Effects of UV-A (320 to 399 Nanometers) on Grazing Pressure of a Marine Heterotrophic Nanoflagellate on Strains of the Unicellular Cyanobacteria Synechococcus spp

In the open ocean, where turbidity is very low, UV radiation may be an important factor regulating interactions among planktonic microorganisms. The effect of exposure to UV radiation on grazing by a commonly isolated marine heterotrophic nanoflagellate, Paraphysomonas bandaiensis, on two strains of the cyanobacteria Synechococcus spp. was investigated. Laboratory cultures were exposed to a range of irradiances of artificially produced UV-B (290 to 319 nm) and UV-A (320 to 399 nm) for up to 10 h. At a UV-B irradiance of 0.19 W m, but not 0.12 W m, grazing mortality of Synechococcus spp. and nanoflagellate-specific grazing rates were reduced compared to mortality and grazing rates with UV-A treatment. Within 6 h of exposure, UV-A alone suppressed grazing mortality at irradiances as low as 3.02 W m. The extent to which grazing mortality and nanoflagellate-specific grazing rates were suppressed by UV-A increased with both irradiance and duration of exposure. Over a 6-h exposure period, differences in grazing mortality were largely attributable to differential survival of nanoflagellates. Over a longer period of exposure, there was impairment by UV-A alone of nanoflagellate-specific grazing rates. Rates of primary productivity of Synechococcus spp. were also reduced by UV-A. The extent to which Synechococcus productivity was reduced, compared to the reduction in Synechococcus grazing mortality, depended on the duration of UV-A exposure. These results support the hypothesis that UV-A alone influences the composition and biomass of marine microbial communities by affecting predator-prey interactions and primary production.     

Variability in protist grazing and growth on different marine Synechococcus isolates

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

The Influence of Preculture Conditions and Food Quality on the Ingestion and Digestion Process of Three Species of Heterotrophic Nanoflagellates

The influence of prey characteristics such as motility and size as well as of predator characteristics such as satiation and preculturing diet on the feeding process of interception feeding heterotrophic nanoflagellates was investigated. Three species of gram-negative bacteria, one species of gram-positive bacteria, two species of cyanobacteria (Synechococcus) and inert latex particles were fed as prey particles for three species of heterotrophic nanoflagellates (Spumella, Ochromonas, Cafeteria). Ingestion rates depended on the satiation of the flagellates and especially on the filling status of the food vacuoles. In addition, the ingestion rates depended on the characteristics of the food particle and were modified by pre-culturing the flagellates on either Pseudomonas putida or Bacillus subtilis. Digestion was found to be particle-specific. Cyanobacteria were excreted a few minutes after ingestion whereas heterotrophic bacteria were stored and digested in the food vacuoles. The spectrum of ingested particles is not identical to that of digested particles and thus neither the diet of the flagellates nor their impact on bacterial communities can be calculated simply from food vacuole content. "Selective digestion" could be shown to be an important selection mechanism concerning natural food particles. The digestion strategies of Cafeteria on the one hand and Spumella and Ochromonas on the other hand may be an important factor to explain protozoan species composition and succession in the field. In addition to bacterial abundance and grazing pressure by metazooplankton, the bacterial speciescomposition as well as biochemical variations within bacterial species may influence protozoan species composition and abundance.     

A suppression subtractive hybridization approach reveals niche-specific genes that may be involved in predator avoidance in marine Synechococcus isolates

Picocyanobacteria of the genus Synechococcus are important contributors to marine primary production and are ubiquitous in the world's oceans. This genus is genetically diverse, and at least 10 discrete lineages or clades have been identified phylogenetically. However, little if anything is known about the genetic attributes which characterize particular lineages or are unique to specific strains. Here, we used a suppression subtractive hybridization (SSH) approach to identify strain- and clade-specific genes in two well-characterized laboratory strains, Synechococcus sp. strain WH8103 (clade III) and Synechococcus sp. strain WH7803 (clade V). Among the genes that were identified as potentially unique to each strain were genes encoding proteins that may be involved in specific predator avoidance, including a glycosyltransferase in strain WH8103 and a permease component of an ABC-type polysaccharide/polyol phosphate export system in WH7803. During this work the genome of one of these strains, WH7803, became available. This allowed assessment of the number of false-positive sequences (i.e., sequences present in the tester genome) present among the SSH-enriched sequences. We found that approximately 9% of the WH8103 sequences were potential false-positive sequences, which demonstrated that caution should be used when this technology is used to assess genomic differences in genetically similar bacterial strains.     

Identification of Heterotrophic Nanoflagellates by Restriction Fragment Length Polymorphism Analysis of Small Subunit Ribosomal DNA

Thirty clones derived from twenty isolates of heterotrophic nanoflagellates originating from a variety of marine and freshwater environments were examined by restriction fragment length polymorphism analysis of small subunit ribosomal RNA genes amplified by the polymerase chain reaction (riboprinting). The data were compared with light and electron microscopical identification of the isolates. On morphological criteria, sixteen of the thirty clones belonged to the genus Paraphysomonas De Saedeleer, seven to the genus Spumella Cienkowski, four to the genus Pteridomonas Penard and three to the genus Cafeteria Fenchel and Patterson. Among these taxa, eleven ribotypes were detected by analysis with the restriction enzymes Hinf I, Hae III, Sau3A I, and Msp I. Differentiation of nanoflagellate taxa by the riboprinting method supported taxonomic classification based on morphology at the generic and species level. The utility of the method for discriminating the 'naked' flagellates and for confirming the identity of polymorphic forms among species of Paraphysomonas is demonstrated.     

Amoebic grazing of freshwater Synechococcus strains rich in phycocyanin

Fifteen strains of naked amoebae were presented with 19 strains of Synechococcus on an agar surface. After 14 days of incubation, each of the 285 combinations yielded one of three responses. 42.1% of combinations showed clearing (digestion) of the Synechococcus (C), 56.5% of combinations showed no clearing of the Synechococcus (N) while 1.4% of combinations showed partial clearing of the Synechococcus (P). In general, the Synechococcus strains showed variability in their susceptibility to digestion by the amoebae and the amoebae showed variability in their ability to digest the Synechococcus strains. There was no evidence for amoebae actively selecting profitable prey and equivalent-sized Synechococcus strains were ingested at the same rate, irrespective of their fate. There was some evidence of 'size-selective' grazing in that amoebae ingested the smaller Synechococcus strains at higher rates than the larger strains. However, there was no correlation between prey size and their ultimate fate. These data suggest that amoebae are not selective with regard to the ingestion of synechococci, but that 'selection' occurs at the digestion stage, i.e. whether the synechococci are digested or not.     

A giant cell surface protein in Synechococcus WH8102 inhibits feeding by a dinoflagellate predator

Diverse strains of the marine planktonic cyanobacterium Synechococcus sp. show consistent differences in their susceptibility to predation. We used mutants of Sargasso Sea strain WH8102 (clade III) to test the hypothesis that cell surface proteins play a role in defence against predation by protists. Predation rates by the heterotrophic dinoflagellate Oxyrrhis marina on mutants lacking the giant SwmB protein were always higher (by 1.6 to 3.9×) than those on wild-type WH8102 cells, and equalled predation rates on a clade I strain (CC9311). In contrast, absence of the SwmA protein, which comprises the S-layer (surface layer of the cell envelope that is external to the outer membrane), had no effect on predation by O. marina. Reductions in predation rate were not due to dissolved substances in Synechococcus cultures, and could not be accounted for by variations in cell hydrophobicity. We hypothesize that SwmB defends Synechococcus WH8102 by interfering with attachment of dinoflagellate prey capture organelles or cell surface receptors. Giant proteins are predicted in the genomes of multiple Synechococcus isolates, suggesting that this defence strategy may be more general. Strategies for resisting predation will contribute to the differential competitive success of different Synechococcus groups, and to the diversity of natural picophytoplankton assemblages.     

Effects of UV radiation on grazing by two marine heterotrophic nanoflagellates on autotrophic picoplankton

Phytoplankton <4-3 µm in diameter, or autotrophic picoplankton,can constitute the majority of the biomass and productivityof photosynthetic organisms in marine and freshwater systems.Indirect evidence has indicated that mortality of autotrophicpicoplankton occurs principally at night in the open ocean,but continuously in coastal water. The predominant view of thefate of autotrophic picoplankton production in the ocean isthat they are consumed by heterotrophic nanoflagellates. A possiblemechanism to explain these observations is that grazing of heterotrophicnanoflagellates on autotrophic picoplankton is inhibited byultraviolet radiation (W), at least in clear open-ocean environments.A series of laboratory experiments was conducted to examinethe effects of UV radiation on the grazing impact of two heterotrophicnanoflagellates on Synechococcus spp., a commonly occurringgenus of autotrophic picoplankton. The two nanoflagellates usedwere Paraphysomonas bandaieensis and Paraphysomonas imperforata.For both nanoflagellates, there was an inverse relationshipbetween the grazing mortality of Synechococcus and UV irradiance.The grazing mortality of Synechococcus was reduced less withP.imperforata than with P.bandaiensis. In some experiments,the effect of UV on the grazing impact of the nanoflagellatepopulations was caused in part by UV-related reductions in nanoflagellatesurvival. However, UV reduced the grazing impact of nanoflagellatesprimarily by reducing the rates of consumption of Synechococcusby individual nanoflagellates, to a degree directly relatedto UV irradiance. The results suggat that UV radiation may bean important selection factor in clear open-ocean water, andthat in order to predict the effect of increasing UV radiationon marine microbial plankton communities, we must consider interactionsbetween trophic levels as well as effects on single trophiclevels.     

Clade-specific 16S ribosomal DNA oligonucleotides reveal the predominance of a single marine Synechococcus clade throughout a stratified water column in the Red Sea

Phylogenetic relationships among members of the marine Synechococcus genus were determined following sequencing of the 16S ribosomal DNA (rDNA) from 31 novel cultured isolates from the Red Sea and several other oceanic environments. This revealed a large genetic diversity within the marine Synechococcus cluster consistent with earlier work but also identified three novel clades not previously recognized. Phylogenetic analyses showed one clade, containing halotolerant isolates lacking phycoerythrin (PE) and including strains capable, or not, of utilizing nitrate as the sole N source, which clustered within the MC-A (Synechococcus subcluster 5.1) lineage. Two copies of the 16S rRNA gene are present in marine Synechococcus genomes, and cloning and sequencing of these copies from Synechococcus sp. strain WH 7803 and genomic information from Synechococcus sp. strain WH 8102 reveal these to be identical. Based on the 16S rDNA sequence information, clade-specific oligonucleotides for the marine Synechococcus genus were designed and their specificity was optimized. Using dot blot hybridization technology, these probes were used to determine the in situ community structure of marine Synechococcus populations in the Red Sea at the time of a Synechococcus maximum during April 1999. A predominance of genotypes representative of a single clade was found, and these genotypes were common among strains isolated into culture. Conversely, strains lacking PE, which were also relatively easily isolated into culture, represented only a minor component of the Synechococcus population. Genotypes corresponding to well-studied laboratory strains also appeared to be poorly represented in this stratified water column in the Red Sea.     

Swimming marine Synechococcus strains with widely different photosynthetic pigment ratios form a monophyletic group

Unicellular marine cyanobacteria are ubiquitous in both coastal and oligotrophic regimes. The contribution of these organisms to primary production and nutrient cycling is substantial on a global scale. Natural populations of marine Synechococcus strains include multiple genetic lineages, but the link, if any, between unique phenotypic traits and specific genetic groups is still not understood. We studied the genetic diversity (as determined by the DNA-dependent RNA polymerase rpoC1 gene sequence) of a set of marine Synechococcus isolates that are able to swim. Our results show that these isolates form a monophyletic group. This finding represents the first example of correspondence between a physiological trait and a phylogenetic group in marine Synechococcus. In contrast, the phycourobilin (PUB)/phycoerythrobilin (PEB) pigment ratios of members of the motile clade varied considerably. An isolate obtained from the California Current (strain CC9703) displayed a pigment signature identical to that of nonmotile strain WH7803, which is considered a model for low-PUB/PEB-ratio strains, whereas several motile strains had higher PUB/PEB ratios than strain WH8103, which is considered a model for high-PUB/PEB-ratio strains. These findings indicate that the PUB/PEB pigment ratio is not a useful characteristic for defining phylogenetic groups of marine Synechococcus strains.     

Dynamic characteristics of Prochlorococcus and Synechococcus consumption by bacterivorous nanoflagellates

We compared the characteristics of ingestion of Prochlorococcus and Synechococcus by the marine heterotrophic nanoflagellate Pseudobodo sp. and by a mixed nanoflagellate culture (around 3 microm in size) obtained from an open sea oligotrophic area. Maximum ingestion rate on Synechococcus (2.7 Syn flagellate(-1) h(-1)) was reached at concentrations of 5 x 10(5) Syn mL(-1) and decreased between 6 x 10(5) and 1.5 x 10(6) Syn mL(-1). In order to validate laboratory data, one set of data on Synechococcus grazing was obtained during a field study in the oligotrophic northeastern Mediterranean Sea. Ingestion rates by heterotrophic nanoflagellates were related to Synechococcus abundance in the water, and the feeding rate showed a clear diel rhythm with consumption being highest during the night, declining during the day hours, and being lowest at dusk. Ingestion rates on Prochlorococcus increased linearly for the whole range of prey density used (i.e., from 1 x 10(3) to 3 x 10(6) Proc mL(-1)), with maximum ingestion of 6.7 Proc flagellate(-1) h(-1). However, for prey concentrations in the range of 10(3)-10(5), which are usually encountered in aquatic systems, ingestion rates were significantly less than on Synechococcus. In our experiments, both Prochlorococcus and Synechococcus proved to be poor food items for support of nanoflagellate growth.     

Effect of Temperature and Prey Type on Nutrient Regeneration by an Antarctic Bacterivorous Protist

Experimental studies were carried out on an Antarctic isolate of the heterotrophic nanoflagellate Paraphysomonas imperforata to examine the efficiency of incorporation and remineralization of nitrogen and phosphorus from bacterial prey. Experiments were carried out over a temperature range from ambient Antarctic temperature (0 degrees C) to 10 degrees C. Temperature had a marked effect on the maximal growth rate of the phagotrophic nanoflagellate. Growth rate in the presence of high prey abundance ranged from 0.6 day(-1) at 0 degrees C to 2.6 day(-1) at 10 degrees C. In contrast, temperature had no discernable effect on the efficiencies of incorporation and remineralization of major nutrients by P. imperforata. The efficiencies of phosphorus and nitrogen incorporation from prey biomass averaged over the temperature range examined were 58 and 39%, respectively, for the two elements. Ammonium and phosphate were the dominant forms of dissolved nitrogen and phosphorus appearing in the culture medium during the grazing phase of the experiments. Overall, dissolved organic nitrogen and phosphorus constituted minor components of these elements released by the grazing activities of the protist. The results of this study indicated that incorporation/remineralization of nitrogen and phosphorus contained in prey was relatively unaffected by culture temperature in this heterotrophic nanoflagellate, although low temperature significantly depressed its growth rate. This finding has important implications for energy utilization and elemental cycling in perennially cold ecosystems and is at odds with conclusions that have been reached in some previous studies regarding the growth efficiency of phagotrophic Antarctic protists.     

Aminopeptidase Activity in Marine Chroococcoid Cyanobacteria

Synechococci are important primary producers in the ocean and can also utilize some components of the dissolved organic matter (DOM). The readily utilizable DOM in seawater is mainly polymeric (e.g., protein, polysaccharide) or phosphorylated and requires hydrolysis prior to uptake. We examined whether synechococci express ectoenzymes to hydrolyze DOM components and considered the possible significance of ectohydrolases for Synechococcus ecology and organic matter cycling in the sea. Five strains of non-nitrogen-fixing synechococci in axenic cultures were tested for enzyme activities with fluorogenic substrates. All strains show ectocellular aminopeptidase activity, but other enzymes were undetectable. The aminopeptidase level was in the range determined for five marine heterotrophic bacterial isolates tested for comparison. Aminopeptidase was not secreted into the medium; the majority (74%; tested in WH 7803) was cell surface bound, and a small fraction was periplasmic. The periplasmic activity was not released by cold osmotic shock of WH 7803. Phenylmethylsulfonyl fluoride and EDTA, inhibitors of serine and metalloproteases, strongly or completely inhibited WH 7803 aminopeptidase. The enzyme seemed constitutive; per-cell activity did not change during incubations in unenriched seawater, bovine serum albumin, or nitrate-replete mineral medium. In natural planktonic assemblages in the Southern California Bight, aminopeptidase activity was correlated with Synechococcus abundance as well as the abundance of other bacteria. Ectocellular aminopeptidase may be common in marine synechococci and play roles in their nitrogen nutrition, particularly in low-nitrate and low-light environments. Since synechococci are much less abundant than heterotrophic bacteria in seawater, the impact of Synechococcus aminopeptidase on proteolysis in the sea is likely to be episodic and restricted to specialized microenvironments.     

Selection and characterization of cyanophage resistance in marine Synechococcus strains

Marine viruses are an important component of the microbial food web, influencing microbial diversity and contributing to bacterial mortality rates. Resistance to cooccurring cyanophages has been reported for natural communities of Synechococcus spp.; however, little is known about the nature of this resistance. This study examined the patterns of infectivity among cyanophage isolates and unicellular marine cyanobacteria (Synechococcus spp.). We selected for phage-resistant Synechococcus mutants, examined the mechanisms of phage resistance, and determined the extent of cross-resistance to other phages. Four strains of Synechococcus spp. (WH7803, WH8018, WH8012, and WH8101) and 32 previously isolated cyanomyophages were used to select for phage resistance. Phage-resistant Synechococcus mutants were recovered from 50 of the 101 susceptible phage-host pairs, and 23 of these strains were further characterized. Adsorption kinetic assays indicate that resistance is likely due to changes in host receptor sites that limit viral attachment. Our results also suggest that receptor mutations conferring this resistance are diverse. Nevertheless, selection for resistance to one phage frequently resulted in cross-resistance to other phages. On average, phage-resistant Synechococcus strains became resistant to eight other cyanophages; however, there was no significant correlation between the genetic similarity of the phages (based on g20 sequences) and cross-resistance. Likewise, host Synechococcus DNA-dependent RNA polymerase (rpoC1) genotypes could not be used to predict sensitivities to phages. The potential for the rapid evolution of multiple phage resistance may influence the population dynamics and diversity of both Synechococcus and cyanophages in marine waters.     

Grazing impact of different heterotrophic nanoflagellates on eukaryotic (Ostreococcus tauri) and prokaryotic picoautotrophs (Prochlorococcus and Synechococcus)

Autotrophic picoplankton (<3 microm) composed of both prokaryotes and eukaryotes are the most abundant primary producers on Earth. In this study we examined the ingestion of the picoeukaryote Ostreococcus tauri by different marine heterotrophic nanoflagellates (HNF) with various morphologies, swimming and feeding behaviours. Cultures of specific bacterivorous nanoflagellates (Rhynchomonas nasuta, Jakoba libera, and a culture of Cafeteria sp./Monosiga sp.) and natural nanoflagellate populations were used as grazers. For comparison with Ostreococcus, we used similar-sized prokaryotes as prey, Prochlorococcus and Synechococcus. We observed large species-specific differences in terms of: use of picoautotrophs among nanoflagellates, time lag between prey addition and prey consumption (0-196 h), grazing rate (0-0.12 h(-1)), growth rate (0-0.3 h(-1)) and maximum abundance of HNF reached in experimental bottles (e.g. from 10(4) to 10(5) cells ml(-1), for a natural coastal population and a Cafeteria sp./Monosiga sp. culture feeding Ostreococcus respectively). Overall, this study shows that the nanoflagellate community composition is conclusive for picoautotrophic community structure and, vice versa, the picoautotrophic community structure favours or inhibits the growth of some nanoflagellate groups.     

An immunological approach to detect phosphate stress in populations and single cells of photosynthetic picoplankton.

In the marine cyanobacterium Synechococcus sp. strain WH7803, PstS is a 32-kDa cell wall-associated phosphate-binding protein specifically synthesized under conditions of restricted inorganic phosphate (P1) availability (D. J. Scanlan, N. H. Mann, and N. G. Carr, Mol. Microbiol. 10:181-191, 1993). We have assessed its use as a potential diagnostic marker for the P status of photosynthetic picoplankton. Expression of PstS in Synechococcus sp. strain WH7803 was observed when the P1 concentration fell below 50 nM, demonstrating that the protein is induced at concentrations of P1 typical of oligotrophic conditions. PstS expression could be specifically detected by use of standard Western blotting (immunoblotting) techniques in natural mesocosm samples under conditions in which the N/P ratio was artificially manipulated to force P depletion. In addition, we have developed an immunofluorescence assay that can detect PstS expression in single Synechococcus cells both in laboratory cultures and natural samples. We show that antibodies raised against PstS cross-react with P-depleted Prochlorococcus cells, extending the use of these antibodies to both major groups of prokaryotic photosynthetic picoplankton. Furthermore, DNA sequencing of a Prochlorococcus pstS homolog demonstrated high amino acid sequence identity (77%) with the marine Synechococcus sp. strain WH7803 protein, including those residues in Escherichia coli PstS known to be directly involved in phosphate binding.     

Flow Cytometric Analysis of Characteristics of Hybridization of Species-Specific Fluorescent Oligonucleotide Probes to rRNA of Marine Nanoflagellates

Identification problems restrict quantitative ecological research on specific nanoflagellates. Identification by specific oligonucleotide probes permits use of flow cytometry for enumeration and measurement of size of nanoflagellates in statistically meaningful samples. Flow cytometry also permits measurement of intensity of probe binding by cells. Five fluorescent probes targeted to different regions of the small subunit rRNA of the common marine flagellate Paraphysomonas vestita all hybridized with cells of this flagellate. Cells fixed with trichloroacetic acid gave detectable signals at a probe concentration of 15 aM and specific fluorescence increased almost linearly to 1.5 fM, but at higher concentrations nonspecific binding increased sharply. Three flagellates, P. vestita, Paraphysomonas imperforata, and Pteridomonas danica, all bound a general eukaryotic probe approximately in proportion to their cell size, but the specific P. vestita probe gave 14 times more fluorescence with P. vestita than with either of the other flagellates. Cell fluorescence increased during the early growth of a batch culture and decreased toward the stationary phase; cell size changed in a comparable manner. Cell fluorescence intensity may allow inferences about growth rate, but whether fluorescence (assumed to reflect ribosome number) merely correlates with cell biomass or changes in a more complex manner remains unresolved.     

Protistan grazing analysis by flow cytometry using prey labeled by in vivo expression of fluorescent proteins

Selective grazing by protists can profoundly influence bacterial community structure, and yet direct, quantitative observation of grazing selectivity has been difficult to achieve. In this investigation, flow cytometry was used to study grazing by the marine heterotrophic flagellate Paraphysomonas imperforata on live bacterial cells genetically modified to express the fluorescent protein markers green fluorescent protein (GFP) and red fluorescent protein (RFP). Broad-host-range plasmids were constructed that express fluorescent proteins in three bacterial prey species, Escherichia coli, Enterobacter aerogenes, and Pseudomonas putida. Micromonas pusilla, an alga with red autofluorescence, was also used as prey. Predator-prey interactions were quantified by using a FACScan flow cytometer and analyzed by using a Perl program described here. Grazing preference of P. imperforata was influenced by prey type, size, and condition. In competitive feeding trials, P. imperforata consumed algal prey at significantly lower rates than FP (fluorescent protein)-labeled bacteria of similar or different size. Within-species size selection was also observed, but only for P. putida, the largest prey species examined; smaller cells of P. putida were grazed preferentially. No significant difference in clearance rate was observed between GFP- and RFP-labeled strains of the same prey species or between wild-type and GFP-labeled strains. In contrast, the common chemical staining method, 5-(4,6-dichloro-triazin-2-yl)-amino fluorescein hydrochloride, depressed clearance rates for bacterial prey compared to unlabeled or RFP-labeled cells.     

Genomic sequence and evolution of marine cyanophage P60: a new insight on lytic and lysogenic phages

The genome of cyanophage P60, a lytic virus which infects marine Synechococcus WH7803, was completely sequenced. The P60 genome contained 47,872 bp with 80 potential open reading frames that were mostly similar to the genes found in lytic phages like T7, phi-YeO3-12, and SIO1. The DNA replication system, consisting of primase-helicase and DNA polymerase, appeared to be more conserved in podoviruses than in siphoviruses and myoviruses, suggesting that DNA replication genes could be the critical elements for lytic phages. Strikingly high sequence similarities in the regions coding for nucleotide metabolism were found between cyanophage P60 and marine unicellular cyanobacteria.     

Nanoflagellate predation on auto- and heterotrophic picoplankton in the oligotrophic Mediterranean Sea

Dynamics of autotrophic and heterotrophic prokaryotes and theirconsumption by nanoflagellates were studied in the euphoticzone at nine stations located from the Levantine Basin (34°E)to the Balearic sea (5°E) in June 1999. Bacterial biomassconstituted the largest proportion of living biomass at allstations. Integrated bacterial production at the furthest eaststation, was sixfold lower than integrated bacterial productionat the furthest west (13 and 75 mg C m^-2 d^-1 respectively).Estimated heterotrophic nanoflagellate bacterivory accountedfor 45–87% of bacterial production. Small protists (<3µm) dominated the bacterivore assemblage and accountedfor more than 90% of the heterotrophic bacterial consumption.Our results indicated that there was no negative selection againstSynechococcus and that both picoplankton groups were grazedaccording to their standing stocks. An estimated consumptionof Synechococcus derived from food vacuole content analysisof nanoflagellates revealed that they consumed from 0.5 to 45%(mean 13%) of Synechococcus stock per day. These data are amongthe first documenting the relative grazing impact of heterotrophicnanoflagellates on bacteria and Synechococcus in situ. Assumingthat there was no selection for or against Prochlorococcus,heterotrophic nanoflagellates could ingest from 1.4 to 21% (mean6%) of Prochlorococcus stock per day. The amount of organiccarbon obtained by heterotrophic nanoflagellates from photosyntheticprokaryotes represented 27% of the total amount of carbon obtainedfrom total prokaryotes