in phylogenetic trees inferred from 16S rRNA gene sequences. The ITSs of three strains (PCC 9511(T), PCC 6307 and PCC 7001) were sequenced and compared with those available for strains Prochlorococcus MED4 (CCMP 1378) and MIT9313 from genome sequencing projects. In spite of large differences in length, sequence and mean DNA base composition, conserved domains important for transcriptional antitermination and folding of the rRNA transcripts were identified in all ITSs. A new group-specific primer permitted ITS amplification even with non-axenic isolates of Prochlorococcus and one OMF-'Synechococcus' strain. Prochlorococcus isolates of the high-light-adapted clade (HL) differed from representatives of the low-light-adapted clade (LL) by the length of their ITS. Restriction fragment length polymorphism (RFLP) of the ITS amplicons revealed three subclusters among the HL strains. Size, sequence data and RFLP of the ITS amplicons will therefore be valuable markers for the identification of different Prochlorococcus genotypes and for their discrimination from other cyanobacterial relatives with which they often co-exist in oceanic ecosystems.

Phycobiliprotein genes of the marine photosynthetic prokaryote Prochlorococcus: evidence for rapid evolution of genetic heterogeneity.

Prochlorococcus is a major photosynthetic prokaryote in nutrient-limited, open ocean environments and an important participant in the global carbon cycle. This phototroph is distinct from other members of the cyanobacterial lineage to which it belongs because it utilizes a chlorophyll a2/b(2) light-harvesting complex as its major antenna, instead of phycobilisomes. Recently, genes encoding the phycobiliprotein phycoerythrin were identified in several Prochlorococcus isolates, thus making it the only extant photosynthetic prokaryote to possess a chlorophyll a/b antenna as well as phycobiliprotein genes. In order to understand the evolution of phycobiliproteins in this genus, the authors have sequenced the phycoerythrin genes of two isolates that are the most deeply branching in the Prochlorococcus lineage and share the highest degree of 16S rDNA sequence similarity to phycobilisome-containing marine SYNECHOCOCCUS: Sequence analyses suggest that within the Prochlorococcus lineage, the selective forces shaping the evolution of the phycoerythrin gene set have not been uniform. Although strains that are most closely related to marine Synechococcus possess genes (cpeB, cpeA) encoding both subunits of phycoerythrin, a more recently evolved strain is shown to lack cpeA and to possess a degenerate form of cpeB. Differences in phycoerythrin gene sequences between Prochlorococcus and Synechococcus appear to be consistent with a model of elevated mutation rates rather than relaxed selection. This suggests that although phycoerythrin is not a major constituent of the light-harvesting apparatus in Prochlorococcus, as it is in Synechococcus, the cpeB and cpeA genes are still under selection, albeit a different type of selection than in Synechococcus. The evolution of the Prochlorococcus light-harvesting antenna complex provides an important system for understanding the origins and scope of phylogenetic diversity in ocean ecosystems.

Photophysiology of the marine cyanobacterium Prochlorococcus: Ecotypic differences among cultured isolates

Cultured isolates of Prochlorococcus from the Mediterranean Sea (MED4) and Sargasso Sea (SS120) have been shown to have dramatically different pigment composition and growth rate responses when grown over a range of irradiances. Moreover, analyses of field populations in the North Atlantic have shown that distinct ecotypes can coexist in the same water column. These and other observations have led to the hypothesis that Prochlorococcus is comprised of genetically distinct ecotypes that collectively expand the range of light intensities over which the genus can thrive. In this paper, we explore this hypothesis by comparing the photophysiology of 10 different Prochlorococcus isolates from diverse oceanographic regimes. We found that the 10 isolates could be grouped into two loose clusters based on their growth response to varying light intensity and their chlorophyll b/a2 (Chl b/a2) ratios. Although both groups photoacclimate when grown over a range of light intensities, isolates with distinctly higher Chl b/a2 ratios (high B/A ecotype) reach maximal growth rates at lower irradiances (Ik,g), have high growth efficiencies (αg), and are inhibited in growth at irradiances where isolates with low Chl b/a2 ratios (low B/A ecotype) are growing maximally. High Chl b/a2 ratios resulted in higher spectrally weighted average Chl a2‐specific absorption coefficient (āchl*), Chl a2 specific light‐harvesting efficiency (αchla), and quantum yield (φm) under low growth irradiances for the isolates of the high B/A ecotype relative to the others. The distinction between the high and low B/A ecotypes is supported by molecular phylogenies constructed using the 16S ribosomal ribonucleic acid (rRNA) gene. The physiological differences between the ecotypes most likely result in different relative distributions in a given water column and in fluctuations in their relative abundances as a function of seasonal dynamics and water‐column stability.