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Microbial interactions in harmful algal bloom (HAB) communities have been examined in marine systems, but are poorly studied in fresh waters. To investigate HAB-microbe interactions, we isolated bacteria with close associations to bloom-forming cyanobacteria, Microcystis spp., during a 2017 bloom in the western basin of Lake Erie. The genomes of five isolates (Exiguobacterium sp. JMULE1, Enterobacter sp. JMULE2, Deinococcus sp. JMULE3, Paenibacillus sp. JMULE4, and Acidovorax sp. JMULE5.) were sequenced on a PacBio Sequel system. These genomes ranged in size from 3.1 Mbp (Exiguobacterium sp. JMULE1) to 5.7 Mbp (Enterobacter sp. JMULE2). The genomes were analyzed for genes relating to critical metabolic functions, including nitrogen reduction and carbon utilization. All five of the sequenced genomes contained genes that could be used in potential signaling and nutrient exchange between the bacteria and cyanobacteria such as Microcystis. Gene expression signatures of algal-derived carbon utilization for two isolates were identified in Microcystis blooms in Lake Erie and Lake Tai (Taihu) at low levels, suggesting these organisms are active and may have a functional role during Microcystis blooms in aggregates, but were largely missing from whole water samples. These findings build on the growing evidence that the bacterial microbiome associated with bloom-forming algae have the functional potential to contribute to nutrient exchange within bloom communities and interact with important bloom formers like Microcystis.

Harmful algal blooms (HABs) threaten ecosystems and human health worldwide. Controlling nitrogen inputs to coastal waters is a common HAB management strategy, as nutrient concentrations often suggest coastal blooms are nitrogen-limited. However, defining best nutrient management practices is a long-standing challenge: in part, because of difficulties in directly tracking the nutritional physiology of harmful species in mixed communities. Using metatranscriptome sequencing and incubation experiments, we addressed this challenge by assaying the in situ physiological ecology of the ecosystem destructive alga, Aureococcus anophagefferens. Here we show that gene markers of phosphorus deficiency were expressed in situ, and modulated by the enrichment of phosphorus, which was consistent with the observed growth rate responses. These data demonstrate the importance of phosphorus in controlling brown-tide dynamics, suggesting that phosphorus, in addition to nitrogen, should be evaluated in the management and mitigation of these blooms. Given that nutrient concentrations alone were suggestive of a nitrogen-limited ecosystem, this study underscores the value of directly assaying harmful algae in situ for the development of management strategies.

The marine archaeon Nanoarchaeum equitans is dependent on direct physical contact with its host, the hyperthermophile Ignicoccus hospitalis. As this interaction is thought to be membrane-associated, involving a myriad of membrane-anchored proteins, proteomic efforts to better characterize this difficult to analyze interface are paramount to uncovering the mechanism of their association. By extending multienzyme digestion strategies that use sample filtration to recover underdigested proteins for reprocessing/consecutive proteolytic digestion, we applied chymotrypsin to redigest the proteinaceous material left over after initial proteolysis with trypsin of sodium dodecyl sulfate (SDS)-extracted I. hospitalis-N. equitans proteins. Using this method, we show that proteins with increased hydrophobic character, including membrane proteins with multiple transmembrane helices, are enriched and recovered in the underdigested fraction. Chymotryptic reprocessing provided significant sequence coverage gains in both soluble and hydrophobic proteins alike, with the latter benefiting more so in terms of membrane protein representation. These gains were despite a large proportion of high-quality peptide spectra remaining unassigned in the underdigested fraction suggesting high levels of protein modification on these often surface-exposed proteins. Importantly, these gains were achieved without applying extensive fractionation strategies usually required for thorough characterization of membrane-associated proteins and were facilitated by the generation of a distinct, complementary set of peptides that aid in both the identification and quantitation of this important, under-represented class of proteins.

Lipids composed of condensed isoprenyl units connected to glycerol backbones by ether linkages are a distinguishing feature of Archaea. Data suggesting that fatty acids with linear hydrocarbon chains are present in some Archaea have been available for decades. However, lack of genomic and biochemical evidence for the metabolic machinery required to synthesize and degrade fatty acids has left the field unclear on this potentially significant biochemical aspect. Because lipids are energy currency and cell signaling molecules, their presence in Archaea is significant for understanding archaeal biology. A recent large-scale bioinformatics analysis reignited the debate as to the importance of fatty acids in Archaea by presenting genetic evidence for the presence of enzymes required for anabolic and catabolic fatty acid metabolism across the archaeal domain. Here, we present direct biochemical evidence from gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy for the presence of fatty acids in two members of the Crenarchaeota,Sulfolobus solfataricusandIgnicoccus hospitalis. This is the first report providing biochemical data for the existence of fatty acids in these Crenarchaeota, opening new discussions on energy balance and the potential for the discovery of new thermostable enzymes for industry.

Interspecies interactions are the basis of microbial community formation and infectious diseases. Systems biology enables the construction of complex models describing such interactions, leading to a better understanding of disease states and communities. However, before interactions between complex organisms can be understood, metabolic and energetic implications of simpler real-world host-microbe systems must be worked out. To this effect, untargeted metabolomics experiments were conducted and integrated with proteomics data to characterize key molecular-level interactions between two hyperthermophilic microbial species, both of which have reduced genomes. Metabolic changes and transfer of metabolites between the archaea Ignicoccus hospitalis and Nanoarcheum equitans were investigated using integrated LC-MS and NMR metabolomics. The study of such a system is challenging, as no genetic tools are available, growth in the laboratory is challenging, and mechanisms by which they interact are unknown. Together with information about relative enzyme levels obtained from shotgun proteomics, the metabolomics data provided useful insights into metabolic pathways and cellular networks of I. hospitalis that are impacted by the presence of N. equitans, including arginine, isoleucine, and CTP biosynthesis. On the organismal level, the data indicate that N. equitans exploits metabolites generated by I. hospitalis to satisfy its own metabolic needs. This finding is based on N. equitans's consumption of a significant fraction of the metabolite pool in I. hospitalis that cannot solely be attributed to increased biomass production for N. equitans. Combining LC-MS and NMR metabolomics datasets improved coverage of the metabolome and enhanced the identification and quantitation of cellular metabolites.

Summary Targeted gene expression using quantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed to track patterns in the expression of genes indicative of nitrogen or phosphorus deficiency in the brown tide-forming alga Aureococcus anophagefferens. During culture experiments, a xanthine/uracil/vitamin C permease (XUV) was upregulated ∼20-fold under nitrogen-deficient conditions relative to a nitrogen-replete control and rapidly returned to nitrogen-replete levels after nitrogen-deficient cells were resupplied with nitrate or ammonium. It was not responsive to phosphorus deficiency. Expression of an inorganic phosphate transporter (PTA3) was enriched ∼10-fold under phosphorus-deficient conditions relative to a phosphorus-replete control, and this signal was rapidly lost upon phosphate resupply. PTA3 was not upregulated by nitrogen deficiency. Natural A. anophagefferens populations from a dense brown tide that occurred in Long Island, NY, in 2009 were assayed for XUV and PTA3 expression and compared with nutrient concentrations over the peak of a bloom. Patterns in XUV expression were consistent with nitrogen-replete growth, never reaching the values observed in N-deficient cultures. PTA3 expression was highest prior to peak bloom stages, reaching expression levels within the range of P-deficient cultures. These data highlight the value of molecular-level assessments of nutrient deficiency and suggest that phosphorus deficiency could play a role in the dynamics of destructive A. anophagefferens blooms.

Metatranscriptomics has emerged as a tool in microbial ecology that can resolve the functional landscape of both prokaryotes and eukaryotes within a community. In this study, we extend the potential of metatranscriptomics to probe active virus infections and virus-host relationships in marine systems. Polyadenylation-selected RNA-seq data were examined from microbial communities in two productive marine environments: a brown tide bloom event dominated by Aureococcus anophagefferens in Quantuck Bay, NY, and a diatom-dominated plankton community in Narragansett Bay, RI. Active infections by diverse giant viruses (NCLDVs) of algal and non-algal hosts were found at both sites. Ongoing infections of A. anophagefferens by a known Mimiviridae (AaV) were observed during both the peak and decline of the bloom. Bloom decline was also accompanied by increased activity for viruses other than AaV, including (+) ssRNA viruses. In Narragansett Bay, increased temporal reso’lution revealed active NCLDVs with both ‘boom-and-bust’ as well as ‘steady-state infection’-like ecologies. Statistical co-occurrence examinations of the dsDNA, ssRNA and dsRNA markers within the data revealed a broad spectrum of statistically strong and significant virus-host relationships that included both known as well as novel interactions. Our approach offers a method for screening the diversity and dynamics of active viral infections in natural systems and develops links between viruses and their potential hosts in situ.SignificanceViruses are important partners in ecosystem scale ecology, yet their study to date is primarily limited to single virus-host infection models in the laboratory or limited to “potential-actions” derived from metagenomics analyses. Using metatranscriptomic sequences from polyadenylated-RNA selected samples, we have simultaneously captured information regarding eukaryotic diversity and active infection by viruses with dsDNA genomes, resulting in a statistical opportunity to predict “who is infecting whom”. This approach further provides concurrent insight regarding viruses with ssRNA and dsRNA genomes, capturing dynamics for the communities of viruses infecting single-celled eukaryotes. Given the central role of these plankton in global scale processes, our efforts result in a transformational step-forward regarding the study of in situ virus-host interactions.

Background Studies of interspecies interactions are inherently difficult due to the complex mechanisms which enable these relationships. A model system for studying interspecies interactions is the marine hyperthermophiles Ignicoccus hospitalis and Nanoarchaeum equitans. Recent independently-conducted ‘omics’ analyses have generated insights into the molecular factors modulating this association. However, significant questions remain about the nature of the interactions between these archaea. Methods We jointly analyzed multiple levels of omics datasets obtained from published, independent transcriptomics, proteomics, and metabolomics analyses. DAVID identified functionally-related groups enriched when I. hospitalis is grown alone or in co-culture with N. equitans. Enriched molecular pathways were subsequently visualized using interaction maps generated using STRING. Results Key findings of our multi-level omics analysis indicated that I. hospitalis provides precursors to N. equitans for energy metabolism. Analysis indicated an overall reduction in diversity of metabolic precursors in the I. hospitalis–N. equitans co-culture, which has been connected to the differential use of ribosomal subunits and was previously unnoticed. We also identified differences in precursors linked to amino acid metabolism, NADH metabolism, and carbon fixation, providing new insights into the metabolic adaptions of I. hospitalis enabling the growth of N. equitans. Conclusions This multi-omics analysis builds upon previously identified cellular patterns while offering new insights into mechanisms that enable the I. hospitalis–N. equitans association. General significance Our study applies statistical and visualization techniques to a mixed-source omics dataset to yield a more global insight into a complex system, that was not readily discernable from separate omics studies.

Biological features can be inferred, based on genomic data, for many microbial lineages that remain uncultured. However, cultivation is important for characterizing an organism's physiology and testing its genome-encoded potential. Here we use single-cell genomics to infer cultivation conditions for the isolation of an ectosymbiotic Nanoarchaeota (‘Nanopusillus acidilobi') and its host (Acidilobus, a crenarchaeote) from a terrestrial geothermal environment. The cells of ‘Nanopusillus' are among the smallest known cellular organisms (100–300 nm). They appear to have a complete genetic information processing machinery, but lack almost all primary biosynthetic functions as well as respiration and ATP synthesis. Genomic and proteomic comparison with its distant relative, the marine Nanoarchaeum equitans illustrate an ancient, common evolutionary history of adaptation of the Nanoarchaeota to ectosymbiosis, so far unique among the Archaea., Many microbial lineages have not yet been cultured, which hampers our understanding of their physiology. Here, Wurch et al. use single-cell genomics to infer cultivation conditions for the isolation of a tiny ectosymbiotic nanoarchaeon and its crenarchaeota host from a geothermal spring.

Harmful algal blooms (HABs) cause significant economic and ecological damage worldwide. Despite considerable efforts, a comprehensive understanding of the factors that promote these blooms has been lacking, because the biochemical pathways that facilitate their dominance relative to other phytoplankton within specific environments have not been identified. Here, biogeochemical measurements showed that the harmful alga Aureococcus anophagefferens outcompeted co-occurring phytoplankton in estuaries with elevated levels of dissolved organic matter and turbidity and low levels of dissolved inorganic nitrogen. We subsequently sequenced the genome of A. anophagefferens and compared its gene complement with those of six competing phytoplankton species identified through metaproteomics. Using an ecogenomic approach, we specifically focused on gene sets that may facilitate dominance within the environmental conditions present during blooms. A. anophagefferens possesses a larger genome (56 Mbp) and has more genes involved in light harvesting, organic carbon and nitrogen use, and encoding selenium- and metal-requiring enzymes than competing phytoplankton. Genes for the synthesis of microbial deterrents likely permit the proliferation of this species, with reduced mortality losses during blooms. Collectively, these findings suggest that anthropogenic activities resulting in elevated levels of turbidity, organic matter, and metals have opened a niche within coastal ecosystems that ideally suits the unique genetic capacity of A. anophagefferens and thus, has facilitated the proliferation of this and potentially other HABs.

Summary Long-SAGE (serial analysis of gene expression) was used to profile the transcriptome of the brown tide-forming alga, Aureococcus anophagefferens, under nutrient replete (control), and nitrogen (N) and phosphorus (P) deficiency to understand how this organism responds at the transcriptional level to varying nutrient conditions. This approach has aided A. anophagefferens genome annotation efforts and identified a suite of genes upregulated by N and P deficiency, some of which have known roles in nutrient metabolism. Genes upregulated under N deficiency include an ammonium transporter, an acetamidase/formamidase and two peptidases. This suggests an ability to utilize reduced N compounds and dissolved organic nitrogen, supporting the hypothesized importance of these N sources in A. anophagefferens bloom formation. There are also a broad suite of P-regulated genes, including an alkaline phosphatase, and two 5′-nucleotidases, suggesting A. anophagefferens may use dissolved organic phosphorus under low phosphate conditions. These N- and P-regulated genes may be important targets for exploring nutrient controls on bloom formation in field populations.

The marine hyperthermophilic crenarchaeon Ignicoccus hospitalis supports the propagation on its surface of Nanoarchaeum equitans, an evolutionarily enigmatic archaeon that resembles highly derived parasitic and symbiotic bacteria. The cellular and molecular mechanisms that enable this interarchaea relationship and the intimate physiologic consequences to I. hospitalis are unknown. Here, we used concerted proteomic and transcriptomic analyses to probe into the functional genomic response of I. hospitalis as N. equitans multiplies on its surface. The expression of over 97% of the genes was detected at mRNA level and over 80% of the predicted proteins were identified and their relative abundance measured by proteomics. These indicate that little, if any, of the host genomic information is silenced during growth in the laboratory. The primary response to N. equitans was at the membrane level, with increases in relative abundance of most protein complexes involved in energy generation as well as that of several transporters and proteins involved in cellular membrane stabilization. Similar upregulation was observed for genes and proteins involved in key metabolic steps controlling nitrogen and carbon metabolism, although the overall biosynthetic pathways were marginally impacted. Proliferation of N. equitans resulted, however, in selective downregulation of genes coding for transcription factors and replication and cell cycle control proteins as I. hospitalis shifted its physiology from its own cellular growth to that of its ectosymbiont/parasite. The combination of these multiomic approaches provided an unprecedented level of detail regarding the dynamics of this interspecies interaction, which is especially pertinent as these organisms are not genetically tractable.

Shotgun mass spectrometry was used to detect proteins in the harmful alga, Aureococcus anophagefferens, and monitor their relative abundance across nutrient replete (control), phosphate-deficient (-P) and -P refed with phosphate (P-refed) conditions. Spectral counting techniques identified differentially abundant proteins and demonstrated that under phosphate deficiency, A. anophagefferens increases proteins involved in both inorganic and organic phosphorus (P) scavenging, including a phosphate transporter, 5'-nucleotidase, and alkaline phosphatase. Additionally, an increase in abundance of a sulfolipid biosynthesis protein was detected in -P and P-refed conditions. Analysis of the polar membrane lipids showed that cellular concentrations of the sulfolipid sulphoquinovosyldiacylglycerol (SQDG) were nearly two-fold greater in the -P condition versus the control condition, while cellular phospholipids were approximately 8-fold less. Transcript and protein abundances were more tightly coupled for gene products involved in P metabolism compared to those involved in a range of other metabolic functions. Comparison of protein abundances between the -P and P-refed conditions identified differences in the timing of protein degradation and turnover. This suggests that culture studies examining nutrient starvation responses will be valuable in interpreting protein abundance patterns for cellular nutritional status and history in metaproteomic datasets.

Recurrent brown tide blooms caused by the harmful alga Alureococcus anophagefferens have decimated coastal ecosystems and shellfisheries along the Eastern U.S and South Africa. The exact mechanisms controlling bloom formation, sustenance, and decline are unclear, however bottom-up factors such as nutrient type and supply are thought to be critical. Traditional assays for studying algal nutrient physiology require bulk community measurements or in situ nutrient perturbations. Although useful, these techniques lack the ability to target individual species in complex, mixed microbial assemblages. The motivation for this thesis is to examine the metabolic strategies utilized by A. anophagefferens for meeting its nitrogen (N) and phosphorus (P) demand at the cellular level using molecular tools that, even in the presence of complex microbial assemblages, can be used to track how nutrients influence the bloom dynamics of A. anophageferens in the environment. Chapter two examines the global transcriptional responses of A. anophagefferens to N and P deficiency. Results demonstrate that A. anophagefferens has the capacity to utilize multiple forms of organic N and P when inorganic forms become unavailable. Chapter three analyzed the global protein changes in response to P deficiency and P re-supply. Consistent with transcript patterns, A. anophagefferens increases protein abundance for a number of genes involved in inorganic and organic P metabolism when inorganic P is deficient. Furthermore, increases in a sulfolipid biosynthesis protein combined with lipid data suggest A. anophagefferens can adjust its P requirement by switching from phospholipids to sulfolipids when inorganic P is unavailable. Analysis of protein abundances from Pdeficient cells that were re-fed inorganic P demonstrates variations in the timing of turnover among various proteins upon release from phosphate deficiency. Chapter four tests the expression patterns of candidate gene markers of nutrient physiology under controlled culture experiments. Results show that expression patterns of a phosphate transporter and xanthine/uracil/vitamin C permease are indicators of P and N deficiency, respectively. Taken together, these findings provide insight into the fundamental and ecological niche space of this harnful algal species with respect to N and P and provide a platform for assaying nutrient controls on natural brown tide blooms.