Characterization of the metalloproteome of Pseudoalteromonas (BB2-AT2): biogeochemical underpinnings for zinc, manganese, cobalt, and nickel cycling in a ubiquitous marine heterotroph.

Pseudoalteromonas (BB2-AT2) is a ubiquitous marine heterotroph, often associated with labile organic carbon sources in the ocean (e.g. phytoplankton blooms and sinking particles). Heterotrophs hydrolyze exported photosynthetic materials, components of the biological carbon pump, with the use of diverse metalloenzymes containing zinc (Zn), manganese (Mn), cobalt (Co), and nickel (Ni). Studies on the metal requirements and cytosolic utilization of metals for marine heterotrophs are scarce, despite their relevance to global carbon cycling. Here, we characterized the Zn, Mn, Co, and Ni metallome of BB2-AT2. We found that the Zn metallome is complex and cytosolic Zn is associated with numerous proteins for transcription (47.2% of the metallome, obtained from singular value decomposition of the metalloproteomic data), translation (33.5%), proteolysis (12.8%), and alkaline phosphatase activity (6.4%). Numerous proteolytic enzymes also appear to be putatively associated with Mn, and to a lesser extent, Co. Putative identification of the Ni-associated proteins, phosphoglucomutase and a protein in the cupin superfamily, provides new insights for Ni utilization in marine heterotrophs. BB2-AT2 relies on numerous transition metals for proteolytic and phosphatase activities, inferring an adaptative potential to metal limitation. Our field observations of increased alkaline phosphatase activity upon addition of Zn in field incubations suggest that such metal limitation operates in sinking particulate material collected from sediment traps. Taken together, this study improves our understanding of the Zn, Mn, Co, and Ni metallome of marine heterotrophic bacteria and provides novel and mechanistic frameworks for understanding the influence of nutrient limitation on biogeochemical cycling

Temperature-induced viral resistance in Emiliania huxleyi (Prymnesiophyceae)

© The Author(s), 2014. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS One 9 (2014): e112134, doi:10.1371/journal.pone.0112134.Annual Emiliania huxleyi blooms (along with other coccolithophorid species) play important roles in the global carbon and sulfur cycles. E. huxleyi blooms are routinely terminated by large, host-specific dsDNA viruses, (Emiliania huxleyi Viruses; EhVs), making these host-virus interactions a driving force behind their potential impact on global biogeochemical cycles. Given projected increases in sea surface temperature due to climate change, it is imperative to understand the effects of temperature on E. huxleyi’s susceptibility to viral infection and its production of climatically active dimethylated sulfur species (DSS). Here we demonstrate that a 3°C increase in temperature induces EhV-resistant phenotypes in three E. huxleyi strains and that successful virus infection impacts DSS pool sizes. We also examined cellular polar lipids, given their documented roles in regulating host-virus interactions in this system, and propose that alterations to membrane-bound surface receptors are responsible for the observed temperature-induced resistance. Our findings have potential implications for global biogeochemical cycles in a warming climate and for deciphering the particular mechanism(s) by which some E. huxleyi strains exhibit viral resistance.This study was supported by funding from the National Science Foundation (OCE-1061883 to KDB, BVM, and OCE-1061876 to GRD) and in part by grants from The Gordon and Betty Moore Foundation (to BVM and KDB)

The mutual interplay between calcification and coccolithovirus infection

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Johns, C. T., Grubb, A. R., Nissimov, J. I., Natale, F., Knapp, V., Mui, A., Fredricks, H. F., Van Mooy, B. A. S., & Bidle, K. D. The mutual interplay between calcification and coccolithovirus infection. Environmental Microbiology, 21(6), (2019): 1896-1915, doi:10.1111/1462-2920.14362.Two prominent characteristics of marine coccolithophores are their secretion of coccoliths and their susceptibility to infection by coccolithoviruses (EhVs), both of which display variation among cells in culture and in natural populations. We examined the impact of calcification on infection by challenging a variety of Emiliania huxleyi strains at different calcification states with EhVs of different virulence. Reduced cellular calcification was associated with increased infection and EhV production, even though calcified cells and associated coccoliths had significantly higher adsorption coefficients than non‐calcified (naked) cells. Sialic acid glycosphingolipids, molecules thought to mediate EhV infection, were generally more abundant in calcified cells and enriched in purified, sorted coccoliths, suggesting a biochemical link between calcification and adsorption rates. In turn, viable EhVs impacted cellular calcification absent of lysis by inducing dramatic shifts in optical side scatter signals and a massive release of detached coccoliths in a subpopulation of cells, which could be triggered by resuspension of healthy, calcified host cells in an EhV‐free, ‘induced media’. Our findings show that calcification is a key component of the E. huxleyi‐EhV arms race and an aspect that is critical both to the modelling of these host–virus interactions in the ocean and interpreting their impact on the global carbon cycle.We thank Liti Haramaty for her guidance and assistance in culturing and infection experiments. This research was funded by the Gordon and Betty Moore Foundation (GBMF3301 to BVM and KDB and GBMF3789 to KDB) and the National Science Foundation (OCE‐1537951 and OCE‐1559179 to KDB)

The multiple fates of sinking particles in the North Atlantic Ocean

Author Posting. © American Geophysical Union, 2015. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Global Biogeochemical Cycles 29 (2015): 1471–1494, doi:10.1002/2014GB005037.The direct respiration of sinking organic matter by attached bacteria is often invoked as the dominant sink for settling particles in the mesopelagic ocean. However, other processes, such as enzymatic solubilization and mechanical disaggregation, also contribute to particle flux attenuation by transferring organic matter to the water column. Here we use observations from the North Atlantic Ocean, coupled to sensitivity analyses of a simple model, to assess the relative importance of particle-attached microbial respiration compared to the other processes that can degrade sinking particles. The observed carbon fluxes, bacterial production rates, and respiration by water column and particle-attached microbial communities each spanned more than an order of magnitude. Rates of substrate-specific respiration on sinking particle material ranged from 0.007 ± 0.003 to 0.173 ± 0.105 day−1. A comparison of these substrate-specific respiration rates with model results suggested sinking particle material was transferred to the water column by various biological and mechanical processes nearly 3.5 times as fast as it was directly respired. This finding, coupled with strong metabolic demand imposed by measurements of water column respiration (729.3 ± 266.0 mg C m−2 d−1, on average, over the 50 to 150 m depth interval), suggested a large fraction of the organic matter evolved from sinking particles ultimately met its fate through subsequent remineralization in the water column. At three sites, we also measured very low bacterial growth efficiencies and large discrepancies between depth-integrated mesopelagic respiration and carbon inputs.U.S. Environmental Protection Agency (EPA) STAR Grant Number: FP-91744301-0; National Science Foundation Grant Numbers OCE-1061883, EF-0424599, OCE-1155438, OCE-1059884, OCE-1031143; Gordon and Betty Moore Foundation Grant Numbers: 3301, 3789; Gordon and Betty Moore Foundation; Woods Hole Oceanographic Institution2016-03-2

Interrogating marine virus-host interactions and elemental transfer with BONCAT and nanoSIMS-based methods

While the collective impact of marine viruses has become more apparent over the last decade, a deeper understanding of virus-host dynamics and the role of viruses in nutrient cycling would benefit from direct observations at the single-virus level. We describe two new complementary approaches - stable isotope probing coupled with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorescence-based biorthogonal non-canonical amino acid tagging (BONCAT) - for studying the activity and biogeochemical influence of marine viruses. These tools were developed and tested using several ecologically relevant model systems (Emiliania huxleyi/EhV207, Synechococcus sp. WH8101/Syn1, and Escherichia coli/T7). By resolving carbon and nitrogen enrichment in viral particles, we demonstrate the power of nanoSIMS tracer experiments in obtaining quantitative estimates for the total number of viruses produced directly from a particular production pathway (by isotopically labeling host substrates). Additionally, we show through laboratory experiments and a pilot field study that BONCAT can be used to directly quantify viral production (via epifluorescence microscopy) with minor sample manipulation and no dependency on conversion factors. This technique can also be used to detect newly synthesized viral proteins. Together these tools will help fill critical gaps in our understanding of the biogeochemical impact of viruses in the ocean

Nitric oxide production and antioxidant function during viral infection of the coccolithophore Emiliania huxleyi

Emiliania huxleyi is a globally important marine phytoplankton that is routinely infected by viruses. Understanding the controls on the growth and demise of E. huxleyi blooms is essential for predicting the biogeochemical fate of their organic carbon and nutrients. In this study, we show that the production of nitric oxide (NO), a gaseous, membrane-permeable free radical, is a hallmark of early-stage lytic infection in E. huxleyi by Coccolithoviruses, both in culture and in natural populations in the North Atlantic. Enhanced NO production was detected both intra- and extra-cellularly in laboratory cultures, and treatment of cells with an NO scavenger significantly reduced viral production. Pre-treatment of exponentially growing E. huxleyi cultures with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) prior to challenge with hydrogen peroxide (H2O2) led to greater cell survival, suggesting that NO may have a cellular antioxidant function. Indeed, cell lysates generated from cultures treated with SNAP and undergoing infection displayed enhanced ability to detoxify H2O2. Lastly, we show that fluorescent indicators of cellular ROS, NO, and death, in combination with classic DNA- and lipid-based biomarkers of infection, can function as real-time diagnostic tools to identify and contextualize viral infection in natural E. huxleyi blooms

Interrogating marine virus-host interactions and elemental transfer with BONCAT and nanoSIMS-based methods

While the collective impact of marine viruses has become more apparent over the last decade, a deeper understanding of virus-host dynamics and the role of viruses in nutrient cycling would benefit from direct observations at the single-virus level. We describe two new complementary approaches - stable isotope probing coupled with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorescence-based biorthogonal non-canonical amino acid tagging (BONCAT) - for studying the activity and biogeochemical influence of marine viruses. These tools were developed and tested using several ecologically relevant model systems (Emiliania huxleyi/EhV207, Synechococcus sp. WH8101/Syn1, and Escherichia coli/T7). By resolving carbon and nitrogen enrichment in viral particles, we demonstrate the power of nanoSIMS tracer experiments in obtaining quantitative estimates for the total number of viruses produced directly from a particular production pathway (by isotopically labeling host substrates). Additionally, we show through laboratory experiments and a pilot field study that BONCAT can be used to directly quantify viral production (via epifluorescence microscopy) with minor sample manipulation and no dependency on conversion factors. This technique can also be used to detect newly synthesized viral proteins. Together these tools will help fill critical gaps in our understanding of the biogeochemical impact of viruses in the ocean

Temperate infection in a virus–host system previously known for virulent dynamics

The blooming cosmopolitan coccolithophore Emiliania huxleyi and its viruses (EhVs) are a model for density-dependent virulent dynamics. EhVs commonly exhibit rapid viral reproduction and drive host death in high-density laboratory cultures and mesocosms that simulate blooms. Here we show that this system exhibits physiology-dependent temperate dynamics at environmentally relevant E. huxleyi host densities rather than virulent dynamics, with viruses switching from a long-term non-lethal temperate phase in healthy hosts to a lethal lytic stage as host cells become physiologically stressed. Using this system as a model for temperate infection dynamics, we present a template to diagnose temperate infection in other virus–host systems by integrating experimental, theoretical, and environmental approaches. Finding temperate dynamics in such an established virulent host–virus model system indicates that temperateness may be more pervasive than previously considered, and that the role of viruses in bloom formation and decline may be governed by host physiology rather than by host–virus densities

Seasonal mixed layer depth shapes phytoplankton physiology, viral production, and accumulation in the North Atlantic

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Diaz, B. P., Knowles, B., Johns, C. T., Laber, C. P., Bondoc, K. G. V., Haramaty, L., Natale, F., Harvey, E. L., Kramer, S. J., Bolaños, L. M., Lowenstein, D. P., Fredricks, H. F., Graff, J., Westberry, T. K., Mojica, K. D. A., Haëntjens, N., Baetge, N., Gaube, P., Boss, E., Carlson, C. A., Behrenfeld, M. J., Van Mooy, B. A. S., Bidle, K. D. Seasonal mixed layer depth shapes phytoplankton physiology, viral production, and accumulation in the North Atlantic. Nature Communications, 12(1), (2021): 6634, https://doi.org/10.1038/s41467-021-26836-1.Seasonal shifts in phytoplankton accumulation and loss largely follow changes in mixed layer depth, but the impact of mixed layer depth on cell physiology remains unexplored. Here, we investigate the physiological state of phytoplankton populations associated with distinct bloom phases and mixing regimes in the North Atlantic. Stratification and deep mixing alter community physiology and viral production, effectively shaping accumulation rates. Communities in relatively deep, early-spring mixed layers are characterized by low levels of stress and high accumulation rates, while those in the recently shallowed mixed layers in late-spring have high levels of oxidative stress. Prolonged stratification into early autumn manifests in negative accumulation rates, along with pronounced signatures of compromised membranes, death-related protease activity, virus production, nutrient drawdown, and lipid markers indicative of nutrient stress. Positive accumulation renews during mixed layer deepening with transition into winter, concomitant with enhanced nutrient supply and lessened viral pressure.This work was made possible by NASA’s Earth Science Program in support of the North Atlantic Aerosol and Marine Ecosystem Study (15-RRNES15-0011 and 0NSSC18K1563 to K.D.B.; NNX15AF30G to M.J.B.), as well as with support from the National Science Foundation (OIA-2021032 to K.D.B., OCE-157943 to C.A.C., and OCE-1756254 to B.A.S.V.M.), the Gordon and Betty Moore Foundation (Award# 3789 to K.G.V.B.), and NASA’s Future Investigators in Space Science and Technology program (FINESST; grant #826380 to K.D.B.; graduate support to BD)