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Publications by author: Chisholm

1. Features of cyanomyovirus gene content and evolution in wild populations and cultured isolates
   Libusha Kelly, Huiming Ding, Katherine H. Huang, Marcia S. Osburne, and Sallie W. Chisholm
   Submitted
   Pubmed: 50000003
   Abstract
   Viruses that infect marine cyanobacteria . cyanophage . often carry genes that have orthologs in their cyanobacterial hosts, and the frequency of these genes can vary with habitat. To explore habitat-influenced genomic diversity more deeply, we used 28 cultured cyanomyoviruses, including 11 new genomes, as reference genomes to identify phage genes in three different ocean habitats. Only about 6-11% of genes were consistently observed in the wild, revealing high gene content variability in these populations. Numerous shared phage/host genes differed in relative frequency in different environments, possibly reflecting environmental selection for these genes in phage genomes. A higher fraction of phage genomes from relatively low-phosphorus environments . the Sargasso and Mediterranean Sea . contained host-like phosphorus assimilation genes compared with those from the N. Pacific Gyre. These genes are upregulated in a model phage/host system when the host is phosphorous starved; the response is mediated by pho box motifs in phage genomes that bind a host regulatory protein. Eleven phage genomes have predicted pho boxes upstream of the phosphate-acquisition genes pstS and phoA, and eight of these have a conserved, hypothetical, cyanophage-specific gene (PhCOG173) between the pho box and pstS. PhCOG173 is also found upstream of other shared phage/host genes, suggesting a unique regulatory role. Pho boxes are also found upstream of high light-inducible protein genes (hli) in phage, suggesting that this motif may play a broader role than simply regulating phosphate-stress responses in infected hosts, or that these hlis may play a role in phosphate stress.
2. Genomes of marine cyanopodoviruses reveal multiple origins of diversity
   Labrie S.J., K. Frois-Moniz, M.S. Osburne, L. Kelly, S.E. Roggensack, M.B. Sullivan, G. Gearin, Q. Zeng, M. Fitzgerald, M.R. Henn and S.W. Chisholm
   Submitted
   Pubmed: 50000002
   Abstract
   The marine cyanobacteria Prochlorococcus and Synechococcus are highly abundant in the global oceans, as are the cyanophage with which they co-evolve. While genomic analyses have been relatively extensive for cyanomyoviruses, only 3 cyanopodoviruses isolated on marine cyanobacteria have been sequenced. Here we present 9 new cyanopodovirus genomes, and analyze them in the context of the broader group. The genomes range from 42.2 to 47.7 kbp, with G+C contents consistent with those of their hosts. They share 12 core genes, and the pan-genome is not close to being fully sampled. The genomes contain 3 variable island regions, with the most hypervariable genes concentrated at one end of the genome. Concatenated core genes phylogeny cluster all but one of the phage into three distinct groups (CPV-I -III). The outlier, P-RSP2, has the smallest genome and lacks RNA polymerase, a hallmark of the Autographivirinae subfamily. The phage in groups CPV-I and CPV-II contain photosynthesis and carbon metabolism associated genes, while group CPV-III and the outlier P-RSP2 do not, suggesting different constraints on their lytic cycles. Four of the phage encode integrases and three have a host integration signature. Metagenomic analyses reveal that cyanopodoviruses may be more abundant in the oceans than previously thought.
3. Transcriptome and proteome dynamics of a light-dark synchronized bacterial cell cycle
   Jacob R. Waldbauer, Sébastien Rodrigue, Maureen L. Coleman, and Sallie W. Chisholm
   PLoS One. 2012;7(8):e43432. Epub 2012 Aug 29
   Download: Additional Raw Data
   Pubmed: 22952681
   Abstract
   Background: Growth of the ocean’s most abundant primary producer, the cyanobacterium Prochlorococcus, is tightly synchronized to the natural 24-hour light-dark cycle. We sought to quantify the relationship between transcriptome and proteome dynamics that underlie this obligate photoautotroph’s highly choreographed response to the daily oscillation in energy supply. Methodology/Principal Findings: Using Illumina RNA-sequencing transcriptomics and mass spectrometry-based quantitative proteomics, we measured timecourses of paired mRNA-protein abundances for 312 genes every 2 hours over a light-dark cycle. These temporal expression patterns reveal strong oscillations in transcript abundance that are broadly damped at the protein level, with mRNA levels varying on average 2.3 times more than the corresponding protein. The single strongest observed protein-level oscillation is in a ribonucleotide reductase, which may reflect a defense strategy against phage infection. The peak in abundance of most proteins also lags that of their transcript by 2-8 hours, and the two are completely antiphase for some genes. While abundant antisense RNA was detected, it apparently does not account for the observed divergences between expression levels. The redirection of flux through central carbon metabolism from daytime carbon fixation to nighttime respiration is associated with quite small changes in relative enzyme abundances. Conclusions/Significance: Our results indicate that expression responses to periodic stimuli that are common in natural ecosystems (such as the diel cycle) can diverge significantly between the mRNA and protein levels. Protein expression patterns that are distinct from those of cognate mRNA have implications for the interpretation of transcriptome and metatranscriptome data in terms of cellular metabolism and its biogeochemical impact.
4. Transcriptome response of high- and low-light-adapted Prochlorococcus strains to changing iron availability.
   Thompson AW, Huang K, Saito MA, Chisholm SW
   ISME J. 2011 Oct;5(10):1580-94.
   Pubmed: 21562599
   Abstract
   Prochlorococcus contributes significantly to ocean primary productivity. The link between primary productivity and iron in specific ocean regions is well established and iron limitation of Prochlorococcus cell division rates in these regions has been shown. However, the extent of ecotypic variation in iron metabolism among Prochlorococcus and the molecular basis for differences is not understood. Here, we examine the growth and transcriptional response of Prochlorococcus strains, MED4 and MIT9313, to changing iron concentrations. During steady state, MIT9313 sustains growth at an order-of-magnitude lower iron concentration than MED4. To explore this difference, we measured the whole-genome transcriptional response of each strain to abrupt iron starvation and rescue. Only four of the 1159 orthologs of MED4 and MIT9313 were differentially expressed in response to iron in both strains. However, in each strain, the expression of over a hundred additional genes changed, many of which are in labile genomic regions, suggesting a role for lateral gene transfer in establishing diversity of iron metabolism among Prochlorococcus. Furthermore, we found that MED4 lacks three genes near the iron-deficiency-induced gene (idiA) that are present and induced by iron stress in MIT9313. These genes are interesting targets for studying the adaptation of natural Prochlorococcus assemblages to local iron conditions as they show more diversity than other genomic regions in environmental metagenomic databases.
5. ProPortal: a resource for integrated systems biology of Prochlorococcus and its phage.
   Libusha Kelly, Katherine H. Huang, Huiming Ding, and Sallie W. Chisholm
   Nucl. Acids Res. (2011) doi: 10.1093/nar/gkr1022
   Google Scholar Search: Google It
   Abstract
   ProPortal (http://proportal.mit.edu/) is a database containing genomic, metagenomic, transcriptomic and field data for the marine cyanobacteriumProchlorococcus. Our goal is to provide a source of cross-referenced data across multiple scales of biological organization—from the genome to the ecosystem—embracing the full diversity of ecotypic variation within this microbial taxon, its sister group, Synechococcus and phage that infect them. The site currently contains the genomes of 13 Prochlorococcus strains, 11Synechococcus strains and 28 cyanophage strains that infect one or both groups. Cyanobacterial and cyanophage genes are clustered into orthologous groups that can be accessed by keyword search or through a genome browser. Users can also identify orthologous gene clusters shared by cyanobacterial and cyanophage genomes. Gene expression data for Prochlorococcus ecotypes MED4 and MIT9313 allow users to identify genes that are up or downregulated in response to environmental stressors. In addition, the transcriptome in synchronized cells grown on a 24-h light–dark cycle reveals the choreography of gene expression in cells in a ‘natural’ state. Metagenomic sequences from the Global Ocean Survey from Prochlorococcus, Synechococcus and phage genomes are archived so users can examine the differences between populations from diverse habitats. Finally, an example of cyanobacterial population data from the field is included.
6. Genomic analysis of oceanic cyanobacterial myoviruses compared with T4-like myoviruses from diverse hosts and environments.
   Sullivan MB, Huang KH, Ignacio-Espinoza JC, Berlin AM, Kelly L, Weigele PR, DeFrancesco AS, Kern SE, Thompson LR, Young S, Yandava C, Fu R, Krastins B, Chase M, Sarracino D, Osburne MS, Henn MR, Chisholm SW
   Environ Microbiol. 2010 Nov
   Pubmed: 20662890
   Abstract
   T4-like myoviruses are ubiquitous, and their genes are among the most abundant documented in ocean systems. Here we compare 26 T4-like genomes, including 10 from non-cyanobacterial myoviruses, and 16 from marine cyanobacterial myoviruses (cyanophages) isolated on diverse Prochlorococcus or Synechococcus hosts. A core genome of 38 virion construction and DNA replication genes was observed in all 26 genomes, with 32 and 25 additional genes shared among the non-cyanophage and cyanophage subsets, respectively. These hierarchical cores are highly syntenic across the genomes, and sampled to saturation. The 25 cyanophage core genes include six previously described genes with putative functions (psbA, mazG, phoH, hsp20, hli03, cobS), a hypothetical protein with a potential phytanoyl-CoA dioxygenase domain, two virion structural genes, and 16 hypothetical genes. Beyond previously described cyanophage-encoded photosynthesis and phosphate stress genes, we observed core genes that may play a role in nitrogen metabolism during infection through modulation of 2-oxoglutarate. Patterns among non-core genes that may drive niche diversification revealed that phosphorus-related gene content reflects source waters rather than host strain used for isolation, and that carbon metabolism genes appear associated with putative mobile elements. As well, phages isolated on Synechococcus had higher genome-wide %G+C and often contained different gene subsets (e.g. petE, zwf, gnd, prnA, cpeT) than those isolated on Prochlorococcus. However, no clear diagnostic genes emerged to distinguish these phage groups, suggesting blurred boundaries possibly due to cross-infection. Finally, genome-wide comparisons of both diverse and closely related, co-isolated genomes provide a locus-to-locus variability metric that will prove valuable for interpreting metagenomic data sets.
7. Structural changes in a marine podovirus associated with release of its genome into Prochlorococcus.
   Liu X, Zhang Q, Murata K, Baker ML, Sullivan MB, Fu C, Dougherty MT, Schmid MF, Osburne MS, Chisholm SW, Chiu W
   Nat Struct Mol Biol. 2010 Jul;17(7):830-6. Epub 2010 Jun 13.
   Pubmed: 20543830
   Abstract
   Podovirus P-SSP7 infects Prochlorococcus marinus, the most abundant oceanic photosynthetic microorganism. Single-particle cryo-electron microscopy yields icosahedral and asymmetrical structures of infectious P-SSP7 with 4.6-A and 9-A resolution, respectively. The asymmetric reconstruction reveals how symmetry mismatches are accommodated among five of the gene products at the portal vertex. Reconstructions of infectious and empty particles show a conformational change of the 'valve' density in the nozzle, an orientation difference in the tail fibers, a disordering of the C terminus of the portal protein and the disappearance of the core proteins. In addition, cryo-electron tomography of P-SSP7 infecting Prochlorococcus showed the same tail-fiber conformation as that in empty particles. Our observations suggest a mechanism whereby, upon binding to the host cell, the tail fibers induce a cascade of structural alterations of the portal vertex complex that triggers DNA release.
8. Short RNA half-lives in the slow-growing marine cyanobacterium Prochlorococcus.
   Steglich C, Lindell D, Futschik M, Rector T, Steen R, Chisholm SW
   Genome Biol. 2010 May 19;11(5):R54.
   Pubmed: 20482874
   Abstract
   ABSTRACT: BACKGROUND: RNA turnover plays an important role in the gene regulation of microorganisms and influences their speed of acclimation to environmental changes. We investigated whole-genome RNA stability of Prochlorococcus, a relatively slow-growing marine cyanobacterium doubling approximately once a day, which is extremely abundant in the oceans. RESULTS: Using a combination of microarrays, quantitative RT-PCR and a new fitting method for determining RNA decay rates, we found a median half-life of 2.4 min and a median decay rate of 2.6 min for expressed genes - two fold faster than that reported for any organism. The shortest transcript half-life (33 seconds) was for a gene of unknown function, while some of the longest (approximately18 min) were for genes with high transcript levels. Genes organized in operons displayed intriguing mRNA decay patterns, such as increased stability, and delayed onset of decay with greater distance from the transcriptional start site. The same phenomenon was observed on a single probe resolution for genes greater than 2 kb. CONCLUSION: We hypothesize that the fast turnover relative to the slow generation time in Prochlorococcus may enable a swift response to environmental changes through rapid recycling of nucleotides, which could be advantageous in nutrient poor oceans. Our growing understanding of RNA half-lives will help the interpretation of the growing bank of metatranscriptomic studies of wild populations of Prochlorococcus. The surprisingly complex decay patterns of large transcripts reported here, and the method developed to describe them, will open new avenues for the investigation and understanding of RNA decay for all organisms.
9. Catalytic promiscuity in the biosynthesis of cyclic peptide secondary metabolites in planktonic marine cyanobacteria.
   Li B, Sher D, Kelly L, Shi Y, Huang K, Knerr PJ, Joewono I, Rusch D, Chisholm SW, van der Donk WA
   Proc Natl Acad Sci U S A. 2010 Jun 8;107(23):10430-5. Epub 2010 May 17.
   Pubmed: 20479271
   Abstract
   Our understanding of secondary metabolite production in bacteria has been shaped primarily by studies of attached varieties such as symbionts, pathogens, and soil bacteria. Here we show that a strain of the single-celled, planktonic marine cyanobacterium Prochlorococcus-which conducts a sizable fraction of photosynthesis in the oceans-produces many cyclic, lanthionine-containing peptides (lantipeptides). Remarkably, in Prochlorococcus MIT9313 a single promiscuous enzyme transforms up to 29 different linear ribosomally synthesized peptides into a library of polycyclic, conformationally constrained products with highly diverse ring topologies. Genes encoding this system are found in variable abundances across the oceans-with a hot spot in a Galapagos hypersaline lagoon-suggesting they play a habitat- and/or community-specific role. The extraordinarily efficient pathway for generating structural diversity enables these cyanobacteria to produce as many secondary metabolites as model antibiotic-producing bacteria, but with much smaller genomes.
10. Temporal dynamics of Prochlorococcus ecotypes in the Atlantic and Pacific oceans.
    Malmstrom RR, Coe A, Kettler GC, Martiny AC, Frias-Lopez J, Zinser ER, Chisholm SW
    ISME J. 2010 May 13.
    Pubmed: 20463762
    Abstract
    To better understand the temporal and spatial dynamics of Prochlorococcus populations, and how these populations co-vary with the physical environment, we followed monthly changes in the abundance of five ecotypes-two high-light adapted and three low-light adapted-over a 5-year period in coordination with the Bermuda Atlantic Time Series (BATS) and Hawaii Ocean Time-series (HOT) programs. Ecotype abundance displayed weak seasonal fluctuations at HOT and strong seasonal fluctuations at BATS. Furthermore, stable 'layered' depth distributions, where different Prochlorococcus ecotypes reached maximum abundance at different depths, were maintained consistently for 5 years at HOT. Layered distributions were also observed at BATS, although winter deep mixing events disrupted these patterns each year and produced large variations in ecotype abundance. Interestingly, the layered ecotype distributions were regularly reestablished each year after deep mixing subsided at BATS. In addition, Prochlorococcus ecotypes each responded differently to the strong seasonal changes in light, temperature and mixing at BATS, resulting in a reproducible annual succession of ecotype blooms. Patterns of ecotype abundance, in combination with physiological assays of cultured isolates, confirmed that the low-light adapted eNATL could be distinguished from other low-light adapted ecotypes based on its ability to withstand temporary exposure to high-intensity light, a characteristic stress of the surface mixed layer. Finally, total Prochlorococcus and Synechococcus dynamics were compared with similar time series data collected a decade earlier at each location. The two data sets were remarkably similar-testimony to the resilience of these complex dynamic systems on decadal time scales.The ISME Journal advance online publication, 13 May 2010; doi:10.1038/ismej.2010.60.
11. UV hyper-resistance in Prochlorococcus MED4 results from a single base pair deletion just upstream of an operon encoding nudix hydrolase and photolyase.
    Osburne MS, Holmbeck BM, Frias-Lopez J, Steen R, Huang K, Kelly L, Coe A, Waraska K, Gagne A, Chisholm SW
    Environ Microbiol. 2010 Mar 23.
    Pubmed: 20345942
    Abstract
    Summary Exposure to solar radiation can cause mortality in natural communities of pico-phytoplankton, both at the surface and to a depth of at least 30 m. DNA damage is a significant cause of death, mainly due to cyclobutane pyrimidine dimer formation, which can be lethal if not repaired. While developing a UV mutagenesis protocol for the marine cyanobacterium Prochlorococcus, we isolated a UV-hyper-resistant variant of high light-adapted strain MED4. The hyper-resistant strain was constitutively upregulated for expression of the mutT-phrB operon, encoding nudix hydrolase and photolyase, both of which are involved in repair of DNA damage that can be caused by UV light. Photolyase (PhrB) breaks pyrimidine dimers typically caused by UV exposure, using energy from visible light in the process known as photoreactivation. Nudix hydrolase (MutT) hydrolyses 8-oxo-dGTP, an aberrant form of GTP that results from oxidizing conditions, including UV radiation, thus impeding mispairing and mutagenesis by preventing incorporation of the aberrant form into DNA. These processes are error-free, in contrast to error-prone SOS dark repair systems that are widespread in bacteria. The UV-hyper-resistant strain contained only a single mutation: a 1 bp deletion in the intergenic region directly upstream of the mutT-phrB operon. Two subsequent enrichments for MED4 UV-hyper-resistant strains from MED4 wild-type cultures gave rise to strains containing this same 1 bp deletion, affirming its connection to the hyper-resistant phenotype. These results have implications for Prochlorococcus DNA repair mechanisms, genome stability and possibly lysogeny.
12. Modeling selective pressures on phytoplankton in the global ocean.
    Bragg JG, Dutkiewicz S, Jahn O, Follows MJ, Chisholm SW
    PLoS One. 2010 Mar 10;5(3):e9569.
    Pubmed: 20224766
    Abstract
    Our view of marine microbes is transforming, as culture-independent methods facilitate rapid characterization of microbial diversity. It is difficult to assimilate this information into our understanding of marine microbe ecology and evolution, because their distributions, traits, and genomes are shaped by forces that are complex and dynamic. Here we incorporate diverse forces-physical, biogeochemical, ecological, and mutational-into a global ocean model to study selective pressures on a simple trait in a widely distributed lineage of picophytoplankton: the nitrogen use abilities of Synechococcus and Prochlorococcus cyanobacteria. Some Prochlorococcus ecotypes have lost the ability to use nitrate, whereas their close relatives, marine Synechococcus, typically retain it. We impose mutations for the loss of nitrogen use abilities in modeled picophytoplankton, and ask: in which parts of the ocean are mutants most disadvantaged by losing the ability to use nitrate, and in which parts are they least disadvantaged? Our model predicts that this selective disadvantage is smallest for picophytoplankton that live in tropical regions where Prochlorococcus are abundant in the real ocean. Conversely, the selective disadvantage of losing the ability to use nitrate is larger for modeled picophytoplankton that live at higher latitudes, where Synechococcus are abundant. In regions where we expect Prochlorococcus and Synechococcus populations to cycle seasonally in the real ocean, we find that model ecotypes with seasonal population dynamics similar to Prochlorococcus are less disadvantaged by losing the ability to use nitrate than model ecotypes with seasonal population dynamics similar to Synechococcus. The model predictions for the selective advantage associated with nitrate use are broadly consistent with the distribution of this ability among marine picocyanobacteria, and at finer scales, can provide insights into interactions between temporally varying ocean processes and selective pressures that may be difficult or impossible to study by other means. More generally, and perhaps more importantly, this study introduces an approach for testing hypotheses about the processes that underlie genetic variation among marine microbes, embedded in the dynamic physical, chemical, and biological forces that generate and shape this diversity.
13. Analysis of high-throughput sequencing and annotation strategies for phage genomes.
    Henn MR, Sullivan MB, Stange-Thomann N, Osburne MS, Berlin AM, Kelly L, Yandava C, Kodira C, Zeng Q, Weiand M, Sparrow T, Saif S, Giannoukos G, Young SK, Nusbaum C, Birren BW, Chisholm SW
    PLoS One. 2010 Feb 5;5(2):e9083.
    Pubmed: 20140207
    Abstract
    BACKGROUND: Bacterial viruses (phages) play a critical role in shaping microbial populations as they influence both host mortality and horizontal gene transfer. As such, they have a significant impact on local and global ecosystem function and human health. Despite their importance, little is known about the genomic diversity harbored in phages, as methods to capture complete phage genomes have been hampered by the lack of knowledge about the target genomes, and difficulties in generating sufficient quantities of genomic DNA for sequencing. Of the approximately 550 phage genomes currently available in the public domain, fewer than 5% are marine phage. METHODOLOGY/PRINCIPAL FINDINGS: To advance the study of phage biology through comparative genomic approaches we used marine cyanophage as a model system. We compared DNA preparation methodologies (DNA extraction directly from either phage lysates or CsCl purified phage particles), and sequencing strategies that utilize either Sanger sequencing of a linker amplification shotgun library (LASL) or of a whole genome shotgun library (WGSL), or 454 pyrosequencing methods. We demonstrate that genomic DNA sample preparation directly from a phage lysate, combined with 454 pyrosequencing, is best suited for phage genome sequencing at scale, as this method is capable of capturing complete continuous genomes with high accuracy. In addition, we describe an automated annotation informatics pipeline that delivers high-quality annotation and yields few false positives and negatives in ORF calling. CONCLUSIONS/SIGNIFICANCE: These DNA preparation, sequencing and annotation strategies enable a high-throughput approach to the burgeoning field of phage genomics.
14. Analysis of high-throughput sequencing and annotation strategies for phage genomes.
    Henn MR, Sullivan MB, Stange-Thomann N, Osburne MS, Berlin AM, Kelly L, Yandava C, Kodira C, Zeng Q, Weiand M, Sparrow T, Saif S, Giannoukos G, Young SK, Nusbaum C, Birren BW, Chisholm SW
    PLoS One. 2010
    Pubmed: 20140207
    Abstract
    Background: Bacterial viruses (phages) play a critical role in shaping microbial populations as they influence both host mortality and horizontal gene transfer. As such, they have a significant impact on local and global ecosystem function and human health. Despite their importance, little is known about the genomic diversity harbored in phages, as methods to capture complete phage genomes have been hampered by the lack of knowledge about the target genomes, and difficulties in generating sufficient quantities of genomic DNA for sequencing. Of the approximately 550 phage genomes currently available in the public domain, fewer than 5% are marine phage.
    METHODOLOGY/PRINCIPAL FINDINGS:To advance the study of phage biology through comparative genomic approaches we used marine cyanophage as a model system. We compared DNA preparation methodologies (DNA extraction directly from either phage lysates or CsCl purified phage particles), and sequencing strategies that utilize either Sanger sequencing of a linker amplification shotgun library (LASL) or of a whole genome shotgun library (WGSL), or 454 pyrosequencing methods. We demonstrate that genomic DNA sample preparation directly from a phage lysate, combined with 454 pyrosequencing, is best suited for phage genome sequencing at scale, as this method is capable of capturing complete continuous genomes with high accuracy. In addition, we describe an automated annotation informatics pipeline that delivers high-quality annotation and yields few false positives and negatives in ORF calling.
    CONCLUSIONS/SIGNIFICANCE: These DNA preparation, sequencing and annotation strategies enable a high-throughput approach to the burgeoning field of phage genomics.
15. The genome and structural proteome of an ocean siphovirus: a new window into the cyanobacterial 'mobilome'
    Sullivan MB, Krastins B, Hughes JL, Kelly L, Chase M, Sarracino D, Chisholm SW
    Environ Microbiol. 2009 Oct 14.
    Pubmed: 19840100
    Abstract
    Summary Prochlorococcus, an abundant phototroph in the oceans, are infected by members of three families of viruses: myo-, podo- and siphoviruses. Genomes of myo- and podoviruses isolated on Prochlorococcus contain DNA replication machinery and virion structural genes homologous to those from coliphages T4 and T7 respectively. They also contain a suite of genes of cyanobacterial origin, most notably photosynthesis genes, which are expressed during infection and appear integral to the evolutionary trajectory of both host and phage. Here we present the first genome of a cyanobacterial siphovirus, P-SS2, which was isolated from Atlantic slope waters using a Prochlorococcus host (MIT9313). The P-SS2 genome is larger than, and considerably divergent from, previously sequenced siphoviruses. It appears most closely related to lambdoid siphoviruses, with which it shares 13 functional homologues. The approximately 108 kb P-SS2 genome encodes 131 predicted proteins and notably lacks photosynthesis genes which have consistently been found in other marine cyanophage, but does contain 14 other cyanobacterial homologues. While only six structural proteins were identified from the genome sequence, 35 proteins were detected experimentally; these mapped onto capsid and tail structural modules in the genome. P-SS2 is potentially capable of integration into its host as inferred from bioinformatically identified genetic machinery int, bet, exo and a 53 bp attachment site. The host attachment site appears to be a genomic island that is tied to insertion sequence (IS) activity that could facilitate mobility of a gene involved in the nitrogen-stress response. The homologous region and a secondary IS-element hot-spot in Synechococcus RS9917 are further evidence of IS-mediated genome evolution coincident with a probable relic prophage integration event. This siphovirus genome provides a glimpse into the biology of a deep-photic zone phage as well as the ocean cyanobacterial prophage and IS element 'mobilome'.
16. Whole genome amplification and de novo assembly of single bacterial cells.
    Rodrigue S, Malmstrom RR, Berlin AM, Birren BW, Henn MR, Chisholm SW
    PLoS One. 2009 Sep 2;4(9):e6864.
    Download: Additional Raw Data
    Pubmed: 19724646
    Abstract
    BACKGROUND: Single-cell genome sequencing has the potential to allow the in-depth exploration of the vast genetic diversity found in uncultured microbes. We used the marine cyanobacterium Prochlorococcus as a model system for addressing important challenges facing high-throughput whole genome amplification (WGA) and complete genome sequencing of individual cells. METHODOLOGY/PRINCIPAL FINDINGS: We describe a pipeline that enables single-cell WGA on hundreds of cells at a time while virtually eliminating non-target DNA from the reactions. We further developed a post-amplification normalization procedure that mitigates extreme variations in sequencing coverage associated with multiple displacement amplification (MDA), and demonstrated that the procedure increased sequencing efficiency and facilitated genome assembly. We report genome recovery as high as 99.6% with reference-guided assembly, and 95% with de novo assembly starting from a single cell. We also analyzed the impact of chimera formation during MDA on de novo assembly, and discuss strategies to minimize the presence of incorrectly joined regions in contigs. CONCLUSIONS/SIGNIFICANCE: The methods describe in this paper will be useful for sequencing genomes of individual cells from a variety of samples.
17. Choreography of the transcriptome, photophysiology, and cell cycle of a minimal photoautotroph, Prochlorococcus.
    Zinser ER, Lindell D, Johnson ZI, Futschik ME, Steglich C, Coleman ML, Wright MA, Rector T, Steen R, McNulty N, Thompson LR, Chisholm SW
    PLoS ONE. 2009;4(4):e5135. Epub 2009 Apr 8.
    Pubmed: 19352512
    Abstract
    The marine cyanobacterium Prochlorococcus MED4 has the smallest genome and cell size of all known photosynthetic organisms. Like all phototrophs at temperate latitudes, it experiences predictable daily variation in available light energy which leads to temporal regulation and partitioning of key cellular processes. To better understand the tempo and choreography of this minimal phototroph, we studied the entire transcriptome of the cell over a simulated daily light-dark cycle, and placed it in the context of diagnostic physiological and cell cycle parameters. All cells in the culture progressed through their cell cycles in synchrony, thus ensuring that our measurements reflected the behavior of individual cells. Ninety percent of the annotated genes were expressed, and 80% had cyclic expression over the diel cycle. For most genes, expression peaked near sunrise or sunset, although more subtle phasing of gene expression was also evident. Periodicities of the transcripts of genes involved in physiological processes such as in cell cycle progression, photosynthesis, and phosphorus metabolism tracked the timing of these activities relative to the light-dark cycle. Furthermore, the transitions between photosynthesis during the day and catabolic consumption of energy reserves at night- metabolic processes that share some of the same enzymes--appear to be tightly choreographed at the level of RNA expression. In-depth investigation of these patterns identified potential regulatory proteins involved in balancing these opposing pathways. Finally, while this analysis has not helped resolve how a cell with so little regulatory capacity, and a 'deficient' circadian mechanism, aligns its cell cycle and metabolism so tightly to a light-dark cycle, it does provide us with a valuable framework upon which to build when the Prochlorococcus proteome and metabolome become available.
18. Identification and Structural Analysis of a Novel Carboxysome Shell Protein with Implications for Metabolite Transport.
    Klein MG, Zwart P, Bagby SC, Cai F, Chisholm SW, Heinhorst S, Cannon GC, Kerfeld CA
    J Mol Biol. 2009 Mar 27.
    Pubmed: 19328811
    Abstract
    Bacterial microcompartments (BMCs) are polyhedral bodies composed entirely of proteins that function as organelles in bacteria; they promote subcellular processes by encapsulating and co-localizing targeted enzymes with their substrates. The best-characterized BMC is the carboxysome, a central part of the carbon-concentrating mechanism that greatly enhances carbon fixation in cyanobacteria and some chemoautotrophs. Here we report the first structural insights into the carboxysome of Prochlorococcus, the numerically dominant cyanobacterium in the world's oligotrophic oceans. Bioinformatic methods, substantiated by analysis of gene expression data, were used to identify a new carboxysome shell component, CsoS1D, in the genome of Prochlorococcus strain MED4; orthologs were subsequently found in all cyanobacteria. Two independent crystal structures of Prochlorococcus MED4 CsoS1D reveal three features not seen in any BMC-domain protein structure solved to date. First, CsoS1D is composed of a fused pair of BMC domains. Second, this double-domain protein trimerizes to form a novel pseudohexameric building block for incorporation into the carboxysome shell, and the trimers further dimerize, forming a two-tiered shell building block. Third, and most strikingly, the large pore formed at the 3-fold axis of symmetry appears to be gated. Each dimer of trimers contains one trimer with an open pore and one whose pore is obstructed due to side-chain conformations of two residues that are invariant among all CsoS1D orthologs. This is the first evidence of the potential for gated transport across the carboxysome shell and reveals a new type of building block for BMC shells.
19. Use of Stable Isotope-Labelled Cells To Identify Active Grazers of Picocyanobacteria In Ocean Surface Waters
    Frias-Lopez J, Thompson A, Waldbauer J, Chisholm SW
    Environ Microbiol. 2009 Feb;11(2):512-25.
    Pubmed: 19196281
    Abstract
    Prochlorococcus and Synechococcus are the two most abundant marine cyanobacteria. They represent a significant fraction of the total primary production of the world oceans and comprise a major fraction of the prey biomass available to phagotrophic protists. Despite relatively rapid growth rates, picocyanobacterial cell densities in open-ocean surface waters remain fairly constant, implying steady mortality due to viral infection and consumption by predators. There have been several studies on grazing by specific protists on Prochlorococcus and Synechococcus in culture, and of cell loss rates due to overall grazing in the field. However, the specific sources of mortality of these primary producers in the wild remain unknown. Here, we use a modification of the RNA stable isotope probing technique (RNA-SIP), which involves adding labelled cells to natural seawater, to identify active predators that are specifically consuming Prochlorococcus and Synechococcus in the surface waters of the Pacific Ocean. Four major groups were identified as having their 18S rRNA highly labelled: Prymnesiophyceae (Haptophyta), Dictyochophyceae (Stramenopiles), Bolidomonas (Stramenopiles) and Dinoflagellata (Alveolata). For the first three of these, the closest relative of the sequences identified was a photosynthetic organism, indicating the presence of mixotrophs among picocyanobacterial predators. We conclude that the use of RNA-SIP is a useful method to identity specific predators for picocyanobacteria in situ, and that the method could possibly be used to identify other bacterial predators important in the microbial food-web.
20. Taxonomic resolution, ecotypes and the biogeography of Prochlorococcus.
    Martiny AC, Tai AP, Veneziano D, Primeau F, Chisholm SW
    Environ Microbiol. 2008 Nov 11.
    Download: Additional Raw Data
    Pubmed: 19021692
    Abstract
    In order to expand our understanding of the diversity and biogeography of Prochlorococcus ribotypes, we PCR-amplified, cloned and sequenced the 16S/23S rRNA ITS region from sites in the Atlantic and Pacific oceans. Ninety-three per cent of the ITS sequences could be assigned to existing Prochlorococcus clades, although many novel subclades were detected. We assigned the sequences to operational taxonomic units using a graduated scale of sequence identity from 80% to 99.5% and correlated Prochlorococcus diversity with respect to environmental variables and dispersal time between the sites. Dispersal time was estimated using a global ocean circulation model. The significance of specific environmental variables was dependent on the degree of sequence identity used to define a taxon: light correlates with broad-scale diversity (90% cut-off), temperature with intermediate scale (95%) whereas no correlation with phosphate was observed. Community structure was correlated with dispersal time between sample sites only when taxa were defined using the finest sequence similarity cut-off. Surprisingly, the concentration of nitrate, which cannot be used as N source by the Prochlorococcus strains in culture, explains some variation in community structure for some definitions of taxa. This study suggests that the spatial distribution of Prochlorococcus ecotypes is shaped by a hierarchy of environmental factors as well dispersal limitation.
21. Modeling The Fitness Consequences of A Cyanophage-Encoded Photosynthesis Gene.
    Bragg JG, Chisholm SW
    PLoS ONE. 2008;3(10):e3550. Epub 2008 Oct 29.
    Pubmed: 18958282
    Abstract
    BACKGROUND: Phages infecting marine picocyanobacteria often carry a psbA gene, which encodes a homolog to the photosynthetic reaction center protein, D1. Host encoded D1 decays during phage infection in the light. Phage encoded D1 may help to maintain photosynthesis during the lytic cycle, which in turn could bolster the production of deoxynucleoside triphosphates (dNTPs) for phage genome replication. METHODOLOGY/PRINCIPAL FINDINGS: To explore the consequences to a phage of encoding and expressing psbA, we derive a simple model of infection for a cyanophage/host pair--cyanophage P-SSP7 and Prochlorococcus MED4--for which pertinent laboratory data are available. We first use the model to describe phage genome replication and the kinetics of psbA expression by host and phage. We then examine the contribution of phage psbA expression to phage genome replication under constant low irradiance (25 microE m(-2) s(-1)). We predict that while phage psbA expression could lead to an increase in the number of phage genomes produced during a lytic cycle of between 2.5 and 4.5% (depending on parameter values), this advantage can be nearly negated by the cost of psbA in elongating the phage genome. Under higher irradiance conditions that promote D1 degradation, however, phage psbA confers a greater advantage to phage genome replication. CONCLUSIONS/SIGNIFICANCE: These analyses illustrate how psbA may benefit phage in the dynamic ocean surface mixed layer.
22. The Challenge of Regulation In A Minimal Photoautotroph: Non-Coding RNAs In Prochlorococcus
    Steglich C, Futschik ME, Lindell D, Voss B, Chisholm SW, Hess WR
    PLoS Genet. 2008 Aug 29;4(8):e1000173.
    Pubmed: 18769676
    Abstract
    Prochlorococcus, an extremely small cyanobacterium that is very abundant in the world's oceans, has a very streamlined genome. On average, these cells have about 2,000 genes and very few regulatory proteins. The limited capability of regulation is thought to be a result of selection imposed by a relatively stable environment in combination with a very small genome. Furthermore, only ten non-coding RNAs (ncRNAs), which play crucial regulatory roles in all forms of life, have been described in Prochlorococcus. Most strains also lack the RNA chaperone Hfq, raising the question of how important this mode of regulation is for these cells. To explore this question, we examined the transcription of intergenic regions of Prochlorococcus MED4 cells subjected to a number of different stress conditions: changes in light qualities and quantities, phage infection, or phosphorus starvation. Analysis of Affymetrix microarray expression data from intergenic regions revealed 276 novel transcriptional units. Among these were 12 new ncRNAs, 24 antisense RNAs (asRNAs), as well as 113 short mRNAs. Two additional ncRNAs were identified by homology, and all 14 new ncRNAs were independently verified by Northern hybridization and 5'RACE. Unlike its reduced suite of regulatory proteins, the number of ncRNAs relative to genome size in Prochlorococcus is comparable to that found in other bacteria, suggesting that RNA regulators likely play a major role in regulation in this group. Moreover, the ncRNAs are concentrated in previously identified genomic islands, which carry genes of significance to the ecology of this organism, many of which are not of cyanobacterial origin. Expression profiles of some of these ncRNAs suggest involvement in light stress adaptation and/or the response to phage infection consistent with their location in the hypervariable genomic islands.
23. Portal Protein Diversity and Phage Ecology
    Sullivan MB, Coleman ML, Quinlivan V, Rosenkrantz JE, Defrancesco AS, Tan G, Fu R, Lee JA, Waterbury JB, Bielawski JP, Chisholm SW
    Environ Microbiol. 2008 Jul 31.
    Pubmed: 18673386
    Abstract
    Oceanic phages are critical components of the global ecosystem, where they play a role in microbial mortality and evolution. Our understanding of phage diversity is greatly limited by the lack of useful genetic diversity measures. Previous studies, focusing on myophages that infect the marine cyanobacterium Synechococcus, have used the coliphage T4 portal-protein-encoding homologue, gene 20 (g20), as a diversity marker. These studies revealed 10 sequence clusters, 9 oceanic and 1 freshwater, where only 3 contained cultured representatives. We sequenced g20 from 38 marine myophages isolated using a diversity of Synechococcus and Prochlorococcus hosts to see if any would fall into the clusters that lacked cultured representatives. On the contrary, all fell into the three clusters that already contained sequences from cultured phages. Further, there was no obvious relationship between host of isolation, or host range, and g20 sequence similarity. We next expanded our analyses to all available g20 sequences (769 sequences), which include PCR amplicons from wild uncultured phages, non-PCR amplified sequences identified in the Global Ocean Survey (GOS) metagenomic database, as well as sequences from cultured phages, to evaluate the relationship between g20 sequence clusters and habitat features from which the phage sequences were isolated. Even in this meta-data set, very few sequences fell into the sequence clusters without cultured representatives, suggesting that the latter are very rare, or sequencing artefacts. In contrast, sequences most similar to the culture-containing clusters, the freshwater cluster and two novel clusters, were more highly represented, with one particular culture-containing cluster representing the dominant g20 genotype in the unamplified GOS sequence data. Finally, while some g20 sequences were non-randomly distributed with respect to habitat, there were always numerous exceptions to general patterns, indicating that phage portal proteins are not good predictors of a phage's host or the habitat in which a particular phage may thrive.
24. Efficient phage-mediated pigment biosynthesis in oceanic cyanobacteria.
    Dammeyer T, Bagby SC, Sullivan MB, Chisholm SW, Frankenberg-Dinkel N
    Curr Biol. 2008 Mar 25;18(6):442-8.
    Pubmed: 18356052
    Abstract
    Although the oceanic cyanobacterium Prochlorococcus harvests light with a chlorophyll antenna [1-3] rather than with the phycobilisomes that are typical of cyanobacteria, some strains express genes that are remnants of the ancestral Synechococcus phycobilisomes [4]. Similarly, some Prochlorococcus cyanophages, which often harbor photosynthesis-related genes [5], also carry homologs of phycobilisome pigment biosynthesis genes [6, 7]. Here, we investigate four such genes in two cyanophages that both infect abundant Prochlorococcus strains [8]: homologs of heme oxygenase (ho1), 15,16-dihydrobiliverdin:ferredoxin oxidoreductase (pebA), ferredoxin (petF) in the myovirus P-SSM2, and a phycocyanobilin:ferredoxin oxidoreductase (pcyA) homolog in the myovirus P-SSM4. We demonstrate that the phage homologs mimic the respective host activities, with the exception of the divergent phage PebA homolog. In this case, the phage PebA single-handedly catalyzes a reaction for which uninfected host cells require two consecutive enzymes, PebA and PebB. We thus renamed the phage enzyme phycoerythrobilin synthase (PebS). This gene, and other pigment biosynthesis genes encoded by P-SSM2 (petF and ho1), are transcribed during infection, suggesting that they can improve phage fitness. Analyses of global ocean metagenomes show that PcyA and Ho1 occur in both cyanobacteria and their phages, whereas the novel PebS-encoding gene is exclusive to phages.
25. Microbial Community Gene Expression In Ocean Surface Waters
    Frias-Lopez J, Shi Y, Tyson GW, Coleman ML, Schuster SC, Chisholm SW, Delong EF
    Proc Natl Acad Sci U S A. 2008 Mar 11;105(10):3805-10. Epub 2008 Mar 3.
    Pubmed: 18316740
    Abstract
    Metagenomics is expanding our knowledge of the gene content, functional significance, and genetic variability in natural microbial communities. Still, there exists limited information concerning the regulation and dynamics of genes in the environment. We report here global analysis of expressed genes in a naturally occurring microbial community. We first adapted RNA amplification technologies to produce large amounts of cDNA from small quantities of total microbial community RNA. The fidelity of the RNA amplification procedure was validated with Prochlorococcus cultures and then applied to a microbial assemblage collected in the oligotrophic Pacific Ocean. Microbial community cDNAs were analyzed by pyrosequencing and compared with microbial community genomic DNA sequences determined from the same sample. Pyrosequencing-based estimates of microbial community gene expression compared favorably to independent assessments of individual gene expression using quantitative PCR. Genes associated with key metabolic pathways in open ocean microbial species-including genes involved in photosynthesis, carbon fixation, and nitrogen acquisition-and a number of genes encoding hypothetical proteins were highly represented in the cDNA pool. Genes present in the variable regions of Prochlorococcus genomes were among the most highly expressed, suggesting these encode proteins central to cellular processes in specific genotypes. Although many transcripts detected were highly similar to genes previously detected in ocean metagenomic surveys, a significant fraction ( approximately 50%) were unique. Thus, microbial community transcriptomic analyses revealed not only indigenous gene- and taxon-specific expression patterns but also gene categories undetected in previous DNA-based metagenomic surveys.
26. Patterns and Implications of Gene Gain and Loss In The Evolution of Prochlorococcus
    Kettler GC, Martiny AC, Huang K, Zucker J, Coleman ML, Rodrigue S, Chen F, Lapidus A, Ferriera S, Johnson J, Steglich C, Church GM, Richardson P, Chisholm SW
    PLoS Genet. 2007 Dec;3(12):e231.
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    Pubmed: 18159947
    Abstract
    Prochlorococcus is a marine cyanobacterium that numerically dominates the mid-latitude oceans and is the smallest known oxygenic phototroph. Numerous isolates from diverse areas of the world's oceans have been studied and shown to be physiologically and genetically distinct. All isolates described thus far can be assigned to either a tightly clustered high-light (HL)-adapted clade, or a more divergent low-light (LL)-adapted group. The 16S rRNA sequences of the entire Prochlorococcus group differ by at most 3%, and the four initially published genomes revealed patterns of genetic differentiation that help explain physiological differences among the isolates. Here we describe the genomes of eight newly sequenced isolates and combine them with the first four genomes for a comprehensive analysis of the core (shared by all isolates) and flexible genes of the Prochlorococcus group, and the patterns of loss and gain of the flexible genes over the course of evolution. There are 1,273 genes that represent the core shared by all 12 genomes. They are apparently sufficient, according to metabolic reconstruction, to encode a functional cell. We describe a phylogeny for all 12 isolates by subjecting their complete proteomes to three different phylogenetic analyses. For each non-core gene, we used a maximum parsimony method to estimate which ancestor likely first acquired or lost each gene. Many of the genetic differences among isolates, especially for genes involved in outer membrane synthesis and nutrient transport, are found within the same clade. Nevertheless, we identified some genes defining HL and LL ecotypes, and clades within these broad ecotypes, helping to demonstrate the basis of HL and LL adaptations in Prochlorococcus. Furthermore, our estimates of gene gain events allow us to identify highly variable genomic islands that are not apparent through simple pairwise comparisons. These results emphasize the functional roles, especially those connected to outer membrane synthesis and transport that dominate the flexible genome and set it apart from the core. Besides identifying islands and demonstrating their role throughout the history of Prochlorococcus, reconstruction of past gene gains and losses shows that much of the variability exists at the "leaves of the tree," between the most closely related strains. Finally, the identification of core and flexible genes from this 12-genome comparison is largely consistent with the relative frequency of Prochlorococcus genes found in global ocean metagenomic databases, further closing the gap between our understanding of these organisms in the lab and the wild.
27. Genome-Wide Expression Dynamics of A Marine Virus and Host Reveal Features of Co-Evolution
    Lindell D, Jaffe JD, Coleman ML, Futschik ME, Axmann IM, Rector T, Kettler G, Sullivan MB, Steen R, Hess WR, Church GM, Chisholm SW
    Nature. 2007 Sep 6;449(7158):83-6.
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    Pubmed: 17805294
    Abstract
    Interactions between bacterial hosts and their viruses (phages) lead to reciprocal genome evolution through a dynamic co-evolutionary process. Phage-mediated transfer of host genes--often located in genome islands--has had a major impact on microbial evolution. Furthermore, phage genomes have clearly been shaped by the acquisition of genes from their hosts. Here we investigate whole-genome expression of a host and phage, the marine cyanobacterium Prochlorococcus MED4 and the T7-like cyanophage P-SSP7, during lytic infection, to gain insight into these co-evolutionary processes. Although most of the phage genome was linearly transcribed over the course of infection, four phage-encoded bacterial metabolism genes formed part of the same expression cluster, even though they are physically separated on the genome. These genes--encoding photosystem II D1 (psbA), high-light inducible protein (hli), transaldolase (talC) and ribonucleotide reductase (nrd)--are transcribed together with phage DNA replication genes and seem to make up a functional unit involved in energy and deoxynucleotide production for phage replication in resource-poor oceans. Also unique to this system was the upregulation of numerous genes in the host during infection. These may be host stress response genes and/or genes induced by the phage. Many of these host genes are located in genome islands and have homologues in cyanophage genomes. We hypothesize that phage have evolved to use upregulated host genes, leading to their stable incorporation into phage genomes and their subsequent transfer back to hosts in genome islands. Thus activation of host genes during infection may be directing the co-evolution of gene content in both host and phage genomes.
28. Code and Context: Prochlorococcus As A Model For Cross-Scale Biology
    Coleman ML, Chisholm SW
    Trends Microbiol. 2007 Sep;15(9):398-407. Epub 2007 Aug 10.
    Pubmed: 17693088
    Abstract
    Prochlorococcus is a simple cyanobacterium that is abundant throughout large regions of the oceans, and has become a useful model for studying the nature and regulation of biological diversity across all scales of complexity. Recent work has revealed that environmental factors such as light, nutrients and predation influence diversity in different ways, changing our image of the structure and dynamics of the global Prochlorococcus population. Advances in metagenomics, transcription profiling and global ecosystem modeling promise to deliver an even greater understanding of this system and further demonstrate the power of cross-scale systems biology.
29. Emergent Biogeography of Microbial Communities In A Model Ocean
    Follows MJ, Dutkiewicz S, Grant S, Chisholm SW
    Science. 2007 Mar 30;315(5820):1843-6.
    Pubmed: 17395828
    Abstract
    A marine ecosystem model seeded with many phytoplankton types, whose physiological traits were randomly assigned from ranges defined by field and laboratory data, generated an emergent community structure and biogeography consistent with observed global phytoplankton distributions. The modeled organisms included types analogous to the marine cyanobacterium Prochlorococcus. Their emergent global distributions and physiological properties simultaneously correspond to observations. This flexible representation of community structure can be used to explore relations between ecosystems, biogeochemical cycles, and climate change.
30. Electron Cryo-Tomography of Prochlorococcus Shows Cytoplasmic and Periplasmic Structural Features
    Marsh, MP; Cruciano, AC; Thompson, LR; Chisholm, SW; Chiu, W
    Biophysical Journal. 2007 ():504A-505A
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31. Influence of Light and Temperature On Prochlorococcus Ecotype Distributions In The Atlantic Ocean
    Zinser, ER; Johnson, ZI; Coe, A; Karaca, E; Veneziano, D; Chisholm, SW
    Limnology and Oceanography. 2007 52(5):2205-2220
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    Abstract
    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 ecotype-specific 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.
32. Culturing The Marine Cyanobacterium Prochlorococcus
    Moore, LR; Coe, A; Zinser, ER; Saito, MA; Sullivan, MB; Lindell, D; Frois-Moniz, K; Waterbury, J; Chisholm, SW
    Limnology and Oceanography-Methods. 2007 5():353-362
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    Abstract
    Prochlorococcus is the numerically dominant primary producer in open ocean ecosystems. Analysis of Prochlorococcus genome sequences from cultured isolates and ocean samples has broadened interest in studying this tiny cell, and efforts are underway to develop it into a model system for studying marine microbial ecology. A critical component of these efforts has been the development of systematic culturing methods that will facilitate the distribution of Prochlorococcus to diverse labs that may be interested in studying it. This paper provides detailed methods for maintaining cultures of Prochlorococcus, including a comparison of growth rates of cells on two artificial seawater media and on a standard medium that uses a natural seawater base. Procedures for agar plating, cryopreservation, obtaining new isolates, and issues associated with culture volume and carbon limitation also are described.
33. Global Gene Expression of Prochlorococcus Ecotypes In Response To Changes In Nitrogen Availability
    Tolonen AC, Aach J, Lindell D, Johnson ZI, Rector T, Steen R, Church GM, Chisholm SW
    Mol Syst Biol. 2006;2:53. Epub 2006 Oct 3.
    Browse this dataset: Here
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    Pubmed: 17016519
    Abstract
    Nitrogen (N) often limits biological productivity in the oceanic gyres where Prochlorococcus is the most abundant photosynthetic organism. The Prochlorococcus community is composed of strains, such as MED4 and MIT9313, that have different N utilization capabilities and that belong to ecotypes with different depth distributions. An interstrain comparison of how Prochlorococcus responds to changes in ambient nitrogen is thus central to understanding its ecology. We quantified changes in MED4 and MIT9313 global mRNA expression, chlorophyll fluorescence, and photosystem II photochemical efficiency (Fv/Fm) along a time series of increasing N starvation. In addition, the global expression of both strains growing in ammonium-replete medium was compared to expression during growth on alternative N sources. There were interstrain similarities in N regulation such as the activation of a putative NtcA regulon during N stress. There were also important differences between the strains such as in the expression patterns of carbon metabolism genes, suggesting that the two strains integrate N and C metabolism in fundamentally different ways.
34. Genome-Wide Analysis of Light Sensing In Prochlorococcus
    Steglich C, Futschik M, Rector T, Steen R, Chisholm SW
    J Bacteriol. 2006 Nov;188(22):7796-806. Epub 2006 Sep 15.
    Browse this dataset: Here
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    Pubmed: 16980454
    Abstract
    Prochlorococcus MED4 has, with a total of only 1,716 annotated protein-coding genes, the most compact genome of a free-living photoautotroph. Although light quality and quantity play an important role in regulating the growth rate of this organism in its natural habitat, the majority of known light-sensing proteins are absent from its genome. To explore the potential for light sensing in this phototroph, we measured its global gene expression pattern in response to different light qualities and quantities by using high-density Affymetrix microarrays. Though seven different conditions were tested, only blue light elicited a strong response. In addition, hierarchical clustering revealed that the responses to high white light and blue light were very similar and different from that of the lower-intensity white light, suggesting that the actual sensing of high light is mediated via a blue-light receptor. Bacterial cryptochromes seem to be good candidates for the blue-light sensors. The existence of a signaling pathway for the redox state of the photosynthetic electron transport chain was suggested by the presence of genes that responded similarly to red and blue light as well as genes that responded to the addition of DCMU [3-(3,4-dichlorophenyl)-1,1-N-N'-dimethylurea], a specific inhibitor of photosystem II-mediated electron transport.
35. Phosphate Acquisition Genes In Prochlorococcus Ecotypes: Evidence For Genome-Wide Adaptation
    Martiny AC, Coleman ML, Chisholm SW
    Proc Natl Acad Sci U S A. 2006 Aug 15;103(33):12552-7. Epub 2006 Aug 8.
    Browse this dataset: Here
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    Pubmed: 16895994
    Abstract
    The cyanobacterium Prochlorococcus is the numerically dominant phototroph in the oligotrophic oceans. This group consists of multiple ecotypes that are physiologically and phylogenetically distinct and occur in different abundances along environmental gradients. Here we examine adaptations to phosphate (P) limitation among ecotypes. First, we used DNA microarrays to identify genes involved in the P-starvation response in two strains belonging to different ecotypes, MED4 (high-light-adapted) and MIT9313 (low-light-adapted). Most of the up-regulated genes under P starvation were unique to one strain. In MIT9313, many ribosomal genes were down-regulated, suggesting a general stress response in this strain. We also observed major differences in regulation. The P-starvation-induced genes comprise two clusters on the chromosome, the first containing the P master regulator phoB and most known P-acquisition genes and the second, absent in MIT9313, containing genes of unknown function. We examined the organization of the phoB gene cluster in 11 Prochlorococcus strains belonging to diverse ecotypes and found high variability in gene content that was not congruent with rRNA phylogeny. We hypothesize that this genome variability is related to differences in P availability in the oceans from which the strains were isolated. Analysis of a metagenomic library from the Sargasso Sea supports this hypothesis; most Prochlorococcus cells in this low-P environment contain the P-acquisition genes seen in MED4, although a number of previously undescribed gene combinations were observed.
36. Prevalence and Evolution of Core Photosystem Ii Genes In Marine Cyanobacterial Viruses and Their Hosts
    Sullivan MB, Lindell D, Lee JA, Thompson LR, Bielawski JP, Chisholm SW
    PLoS Biol. 2006 Jul;4(8):e234.
    Pubmed: 16802857
    Abstract
    Cyanophages (cyanobacterial viruses) are important agents of horizontal gene transfer among marine cyanobacteria, the numerically dominant photosynthetic organisms in the oceans. Some cyanophage genomes carry and express host-like photosynthesis genes, presumably to augment the host photosynthetic machinery during infection. To study the prevalence and evolutionary dynamics of this phenomenon, 33 cultured cyanophages of known family and host range and viral DNA from field samples were screened for the presence of two core photosystem reaction center genes, psbA and psbD. Combining this expanded dataset with published data for nine other cyanophages, we found that 88% of the phage genomes contain psbA, and 50% contain both psbA and psbD. The psbA gene was found in all myoviruses and Prochlorococcus podoviruses, but could not be amplified from Prochlorococcus siphoviruses or Synechococcus podoviruses. Nearly all of the phages that encoded both psbA and psbD had broad host ranges. We speculate that the presence or absence of psbA in a phage genome may be determined by the length of the latent period of infection. Whether it also carries psbD may reflect constraints on coupling of viral- and host-encoded PsbA-PsbD in the photosynthetic reaction center across divergent hosts. Phylogenetic clustering patterns of these genes from cultured phages suggest that whole genes have been transferred from host to phage in a discrete number of events over the course of evolution (four for psbA, and two for psbD), followed by horizontal and vertical transfer between cyanophages. Clustering patterns of psbA and psbD from Synechococcus cells were inconsistent with other molecular phylogenetic markers, suggesting genetic exchanges involving Synechococcus lineages. Signatures of intragenic recombination, detected within the cyanophage gene pool as well as between hosts and phages in both directions, support this hypothesis. The analysis of cyanophage psbA and psbD genes from field populations revealed significant sequence diversity, much of which is represented in our cultured isolates. Collectively, these findings show that photosynthesis genes are common in cyanophages and that significant genetic exchanges occur from host to phage, phage to host, and within the phage gene pool. This generates genetic diversity among the phage, which serves as a reservoir for their hosts, and in turn influences photosystem evolution.
37. Sequencing Genomes From Single Cells By Polymerase Cloning
    Zhang K, Martiny AC, Reppas NB, Barry KW, Malek J, Chisholm SW, Church GM
    Nat Biotechnol. 2006 Jun;24(6):680-6. Epub 2006 May 28.
    Pubmed: 16732271
    Abstract
    Genome sequencing currently requires DNA from pools of numerous nearly identical cells (clones), leaving the genome sequences of many difficult-to-culture microorganisms unattainable. We report a sequencing strategy that eliminates culturing of microorganisms by using real-time isothermal amplification to form polymerase clones (plones) from the DNA of single cells. Two Escherichia coli plones, analyzed by Affymetrix chip hybridization, demonstrate that plonal amplification is specific and the bias is randomly distributed. Whole-genome shotgun sequencing of Prochlorococcus MIT9312 plones showed 62% coverage of the genome from one plone at a sequencing depth of 3.5x, and 66% coverage from a second plone at a depth of 4.7x. Genomic regions not revealed in the initial round of sequencing are recovered by sequencing PCR amplicons derived from plonal DNA. The mutation rate in single-cell amplification is <2 x 10(5), better than that of current genome sequencing standards. Polymerase cloning should provide a critical tool for systematic characterization of genome diversity in the biosphere.
38. Genomic Islands and The Ecology and Evolution of Prochlorococcus
    Coleman ML, Sullivan MB, Martiny AC, Steglich C, Barry K, Delong EF, Chisholm SW
    Science. 2006 Mar 24;311(5768):1768-70.
    Pubmed: 16556843
    Abstract
    Prochlorococcus ecotypes are a useful system for exploring the origin and function of diversity among closely related microbes. The genetic variability between phenotypically distinct strains that differ by less that 1% in 16S ribosomal RNA sequences occurs mostly in genomic islands. Island genes appear to have been acquired in part by phage-mediated lateral gene transfer, and some are differentially expressed under light and nutrient stress. Furthermore, genome fragments directly recovered from ocean ecosystems indicate that these islands are variable among cooccurring Prochlorococcus cells. Genomic islands in this free-living photoautotroph share features with pathogenicity islands of parasitic bacteria, suggesting a general mechanism for niche differentiation in microbial species.
39. Niche Partitioning Among Prochlorococcus Ecotypes Along Ocean-Scale Environmental Gradients
    Johnson ZI, Zinser ER, Coe A, McNulty NP, Woodward EM, Chisholm SW
    Science. 2006 Mar 24;311(5768):1737-40.
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    Pubmed: 16556835
    Abstract
    Prochlorococcus is the numerically dominant phytoplankter in the oligotrophic oceans, accounting for up to half of the photosynthetic biomass and production in some regions. Here, we describe how the abundance of six known ecotypes, which have small subunit ribosomal RNA sequences that differ by less than 3%, changed along local and basin-wide environmental gradients in the Atlantic Ocean. Temperature was significantly correlated with shifts in ecotype abundance, and laboratory experiments confirmed different temperature optima and tolerance ranges for cultured strains. Light, nutrients, and competitor abundances also appeared to play a role in shaping different distributions.
40. Measurement of Prochlorococcus Ecotypes Using Real-Time Polymerase Chain Reaction Reveals Different Abundances of Genotypes With Similar Light Physiologies
    Ahlgren NA, Rocap G, Chisholm SW
    Environ Microbiol. 2006 Mar;8(3):441-54.
    Pubmed: 16478451
    Abstract
    Prochlorococcus is a marine cyanobacterium which is found at high abundances in world's tropical and subtropical oligotrophic oceans. The genus Prochlorococcus can be divided into two major groups based on light physiology. Both of these groups can be further subdivided into genetically distinct lineages, or ecotypes. Real-time polymerase chain reaction (PCR) assays based on sequence differences in the 16S-23S rDNA internal transcribed spacer or the 23S rDNA were developed to examine the distribution of each ecotype in the field. The real-time PCR assays enabled linear quantification of concentrations ranging from 10 to 4 x 10(5) cells ml(-1). These assays were applied to a stratified water column in the Sargasso Sea. The majority of Prochlorococcus cells above 110 m belonged to the one of the low chlorophyll b/a ratio (high-light adapted) ecotypes, while two types of high chlorophyll b/a ratio (low-light adapted) cells dominated below 110 m. The other three types were found at significantly lower numbers or not detected at all. Differences in the abundance of ecotypes within the major light physiology groupings suggest that other factors, such as nutrient utilization and differential mortality, are driving their relative distributions. Real-time PCR assays will enable further exploration of these factors and temporal and geographic variability in ecotype abundance.
41. Community Genomics Among Stratified Microbial Assemblages In The Ocean's Interior
    DeLong, EF; Preston, CM; Mincer, T; Rich, V; Hallam, SJ; Frigaard, NU; Martinez, A; Sullivan, MB; Edwards, R; Brito, BR; Chisholm, SW; Karl, DM
    Science. 2006 311(5760):496-503
    Pubmed: 16439655
    Abstract
    Microbial life predominates in the ocean, yet little is known about its genomic variability, especially along the depth continuum. We report here genomic analyses of planktonic microbial communities in the North Pacific Subtropical Gyre, from the ocean's surface to near-sea floor depths. Sequence variation in microbial community genes reflected vertical zonation of taxonomic groups, functional gene repertoires, and metabolic potential. The distributional patterns of microbial genes suggested depth-variable community trends in carbon and energy metabolism, attachment and motility, gene mobility, and host-viral interactions. Comparative genomic analyses of stratified microbial communities have the potential to provide significant insight into higher-order community organization and dynamics.
42. Prochlorococcus Ecotype Abundances In The North Atlantic Ocean As Revealed By An Improved Quantitative Pcr Method
    Zinser ER, Coe A, Johnson ZI, Martiny AC, Fuller NJ, Scanlan DJ, Chisholm SW
    Appl Environ Microbiol. 2006 Jan;72(1):723-32.
    Pubmed: 16391112
    Abstract
    The cyanobacterium Prochlorococcus numerically dominates the photosynthetic community in the tropical and subtropical regions of the world's oceans. Six evolutionary lineages of Prochlorococcus have been described, and their distinctive physiologies and genomes indicate that these lineages are "ecotypes" and should have different oceanic distributions. Two methods recently developed to quantify these ecotypes in the field, probe hybridization and quantitative PCR (QPCR), have shown that this is indeed the case. To facilitate a global investigation of these ecotypes, we modified our QPCR protocol to significantly increase its speed, sensitivity, and accessibility and validated the method in the western and eastern North Atlantic Ocean. We showed that all six ecotypes had distinct distributions that varied with depth and location, and, with the exception of the deeper waters at the western North Atlantic site, the total Prochlorococcus counts determined by QPCR matched the total counts measured by flow cytometry. Clone library analyses of the deeper western North Atlantic waters revealed ecotypes that are not represented in the culture collections with which the QPCR primers were designed, explaining this discrepancy. Finally, similar patterns of relative ecotype abundance were obtained in QPCR and probe hybridization analyses of the same field samples, which could allow comparisons between studies.
43. Photosynthesis Genes In Marine Viruses Yield Proteins During Host Infection
    Lindell D, Jaffe JD, Johnson ZI, Church GM, Chisholm SW
    Nature. 2005 Nov 3;438(7064):86-9. Epub 2005 Oct 12.
    Pubmed: 16222247
    Abstract
    Cyanobacteria, and the viruses (phages) that infect them, are significant contributors to the oceanic 'gene pool'. This pool is dynamic, and the transfer of genetic material between hosts and their phages probably influences the genetic and functional diversity of both. For example, photosynthesis genes of cyanobacterial origin have been found in phages that infect Prochlorococcus and Synechococcus, the numerically dominant phototrophs in ocean ecosystems. These genes include psbA, which encodes the photosystem II core reaction centre protein D1, and high-light-inducible (hli) genes. Here we show that phage psbA and hli genes are expressed during infection of Prochlorococcus and are co-transcribed with essential phage capsid genes, and that the amount of phage D1 protein increases steadily over the infective period. We also show that the expression of host photosynthesis genes declines over the course of infection and that replication of the phage genome is a function of photosynthesis. We thus propose that the phage genes are functional in photosynthesis and that they may be increasing phage fitness by supplementing the host production of these proteins.
44. Three Prochlorococcus Cyanophage Genomes: Signature Features and Ecological Interpretations
    Sullivan MB, Coleman ML, Weigele P, Rohwer F, Chisholm SW
    PLoS Biol. 2005 May;3(5):e144. Epub 2005 Apr 19.
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    Pubmed: 15828858
    Abstract
    The oceanic cyanobacteria Prochlorococcus are globally important, ecologically diverse primary producers. It is thought that their viruses (phages) mediate population sizes and affect the evolutionary trajectories of their hosts. Here we present an analysis of genomes from three Prochlorococcus phages: a podovirus and two myoviruses. The morphology, overall genome features, and gene content of these phages suggest that they are quite similar to T7-like (P-SSP7) and T4-like (P-SSM2 and P-SSM4) phages. Using the existing phage taxonomic framework as a guideline, we examined genome sequences to establish "core" genes for each phage group. We found the podovirus contained 15 of 26 core T7-like genes and the two myoviruses contained 43 and 42 of 75 core T4-like genes. In addition to these core genes, each genome contains a significant number of "cyanobacterial" genes, i.e., genes with significant best BLAST hits to genes found in cyanobacteria. Some of these, we speculate, represent "signature" cyanophage genes. For example, all three phage genomes contain photosynthetic genes (psbA, hliP) that are thought to help maintain host photosynthetic activity during infection, as well as an aldolase family gene (talC) that could facilitate alternative routes of carbon metabolism during infection. The podovirus genome also contains an integrase gene (int) and other features that suggest it is capable of integrating into its host. If indeed it is, this would be unprecedented among cultured T7-like phages or marine cyanophages and would have significant evolutionary and ecological implications for phage and host. Further, both myoviruses contain phosphate-inducible genes (phoH and pstS) that are likely to be important for phage and host responses to phosphate stress, a commonly limiting nutrient in marine systems. Thus, these marine cyanophages appear to be variations of two well-known phages-T7 and T4-but contain genes that, if functional, reflect adaptations for infection of photosynthetic hosts in low-nutrient oceanic environments.
45. Release of Dissolved Organic Matter By Prochlorococcus
    Bertilsson, S; Berglund, O; Pullin, MJ; Chisholm, SW
    Vie Et Milieu-Life and Environment. 2005 55(3-4):225-231
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    Abstract
    Phytoplankton release a variable fraction (0 to > 80%) of the photosynthetically fixed inorganic carbon as extracellular dissolved organic compounds. Despite this wide range and the potential effects on carbon fluxes and food p webs, the knowledge on how the environment and phytoplankton species involved influence this process is incomplete. Notably, there are no estimates for release of dissolved organic carbon by Prochlorococcus, a marine cyanobacterium estimated to be the numerically dominant oxygenic phototroph in the oceans. Here we report extracellular release of dissolved organic compounds from two axenic Prochlorococcus strains representing different ecotypes (MED4 and MIT9312) cultured under nutrient replete and phosphorus limited conditions. Independent assays based on C-14-bicarbonate tracers and analyses of particulate and dissolved organic carbon suggest that the release of dissolved organic carbon ranged from 9 to 24% of the total assimilated inorganic carbon with slightly lower values for phosphorus limited cultures. Between 4 and 20% of the released organic matter consisted of low molecular weight carboxylic acids, compounds known to be highly labile substrates for heterotrophic bacteria. In oligotrophic oceans where Prochlorococcus is a dominant contributor to primary production and input of terrigenous organic matter is negligible, this process is likely a significant contributor to the pool of organic substrates available for microbial heterotrophs.
46. Quantitative Proteomics of Prochlorococcus Marinus
    Leptos, Kyriacos C.; Jaffe, Jacob D.; Zinser, Erik; Lindell, Debbie; Chisholm, Sallie W.; Church, George M.
    Cancer Genomics & Proteomics. 2005 2(3):178-
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47. Patterns and Regulation of Clade-Specific Diversity of Prochlorococcus In The Atlantic Ocean
    Johnson, Z.; Zinser, E.; Coe, A.; Chisholm, S. W.
    Abstracts of The General Meeting of The American Society For Microbiology. 2005 105():379-
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48. Properties of Overlapping Genes Are Conserved Across Microbial Genomes
    Johnson ZI, Chisholm SW
    Genome Res. 2004 Nov;14(11):2268-72.
    Pubmed: 15520290
    Abstract
    There are numerous examples from the genomes of viruses, mitochondria, and chromosomes that adjacent genes can overlap, sharing at least one nucleotide. Overlaps have been hypothesized to be involved in genome size minimization and as a regulatory mechanism of gene expression. Here we show that overlapping genes are a consistent feature (approximately one-third of all genes) across all microbial genomes sequenced to date, have homologs in more microbes than do non-overlapping genes, and are therefore likely more conserved. In addition, the size, phase (reading frame offset), and distribution, among other characteristics, of overlapping genes are most consistent with the hypothesis that overlaps function in the regulation of gene expression. The upstream sequences and conservation of overlapping orthologs of two model organisms from the genus Prochlorococcus that have significantly different GC-content, and therefore different nucleotide sequences for orthologs, are also consistent with small overlapping sequence regions and programmed shifts in reading frame as a common mechanism in the regulation of microbial gene expression.
49. Transfer of Photosynthesis Genes To and From Prochlorococcus Viruses
    Lindell D, Sullivan MB, Johnson ZI, Tolonen AC, Rohwer F, Chisholm SW
    Proc Natl Acad Sci U S A. 2004 Jul 27;101(30):11013-8. Epub 2004 Jul 15.
    Pubmed: 15256601
    Abstract
    Comparative genomics gives us a new window into phage-host interactions and their evolutionary implications. Here we report the presence of genes central to oxygenic photosynthesis in the genomes of three phages from two viral families (Myoviridae and Podoviridae) that infect the marine cyanobacterium Prochlorococcus. The genes that encode the photosystem II core reaction center protein D1 (psbA), and a high-light-inducible protein (HLIP) (hli) are present in all three genomes. Both myoviruses contain additional hli gene types, and one of them encodes the second photosystem II core reaction center protein D2 (psbD), whereas the other encodes the photosynthetic electron transport proteins plastocyanin (petE) and ferredoxin (petF). These uninterrupted, full-length genes are conserved in their amino acid sequence, suggesting that they encode functional proteins that may help maintain photosynthetic activity during infection. Phylogenetic analyses show that phage D1, D2, and HLIP proteins cluster with those from Prochlorococcus, indicating that they are of cyanobacterial origin. Their distribution among several Prochlorococcus clades further suggests that the genes encoding these proteins were transferred from host to phage multiple times. Phage HLIPs cluster with multicopy types found exclusively in Prochlorocococus, suggesting that phage may be mediating the expansion of the hli gene family by transferring these genes back to their hosts after a period of evolution in the phage. These gene transfers are likely to play a role in the fitness landscape of hosts and phages in the surface oceans.
50. Cyanophages Infecting The Oceanic Cyanobacterium Prochlorococcus
    Sullivan MB, Waterbury JB, Chisholm SW
    Nature. 2003 Aug 28;424(6952):1047-51.
    Pubmed: 12944965
    Abstract
    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.
51. Genome Divergence In Two Prochlorococcus Ecotypes Reflects Oceanic Niche Differentiation
    Rocap G, Larimer FW, Lamerdin J, Malfatti S, Chain P, Ahlgren NA, Arellano A, Coleman M, Hauser L, Hess WR, Johnson ZI, Land M, Lindell D, Post AF, Regala W, Shah M, Shaw SL, Steglich C, Sullivan MB, Ting CS, Tolonen A, Webb EA, Zinser ER, Chisholm SW
    Nature. 2003 Aug 28;424(6952):1042-7. Epub 2003 Aug 13.
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    Pubmed: 12917642
    Abstract
    The marine unicellular cyanobacterium Prochlorococcus is the smallest-known oxygen-evolving autotroph. It numerically dominates the phytoplankton in the tropical and subtropical oceans, and is responsible for a significant fraction of global photosynthesis. Here we compare the genomes of two Prochlorococcus strains that span the largest evolutionary distance within the Prochlorococcus lineage and that have different minimum, maximum and optimal light intensities for growth. The high-light-adapted ecotype has the smallest genome (1,657,990 base pairs, 1,716 genes) of any known oxygenic phototroph, whereas the genome of its low-light-adapted counterpart is significantly larger, at 2,410,873 base pairs (2,275 genes). The comparative architectures of these two strains reveal dynamic genomes that are constantly changing in response to myriad selection pressures. Although the two strains have 1,350 genes in common, a significant number are not shared, and these have been differentially retained from the common ancestor, or acquired through duplication or lateral transfer. Some of these genes have obvious roles in determining the relative fitness of the ecotypes in response to key environmental variables, and hence in regulating their distribution and abundance in the oceans.
52. Elemental Composition of Marine Prochlorococcus and Synechococcus: Implications For The Ecological Stoichiometry of The Sea
    Bertilsson, S; Berglund, O; Karl, DM; Chisholm, SW
    Limnology and Oceanography. 2003 48(5):1721-1731
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    Abstract
    The elemental composition of marine cyanobacteria is an important determinant of the ecological stoichiometry in low-latitude marine biomes. We analyzed the cellular carbon (C), nitrogen (N), and phosphorus (P) contents of Prochlorococcus (MED4) and Synechococcus (WH8103 and WH8012) under nutrient-replete and P-starved conditions. Under nutrient-replete conditions, C, N, and P quotas (femtogram cell(-1)) of the three strains were 46 +/- 4, 9.4 +/- 0.9, and 1.0 +/- 0.2 for MED4; 92 +/- 13, 20 +/- 3, and 1.8 +/- 0.1 for WH8012; and 213 +/- 7, 50 +/- 2, 3.3 +/- 10.5 for WH8103. In P-limited cultures, they were 61 +/- 2, 9.6 +/- 0.1, and 0.3 +/- 0.1 for MED4; 132 +/- 6, 21 +/- 2, and 0.5 +/- 0.2 for WH8012; and 244 +/- 21, 40 +/- 4, and 0.8 +/- 0.01 for WH8103. P limitation had no effect on the N cell quota of MED4 and WH8012 but reduced the N content of WH8103. The cellular C quota was consistently higher in P-limited than in nutrient-replete cultures. All three strains had higher C: P and N: P ratios than the Redfield ratio under both nutrient-replete and P-limited conditions. The C:N molar ratios ranged 5-5.7 in replete cultures and 7.1-7.5 in P-limited cultures; C: P ranged 121-165 in the replete cultures and 464-779 under P limitation; N:P ranged 21-33 in the replete cultures and 59-109 under P limitation. Our results suggest that Prochlorococcus and Syneehococcus may have relatively low P requirements in the field, and thus the particulate organic matter they produce would differ from the Redfield ratio (106C: 16N: 1P) often assumed for the production of new particulate organic matter in the sea.
53. Isoprene Production By Prochlorococcus, A Marine Cyanobacterium, and Other Phytoplankton
    Shaw, SL; Chisholm, SW; Prinn, RG
    Marine Chemistry. 2003 80(4):PII S0304-4203(02)00101-9-
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    Abstract
    The oceans are a small source of light (C-2-C-6) non-methane hydrocarbons (NMHC), which influence the photo-oxidant chemistry of the remote marine atmosphere. Previous work has shown that water column sources of alkenes include photochemical processes, and that various phytoplankton species can produce isoprene. However, only a few phytoplankton species have been studied, and no assessment has been performed of the effects of other pelagic microorganisms on NMHC cycling. The dependence of phytoplanktonic isoprene production on light, temperature, and organism size has also not been investigated. In this work, laboratory cultures of five different marine phytoplankton species (Prochlorococcus, Synechococcus, Micromonas pusilla, Pelagomonas calceolata, and Emiliania huxleyi) were examined for NMHC production capabilities. All species were found to produce isoprene at constant rates during the balanced exponential growth phase; rates ranged from 1 X 10(-21) to 4 X 10(-19) mol cell(-1) day(-1) over all cell species and growth conditions tested. No other NMHC was consistently produced or consumed by these cells. The presence of heterotrophic bacteria in phytoplankton cultures had no effect on isoprene production rates per phytoplankton cell. A positive allomettic relationship was observed between isoprene production rate and cell volume; highest production rates were found for the largest cell, E. huxleyi, and lowest rates for Prochlorococcus, the smallest cell. Isoprene production was a function of light intensity and temperature in Prochlorococcus, with patterns that were similar to those between growth rate and these environmental variables. The maximum production with light intensity occurred in the photoinhibited regime, and the maximum with temperature was at the maximum of growth rate for this species, near 23 degreesC. Nanoflagellate grazing by Cafeteria roenbergensis on, and phage infection of, Prochlorococcus controlled total isoprene produced in the flask by controlling cell abundances. Phage infection also decreased the isoprene production rate per cell during latent period of infection as compared to healthy cells. With certain assumptions, combining the measured laboratory isoprene production rates with observed water column phytoplankton abundances resulted in a maximum estimated sea-to-air flux of isoprene that was on the same order of magnitude as previously reported values determined using in situ measured seawater and atmospheric measurements. (C) 2002 Elsevier Science B.V. All rights reserved.
54. Genomic and Physiological Analyses of Environmental Stress Response Mechanisms In Two Closely Related Marine Cyanobacteria.
    Ting, C. S.; Chisholm, S. W.; King, J.
    Abstracts of The General Meeting of The American Society For Microbiology. 2003 103():N-103
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    Abstract
    Prochlorococcus and Synechococcus are the most abundant photosynthetic prokaryotes in the oceans and are important contributors to global primary production. A key factor in understanding their ecological distribution is knowledge of their general abiotic stress response mechanisms. The availability of whole genome sequences for marine Synechococcus (isolate WH 8102) and Prochlorococcus (isolates MED4, MIT 9313) (http://www.jgi.doe.gov/JGI_microbial/html/index.html), allowed us to explore if these closely related cyanobacteria have evolved different strategies for responding to environmental stress, particularly at the level of regulation of gene expression. As a first step, we have characterized their response to temperature stress and have focused on a key group of cellular stress proteins, the molecular chaperones. The results indicate that protein synthesis rates in heat stressed Pro MED4 cells are comparable to controls and do not decrease following short (20 min) or long (6 hrs) exposures to elevated temperatures. Notably, fewer cellular proteins are induced in Pro MED4 compared to marine Synechococcus in response to heat stress, and these stress-induced proteins constitute only a minor fraction of the newly synthesized polypeptides. This is not attributable to the absence of genes encoding major molecular chaperones and proteases in Pro MED4. Comparative genome analyses indicate that Pro MED4 possesses a similar suite of genes encoding major molecular chaperones as in Syn WH8102, and has multiple copies of groEL, dnaK and dnaJ genes. Mass spectrometry analysis has established that GroEL1, and not GroEL2, is a major heat-induced chaperone in Pro MED4. These results support the idea that although Prochlorococcus and Synechococcus are closely related and often co-occur in the same ocean habitat, they have evolved differences in their stress-induced transcriptional activation and repression mechanisms. This in turn could result in dissimilarities in their ability to maintain productive protein folding, stabilization, and/or degradation reactions at elevated temperatures.
55. Correlation of the cytochrome c (550) content of cyanobacterial Photosystem II with the EPR properties of the oxygen-evolving complex.
    Lakshmi KV, Reifler MJ, Chisholm DA, Wang JY, Diner BA, Brudvig GW
    Photosynth Res. 2002;72(2):175-89.
    Pubmed: 16228516
    Abstract
    The Mn(4) cluster of PS II advances through a series of oxidation states (S states) that catalyze the breakdown of water to dioxygen in the oxygen-evolving complex. The present study describes the engineering and purification of highly active PS II complexes from mesophilic His-tagged Synechocystis PCC 6803 and purification of PS II core complexes from thermophilic wild-type Synechococcus lividus with high levels of the extrinsic polypeptide, cytochrome c (550). The g = 4.1 S(2) state EPR signal, previously not characterized in untreated cyanobacterial PS II, is detected in high yields in these PS II preparations. We present a complete characterization of the g = 4.1 state in cyanobacterial His-tagged Synechocystis PCC 6803 PS II and S. lividus PS II. Also presented are a determination of the stoichiometry of cytochrome c (550) bound to His-tagged Synechocystis PCC 6803 PS II and analytical ultracentrifugation results which indicate that cytochrome c (550) is a monomer in solution. The temperature-dependent multiline to g = 4.1 EPR signal conversion observed for the S(2) state in cyanobacterial PS II with high cytochrome c (550) content is very similar to that previously found for spinach PS II. In spinach PS II, the formation of the S(2) state g = 4.1 EPR signal has been found to correlate with the binding of the extrinsic 17 and 23 kDa polypeptides. The finding of a similar correlation in cyanobacterial PS II with the binding of cytochrome c (550) suggests a functional homology between cytochrome c (550) and the 17 and 23 kDa extrinsic proteins of spinach PS II.
56. Is Ocean Fertilization Credible and Creditable? Response
    Chisholm, SW; Falkowski, PG; Cullen, JJ
    Science. 2002 296(5567):467-468
    Pubmed: 11965672
    
57. Resolution of Prochlorococcus and Synechococcus Ecotypes By Using 16S-23S Ribosomal Dna Internal Transcribed Spacer Sequences
    Rocap G, Distel DL, Waterbury JB, Chisholm SW
    Appl Environ Microbiol. 2002 Mar;68(3):1180-91.
    Pubmed: 11872466
    Abstract
    Cultured isolates of the marine cyanobacteria Prochlorococcus and Synechococcus vary widely in their pigment compositions and growth responses to light and nutrients, yet show greater than 96% identity in their 16S ribosomal DNA (rDNA) sequences. In order to better define the genetic variation that accompanies their physiological diversity, sequences for the 16S-23S rDNA internal transcribed spacer (ITS) region were determined in 32 Prochlorococcus isolates and 25 Synechococcus isolates from around the globe. Each strain examined yielded one ITS sequence that contained two tRNA genes. Dramatic variations in the length and G+C content of the spacer were observed among the strains, particularly among Prochlorococcus strains. Secondary-structure models of the ITS were predicted in order to facilitate alignment of the sequences for phylogenetic analyses. The previously observed division of Prochlorococcus into two ecotypes (called high and low-B/A after their differences in chlorophyll content) were supported, as was the subdivision of the high-B/A ecotype into four genetically distinct clades. ITS-based phylogenies partitioned marine cluster A Synechococcus into six clades, three of which can be associated with a particular phenotype (motility, chromatic adaptation, and lack of phycourobilin). The pattern of sequence divergence within and between clades is suggestive of a mode of evolution driven by adaptive sweeps and implies that each clade represents an ecologically distinct population. Furthermore, many of the clades consist of strains isolated from disparate regions of the world's oceans, implying that they are geographically widely distributed. These results provide further evidence that natural populations of Prochlorococcus and Synechococcus consist of multiple coexisting ecotypes, genetically closely related but physiologically distinct, which may vary in relative abundance with changing environmental conditions.
58. Cyanobacterial Photosynthesis In The Oceans: The Origins and Significance of Divergent Light-Harvesting Strategies
    Ting CS, Rocap G, King J, Chisholm SW
    Trends Microbiol. 2002 Mar;10(3):134-42.
    Pubmed: 11864823
    Abstract
    Prochlorococcus and Synechococcus are abundant unicellular cyanobacteria and major participants in global carbon cycles. Although they are closely related and often coexist in the same ocean habitat, they possess very different photosynthetic light-harvesting antennas. Whereas Synechococcus and the majority of cyanobacteria use phycobilisomes, Prochlorococcus has evolved to use a chlorophyll a(2)/b(2) light-harvesting complex. Here, we present a scenario to explain how the Prochlorococcus antenna might have evolved in an ancestral cyanobacterium in iron-limited oceans, resulting in the diversification of the Prochlorococcus and marine Synechococcus lineages from a common phycobilisome-containing ancestor. Differences in the absorption properties and cellular costs between chlorophyll a(2)/b(2) and phycobilisome antennas in extant Prochlorococcus and Synechococcus appear to play a role in differentiating their ecological niches in the ocean environment.
59. Copper Toxicity and Cyanobacteria Ecology In The Sargasso Sea
    Mann, EL; Ahlgren, N; Moffett, JW; Chisholm, SW
    Limnology and Oceanography. 2002 47(4):976-988
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    Abstract
    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 Prochlorococus its 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 high- and low-light adapted ecotypes were inhibited at free Cu2+ concentrations that had no effect on Synechococcus. However, the high-light adapted strains were more copper resistant than their low-light adapted Counterparts. When copper was added to Prochlorococcus front 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 G(1) and early S phases of the cell cycle. Prochlorococcus in shallow mixed layers were less sensitive to copper and were probably members of the copper-resistant high-fight adapted ecotype. Synechoccus 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.
60. Utilization of Different Nitrogen Sources By The Marine Cyanobacteria Prochlorococcus and Synechococcus
    Moore, LR; Post, AF; Rocap, G; Chisholm, SW
    Limnology and Oceanography. 2002 47(4):989-996
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    Abstract
    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 NO2-, but all others did not. Whole genome analysis of the low-B/A Prochlorococcus MED4 revealed that the genes required for NO3- 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 NO2- 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.
61. Cross-Scale Ecological Dynamics and Microbial Size Spectra In Marine Ecosystems
    Rinaldo, A; Maritan, A; Cavender-Bares, KK; Chisholm, SW
    Proceedings of The Royal Society B-Biological Sciences. 2002 269(1504):2051-2059
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    Abstract
    Evaluating the component features of 'scaling' planktonic size spectra, commonly observed in marine ecosystems, is crucial for understanding the ecological and evolutionary processes from which they emerge. Here, we develop a theoretical framework that describes such spectra in terms of the size distributions of individual species, and test it against actual datasets of microbial size spectra from the Atlantic Ocean. We describe characteristics of size probability distributions of component species that are sufficient to support the observational evidence and infer that, when a power law describes the community size spectrum (thus suggesting critical self-organization of microbial ecosystem structure and function), a related power law links the total number of individuals of a given species to its mean size.
62. Cobalt Limitation and Uptake In Prochlorococcus
    Saito, MA; Moffett, JW; Chisholm, SW; Waterbury, JB
    Limnology and Oceanography. 2002 47(6):1629-1636
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    Abstract
    Processes that enable marine phytoplankton to acquire trace metals are fundamental to our understanding of primary productivity and global carbon biogeochemical cycling. Here we show that the abundant marine cyanobacterium, Prochlorococcus strain MED4-Ax, has an absolute cobalt requirement and that zinc cannot substitute for cobalt in the growth medium, as is the case in some other phytoplankton species. When resuspended into fresh medium, uptake of cobalt into the cell occurs as free cobalt (Co2+). In contrast, cultures augmented with conditioned medium assimilated cobalt significantly faster than those in fresh medium, leading to the hypothesis that Prochlorococcus produced organic cobalt ligands in the conditioned medium. This work suggests that the availability of cobalt might influence the composition of phytoplankton assemblages in the open ocean.
63. The Photosynthetic Apparatus of Prochlorococcus: Insights Through Comparative Genomics
    Hess WR, Rocap G, Ting CS, Larimer F, Stilwagen S, Lamerdin J, Chisholm SW
    Photosynth Res. 2001;70(1):53-71.
    Pubmed: 16228362
    Abstract
    Within the vast oceanic gyres, a significant fraction of the total chlorophyll belongs to the light-harvesting antenna systems of a single genus, Prochlorococcus. This organism, discovered only about 10 years ago, is an extremely small, Chl b-containing cyanobacterium that sometimes constitutes up to 50% of the photosynthetic biomass in the oceans. Various Prochlorococcus strains are known to have significantly different conditions for optimal growth and survival. Strains which dominate the surface waters, for example, have an irradiance optimum for photosynthesis of 200 mumol photons m(-2) s(-1), whereas those that dominate the deeper waters photosynthesize optimally at 30-50 mumol photons m(-2) s(-1). These high and low light adapted 'ecotypes' are very closely related - less than 3% divergent in their 16S rRNA sequences - inviting speculation as to what features of their photosynthetic mechanisms might account for the differences in photosynthetic performance. Here, we compare information obtained from the complete genome sequences of two Prochlorococcus strains, with special emphasis on genes for the photosynthetic apparatus. These two strains, Prochlorococcus MED4 and MIT 9313, are representatives of high- and low-light adapted ecotypes, characterized by their low or high Chl b/a ratio, respectively. Both genomes appear to be significantly smaller (1700 and 2400 kbp) than those of other cyanobacteria, and the low-light-adapted strain has significantly more genes than its high light counterpart. In keeping with their comparative light-dependent physiologies, MED4 has many more genes encoding putative high-light-inducible proteins (HLIP) and photolyases to repair UV-induced DNA damage, whereas MIT 9313 possesses more genes associated with the photosynthetic apparatus. These include two pcb genes encoding Chl-binding proteins and a second copy of the gene psbA, encoding the Photosystem II reaction center protein D1. In addition, MIT 9313 contains a gene cluster to produce chromophorylated phycoerythrin. The latter represents an intermediate form between the phycobiliproteins of non-Chl b containing cyanobacteria and an extremely modified beta phycoerythrin as the sole derivative of phycobiliproteins still present in MED4. Intriguing features found in both Prochlorococcus strains include a gene cluster for Rubisco and carboxysomal proteins that is likely of non-cyanobacterial origin and two genes for a putative varepsilon and beta lycopene cyclase, respectively, explaining how Prochlorococcus may synthesize the alpha branch of carotenoids that are common in green organisms but not in other cyanobacteria.
64. Phycobiliprotein Genes of The Marine Photosynthetic Prokaryote Prochlorococcus: Evidence For Rapid Evolution of Genetic Heterogeneity
    Ting CS, Rocap G, King J, Chisholm SW
    Microbiology. 2001 Nov;147(Pt 11):3171-82.
    Pubmed: 11700369
    Abstract
    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.
65. Oceans - Dis-Crediting Ocean Fertilization
    Chisholm, SW; Falkowski, PG; Cullen, JJ
    Science. 2001 294(5541):309-310
    Pubmed: 11598285
    
66. Microbial Size Spectra From Natural and Nutrient Enriched Ecosystems
    Cavender-Bares, KK; Rinaldo, A; Chisholm, SW
    Limnology and Oceanography. 2001 46(4):778-789
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    Abstract
    Microbial size spectra, including bacteria through nanophytoplankton, were measured by use of how cytometry across the western north Atlantic Ocean and during two nutrient enrichment studies: bottle enrichments in the Sargasso Sea and an in situ iron enrichment in the equatorial Pacific (IronEx II). Spectral shapes, or the relative conformity to a function described by a power law, ranged from smooth and log linear during the spring bloom in the Sargasso Sea to being distinctly non-log linear in coastal waters. Overall, the individual spectra within large regions characterized by similar ecological conditions showed remarkable consistency, inviting speculation that powerful organizing mechanisms are at work in these communities. Moreover, the ensemble average of all of the spectra along the transect displays clear power-law behavior. Slopes ranged from -1.0, in which biomass was equally distributed between all size classes, to -1.4, in which proportionally more biomass was contained in smaller size classes; there was no clear relationship between nutrient concentrations and spectral slopes over the entire data set. Species succession in nutrient-enriched bottles caused spectra to evolve from relatively smooth power laws to distributions showing preferred sizes (i.e., nonlinear on a log-log plot). The IronEx II spectra, however, remained similar over the course of the experiment. It could be that the elimination of bottle effects in this experiment buffered the system in ways that maintained the size structure of the microbial community over the size range we measured. Our results suggest conditions that lead to log-linear size distributions; these should be verified over a broader range of scales and environments.
67. Nutrient Gradients In The Western North Atlantic Ocean: Relationship To Microbial Community Structure and Comparison To Patterns In The Pacific Ocean
    Cavender-Bares, KK; Karl, DM; Chisholm, SW
    Deep-Sea Research Part I-Oceanographic Research Papers. 2001 48(11):2373-2395
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    Abstract
    Studies of nitrogen and phosphorus dynamics in the oligotrophic surface waters of the western North Atlantic Ocean have been constrained because ambient concentrations are typically at or below the detection limits of standard colorometric methods, except during periods of deep vertical mixing. Here we report the application of high-sensitivity analytical methods-determinations of nitrate plus nitrite (N + N) by chemiluminescence and soluble reactive phosphorus (SR-P) by the magnesium induced co-precipitation (MAGIC) protocol-to surface waters along a transect from the Sargasso Sea at 26 degreesN through the Gulf Stream at 37 degreesN, including sampling at the JGOFS Bermuda Atlantic Time-series Study (BATS) station. The results were compared with data from the BATS program, and the HOT station in the Pacific Ocean, permitting cross-ecosystem comparisons. Microbial populations were analyzed along the transect, and an attempt was made to interpret their distributions in the context of the measured nutrient concentrations. Surface concentrations of N+N and SRP during the March 1998 transect separated into 3 distinct regions, with the boundaries corresponding roughly to the locations of the BATS station (similar to 31 degreesN) and the Gulf Stream (similar to 37 degreesN). Although N+N and SRP co-varied, the [N+N]:[SR-P] molar ratios increased systematically from similar to1 to 10 in the southern segment, remained relatively constant at similar to 40-50 between 31 degreesN and 37 degreesN, then decreased again systematically to ratios < 10 north of the Gulf Stream. Dissolved organic N (DON) and P (DOP) dominated (greater than or equal to 90%) the total dissolved N (TDN) and P (TDP) pools except in the northern portion of the transect. The [DON]: [DOP] molar ratios were relatively invariant (similar to 30-60) across the entire transect. Heterotrophic prokaryotes (operationally defined as "bacteria"), Prochlorococcus, Synechococcus, ultra-and nanophytoplaakton, cryptophytes, and coccolithophores were enumerated by flow cytometry. The abundance of bacteria was well correlated with the concentration of SRP, and that of the ultra- and nanophytoplankton was well correlated with the concentration of N+N. The only group whose concentration was correlated with temperature was Prochlorococcus, and its abundance was unrelated to the concentrations of nutrients measured at the surface. We combined our transect results with time-series measurements from the BATS site and data from select depth profiles, and contrasted these North Atlantic data sets with time-series of N and P nutrient measurements from a station in the North Pacific subtropical gyre near Hawaii [Hawaii Ocean Time-series (HOT) site]. Two prominent differences are readily observed from this comparison. The [N + N]: [SRP] molar ratios are much less than 16: 1 during stratified periods in surface waters at the BATS site, as is the case at the HOT site year round. However, following deep winter mixing, this ratio is much higher than 16, 1 at BATS. Also, SRP concentrations in the upper 100 m at BATS fall in the range 1-10 nM during stratified periods, which is at least one order of magnitude lower than at the HOT site. That two ecosystems with comparable rates of primary and export production would differ so dramatically in their nutrient dynamics is intriguing, and highlights the need for detailed cross ecosystem comparisons. (C) 2001 Elsevier Science Ltd. All rights reserved.
68. Phytoplankton Population Dynamics At The Bermuda Atlantic Time-Series Station In The Sargasso Sea
    DuRand, MD; Olson, RJ; Chisholm, SW
    Deep-Sea Research Part Ii-Topical Studies In Oceanography. 2001 48(8-9):1983-2003
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    Abstract
    Phytoplankton populations were analyzed using flow cytometry in monthly samples at the Bermuda Atlantic Time-series Study (BATS) station in the Sargasso Sea from 1989-1994 for picoplankton (Synechococcus and Prochlorococcus) and from 1992-1994 for eukaryotic phytoplankton in order to better understand the mechanisms that dictate seasonal and inter-annual patterns in the phytoplankton community. The eukaryotic phytoplankton were dominated by populations of small nanoplankton (mostly 2-4 mum diameter), though populations of coccolithophores and sometimes pennate diatoms also could be distinguished. Flow cytometric measurements of population abundances, individual cell light scattering (which can be related to cell. size), and chlorophyll fluorescence were made. Synechococcus and the eukaryotic phytoplankton reached their greatest concentrations during the spring bloom each year when the water column was deeply mixed and nutrients were detectable in surface waters. The maximum cell concentration for Prochlorococcus was in the summer and fall of each year, with a deeper sub-surface maximum than Synechococcus. Picoplankton chlorophyll fluorescence and estimated cell size were greater at depth than near the surface, and were lowest in midsummer for both Synechococcus and Prochlorococcus. In the summer and fall, Prochlorococcus cells were often smallest at mid-depth, even when fluorescence per cell and cell concentration were lower at the surface. For the eukaryotes (including coccolithophores), cell concentrations were high during the spring in both 1992 and 1993, and in fall 1992. At these times, mean cell size and fluorescence were low. Improved size and carbon estimates were made and it was found that the estimated contribution of phytoplankton carbon to total particulate organic carbon, integrated over the upper 200 m, averaged 33% (range 21-43%) with no pronounced seasonal pattern. (C) 2001 Elsevier Science Ltd. All rights reserved.
69. Photoacclimation Kinetics of Single-Cell Fluorescence In Laboratory and Field Populations of Prochlorococcus
    Dusenberry, JA; Olson, RJ; Chisholm, SW
    Deep-Sea Research Part I-Oceanographic Research Papers. 2001 48(6):1443-1458
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    Abstract
    Picophytoplankton show promise as dynamic tracers for vertical mixing if an appropriate index of photoacclimative state and the kinetics of change in that index can be determined. To this end, several light shift incubations were carried out with laboratory cultures of Prochlorococcus MED4 and natural populations of Prochlorococcus at sea. These time series revealed systematic changes in chlorophyll fluorescence in response to changes in incident light intensity. Prochlorococcus also exhibited strong diel patterns in fluorescence and light scattering resulting from the phasing of cell division to the daily photoperiod, In order to obtain a parameter that was insensitive to the daily photoperiod, Prochlorococcus red fluorescence was normalized to the 0.33 power of forward angle light scattering, an empirically derived normalization factor. We propose that this normalized fluorescence could be an appropriate measure of photoacclimation in Prochlorococcus, suitable for use in inverse modeling of mixing dynamics. A logistic model for photoacclimation was found to fit the experimental data well, although there was some systematic deviation from the data for populations shifted to high irradiances. Estimates of photoacclimative rates ranged from 0.9 to 2 d(-1). (C) 2001 Elsevier Science Ltd. All rights reserved.
70. Niche Adaptation of Prochlorococcus Ecotypes: Insights Through Comparative Genomics
    Hess, W. R.; Rocap, G.; Steglich, C.; Post, A.; Ting, C. S.; Chisholm, S. W.
    Journal of Phycology. 2001 37(3 Supplement):23-24
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71. Phycoerythrin and The Photosynthetic Apparatus of Prochlorococcus: Insights Through Comparative Genomics
    Hess, W. R.; Rocap, G.; Chisholm, S. W.
    Photosynthesis Research. 2001 69(1-3):271-
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72. Oceanography - Stirring Times In The Southern Ocean
    Chisholm, SW
    Nature. 2000 407(6805):685-687
    Pubmed: 11048702
    
73. In Situ Hybridization of Prochlorococcus and Synechococcus (Marine Cyanobacteria) Spp. With Rrna-Targeted Peptide Nucleic Acid Probes
    Worden AZ, Chisholm SW, Binder BJ
    Appl Environ Microbiol. 2000 Jan;66(1):284-9.
    Pubmed: 10618237
    Abstract
    A simple method for whole-cell hybridization using fluorescently labeled rRNA-targeted peptide nucleic acid (PNA) probes was developed for use in marine cyanobacterial picoplankton. In contrast to established protocols, this method is capable of detecting rRNA in Prochlorococcus, the most abundant unicellular marine cyanobacterium. Because the method avoids the use of alcohol fixation, the chlorophyll content of Prochlorococcus cells is preserved, facilitating the identification of these cells in natural samples. PNA probe-conferred fluorescence was measured flow cytometrically and was always significantly higher than that of the negative control probe, with positive/negative ratio varying between 4 and 10, depending on strain and culture growth conditions. Prochlorococcus cells from open ocean samples were detectable with this method. RNase treatment reduced probe-conferred fluorescence to background levels, demonstrating that this signal was in fact related to the presence of rRNA. In another marine cyanobacterium, Synechococcus, in which both PNA and oligonucleotide probes can be used in whole-cell hybridizations, the magnitude of fluorescence from the former was fivefold higher than that from the latter, although the positive/negative ratio was comparable for both probes. In Synechococcus cells growing at a range of growth rates (and thus having different rRNA concentrations per cell), the PNA- and oligonucleotide-derived signals were highly correlated (r = 0.99). The chemical nature of PNA, the sensitivity of PNA-RNA binding to single-base-pair mismatches, and the preservation of cellular integrity by this method suggest that it may be useful for phylogenetic probing of whole cells in the natural environment.
74. Field Observations of Oceanic Mixed Layer Dynamics and Picophytoplankton Photoacclimation
    Dusenberry, JA; Olson, RJ; Chisholm, SW
    Journal of Marine Systems. 2000 24(3-4):221-232
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    Abstract
    To explore the relationship between mixing dynamics in the surface ocean and picophytoplankton optical properties, we measured the distributions of single cell fluorescence in Prochlorococcus populations throughout a diel cycle. Two time series were conducted in the N. Atlantic, and both showed a shoaling of the mixed layer, due to surface warming or a rain-formed surface layer. These dynamics, coupled with the diel cycle of solar irradiance, drove the development of a depth gradient in mean red fluorescence of Prochlorococcus, due to photoacclimation, in the newly-stratified layer. Furthermore, the frequency distribution of single-cell fluorescence within field populations appears to have responded to changing mixing and photoacclimation dynamics, with photoacclimation in the absence of strong mixing generally resulting in a reduced variance in fluorescence within sample populations. Nighttime mixing in the absence of photoacclimation reversed this process and resulted in increased variation of single-cell fluorescence. Departures from normality in observed distributions suggest vertical mixing time-scales that are slightly longer than time-scales of photoacclimation. The behavior of the mean and variance, and possibly the third and fourth moments, of single-cell optical properties is consistent with photoacclimation in response to the physical dynamics. (C) 2000 Elsevier Science B.V. All rights reserved.
75. Iron Limits The Cell Division Rate of Prochlorococcus In The Eastern Equatorial Pacific
    Mann, EL; Chisholm, SW
    Limnology and Oceanography. 2000 45(5):1067-1076
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    Abstract
    Prochlorococcus, a small, unicellular cyanobacterium, is an important member of the phytoplankton community in the eastern equatorial Pacific. When these waters were enriched with iron during IronEx II, the chlorophyll per cell and cell size of Prochlorococcus increased, implying that they were iron limited. The extent of this limitation was unclear, however, and the number of Prochlorococcus remained constant. To examine whether cell division rates were stimulated significantly by iron, we used a cell cycle analysis approach to measure them in and out of the Fe-enriched patch and in Fe-enriched bottles. The cell division rate increased from 0.6 to 1.1 d(-1) over 6 d of exposure to the elevated iron concentrations in the patch. Cells incubated in bottles with additional iron had rates of 1.4 d(-1) or two doublings per day. Prochlorococcus mortality rates, measured independently, nearly doubled after the addition of iron. This marched the increase in the cell division rate and maintained a relatively constant population size. Thus the cell division rates of even the smallest phytoplankton in the equatorial Pacific are significantly iron limited, but biomass is constrained by both iron limitation and microzooplankton grazing. The differential response of individual phytoplankton groups to the addition of iron during IronEx II was at least partially a result of differential mortality rates over the time course of the experiment. How the community would respond to sustained fertilization, however, is not obvious.
76. The Effectiveness of The Consed Autofinish Program In Automating The Finishing Process For Microbial Genomes Derived By Whole Genome Shotgun
    Arellano, A.; Trong, S.; Chain, P.; Do, L.; Stilwagen, S.; Burkhart-Schultz, K.; Janecki, T.; Erler, A.; Mariero, L.; Sanders, C.; Johnson, G.; Sakaldasis, G.; Regala, W.; Rivera, A.; Bhakta, R.; Golinveaux, K.; Tran, M.; Lao, V.; Pitesky, M.; Duarte, S.; Kobayashi, A.; Larimer, F.; Arp, D.; Chisholm, S.; Harwood, Caroline; Meeks, J.; Lamerdin, J. E.
    International Genome Sequencing and Analysis Conference. 2000 12():48-
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    Abstract
    The JGI has completed draft sequencing for the genomes of Nitrosomonas europea 2.97 MB, Prochlorococcus marinus 1.67MB, Prochlorococcus marinus MED4 2.72Mb, Rhodopseudomonas palustris 5.49MB, and Nostoc punctiforme 9.22MB. We use the Consed Autofinish program (David Gordon, University of Washington) as an automated initial phase in our finishing process to select walking primers for double stranding and gap closure. These microbial genomes exhibit large variations in size, GC content, and the number and extent of gene duplications and IS elements, and the results of Consed Autofinish primer walks vary greatly depending on the content of the sequence. For instance, P. marinus went from 35 contigs down to 4 in the first pass, but N. europeae, -which has 80 identified IS elements averaging >1Kb in size and 2 recently duplicated operons- is still in 70 major contigs after 3 rounds of automated primer walking. We have now optimized the Autofinish output via scripts that screen out repeat regions early on in the process. We will present our efficiency in using Consed Autofinish as a high throughput, automated approach to finishing whole genome shotgun sequencing projects.
77. Annotation of Microbial Genomes For High-Throughput Sequencing
    Larimer, F.; Land, M.; Hyatt, D.; Shah, M.; Arellano, A.; Stilwagen, S.; Do, L.; Chain, P.; Kobayashi, A.; Arp, D.; Hooper, A.; Chisholm, S.; Rocap, G.; Harwood, C.; Meeks, J.; Weinstock, G.; Uberbacher, E.; Branscomb, E.; Lamerdin, J.
    International Genome Sequencing and Analysis Conference. 2000 12():104-
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    Abstract
    The Microbial Sequencing Program of the Department of Energy's Joint Genome Institute has exercised a policy of early release of sequence, including, the release of draft-level annotation when sequencing reaches 8-fold coverage. Currently, extensive draft annotation is provided for Nitrosomonas europaea, Prochlorococcus marinus, Rhodopseudomonas palustris, Nostoc punctiforme, and Enterococcus faecium. The draft annotation pipeline is highly automated, utilizing three gene calling algorithms (Generation, Glimmer2, and Critica) in an iterative procedure that maximizes both the sensitivity of detection and the accuracy of the 'final' gene model. Blast/BEAUTY search results, Pfam hmm profile matches and COGs matches are generated for the translations of each gene model. Nucleotide content and skew is provided for each gene. tRNA genes are detected using tRNAscan-SE; rRNAs and miscellaneous structural RNAs are also indicated. Draft annotation can be accessed through the individual organism pages at the JGI Microbial Sequencing web site (http://spider.jgi-psf.org/JGI_microbial/html/) or the ORNL Microbial genome resource web page (http://genome.ornl.gov/microbial/). Early views of sequence and annotation have proven invaluable to the scientific user community to direct and focus ongoing research. This scheme for annotation of original sequence is being applied to the Genome Channel/Genome Catalog environment for refreshing annotation of completed genomes.
78. Genomic Sequencing and Analysis of The Marine Cyanobacterium, Prochlorococcus Marinus (Med4)
    Stilwagen, S.; Arellano, A.; Burkhart-Schultz, K.; Janecki, T.; Erler, A.; Mariero, L.; Attix, T.; Sanders, C.; Do, L.; Johnson, G.; Sakaldasis, G.; Regala, W.; Duarte, S.; Kobayashi, A.; Larimer, F.; Chisholm, S.; Rocap, G.; Lamerdin, J. E.
    International Genome Sequencing and Analysis Conference. 2000 12():92-93
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    Abstract
    The Joint Genome Institute (JGI) has sequenced the unicellular Cyanobacterium, Prochlorococcus marinus (MED4), as part of a DOE initiative to explore the role of microorganisms in global carbon sequestration. Prochlorococcus species are found in oligotrophic regions of the world's temperate oceans. Due to their extremely small size and broad global distribution, they are thought to be the most abundant photosynthetic organisms on the planet. Prochlorococcus species have adapted to a wide range of light conditions found at different levels of the water column in the open oceans. This may be in part due to their unusual photosynthetic pigment composition relative to other known marine cyanobacteria. The complete 1.65 Mb genome sequence of P. marinus(MED4), a high-light adapted strain, was determined using a whole genome shotgun strategy. Minimally overlapping long range PCR products that spanned the entire genome were generated and fingerprinted to aid in the verification of the final assembly. The initial analyses of the genomic sequence have identified 1689 putative encoding genes, 36 tRNAs and one RNA cluster. The JGI is also currently sequencing a low-light adapted strain of P. marinus, (MIT9313) as well as the closely related Cyanobacterium, marine Synechococcus WH8102. Future comparative analyses of these genomes should allow the identification of environmental variables that exert the strongest selection pressures on these co-adapted species in the open oceans.
79. Differential Response of Equatorial Pacific Phytoplankton To Iron Fertilization
    Cavender-Bares, KK; Mann, EL; Chisholm, SW; Ondrusek, ME; Bidigare, RR
    Limnology and Oceanography. 1999 44(2):237-246
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    Abstract
    Recent unenclosed iron-fertilization experiments in the equatorial Pacific Ocean have shown that phytoplankton biomass can be increased substantially by the addition of iron. Analyses of size-fractionated chlorophyll indicate that much of the increase during the most recent fertilization experiment, IronEx II, occurred in the >10-mu m size fraction. We used flow cytometry, combined with taxon-specific pigment measurements by high-performance liquid chromatography (HPLC), to analyze the responses of five different groups of phytoplankton: Prochlorococcus, Synechococcus, ultraplankton, nanoplankton, and pennate diatoms. These results are unique in the suite of measurements from the IronEx studies in that they simultaneously examine individual cell properties, which are grazer independent, and population dynamics, which reflect the net result of growth and grazing. Our results show that the overall increase of chlorophyll a (Chl a) in the patch was due in part to increases in chlorophyll content per cell and in part to increases in cell numbers of specific groups. Cellular fluorescence was stimulated by iron addition in all five groups to a qualitatively similar degree and was correlated with taxon-specific changes in cellular pigments. In terms of net cell growth, however, these groups responded very differently. The groups that dominated the community before the addition of iron increased at most twofold in cell number; Prochlorococcus actually decreased. In contrast, the initially rare pennate diatoms increased 15-fold in number by the peak of the iron-induced bloom. Within 1 week, this differential response led to a dramatic change in the phytoplankton community structure, from one dominated by picoplankton to one dominated by large diatoms. It is not known whether this shift would be sustained over extended periods of fertilization, a response that would ultimately change the structure of the food web.
80. Seasonal and Depth Variation In Microbial Size Spectra At The Bermuda Atlantic Time Series Station
    Gin, KYH; Chisholm, SW; Olson, RJ
    Deep-Sea Research Part I-Oceanographic Research Papers. 1999 46(7):1221-1245
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    Abstract
    Dual-beam flow cytometry was used to generate concentration and biomass size spectra (derived from light scatter signals) of bacteria and phytoplankton at the Bermuda Atlantic Time Series (USJGOFS) station in the oligotrophic Sargasso Sea. The size structure of the phytoplankon was characterized by an average slope of -1.8 for the normalized cell concentration spectrum. When bacteria were included, the average slope was -1.9, very close to the point at which there would be an equal amount of biomass in equal sized logarithmic classes (slope = -2.0). Nanoplankton were the major biomass fraction (about 55-85%) in the upper 100 m of the water column where total biomass levels are highest. At greater depths, where total biomass is lower, the relative proportion of picoplankton (especially bacteria) increases (to about 70-90%). Microplankton generally were less than 20% of the microbial community biomass, The size spectra indicate the importance of picophytoplankton at the chlorophyll maximum, consistent with the competitive advantage of small cells in light-limited conditions. Most of the seasonal variability in biomass occured in the nanoplankton fraction, whereas bacteria biomass remained relatively constant. In the spring, increases in the nano- and picoplankton were observed which could be attributed to small increases in nutrient concentrations in the surface layer. Late summer stratification and the subsequent depletion of nutrients from surface waters resulted in a decline in the nano/micro fraction and thus the mean cell size of phytoplankton. Overall, the bacterial contribution to total microbial biomass integrated over the euphotic zone was about 12%, a finding that is lower than that of most other studies. This can be attributed to methodological differences between flow cytometry and microscopy, as well as the choice of cell volume to biomass conversion factors. (C) 1999 Elsevier Science Ltd. All rights reserved.
81. Photophysiology of The Marine Cyanobacterium Prochlorococcus: Ecotypic Differences Among Cultured Isolates
    Moore, LR; Chisholm, SW
    Limnology and Oceanography. 1999 44(3):628-638
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    Abstract
    Cultured isolates of Prochlorococus 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/a(2) (Chl b/a(2)) ratios. Although both groups photoacclimate when grown over a range of light intensities, isolates with distinctly higher Chl b/a(2) ratios (high B/A ecotype) reach maximal growth rates at lower irradiances (I-k,I-g), have high growth efficiencies (OLS), and are inhibited in growth at irradiances where isolates with low Chl b/a(2) ratios (low B/A ecotype) are growing maximally. High Chl b/a(2) ratios resulted in higher spectrally weighted average chi at-specific absorption coefficient ((a) over bar)(chl)*, Chl a(2)-specific light-harvesting efficiency (alpha(chla)), and quantum yield (phi(m)) 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.
82. Molecular Phylogeny of Prochlorococcus Ecotypes
    Rocap, Gabrielle; Moore, Lisa R.; Chisholm, Sallie W.
    Bulletin De L'Institut Oceanographique (Monaco). 1999 0(SPEC. ISSUE 19):107-115
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    Abstract
    Prochlorococcus is the dominant phototroph in the temperate oceans. It is found throughout the euphotic zone, at light intensities spanning over four orders of magnitude. This growth range is larger than observed for any single culture of Prochlorococcus and is likely due to the co-existence of multiple ecotypes adapted for optimal growth at different light intensities. Here we report the 16S rDNA sequences of four Prochlorococcus isolates from the Pacific Ocean, MIT9201, MIT9202, MIT9211 and MIT9215, which have been physiologically characterized as high or low B/A ecotypes based on their chl b/a2 ratios and light-dependent growth responses. The three low B/A isolates are closely related and cluster together with other low B/A isolates in a previously identified "high-light adapted" clade. The fourth Pacific isolate, a high B/A ecotype, is not a member of this clade. Sequences specific to each physiological type will be useful in probing field populations to determine the spatial and temporal distribution of the ecotypes of Prochlorococcus.
83. Physiology and Molecular Phylogeny of Coexisting Prochlorococcus Ecotypes
    Moore LR, Rocap G, Chisholm SW
    Nature. 1998 Jun 4;393(6684):464-7.
    Pubmed: 9624000
    Abstract
    The cyanobacterium Prochlorococcus is the dominant oxygenic phototroph in the tropical and subtropical regions of the world's oceans. It can grow at a range of depths over which light intensities can vary by up to 4 orders of magnitude. This broad depth distribution has been hypothesized to stem from the coexistence of genetically different populations adapted for growth at high- and low-light intensities. Here we report direct evidence supporting this hypothesis, which has been generated by isolating and analysing distinct co-occurring populations of Prochlorococcus at two locations in the North Atlantic. Co-isolates from the same water sample have very different light-dependent physiologies, one growing maximally at light intensities at which the other is completely photoinhibited. Despite this ecotypic differentiation, the co-isolates have 97% similarity in their 16S ribosomal RNA sequences, demonstrating that molecular microdiversity, commonly observed in microbial systems, can be due to the coexistence of closely related, physiologically distinct populations. The coexistence and distribution of multiple ecotypes permits the survival of the population as a whole over a broader range of environmental conditions than would be possible for a homogeneous population.
84. Rapid Diversification of Marine Picophytoplankton With Dissimilar Light-Harvesting Structures Inferred From Sequences of Prochlorococcus and Synechococcus (Cyanobacteria)
    Urbach E, Scanlan DJ, Distel DL, Waterbury JB, Chisholm SW
    J Mol Evol. 1998 Feb;46(2):188-201.
    Pubmed: 9452521
    Abstract
    Cultured isolates of the unicellular planktonic cyanobacteria Prochlorococcus and marine Synechococcus belong to a single marine picophytoplankton clade. Within this clade, two deeply branching lineages of Prochlorococcus, two lineages of marine A Synechococcus and one lineage of marine B Synechococcus exhibit closely spaced divergence points with low bootstrap support. This pattern is consistent with a near-simultaneous diversification of marine lineages with divinyl chlorophyll b and phycobilisomes as photosynthetic antennae. Inferences from 16S ribosomal RNA sequences including data for 18 marine picophytoplankton clade members were congruent with results of psbB and petB and D sequence analyses focusing on five strains of Prochlorococcus and one strain of marine A Synechococcus. Third codon position and intergenic region nucleotide frequencies vary widely among members of the marine picophytoplankton group, suggesting that substitution biases differ among the lineages. Nonetheless, standard phylogenetic methods and newer algorithms insensitive to such biases did not recover different branching patterns within the group, and failed to cluster Prochlorococcus with chloroplasts or other chlorophyll b-containing prokaryotes. Prochlorococcus isolated from surface waters of stratified, oligotrophic ocean provinces predominate in a lineage exhibiting low G + C nucleotide frequencies at highly variable positions.
85. A Dual Sheath Flow Cytometer For Shipboard Analyses of Phytoplankton Communities From The Oligotrophic Oceans
    Cavender-Bares, KK; Frankel, SL; Chisholm, SW
    Limnology and Oceanography. 1998 43(6):1383-1388
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    Abstract
    Flow cytometry has been used to study phytoplankton since the early 1980s. Because of the wide range of cell sizes and concentrations that occur in natural samples, multiple instrument configurations have been required to characterize pico, ultra-, and nanophytoplankton. Lengthy changeover times between configurations have made synoptic analyses of phytoplankton communities extremely cumbersome and therefore rare. To overcome this problem, we have added dual-sheath capability and adjustable optics to a commercially available flow cytometer. These additions expand the instrument's capabilities during routine operation. The dual-sheath cytometer can characterize both the submicron picoplankter Prochlorococcus at sample flow rates of 10 mu l min(-1) and, after a 1-min changeover, ultraphytoplankton and nanophytoplankton at sample flow rates >1 ml min(-1). These capabilities make possible real-time analysis of phytoplankton size spectra at sea.
86. Winter Presence of Prochlorococcus In The East China Sea
    Jiao, NZ; Yang, YH; Mann, E; Chisholm, SW; Chen, NH
    Chinese Science Bulletin. 1998 43(10):877-878
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87. Genetic Diversity In Prochlorococcus Populations Flow Cytometrically Sorted From The Sargasso Sea and Gulf Stream
    Urbach, E; Chisholm, SW
    Limnology and Oceanography. 1998 43(7):1615-1630
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    Abstract
    An investigation into the genetic structure of Prochlorococcus picophytoplankton in depth profiles from the Sargasso Sea and the Gulf Stream revealed a high degree of genetic heterogeneity within local populations. partial sequencing of cloned polymerase chain reaction products amplified from flow cytometrically sorted cells was used to identify 6-21 different Prochlorococcus petB/D alleles recovered from each of eight sorted samples, with 68 alleles identified among 187 clones in the combined data set. Rarefaction analyses indicated that many additional unsampled alleles were present at each level of sample aggregation. Overlapping sets of alleles were recovered from Prochlorococcus populations in the two water columns, from different depths within each water column, and from flow cytometrically distinguishable subpopulations sorted from the same water sample, suggesting that each of these populations drew their membership from a single gene pool. Consistent with results of autecological studies, members of a high-light-adapted Prochlorococcus clade predominated in clone libraries from surface waters. It thus appears that wild Prochlorococcus populations consist of individuals drawn from a variety of evolutionary Lineages and that populations at different depths and in two distinct hydrographic regimes recruit their members from the same gene pool. Natural selection favors the predominance of high-light-adapted genotypes in near-surface populations drawn from this gene pool.
88. Dynamics of Picophytoplankton, Ultraphytoplankton and Bacteria In The Central Equatorial Pacific
    Binder, BJ; Chisholm, SW; Olson, RJ; Frankel, SL; Worden, AZ
    Deep-Sea Research Part Ii-Topical Studies In Oceanography. 1996 43(4-6):907-931
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    Abstract
    Pico- and ultraplankton are known to contribute significantly to overall biomass and primary productivity in the ''high nutrient-low chlorophyll'' waters of the equatorial Pacific. In order to understand the dynamics of this community on ecologically relevant time-scales, we examined the abundance, distribution and cellular characteristics of Prochlorococcus, Synechococcus, eukaryotic ultraphytoplankton and heterotrophic bacteria during two 20-day time-series at 0 degrees N, 140 degrees W in the spring and fall of 1992 (JGOFS time-series cruises, TS-I and TS-II). Prochlorococcus was numerically dominant among the autotrophic groups considered, with mean cell concentrations in surface waters on the order of 1.4 x 10(5) cells ml(-1). Synechococcus and ultraphytoplankton abundances were 17-30-fold lower than those of Prochlorococcus, and heterotrophic bacterial abundances were 5-7-fold higher (during TS-I and TS-II, respectively). Daily cell abundances for all groups varied by factors of 1.5-2 within each time-series. Depth-integrated Prochlorococcus abundance averaged over each time-series was 25% lower during TS-II relative to TS-I; ultraphytoplankton abundance was 42% higher during the same period. Prochlorococcus and ultraphytoplankton both contributed significantly to the estimated total autotrophic biomass; Synechococcus contributed relatively little. Estimated total photosynthetic pico- plus ultraplankton biomass was on average 30% higher than heterotrophic bacterial biomass. Changes in the fluorescence and light scatter properties of individual Prochlorococcus cells were observed during the passage of a tropical instability wave during TS-II, and are hypothesized to reflect a physiological response among these cells to that event. Examination of bulk properties alone (e.g. cell numbers or total red fluorescence) would not have revealed these physiological changes. Lower bounds for Prochlorococcus-specific growth rates were calculated based on the DNA distributions of these populations at dusk. These rates were maximal at 15 or 30 m depth, where they approached one doubling per day. Changes in Prochlorococcus forward angle light scatter (FALS) from dawn to dusk were well correlated with these estimates of specific growth rate, an observation that allowed us to relate measurements of FALS to cell volume for Prochlorococcus. Copyright (C) 1996 Elsevier Science Ltd.
89. Iron-Enrichment Bottle Experiments In The Equatorial Pacific: Responses of Individual Phytoplankton Cells
    Zettler, ER; Olson, RJ; Binder, BJ; Chisholm, SW; Fitzwater, SE; Gordon, RM
    Deep-Sea Research Part Ii-Topical Studies In Oceanography. 1996 43(4-6):1017-1029
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    Abstract
    Iron-enrichment bottle experiments were monitored using how cytometry to investigate the hypothesis that phytoplankton in the equatorial Pacific are iron-limited. Iron-enriched Synechococcus, ultraphytoplankton, nanophytoplankton, pennate diatoms, and coccolithophorids had higher fluorescence and/or forward light scatter per cell than control cells; for Prochlorococcus the trends were the same although the differences were not significant. This suggests that most phytoplankton cells were physiologically affected by the low iron concentrations in this region. However, only pennate diatoms showed significant increases in cell concentrations due to iron enrichment. The sum of chlorophyll fluorescences of individual cells measured by flow cytometry yielded patterns similar to those of extracted bulk chlorophyll, with increases of up to 10-fold in iron-enriched bottles but at most 3-fold in control bottles; pennate diatoms accounted for most of the increase in chlorophyll in iron-enriched bottles. Copyright (C) 1996 Elsevier Science Ltd.
90. Growth of Prochlorococcus, a Photosynthetic Prokaryote, in the Equatorial Pacific Ocean.
    Vaulot D, Marie D, Olson RJ, Chisholm SW
    Science. 1995 Jun 9;268(5216):1480-1482.
    Pubmed: 17843668
    Abstract
    The cell cycle of Prochlorococcus, a prokaryote that accounts for a sizable fraction of the photosynthetic biomass in the eastern equatorial Pacific, progressed in phase with the daily light cycle. DNA replication occurred in the afternoon and cell division occurred at night. Growth rates were maximal (about one doubling per day) at 30 meters and decreased toward the surface and the bottom of the ocean. Estimated Prochlorococcus production varied between 174 and 498 milligrams of carbon per square meter per day and accounted for 5 to 19 percent of total gross primary production at the equator. Because Prochlorococcus multiplies close to its maximum possible rate, it is probably not severely nutrient-limited in this region of the oceans.
91. Ecosystem Experiments
    CARPENTER, SR; CHISHOLM, SW; KREBS, CJ; SCHINDLER, DW; WRIGHT, RF
    Science. 1995 269(5222):324-327
    Pubmed: 17841247
    Abstract
    Experimental manipulations of entire ecosystems have been conducted in lakes, catchments, streams, and open terrestrial and marine environments. Experiments have addressed applied problems of ecosystem management and complex responses of communities and ecosystems to perturbations. in the course of some experiments, environmental indicators acid models have been developed and tested. Surprising results with implications for ecological understanding and management are common.
92. Cell-Cycle Regulation In Marine Synechococcus Sp Strains
    BINDER, BJ; CHISHOLM, SW
    Applied and Environmental Microbiology. 1995 61(2):708-717
    Pubmed: 16534938
    Abstract
    The cell cycle behavior of four marine strains of the unicellular cyanobacterium Synechococcus sp. was analyzed by examining the DNA frequency distributions of exponentially growing and dark-blocked populations and by considering the patterns of change in these distributions during growth under a diel light-dark cycle. The two modes of cell cycle regulation previously identified in a freshwater and coastal marine Synechococcus isolate, respectively, were represented among the three open-ocean strains we examined. The first of these modes of regulation is consistent with the slow-growth case of the widely accepted prokaryotic cell cycle paradigm. The second appears to involve asynchronous initiation of chromosome replication, the presence of multiple chromosome copies at low growth rates, and variability in chromosome copy number among cells in the population. These characteristics suggest the involvement of a large probabilistic component in cell cycle regulation which could make the application of cell cycle-based estimators of in situ growth rate to Synechococcus populations problematic.
93. The Iron Hypothesis - Basic Research Meets Environmental-Policy
    CHISHOLM, SW
    Reviews of Geophysics. 1995 33():1277-1286
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94. Comparative Physiology of Synechococcus and Prochlorococcus - Influence of Light and Temperature On Growth, Pigments, Fluorescence and Absorptive Properties
    MOORE, LR; GOERICKE, R; CHISHOLM, SW
    Marine Ecology-Progress Series. 1995 116(1-3):259-275
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    Abstract
    Prochlorococcus marinus is abundant and widespread throughout the world's oceans and always co-occurs geographically with the marine cyanobacterium Synechococcus. In the Atlantic Ocean, these 2 picoplankters exhibit different spatial and seasonal distributions. In order to better understand the ecology of these species, we measured growth and photoacclimation responses including fluorescence excitation [F*(ph)(lambda)] and in vivo absorption [a*(ph)(lambda)] spectra over a range of growth irradiances for P. marinus (clone SS120) and Synechococcus WH8103, both isolated from the Sargasso Sea. To explore the physiological diversity of P. marinus, we measured the physiological responses of another P. marinus clone, MED4, isolated from the Mediterranean Sea. Growth rate as a function of temperature was also examined for all 3 clones. P. marinus SS120 and Synechococcus WH8103 have different temperature optima for growth, but these do not explain the different latitudinal distributions in the North Atlantic. P. marinus SS120 is adapted for growth at low light intensities relative to Synechococcus WH8103, which is consistent with the relative depth distribution of P. marinus and Synechococcus in the field. The light-dependent growth response of P. marinus MED4 is more similar to Synechococcus WH8103 than to P. marinus SS120. The unique pigment content of P. marinus (which contain divinyl chlorophylls a and b) results in maximal absorbance in the blue wavelengths. The high total chl b/chl a ratio of P. marinus SS120 enables it to absorb more light, grow faster than Synechococcus WH8103 (and P. marinus MED4) at low light intensities, and presumably to outcompete Synechococcus in the deep euphotic zone. At high growth irradiances, P. marinus SS120 contains measureable amounts of normal (monovinyl) chl b, whereas this pigment was not found in P. marinus MED4 at any growth irradiance. Photoacclimative changes in pigment ratios, and not package effect, account for most of the changes in a*(ph)(lambda) and F*(ph)(lambda) With Light intensity for all 3 picoplankters. At high light intensities, zeaxanthin contributes substantially to a*(ph)(lambda) in the blue, but appears to transfer little or no excitation energy to the reaction centers, based on F*(ph)(lambda) measurements. For P. marinus, high absorption in the blue due to divinyl chl a and b relative to normal chi a and b, absorption due to zeaxanthin, and small cell size result in unusually high a*(ph) (blue) relative to a*(ph) (red).
95. Testing The Iron Hypothesis In Ecosystems of The Equatorial Pacific-Ocean
    MARTIN, JH; COALE, KH; JOHNSON, KS; FITZWATER, SE; GORDON, RM; TANNER, SJ; HUNTER, CN; ELROD, VA; NOWICKI, JL; COLEY, TL; BARBER, RT; LINDLEY, S; WATSON, AJ; VANSCOY, K; LAW, CS; LIDDICOAT, MI; LING, R; STANTON, T; STOCKEL, J; COLLINS, C; ANDERSON, A; BIDIGARE, R; ONDRUSEK, M; LATASA, M; MILLERO, FJ; LEE, K; YAO, W; ZHANG, JZ; FRIEDERICH, G; SAKAMOTO, C; CHAVEZ, F; BUCK, K; KOLBER, Z; GREENE, R; FALKOWSKI, P; CHISHOLM, SW; HOGE, F; SWIFT, R; YUNGEL, J; TURNER, S; NIGHTINGALE, P; HATTON, A; LISS, P; TINDALE, NW
    Nature. 1994 371(6493):123-129
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    Abstract
    The idea that iron might limit phytoplankton growth in large regions of the ocean has been tested by enriching an area of 64 km(2) in the open equatorial Pacific Ocean with iron. This resulted in a doubling of plant biomass, a threefold increase In chlorophyll and a fourfold increase in plant production. Similar increases were found in a chlorophyll-rich plume downstream of the Galapagos Islands, which was naturally enriched in iron. These findings indicate that iron limitation can control rates of phytoplankton productivity and biomass in the ocean.
96. Simulating Bacterial Clustering Around Phytoplankton Cells In A Turbulent Ocean
    BOWEN, JD; STOLZENBACH, KD; CHISHOLM, SW
    Limnology and Oceanography. 1993 38(1):36-51
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    Abstract
    The hypothesis that bacteria can cluster around phytoplankton cells in the turbulent mixed layer was tested with a model that simulates bacterial chemotaxis toward a neutrally buoyant phytoplankton cell exuding dissolved organic C. The model results indicate that bacteria can attain population densities orders of magnitude above background levels in microzones occupying <0.1% of the fluid volume surrounding each phytoplankton cell. The simulation results indicate that at turbulence intensities expected in the upper mixed layer of the ocean (shear rates of approximately 0.15 s-1) bacteria initially approach phytoplankton through random swimming and relative fluid motions. Chemotactic response serves to prolong a bacteria's stay near the phytoplankter before it is carried away by random swimming and fluid motions. At these shear rates, up to 20% of the chemotactic bacteria population could be clustered around exuding phytoplankton cells, even though individual bacteria stay in a cluster less than a minute. For these conditions the time-averaged exudate exposure of the bacterial population could be 10 times higher than that of a nonchemotactic population. Exudate exposures in unsteady shearing were found to equal or exceed the corresponding steady shear values. Although unsteady bursts of turbulent mixing in the oceanic surface layer should disperse clusters, intervening calm periods are long enough to allow clusters to reform. The model indicates that bacterial clustering is unlikely to have a significant effect on phytoplankton nutrient uptake or on the fate of bacterial secondary production in the microbial food web.
97. Prochlorophytes
    Urbach E, Robertson DL, Chisholm SW
    Nature. 1992 Jan 16;355(6357):267-70.
    Pubmed: 1731225
    Abstract
    The taxonomic group Prochlorales (Lewin 1977) Burger-Wiersma, Stal and Mur 1989 was established to accommodate a set of prokaryotic oxygenic phototrophs which, like plant, green algal and euglenoid chloroplasts, contain chlorophyll b instead of phycobiliproteins. Prochlorophytes were originally proposed (with concomitant scepticism) to be a monophyletic group sharing a common ancestry with these 'green' chloroplasts. Results from molecular sequence phylogenies, however, have suggested that Prochlorothrix hollandica is not on a lineage that leads to plastids. Our results from 16S ribosomal RNA sequence comparisons, which include new sequences from the marine picoplankter Prochlorococcus marinus and the Lissoclinum patella symbiont Prochloron sp., indicate that prochlorophytes are polyphyletic within the cyanobacterial radiation, and suggest that none of the known species is specifically related to chloroplasts. This implies that the three prochlorophytes and the green chloroplast ancestor acquired chlorophyll b and its associated structural proteins in convergent evolutionary events. We report further that the 16S rRNA gene sequence from Prochlorococcus is very similar to those of open ocean Synechococcus strains (marine cluster A), and to a family of 16S rRNA genes shotgun-cloned from plankton in the north Atlantic and Pacific Oceans.
98. Patterns of Cell-Size Change In A Marine Centric Diatom - Variability Evolving From Clonal Isolates
    ARMBRUST, EV; CHISHOLM, SW
    Journal of Phycology. 1992 28(2):146-156
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    Abstract
    Isolates of the centric diatom, Thalassiosira weissflogii Grun., were maintained in exponential growth under constant, favorable conditions for nearly 2 years. During this interval, each culture underwent periodic increases and decreases in mean cell size, a behavior predicted for diatom populations alternating between sexual and asexual reproduction, respectively. The overall patterns of cell size change displayed by each culture, however, were unique. The maximum size of newly enlarged cells varied among isolates and within a given isolate over time. Consequently, both the timing and rate of increase in mean cell size also varied despite the fact that the minimum average cell size obtained by the various cultures was relatively constant. The most consistent feature among the isolates was the rate of decrease in mean cell size, a value determined by the physical constraints of the diatom frustule during mitotic divisions. We hypothesize that the extent of the variability exhibited by these cultures results from the fact that an inherent feature of diatom populations is a constantly changing genetic composition.
99. What Limits Phytoplankton Growth
    CHISHOLM, SW
    Oceanus. 1992 35(3):36-46
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100. Prochlorococcus-Marinus Nov Gen-Nov Sp - An Oxyphototrophic Marine Prokaryote Containing Divinyl Chlorophyll-A and Chlorophyll-B
     CHISHOLM, SW; FRANKEL, SL; GOERICKE, R; OLSON, RJ; PALENIK, B; WATERBURY, JB; WESTJOHNSRUD, L; ZETTLER, ER
     Archives of Microbiology. 1992 157(3):297-300
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     Abstract
     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.
101. What Controls Phytoplankton Production In Nutrient-Rich Areas of The Open Sea - American-Society-of-Limnology-and-Oceanography Symposium - 22-24 February 1991 San-Marcos, California - Preface
     CHISHOLM, SW; MOREL, FMM
     Limnology and Oceanography. 1991 36(8):U1507-U1511
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102. Combination of 16S Ribosomal-RNA-Targeted Oligonucleotide Probes With Flow-Cytometry For Analyzing Mixed Microbial-Populations
     AMANN, RI; BINDER, BJ; OLSON, RJ; CHISHOLM, SW; DEVEREUX, R; STAHL, DA
     Applied and Environmental Microbiology. 1990 56(6):1919-1925
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103. Role of Light and The Cell-Cycle On The Induction of Spermatogenesis In A Centric Diatom
     ARMBRUST, EV; CHISHOLM, SW
     Journal of Phycology. 1990 26(3):470-478
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     Abstract
     The centric diatom, Thalassiosira weissflogii Grun., can be induced to undergo spermatogenesis by exposing cells maintained at saturating levels of continuous light to either dim light or darkness. Using flow cytometry to determine the relative DNA and chlorophyll content per cell, the number of cells within a population that responded to an induction signal was measured. From 0 to over 90% of a population differentiated into male gametes depending upon both the induction trigger and the population examined, regardless of the average cell size of the population. Through the use of synchronized cultures, we demonstrated that responsiveness to an induction trigger was a function of cell cycle stage; cells in early G1 were not yet committed to complete mitosis and were induced to form male gametes, whereas cells further along in their cell cycle were unresponsive to these same cues. A simple model combining the influence of light on the mitotic cell cycle and on the induction of spermatogenesis is proposed to explain the observed diversity in population responses to changes in light conditions.
104. Relationship Between Dna-Cycle and Growth-Rate In Synechococcus Sp Strain Pcc6301
     BINDER, BJ; CHISHOLM, SW
     Journal of Bacteriology. 1990 172(5):2313-2319
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105. Silicon Availability and Cell-Cycle Progression In Marine Diatoms
     BRZEZINSKI, MA; OLSON, RJ; CHISHOLM, SW
     Marine Ecology-Progress Series. 1990 67(1):83-96
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106. A High-Sensitivity Flow Cytometer For Studying Picoplankton
     FRANKEL, SL; BINDER, BJ; CHISHOLM, SW; SHAPIRO, HM
     Limnology and Oceanography. 1990 35(5):1164-1169
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107. Pigments, Size, and Distribution of Synechococcus In The North-Atlantic and Pacific Oceans
     OLSON, RJ; CHISHOLM, SW; ZETTLER, ER; ARMBRUST, EV
     Limnology and Oceanography. 1990 35(1):45-58
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108. Spatial and Temporal Distributions of Prochlorophyte Picoplankton In The North-Atlantic Ocean
     OLSON, RJ; CHISHOLM, SW; ZETTLER, ER; ALTABET, MA; DUSENBERRY, JA
     Deep-Sea Research Part A-Oceanographic Research Papers. 1990 37(6):1033-1051
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109. Effect of Light On The Cell-Cycle of A Marine Synechococcus Strain
     ARMBRUST, EV; BOWEN, JD; OLSON, RJ; CHISHOLM, SW
     Applied and Environmental Microbiology. 1989 55(2):425-432
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110. Use of A Neural Net Computer-System For Analysis of Flow Cytometric Data of Phytoplankton Populations
     FRANKEL, DS; OLSON, RJ; FRANKEL, SL; CHISHOLM, SW
     Cytometry. 1989 10(5):540-550
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111. Change In Photosynthetic Capacity Over The Cell-Cycle In Light Dark-Synchronized Amphidinium-Carteri Is Due Solely To The Photocycle
     GERATH, MW; CHISHOLM, SW
     Plant Physiology. 1989 91(3):999-1005
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112. Chlorophyll Fluorescence From Single Cells - Interpretation of Flow Cytometric Signals
     SOSIK, HM; CHISHOLM, SW; OLSON, RJ
     Limnology and Oceanography. 1989 34(8):1749-1761
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113. A Novel Free-Living Prochlorophyte Abundant In The Oceanic Euphotic Zone
     CHISHOLM, SW; OLSON, RJ; ZETTLER, ER; GOERICKE, R; WATERBURY, JB; WELSCHMEYER, NA
     Nature. 1988 334(6180):340-343
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114. Food Selection By Copepods - Discrimination On The Basis of Food Quality
     COWLES, TJ; OLSON, RJ; CHISHOLM, SW
     Marine Biology. 1988 100(1):41-49
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115. Analysis of Synechococcus Pigment Types In The Sea Using Single and Dual Beam Flow-Cytometry
     OLSON, RJ; CHISHOLM, SW; ZETTLER, ER; ARMBRUST, EV
     Deep-Sea Research Part A-Oceanographic Research Papers. 1988 35(3):425-440
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116. Flow Cytometric Analysis of Spermatogenesis In The Diatom Thalassiosira-Weissflogii (Bacillariophyceae)
     VAULOT, D; CHISHOLM, SW
     Journal of Phycology. 1987 23(1):132-137
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117. A Simple-Model of The Growth of Phytoplankton Populations In Light Dark Cycles
     VAULOT, D; CHISHOLM, SW
     Journal of Plankton Research. 1987 9(2):345-366
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118. Cell-Cycle Response To Nutrient Starvation In 2 Phytoplankton Species, Thalassiosira-Weissflogii and Hymenomonas-Carterae
     VAULOT, D; OLSON, RJ; MERKEL, S; CHISHOLM, SW
     Marine Biology. 1987 95(4):625-630
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119. Effects of Light and Nitrogen Limitation On The Cell-Cycle of The Dinoflagellate Amphidinium-Carteri
     OLSON, RJ; CHISHOLM, SW
     Journal of Plankton Research. 1986 8(4):785-793
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120. Effects of Environmental Stresses On The Cell-Cycle of 2 Marine-Phytoplankton Species
     OLSON, RJ; VAULOT, D; CHISHOLM, SW
     Plant Physiology. 1986 80(4):918-925
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121. Patterns of Individual Cell-Growth In Marine Centric Diatoms
     OLSON, RJ; WATRAS, C; CHISHOLM, SW
     Journal of General Microbiology. 1986 132():1197-1204
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122. Light and Dark Control of The Cell-Cycle In 2 Marine-Phytoplankton Species
     VAULOT, D; OLSON, RJ; CHISHOLM, SW
     Experimental Cell Research. 1986 167(1):38-52
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123. Effect of Temperature On Growth and Ingestion Rates of Favella Sp
     AELION, CM; CHISHOLM, SW
     Journal of Plankton Research. 1985 7(6):821-830
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124. Marine-Phytoplankton Distributions Measured Using Shipboard Flow-Cytometry
     OLSON, RJ; VAULOT, D; CHISHOLM, SW
     Deep-Sea Research Part A-Oceanographic Research Papers. 1985 32(10):1273-1280
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125. The Effect of Zooplankton Grazing On Estuarine Blooms of The Toxic Dinoflagellate Gonyaulax-Tamarensis
     WATRAS, CJ; GARCON, VC; OLSON, RJ; CHISHOLM, SW; ANDERSON, DM
     Journal of Plankton Research. 1985 7(6):891-908
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126. Importance of Life-Cycle Events In The Population-Dynamics of Gonyaulax-Tamarensis
     ANDERSON, DM; CHISHOLM, SW; WATRAS, CJ
     Marine Biology. 1983 76(2):179-189
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127. Cuso4 Treatment of Nuisance Algal Blooms In Drinking-Water Reservoirs
     MCKNIGHT, DM; CHISHOLM, SW; HARLEMAN, DRF
     Environmental Management. 1983 7(4):311-320
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128. An Inexpensive Flow Cytometer For The Analysis of Fluorescence Signals In Phytoplankton - Chlorophyll and Dna Distributions
     OLSON, RJ; FRANKEL, SL; CHISHOLM, SW; SHAPIRO, HM
     Journal of Experimental Marine Biology and Ecology. 1983 68(2):129-144
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129. Effects of Photocycles and Periodic Ammonium Supply On 3 Marine-Phytoplankton Species .1. Cell-Division Patterns
     OLSON, RJ; CHISHOLM, SW
     Journal of Phycology. 1983 19(4):522-528
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130. Effects of Photocycles and Periodic Ammonium Supply On 3 Marine-Phytoplankton Species .2. Ammonium Uptake and Assimilation
     WHEELER, PA; OLSON, RJ; CHISHOLM, SW
     Journal of Phycology. 1983 19(4):528-533
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131. The Calculation of Insitu Growth-Rates of Phytoplankton Populations From Fractions of Cells Undergoing Mitosis - A Clarification
     MCDUFF, RE; CHISHOLM, SW
     Limnology and Oceanography. 1982 27(4):783-788
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132. Regulation of Growth In An Estuarine Clone of Gonyaulax-Tamarensis Lebour - Salinity-Dependent Temperature Responses
     WATRAS, CJ; CHISHOLM, SW; ANDERSON, DM
     Journal of Experimental Marine Biology and Ecology. 1982 62(1):25-37
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133. Persistence of Cell-Division Phasing In Marine-Phytoplankton In Continuous Light After Entrainment To Light-Dark Cycles
     CHISHOLM, SW; BRAND, LE
     Journal of Experimental Marine Biology and Ecology. 1981 51(2-3):107-118
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134. Temporal Patterns of Cell-Division In Unicellular Algae
     CHISHOLM, SW
     Canadian Bulletin of Fisheries and Aquatic Sciences. 1981 (210):150-181
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135. Effects of Copper Toxicity On Silicic-Acid Uptake and Growth In Thalassiosira-Pseudonana
     RUETER, JG; CHISHOLM, SW; MOREL, FMM
     Journal of Phycology. 1981 17(3):270-278
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136. Phytoplankton Lipids - Interspecific Differences and Effects of Nitrate, Silicate and Light-Dark Cycles
     SHIFRIN, NS; CHISHOLM, SW
     Journal of Phycology. 1981 17(4):374-384
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137. Changes In The Soluble Silicon Pool Size In The Marine Diatom Thalassiosira-Weisflogii
     BINDER, BJ; CHISHOLM, SW
     Marine Biology Letters. 1980 1(4):205-212
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138. Influence of Environmental-Factors and Population Composition On The Timing of Cell-Division In Thalassiosira-Fluviatilis (Bacillariophyceae) Grown On Light
     CHISHOLM, SW; COSTELLO, JC
     Journal of Phycology. 1980 16(3):375-383
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139. Comparison of 2 Methods For Measuring Phosphate-Uptake By Monochrysis-Lutheri Droop Grown In Continuous Culture
     BURMASTER, DE; CHISHOLM, SW
     Journal of Experimental Marine Biology and Ecology. 1979 39(2):187-202
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140. Sunday - Simulation-Model of An Arctic Daphnia Population
     STROSS, RG; NOBBS, PA; CHISHOLM, SW
     Oikos. 1979 32(3):349-362
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141. Silicic-Acid Incorporation In Marine Diatoms On Light-Dark Cycles - Use As An Assay For Phased Cell-Division
     CHISHOLM, SW; AZAM, F; EPPLEY, RW
     Limnology and Oceanography. 1978 23(3):518-529
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142. Marine Alga Platymonas Sp Accumulates Silicon Without Apparent Requirement
     FUHRMAN, JA; CHISHOLM, SW; GUILLARD, RRL
     Nature. 1978 272(5650):244-246
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143. Particulate Organic-Matter In Surface Waters off Southern-California and Its Relationship To Phytoplankton
     EPPLEY, RW; HARRISON, WG; CHISHOLM, SW; STEWART, E
     Journal of Marine Research. 1977 35(4):671-696
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144. Silicic-Acid Uptake and Incorporation By Natural Marine-Phytoplankton Populations
     AZAM, F; CHISHOLM, SW
     Limnology and Oceanography. 1976 21(3):427-435
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145. Phosphate Uptake Kinetics In Euglena-Gracilis (Z) (Euglenophyceae) Grown On Light-Dark Cycles .1. Synchronized Batch Cultures
     CHISHOLM, SW; STROSS, RG
     Journal of Phycology. 1976 12(2):210-217
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146. Phosphate Uptake Kinetics In Euglena-Gracilis (Z) (Euglenophyceae) Grown In Light-Dark Cycles .2. Phased Po4-Limited Cultures
     CHISHOLM, SW; STROSS, RG
     Journal of Phycology. 1976 12(2):217-222
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147. Phased Cell-Division In Natural-Populations of Marine Dinoflagellates From Shipboard Cultures
     WEILER, CS; CHISHOLM, SW
     Journal of Experimental Marine Biology and Ecology. 1976 25(3):239-247
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148. Simulation of Algal Growth and Competition In A Phosphate-Limited Cyclostat
     CHISHOLM, SW; NOBBS, PA; STROSS, RG
     Abstracts of Papers of The American Chemical Society. 1975 (169):42-42
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149. Environmental and Intrinsic Control of Filtering and Feeding Rates In Arctic Daphnia
     CHISHOLM, SW; STROSS, RG; NOBBS, PA
     Journal of The Fisheries Research Board of Canada. 1975 32(2):219-226
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150. Light-Dark-Phased Cell-Division In Euglena-Gracilis (Z) (Euglenophyceae) In Po4-Limited Continuous Culture
     CHISHOLM, SW; STROSS, RG; NOBBS, PA
     Journal of Phycology. 1975 11(4):367-373
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151. Seasonal-Variation of Manganese In A Eutrophic Lake
     HOWARD, HH; CHISHOLM, SW
     American Midland Naturalist. 1975 93(1):188-197
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152. Causes of Daily Rhythms In Photosynthetic Rates of Phytoplankton
     STROSS, RG; CHISHOLM, SW; DOWNING, TA
     Biological Bulletin. 1973 145(1):200-209
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