Search

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., (© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Coccolithoviruses (EhVs) are large, double-stranded DNA-containing viruses that infect the single-celled, marine coccolithophore Emiliania huxleyi. Given the cosmopolitan nature and global importance of E. huxleyi as a bloom-forming, calcifying, photoautotroph, E. huxleyi-EhV interactions play a key role in oceanic carbon biogeochemistry. Virally-encoded glycosphingolipids (vGSLs) are virulence factors that are produced by the activity of virus-encoded serine palmitoyltransferase (SPT). Here, we characterize the dynamics, diversity and catalytic production of vGSLs in an array of EhV strains in relation to their SPT sequence composition and explore the hypothesis that they are a determinant of infectivity and host demise. vGSL production and diversity was positively correlated with increased virulence, virus replication rate and lytic infection dynamics in laboratory experiments, but they do not explain the success of less-virulent EhVs in natural EhV communities. The majority of EhV-derived SPT amplicon sequences associated with infected cells in the North Atlantic derived from slower infecting, less virulent EhVs. Our lab-, field- and mathematical model-based data and simulations support ecological scenarios whereby slow-infecting, less-virulent EhVs successfully compete in North Atlantic populations of E. huxleyi, through either the preferential removal of fast-infecting, virulent EhVs during active infection or by having access to a broader host range., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Metacaspases are cysteine specific proteases implicated in cell-signalling, stress acclimation and programmed cell death (PCD) pathways in plants, fungi, protozoa, bacteria and algae. We investigated metacaspase-like gene expression and biochemical activity in the bloom-forming, N 2 -fixing, marine cyanobacterium Trichodesmium, which undergoes PCD under low iron and high-light stress. We examined these patterns with respect to in-silico analyses of protein domain architectures that revealed a diverse array of regulatory domains within Trichodesmium metacaspases-like (TeMC) proteins. Experimental manipulations of laboratory cultures and oceanic surface blooms of Trichodesmium from the South Pacific Ocean triggered PCD under Fe-limitation and high light along with enhanced TeMC activity and upregulated expression of diverse TeMC representatives containing different regulatory domains. Furthermore, TeMC activity was significantly and positively correlated with caspase-like activity, which has been routinely observed to increase with PCD induction in Trichodesmium. Although both TeMC and caspase-like activities were stimulated upon PCD induction, inhibitor treatments of these proteolytic activities provided further evidence of largely distinct substrate specificities, even though some inhibitory crossover was observed. Our findings are the first results linking metacaspase expression and activity in PCD induced mortality in Trichodesmium. Yet, the role/s and specific activities of these different proteins remain to be elucidated., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The model haloarchaeon, Haloferax volcanii possess an extremely high, and highly specific, basal caspase activity in exponentially growing cells that closely resembles caspase-4. This activity is specifically inhibited by the pan-caspase inhibitor, z-VAD-FMK, and has no cross-reactivity with other known protease families. Although it is one of the dominant cellular proteolytic activities in exponentially growing H. volcanii cells, the interactive cellular roles remain unknown and the protein(s) responsible for this activity remain elusive. Here, biochemical purification and in situ trapping with caspase targeted covalent inhibitors combined with genome-enabled proteomics, structural analysis, targeted gene knockouts and treatment with canavanine demonstrated a catalytic linkage between caspase activity and thermosomes, proteasomes and cdc48b, a cell division protein and proteasomal degradation facilitating ATPase, as part of an 'interactase' of stress-related protein complexes with an established link to the unfolded protein response (UPR). Our findings provide novel cellular and biochemical context for the observed caspase activity in Archaea and add new insight to understanding the role of this activity, implicating their possible role in the establishment of protein stress and ER associated degradation pathways in Eukarya., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Emiliania huxleyi produces calcium carbonate (CaCO 3 ) coccoliths and transparent exopolymer particles (TEP), sticky, acidic carbohydrates that facilitate aggregation. E. huxleyi's extensive oceanic blooms are often terminated by coccolithoviruses (EhVs) with the transport of cellular debris and associated particulate organic carbon (POC) to depth being facilitated by TEP-bound 'marine snow' aggregates. The dynamics of TEP production and particle aggregation in response to EhV infection are poorly understood. Using flow cytometry, spectrophotometry and FlowCam visualization of alcian blue (AB)-stained aggregates, we assessed TEP production and the size spectrum of aggregates for E. huxleyi possessing different degrees of calcification and cellular CaCO 3 :POC mass ratios, when challenged with two EhVs (EhV207 and EhV99B1). FlowCam imaging also qualitatively assessed the relative amount of AB-stainable TEP (i.e., blue:red ratio of each particle). We show significant increases in TEP during early phase EhV207-infection (∼ 24 h) of calcifying strains and a shift towards large aggregates following EhV99B1-infection. We also observed the formation of large aggregates with low blue:red ratios, suggesting that other exopolymer substances contribute towards aggregation. Our findings show the potential for virus infection and the associated response of their hosts to impact carbon flux dynamics and provide incentive to explore these dynamics in natural populations., (© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

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 labelling 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., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Coccolithoviruses employ a suite of glycosphingolipids (GSLs) to successfully infect the globally important coccolithophore Emiliania huxleyi. Lipid rafts, chemically distinct membrane lipid microdomains that are enriched in GSLs and are involved in sensing extracellular stimuli and activating signalling cascades through protein-protein interactions, likely play a fundamental role in host-virus interactions. Using combined lipidomics, proteomics and bioinformatics, we isolated and characterized the lipid and protein content of lipid rafts from control E. huxleyi cells and those infected with EhV86, the type strain for Coccolithovirus. Lipid raft-enriched fractions were isolated and purified as buoyant, detergent-resistant membranes (DRMs) in OptiPrep density gradients. Transmission electron microscopy of vesicle morphology, polymerase chain reaction amplification of the EhV major capsid protein gene and immunoreactivity to flotillin antisera served as respective physical, molecular and biochemical markers. Subsequent lipid characterization of DRMs via high performance liquid chromatography-triple quadrapole mass spectrometry revealed four distinct GSL classes. Parallel proteomic analysis confirmed flotillin as a major lipid raft protein, along with a variety of proteins affiliated with host defence, programmed cell death and innate immunity pathways. The detection of an EhV86-encoded C-type lectin-containing protein confirmed that infection occurs at the interface between lipid rafts and cellular stress/death pathways via specific GSLs and raft-associated proteins., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Viruses play a key role in controlling the population dynamics of algae, including Emiliania huxleyi, a globally distributed haptophyte with calcite coccoliths that comprise ca. 50% of the sinking carbonate flux from the surface ocean. Emiliania huxleyi viruses (EhVs) routinely infect and terminate E. huxleyi blooms. EhVs are surrounded by a lipid envelope, which we found to be comprised largely of glycosphingolipids (GSLs) with lesser amounts of polar glycerolipids. Infection appears to involve membrane fusion between the virus and host, and we hypothesized that specific polar lipids may facilitate virus attachment. We identified three novel intact polar lipids in E. huxleyi strain CCMP 374 and EhV86, including a GSL with a monosaccharide sialic acid headgroup (sGSL); for all 11 E. huxleyi strains we tested, there was a direct relationship between sGSL content and sensitivity to infection by EhV1, EhV86 and EhV163. In mesocosms, the E. huxleyi population with greatest initial sGSL content had the highest rate of virus-induced mortality. We propose potential physiological roles for sGSL that would be beneficial for growth but leave cells susceptible to infection, thus furthering the discussion of Red Queen-based co-evolution and the cost(s) of sensitivity and resistance in the dynamic E. huxleyi-EhV system., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The cosmopolitan coccolithophore Emiliania huxleyi is characterized by a strongly differentiated haplodiplontic life cycle consisting of a diploid phase, generally bearing coccoliths (calcified) but that can be also non-calcified, and a non-calcified biflagellated haploid phase. Given most studies have focused on the bloom-producing calcified phase, there is little-to-no information about non-calcified cells in nature. Using field mesocoms as experimental platforms, we quantitatively surveyed calcified and non-calcified cells using the combined calcareous detection fluorescent in situ hybridization (COD-FISH) method and qualitatively screened for haploid specific transcripts using reverse transcription-PCR during E. huxleyi bloom successions. Diploid, calcified cells formed dense blooms that were followed by the massive proliferation of E. huxleyi viruses (EhVs), which caused bloom demise. Non-calcified cells were also detected throughout the experiment, accounting for a minor fraction of the population but becoming progressively more abundant during mid-late bloom periods concomitant with EhV burst. Non-calcified cell growth also paralleled a distinct window of haploid-specific transcripts and the appearance of autotrophic flagellates morphologically similar to haploid cells, both of which are suggestive of meiosis and sexual life cycling during natural blooms of this prominent marine phytoplankton species., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Low iron (Fe) availability critically limits diatom distribution and productivity in vast regions of the modern ocean, such as open-ocean, high nutrient low chlorophyll areas and coastal regimes characterized as Fe limitation 'mosaics'. While unique strategies of Fe uptake and storage confer competitive advantages to pennate diatoms, the molecular determinants of low Fe acclimation are largely unknown in centric diatoms. We combined genome-wide and targeted comparative transcriptomic analysis with diagnostic biochemistry and in vivo cell staining as a platform to identify the suite of genes involved in acclimation to Fe and associated oxidative stress in Thalassiosira pseudonana. A total of 1312 genes, nearly 12% of the total genome content, responded to Fe starvation in growing cells characterized by low photosynthetic efficiency and enhanced oxidative stress, caspase activity and metacaspase expression. While 82% of the most highly upregulated genes were also represented in EST libraries derived from diverse diatoms grown under various stress conditions (e.g. silicon, CO(2) and nitrogen limitation), our analysis suggests that T. pseudonana mounts a unique molecular response to Fe starvation that includes a number of genes distinct from those of the model pennate diatom, Phaeodactylum tricornutum, which diverged ~90 million years ago. Homologues to ~50% of the upregulated genes were also identified in a metatranscriptome of eukaryotic phytoplankton communities from a chronically Fe-limited region in the Northeast Pacific. Furthermore, we provide experimental evidence that a subset of putative death-related genes participate in the cellular acclimation to low Fe and associated oxidative damage, suggesting that they co-evolved with other metabolic pathways and play adaptive roles in the success of diatoms., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

An enigmatic feature of microbial evolution is the emergence of programmed cell death (PCD), a genetically controlled form of cell suicide triggered by environmental stimuli. Archaea, the second major prokaryotic domain of life, have been notably absent from the PCD inheritance discussion, due to a lack of genetic homologues. Using the model haloarchaeon Haloferax volcanii, we document extremely high caspase-specific activity and expression of immunoreactive proteins to human caspase 8 antisera, both of which were induced by salt stress and death and were abolished by in vivo addition of a broad-spectrum caspase inhibitor. Caspase inhibition severely impaired cell growth under low and high salt stress, demonstrating a critical role in the cellular stress response. In silico analysis of the H. volcanii proteome identified a subset of 18 potential target proteins containing a signature tetrapeptide caspase cleavage motif (IETD), some with putative roles in allosteric regulation, signal transduction, osmotic stress and cell communication. Detection of similarly high activity and expression in other haloarchaea (Halorubrum and Haloarcula) and in diverse members of Euryarchaeota (the methanogen Methanosarcina acetivorans and the hyperthermophile Pyrococcus furiosus) and Crenarchaeota (the acidophile Sulfolobus solfataricus) argue for a broad representation within the archaeal domain. By playing a role in normal cell function, caspase-like proteases in Archaea appear to have co-evolved with other metabolic pathways, broadening their biological roles beyond apoptosis and cell death.