Characterization of the Fe metalloproteome of a ubiquitous marine heterotroph, Pseudoalteromonas (BB2-AT2): multiple bacterioferritin copies enable significant Fe storage

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Mazzotta, M. G., McIlvin, M. R., & Saito, M. A. Characterization of the Fe metalloproteome of a ubiquitous marine heterotroph, Pseudoalteromonas (BB2-AT2): multiple bacterioferritin copies enable significant Fe storage. Metallomics, (2020), doi:10.1039/d0mt00034e.Fe is a critical nutrient to the marine biological pump, which is the process that exports photosynthetically fixed carbon in the upper ocean to the deep ocean. Fe limitation controls photosynthetic activity in major regions of the oceans, and the subsequent degradation of exported photosynthetic material is facilitated particularly by marine heterotrophic bacteria. Despite their importance in the carbon cycle and the scarcity of Fe in seawater, the Fe requirements, storage and cytosolic utilization of these marine heterotrophs has been less studied. Here, we characterized the Fe metallome of Pseudoalteromonas (BB2-AT2). We found that with two copies of bacterioferritin (Bfr), Pseudoalteromonas possesses substantial capacity for luxury uptake of Fe. Fe : C in the whole cell metallome was estimated (assuming C : P stoichiometry ∼51 : 1) to be between ∼83 μmol : mol Fe : C, ∼11 fold higher than prior marine bacteria surveys. Under these replete conditions, other major cytosolic Fe-associated proteins were observed including superoxide dismutase (SodA; with other metal SOD isoforms absent under Fe replete conditions) and catalase (KatG) involved in reactive oxygen stress mitigation and aconitase (AcnB), succinate dehydrogenase (FrdB) and cytochromes (QcrA and Cyt1) involved in respiration. With the aid of singular value decomposition (SVD), we were able to computationally attribute peaks within the metallome to specific metalloprotein contributors. A putative Fe complex TonB transporter associated with the closely related Alteromonas bacterium was found to be abundant within the Pacific Ocean mesopelagic environment. Despite the extreme scarcity of Fe in seawater, the marine heterotroph Pseudoalteromonas has expansive Fe storage capacity and utilization strategies, implying that within detritus and sinking particles environments, there is significant opportunity for Fe acquisition. Together these results imply an evolved dedication of marine Pseudoalteromonas to maintaining an Fe metalloproteome, likely due to its dependence on Fe-based respiratory metabolism.M. G. M. was supported by the Camille and Henry Dreyfus Foundation Environmental Chemistry Postdoctoral Fellowship. We thank Kay Bidle (Rutgers University) for providing a culture of Pseudoalteromonas (BB2-AT2). We also thank Dawn Moran (WHOI) and Noelle Held (WHOI-MIT) for culturing assistance. We appreciate the Captain and Crew of the R/V Kilo Moana, and the many involved in the METZYME expedition sampling efforts. Discussions with Kevin Waldron (Newcastle University), Alison Butler (University of California, Santa Barbara), Lauren Manck (Scripps Institution of Oceanography), Randie Bundy (University of Washington) and Jake Gebbie (WHOI) were much appreciated. Funding for this research was provided by the Gordon and Betty Moore Foundation (3782), and NSF-OCE 1658030, 1736599, 1657766, 1924554, 1850719, 1924554

Oxygen isotopic composition of nitrate and nitrite produced by nitrifying cocultures and natural marine assemblages

Author Posting. © Association for the Sciences of Limnology and Oceanography, 2012. 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 57 (2012): 1361-1375, doi:10.4319/lo.2012.57.5.1361.The δ18O value of nitrate produced during nitrification (δ18ONO3,nit) was measured in experiments designed to mimic oceanic conditions, involving cocultures of ammonia-oxidizing bacteria or ammonia-oxidizing archaea and nitrite-oxidizing bacteria, as well as natural marine assemblages. The estimates of ranged from −1.5‰ ± 0.1‰ to +1.3‰ ± 1.4‰ at δ18O values of water (H2O) and dissolved oxygen (O2) of 0‰ and 24.2‰ vs. Vienna Standard Mean Ocean Water, respectively. Additions of 18O-enriched H2O allowed us to evaluate the effects of oxygen (O) isotope fractionation and exchange on . Kinetic isotope effects for the incorporation of O atoms were the most important factors for setting overall values relative to the substrates (O2 and H2O). These isotope effects ranged from +10‰ to +22‰ for ammonia oxidation (O2 plus H2O incorporation) and from +1‰ to +27‰ for incorporation of H2O during nitrite oxidation. values were also affected by the amount and duration of nitrite accumulation, which permitted abiotic O atom exchange between nitrite and H2O. Coculture incubations where ammonia oxidation and nitrite oxidation were tightly coupled showed low levels of nitrite accumulation and exchange (3% ± 4%). These experiments had values of −1.5‰ to +0.7‰. Field experiments had greater accumulation of nitrite and a higher amount of exchange (22% to 100%), yielding an average value of +1.9‰ ± 3.0‰. Low levels of biologically catalyzed exchange in coculture experiments may be representative of nitrification in much of the ocean where nitrite accumulation is low. Abiotic oxygen isotope exchange may be important where nitrite does accumulate, such as oceanic primary and secondary nitrite maxima.This research was funded by the National Science Foundation Chemical Oceanography grants 05-26277 and 09- 610998 to K.L.C

Unique patterns and biogeochemical relevance of two-component sensing in marine bacteria.

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 License. The definitive version was published in mSystems 4(1), (2019): 4:e00317-18, doi:10.1128/mSystems.00317-18.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.We thank Joe Jennings at Oregon State University and Chris Dupont at the J. Craig Venter Institute for providing nutrient and metagenomic analyses, respectively, for the KM1128 METZYME research expedition. We also thank our anonymous reviewers for their thoughtful comments. This material is based on work supported by a National Science Foundation Graduate Research Fellowship under grant number 1122274 (N. A. Held). It was also supported by the Gordon and Betty Moore Foundation (grant number 3782 [M. Saito]) and by the National Science Foundation (grant numbers OCE-1657766, EarthCube 1639714, OCE-1658030, and OCE-1260233)

Divergent responses of Atlantic coastal and oceanic Synechococcus to iron limitation

Author Posting. © The Author(s), 2015. This is the author's version of the work. It is posted here by permission of National Academy of Sciences for personal use, not for redistribution. The definitive version was published in Proceedings of the National Academy of Sciences of the United States of America 112 (2015): 9944-9949, doi:10.1073/pnas.1509448112.Marine Synechococcus are some of the most diverse and ubiquitous phytoplankton, and iron (Fe) is an essential micronutrient that limits productivity in many parts of the ocean. To investigate how coastal and oceanic Atlantic Synechococcus strains acclimate to Fe availability, we compared the growth, photophysiology, and quantitative proteomics of two Synechococcus strains from different Fe regimes. Synechococcus strain WH8102, from a region in the southern Sargasso Sea that receives substantial dust deposition, showed impaired growth and photophysiology as Fe declined, yet utilized few acclimation responses. Coastal WH8020, from the dynamic, seasonally variable New England shelf, displayed a multi-tiered, hierarchical cascade of acclimation responses with different Fe thresholds. The multi-tiered response included changes in Fe acquisition, storage, and photosynthetic proteins, substitution of flavodoxin for ferredoxin, and modified photophysiology, all while maintaining remarkably stable growth rates over a range of Fe concentrations. Modulation of two distinct ferric uptake regulator (Fur) proteins that coincided with the multi-tiered proteome response was found, implying the coastal strain has different regulatory threshold responses to low Fe availability. Low nitrogen (N) and phosphorus (P) availability in the open ocean may favor the loss of Fe response genes when Fe availability is consistent over time, whereas these genes are retained in dynamic environments where Fe availability fluctuates and N and P are more abundant.This work was supported by a National Science Foundation Postdoctoral Research Fellowship in Biology to K.R.M.M. (NSF 1103575), National Science Foundation Oceanography grants OCE-1220484, OCE-0928414, OCE-1233261, OCE- 1155566, OCE-1131387, and OCE-0926092, as well as Gordon and Betty Moore Foundation grants 3782 and 3934

Element-selective targeting of nutrient metabolites in environmental samples by inductively coupled plasma mass spectrometry and electrospray ionization mass spectrometry

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Li, J., Boiteau, R. M., Babcock-Adams, L., Acker, M., Song, Z., McIlvin, M. R., & Repeta, D. J. Element-selective targeting of nutrient metabolites in environmental samples by inductively coupled plasma mass spectrometry and electrospray ionization mass spectrometry. Frontiers in Marine Science, 8, (2021): 630494, https://doi.org/10.3389/fmars.2021.630494.Metabolites that incorporate elements other than carbon, nitrogen, hydrogen and oxygen can be selectively detected by inductively coupled mass spectrometry (ICPMS). When used in parallel with chromatographic separations and conventional electrospray ionization mass spectrometry (ESIMS), ICPMS allows the analyst to quickly find, characterize and identify target metabolites that carry nutrient elements (P, S, trace metals; “nutrient metabolites”), which are of particular interest to investigations of microbial biogeochemical cycles. This approach has been applied to the study of siderophores and other trace metal organic ligands in the ocean. The original method used mass search algorithms that relied on the ratio of stable isotopologues of iron, copper and nickel to assign mass spectra collected by ESIMS to metabolites carrying these elements detected by ICPMS. However, while isotopologue-based mass assignment algorithms were highly successful in characterizing metabolites that incorporate some trace metals, they do not realize the whole potential of the ICPMS/ESIMS approach as they cannot be used to assign the molecular ions of metabolites with monoisotopic elements or elements for which the ratio of stable isotopes is not known. Here we report a revised ICPMS/ESIMS method that incorporates a number of changes to the configuration of instrument hardware that improves sensitivity of the method by a factor of 4–5, and allows for more accurate quantitation of metabolites. We also describe a new suite of mass search algorithms that can find and characterize metabolites that carry monoisotopic elements. We used the new method to identify siderophores in a laboratory culture of Vibrio cyclitrophicus and a seawater sample collected in the North Pacific Ocean, and to assign molecular ions to monoisotopic cobalt and iodine nutrient metabolites in extracts of a laboratory culture of the marine cyanobacterium Prochorococcus MIT9215.This work was generously supported by the National Science Foundation grant OCE-1829761 to RB and OCE-1356747 and -1736280 to DR. DR also received generous support from the Simons Foundation Life Sciences Project Award 49476

Siderophore-Based Microbial Adaptations to Iron Scarcity Across the Eastern Pacific Ocean

Nearly all iron dissolved in the ocean is complexed by strong organic ligands of unknown composition. The effect of ligand composition on microbial iron acquisition is poorly understood, but amendment experiments using model ligands show they can facilitate or impede iron uptake depending on their identity. Here we show that siderophores, organic compounds synthesized by microbes to facilitate iron uptake, are a dynamic component of the marine ligand pool in the eastern tropical Pacific Ocean. Siderophore concentrations in iron-deficient waters averaged 9 pM, up to fivefold higher than in iron-rich coastal and nutrient-depleted oligotrophic waters, and were dominated by amphibactins, amphiphilic siderophores with cell membrane affinity. Phylogenetic analysis of amphibactin biosynthetic genes suggests that the ability to produce amphibactins has transferred horizontally across multiple Gammaproteobacteria, potentially driven by pressures to compete for iron. In coastal and oligotrophic regions of the eastern Pacific Ocean, amphibactins were replaced with lower concentrations (1-2 pM) of hydrophilic ferrioxamine siderophores. Our results suggest that organic ligand composition changes across the surface ocean in response to environmental pressures. Hydrophilic siderophores are predominantly found across regions of the ocean where iron is not expected to be the limiting nutrient for the microbial community at large. However, in regions with intense competition for iron, some microbes optimize iron acquisition by producing siderophores that minimize diffusive losses to the environment. These siderophores affect iron bioavailability and thus may be an important component of the marine iron cycle

Divergent Responses of Atlantic Coastal and Oceanic Synechococcus to Iron Limitation

Marine Synechococcus are some of the most diverse and ubiquitous phytoplankton, and iron (Fe) is an essential micronutrient that limits productivity in many parts of the ocean. To investigate how coastal and oceanic Atlantic Synechococcus strains acclimate to Fe availability, we compared the growth, photophysiology, and quantitative proteomics of two Synechococcus strains from different Fe regimes. Synechococcus strain WH8102, from a region in the southern Sargasso Sea that receives substantial dust deposition, showed impaired growth and photophysiology as Fe declined, yet used few acclimation responses. Coastal WH8020, from the dynamic, seasonally variable New England shelf, displayed a multitiered, hierarchical cascade of acclimation responses with different Fe thresholds. The multitiered response included changes in Fe acquisition, storage, and photosynthetic proteins, substitution of flavodoxin for ferredoxin, and modified photophysiology, all while maintaining remarkably stable growth rates over a range of Fe concentrations. Modulation of two distinct ferric uptake regulator (Fur) proteins that coincided with the multitiered proteome response was found, implying the coastal strain has different regulatory threshold responses to low Fe availability. Low nitrogen (N) and phosphorus (P) availability in the open ocean may favor the loss of Fe response genes when Fe availability is consistent over time, whereas these genes are retained in dynamic environments where Fe availability fluctuates and N and P are more abundant

Methionine synthase interreplacement in diatom cultures and communities : implications for the persistence of B12 use by eukaryotic phytoplankton

Author Posting. © Association for the Sciences of Limnology and Oceanography, 2013. 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 58 (2013): 1431–1450, doi:10.4319/lo.2013.58.4.1431.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.This work was supported by National Science Foundation (NSF) Antarctic Sciences awards 0732665, 1103503, and 0732822; NSF Division of Ocean Science awards 0752291, 0928414, and 1031271; The Gordon and Betty Moore Foundation; Center for Microbial Oceanography Research and Education; an NSF Graduate Research Fellowship (2007037200); and an Environmental Protection Agency Science To Achieve Results (EPA-STAR) Fellowship to E.M.B. (F6E720324)

NADPH-dependent extracellular superoxide production is vital to photophysiology in the marine diatom Thalassiosira oceanica

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Diaz, J. M., Plummer, S., Hansel, C. M., Andeer, P. F., Saito, M. A., & McIlvin, M. R. NADPH-dependent extracellular superoxide production is vital to photophysiology in the marine diatom Thalassiosira oceanica. Proceedings of the National Academy of Sciences of the United States of America, 116 (33), (2019): 16448-16453, doi: 10.1073/pnas.1821233116.Reactive oxygen species (ROS) like superoxide drive rapid transformations of carbon and metals in aquatic systems and play dynamic roles in biological health, signaling, and defense across a diversity of cell types. In phytoplankton, however, the ecophysiological role(s) of extracellular superoxide production has remained elusive. Here, the mechanism and function of extracellular superoxide production by the marine diatom Thalassiosira oceanica are described. Extracellular superoxide production in T. oceanica exudates was coupled to the oxidation of NADPH. A putative NADPH-oxidizing flavoenzyme with predicted transmembrane domains and high sequence similarity to glutathione reductase (GR) was implicated in this process. GR was also linked to extracellular superoxide production by whole cells via quenching by the flavoenzyme inhibitor diphenylene iodonium (DPI) and oxidized glutathione, the preferred electron acceptor of GR. Extracellular superoxide production followed a typical photosynthesis-irradiance curve and increased by 30% above the saturation irradiance of photosynthesis, while DPI significantly impaired the efficiency of photosystem II under a wide range of light levels. Together, these results suggest that extracellular superoxide production is a byproduct of a transplasma membrane electron transport system that serves to balance the cellular redox state through the recycling of photosynthetic NADPH. This photoprotective function may be widespread, consistent with the presence of putative homologs to T. oceanica GR in other representative marine phytoplankton and ocean metagenomes. Given predicted climate-driven shifts in global surface ocean light regimes and phytoplankton community-level photoacclimation, these results provide implications for future ocean redox balance, ecological functioning, and coupled biogeochemical transformations of carbon and metals.This work was supported by a postdoctoral fellowship from the Ford Foundation (to J.M.D.), the National Science Foundation (NSF) under grants OCE 1225801 (to J.M.D.) and OCE 1246174 (to C.M.H.), a Junior Faculty Seed Grant from the University of Georgia Research Foundation (to J.M.D.), and a National Science Foundation Graduate Research Fellowship (to S.P.). The FIRe was purchased through a NSF equipment improvement grant (1624593).The authors thank Melissa Soule for assistance with LC/MS/MS analysis of peptide samples