Brook L. Nunn

Comparative metaproteomics reveals ocean-scale shifts in microbial nutrient utilization and energy transduction

Bacteria and Archaea play critical roles in marine energy fluxes and nutrient cycles by incorporating and redistributing dissolved organic matter and inorganic nutrients in the oceans. How these microorganisms do this work at the level of the expressed protein is known only from a few studies of targeted lineages. We used comparative membrane metaproteomics to identify functional responses of communities to different nutrient concentrations on an oceanic scale. Comparative analyses of microbial membrane fractions revealed shifts in nutrient utilization and energy transduction along an environmental gradient in South Atlantic surface waters, from a low-nutrient gyre to a highly productive coastal upwelling region. The dominant membrane proteins identified (19%) were TonB-dependent transporters (TBDTs), which are known to utilize a proton motive force to transport nutrients across the outer membrane of Gram-negative bacteria. The ocean-wide importance of TonB-dependent nutrient acquisition in marine bacteria was unsuspected. Diverse light-harvesting rhodopsins were detected in membrane proteomes from every sample. Proteomic evidence of both TBDTs and rhodopsins in the same lineages suggest that phototrophic bacterioplankton have the potential to use energy from light to fuel transport activities. We also identified viral proteins in every sample and archaeal ammonia monooxygenase proteins in the upwelling region, suggesting that Archaea are important nitrifiers in nutrient-rich surface waters.

Metabolomics and proteomics reveal impacts of chemically mediated competition on marine plankton

Significance Microscopic marine algae (phytoplankton) are responsible for much of Earths photosynthesis, serving as the base of a massive food web supporting fisheries. Phytoplankton compete for limiting resources, with some species producing noxious compounds that kill competitors or inhibit their growth. The red-tide dinoflagellate Karenia brevis is one such allelopathic species, causing growth suppression of other phytoplankton and negatively impacting coastal ecosystems. Metabolomic and proteomic approaches were used to characterize the sublethal physiological impacts of K. brevis allelopathy on two competing phytoplankton, providing insights into the physiological mechanisms by which allelopathy occurs and the metabolic pathways that enable resistance in co-occurring competitors. Competition is a major force structuring marine planktonic communities. The release of compounds that inhibit competitors, a process known as allelopathy, may play a role in the maintenance of large blooms of the red-tide dinoflagellate Karenia brevis, which produces potent neurotoxins that negatively impact coastal marine ecosystems. K. brevis is variably allelopathic to multiple competitors, typically causing sublethal suppression of growth. We used metabolomic and proteomic analyses to investigate the role of chemically mediated ecological interactions between K. brevis and two diatom competitors, Asterionellopsis glacialis and Thalassiosira pseudonana. The impact of K. brevis allelopathy on competitor physiology was reflected in the metabolomes and expressed proteomes of both diatoms, although the diatom that co-occurs with K. brevis blooms (A. glacialis) exhibited more robust metabolism in response to K. brevis. The observed partial resistance of A. glacialis to allelopathy may be a result of its frequent exposure to K. brevis blooms in the Gulf of Mexico. For the more sensitive diatom, T. pseudonana, which may not have had opportunity to evolve resistance to K. brevis, allelopathy disrupted energy metabolism and impeded cellular protection mechanisms including altered cell membrane components, inhibited osmoregulation, and increased oxidative stress. Allelopathic compounds appear to target multiple physiological pathways in sensitive competitors, demonstrating that chemical cues in the plankton have the potential to alter large-scale ecosystem processes including primary production and nutrient cycling.

Diatom proteomics reveals unique acclimation strategies to mitigate Fe limitation.

Phytoplankton growth rates are limited by the supply of iron (Fe) in approximately one third of the open ocean, with major implications for carbon dioxide sequestration and carbon (C) biogeochemistry. To date, understanding how alteration of Fe supply changes phytoplankton physiology has focused on traditional metrics such as growth rate, elemental composition, and biophysical measurements such as photosynthetic competence (Fv/Fm). Researchers have subsequently employed transcriptomics to probe relationships between changes in Fe supply and phytoplankton physiology. Recently, studies have investigated longer-term (i.e. following acclimation) responses of phytoplankton to various Fe conditions. In the present study, the coastal diatom, Thalassiosira pseudonana, was acclimated (10 generations) to either low or high Fe conditions, i.e. Fe-limiting and Fe-replete. Quantitative proteomics and a newly developed proteomic profiling technique that identifies low abundance proteins were employed to examine the full complement of expressed proteins and consequently the metabolic pathways utilized by the diatom under the two Fe conditions. A total of 1850 proteins were confidently identified, nearly tripling previous identifications made from differential expression in diatoms. Given sufficient time to acclimate to Fe limitation, T. pseudonana up-regulates proteins involved in pathways associated with intracellular protein recycling, thereby decreasing dependence on extracellular nitrogen (N), C and Fe. The relative increase in the abundance of photorespiration and pentose phosphate pathway proteins reveal novel metabolic shifts, which create substrates that could support other well-established physiological responses, such as heavily silicified frustules observed for Fe-limited diatoms. Here, we discovered that proteins and hence pathways observed to be down-regulated in short-term Fe starvation studies are constitutively expressed when T. pseudonana is acclimated (i.e., nitrate and nitrite transporters, Photosystem II and Photosystem I complexes). Acclimation of the diatom to the desired Fe conditions and the comprehensive proteomic approach provides a more robust interpretation of this dynamic proteome than previous studies.

Shotgun proteomics reveals physiological response to ocean acidification in Crassostrea gigas.

BackgroundOcean acidification as a result of increased anthropogenic CO2 emissions is occurring in marine and estuarine environments worldwide. The coastal ocean experiences additional daily and seasonal fluctuations in pH that can be lower than projected end-of-century open ocean pH reductions. In order to assess the impact of ocean acidification on marine invertebrates, Pacific oysters (Crassostrea gigas) were exposed to one of four different pCO2 levels for four weeks: 400 μatm (pH 8.0), 800 μatm (pH 7.7), 1000 μatm (pH 7.6), or 2800 μatm (pH 7.3).ResultsAt the end of the four week exposure period, oysters in all four pCO2 environments deposited new shell, but growth rate was not different among the treatments. However, micromechanical properties of the new shell were compromised by elevated pCO2. Elevated pCO2 affected neither whole body fatty acid composition, nor glycogen content, nor mortality rate associated with acute heat shock. Shotgun proteomics revealed that several physiological pathways were significantly affected by ocean acidification, including antioxidant response, carbohydrate metabolism, and transcription and translation. Additionally, the proteomic response to a second stress differed with pCO2, with numerous processes significantly affected by mechanical stimulation at high versus low pCO2 (all proteomics data are available in the ProteomeXchange under the identifier PXD000835).ConclusionsOyster physiology is significantly altered by exposure to elevated pCO2, indicating changes in energy resource use. This is especially apparent in the assessment of the effects of pCO2 on the proteomic response to a second stress. The altered stress response illustrates that ocean acidification may impact how oysters respond to other changes in their environment. These data contribute to an integrative view of the effects of ocean acidification on oysters as well as physiological trade-offs during environmental stress.

Sulfur oxidizers dominate carbon fixation at a biogeochemical hot spot in the dark ocean

Bacteria and archaea in the dark ocean (>200 m) comprise 0.3–1.3 billion tons of actively cycled marine carbon. Many of these microorganisms have the genetic potential to fix inorganic carbon (autotrophs) or assimilate single-carbon compounds (methylotrophs). We identified the functions of autotrophic and methylotrophic microorganisms in a vent plume at Axial Seamount, where hydrothermal activity provides a biogeochemical hot spot for carbon fixation in the dark ocean. Free-living members of the SUP05/Arctic96BD-19 clade of marine gamma-proteobacterial sulfur oxidizers (GSOs) are distributed throughout the northeastern Pacific Ocean and dominated hydrothermal plume waters at Axial Seamount. Marine GSOs expressed proteins for sulfur oxidation (adenosine phosphosulfate reductase, sox (sulfur oxidizing system), dissimilatory sulfite reductase and ATP sulfurylase), carbon fixation (ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO)), aerobic respiration (cytochrome c oxidase) and nitrogen regulation (PII). Methylotrophs and iron oxidizers were also active in plume waters and expressed key proteins for methane oxidation and inorganic carbon fixation (particulate methane monooxygenase/methanol dehydrogenase and RuBisCO, respectively). Proteomic data suggest that free-living sulfur oxidizers and methylotrophs are among the dominant primary producers in vent plume waters in the northeastern Pacific Ocean.

Tandem Mass Spectrometry Investigation of ADP-ribosylated Kemptide

Bacterial adenosine diphosphate-ribosyltransferases (ADPRTs) are toxins that play a significant role in pathogenicity by inactivating host proteins through covalent addition of ADP-ribose. In this study we used ADP-ribosylated Kemptide (LRRASLG) as a standard to examine the effectiveness of three common tandem mass spectrometry fragmentation methods for assignment of amino acid sequence and site of modification. Fragmentation mechanisms investigated include low-energy collision-induced dissociation (CID), infrared multiphoton dissociation (IRMPD), and electron-capture dissociation (ECD); all were performed on a hybrid linear ion trap Fourier transform ion cyclotron resonance mass spectrometer. We show that ECD, but neither CID nor IRMPD, of ADP-ribosylated Kemptide produces tandem mass spectra that are interpretable with regard to amino acid sequence assignment and site of modification. Examination of CID and IRMPD tandem mass spectra of ADP-ribosylated Kemptide revealed that fragmentation was primarily focused to the ADP-ribose region, generating several potential diagnostic ions for use in discovery of ADP-ribosylated proteins. Because of the lower relative sensitivity of ECD during data-dependent acquisition to CID, we suggest a 2-fold strategy where CID and IRMPD are first used to detect ADP-ribosylated peptides, followed by sequence assignment and location of modification by ECD analysis.

Hydrolysis patterns and the production of peptide intermediates during protein degradation in marine systems

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) was used to evaluate the degradation of the protein bovine serum albumin (BSA) added to seawater. The production of peptides during degradation, the size of the peptides produced and the within-protein locations of protease attack were all monitored in an effort to evaluate whether specific types of proteases or specific peptide bond locations were targeted during BSA degradation. Analysis of products from the bacterial degradation of proteins in both seawater and sediment pore water revealed the creation and release of peptide intermediates into the medium. Peptides observed in seawater degradation experiments were all less than 40 amino acids long, whereas sedimentary digestions of the same protein generated peptide fragments in the pore water ranging from 110 to 508 amino acids in length. Neither of the environments demonstrated recurring cleavages adjacent to one amino acid or functional group that might be indicative of the action of a single protease. Instead, it appears that a mixture of bacterial proteases were involved in the degradation of protein. The MALDI-TOF-MS method presented, in combination with these results, can help us delineate some of the processes responsible in the initial stages of degradation and better understand how proteins are partitioned between dissolved and solid phases in marine systems.

Acquisition of Iron by Alkaliphilic Bacillus Species

ABSTRACT The biochemical and molecular mechanisms used by alkaliphilic bacteria to acquire iron are unknown. We demonstrate that alkaliphilic (pH > 9) Bacillus species are sensitive to artificial iron (Fe3+) chelators and produce iron-chelating molecules. These alkaliphilic siderophores contain catechol and hydroxamate moieties, and their synthesis is stimulated by manganese(II) salts and suppressed by FeCl3 addition. Purification and mass spectrometric characterization of the siderophore produced by Caldalkalibacillus thermarum failed to identify any matches to previously observed fragmentation spectra of known siderophores, suggesting a novel structure.

Proteomics of Colwellia psychrerythraea at subzero temperatures - a life with limited movement, flexible membranes and vital DNA repair.

The mechanisms that allow psychrophilic bacteria to remain metabolically active at subzero temperatures result from form and function of their proteins. We present first proteomic evidence of physiological changes of the marine psychrophile Colwellia psychrerythraea 34H (Cp34H) after exposure to subzero temperatures (-1, and -10°C in ice) through 8 weeks. Protein abundance was compared between different treatments to understand the effects of temperature and time, independently and jointly, within cells transitioning to, and being maintained in ice. Parallel [3H]-leucine and [3H]-thymidine incubations indicated active protein and DNA synthesis to -10°C. Mass spectrometry-based proteomics identified 1763 proteins across four experimental treatments. Proteins involved in osmolyte regulation and polymer secretion were found constitutively present across all treatments, suggesting that they are required for metabolic success below 0°C. Differentially abundant protein groups indicated a reallocation of resources from DNA binding to DNA repair and from motility to chemo-taxis and sensing. Changes to iron and nitrogen metabolism, cellular membrane structures, and protein synthesis and folding were also revealed. By elucidating vital strategies during life in ice, this study provides novel insight into the extensive molecular adaptations that occur in cold-adapted marine organisms to sustain cellular function in their habitat.

Critical decisions in metaproteomics: achieving high confidence protein annotations in a sea of unknowns

Critical decisions in metaproteomics: achieving high confidence protein annotations in a sea of unknowns