Moran Dawn M.

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Moran

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Dawn M.

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Needles in the blue sea : sub-species specificity in targeted protein biomarker analyses within the vast oceanic microbial metaproteome

( 2015-03-27) Saito, Mak A. ; Dorsk, Alexander ; Post, Anton F. ; McIlvin, Matthew R. ; Rappe, Michael S. ; DiTullio, Giacomo R. ; Moran, Dawn M.

Proteomics has great potential for studies of marine microbial biogeochemistry, yet high microbial diversity in many locales presents us with unique challenges. We addressed this challenge with a targeted metaproteomics workflow for NtcA and P-II, two nitrogen regulatory proteins, and demonstrated its application for cyanobacterial taxa within microbial samples from the Central Pacific Ocean. Using METATRYP, an open-source Python toolkit, we examined the number of shared (redundant) tryptic peptides in representative marine microbes, with the number of tryptic peptides shared between different species typically being 1% or less. The related cyanobacteria Prochlorococcus and Synechococcus shared an average of 4.8+1.9% of their tryptic peptides, while shared intraspecies peptides were higher, 13+15% shared peptides between 12 Prochlorococcus genomes. An NtcA peptide was found to target multiple cyanobacteria species, whereas a P-II peptide showed specificity to the high-light Prochlorococcus ecotype. Distributions of NtcA and P-II in the Central Pacific Ocean were similar except at the Equator likely due to differential nitrogen stress responses between Prochlorococcus and Synechococcus. The number of unique tryptic peptides coded for within three combined oceanic microbial metagenomes was estimated to be ~4x107, 1000-fold larger than an individual microbial proteome and 27-fold larger than the human proteome, yet still 20 orders of magnitude lower than the peptide diversity possible in all protein space, implying that peptide mapping algorithms should be able to withstand the added level of complexity in metaproteomic samples.
Environmental distribution and persistence of Quahog Parasite Unknown (QPX)

(Inter-Research, 2008-09-24) Gast, Rebecca J. ; Moran, Dawn M. ; Audemard, Corinne ; Lyons, M. Maille ; DeFaveri, Jacquelin ; Reece, Kimberly S. ; Leavitt, Dale F. ; Smolowitz, Roxanna M.

Quahog Parasite Unknown (QPX) is the cause of mass mortality events of hard clams Mercenaria mercenaria from Virginia, USA, to New Brunswick, Canada. Aquaculture areas in Massachusetts, USA, have been particularly hard hit. The parasite has been shown to be a directly infective organism, but it is unclear whether it could exist or persist outside of its clam host. We used molecular methods to examine water, sediment, seaweeds, seagrass and various invertebrates for the presence of QPX. Sites in Virginia and Massachusetts were selected based upon the incidence of QPX-induced clam die-offs, and they were monitored seasonally. QPX was detectable in almost all of our different sample types from Massachusetts, indicating that the parasite was widely distributed in the environment. Significantly more samples from Massachusetts were positive than from Virginia, and there was a seasonal pattern to the types of samples positive from Massachusetts. The data suggest that, although it may be difficult to completely eradicate QPX from the environment, it may be possible to keep the incidence of disease under control through good plot husbandry and the removal of infected and dying clams.
Dissolved and particulate trace metal micronutrients under the McMurdo Sound seasonal sea ice : basal sea ice communities as a capacitor for iron

(Frontiers Media, 2013-10-30) Noble, Abigail E. ; Moran, Dawn M. ; Allen, Andrew E. ; Saito, Mak A.

Dissolved and particulate metal concentrations are reported from three sites beneath and at the base of the McMurdo Sound seasonal sea ice in the Ross Sea of Antarctica. This dataset provided insight into Co and Mn biogeochemistry, supporting a previous hypothesis for water column mixing occurring faster than scavenging. Three observations support this: first, Mn-containing particles with Mn/Al ratios in excess of the sediment were present in the water column, implying the presence of bacterial Mn-oxidation processes. Second, dissolved and labile Co were uniform with depth beneath the sea ice after the winter season. Third, dissolved Co:PO3−4 ratios were consistent with previously observed Ross Sea stoichiometry, implying that over-winter scavenging was slow relative to mixing. Abundant dissolved Fe and Mn were consistent with a winter reserve concept, and particulate Al, Fe, Mn, and Co covaried, implying that these metals behaved similarly. Elevated particulate metals were observed in proximity to the nearby Islands, with particulate Fe/Al ratios similar to that of nearby sediment, consistent with a sediment resuspension source. Dissolved and particulate metals were elevated at the shallowest depths (particularly Fe) with elevated particulate P/Al and Fe/Al ratios in excess of sediments, demonstrating a sea ice biomass source. The sea ice biomass was extremely dense (chl a >9500 μg/L) and contained high abundances of particulate metals with elevated metal/Al ratios. A hypothesis for seasonal accumulation of bioactive metals at the base of the McMurdo Sound sea ice by the basal algal community is presented, analogous to a capacitor that accumulates iron during the spring and early summer. The release and transport of particulate metals accumulated at the base of the sea ice by sloughing is discussed as a potentially important mechanism in providing iron nutrition during polynya phytoplankton bloom formation and could be examined in future oceanographic expeditions.
Irreversibly increased nitrogen fixation in Trichodesmium experimentally adapted to elevated carbon dioxide

(Nature Publishing Group, 2015-09-01) Hutchins, David A. ; Walworth, Nathan G. ; Webb, Eric A. ; Saito, Mak A. ; Moran, Dawn M. ; McIlvin, Matthew R. ; Gale, Jasmine ; Fu, Fei-Xue

Nitrogen fixation rates of the globally distributed, biogeochemically important marine cyanobacterium Trichodesmium increase under high carbon dioxide (CO2) levels in short-term studies due to physiological plasticity. However, its long-term adaptive responses to ongoing anthropogenic CO2 increases are unknown. Here we show that experimental evolution under extended selection at projected future elevated CO2 levels results in irreversible, large increases in nitrogen fixation and growth rates, even after being moved back to lower present day CO2 levels for hundreds of generations. This represents an unprecedented microbial evolutionary response, as reproductive fitness increases acquired in the selection environment are maintained after returning to the ancestral environment. Constitutive rate increases are accompanied by irreversible shifts in diel nitrogen fixation patterns, and increased activity of a potentially regulatory DNA methyltransferase enzyme. High CO2-selected cell lines also exhibit increased phosphorus-limited growth rates, suggesting a potential advantage for this keystone organism in a more nutrient-limited, acidified future ocean.
Development and application of a monoclonal-antibody technique for counting Aureococcus anophagefferens, an alga causing recurrent brown tides in the Mid-Atlantic United States

(American Society for Microbiology, 2003-09) Caron, David A. ; Dennett, Mark R. ; Moran, Dawn M. ; Schaffner, Rebecca A. ; Lonsdale, Darcy J. ; Gobler, Christopher J. ; Nuzzi, Robert ; McLean, Tim I.

A method was developed for the rapid detection and enumeration of Aureococcus anophagefferens, the cause of harmful algal blooms called "brown tides" in estuaries of the Mid-Atlantic United States. The method employs a monoclonal antibody (MAb) and a colorimetric, enzyme-linked immunosorbent assay format. The MAb obtained exhibits high reactivity with A. anophagefferens and very low cross-reactivities with a phylogenetically diverse array of other protists and bacteria. Standard curves are constructed for each 96-well microtiter plate by using known amounts of a preserved culture of A. anophagefferens. This approach allows estimation of the abundance of the alga in natural samples. The MAb method was compared to an existing method that employs polyclonal antibodies and epifluorescence microscopy and to direct microscopic counts of A. anophagefferens in samples with high abundances of the alga. The MAb method provided increased quantitative accuracy and greatly reduced sample processing time. A spatial survey of several Long Island estuaries in May 2000 using this new approach documented a range of abundances of A. anophagefferens in these bays spanning nearly 3 orders of magnitude.
DGGE-based detection method for Quahog Parasite Unknown (QPX)

(Inter-Research, 2006-06-12) Gast, Rebecca J. ; Cushman, E. ; Moran, Dawn M. ; Uhlinger, Kevin R. ; Leavitt, Dale F. ; Smolowitz, Roxanna M.

Quahog Parasite Unknown (QPX) is a significant cause of hard clam Mercenaria mercenaria mortality along the northeast coast of the United States. It infects both wild and cultured clams, often annually in plots that are heavily farmed. Subclinically infected clams can be identified by histological examination of the mantle tissue, but there is currently no method available to monitor the presence of QPX in the environment. Here, we report on a polymerase chain reaction (PCR)-based method that will facilitate the detection of QPX in natural samples and seed clams. With our method, between 10 and 100 QPX cells can be detected in 1 l of water, 1 g of sediment and 100 mg of clam tissue. Denaturing gradient gel electrophoresis (DGGE) is used to establish whether the PCR products are the same as those in the control QPX culture. We used the method to screen 100 seed clams of 15 mm, and found that 10 to 12% of the clams were positive for the presence of the QPX organism. This method represents a reliable and sensitive procedure for screening both environmental samples and potentially contaminated small clams.
The acceleration of dissolved cobalt's ecological stoichiometry due to biological uptake, remineralization, and scavenging in the Atlantic Ocean

(Copernicus Publications on behalf of the European Geosciences Union, 2017-10-20) Saito, Mak A. ; Noble, Abigail E. ; Hawco, Nicholas J. ; Twining, Benjamin S. ; Ohnemus, Daniel C. ; John, Seth G. ; Lam, Phoebe J. ; Conway, Tim M. ; Johnson, Rod ; Moran, Dawn M. ; McIlvin, Matthew R.

The stoichiometry of biological components and their influence on dissolved distributions have long been of interest in the study of the oceans. Cobalt has the smallest oceanic inventory of inorganic micronutrients and hence is particularly vulnerable to influence by internal oceanic processes including euphotic zone uptake, remineralization, and scavenging. Here we observe not only large variations in dCo : P stoichiometry but also the acceleration of those dCo : P ratios in the upper water column in response to several environmental processes. The ecological stoichiometry of total dissolved cobalt (dCo) was examined using data from a US North Atlantic GEOTRACES transect and from a zonal South Atlantic GEOTRACES-compliant transect (GA03/3_e and GAc01) by Redfieldian analysis of its statistical relationships with the macronutrient phosphate. Trends in the dissolved cobalt to phosphate (dCo : P) stoichiometric relationships were evident in the basin-scale vertical structure of cobalt, with positive dCo : P slopes in the euphotic zone and negative slopes found in the ocean interior and in coastal environments. The euphotic positive slopes were often found to accelerate towards the surface and this was interpreted as being due to the combined influence of depleted phosphate, phosphorus-sparing (conserving) mechanisms, increased alkaline phosphatase metalloenzyme production (a zinc or perhaps cobalt enzyme), and biochemical substitution of Co for depleted Zn. Consistent with this, dissolved Zn (dZn) was found to be drawn down to only 2-fold more than dCo, despite being more than 18-fold more abundant in the ocean interior. Particulate cobalt concentrations increased in abundance from the base of the euphotic zone to become ∼  10 % of the overall cobalt inventory in the upper euphotic zone with high stoichiometric values of ∼  400 µmol Co mol−1 P. Metaproteomic results from the Bermuda Atlantic Time-series Study (BATS) station found cyanobacterial isoforms of the alkaline phosphatase enzyme to be prevalent in the upper water column, as well as a sulfolipid biosynthesis protein indicative of P sparing. The negative dCo : P relationships in the ocean interior became increasingly vertical with depth, and were consistent with the sum of scavenging and remineralization processes (as shown by their dCo : P vector sums). Attenuation of the remineralization with depth resulted in the increasingly vertical dCo : P relationships. Analysis of particulate Co with particulate Mn and particulate phosphate also showed positive linear relationships below the euphotic zone, consistent with the presence and increased relative influence of Mn oxide particles involved in scavenging. Visualization of dCo : P slopes across an ocean section revealed hotspots of scavenging and remineralization, such as at the hydrothermal vents and below the oxygen minimum zone (OMZ) region, respectively, while that of an estimate of Co* illustrated stoichiometrically depleted values in the mesopelagic and deep ocean due to scavenging. This study provides insights into the coupling between the dissolved and particulate phase that ultimately creates Redfield stoichiometric ratios, demonstrating that the coupling is not an instantaneous process and is influenced by the element inventory and rate of exchange between phases. Cobalt's small water column inventory and the influence of external factors on its biotic stoichiometry can erode its limited inertia and result in an acceleration of the dissolved stoichiometry towards that of the particulate phase in the upper euphotic zone. As human use of cobalt grows exponentially with widespread adoption of lithium ion batteries, there is a potential to affect the limited biogeochemical inertia of cobalt and its resultant ecology in the oceanic euphotic zone.
Methionine synthase interreplacement in diatom cultures and communities : implications for the persistence of B12 use by eukaryotic phytoplankton

(Association for the Sciences of Limnology and Oceanography, 2013-07) Bertrand, Erin M. ; Moran, Dawn M. ; McIlvin, Matthew R. ; Hoffman, Jeffrey M. ; Allen, Andrew E. ; Saito, Mak A.

Three proteins related to vitamin B12 metabolism in diatoms were quantified via selected reaction monitoring mass spectrometry: B12-dependent and B12-independent methionine synthase (MetH, MetE) and a B12 acquisition protein (CBA1). B12-mediated interreplacement of MetE and MetH metalloenzymes was observed in Phaeodactylum tricornutum where MetH abundance was highest (0.06 fmol µg−1 protein) under high B12 and MetE abundance increased to 3.25 fmol µg−1 protein under low B12 availability. Maximal MetE abundance was 60-fold greater than MetH, consistent with the expected ∼ 50–100-fold larger turnover number for MetH. MetE expression resulted in 30-fold increase in nitrogen and 40-fold increase in zinc allocated to methionine synthase activity under low B12. CBA1 abundance was 6-fold higher under low-B12 conditions and increased upon B12 resupply to starved cultures. While biochemical pathways that supplant B12 requirements exist and are utilized by organisms such as land plants, B12 use persists in eukaryotic phytoplankton. This study suggests that retention of B12 utilization by phytoplankton results in resource conservation under conditions of high B12 availability. MetE and MetH abundances were also measured in diatom communities from McMurdo Sound, verifying that both these proteins are expressed in natural communities. These protein measurements are consistent with previous studies suggesting that B12 availability influences Antarctic primary productivity. This study illuminates controls on expression of B12-related proteins, quantitatively assesses the metabolic consequences of B12 deprivation, and demonstrates that mass spectrometry–based protein measurements yield insight into the functioning of marine microbial communities.
Defining DNA-based operational taxonomic units for microbial-eukaryote ecology

( 2009-06-19) Caron, David A. ; Countway, Peter D. ; Savai, Pratik ; Gast, Rebecca J. ; Schnetzer, Astrid ; Moorthi, Stefanie D. ; Dennett, Mark R. ; Moran, Dawn M. ; Jones, Adriane C.

DNA sequence information has been increasingly used in ecological research on microbial eukaryotes. Sequence-based approaches have included studies of the total diversity of selected ecosystems, the autecology of ecologically relevant species, and the identification and enumeration of species of interest to human health. It is still uncommon, however, to delineate protistan species based on their genetic signatures. The reluctance to assign species-level designations based on DNA sequences is partly a consequence of the limited amount of sequence information presently available for many free-living microbial eukaryotes, and partly the problematic nature and debate surrounding the microbial species concept. Despite the difficulties inherent in assigning species names to DNA sequences, there is a growing need to attach meaning to the burgeoning amount of sequence information entering the literature, and a growing desire to apply this information in ecological studies. We describe a computer-based tool that assigns DNA sequences from environmental databases to operational taxonomic units at approximate species-level distinctions. The approach provides a practical method for ecological studies of microbial eukaryotes (primarily protists) by enabling semiautomated analysis of large numbers of samples spanning great taxonomic breadth. Derivation of the algorithm was based on an analysis of complete small subunit ribosomal RNA (18S) gene sequences and partial gene sequences obtained from GenBank for morphologically described protistan species. The program was tested using environmental 18S data sets from two oceanic ecosystems. A total of 388 operational taxonomic units were observed among 2,207 sequences obtained from samples collected in the western North Atlantic and eastern North Pacific.
Examination of microbial proteome preservation techniques applicable to autonomous environmental sample collection

(Frontiers Media, 2011-11-07) Saito, Mak A. ; Bulygin, Vladimir V. ; Moran, Dawn M. ; Taylor, Craig D. ; Scholin, Christopher A.

Improvements in temporal and spatial sampling frequency have the potential to open new windows into the understanding of marine microbial dynamics. In recent years, efforts have been made to allow automated samplers to collect microbial biomass for DNA/RNA analyses from moored observatories and autonomous underwater vehicles. Measurements of microbial proteins are also of significant interest given their biogeochemical importance as enzymes that catalyze reactions and transporters that interface with the environment. We examined the influence of five preservatives solutions (SDS-extraction buffer, ethanol, trichloroacetic acid, B-PER, and RNAlater) on the proteome integrity of the marine cyanobacterium Synechococcus WH8102 after 4 weeks of storage at room temperature. Four approaches were used to assess degradation: total protein recovery, band integrity on an SDS detergent polyacrylamide electrophoresis (SDS-PAGE) gel, and number of protein identifications and relative abundances by 1-dimensional LC–MS/MS proteomic analyses. Total protein recoveries from the preserved samples were lower than the frozen control due to processing losses, which could be corrected for with internal standardization. The trichloroacetic acid preserved sample showed significant loss of protein band integrity on the SDS-PAGE gel. The RNAlater preserved sample showed the highest number of protein identifications (103% relative to the control; 520 ± 31 identifications in RNAlater versus 504 ± 4 in the control), equivalent to the frozen control. Relative abundances of individual proteins in the RNAlater treatment were quite similar to that of the frozen control (average ratio of 1.01 ± 0.27 for the 50 most abundant proteins), while the SDS-extraction buffer, ethanol, and B-PER all showed significant decreases in both number of identifications and relative abundances of individual proteins. Based on these findings, RNAlater was an effective proteome preservative, although further study is warranted on additional marine microbes.
Microbial functional diversity across biogeochemical provinces in the central Pacific Ocean

(National Academy of Sciences, 2022-09-13) Saunders, Jaclyn K. ; McIlvin, Matthew R. ; Dupont, Christopher L. ; Kaul, Drishti ; Moran, Dawn M. ; Horner, Tristan J. ; Laperriere, Sarah ; Webb, Eric A. ; Bosak, Tanja ; Santoro, Alyson E. ; Saito, Mak A.

Enzymes catalyze key reactions within Earth’s life-sustaining biogeochemical cycles. Here, we use metaproteomics to examine the enzymatic capabilities of the microbial community (0.2 to 3 µm) along a 5,000-km-long, 1-km-deep transect in the central Pacific Ocean. Eighty-five percent of total protein abundance was of bacterial origin, with Archaea contributing 1.6%. Over 2,000 functional KEGG Ontology (KO) groups were identified, yet only 25 KO groups contributed over half of the protein abundance, simultaneously indicating abundant key functions and a long tail of diverse functions. Vertical attenuation of individual proteins displayed stratification of nutrient transport, carbon utilization, and environmental stress. The microbial community also varied along horizontal scales, shaped by environmental features specific to the oligotrophic North Pacific Subtropical Gyre, the oxygen-depleted Eastern Tropical North Pacific, and nutrient-rich equatorial upwelling. Some of the most abundant proteins were associated with nitrification and C1 metabolisms, with observed interactions between these pathways. The oxidoreductases nitrite oxidoreductase (NxrAB), nitrite reductase (NirK), ammonia monooxygenase (AmoABC), manganese oxidase (MnxG), formate dehydrogenase (FdoGH and FDH), and carbon monoxide dehydrogenase (CoxLM) displayed distributions indicative of biogeochemical status such as oxidative or nutritional stress, with the potential to be more sensitive than chemical sensors. Enzymes that mediate transformations of atmospheric gases like CO, CO2, NO, methanethiol, and methylamines were most abundant in the upwelling region. We identified hot spots of biochemical transformation in the central Pacific Ocean, highlighted previously understudied metabolic pathways in the environment, and provided rich empirical data for biogeochemical models critical for forecasting ecosystem response to climate change.
Mechanisms of increased Trichodesmium fitness under iron and phosphorus co-limitation in the present and future ocean

(Nature Publishing Group, 2016-06-27) Walworth, Nathan G. ; Fu, Fei-Xue ; Webb, Eric A. ; Saito, Mak A. ; Moran, Dawn M. ; Mcllvin, Matthew R. ; Lee, Michael D. ; Hutchins, David A.

Nitrogen fixation by cyanobacteria supplies critical bioavailable nitrogen to marine ecosystems worldwide; however, field and lab data have demonstrated it to be limited by iron, phosphorus and/or CO2. To address unknown future interactions among these factors, we grew the nitrogen-fixing cyanobacterium Trichodesmium for 1 year under Fe/P co-limitation following 7 years of both low and high CO2 selection. Fe/P co-limited cell lines demonstrated a complex cellular response including increased growth rates, broad proteome restructuring and cell size reductions relative to steady-state growth limited by either Fe or P alone. Fe/P co-limitation increased abundance of a protein containing a conserved domain previously implicated in cell size regulation, suggesting a similar role in Trichodesmium. Increased CO2 further induced nutrient-limited proteome shifts in widespread core metabolisms. Our results thus suggest that N2-fixing microbes may be significantly impacted by interactions between elevated CO2 and nutrient limitation, with broad implications for global biogeochemical cycles in the future ocean.
Adaptive responses of marine diatoms to zinc scarcity and ecological implications

(Nature Research, 2022-04-14) Kellogg, Riss ; Moosburner, Mark A. ; Cohen, Natalie R. ; Hawco, Nicholas J. ; McIlvin, Matthew R. ; Moran, Dawn M. ; DiTullio, Giacomo R. ; Subhas, Adam V. ; Saito, Mak A.

Scarce dissolved surface ocean concentrations of the essential algal micronutrient zinc suggest that Zn may influence the growth of phytoplankton such as diatoms, which are major contributors to marine primary productivity. However, the specific mechanisms by which diatoms acclimate to Zn deficiency are poorly understood. Using global proteomic analysis, we identified two proteins (ZCRP-A/B, Zn/Co Responsive Protein A/B) among four diatom species that became abundant under Zn/Co limitation. Characterization using reverse genetic techniques and homology data suggests putative Zn/Co chaperone and membrane-bound transport complex component roles for ZCRP-A (a COG0523 domain protein) and ZCRP-B, respectively. Metaproteomic detection of ZCRPs along a Pacific Ocean transect revealed increased abundances at the surface (<200 m) where dZn and dCo were scarcest, implying Zn nutritional stress in marine algae is more prevalent than previously recognized. These results demonstrate multiple adaptive responses to Zn scarcity in marine diatoms that are deployed in low Zn regions of the Pacific Ocean.
Hydrothermal trace metal release and microbial metabolism in the northeastern Lau Basin of the South Pacific Ocean

(European Geosciences Union, 2021-10-06) Cohen, Natalie R. ; Noble, Abigail E. ; Moran, Dawn M. ; McIlvin, Matthew R. ; Goepfert, Tyler J. ; Hawco, Nicholas J. ; German, Christopher R. ; Horner, Tristan J. ; Lamborg, Carl H. ; McCrow, John P. ; Allen, Andrew E. ; Saito, Mak A.

Bioactive trace metals are critical micronutrients for marine microorganisms due to their role in mediating biological redox reactions, and complex biogeochemical processes control their distributions. Hydrothermal vents may represent an important source of metals to microorganisms, especially those inhabiting low-iron waters, such as in the southwest Pacific Ocean. Previous measurements of primordial 3He indicate a significant hydrothermal source originating in the northeastern (NE) Lau Basin, with the plume advecting into the southwest Pacific Ocean at 1500–2000 m depth (Lupton et al., 2004). Studies investigating the long-range transport of trace metals associated with such dispersing plumes are rare, and the biogeochemical impacts on local microbial physiology have not yet been described. Here we quantified dissolved metals and assessed microbial metaproteomes across a transect spanning the tropical and equatorial Pacific with a focus on the hydrothermally active NE Lau Basin and report elevated iron and manganese concentrations across 441 km of the southwest Pacific. The most intense signal was detected near the Mangatolo Triple Junction (MTJ) and Northeast Lau Spreading Center (NELSC), in close proximity to the previously reported 3He signature. Protein content in distal-plume-influenced seawater, which was high in metals, was overall similar to background locations, though key prokaryotic proteins involved in metal and organic uptake, protein degradation, and chemoautotrophy were abundant compared to deep waters outside of the distal plume. Our results demonstrate that trace metals derived from the NE Lau Basin are transported over appreciable distances into the southwest Pacific Ocean and that bioactive chemical resources released from submarine vent systems are utilized by surrounding deep-sea microbes, influencing both their physiology and their contributions to ocean biogeochemical cycling.
Metabolic versatility of the nitrite-oxidizing bacterium Nitrospira marina and its proteomic response to oxygen-limited conditions

(Springer Nature, 2020-11-23) Bayer, Barbara ; Saito, Mak A. ; McIlvin, Matthew R. ; Lücker, Sebastian ; Moran, Dawn M. ; Lankiewicz, Thomas S. ; Dupont, Christopher L. ; Santoro, Alyson E.

The genus Nitrospira is the most widespread group of nitrite-oxidizing bacteria and thrives in diverse natural and engineered ecosystems. Nitrospira marina Nb-295T was isolated from the ocean over 30 years ago; however, its genome has not yet been analyzed. Here, we investigated the metabolic potential of N. marina based on its complete genome sequence and performed physiological experiments to test genome-derived hypotheses. Our data confirm that N. marina benefits from additions of undefined organic carbon substrates, has adaptations to resist oxidative, osmotic, and UV light-induced stress and low dissolved pCO2, and requires exogenous vitamin B12. In addition, N. marina is able to grow chemoorganotrophically on formate, and is thus not an obligate chemolithoautotroph. We further investigated the proteomic response of N. marina to low (∼5.6 µM) O2 concentrations. The abundance of a potentially more efficient CO2-fixing pyruvate:ferredoxin oxidoreductase (POR) complex and a high-affinity cbb3-type terminal oxidase increased under O2 limitation, suggesting a role in sustaining nitrite oxidation-driven autotrophy. This putatively more O2-sensitive POR complex might be protected from oxidative damage by Cu/Zn-binding superoxide dismutase, which also increased in abundance under low O2 conditions. Furthermore, the upregulation of proteins involved in alternative energy metabolisms, including Group 3b [NiFe] hydrogenase and formate dehydrogenase, indicate a high metabolic versatility to survive conditions unfavorable for aerobic nitrite oxidation. In summary, the genome and proteome of the first marine Nitrospira isolate identifies adaptations to life in the oxic ocean and provides insights into the metabolic diversity and niche differentiation of NOB in marine environments.
Co-occurrence of fe and P stress in natural populations of the marine diazotroph Trichodesmium

(European Geosciences Union, 2020-05-12) Held, Noelle A. ; Webb, Eric A. ; McIlvin, Matthew R. ; Hutchins, David A. ; Cohen, Natalie R. ; Moran, Dawn M. ; Kunde, Korinna ; Lohan, Maeve C. ; Mahaffey, Claire ; Woodward, E. Malcolm S. ; Saito, Mak A.

Trichodesmium is a globally important marine microbe that provides fixed nitrogen (N) to otherwise N-limited ecosystems. In nature, nitrogen fixation is likely regulated by iron or phosphate availability, but the extent and interaction of these controls are unclear. From metaproteomics analyses using established protein biomarkers for nutrient stress, we found that iron–phosphate co-stress is the norm rather than the exception for Trichodesmium colonies in the North Atlantic Ocean. Counterintuitively, the nitrogenase enzyme was more abundant under co-stress as opposed to single nutrient stress. This is consistent with the idea that Trichodesmium has a specific physiological state during nutrient co-stress. Organic nitrogen uptake was observed and occurred simultaneously with nitrogen fixation. The quantification of the phosphate ABC transporter PstA combined with a cellular model of nutrient uptake suggested that Trichodesmium is generally confronted by the biophysical limits of membrane space and diffusion rates for iron and phosphate acquisition in the field. Colony formation may benefit nutrient acquisition from particulate and organic sources, alleviating these pressures. The results highlight that to predict the behavior of Trichodesmium, both Fe and P stress must be evaluated simultaneously.
Colony formation in Phaeocystis antarctica : connecting molecular mechanisms with iron biogeochemistry

(Copernicus Publications on behalf of the European Geosciences Union, 2018-08-21) Bender, Sara J. ; Moran, Dawn M. ; McIlvin, Matthew R. ; Zheng, Hong ; McCrow, John P. ; Badger, Jonathan ; DiTullio, Giacomo R. ; Allen, Andrew E. ; Saito, Mak A.

Phaeocystis antarctica is an important phytoplankter of the Ross Sea where it dominates the early season bloom after sea ice retreat and is a major contributor to carbon export. The factors that influence Phaeocystis colony formation and the resultant Ross Sea bloom initiation have been of great scientific interest, yet there is little known about the underlying mechanisms responsible for these phenomena. Here, we present laboratory and field studies on Phaeocystis antarctica grown under multiple iron conditions using a coupled proteomic and transcriptomic approach. P. antarctica had a lower iron limitation threshold than a Ross Sea diatom Chaetoceros sp., and at increased iron nutrition (>120pM Fe') a shift from flagellate cells to a majority of colonial cells in P. antarctica was observed, implying a role for iron as a trigger for colony formation. Proteome analysis revealed an extensive and coordinated shift in proteome structure linked to iron availability and life cycle transitions with 327 and 436 proteins measured as significantly different between low and high iron in strains 1871 and 1374, respectively. The enzymes flavodoxin and plastocyanin that can functionally replace iron metalloenzymes were observed at low iron treatments consistent with cellular iron-sparing strategies, with plastocyanin having a larger dynamic range. The numerous isoforms of the putative iron-starvation-induced protein (ISIP) group (ISIP2A and ISIP3) had abundance patterns coinciding with that of either low or high iron (and coincident flagellate or the colonial cell types in strain 1871), implying that there may be specific iron acquisition systems for each life cycle type. The proteome analysis also revealed numerous structural proteins associated with each cell type: within flagellate cells actin and tubulin from flagella and haptonema structures as well as a suite of calcium-binding proteins with EF domains were observed. In the colony-dominated samples a variety of structural proteins were observed that are also often found in multicellular organisms including spondins, lectins, fibrillins, and glycoproteins with von Willebrand domains. A large number of proteins of unknown function were identified that became abundant at either high or low iron availability. These results were compared to the first metaproteomic analysis of a Ross Sea Phaeocystis bloom to connect the mechanistic information to the in situ ecology and biogeochemistry. Proteins associated with both flagellate and colonial cells were observed in the bloom sample consistent with the need for both cell types within a growing bloom. Bacterial iron storage and B12 biosynthesis proteins were also observed consistent with chemical synergies within the colony microbiome to cope with the biogeochemical conditions. Together these responses reveal a complex, highly coordinated effort by P. antarctica to regulate its phenotype at the molecular level in response to iron and provide a window into the biology, ecology, and biogeochemistry of this group.
Unique patterns and biogeochemical relevance of two-component sensing in marine bacteria.

(American Society for Microbiology Journals, 2019-01-11) Held, Noelle A. ; McIlvin, Matthew R. ; Moran, Dawn M. ; Laub, Michael T. ; Saito, Mak A.

Two-component sensory (TCS) systems link microbial physiology to the environment and thus may play key roles in biogeochemical cycles. In this study, we surveyed the TCS systems of 328 diverse marine bacterial species. We identified lifestyle traits such as copiotrophy and diazotrophy that are associated with larger numbers of TCS system genes within the genome. We compared marine bacterial species with 1,152 reference bacterial species from a variety of habitats and found evidence of extra response regulators in marine genomes. Examining the location of TCS genes along the circular bacterial genome, we also found that marine bacteria have a large number of “orphan” genes, as well as many hybrid histidine kinases. The prevalence of “extra” response regulators, orphan genes, and hybrid TCS systems suggests that marine bacteria break with traditional understanding of how TCS systems operate. These trends suggest prevalent regulatory networking, which may allow coordinated physiological responses to multiple environmental signals and may represent a specific adaptation to the marine environment. We examine phylogenetic and lifestyle traits that influence the number and structure of two-component systems in the genome, finding, for example, that a lack of two-component systems is a hallmark of oligotrophy. Finally, in an effort to demonstrate the importance of TCS systems to marine biogeochemistry, we examined the distribution of Prochlorococcus/Synechococcus response regulator PMT9312_0717 in metaproteomes of the tropical South Pacific. We found that this protein’s abundance is related to phosphate concentrations, consistent with a putative role in phosphate regulation.
Low cobalt inventories in the Amundsen and Ross seas driven by high demand for labile cobalt uptake among native phytoplankton communities

(European Geosciences Union, 2023-10-04) Chmiel, Rebecca J. ; Kell, Riss M. ; Rao, Deepa ; Moran, Dawn M. ; DiTullio, Giacomo R. ; Saito, Mak A.

Cobalt (Co) is a scarce but essential micronutrient for marine plankton in the Southern Ocean and coastal Antarctic seas, where dissolved cobalt (dCo) concentrations can be extremely low. This study presents total dCo and labile dCo distributions measured via shipboard voltammetry in the Amundsen Sea, the Ross Sea and Terra Nova Bay during the CICLOPS (Cobalamin and Iron Co-Limitation of Phytoplankton Species) expedition. A significantly smaller dCo inventory was observed during the 2017/2018 CICLOPS expedition compared to two 2005/2006 expeditions to the Ross Sea conducted over a decade earlier. The dCo inventory loss (∼ 10–20 pM) was present in both the surface and deep ocean and was attributed to the loss of labile dCo, resulting in the near-complete complexation of dCo by strong ligands in the photic zone. A changing dCo inventory in Antarctic coastal seas could be driven by the alleviation of iron (Fe) limitation in coastal areas, where the flux of Fe-rich sediments from melting ice shelves and deep sediment resuspension may have shifted the region towards vitamin B12 and/or zinc (Zn) limitation, both of which are likely to increase the demand for Co among marine plankton. High demand for Zn by phytoplankton can result in increased Co and cadmium (Cd) uptake because these metals often share the same metal uptake transporters. This study compared the magnitudes and ratios of Zn, Cd and Co uptake (ρ) across upper-ocean profiles and the observed order-of-magnitude uptake trends (ρZn > ρCd > ρCo) that paralleled the trace metal concentrations in seawater. High rates of Co and Zn uptake were observed throughout the region, and the speciation of available Co and Zn appeared to influence trends in dissolved metal : phosphate stoichiometry and uptake rates over depth. Multi-year loss of the dCo inventory throughout the water column may be explained by an increase in Co uptake into particulate organic matter and subsequently an increased flux of Co into sediments via sinking and burial. This perturbation of the Southern Ocean Co biogeochemical cycle could signal changes in the nutrient limitation regimes, phytoplankton bloom composition and carbon sequestration sink of the Southern Ocean.
Persistent equatorial Pacific iron limitation under ENSO forcing

(Nature Research, 2023-08-16) Browning, Thomas J. ; Saito, Mak A. ; Garaba, Shungudzemwoyo P. ; Wang, Xuechao ; Achterberg, Eric P. ; Moore, C. Mark ; Engel, Anja ; Mcllvin, Matthew R. ; Moran, Dawn ; Voss, Daniela ; Zielinski, Oliver ; Tagliabue, Alessandro

Projected responses of ocean net primary productivity to climate change are highly uncertain1. Models suggest that the climate sensitivity of phytoplankton nutrient limitation in the low-latitude Pacific Ocean plays a crucial role1,2,3, but this is poorly constrained by observations4. Here we show that changes in physical forcing drove coherent fluctuations in the strength of equatorial Pacific iron limitation through multiple El Niño/Southern Oscillation (ENSO) cycles, but that this was overestimated twofold by a state-of-the-art climate model. Our assessment was enabled by first using a combination of field nutrient-addition experiments, proteomics and above-water hyperspectral radiometry to show that phytoplankton physiological responses to iron limitation led to approximately threefold changes in chlorophyll-normalized phytoplankton fluorescence. We then exploited the >18-year satellite fluorescence record to quantify climate-induced nutrient limitation variability. Such synoptic constraints provide a powerful approach for benchmarking the realism of model projections of net primary productivity to climate changes.

Moran Dawn M.

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