Archaea (archaebacteria) constitute one of the three major evolutionary lineages of life on Earth. Previously these prokaryotes were thought to predominate in only a few unusual and disparate niches, characterized by hypersaline, extremely hot, or strictly anoxic conditions. Recently, novel (uncultivated) phylotypes of Archaea have been detected in coastal and subsurface marine waters, but their abundance, distribution, physiology and ecology remain largely undescribed. Here we report exceptionally high archaeal abundance in frigid marine surface waters of Antarctica. Pelagic Archaea constituted up to 34% of the prokaryotic biomass in coastal Antarctic surface waters, and they were also abundant in a variety of other cold, pelagic marine environments. Because they can make up a significant fraction of picoplankton biomass in the vast habitats encompassed by cold and deep marine waters, these pelagic Archaea represent an unexpectedly abundant component of the Earth's biota.

Alkaline phosphatase (AP) activity in marine and freshwater phytoplankton has been associated with phosphorus (P) limitation whereby the enzyme functions in the breakdown of exogenous organic P con~pounds to utilizable inorganic forms. Current enzyme assays to determine the P status of the phytoplankton measure only the AP activity of the whole community and do not yield information on ~ndiv~dual species. A new insoluble fluorogenic substrate for AP, termed ELF (EnzymeLabeled Fluorescence), yields a stable, highly fluorescent precipitate at the site of enzyme act~vity and thus has the capability to determine the P status of indimdual cells. In t h~s study, ELF was utilized for in situ detection and quantification of AP in marine phytoplankton cultures and a comparison was made between the insoluble ELF substrate and several soluble AP substrates [3-0-methylfluorescein phosphate (MFP), 3,6-fluorescein diphosphate (FDP) and Attophos]. Non-axenic batch cultures of Alexandrium fundyense, Arnphidln~um sp, and Isochrysis galbana were grown in different media types using orthophosphate as an inorganic source and sodium-glycerophosphate as an organic source, with final phosphate concentrations ranging from 38.3 to 3 p M (i.e. f/2, f/40, f/80, plus ambient P). Epifluorescence microscopy was used to determine if and where the cells were labeled with ELF, while flow cytometry was used to quantify the amount of ELF retained on individual cells. The detection of the soluble substrates utilized a multiwell fluorescence plate reader (CytofluorTh4). Only cells grown in low phosphate concentrations (f/40, f/80) exhibited the bright green fluorescence signal of the ELF precipItate. This signal was always observed for P-starved Amphidiniurn sp. and I galbana cells, but was seen in some A, fundyense cells only during the late stationary phase. Cells grown in high phosphate concentrations (i.e. at f/2 levels) showed no ELF fluorescence. Slightly positive soluble substrate assays suggest that these species may have produced small amounts of AP constitutively that were not detected with the precipitable substrate. Similar results were obtained when the cultures were analyzed by flow cytometry. Except for A. fundyense, cells grown In low phosphate concentrations showed high ELF fluorescence. However, no positive ELF fluorescence was detected with the Cytofluor for all 3 species due to lack of instrument sensitivity. Comparable analysis using the soluble substrates MFP, FDP, and Att~phos-l-~' on the Cytofluor showed little activity for A , fundyense, but high fluorescence for P-starved Amphldiniun? sp. and I. galbana. Insoluble ELF thus provides a means to detect and quantify AP in individual cells using visual observations or flow cytometry. This technique offers a new level of resolution and sensitivity at the single cell level that can provide insights into the P nutrition of phytoplankton and other microorganisms in natural waters.

Wc compared the impact of structural changes in the light-harvesting machinery of two bloom-forming dinoflagellates (Prorocentrum minimum and Heterocupsa pygmaen) by quantifyi lg photophysiological changes in order to define the alternative mechanisms of photoacclimation. There arc species-specific and photoacclimative differences in the abundance and composition of functional light-harvesting complexes (including photosystem I-and 2-enriched bands) which are rehected in spectral changes in absorption, and photosystem 2 (PS2) fluorescence cxcitation spectra of whole cells, thylakoid micelles, and discrete photosyntheti I: pigment-protein complexes. DCMUinduced variations in fluorescence excitation spectra are evident in whole eels but not in isolated thylakoid micelles or functional chromoproteins; such variations indicate that intracellular self-shading which causes variable fluorescence is eliminated in subcellular preparations, We developed a scaling proceclurc of PS2-related in vivo fluorescence excitation spectra relative to Chl u-specific absorption coefficients and used the chromoprotein differences to illustratc how the chromoprotein composition alters cellular fluorescence properties. We also examined the energy transfer efficiency of photosynthetic pigments and the photoprotective role and location of diadinoxanthin.

We have identified a major 32-kDa protein in the dinoflagellate Gonyaulax polyedra as a peridinin-chlorophyll a-binding protein (PCP), based on micro-sequence data and immunological cross-reaction with antibodies raised against PCP from another dinoflagellate species. A cDNA for this protein, identified by a PCR-based cloning strategy, encoded all 68 of the amino acids microsequenced, thus confirming the identity of the clone. The PCP gene is highly expressed at both the mRNA and protein levels, and only PCP transcripts corresponding in size to the cDNA sequence were detected. Slot blot analyses show that there are roughly 5000 copies of the PCP gene in Gonyaulax, making this gene one of the most highly repeated protein-coding genes ever reported, yet the sequence of the different gene copies in the genome appears extraordinarily well conserved as judged by Southern blot analyses. The gene, as indicated by Southern blot and PCR data, is suggested to be present in 5000 intronless copies arranged head to tail in the genome, separated by conserved 1-kb spacers. Based on the conserved sequence of the spacer region, its presence next to each of the PCP coding sequences, and the uniform size of the PCP transcript, we propose that this region represents a dinoflagellate transcriptional promoter. This putative promoter region contains none of the sequence elements for DNA-binding proteins involved in transcriptional initiation reported in other organisms.

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