RESUMO
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.
Assuntos
Proteínas de Membrana/química , Nanoarchaeota/metabolismo , Proteoma/química , Proteômica/métodos , Cromatografia Líquida , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas , Nanoarchaeota/químicaRESUMO
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 â¼20-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 â¼10-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.
Assuntos
Proteínas de Algas/genética , Proteínas de Membrana Transportadoras/genética , Phaeophyceae/genética , Proteínas de Transporte de Fosfato/genética , Estramenópilas/genética , Proteínas de Algas/metabolismo , Regulação da Expressão Gênica , Proliferação Nociva de Algas , Proteínas de Membrana Transportadoras/metabolismo , Nitratos/metabolismo , Nitrogênio/metabolismo , Phaeophyceae/metabolismo , Proteínas de Transporte de Fosfato/metabolismo , Fosfatos/metabolismo , Fósforo/metabolismo , Estramenópilas/metabolismo , Xantina/metabolismoRESUMO
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.
Assuntos
Ecossistema , Eucariotos/genética , Genômica/métodos , Sequência de Aminoácidos , Bactérias/metabolismo , Bactérias/efeitos da radiação , Biodegradação Ambiental/efeitos da radiação , Enzimas/metabolismo , Eucariotos/enzimologia , Genoma/genética , Luz , Filogenia , Fitoplâncton/genética , Fitoplâncton/efeitos da radiação , Proteínas/química , Especificidade da EspécieRESUMO
Here we describe a publicly available environmental DNA (eDNA) sequence dataset, consisting of samples collected from a National Oceanic and Atmospheric Administration (NOAA) Great Lakes Environmental Research Laboratory (GLERL) on Lake Erie. We sequenced samples drawn from before, during, and after a 2019 Microcystis harmful algal bloom (HAB) using 3rd generation sequencing with the Oxford Nanopore MinION device. We classified the eDNA reads taxonomically, and estimated the abundances of all taxa in each sample. While the taxonomic data showed evidence of significant human and E. coli contamination, we found abundant Mycrocystis, especially in the samples drawn from bloom environments. The raw sequence data are available in the Sequence Read Archive (SRA) under accession number PRJNA812770. HABs pose a significant and increasing risk, both to human health and to the Blue Economy, and genomic approaches to early detection promise to help mitigate these risks. As such, this dataset could be of interest to freshwater ecology research teams, or any stakeholders interested in the detection and mitigation of HABs.
RESUMO
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.
Assuntos
Perfilação da Expressão Gênica , Nitrogênio/metabolismo , Phaeophyceae/genética , Fósforo/metabolismo , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica , Biblioteca Gênica , Phaeophyceae/fisiologia , Transcrição GênicaRESUMO
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.
Assuntos
DNA Bacteriano/genética , Genoma Bacteriano , Proliferação Nociva de Algas/fisiologia , Metagenoma , Microcystis/genética , Percepção de Quorum/genética , Carbono/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Lagos/microbiologia , Microbiota/genética , Microcystis/classificação , Microcystis/metabolismo , Nitrogênio/metabolismo , Oxirredução , Filogenia , Transdução de Sinais , Estados UnidosRESUMO
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.
RESUMO
Establishing virus-host relationships has historically relied on culture-dependent approaches. Here we report on the use of marine metatranscriptomics to probe virus-host relationships. Statistical co-occurrence analyses of dsDNA, ssRNA and dsRNA viral markers of polyadenylation-selected RNA sequences from microbial communities dominated by Aureococcus anophagefferens (Quantuck Bay, NY), and diatoms (Narragansett Bay, RI) show active infections by diverse giant viruses (NCLDVs) associated with algal and nonalgal hosts. Ongoing infections of A. anophagefferens by a known Mimiviridae (AaV) occur during bloom peak and decline. Bloom decline is also accompanied by increased activity of viruses other than AaV, including (+) ssRNA viruses. In Narragansett Bay, increased temporal resolution reveals active NCLDVs with both 'boom-and-bust' and 'steady-state infection'-like ecologies that include known as well as novel virus-host interactions. Our approach offers a method for screening active viral infections and develops links between viruses and their potential hosts in situ. Our observations further demonstrate that previously unknown virus-host relationships in marine systems are abundant.
Assuntos
Genômica/métodos , Vírus Gigantes/genética , Proliferação Nociva de Algas , Interações Hospedeiro-Patógeno , Estramenópilas/virologia , Mimiviridae/fisiologia , New York , Poliadenilação , Rhode Island , Água do Mar/virologiaRESUMO
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.
Assuntos
Desulfurococcaceae/fisiologia , Interações Microbianas , Nanoarchaeota/genética , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Membrana Celular/metabolismo , Desulfurococcaceae/crescimento & desenvolvimento , Expressão Gênica , Genoma Arqueal , Nanoarchaeota/crescimento & desenvolvimento , Nanoarchaeota/metabolismo , ProteômicaRESUMO
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.
Assuntos
Phaeophyceae/metabolismo , Fósforo/deficiência , Proteoma/metabolismo , Movimentos da Água , Regulação da Expressão Gênica/efeitos dos fármacos , Phaeophyceae/efeitos dos fármacos , Phaeophyceae/genética , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/metabolismo , Fósforo/farmacologia , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
The abundant and widespread coccolithophore Emiliania huxleyi plays an important role in mediating CO2 exchange between the ocean and the atmosphere through its impact on marine photosynthesis and calcification. Here, we use long serial analysis of gene expression (SAGE) to identify E. huxleyi genes responsive to nitrogen (N) or phosphorus (P) starvation. Long SAGE is an elegant approach for examining quantitative and comprehensive gene expression patterns without a priori knowledge of gene sequences via the detection of 21-bp nucleotide sequence tags. E. huxleyi appears to have a robust transcriptional-level response to macronutrient deficiency, with 42 tags uniquely present or up-regulated twofold or greater in the N-starved library and 128 tags uniquely present or up-regulated twofold or greater in the P-starved library. The expression patterns of several tags were validated with reverse transcriptase PCR. Roughly 48% of these differentially expressed tags could be mapped to publicly available genomic or expressed sequence tag (EST) sequence data. For example, in the P-starved library a number of the tags mapped to genes with a role in P scavenging, including a putative phosphate-repressible permease and a putative polyphosphate synthetase. In short, the long SAGE analyses have (i) identified many new differentially regulated gene sequences, (ii) assigned regulation data to EST sequences with no database homology and unknown function, and (iii) highlighted previously uncharacterized aspects of E. huxleyi N and P physiology. To this end, our long SAGE libraries provide a new public resource for gene discovery and transcriptional analysis in this biogeochemically important marine organism.