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Apple, JK, Strom SL, Palenik B, Brahamsha B.  2011.  Variability in protist grazing and growth on different marine Synechococcus isolates. Applied and Environmental Microbiology. 77:3074-3084.   10.1128/aem.02241-10   AbstractWebsite

Grazing mortality of the marine phytoplankton Synechococcus is dominated by planktonic protists, yet rates of consumption and factors regulating grazer-Synechococcus interactions are poorly understood. One aspect of predator-prey interactions for which little is known are the mechanisms by which Synechococcus avoids or resists predation and, in turn, how this relates to the ability of Synechococcus to support growth of protist grazer populations. Grazing experiments conducted with the raptorial dinoflagellate Oxyrrhis marina and phylogenetically diverse Synechococcus isolates (strains WH8102, CC9605, CC9311, and CC9902) revealed marked differences in grazing rates-specifically that WH8102 was grazed at significantly lower rates than all other isolates. Additional experiments using the heterotrophic nanoflagellate Goniomonas pacifica and the filter-feeding tintinnid ciliate Eutintinnis sp. revealed that this pattern in grazing susceptibility among the isolates transcended feeding guilds and grazer taxon. Synechococcus cell size, elemental ratios, and motility were not able to explain differences in grazing rates, indicating that other features play a primary role in grazing resistance. Growth of heterotrophic protists was poorly coupled to prey ingestion and was influenced by the strain of Synechococcus being consumed. Although Synechococcus was generally a poor-quality food source, it tended to support higher growth and survival of G. pacifica and O. marina relative to Eutintinnis sp., indicating that suitability of Synechococcus varies among grazer taxa and may be a more suitable food source for the smaller protist grazers. This work has developed tractable model systems for further studies of grazer-Synechococcus interactions in marine microbial food webs.

Armbrust, EV, Berges JA, Bowler C, Green BR, Martinez D, Putnam NH, Zhou SG, Allen AE, Apt KE, Bechner M, Brzezinski MA, Chaal BK, Chiovitti A, Davis AK, Demarest MS, Detter JC, Glavina T, Goodstein D, Hadi MZ, Hellsten U, Hildebrand M, Jenkins BD, Jurka J, Kapitonov VV, Kroger N, Lau WWY, Lane TW, Larimer FW, Lippmeier JC, Lucas S, Medina M, Montsant A, Obornik M, Parker MS, Palenik B, Pazour GJ, Richardson PM, Rynearson TA, Saito MA, Schwartz DC, Thamatrakoln K, Valentin K, Vardi A, Wilkerson FP, Rokhsar DS.  2004.  The genome of the diatom Thalassiosira pseudonana: Ecology, evolution, and metabolism. Science. 306:79-86.   10.1126/science.1101156   AbstractWebsite

Diatoms are unicellular algae with plastids acquired by secondary endosymbiosis. They are responsible for similar to20% of global carbon fixation. We report the 34 million-base pair draft nuclear genome of the marine diatom Thalassiosira pseudonana and its 129 thousand-base pair ptastid and 44 thousand-base pair mitochondrial genomes. Sequence and optical restriction mapping revealed 24 diploid nuclear chromosomes. We identified novel genes for silicic acid transport and formation of silica-based cell walls, high-affinity iron uptake, biosynthetic enzymes for several types of polyunsaturated fatty acids, use of a range of nitrogenous compounds, and a complete urea cycle, all attributes that allow diatoms to prosper in aquatic environments.

B
Bradley, JM, Hill N, Le Brun NE, Stuart RK, Palenik B.  2014.  Spectroscopic investigation of iron mineralisation by a cyanobacterial ferritin. Journal of Biological Inorganic Chemistry. 19:S291-S291. AbstractWebsite
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Bradley, JM, Svistunenko DA, Pullin J, Hill N, Stuart RK, Palenik B, Wilson MT, Hemmings AM, Moore GR, Le Brun NE.  2019.  Reaction of O-2 with a diiron protein generates a mixed-valent Fe2+/Fe3+ center and peroxide. Proceedings of the National Academy of Sciences of the United States of America. 116:2058-2067.   10.1073/pnas.1809913116   AbstractWebsite

The gene encoding the cyanobacterial ferritin SynFtn is up-regulated in response to copper stress. Here, we show that, while SynFtn does not interact directly with copper, it is highly unusual in several ways. First, its catalytic diiron ferroxidase center is unlike those of all other characterized prokaryotic ferritins and instead resembles an animal H-chain ferritin center. Second, as demonstrated by kinetic, spectro-scopic, and high-resolution X-ray crystallographic data, reaction of O-2 with the di-Fe2+ center results in a direct, one-electron oxidation to a mixed-valent Fe2+/Fe3+ form. Iron-O-2 chemistry of this type is currently unknown among the growing family of proteins that bind a diiron site within a four alpha-helical bundle in general and ferritins in particular. The mixed-valent form, which slowly oxidized to the more usual di-Fe3+ form, is an intermediate that is continually generated during mineralization. Peroxide, rather than superoxide, is shown to be the product of O-2 reduction, implying that ferroxidase centers function in pairs via long-range electron transfer through the protein resulting in reduction of O-2 bound at only one of the centers. We show that electron transfer is mediated by the transient formation of a radical on Tyr40, which lies similar to 4 angstrom from the diiron center. As well as demonstrating an expansion of the iron-O-2 chemistry known to occur in nature, these data are also highly relevant to the question of whether all ferritins mineralize iron via a common mechanism, providing unequivocal proof that they do not.

de la Broise, D, Palenik B.  2007.  Immersed in situ microcosms: A tool for the assessment of pollution impact on phytoplankton. Journal of Experimental Marine Biology and Ecology. 341:274-281.   10.1016/j.jembe.2006.10.045   AbstractWebsite

In situ phytoplankton microcosms were developed and characterized for use in toxicity testing. The microcosms contained 225 mu m filtered seawater maintained in 1 liter glass bottles attached to a plastic frame and immersed at 3 in under the sea surface. Synechococcus and picoeukaryote population dynamics in microcosms and the surrounding water were compared. A bloom-like behaviour observed for Synechococcus in these phytoplankton microcosms was avoided when 10% of the culture volume was replaced, every two days, by filtered seawater. After 2 weeks, no significant difference in Synechococcus and picoeukaryotes cell counts was observed in microcosms compared to the surrounding free seawater. Synechococcus fluorescence at 545 nm (phycoerythrobilin) fluctuated with a similar pattern in such microcosms and in free seawater and were shown to be correlated to light intensity fluctuations over a two week experiment. The in situ microcosms were used to study the impact of low copper additions. Synechococcus populations were dramatically decreased by copper addition, while picoeukaryote populations were increased simultaneously. Our data show that drastic changes in species composition can occur at copper concentrations encountered in polluted coastal areas. (c) 2006 Elsevier B.V. All rights reserved.

C
Chen, X, Su Z, Dam P, Palenik B, Xu Y, Jiang T.  2004.  Operon prediction by comparative genomics: an application to the Synechococcus sp WH8102 genome. Nucleic Acids Research. 32:2147-2157.   10.1093/nar/gkh510   AbstractWebsite

We present a computational method for operon prediction based on a comparative genomics approach. A group of consecutive genes is considered as a candidate operon if both their gene sequences and functions are conserved across several phylogenetically related genomes. In addition, various supporting data for operons are also collected through the application of public domain computer programs, and used in our prediction method. These include the prediction of conserved gene functions, promoter motifs and terminators. An apparent advantage of our approach over other operon prediction methods is that it does not require many experimental data (such as gene expression data and pathway data) as input. This feature makes it applicable to many newly sequenced genomes that do not have extensive experimental information. In order to validate our prediction, we have tested the method on Escherichia coli K12, in which operon structures have been extensively studied, through a comparative analysis against Haemophilus influenzae Rd and Salmonella typhimurium LT2. Our method successfully predicted most of the 237 known operons. After this initial validation, we then applied the method to a newly sequenced and annotated microbial genome, Synechococcus sp. WH8102, through a comparative genome analysis with two other cyanobacterial genomes, Prochlorococcus marinus sp. MED4 and P.marinus sp. MIT9313. Our results are consistent with previously reported results and statistics on operons in the literature.

Chisholm, SW, Frankel SL, Goericke R, Olson RJ, Palenik B, Waterbury JB, Westjohnsrud L, Zettler ER.  1992.  Prochlorococcus marinus nov. gen. nov. sp.: An oxyphototrophic marine prokaryote containing divinyl chlorophyll a and b . Archives of Microbiology. 157:297-300.   10.1007/bf00245165   AbstractWebsite

Several years ago, prochlorophyte picoplankton were discovered in the N. Atlantic. They have since been found to be abundant within the euphotic zone of the world's tropical and temperate oceans. The cells are extremely small, lack phycobiliproteins. and contain divinyl chlorophyll a and b as their primary photosynthetic pigments. Phylogenies constructed from DNA sequence data indicate that these cells are more closely related to a cluster of marine cyanobacteria than to their prochlorophyte 'relatives' Prochlorothrix and Prochloron. Several strains of this organism have recently been brought into culture, and herewith are given the name Prochlorococcus marinus.

Collier, JL, Palenik B.  2003.  Phycoerythrin-containing picoplankton in the Southern California Bight. Deep-Sea Research Part Ii-Topical Studies in Oceanography. 50:2405-2422.   10.1016/s0967-0645(03)00127-9   AbstractWebsite

Flow cytometry was used to examine the distribution of phycoerythrin-rich picophytoplankton. referred to here as Synechococcus, off the Southern California coast during six California Cooperative oceanic Fisheries Investigations (CalCOFI) cruises. Depth profiles revealed that Synechococcus was most abundant in the surface mixed layer, gradually disappearing with depth below the thermocline. Within the surface mixed layer, Synechococcus abundance was generally greater and more variable at stations shoreward of the California Current than at stations offshore of it. In waters associated with the California Current not impacted by upwelling, Synechococcus abundance increased with increasing bulk chlorophyll. In contrast, Synechococcus abundance declined with increasing bulk chlorophyll at stations that were impacted by upwelling. Synechococcus at stations impacted by upwelling also had more phycoerythrin per cell than at non-upwelling stations. Offshore of the California Current, Synechococcus cells in waters intruding from the Central North Pacific displayed higher side-scatter relative to forward scatter than did Synechococcus cells elsewhere in the region. Flow cytometrically distinct Synechococcus cell types were also detected below the thermocline at most of the stations where depth profiles were analyzed. These patterns in Synechococcus abundance and cellular characteristics might reflect physiological and/or genetic differences among Synechococcus associated with the various water masses that comprise the CalCOFI region. The data presented here provide a framework from which to launch more detailed and mechanistic studies examining the role of Synechococcus in the CalCOFI ecosystem. (C) 2003 Elsevier Ltd. All rights reserved.

Collier, JL, Brahamsha B, Palenik B.  1999.  The marine cyanobacterium Synechococcus sp. WH7805 requires urease (urea amidohydrolase, EC 3.5.1.5) to utilize urea as a nitrogen source: molecular-genetic and biochemical analysis of the enzyme. Microbiology-Sgm. 145:447-459.   10.1099/13500872-145-2-447   AbstractWebsite

Cyanobacteria assigned to the genus Synechococcus are an important component of oligotrophic marine ecosystems, where their growth may be constrained by low availability of fixed nitrogen. Urea appears to be a major nitrogen resource in the sea, but little molecular information exists about its utilization by marine organisms, including Synechococcus. Oligonucleotide primers were used to amplify a conserved fragment of the urease (urea amidohydrolase, EC 3.5.1.5) coding region from cyanobacteria. A 5.7 kbp region of the genome of the unicellular marine cyanobacterium Synechococcus sp. strain WH7805 was then cloned, and genes encoding three urease structural subunits and four urease accessory proteins were sequenced and identified by homology. The WH7805 urease had a predicted subunit composition typical of bacterial ureases, but the organization of the WH7805 urease genes was unique. Biochemical characteristics of the WH7805 urease enzyme were consistent with the predictions of the sequence data. Physiological data and sequence analysis both suggested that the urease operon may be nitrogen-regulated by the ntcA system in WH7805. Inactivation of the large subunit of urease, ureC, prevented WH7805 and Synechococcus WH8102 from growing on urea, demonstrating that the urease genes cloned are essential to the ability of these cyanobacteria to utilize urea as a nitrogen source.

D
Davis, AK, Hildebrand M, Palenik B.  2005.  A stress-induced protein associated with the girdle band region of the diatom Thalassiosira pseudonana (Bacillariophyta). Journal of Phycology. 41:577-589.   10.1111/j.1529-8817.2005.00076.x   AbstractWebsite

We report the characterization of a cell-surface protein isolated from the centric diatom Thalassiosira pseudonana Hasle and Heimdal. This protein has an apparent molecular weight of 150 kDa, is highly acidic, and is intimately associated with the cell wall. Although originally identified in cells experiencing copper toxicity, it is also induced by silicon and iron limitation but not by phosphate or nitrate limitation. Using immunofluorescence techniques, the 150-kDa protein was localized to the girdle band region and covered the elongated girdle band region of morphologically aberrant cells suffering from copper toxicity. Although having biochemical similarities to girdle band associated proteins identified in pennate diatoms known as pleuralins, the 150-kDa protein is not a sequence homolog and is predicted to have a number of unique features, such as chitin binding domains and a possible RGD cell attachment motif. Results presented here suggest that this protein is normally cell cycle regulated and may be involved in stabilizing cells during the division process.

Davis, AK, Hildebrand M, Palenik B.  2006.  Gene expression induced by copper stress in the diatom Thalassiosira pseudonana. Eukaryotic Cell. 5:1157-1168.   10.1128/ec.00042-06   AbstractWebsite

Utilizing a PCR-based subtractive cDNA approach, we demonstrated that the marine diatom Thalassiosira pseudonana exhibits a rapid response at the gene level to elevated concentrations of copper and that this response attenuates over 24 h of continuous exposure. A total of 16 copper-induced genes were identified, 11 of which were completely novel; however, many of the predicted amino acid sequences had characteristics suggestive of roles in ameliorating copper toxicity. Most of the novel genes were not equivalently induced by H2O2- or Cd-induced stress, indicating specificity in response. Two genes that could be assigned functions based on homology were also induced under conditions of general cellular stress. Half of the identified genes were located within two inverted repeats in the genome, and novel genes in one inverted repeat had mRNA levels induced by similar to 500- to 2,000-fold by exposure to copper for 1 h. Additionally, some of the inverted repeat genes demonstrated a dose-dependent response to Cu, but not Cd, and appear to belong to a multigene family. This multigene family may be the diatom functional homolog of metallothioneins.

Davis, AK, Palenik B.  2008.  Characterization of a modular, cell-surface protein and identification of a new gene family in the diatom Thalassiosira pseudonana. Protist. 159:195-207.   10.1016/j.protis.2007.09.006   AbstractWebsite

We report the characterization of a cell-surface protein isolated from copper-stressed cells of the centric diatom Thalassiosira pseudonana Hasle and Heimdal (CCMP 1335). This protein has an apparent molecular weight of 100kDa and is highly acidic. The 100kDa protein (p100) sequence is comprised almost entirely of a novel domain termed TpRCR for T pseudonana repetitive cysteine-rich domain, that is repeated 8 times and that contains conserved aromatic, acidic, and potential metal-binding amino acids. The analysis of the T pseudonana genome suggests that p100 belongs to a large family of modular proteins that consist of a variable number of TpRCR domain repeats. Based on cell surface biotinylation and antibody data, p100 appears to migrate more rapidly with SDS-PAGE when extracted from cells exposed to high levels of copper; however, the discovery of a large family of TpRCR domain-containing proteins leaves open the possibility that the antibody may be crossreacting with members of this protein family that are responding differently to copper. The response of the gene encoding p100 at the mRNA level during synchronized progression through the normal cell cycle is similar to previously characterized genes in T pseudonana encoding cell wall proteins called silaffins. (c) 2007 Elsevier GmbH. All rights reserved.

Dufresne, A, Ostrowski M, Scanlan DJ, Garczarek L, Mazard S, Palenik BP, Paulsen IT, de Marsac NT, Wincker P, Dossat C, Ferriera S, Johnson J, Post AF, Hess WR, Partensky F.  2008.  Unraveling the genomic mosaic of a ubiquitous genus of marine cyanobacteria. Genome Biology. 9   10.1186/gb-2008-9-5-r90   AbstractWebsite

Background: The picocyanobacterial genus Synechococcus occurs over wide oceanic expanses, having colonized most available niches in the photic zone. Large scale distribution patterns of the different Synechococcus clades (based on 16S rRNA gene markers) suggest the occurrence of two major lifestyles ('opportunists'/'specialists'), corresponding to two distinct broad habitats ('coastal'/'open ocean'). Yet, the genetic basis of niche partitioning is still poorly understood in this ecologically important group. Results: Here, we compare the genomes of 11 marine Synechococcus isolates, representing 10 distinct lineages. Phylogenies inferred from the core genome allowed us to refine the taxonomic relationships between clades by revealing a clear dichotomy within the main subcluster, reminiscent of the two aforementioned lifestyles. Genome size is strongly correlated with the cumulative lengths of hypervariable regions (or 'islands'). One of these, encompassing most genes encoding the light-harvesting phycobilisome rod complexes, is involved in adaptation to changes in light quality and has clearly been transferred between members of different Synechococcus lineages. Furthermore, we observed that two strains (RS9917 and WH5701) that have similar pigmentation and physiology have an unusually high number of genes in common, given their phylogenetic distance. Conclusion: We propose that while members of a given marine Synechococcus lineage may have the same broad geographical distribution, local niche occupancy is facilitated by lateral gene transfers, a process in which genomic islands play a key role as a repository for transferred genes. Our work also highlights the need for developing picocyanobacterial systematics based on genome-derived parameters combined with ecological and physiological data.

Dupont, CL, Johnson DA, Phillippy K, Paulsen IT, Brahamsha B, Palenik B.  2012.  Genetic identification of a high-affinity Ni transporter and the transcriptional response to Ni deprivation in Synechococcus sp. strain WH8102. Applied and Environmental Microbiology. 78:7822-7832.   10.1128/aem.01739-12   AbstractWebsite

One biological need for Ni in marine cyanobacteria stems from the utilization of the Ni metalloenzyme urease for the assimilation of urea as a nitrogen source. In many of the same cyanobacteria, including Synechococcus sp. strain WH8102, an additional and obligate nutrient requirement for Ni results from usage of a Ni superoxide dismutase (Ni-SOD), which is encoded by sodN. To better understand the effects of Ni deprivation on WH8102, parallel microarray-based analysis of gene expression and gene knockout experiments were conducted. The global transcriptional response to Ni deprivation depends upon the nitrogen source provided for growth; fewer than 1% of differentially expressed genes for Ni deprivation on ammonium or urea were concordantly expressed. Surprisingly, genes for putative Ni transporters, including one colocalized on the genome with sodN, sodT, were not induced despite an increase in Ni transport. Knockouts of the putative Ni transporter gene sodT appeared to be lethal in WH8102, so the genes for sodT and sodN in WH8102 were interrupted with the gene for Fe-SOD, sodB, and its promoter from Synechococcus sp. strain WH7803. The sodT:: sodB exconjugants were unable to grow at low Ni concentrations, confirming that SodT is a Ni transporter. The sodN::sodB exconjugants displayed higher growth rates at low Ni concentrations than did the wild type, presumably due to a relaxed competition between urease and Ni-SOD for Ni. Both sodT::sodB and sodN::sodB lines exhibited an impaired ability to grow at low Fe concentrations. We propose a posttranslational allosteric SodT regulation involving the binding of Ni to a histidine-rich intracellular protein loop.

Dupont, CL, Yang S, Palenik B, Bourne PE.  2006.  Modern proteomes contain putative imprints of ancient shifts in trace metal geochemistry. Proceedings of the National Academy of Sciences of the United States of America. 103:17822-17827.   10.1073/pnas.0605798103   AbstractWebsite

Because of the rise in atmospheric oxygen 2.3 billion years ago (Gya) and the subsequent changes in oceanic redox state over the last 2.3-1 Gya, trace metal bioavailability in marine environments has changed dramatically. Although theorized to have influenced the biological usage of metals leaving discernable genomic signals, a thorough and quantitative test of this hypothesis has been lacking. Using structural bioinformatics and whole-genome sequences, the Fe-, Zn-, Mn-, and Co-binding metallomes of 23 Archaea, 233 Bacteria, and 57 Eukarya were constructed. These metallomes reveal that the overall abundances of these metal-binding structures scale to proteome size as power laws with a unique set of slopes for each Superkingdom of Life. The differences in the power describing the abundances of Fe-, Mrl Zn-, and Co-binding proteins in the proteomes of Prokaryotes and Eukaryotes are similar to the theorized changes in the abundances of these metals after the oxygenation of oceanic deep waters. This phenomenon suggests that Prokarya and Eukarya evolved in anoxic and oxic environments, respectively, a hypothesis further supported by structures and functions of Fe-binding proteins in each Superkingdom. Also observed is a proliferation in the diversity of Zn-binding protein structures involved in protein-DNA and protein-protein interactions within Eukarya, an event unlikely to occur in either an anoxic or euxinic environment where Zn concentrations would be vanishingly low. We hypothesize that these conserved trends are proteomic imprints of changes in trace metal bioavailability in the ancient ocean that highlight a major evolutionary shift in biological trace metal usage.

Dupont, CL, Buck KN, Palenik B, Barbeau K.  2010.  Nickel utilization in phytoplankton assemblages from contrasting oceanic regimes. Deep-Sea Research Part I-Oceanographic Research Papers. 57:553-566.   10.1016/j.dsr.2009.12.014   AbstractWebsite

In most oceanic environments, dissolved nickel (Ni) concentrations are drawn clown in surface waters with increasing concentrations at depth, implying a role for biology in the geochemical distribution of Ni Studies with phytoplankton isolates from the surface ocean have established the biochemical roles of Ni in the assimilation of urea and oxidative defense To determine if these requirements are relevant in natural marine planktonic assemblages, bottle-based fertilization experiments were used to test the effects of low-level additions of Ni. urea, or both Ni and urea to surface waters at several locations offshore of Peru and California, as well as in the Gulf of California Urea and Ni(+) urea additions consistently promoted phytoplankton growth relative to control and +Ni treatments, except in a coastal upwelling site and Peruvian water. No effect was observed in the upwelling site, but in Peruvian waters urea additions resulted in increased phytoplankton pigments and phosphate drawdown only when Ni was added concurrently, suggesting a biochemically dependent Ni-urea colimitation In the Gulf of California, Ni additions without urea resulted in increased abundances of cyanobacteria, picoeukaryotes, and the corresponding pigments As urea additions showed the overall phytoplankton community was also urea-limited, it appears that the cyanobactena and potentially the picoeukaryotes were colimited by Ni and urea in a biochemically independent fashion. In parallel, radiotracer-based uptake experiments were used to study the kinetics and spatial variation of biological Ni assimilation. In these experiments, the added radiotracer rarely equilibrated with the natural Ni present, precluding estimates a determination of in situ Ni uptake rates and suggesting that much of the natural Ni was not bioavailable. The lack of equilibration likely did not preclude the measurement of community Ni uptake kinetics, nor the comparison of measured rates between locations The highest V(max)K(p)(-1) values, which reflect a competitive advantage in Ni acquisition at low concentrations, were observed in stratified nitrogen-deplete communities, potentially linking Ni and nitrogen biogeochemistry in a manner consistent with the biochemical utilization of Ni. Overall, uptake rates were higher in the euphotic rather than non-euphotic zone communities, directly reconciling the nutrient-like depth profile of Ni The Ni uptake rates observed at the nitrate-replete Fe-deplete Peru stations were an order of magnitude lower than the other sites This result agrees with calculations suggesting that saturation of the cell surface with Ni and iron (Fe) transporters may limit uptake rates in low Fe waters. (C) 2010 Elsevier Ltd. All rights reserved

Dupont, CL, Barbeau K, Palenik B.  2008.  Ni uptake and limitation in marine Synechococcus strains. Applied and Environmental Microbiology. 74:23-31.   10.1128/aem.01007-07   AbstractWebsite

Ni accumulation and utilization were studied in two strains of marine Synechococcus, isolated from both coastal (CC9311; clade I) and open-ocean (WTH8102; clade III) environments, for which complete genome sequences are available. Both strains have genes encoding an Ni-containing urease and when grown on urea without Ni become Ni-N colimited. The Ni requirements of these strains also depend upon the genomic complement of genes encoding superoxide dismutase (SOD). WH8102, with a gene encoding only an Ni-SOD, has a novel obligate requirement for Ni, regardless of the N source. Reduced SOD activity in Ni-depleted cultures of VM8102 supports the link of this strain's Ni requirement to Ni-SOD. The genome of CC9311 contains a gene for a Cu/Zn-SOD in addition to a predicted pair of Ni-SODs, yet this strain cannot grow without Ni on NO3- and can grow only slowly on NH4+ without Ni, implying that the Cu/Zn-SOD cannot completely replace Ni-SOD in marine cyanobacteria. CC9311 does have a greater tolerance for Ni starvation. Both strains increase their Ni uptake capabilities and actively bioconcentrate Ni in response to decreasing extracellular and intracellular Ni. The changes in Ni uptake rates were more pronounced in WH8102 than in CC9311 and for growth on urea or nitrate than for growth on ammonia. These results, combined with an analysis of fully sequenced marine cyanobacterial genomes, suggest that the growth of many marine Synechococcus and all Prochlorococcus strains is dependent upon Ni.

Dupont, CL, McCrow JP, Valas R, Moustafa A, Walworth N, Goodenough U, Roth R, Hogle SL, Bai J, Johnson ZI, Mann E, Palenik B, Barbeau KA, Craig Venter J, Allen AE.  2015.  Genomes and gene expression across light and productivity gradients in eastern subtropical Pacific microbial communities. ISME J. 9:1076-1092.: International Society for Microbial Ecology   10.1038/ismej.2014.198   Abstract

Transitions in community genomic features and biogeochemical processes were examined in surface and subsurface chlorophyll maximum (SCM) microbial communities across a trophic gradient from mesotrophic waters near San Diego, California to the oligotrophic Pacific. Transect end points contrasted in thermocline depth, rates of nitrogen and CO2 uptake, new production and SCM light intensity. Relative to surface waters, bacterial SCM communities displayed greater genetic diversity and enrichment in putative sulfur oxidizers, multiple actinomycetes, low-light-adapted Prochlorococcus and cell-associated viruses. Metagenomic coverage was not correlated with transcriptional activity for several key taxa within Bacteria. Low-light-adapted Prochlorococcus, Synechococcus, and low abundance gamma-proteobacteria enriched in the>3.0-[mu]m size fraction contributed disproportionally to global transcription. The abundance of these groups also correlated with community functions, such as primary production or nitrate uptake. In contrast, many of the most abundant bacterioplankton, including SAR11, SAR86, SAR112 and high-light-adapted Prochlorococcus, exhibited low levels of transcriptional activity and were uncorrelated with rate processes. Eukaryotes such as Haptophytes and non-photosynthetic Aveolates were prevalent in surface samples while Mamielles and Pelagophytes dominated the SCM. Metatranscriptomes generated with ribosomal RNA-depleted mRNA (total mRNA) coupled to in vitro polyadenylation compared with polyA-enriched mRNA revealed a trade-off in detection eukaryotic organelle and eukaryotic nuclear origin transcripts, respectively. Gene expression profiles of SCM eukaryote populations, highly similar in sequence identity to the model pelagophyte Pelagomonas sp. CCMP1756, suggest that pelagophytes are responsible for a majority of nitrate assimilation within the SCM.

Dupont, CL, Neupane K, Shearer J, Palenik B.  2008.  Diversity, function and evolution of genes coding for putative Ni-containing superoxide dismutases. Environmental Microbiology. 10:1831-1843.   10.1111/j.1462-2920.2008.01604.x   AbstractWebsite

We examined the phylogenetic distribution, functionality and evolution of the sodN gene family, which has been shown to code for a unique Ni-containing isoform of superoxide dismutase (Ni-SOD) in Streptomyces. Many of the putative sodN sequences retrieved from public domain genomic and metagenomic databases are quite divergent from structurally and functionally characterized Ni-SOD. Structural bioinformatics studies verified that the divergent members of the sodN protein family code for similar three-dimensional structures and identified evolutionarily conserved amino acid residues. Structural and biochemical studies of the N-terminus 'Ni-hook' motif coded for by the putative sodN sequences confirmed both Ni (II) ligating and superoxide dismutase activity. Both environmental and organismal genomes expanded the previously noted phylogenetic distribution of sodN, and the sequences form four well-separated clusters, with multiple subclusters. The phylogenetic distribution of sodN suggests that the gene has been acquired via horizontal gene transfer by numerous organisms of diverse phylogenetic background, including both Eukaryotes and Prokaryotes. The presence of sodN correlates with the genomic absence of the gene coding for Fe-SOD, a structurally and evolutionarily distinct isoform of SOD. Given the low levels of Fe found in the marine environment from where many sequences were attained, we suggest that the replacement of Fe-SOD with Ni-SOD may be an evolutionary adaptation to reduce iron requirements.

Dyhrman, ST, Palenik B.  1999.  Phosphate stress in cultures and field populations of the dinoflagellate Prorucentrum minimum detected by a single-cell alkaline phosphatase assay. Applied and Environmental Microbiology. 65:3205-3212. AbstractWebsite

Alkaline phosphatase activity is a common marker of phosphate stress in many phytoplankton, but it has been difficult to attribute alkaline phosphatase activity to specific organisms or groups of phytoplankton in the field with traditional biochemical procedures, A ne cv alkaline phosphatase substrate, ELF-97 (enzyme-labeled fluorescence), shows promise in this regard. When a phosphate group is cleaved from the ELF-97 reagent, the remaining molecule precipitates near the site of enzyme activity, thus fluorescently tagging cells with alkaline phosphatase activity. We characterized ELF-97 labeling in axenic cultures of a common dinoflagellate, Prorocentrum minimum, in order to understand ELF-97 labeling dynamics when phosphate nutrition varies. Enzyme activity, as detected by ELF-97 labeling, appears to be induced in late-log- or early-stationary-phase cultures if cells are grown in low-phosphate media and is lost when phosphate-stressed cells are refed with phosphate, ELF-97 appears to label an inducible intracellular alkaline phosphatase in P, minimum based on confocal microscopy studies. This may limit the use of this reagent to organisms that lack high levels of constitutive intracellular phosphatases, After laboratory cultures were characterized, ELF-97 was used to assay field populations of P, minimum in Narragansett Bay during two 1-week periods, and 12 to 100% of the P. minimum cells were labeled. The level of cell labeling was reduced by 3 days of incubation with added inorganic phosphate. Our results indicate that ELF-97 is an excellent new tool for monitoring phytoplankton phosphate stress in the environment when the data are supported by appropriate laboratory studies.

Dyhrman, ST, Palenik BP.  1997.  The identification and purification of a cell-surface alkaline phosphatase from the dinoflagellate Prorocentrum minimum (Dinophyceae). Journal of Phycology. 33:602-612.   10.1111/j.0022-3646.1997.00602.x   AbstractWebsite

Two cell-surface proteins were identified in the dinoflagellate Prorocentrum minimum (Pavillard) Schiller strain CCMP 1329 that are evident in phosphate-limited cultures, but not in nitrate-limited cultures or cultures growing-exponentially in complete media. These proteins were detected by labeling cell-surface proteins with the biotinylating reagent succinimidyl 6-(biotinamido) hexanoate. One protein, of approximately 200,000 daltons was purified using differential centrifugation, detergent extraction, and gel filtration chromatography. This purified protein was able to hydrolyze orthophosphate groups from p-nitrophenylphosphate at pH 8, indicating it is an alkaline phosphatase, although it is larger than other alkaline phosphatases isolated to date from most microorganisms. This protein may be induced to help P. minimum cleave orthophosphate groups from organic forms of phosphate in marine environments. Ultimately, this protein could represent a unique antigen for developing an antibody probe for examining the relationships between phosphate stress and bloom formation in P. minimum, and perhaps other dinoflagellates, in the field.

Dyhrman, ST, Palenik B.  2003.  Characterization of ectoenzyme activity and phosphate-regulated proteins in the coccolithophorid Emiliania huxleyi. Journal of Plankton Research. 25:1215-1225.   10.1093/plankt/fbg086   AbstractWebsite

Three phosphate-regulated proteins in the coccolithophorid Emiliania huxleyi were detected by the biotinylation of cell-surface proteins. Two of these phosphate-regulated proteins have reduced denatured molecular weights near I 10 000 Do (118 078 and 110 541, respectively), while the third, and most abundant, is 69 087 Da. Induction of the three proteins and the common marker of phosphate stress, alkaline phosphatase activity, occur in the presence of <0.25 mu M inorganic phosphate in batch culture. Phosphate-regulated proteins and enzyme activity differed among E. huxleyi strains. Alkaline phosphatase is an enzyme commonly induced by phytoplankton in response to phosphate stress in order for cells to scavenge inorganic phosphate from organic sources. In E. huxleyi, this enzyme activity and the phosphate-regulated proteins are rapidly lost when phosphate is added back to phosphate-stressed cultures. This contrasts with the slower loss of alkaline phosphatase activity in the dinoflagellate Prorocentrum minimum. The presence of the three phosphate-regulated proteins and enzyme activity appear to differ somewhat among E. huxleyi strains. Based on these differences between strains, kinetic data, growth experiments and enzyme activities, the 69 087 Da protein may be a phosphatase with a high specificity for 5'-nucleotides.

Dyhrman, ST, Palenik B.  2001.  A single-cell immunoassay for phosphate stress in the dinoflagellate Prorocentrum minimum (Dinophyceae). Journal of Phycology. 37:400-410.   10.1046/j.1529-8817.2001.037003400.x   AbstractWebsite

Current techniques for studying phytoplankton physiology in the field, such as measurements of biochemical activities, nutrient addition bioassays, and determination of photosynthetic efficiency, are useful for assessing the physiology of the bulk community but suffer from a lack of specificity. This would be improved by the development of single-cell methods for monitoring in situ physiology, Here we develop and test an antibody-based assay for identifying phosphate stress in the model dinoflagellate Prorocentrum minimum (Pavillard) Schiller, Antiserum was raised against a cell-surface alkaline phosphatase purified from P, minimum. Western screening indicated that the antiserum reacted with phosphate-stressed cells but not nitrate-stressed or phosphate-replete cells in culture. Immunodepletion confirmed the identification of this protein as an alkaline phosphatase, Based on Western blots, the antiserum appeared to be specific for phosphate-regulated proteins in P, minimum because there is no discernible cross-reaction with closely related P, micans. A whole-cell immunofluorescence assay was used to identify phosphate stress in field populations of P, minimum from Narragansett Bay, Rhode Island. The percentage of labeled P, minimum cells in this environment during the summer of 1998 decreased through time as the inorganic phosphate concentration increased. The percentage of antibody-labeled cells significantly correlated with the percentage of ELF-97-labeled cells determined as another single-cell assay of phosphate stress. This is the first antibody-based method developed for monitoring cell-specific physiology in a dinoflagellate, and the method described here may serve as a model for developing similar tools in other species of phytoplankton.

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Ferris, MJ, Palenik B.  1998.  Niche adaptation in ocean cyanobacteria. Nature. 396:226-228.   10.1038/24297   AbstractWebsite

Small unicellular cyanobacteria (Synechococcus and Prochlorococcus ) are the most abundant photosynthetic microorganisms in the world's oceans, yet we know little of the genetic structure of these populations. Here we show that distinct clades of Prochlorococcus, and possibly of Synechococcus, are adapted to their specific surface and deep oceanic niches.

Foflonker, F, Price DC, Qiu H, Palenik B, Wang SY, Bhattacharya D.  2015.  Genome of the halotolerant green alga Picochlorum sp reveals strategies for thriving under fluctuating environmental conditions. Environmental Microbiology. 17:412-426.   10.1111/1462-2920.12541   AbstractWebsite

An expected outcome of climate change is intensification of the global water cycle, which magnifies surface water fluxes, and consequently alters salinity patterns. It is therefore important to understand the adaptations and limits of microalgae to survive changing salinities. To this end, we sequenced the 13.5Mbp genome of the halotolerant green alga PicochlorumSENEW3 (SE3) that was isolated from a brackish water pond subject to large seasonal salinity fluctuations. PicochlorumSE3 encodes 7367 genes, making it one of the smallest and most gene dense eukaryotic genomes known. Comparison with the pico-prasinophyte Ostreococcus tauri, a species with a limited range of salt tolerance, reveals the enrichment of transporters putatively involved in the salt stress response in PicochlorumSE3. Analysis of cultures and the protein complement highlight the metabolic flexibility of PicochlorumSE3 that encodes genes involved in urea metabolism, acetate assimilation and fermentation, acetoin production and glucose uptake, many of which form functional gene clusters. Twenty-four cases of horizontal gene transfer from bacterial sources were found in PicochlorumSE3 with these genes involved in stress adaptation including osmolyte production and growth promotion. Our results identify PicochlorumSE3 as a model for understanding microalgal adaptation to stressful, fluctuating environments.