Ecology and population structure of Vibrionaceae in the coastal ocean

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole oceanographic Institution February 2010Extensive genetic diversity has been discovered in the microbial world, yet mechanisms that shape and maintain this diversity remain poorly understood. This thesis investigates to what extent populations of the gamma-proteobacterial family, Vibrionaceae, are ecologically specialized by investigating the distribution across a wide range of environmental categories, such as marine invertebrates or particles in the water column. Additionally, it seeks to determine whether in situ population distributions directly result from a competitive advantage over other Vibrio populations. This was investigated by in vitro competition assays on mixtures of native, sterilized particles. Generalist populations were found to dominate the associations with marine invertebrates, consistent with a model of high migration dominated population assembly. A majority of populations occurred broadly within and among the different types of invertebrates sampled, with one population being a near perfect generalist with regard to seasons, host taxa and body regions. High variability across host individuals, consistent with a scenario of stochastic clonal expansion, was especially pronounced in crab and zooplankton samples. Specialization, demonstrated by specific and reproducible association with different particle types in the water column, is more common than specialization within invertebrate hosts. Co-existing Vibrio species show strong preferences for different types of particulate matter in the water column suggesting that competition for limited resources influences their evolution. While populations show different growth profiles on particle derived substrates, relative growth advantages of specialist populations in competition with other Vibrio populations on native particles may not be sufficient to explain observed environmental distributions. Instead, populations may gain an advantage on these particles by colonizing the living plant or zooplankton prior to death and degradation into particulate matter. In summary, although vibrios are known commensals of marine invertebrates, evidence suggests that population structure within animals is fairly weak compared to suspended particles in the water column. This highlights the importance of comparing multiple environmental categories and migration among them to investigate population structure and adaptation.I would like to thank the Linden Fellowship for their support during the Fall, 2003- Spring, 2004 and the Martin Family Society of Fellows for Sustainability for their support during the Fall 2005. Work in this thesis was also made possible by support from the following agencies: Woods Hole Center for Oceans and Human Health, which is funded jointly by the National Science Foundation (OCE-0430724) and the National Institute for Environmental Health Sciences (P50 ES012742); DOE-Chicago (DE-FG02- 08ER64516); The Gordon and Betty Moore Foundation (Letter Agreement 1108)

High resolution time series reveals cohesive but short-lived communities in coastal plankton

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Nature Communications 9 (2018): 266, doi:10.1038/s41467-017-02571-4.Because microbial plankton in the ocean comprise diverse bacteria, algae, and protists that are subject to environmental forcing on multiple spatial and temporal scales, a fundamental open question is to what extent these organisms form ecologically cohesive communities. Here we show that although all taxa undergo large, near daily fluctuations in abundance, microbial plankton are organized into clearly defined communities whose turnover is rapid and sharp. We analyze a time series of 93 consecutive days of coastal plankton using a technique that allows inference of communities as modular units of interacting taxa by determining positive and negative correlations at different temporal frequencies. This approach shows both coordinated population expansions that demarcate community boundaries and high frequency of positive and negative associations among populations within communities. Our analysis thus highlights that the environmental variability of the coastal ocean is mirrored in sharp transitions of defined but ephemeral communities of organisms.This work was supported by grants from the U.S. National Science Foundation (OCE-1441943) to M.F.P. and the U.S. Department of Energy (DE-SC0008743) to M.F.P. and E.J.A. A.M.M.-P. was partially supported by the Ramon Areces foundation through a postdoctoral fellowship. D.J.M. was supported by the U.S. National Science Foundation (OCE-1314642) and National Institute of Environmental Health Sciences (1P01ES021923-01) through the Woods Hole Center for Oceans and Human Health

Natural Bacterial Communities Serve as Quantitative Geochemical Biosensors

Biological sensors can be engineered to measure a wide range of environmental conditions. Here we show that statistical analysis of DNA from natural microbial communities can be used to accurately identify environmental contaminants, including uranium and nitrate at a nuclear waste site. In addition to contamination, sequence data from the 16S rRNA gene alone can quantitatively predict a rich catalogue of 26 geochemical features collected from 93 wells with highly differing geochemistry characteristics. We extend this approach to identify sites contaminated with hydrocarbons from the Deepwater Horizon oil spill, finding that altered bacterial communities encode a memory of prior contamination, even after the contaminants themselves have been fully degraded. We show that the bacterial strains that are most useful for detecting oil and uranium are known to interact with these substrates, indicating that this statistical approach uncovers ecologically meaningful interactions consistent with previous experimental observations. Future efforts should focus on evaluating the geographical generalizability of these associations. Taken as a whole, these results indicate that ubiquitous, natural bacterial communities can be used as in situ environmental sensors that respond to and capture perturbations caused by human impacts. These in situ biosensors rely on environmental selection rather than directed engineering, and so this approach could be rapidly deployed and scaled as sequencing technology continues to become faster, simpler, and less expensive

Transcriptome analyses of the Giardia lamblia life cycle

Author Posting. © The Author(s), 2010. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Molecular and Biochemical Parasitology 174 (2010): 62-65, doi:10.1016/j.molbiopara.2010.05.010.We quantified mRNA abundance from 10 stages in the Giardia lamblia life cycle in vitro using Serial Analysis of Gene Expression (SAGE). 163 abundant transcripts were expressed constitutively. 71 transcripts were upregulated specifically during excystation and 42 during encystation. Nonetheless, the transcriptomes of cysts and trophozoites showed major differences. SAGE detected co-expressed clusters of 284 transcripts differentially expressed in cysts and excyzoites and 287 transcripts in vegetative trophozoites and encysting cells. All clusters included known genes and pathways as well as proteins unique to Giardia or diplomonads. SAGE analysis of the Giardia life cycle identified a number of kinases, phosphatases, and DNA replication proteins involved in excystation and encystation, which could be important for examining the roles of cell signaling in giardial differentiation. Overall, these data pave the way for directed gene discovery and a better understanding of the biology of Giardia lamblia.BJD, DSR, and FDG were supported by NIH grants AI42488, GM61896, DK35108, and AI051687. DP and SGS were supported by grants from the Swedish Natural Science Research Council, the Swedish Medical Research Council, and the Karolinska Institutet. AGM, SRB, SPP, and MJC were supported by NIH grant AI51089 and by the Marine Biological Laboratory’s Program in Global Infectious Diseases, funded by the Ellison Medical Foundation

Protein phosphatase 2A plays a crucial role in Giardia lamblia differentiation

Author Posting. © The Authors, 2006. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Molecular and Biochemical Parasitology 152 (2007): 80-89, doi:10.1016/j.molbiopara.2006.12.001.The ability of Giardia lamblia to undergo two distinct differentiations in response to physiologic stimuli is central to its pathogenesis. The giardial cytoskeleton changes drastically during encystation and excystation. However, the signal transduction pathways mediating these transformations are poorly understood. We tested the hypothesis that PP2A, a highly conserved serine/threonine protein phosphatase, might be important in giardial differentiation. We found that in vegetatively growing trophozoites, gPP2A-C protein localizes to basal bodies/centrosomes, and to cytoskeletal structures unique to Giardia: the ventral disk, and the dense rods of the anterior, posterior-lateral, and caudal flagella. During encystation, gPP2A-C protein disappears from only the anterior flagellar dense rods. During excystation, gPP2A-C localizes to the cyst wall in excysting cysts but is not found in the wall of cysts with emerging excyzoites. Transcriptome and immunoblot analyses indicated that gPP2A-C mRNA and protein are upregulated in mature cysts and during the early stage of excystation that models passage through the host stomach. Stable expression of gPP2A-C antisense RNA did not affect vegetative growth, but strongly inhibited the formation of encystation secretory vesicles (ESV) and water-resistant cysts. Moreover, the few cysts that formed were highly defective in excystation. Thus, gPP2A-C localizes to universal cytoskeletal structures and to structures unique to Giardia. It is also important for encystation and excystation, crucial giardial transformations that entail entry into and exit from dormancy.This work was funded by NIH grants GM61896, AI51687, AI42488, and DK35108. Dr. A.G. McArthur was supported by NIH grant AI51089 and the Marine Biological Laboratory’s Program in Global Infectious Diseases, funded by the Ellison Medical Foundation

Non-Invasive Mapping of the Gastrointestinal Microbiota Identifies Children with Inflammatory Bowel Disease

Background: Pediatric inflammatory bowel disease (IBD) is challenging to diagnose because of the non-specificity of symptoms; an unequivocal diagnosis can only be made using colonoscopy, which clinicians are reluctant to recommend for children. Diagnosis of pediatric IBD is therefore frequently delayed, leading to inappropriate treatment plans and poor outcomes. We investigated the use of 16S rRNA sequencing of fecal samples and new analytical methods to assess differences in the microbiota of children with IBD and other gastrointestinal disorders. Methodology/Principal Findings: We applied synthetic learning in microbial ecology (SLiME) analysis to 16S sequencing data obtained from i) published surveys of microbiota diversity in IBD and ii) fecal samples from 91 children and young adults who were treated in the gastroenterology program of Children’s Hospital (Boston, USA). The developed method accurately distinguished control samples from those of patients with IBD; the area under the receiver-operating-characteristic curve (AUC) value was 0.83 (corresponding to 80.3% sensitivity and 69.7% specificity at a set threshold). The accuracy was maintained among data sets collected by different sampling and sequencing methods. The method identified taxa associated with disease states and distinguished patients with Crohn’s disease from those with ulcerative colitis with reasonable accuracy. The findings were validated using samples from an additional group of 68 patients; the validation test identified patients with IBD with an AUC value of 0.84 (e.g. 92% sensitivity, 58.5% specificity). Conclusions/Significance: Microbiome-based diagnostics can distinguish pediatric patients with IBD from patients with similar symptoms. Although this test can not replace endoscopy and histological examination as diagnostic tools, classification based on microbial diversity is an effective complementary technique for IBD detection in pediatric patients.Natural Sciences and Engineering Research Council of Canada (Award NSERC PGS D)National Institutes of Health (U.S.) (1-R21-A1084032-01A1

A new family of giardial cysteine-rich non-VSP protein genes and a novel cyst protein

© 2006 Davids et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The definitive version was published in PLoS ONE 1 (2006): e44, doi:10.1371/journal.pone.0000044.Since the Giardia lamblia cyst wall is necessary for survival in the environment and host infection, we tested the hypothesis that it contains proteins other than the three known cyst wall proteins. Serial analysis of gene expression during growth and encystation revealed a gene, “HCNCp” (High Cysteine Non-variant Cyst protein), that was upregulated late in encystation, and that resembled the classic Giardia variable surface proteins (VSPs) that cover the trophozoite plasmalemma. HCNCp is 13.9% cysteine, with many “CxxC” tetrapeptide motifs and a transmembrane sequence near the C-terminus. However, HCNCp has multiple “CxC” motifs rarely found in VSPs, and does not localize to the trophozoite plasmalemma. Moreover, the HCNCp C-terminus differed from the canonical VSP signature. Full-length epitope-tagged HCNCp expressed under its own promoter was upregulated during encystation with highest expression in cysts, including 42 and 21 kDa C-terminal fragments. Tagged HCNCp targeted to the nuclear envelope in trophozoites, and co-localized with cyst proteins to encystation-specific secretory vesicles during encystation. HCNCp defined a novel trafficking pathway as it localized to the wall and body of cysts, while the cyst proteins were exclusively in the wall. Unlike VSPs, HCNCp is expressed in at least five giardial strains and four WB subclones expressing different VSPs. Bioinformatics identified 60 additional large high cysteine membrane proteins (HCMp) containing ≥20 CxxC/CxC's lacking the VSP-specific C-terminal CRGKA. HCMp were absent or rare in other model or parasite genomes, except for Tetrahymena thermophila with 30. MEME analysis classified the 61 gHCMp genes into nine groups with similar internal motifs. Our data suggest that HCNCp is a novel invariant cyst protein belonging to a new HCMp family that is abundant in the Giardia genome. HCNCp and the other HCMp provide a rich source for developing parasite-specific diagnostic reagents, vaccine candidates, and subjects for further research into Giardia biology

Dynamics of microbial populations mediating biogeochemical cycling in a freshwater lake

Background Microbial processes are intricately linked to the depletion of oxygen in in-land and coastal water bodies, with devastating economic and ecological consequences. Microorganisms deplete oxygen during biomass decomposition, degrading the habitat of many economically important aquatic animals. Microbes then turn to alternative electron acceptors, which alter nutrient cycling and generate potent greenhouse gases. As oxygen depletion is expected to worsen with altered land use and climate change, understanding how chemical and microbial dynamics impact dead zones will aid modeling efforts to guide remediation strategies. More work is needed to understand the complex interplay between microbial genes, populations, and biogeochemistry during oxygen depletion. Results Here, we used 16S rRNA gene surveys, shotgun metagenomic sequencing, and a previously developed biogeochemical model to identify genes and microbial populations implicated in major biogeochemical transformations in a model lake ecosystem. Shotgun metagenomic sequencing was done for one time point in Aug., 2013, and 16S rRNA gene sequencing was done for a 5-month time series (Mar.–Aug., 2013) to capture the spatiotemporal dynamics of genes and microorganisms mediating the modeled processes. Metagenomic binning analysis resulted in many metagenome-assembled genomes (MAGs) that are implicated in the modeled processes through gene content similarity to cultured organism and the presence of key genes involved in these pathways. The MAGs suggested some populations are capable of methane and sulfide oxidation coupled to nitrate reduction. Using the model, we observe that modulating these processes has a substantial impact on overall lake biogeochemistry. Additionally, 16S rRNA gene sequences from the metagenomic and amplicon libraries were linked to processes through the MAGs. We compared the dynamics of microbial populations in the water column to the model predictions. Many microbial populations involved in primary carbon oxidation had dynamics similar to the model, while those associated with secondary oxidation processes deviated substantially. Conclusions This work demonstrates that the unique capabilities of resident microbial populations will substantially impact the concentration and speciation of chemicals in the water column, unless other microbial processes adjust to compensate for these differences. It further highlights the importance of the biological aspects of biogeochemical processes, such as fluctuations in microbial population dynamics. Integrating gene and population dynamics into biogeochemical models has the potential to improve predictions of the community response under altered scenarios to guide remediation efforts

Dynamics of microbial populations mediating biogeochemical cycling in a freshwater lake

Abstract Background Microbial processes are intricately linked to the depletion of oxygen in in-land and coastal water bodies, with devastating economic and ecological consequences. Microorganisms deplete oxygen during biomass decomposition, degrading the habitat of many economically important aquatic animals. Microbes then turn to alternative electron acceptors, which alter nutrient cycling and generate potent greenhouse gases. As oxygen depletion is expected to worsen with altered land use and climate change, understanding how chemical and microbial dynamics impact dead zones will aid modeling efforts to guide remediation strategies. More work is needed to understand the complex interplay between microbial genes, populations, and biogeochemistry during oxygen depletion. Results Here, we used 16S rRNA gene surveys, shotgun metagenomic sequencing, and a previously developed biogeochemical model to identify genes and microbial populations implicated in major biogeochemical transformations in a model lake ecosystem. Shotgun metagenomic sequencing was done for one time point in Aug., 2013, and 16S rRNA gene sequencing was done for a 5-month time series (Mar.–Aug., 2013) to capture the spatiotemporal dynamics of genes and microorganisms mediating the modeled processes. Metagenomic binning analysis resulted in many metagenome-assembled genomes (MAGs) that are implicated in the modeled processes through gene content similarity to cultured organism and the presence of key genes involved in these pathways. The MAGs suggested some populations are capable of methane and sulfide oxidation coupled to nitrate reduction. Using the model, we observe that modulating these processes has a substantial impact on overall lake biogeochemistry. Additionally, 16S rRNA gene sequences from the metagenomic and amplicon libraries were linked to processes through the MAGs. We compared the dynamics of microbial populations in the water column to the model predictions. Many microbial populations involved in primary carbon oxidation had dynamics similar to the model, while those associated with secondary oxidation processes deviated substantially. Conclusions This work demonstrates that the unique capabilities of resident microbial populations will substantially impact the concentration and speciation of chemicals in the water column, unless other microbial processes adjust to compensate for these differences. It further highlights the importance of the biological aspects of biogeochemical processes, such as fluctuations in microbial population dynamics. Integrating gene and population dynamics into biogeochemical models has the potential to improve predictions of the community response under altered scenarios to guide remediation efforts