The metabolic demands of endosymbiotic chemoautotrophic metabolism on host physiological capacities

While chemoautotrophic endosymbioses of hydrothermal vents and other reducing environments have been well studied, little attention has been paid to the magnitude of the metabolic demands placed upon the host by symbiont metabolism and the adaptations necessary to meet such demands. Here we make the first attempt at such an evaluation, and show that moderate to high rates of chemoautotrophic or methanotrophic metabolism impose oxygen uptake and proton equivalent elimination demands upon the hosts that are much higher than is typical for the non-symbiotic annelid, bivalve and gastropod lineages to which they are related. The properties of the hosts are described and compared to determine which properties are associated with and predictive of the highest rates. We suggest that the high oxygen demand of these symbionts is perhaps the most limiting flux for the symbioses. Among the consequences of such demands has been the widespread presence of circulating and/or tissue hemoglobins in these symbioses that are necessary to support high metabolic rates in thioautotrophic endosymbioses. We also compare photoautotrophic with chemoautotrophic and methanotrophic endosymbioses to evaluate the differences and similarities in physiologies. These analyses suggest that the high demand for oxygen by chemoautotrophic and methanotrophic symbionts is likely a major factor precluding their endosymbiosis with cnidarians.Organismic and Evolutionary Biolog

Comparative genomics of vesicomyid clam (Bivalvia: Mollusca) chemosynthetic symbionts

<p>Abstract</p> <p>Background</p> <p>The Vesicomyidae (Bivalvia: Mollusca) are a family of clams that form symbioses with chemosynthetic gamma-proteobacteria. They exist in environments such as hydrothermal vents and cold seeps and have a reduced gut and feeding groove, indicating a large dependence on their endosymbionts for nutrition. Recently, two vesicomyid symbiont genomes were sequenced, illuminating the possible nutritional contributions of the symbiont to the host and making genome-wide evolutionary analyses possible.</p> <p>Results</p> <p>To examine the genomic evolution of the vesicomyid symbionts, a comparative genomics framework, including the existing genomic data combined with heterologous microarray hybridization results, was used to analyze conserved gene content in four vesicomyid symbiont genomes. These four symbionts were chosen to include a broad phylogenetic sampling of the vesicomyid symbionts and represent distinct chemosynthetic environments: cold seeps and hydrothermal vents.</p> <p>Conclusion</p> <p>The results of this comparative genomics analysis emphasize the importance of the symbionts' chemoautotrophic metabolism within their hosts. The fact that these symbionts appear to be metabolically capable autotrophs underscores the extent to which the host depends on them for nutrition and reveals the key to invertebrate colonization of these challenging environments.</p

A molluscan class struggle: exploring the surprisingly uneven distribution of chemosymbiosis among two major mollusk groups

Many bivalves and gastropods from marine reducing environments such as deep ocean hydrothermal vents and seeps host chemosynthetic bacteria in a nutritional symbiosis. Despite their functional similarities, the distribution of chemosymbiosis in these two mollusk classes is surprisingly uneven: the number of bivalve species known to host chemosynthetic symbionts is more than twenty times that of gastropods, and chemosymbiotic bivalves are reported from a far greater diversity of marine habitats. Here we explore the potential drivers for this trend, including but not limited to physiological differences, habitat characteristics, and sampling bias. Sampling bias likely contributes to the magnitude of the observed discrepancy, but we posit that the phenomenon itself reveals how intrinsic (e.g. morphology) and extrinsic (e.g. organic matter availability) factors might have shaped the distribution of extant gastropod and bivalve associations. These observations also serve as an impetus for increasing investigation into gastropods and other mollusks from chemically reducing environments to better understand the evolution and ecology of chemosymbiosis among molluscan hosts

Vortex fluidics-mediated DNA rescue from formalin-fixed museum specimens.

DNA from formalin-preserved tissue could unlock a vast repository of genetic information stored in museums worldwide. However, formaldehyde crosslinks proteins and DNA, and prevents ready amplification and DNA sequencing. Formaldehyde acylation also fragments the DNA. Treatment with proteinase K proteolyzes crosslinked proteins to rescue the DNA, though the process is quite slow. To reduce processing time and improve rescue efficiency, we applied the mechanical energy of a vortex fluidic device (VFD) to drive the catalytic activity of proteinase K and recover DNA from American lobster tissue (Homarus americanus) fixed in 3.7% formalin for &gt;1-year. A scan of VFD rotational speeds identified the optimal rotational speed for recovery of PCR-amplifiable DNA and while 500+ base pairs were sequenced, shorter read lengths were more consistently obtained. This VFD-based method also effectively recovered DNA from formalin-preserved samples. The results provide a roadmap for exploring DNA from millions of historical and even extinct species

Exploring the limit of metazoan thermal tolerance via comparative proteomics: thermally induced changes in protein abundance by two hydrothermal vent polychaetes

Temperatures around hydrothermal vents are highly variable, ranging from near freezing up to 300°C. Nevertheless, animals thrive around vents, some of which live near the known limits of animal thermotolerance. Paralvinella sulfincola, an extremely thermotolerant vent polychaete, and Paralvinella palmiformis, a cooler-adapted congener, are found along the Juan de Fuca Ridge in the northwestern Pacific. We conducted shipboard high-pressure thermotolerance experiments on both species to characterize the physiological adaptations underlying P. sulfincola's pronounced thermotolerance. Quantitative proteomics, expressed sequence tag (EST) libraries and glutathione assays revealed that P. sulfincola (i) exhibited an upregulation in the synthesis and recycling of glutathione with increasing temperature, (ii) downregulated nicotinamide adenine dinucleotide (NADH) and succinate dehydrogenases (key enzymes in oxidative phosphorylation) with increasing temperature, and (iii) maintained elevated levels of heat shock proteins (HSPs) across all treatments. In contrast, P. palmiformis exhibited more typical responses to increasing temperatures (e.g. increasing HSPs at higher temperatures). These data reveal differences in how a mesotolerant and extremely thermotolerant eukaryote respond to thermal stress, and suggest that P. sulfincola's capacity to mitigate oxidative stress via increased synthesis of antioxidants and decreased flux through the mitochondrial electron transport chain enable pronounced thermotolerance. Ultimately, oxidative stress may be the key factor in limiting all metazoan thermotolerance.Organismic and Evolutionary Biolog

Characterizing the Distribution and Rates of Microbial Sulfate Reduction at Middle Valley Hydrothermal Vents

Few studies have directly measured sulfate reduction at hydrothermal vents, and relatively little is known about how environmental or ecological factors influence rates of sulfate reduction in vent environments. A better understanding of microbially mediated sulfate reduction in hydrothermal vent ecosystems may be achieved by integrating ecological and geochemical data with metabolic rate measurements. Here we present rates of microbially mediated sulfate reduction from three distinct hydrothermal vents in the Middle Valley vent field along the Juan de Fuca Ridge, as well as assessments of bacterial and archaeal diversity, estimates of total biomass and the abundance of functional genes related to sulfate reduction, and in situ geochemistry. Maximum rates of sulfate reduction occurred at $90^{\circ}C$ in all three deposits. Pyrosequencing and functional gene abundance data reveal differences in both biomass and community composition among sites, including differences in the abundance of known sulfate reducing bacteria. The abundance of sequences for Thermodesulfovibro-like organisms and higher sulfate reduction rates at elevated temperatures, suggests that Thermodesulfovibro-like organisms may play a role in sulfate reduction in warmer environments. The rates of sulfate reduction presented here suggest that - within anaerobic niches of hydrothermal deposits - heterotrophic sulfate reduction may be quite common and can contribute to secondary productivity, underscoring the potential role of this process in both sulfur and carbon cycling at vents.Organismic and Evolutionary Biolog

Electron Uptake by Iron-Oxidizing Phototrophic Bacteria

Oxidation–reduction reactions underlie energy generation in nearly all life forms. Although most organisms use soluble oxidants and reductants, some microbes can access solid-phase materials as electron-acceptors or -donors via extracellular electron transfer. Many studies have focused on the reduction of solid-phase oxidants. Far less is known about electron uptake via microbial extracellular electron transfer, and almost nothing is known about the associated mechanisms. Here we show that the iron-oxidizing photoautotroph Rhodopseudomonas palustris TIE-1 accepts electrons from a poised electrode, with carbon dioxide as the sole carbon source/electron acceptor. Both electron uptake and ruBisCo form I expression are stimulated by light. Electron uptake also occurs in the dark, uncoupled from photosynthesis. Notably, the pioABC operon, which encodes a protein system essential for photoautotrophic growth by ferrous iron oxidation, influences electron uptake. These data reveal a previously unknown metabolic versatility of photoferrotrophs to use extracellular electron transfer for electron uptake.Engineering and Applied SciencesOrganismic and Evolutionary Biolog

Toward establishing model organisms for marine protists : Successful transfection protocols for Parabodo caudatus (Kinetoplastida: Excavata)

Author Posting. © The Author(s), 2017. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in Environmental Microbiology 19 (2017): 3487-3499, doi:10.1111/1462-2920.13830.We developed protocols for, and demonstrated successful transfection of, the free-living kinetoplastid flagellate Parabodo caudatus with three plasmids carrying a fluorescence reporter gene (pEF-GFP with the EF1 alpha promoter, pUB-GFP with Ubiquitin C promoter, and pEYFP37 Mitotrap with CMV promoter). We evaluated three electroporation approaches: 1) a square-wave electroporator designed for eukaryotes, 2) a novel microfluidic transfection system employing hydrodynamically-controlled electric field waveforms, and 3) a traditional exponential decay electroporator. We found the microfluidic device provides a simple and efficient platform to quickly test a wide range of electric field parameters to find the optimal set of conditions for electroporation of target species. It also allows for processing large sample volumes (> 10 ml) within minutes, increasing throughput 100 times over cuvettes. Fluorescence signal from the reporter gene was detected a few hours after transfection and persisted for 3 days in cells transformed by pEF-GFP and pUB-GFP plasmids and for at least 5 days post-transfection for cells transformed with pEYFP-Mitotrap. Expression of the reporter genes (GFP and YFP) was also confirmed using reverse transcription-PCR (RT-PCR). This work opens the door for further efforts with this taxon and close relatives toward establishing model systems for genome editing.This project was funded by the Gordon and Betty Moore Foundation through Grant GBMF4963 to V. Edgcomb, P. Girguis, and C. Buie

Anaerobic Oxidation of Short-Chain Alkanes in Hydrothermal Sediments: Potential Influences on Sulfur Cycling and Microbial Diversity

Short-chain alkanes play a substantial role in carbon and sulfur cycling at hydrocarbon-rich environments globally, yet few studies have examined the metabolism of ethane $(C_2)$, propane $(C_3)$, and butane $(C_4)$ in anoxic sediments in contrast to methane $(C_1)$. In hydrothermal vent systems, short-chain alkanes are formed over relatively short geological time scales via thermogenic processes and often exist at high concentrations. The sediment-covered hydrothermal vent systems at Middle Valley (MV, Juan de Fuca Ridge) are an ideal site for investigating the anaerobic oxidation of $C_1–C_4$ alkanes, given the elevated temperatures and dissolved hydrocarbon species characteristic of these metalliferous sediments. We examined whether MV microbial communities oxidized $C_1–C_4$ alkanes under mesophilic to thermophilic sulfate-reducing conditions. Here we present data from discrete temperature (25, 55, and $75^{\circ}C$) anaerobic batch reactor incubations of MV sediments supplemented with individual alkanes. Co-registered alkane consumption and sulfate reduction (SR) measurements provide clear evidence for $C_1–C_4$ alkane oxidation linked to SR over time and across temperatures. In these anaerobic batch reactor sediments, 16S ribosomal RNA pyrosequencing revealed that Deltaproteobacteria, particularly a novel sulfate-reducing lineage, were the likely phylotypes mediating the oxidation of $C_2–C_4$ alkanes. Maximum $C_1–C_4$ alkane oxidation rates occurred at $55^{\circ}C$, which reflects the mid-core sediment temperature profile and corroborates previous studies of rate maxima for the anaerobic oxidation of methane (AOM). Of the alkanes investigated, $C_3$ was oxidized at the highest rate over time, then $C_4$, $C_2$, and $C_1$, respectively. The implications of these results are discussed with respect to the potential competition between the anaerobic oxidation of $C_2–C_4$ alkanes with AOM for available oxidants and the influence on the fate of $C_1$ derived from these hydrothermal systems.Molecular and Cellular BiologyOrganismic and Evolutionary Biolog

The Ecological Physiology of Earth's Second Oxygen Revolution

Living animals display a variety of morphological, physiological, and biochemical characters that enable them to live in low-oxygen environments. These features and the organisms that have evolved them are distributed in a regular pattern across dioxygen (O2) gradients associated with modern oxygen minimum zones. This distribution provides a template for interpreting the stratigraphic covariance between inferred Ediacaran-Cambrian oxygenation and early animal diversification. Although Cambrian oxygen must have reached 10--20% of modern levels, sufficient to support the animal diversity recorded by fossils, it may not have been much higher than this. Today’s levels may have been approached only later in the Paleozoic Era. Nonetheless, Ediacaran-Cambrian oxygenation may have pushed surface environments across the low, but critical, physiological thresholds required for large, active animals, especially carnivores. Continued focus on the quantification of the partial pressure of oxygen (pO2) in the Proterozoic will provide the definitive tests of oxygen-based coevolutionary hypotheses.Earth and Planetary SciencesOrganismic and Evolutionary Biolog