Protein regulation in Trichodesmium and other marine bacteria: observational and interpretive biomarkers of biogeochemical processes

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemical Oceanography and Microbial Biogeochemistry at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution February 2020.Marine microbes play key roles in global biogeochemistry by mediating chemical transformations and linking nutrient cycles to one another. A major goal in oceanography is to predict the activity of marine microbes across disparate ocean ecosystems. Towards this end, molecular biomarkers are important tools in chemical oceanography because they allow for both the observation and interpretation of microbial behavior. In this thesis, I use molecular biomarkers to develop a holistic, systems biology approach to the study of marine microbes. I begin by identifying unique patterns in the biochemical sensory systems of marine bacteria and suggest that these represent a specific adaptation to the marine environment. Building from this, I focus on the prevalent marine nitrogen fixer Trichodesmium, whose activity affects global nitrogen, carbon, phosphorus, and trace metal cycles. A metaproteomic survey of Trichodesmium populations identified simultaneous iron and phosphate co-stress throughout the tropical and subtropical oceans, demonstrating that this is caused by the biophysical limits of membrane space and nutrient diffusion. Tackling the problem at a smaller scale, I investigated the metaproteomes of individual Trichodesmium colonies captured from a single field site, and identified significant variability related to iron acquisition from mineral particles. Next, I investigated diel proteomes of cultured Trichodesmium erythraeum sp. IMS101 to highlight its physiological complexity and understand how and why nitrogen fixation occurs in the day, despite the incompatibly of the nitrogenase enzyme with oxygen produced in photosynthesis. This thesis develops a fundamental understanding of how Trichodesmium and other organisms affect, and are affected by, their surroundings. It indicates that a reductionist approach in which environmental drivers are considered independently may not capture the full complexity of microbechemistry interactions. Future work can focus on benchmarking and calibration of the protein biomarkers identified here, as well as continued connection of systems biology frameworks to the study of ocean chemistry.This work was supported by an MIT Walter A. Rosenblith Presidential Fellowship and a National Science Foundation Graduate Research Program Fellowship under grant number 1122274 [N.Held]. This work was also supported by the WHOI Ocean Ventures fund [N.Held], Gordon and Betty Moore Foundation grant number 3782 [M.Saito], National Science Foundation grant numbers OCE-1657766 [M.Saito], EarthCube-1639714 [M.Saito], OCE-1658030 [M.Saito], and OCE-1260233 [M.Saito], and funding from the UK Natural Environment Research Council (NERC) under grants awarded to C.M. (NE/N001079/1) and M.L. (NE/N001125/1). This thesis was completed during a writing residency at the Turkeyland Cove Foundation

Metaproteomes of Trichodesmium from samples collected in North Atlantic surface waters, station BATS, and station ALOHA between 2000 and 2018

Dataset: Trichodesmium field metaproteomes - protein spectral counts in alternative formatMetaproteomes of Trichodesmium from samples collected in North Atlantic surface waters, at station BATS (Bermuda Atlantic Time-series Study), and station ALOHA (A Long-Term Oligotrophic Habitat Assessment) between 2000 and 2018. For a complete list of measurements, refer to the full dataset description in the supplemental file 'Dataset_description.pdf'. The most current version of this dataset is available at: https://www.bco-dmo.org/dataset/787078Gordon and Betty Moore Foundation (GBMF) GBMF3934, Gordon and Betty Moore Foundation: Marine Microbiology Initiative (MMI) GBMF3782, NSF Division of Ocean Sciences (NSF OCE) OCE-1657766, NSF Division of Ocean Sciences (NSF OCE) OCE-185071

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)

Co-occurrence of Fe and P stress in natural populations of the marine diazotroph Trichodesmium

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

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

Diel proteomes of cultured Trichodesmium erythraeum sp. IMS101 from laboratory experiments conducted in November of 2018

Dataset: Diel proteomes Trichodesmium IMS101Diel proteomes of cultured Trichodesmium erythraeum sp. IMS101 from laboratory experiments conducted in November of 2018. For a complete list of measurements, refer to the full dataset description in the supplemental file 'Dataset_description.pdf'. The most current version of this dataset is available at: https://www.bco-dmo.org/dataset/783873Gordon and Betty Moore Foundation (GBMF) GBMF3934, Gordon and Betty Moore Foundation: Marine Microbiology Initiative (MMI) GBMF378

Data from: Microbial population dynamics decouple growth response from environmental nutrient concentration

To explore the diversity of microbial growth responses, we have compiled 247 measurements of half-saturation concentrations K and maximum growth rates gmax from previously-published studies (see Methods). The data includes a wide range of resources, including sources of carbon, nitrogen, phosphorus, metals, and vitamins, with phosphate, glucose, and nitrate having the largest number of measurements due to their emphasis in marine and laboratory systems. Organisms include prokaryotes and eukaryotes as well as autotrophs and heterotrophs. This data has been analyzed in a companion research article. For data plots and a discussion of results, please refer to: Fink, Justus Wilhelm, Noelle A. Held, and Michael Manhart. "Microbial Population Dynamics Decouple Growth Response from Environmental Nutrient Concentration." Proceedings of the National Academy of Sciences 120, no. 2 (January 10, 2023). https://doi.org/10.1073/pnas.2207295120.The datafile can be opened with Microsoft Excel or the free open-source alternative Libre Office. To open the dataset in Python, we recommend the package pandas. We give a short example how to load this dataset with pandas in the accompanying file 'README.md'. Funding provided by: Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen ForschungCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100001711Award Number: PZ00P3_180147Funding provided by: Simons FoundationCrossref Funder Registry ID: http://dx.doi.org/10.13039/100000893Award Number: 542379Funding provided by: Eidgenössische Technische Hochschule ZürichCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100003006Award Number: SEED-26 21-2We collected measurements of Monod model parameters (half-saturation concentration K and maximum growth rate gmax) through a targeted literature search that included prior surveys and reviews [1, 2], the phytoplankton trait database [3], as well as original research papers. In all but two cases, we traced data from surveys and reviews back to their original papers, which we report in Dataset S1 (sheet 1). We included only experiments that directly measured population growth rates, rather than nutrient uptake rates or respiration. We excluded measurements where the actual limiting resource was unclear, such as measurements in rich medium with added glucose. Where possible, we checked the raw data of growth rate over resource concentrations to determine if the focal resource concentration was measured up to saturation and had sufficient sampling of concentrations around $K$. For a subset of measurements of E.coli on glucose, we also checked for the concentration of a nitrogen source to determine the relative impact of colimitation (Dataset S1, sheet 2). To assess the effect of pre-acclimation, we focused on a subset of isolates with paired measurements under different regimes of acclimation (Dataset S1, sheet 3). If the original K value was reported as weight per volume, we converted these into units of micromolar (µM) using the calculated molecular weight of the compound's chemical formula. We preserved significant digits from the original studies. [1] Kovárová-Kovar, Karin, and Thomas Egli. "Growth Kinetics of Suspended Microbial Cells: From Single-Substrate-Controlled Growth to Mixed-Substrate Kinetics." Microbiology and Molecular Biology Reviews 62, no. 3 (1998): 646–66. https://doi.org/10.1128/MMBR.62.3.646-666.1998 [2] Owens, J.D., and J.D. Legan. "Determination of the Monod Substrate Saturation Constant for Microbial Growth." FEMS Microbiology Letters 46, no. 4 (1987): 419–32. https://doi.org/10.1111/j.1574-6968.1987.tb02478.x. [3] Edwards, Kyle F., Christopher A. Klausmeier, and Elena Litchman. "Nutrient Utilization Traits of Phytoplankton." Ecology 96, no. 8 (2015): 2311–2311. https://doi.org/10.1890/14-2252.1

Direct physical interaction involving CD40 ligand on T cells and CD40 on B cells is required to propagate MMTV.

The propagation of mouse mammary tumor virus (MMTV) has been analyzed in mice defective for expression of CD40 ligand (CD40L). Mice with endogenous viral superantigen (SAG) delete T cells with cognate V beta independent of CD40L expression. Nevertheless, CD40L-mice do not show deletion of cognate T cells after being exposed to infectious MMTV and have greatly diminished viral replication. The response of CD40L- T cells to SAG in vitro is also impaired, but can be reconstituted by adding B cells activated by recombinant CD40L to express costimulatory molecules. Thus, direct CD40L-dependent B cell activation appears to be a critical step in the life cycle of MMTV. The initial step in SAG-dependent T cell activation, and hence the MMTV life cycle, may be mediated by non-B cells, because splenocytes from B cell-deficient SAG-transgenic mice are able to activate cognate T cells

Addressing Co-occurring Suicidal Thoughts and Behaviors and Posttraumatic Stress Disorder in Evidence-Based Psychotherapies for Adults: A Systematic Review

Posttraumatic stress disorder (PTSD) is a well-established risk factor for suicidal thoughts and behaviors. Historically, guidelines for treating PTSD have recommended against the use of trauma-focused therapies with patients who are high-risk for suicide likely due to concerns about potential suicide-related iatrogenesis, specifically the “triggering” of suicidal behaviors. This systematic review examines evidence for the impact of treatments specifically designed to treat PTSD or suicide on both PTSD- and suicide-related outcomes. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed and a total of 33 articles met full inclusion criteria, of which 23 examined PTSD treatments, 4 examined suicide-focused treatments, and 6 examined combined treatments. PTSD and combined treatments reduced both PTSD- and suicide-related outcomes, with most studies examining Cognitive Processing Therapy or Prolonged Exposure. Suicide-focused treatments (e.g., cognitive therapies for suicide prevention) also reduced suicide-related outcomes, but findings were mixed for their impact on PTSD-related outcomes. Overall, PTSD treatments had the most support, primarily due to a larger number of studies examining their outcomes. This supports current clinical guidelines, which suggest utilizing PTSD treatments for individuals at risk for suicide and who have PTSD. Suicide-focused and combined treatments also appeared to be promising formats although additional research is needed. Future research should seek to compare the effectiveness of the approaches to the treatment of PTSD and suicidal thoughts and behaviors concurrently, as well as to inform guidelines aimed at supporting decisions about the selection of an appropriate treatment approach