Contrasting community versus population-based estimates of grazing and virus-induced mortality of phytoplankton.

In this study, grazing and virus-induced mortality of phytoplankton was investigated in a freshwater pond at the University of Toronto Mississauga, Canada, during September 2009. The modified dilution assay, which partitions phytoplankton mortality into virus and grazing-induced fractions, was used along with newly designed, taxon-specific quantitative polymerase chain reaction (qPCR) assays that target psbA gene fragments to estimate growth and mortality rates for both the entire phytoplankton community and four distinct phytoplankton populations. Community mortality was estimated via fluorometric determination of chlorophyll a (Chl a) concentrations, whereas the relative mortality of individual phytoplankton populations was estimated via qPCR. The sources and amounts of mortality for individual phytoplankton populations differed from those of the whole community, as well as from each other. Grazing was found to be the only significant source of mortality for the community (0.32 day(-1)), and the Prymnesiales (1.65 day(-1)) and Chroococcales (2.79 day(-1)) populations studied. On the other hand, the Chlamydomonadales population examined experienced both significant grazing (1.01 day(-1)) and viral lysis (0.96 day(-1)), while the Chlorellales population only experienced significant mortality as a result of viral lysis (1.38 day(-1)). Our results demonstrate that the combination of qPCR and the modified dilution method can be used to estimate both viral lysis and grazing pressure on several individual phytoplankton populations within a community simultaneously. Further, previously noted limitations of the modified dilution method associated with the dilution of specific phytoplankton populations at low abundances can be overcome with the qPCR-based approach. Most importantly, this study demonstrates that when used alone, whole community-based methods of assessing mortality can overlook valuable information about carbon flow in aquatic microbial food webs.

Quantification of virus genes provides evidence for seed-bank populations of phycodnaviruses in Lake Ontario, Canada.

Using quantitative PCR, the abundances of six phytoplankton viruses DNA polymerase (polB) gene fragments were estimated in water samples collected from Lake Ontario, Canada over 26 months. Four of the polB fragments were most related to marine prasinoviruses, while the other two were most closely related to cultivated chloroviruses. Two Prasinovirus-related genes reached peak abundances of >1000 copies ml(-1) and were considered 'high abundance', whereas the other two Prasinovirus-related genes peaked at abundances <1000 copies ml(-1) and were considered 'low abundance'. Of the genes related to chloroviruses, one peaked at ca 1600 copies ml(-1), whereas the other reached only ca 300 copies ml(-1). Despite these differences in peak abundance, the abundances of all genes monitored were lowest during the late fall, winter and early spring; during these months the high abundance genes persisted at 100-1000 copies ml(-1) while the low abundance Prasinovirus- and Chlorovirus-related genes persisted at fewer than ca 100 copies ml(-1). Clone libraries of psbA genes from Lake Ontario revealed numerous Chlorella-like algae and two prasinophytes demonstrating the presence of candidate hosts for all types of viruses monitored. Our results corroborate recent metagenomic analyses that suggest that aquatic virus communities are composed of only a few abundant populations and many low abundance populations. Thus, we speculate that an ecologically important characteristic of phycodnavirus communities is seed-bank populations with members that can become numerically dominant when their host abundances reach appropriate levels.

Quantitative PCR reveals transient and persistent algal viruses in Lake Ontario, Canada.

To determine if different algal viruses (Phycodnaviridae) share common patterns of seasonal abundance, quantitative PCR methods were developed and applied to monitor the abundances of three different viruses in Lake Ontario, Canada over 13 months. Throughout the year, the abundances of two different phycodnavirus polB gene fragments (LO1b-49 and LO1a-68) varied by more than two orders of magnitude, peaked during the autumn months, and were lowest during the summer. The seasonal abundance patterns of these two virus genes were similar and both were detected in almost every sample, but LO1b-49 was consistently an order of magnitude more abundant than LO1a-68. LO1b-49 reached a maximum abundance of 5413 +/- 312 genes ml(-1), whereas LO1a-68's abundance peaked at only 881 +/- 113 genes ml(-1). Another phycodnavirus polB fragment that was monitored (LO1b-16) was detected in only a few samples, but reached a higher maximum concentration (6771 +/- 879 genes ml(-1)) than either LO1b-49 or LO1a-68. The results of this year-long investigation of virus gene abundances suggests that Lake Ontario's phycodnavirus community is composed of persistent viruses detectable throughout the year and transient viruses present in only a few sporadic samples. The results also suggest that some persistent algal viruses are able to survive at relatively low abundances through several seasons.

MPYS, a novel membrane tetraspanner, is associated with major histocompatibility complex class II and mediates transduction of apoptotic signals.

Although the best-defined function of type II major histocompatibility complex (MHC-II) is presentation of antigenic peptides to T lymphocytes, these molecules can also transduce signals leading alternatively to cell activation or apoptotic death. MHC-II is a heterodimer of two transmembrane proteins, each containing a short cytoplasmic tail that is dispensable for transduction of death signals. This suggests the function of an undefined MHC-II-associated transducer in signaling the death response. Here we describe a novel plasma membrane tetraspanner (MPYS) that is associated with MHC-II and mediates its transduction of death signals. MPYS is unusual among tetraspanners in containing an extended C-terminal cytoplasmic tail (approximately 140 amino acids) with multiple embedded signaling motifs. MPYS is tyrosine phosphorylated upon MHC-II aggregation and associates with inositol lipid and tyrosine phosphatases. Finally, MHC class II-mediated cell death signaling requires MPYS-dependent activation of the extracellular signal-regulated kinase signaling pathway.

Temporal patterns of nitrogenase gene (nifH) expression in the oligotrophic North Pacific Ocean.

Dinitrogen (N(2))-fixing microorganisms (diazotrophs) play important roles in ocean biogeochemistry and plankton productivity. In this study, we examined the presence and expression of specific planktonic nitrogenase genes (nifH) in the upper ocean (0 to 175 m) at Station ALOHA in the oligotrophic North Pacific Ocean. Clone libraries constructed from reverse-transcribed PCR-amplified mRNA revealed six unique phylotypes. Five of the nifH phylotypes grouped with sequences from unicellular and filamentous cyanobacteria, and one of the phylotypes clustered with gamma-proteobacteria. The cyanobacterial nifH phylotypes retrieved included two sequence types that phylogenetically grouped with unicellular cyanobacteria (termed groups A and B), several sequences closely related (97 to 99%) to Trichodesmium spp. and Katagnymene spiralis, and two previously unreported phylotypes clustering with heterocyst-forming nifH cyanobacteria. Temporal patterns of nifH expression were evaluated using reverse-transcribed quantitative PCR amplification of nifH gene transcripts. The filamentous and presumed unicellular group A cyanobacterial phylotypes exhibited elevated nifH transcription during the day, while members of the group B (closely related to Crocosphaera watsonii) unicellular phylotype displayed greater nifH transcription at night. In situ nifH expression by all of the cyanobacterial phylotypes exhibited pronounced diel periodicity. The gamma-proteobacterial phylotype had low transcript abundance and did not exhibit a clear diurnal periodicity in nifH expression. The temporal separation of nifH expression by the various phylotypes suggests that open ocean diazotrophic cyanobacteria have unique in situ physiological responses to daily fluctuations of light in the upper ocean.

Nearly identical bacteriophage structural gene sequences are widely distributed in both marine and freshwater environments.

Primers were designed to amplify a 592-bp region within a conserved structural gene (g20) found in some cyanophages. The goal was to use this gene as a proxy to infer genetic richness in natural cyanophage communities and to determine if sequences were more similar in similar environments. Gene products were amplified from samples from the Gulf of Mexico, the Arctic, Southern, and Northeast and Southeast Pacific Oceans, an Arctic cyanobacterial mat, a catfish production pond, lakes in Canada and Germany, and a depth of ca. 3,246 m in the Chuckchi Sea. Amplicons were separated by denaturing gradient gel electrophoresis, and selected bands were sequenced. Phylogenetic analysis revealed four previously unknown groups of g20 clusters, two of which were entirely found in freshwater. Also, sequences with >99% identities were recovered from environments that differed greatly in temperature and salinity. For example, nearly identical sequences were recovered from the Gulf of Mexico, the Southern Pacific Ocean, an Arctic freshwater cyanobacterial mat, and Lake Constance, Germany. These results imply that closely related hosts and the viruses infecting them are distributed widely across environments or that horizontal gene exchange occurs among phage communities from very different environments. Moreover, the amplification of g20 products from deep in the cyanobacterium-sparse Chuckchi Sea suggests that this primer set targets bacteriophages other than those infecting cyanobacteria.