Diel transcriptional response of a California Current plankton microbiome to light, low iron, and enduring viral infection.

Phytoplankton and associated microbial communities provide organic carbon to oceanic food webs and drive ecosystem dynamics. However, capturing those dynamics is challenging. Here, an in situ, semi-Lagrangian, robotic sampler profiled pelagic microbes at 4 h intervals over ~2.6 days in North Pacific high-nutrient, low-chlorophyll waters. We report on the community structure and transcriptional dynamics of microbes in an operationally large size class (>5 µm) predominantly populated by dinoflagellates, ciliates, haptophytes, pelagophytes, diatoms, cyanobacteria (chiefly Synechococcus), prasinophytes (chiefly Ostreococcus), fungi, archaea, and proteobacteria. Apart from fungi and archaea, all groups exhibited 24-h periodicity in some transcripts, but larger portions of the transcriptome oscillated in phototrophs. Periodic photosynthesis-related transcripts exhibited a temporal cascade across the morning hours, conserved across diverse phototrophic lineages. Pronounced silica:nitrate drawdown, a high flavodoxin to ferredoxin transcript ratio, and elevated expression of other Fe-stress markers indicated Fe-limitation. Fe-stress markers peaked during a photoperiodically adaptive time window that could modulate phytoplankton response to seasonal Fe-limitation. Remarkably, we observed viruses that infect the majority of abundant taxa, often with total transcriptional activity synchronized with putative hosts. Taken together, these data reveal a microbial plankton community that is shaped by recycled production and tightly controlled by Fe-limitation and viral activity.

Mortality of sea lions along the central California coast linked to a toxic diatom bloom.

Over 400 California sea lions (Zalophus californianus) died and many others displayed signs of neurological dysfunction along the central California coast during May and June 1998. A bloom of Pseudo-nitzschia australis (diatom) was observed in the Monterey Bay region during the same period. This bloom was associated with production of domoic acid (DA), a neurotoxin that was also detected in planktivorous fish, including the northern anchovy (Engraulis mordax), and in sea lion body fluids. These and other concurrent observations demonstrate the trophic transfer of DA resulting in marine mammal mortality. In contrast to fish, blue mussels (Mytilus edulus) collected during the DA outbreak contained no DA or only trace amounts. Such findings reveal that monitoring of mussel toxicity alone does not necessarily provide adequate warning of DA entering the food web at levels sufficient to harm marine wildlife and perhaps humans.

Isolation and sequence analysis of the orotidine-5'-phosphate decarboxylase gene (URA3) of Candida utilis. Comparison with the OMP decarboxylase gene family.

The URA3 gene of Candida utilis encoding orotidine-5'-phosphate decarboxylase enzyme was isolated by complementation in Escherichia coli pyrF mutation. The deduced amino-acid sequence is highly similar to that of the Ura3 proteins from other yeast and fungal species. An extensive analysis of the family of orotidine-5'-phosphate decarboxylase is shown. The URA3 gene of C. utilis was able to complement functionally the ura3 mutation of Saccharomyces cerevisiae.

Cloning and sequence analysis of the gene encoding invertase (INV1) from the yeast Candida utilis.

The gene INV1 encoding invertase from the yeast Candida utilis has been cloned using a homologous PCR hybridization probe, amplified with two sets of degenerate primers designed considering sequence comparisons between yeast invertases. The cloned gene was sequenced and found to encode a polypeptide of 533 amino acids that contain a 26 amino-acid signal peptide and 12 potential N-glycosylation sites. The nucleotide sequences of the 5' and 3' non-coding regions were found to contain motifs probably involved in initiation, regulation and termination of gene transcription. The amino-acid sequence shows significant identity with other yeast, bacterial and plant beta-fructofuranosidases. The INV1 gene from C. utilis was able to complement functionally the suc2 mutation of S. cerevisiae.

Development of an integrative DNA transformation system for the yeast Candida utilis.

We report here the development of an auxotrophic transformation system for the food yeast Candida utilis. To facilitate molecular studies in Candida utilis, we isolated auxotrophic strains for uracil biosynthesis by the combination of NTG-mutagenesis and 5-fluorotic acid (FOA) selection. The ura-mutation could be functionally complemented by the homologous URA3 gene. We used both, LiAc and electroporation methods to direct insertions at the ura3 locus through homologous recombination.

Purification and characterization of an invertase from Candida utilis: comparison with natural and recombinant yeast invertases.

A periplasmic invertase from the yeast Candida utilis was purified to homogeneity from cells fully derepressed for invertase synthesis. The enzyme was purified by successive Sephacryl S-300, and affinity chromatography and shown to be a dimeric glycoprotein composed of two identical monomer subunits with an apparent molecular mass of 150 kDa. After EndoH treatment, the deglycosylated protein showed an apparent molecular weight of 60 kDa. The apparent K(m) values for sucrose and raffinose were 11 and 150 mM, respectively, similar to those reported in Saccharomyces cerevisiae. The range of optimum temperature was 60-75 degrees C. The optimum pH was 5.5 and the enzyme was stable over pH range 3-6.

A massive phytoplankton bloom induced by an ecosystem-scale iron fertilization experiment in the equatorial Pacific Ocean.

The seeding of an expanse of surface waters in the equatorial Pacific Ocean with low concentrations of dissolved iron triggered a massive phytoplankton bloom which consumed large quantities of carbon dioxide and nitrate that these microscopic plants cannot fully utilize under natural conditions. These and other observations provide unequivocal support for the hypothesis that phytoplankton growth in this oceanic region is limited by iron bioavailability.

Methyl bromide: ocean sources, ocean sinks, and climate sensitivity.

The oceans play an important role in the geochemical cycle of methyl bromide (CH3Br), the major carrier of O3-destroying bromine to the stratosphere. The quantity of CH3Br produced annually in seawater is comparable to the amount entering the atmosphere each year from natural and anthropogenic sources. The production mechanism is unknown but may be biological. Most of this CH3Br is consumed in situ by hydrolysis or reaction with chloride. The size of the fraction which escapes to the atmosphere is poorly constrained; measurements in seawater and the atmosphere have been used to justify both a large oceanic CH3Br flux to the atmosphere and a small net ocean sink. Since the consumption reactions are extremely temperature-sensitive, small temperature variations have large effects on the CH3Br concentration in seawater, and therefore on the exchange between the atmosphere and the ocean. The net CH3Br flux is also sensitive to variations in the rate of CH3Br production. We have quantified these effects using a simple steady state mass balance model. When CH3Br production rates are linearly scaled with seawater chlorophyll content, this model reproduces the latitudinal variations in marine CH3Br concentrations observed in the east Pacific Ocean by Singh et al. [1983] and by Lobert et al. [1995]. The apparent correlation of CH3Br production with primary production explains the discrepancies between the two observational studies, strengthening recent suggestions that the open ocean is a small net sink for atmospheric CH3Br, rather than a large net source. The Southern Ocean is implicated as a possible large net source of CH3Br to the atmosphere. Since our model indicates that both the direction and magnitude of CH3Br exchange between the atmosphere and ocean are extremely sensitive to temperature and marine productivity, and since the rate of CH3Br production in the oceans is comparable to the rate at which this compound is introduced to the atmosphere, even small perturbations to temperature or productivity can modify atmospheric CH3Br. Therefore atmospheric CH3Br should be sensitive to climate conditions. Our modeling indicates that climate-induced CH3Br variations can be larger than those resulting from small (+/- 25%) changes in the anthropogenic source, assuming that this source comprises less than half of all inputs. Future measurements of marine CH3Br, temperature, and primary production should be combined with such models to determine the relationship between marine biological activity and CH3Br production. Better understanding of the biological term is especially important to assess the importance of non-anthropogenic sources to stratospheric ozone loss and the sensitivity of these sources to global climate change.

Ribosomal DNA sequences discriminate among toxic and non-toxic Pseudonitzschia species.

Cultured isolates of Pseudonitzschia australis Frenguelli, P. delicatissima (Cleve) Heiden, P. americana (Hasle) Fryxell, P. pungens (Grunow) Hasle, and P. pungens f. multiseries (Hasle) Hasle from Monterey Bay, California, were compared on the basis of their large-subunit ribosomal RNA gene (LsrDNA). Pseudonitzschia australis, P. pungens f. multiseries, and P. delicatissima were previously shown to produce the neurotoxin domoic acid; the remaining isolates are considered non-toxic. For each isolate approximately 800 base pairs of LsrDNA, encompassing both evolutionarily conserved and evolutionarily variable regions of the molecule, were amplified using the polymerase chain reaction (PCR) and sequenced. Phylogenetic trees generated by parsimony analysis of aligned sequences afford a preliminary view of the organisms genetic relationships. Species defined by morphological criteria are also distinguishable by LsrDNA sequence. Organisms known or suspected to produce domoic acid cluster at different termini on the phylogenetic tree. Two genetically distinct strains of P. australis and P. pungens were identified. Development of a restriction fragment length polymorphism (RFLP) assay of the LsrDNA is described. The RFLP assay discriminates each species, including distinguished strains of P. australis and P. pungens. The restriction test provides a rapid and convenient method for screening isolates' LsrDNA, facilitating further tests of the apparent positive correlation between Pseudonitzschia species' ribosomal gene signatures, morphology, and capacity to produce domoic acid.

Biological consequences of el nino.

Observations of the 1982-1983 El Niño make it possible to relate the anomalous ocean conditions to specific biological responses. In October 1982 upwelling ecosystems in the eastern equatorial Pacific began a series of transitions from the normal highly productive condition to greatly reduced productivity. The highly productive condition had returned by July 1983. Nutrients, phytoplankton biomass, and primary productivity are clearly regulated by the physical changes of El Niño. Evidence from 1982 and 1983 also suggests effects on higher organisms such as fish, seabirds, and marine mammals, but several more years of observation are required to accurately determine the magnitude of the consequences on these higher trophic levels.