Arsenate Resistance in the Unicellular Marine Diazotroph Crocosphaera watsonii

The toxic arsenate ion can behave as a phosphate analog, and this can result in arsenate toxicity especially in areas with elevated arsenate to phosphate ratios like the surface waters of the ocean gyres. In these systems, cellular arsenate resistance strategies would allow phytoplankton to ameliorate the effects of arsenate transport into the cell. Despite the potential coupling between arsenate and phosphate cycling in oligotrophic marine waters, relatively little is known about arsenate resistance in the nitrogen-fixing marine cyanobacteria that are key components of the microbial community in low nutrient systems. The unicellular diazotroph, Crocosphaera watsonii WH8501, was able to grow at reduced rates with arsenate additions up to 30 nM, and estimated arsenate to phosphate ratios of 6:1. The genome of strain WH8501 contains homologs for arsA, arsH, arsB, and arsC, allowing for the reduction of arsenate to arsenite and the pumping of arsenite out of the cell. The short-term addition of arsenate to the growth medium had no effect on nitrogen fixation. However, arsenate addition did result in the up-regulation of the arsB gene with increasing arsenate concentrations, indicating the induction of the arsenate detoxification response. The arsB gene was also up-regulated by phosphorus stress in concert with a gene encoding the high-affinity phosphate binding protein pstS. Both genes were down-regulated when phosphate was re-fed to phosphorus-stressed cells. A field survey of surface water from the low phosphate western North Atlantic detected expression of C. watsonii arsB, suggestive of the potential importance of arsenate resistance strategies in this and perhaps other systems

Arsenate resistance in the unicellular marine diazotroph Crocosphaera watsonii

© The Author(s), 2011. This is an open-access article subject to a non-exclusive license between the authors and Frontiers Media SA, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited. The definitive version was published in Frontiers in Microbiology 2 (2011): 214, doi:10.3389/fmicb.2011.00214.The toxic arsenate ion can behave as a phosphate analog, and this can result in arsenate toxicity especially in areas with elevated arsenate to phosphate ratios like the surface waters of the ocean gyres. In these systems, cellular arsenate resistance strategies would allow phytoplankton to ameliorate the effects of arsenate transport into the cell. Despite the potential coupling between arsenate and phosphate cycling in oligotrophic marine waters, relatively little is known about arsenate resistance in the nitrogen-fixing marine cyanobacteria that are key components of the microbial community in low nutrient systems. The unicellular diazotroph, Crocosphaera watsonii WH8501, was able to grow at reduced rates with arsenate additions up to 30 nM, and estimated arsenate to phosphate ratios of 6:1. The genome of strain WH8501 contains homologs for arsA, arsH, arsB, and arsC, allowing for the reduction of arsenate to arsenite and the pumping of arsenite out of the cell. The short-term addition of arsenate to the growth medium had no effect on nitrogen fixation. However, arsenate addition did result in the up-regulation of the arsB gene with increasing arsenate concentrations, indicating the induction of the arsenate detoxification response. The arsB gene was also up-regulated by phosphorus stress in concert with a gene encoding the high-affinity phosphate binding protein pstS. Both genes were down-regulated when phosphate was re-fed to phosphorus-stressed cells. A field survey of surface water from the low phosphate western North Atlantic detected expression of C. watsonii arsB, suggestive of the potential importance of arsenate resistance strategies in this and perhaps other systems.This research was funded in part by the National Science Foundation #OCE-0451419, and the Center for Microbial Oceanography: Research and Education

Metatranscriptome analyses indicate resource partitioning between diatoms in the field

Diverse communities of marine phytoplankton carry out half of global primary production. The vast diversity of the phytoplankton has long perplexed ecologists because these organisms coexist in an isotropic environment while competing for the same basic resources (e.g., inorganic nutrients). Differential niche partitioning of resources is one hypothesis to explain this “paradox of the plankton,” but it is difficult to quantify and track variation in phytoplankton metabolism in situ. Here, we use quantitative metatranscriptome analyses to examine pathways of nitrogen (N) and phosphorus (P) metabolism in diatoms that cooccur regularly in an estuary on the east coast of the United States (Narragansett Bay). Expression of known N and P metabolic pathways varied between diatoms, indicating apparent differences in resource utilization capacity that may prevent direct competition. Nutrient amendment incubations skewed N/P ratios, elucidating nutrient-responsive patterns of expression and facilitating a quantitative comparison between diatoms. The resource-responsive (RR) gene sets deviated in composition from the metabolic profile of the organism, being enriched in genes associated with N and P metabolism. Expression of the RR gene set varied over time and differed significantly between diatoms, resulting in opposite transcriptional responses to the same environment. Apparent differences in metabolic capacity and the expression of that capacity in the environment suggest that diatom-specific resource partitioning was occurring in Narragansett Bay. This high-resolution approach highlights the molecular underpinnings of diatom resource utilization and how cooccurring diatoms adjust their cellular physiology to partition their niche space.American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowshi

Alkaline phosphatase activity in the phytoplankton communities of Monterey Bay and San Francisco Bay

Author Posting. © American Society of Limnology and Oceanography, 2006. This is the author's version of the work. It is posted here by permission of American Society of Limnology and Oceanography for personal use, not for redistribution. The definitive version was published in Limnology and Oceanography 51 (2006): 874–883.Enzyme-labeled fluorescence (ELF) and bulk alkaline phosphatase (AP) activity enzyme assays were used to evaluate the phosphorus (P) status of phytoplankton communities in San Francisco and Monterey bays. Both regions exhibit spatial and temporal variability in bulk AP activity with maximum activities during the early spring and summer periods of high biological productivity. ELF analysis revealed pronounced differences in the makeup of organisms responsible for AP activity in these two environments. In Monterey Bay dinoflagellates are responsible for the bulk of the AP activity. Diatoms infrequently exhibited AP activity. Dinoflagellates that comprised only 14% of all cells counted in Monterey Bay accounted for 78% of AP-producing cells examined. The presence of AP activity in this group suggests that changes in P sources, concentrations, and bioavailability could disproportionably influence this group relative to diatoms in Monterey Bay. In San Francisco Bay, AP production, indicated by ELF, was associated primarily with bacteria attached to suspended particles, potentially used to hydrolyze organic compounds for carbon, rather than to satisfy P requirements. Our results highlight the importance of organic P as a bioavailable nutrient source in marine ecosystems and as a component of the marine P cycle

Microbes and the marine phosphorus cycle

Author Posting. © Oceanography Society, 2007. This article is posted here by permission of Oceanography Society for personal use, not for redistribution. The definitive version was published in Oceanography 20, 2 (2007): 110-116.Phosphorus (P) is fundamental to life, and years of study in marine systems have built a broad understanding of the marine P cycle. Various aspects of marine P biogeochemistry have been reviewed previously (Benitez-Nelson, 2000; Paytan and McLaughlin, 2007). Here, we focus on recent advances in our understanding of marine P and the interactions between microbes and the P cycle. These advances come from a variety of disciplines, but generally highlight three main themes: (1) ocean microbes are adapted for surviving in a variable P environment, (2) the dissolved organic phosphorus (DOP) pool likely plays a critical role in driving growth, metabolism, and community composition of ocean microorganisms, and (3) P is very rapidly cycled, which highlights its importance in marine systems

Cross-Basin Comparison of Phosphorus Stress and Nitrogen Fixation in Trichodesmium

We investigated the phosphorus (P) status and N2 fixation rates of Trichodesmium populations from the North Pacific, western South Pacific, and western North Atlantic. Colonies of Trichodesmium were collected and analyzed for endogenous alkaline phosphatase (AP) activity using enzyme-labeled fluorescence ( ELF) and for nitrogenase activity using acetylene reduction. AP hydrolyzes dissolved inorganic phosphate (DIP) from dissolved organic phosphorus and is active in Trichodesmium colonies experiencing P stress. Across multiple stations in the subtropical North and South Pacific, there was low to moderate ELF labeling in Trichodesmium, although labeling was present in other taxa. In contrast, Trichodesmium ELF labeling in the North Atlantic ranged from low to high. Low ELF labeling corresponded with high DIP concentrations while high ELF labeling occurred only at North Atlantic stations with DIP concentrations \u3c = 40 nmol L-1, indicating that Trichodesmium was not experiencing dramatic P stress in the Pacific Ocean while P stress was evident in the western North Atlantic. However, nitrogenase activity was significantly higher in the P-stressed western North Atlantic than in the Pacific Ocean (0.40-1.30 compared to 0.01-0.46 nmol C2H4 h-1 colony-1. These data underscore the differential basin-level importance of P availability to Trichodesmium and suggest that factors other than P are constraining their N2 fixation rates in the Pacific

Polyphosphate dynamics at Station ALOHA, North Pacific subtropical gyre

Author Posting. © Association for the Sciences of Limnology and Oceanography, 2015. This article is posted here by permission of Association for the Sciences of Limnology and Oceanography for personal use, not for redistribution. The definitive version was published in Limnology and Oceanography 61 (2016): 227–239, doi:10.1002/lno.10206.Polyphosphate (polyP) was examined within the upper water column (≤ 150 m) of Station ALOHA (22° 45′N, 158° 00′W) during two cruises conducted in May–June 2013 and September 2013. Phosphorus molar ratios of particulate polyP to total particulate phosphorus (TPP) were relatively low, similar to previously reported values from the temperate western North Atlantic, and did not exhibit strong vertical gradients, reflecting a lack of polyP recycling relative to other forms of TPP with depth. Furthermore, relationships among polyP:TPP, soluble reactive phosphorus (SRP), and alkaline phosphatase activity (APA) were also consistent with previous observations from the Atlantic Ocean. To ascertain potential mechanisms of biological polyP production and utilization, surface seawater was incubated following nutrient additions. Results were consistent with polyP:TPP enrichment under opposite extremes of APA, suggesting diverse polyP accumulation/retention mechanisms. Addition of exogenous polyP (45 ± 5 P atoms) to field incubations did not increase chlorophyll content relative to controls, suggesting that polyP was not bioavailable to phytoplankton at Station ALOHA. To clarify this result, phytoplankton cultures were screened for the ability to utilize exogenous polyP. PolyP bioavailability was variable among model diatoms of the genus Thalassiosira, yet chain length did not influence polyP bioavailability. Thus, microbial community composition may influence polyP dynamics in the ocean, and vice versa.This work was supported by a Postdoctoral Fellowship from the Ford Foundation (JMD), the National Science Foundation under grants OCE 1225801 (JMD), OCE 1316036 (STD), EF 04-24599 (DMK), the Woods Hole Oceanographic Coastal Ocean Institute, the Center for Microbial Oceanography: Research and Education, and the Gordon and Betty Moore Foundation (DMK). Additional support was provided by grants from the Simons Foundation to DMK and STD

Virus-host relationships of marine single-celled eukaryotes resolved from metatranscriptomics

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

Identifying reference genes with stable expression from high throughput sequence data

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Microbiology 3 (2012): 385, doi:10.3389/fmicb.2012.00385.Genes that are constitutively expressed across multiple environmental stimuli are crucial to quantifying differentially expressed genes, particularly when employing quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) assays. However, the identification of these potential reference genes in non-model organisms is challenging and is often guided by expression patterns in distantly related organisms. Here, transcriptome datasets from the diatom Thalassiosira pseudonana grown under replete, phosphorus-limited, iron-limited, and phosphorus and iron co-limited nutrient regimes were analyzed through literature-based searches for homologous reference genes, k-means clustering, and analysis of sequence counts (ASC) to identify putative reference genes. A total of 9759 genes were identified and screened for stable expression. Literature-based searches surveyed 18 generally accepted reference genes, revealing 101 homologs in T. pseudonana with variable expression and a wide range of mean tags per million. k-means analysis parsed the whole transcriptome into 15 clusters. The two most stable clusters contained 709 genes, but still had distinct patterns in expression. ASC analyses identified 179 genes that were stably expressed (posterior probability < 0.1 for 1.25 fold change). Genes known to have a stable expression pattern across the test treatments, like actin, were identified in this pool of 179 candidate genes. ASC can be employed on data without biological replicates and was more robust than the k-means approach in isolating genes with stable expression. The intersection of the genes identified through ASC with commonly used reference genes from the literature suggests that actin and ubiquitin ligase may be useful reference genes for T. pseudonana and potentially other diatoms. With the wealth of transcriptome sequence data becoming available, ASC can be easily applied to transcriptome datasets from other phytoplankton to identify reference genes.This research was funded by the National Science Foundation grant #OCE-0723667 (to Sonya T. Dyhrman, Mak A. Saito, Bethany D. Jenkins, and Tatiana A. Rynearson). Harriet Alexander is funded under a National Defense Science and Engineering Graduate (NDSEG) Fellowship