Publications

@article{nokey,

title = {Growth rate-dependent coordination of catabolism and anabolism in the archaeon Methanococcus maripaludis under phosphate limitation},

author = {Wenyu Gu and Albert L. Mueller and Jörg Deutzmann and James R. Williamson and Alfred M. Spormann},

url = {https://rdcu.be/cRuS4},

doi = {10.1038/s41396-022-01278-9},

year  = {2022},

date = {2022-07-02},

urldate = {2022-07-02},

journal = {ISME J},

abstract = {Catabolic and anabolic processes are finely coordinated in microorganisms to provide optimized fitness under varying environmental conditions. Understanding this coordination and the resulting physiological traits reveals fundamental strategies of microbial acclimation. Here, we characterized the system-level physiology of Methanococcus maripaludis, a niche-specialized methanogenic archaeon, at different dilution rates ranging from 0.09 to 0.003 h−1 in chemostat experiments under phosphate (i.e., anabolic) limitation. Phosphate was supplied as the limiting nutrient, while formate was supplied in excess as the catabolic substrate and carbon source. We observed a decoupling of catabolism and anabolism resulting in lower biomass yield relative to catabolically limited cells at the same dilution rates. In addition, the mass abundance of several coarse-grained proteome sectors (i.e., combined abundance of proteins grouped based on their function) exhibited a linear relationship with growth rate, mostly ribosomes and their biogenesis. Accordingly, cellular RNA content also correlated with growth rate. Although the methanogenesis proteome sector was invariant, the metabolic capacity for methanogenesis, measured as methane production rates immediately after transfer to batch culture, correlated with growth rate suggesting translationally independent regulation that allows cells to only increase catabolic activity under growth-permissible conditions. These observations are in stark contrast to the physiology of M. maripaludis under formate (i.e., catabolic) limitation, where cells keep an invariant proteome including ribosomal content and a high methanogenesis capacity across a wide range of growth rates. Our findings reveal that M. maripaludis employs fundamentally different strategies to coordinate global physiology during anabolic phosphate and catabolic formate limitation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Low-Cost Clamp-On Photometers (ClampOD) and Tube Photometers (TubeOD) for Online Cell Density Determination},

author = {Jörg S. Deutzmann and Grace Callander and Wenyu Gu and Albert L. Mueller and Alexandra L. McCully and Jenna Kim Ahn and Frauke Kracke and Alfred M. Spormann},

editor = {Frank Schreiber},

url = {https://www.frontiersin.org/article/10.3389/fmicb.2021.790576},

doi = {10.3389/fmicb.2021.790576},

issn = {1664-302X},

year  = {2022},

date = {2022-01-13},

urldate = {2022-01-13},

journal = {Frontiers in Microbiology},

volume = {12},

abstract = {Optical density (OD) measurement is the gold standard to estimate microbial cell density in aqueous systems. Recording microbial growth curves is essential to assess substrate utilization, gauge sensitivity to inhibitors or toxins, or determine the perfect sampling point. Manual sampling for cuvette-photometer-based measurements can cause disturbances and impact growth, especially for strictly anaerobic or thermophilic microbes. For slow growing microbes, manual sampling can cause data gaps that complicate analysis. Online OD measurement systems provide a solution, but are often expensive and ill-suited for applications such as monitoring microbial growth in custom or larger anaerobic vessels. Furthermore, growth measurements of thermophilic cultures are limited by the heat sensitivity of complex electronics. Here, we present two simple, low-cost, self-assembled photometers—a “TubeOD” for online measurement of anaerobic and thermophilic cultures in Hungate tubes and a “ClampOD” that can be attached to virtually any transparent growth vessel. Both OD-meters can be calibrated in minutes. We detail the manufacturing and calibration procedure and demonstrate continuous acquisition of high quality cell density data of a variety of microbes, including strict anaerobes, a thermophile, and gas-utilizing strains in various glassware. When calibrated and operated within their detection limits (ca. 0.3–90% of the photosensor voltage range), these self-build OD-meters can be used for continuous measurement of microbial growth in a variety of applications, thereby, simplifying and enhancing everyday lab operations.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Lamaison2021,

title = {Designing a Zn–Ag Catalyst Matrix and Electrolyzer System for CO2 Conversion to CO and Beyond},

author = {Sarah Lamaison and David Wakerley and Frauke Kracke and Thomas Moore and Lan Zhou and Dong Un Lee and Lei Wang and McKenzie A. Hubert and Jaime E. Aviles Acosta and John M. Gregoire and Eric B. Duoss and Sarah Baker and Victor A. Beck and Alfred M. Spormann and Marc Fontecave and Christopher Hahn and Thomas F. Jaramillo},

url = {https://doi.org/10.1002/adma.202103963},

doi = {10.1002/adma.202103963},

year  = {2021},

date = {2021-10-21},

urldate = {2021-10-21},

journal = {Advanced Materials},

volume = {2021},

number = {2103963},

abstract = {CO2 emissions can be transformed into high-added-value commodities through CO2 electrocatalysis; however, efficient low-cost electrocatalysts are needed for global scale-up. Inspired by other emerging technologies, the authors report the development of a gas diffusion electrode containing highly dispersed Ag sites in a low-cost Zn matrix. This catalyst shows unprecedented Ag mass activity for CO production: −614 mA cm−2 at 0.17 mg of Ag. Subsequent electrolyte engineering demonstrates that halide anions can further improve stability and activity of the Zn–Ag catalyst, outperforming pure Ag and Au. Membrane electrode assemblies are constructed and coupled to a microbial process that converts the CO to acetate and ethanol. Combined, these concepts present pathways to design catalysts and systems for CO2 conversion toward sought-after products.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Ruth2018,

title = {Enzymatic Hydrogen Electrosynthesis at Enhanced Current Density Using a Redox Polymer},

author = {John C. Ruth and Fabian M. Schwarz and Volker Müller and Alfred M. Spormann},

url = {https://doi.org/10.3390/catal11101197},

doi = {10.3390/catal11101197},

year  = {2021},

date = {2021-09-30},

urldate = {2021-09-30},

journal = {Catalysts},

volume = {11},

number = {1197},

abstract = {High-temperature tolerant enzymes offer multiple advantages over enzymes from mesophilic organisms for the industrial production of sustainable chemicals due to high specific activities and stabilities towards fluctuations in pH, heat, and organic solvents. The production of molecular hydrogen (H2) is of particular interest because of the multiple uses of hydrogen in energy and chemicals applications, and the ability of hydrogenase enzymes to reduce protons to H2 at a cathode. We examined the activity of Hydrogen-Dependent CO2 Reductase (HDCR) from the thermophilic bacterium Thermoanaerobacter kivui when immobilized in a redox polymer, cobaltocene-functionalized polyallylamine (Cc-PAA), on a cathode for enzyme-mediated H2 formation from electricity. The presence of Cc-PAA increased reductive current density 340-fold when used on an electrode with HDCR at 40 °C, reaching unprecedented current densities of up to 3 mA·cm−2 with minimal overpotential and high faradaic efficiency. In contrast to other hydrogenases, T. kivui HDCR showed substantial reversibility of CO-dependent inactivation, revealing an opportunity for usage in gas mixtures containing CO, such as syngas. This study highlights the important potential of combining redox polymers with novel enzymes from thermophiles for enhanced electrosynthesis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Kracke2021,

title = {Efficient Hydrogen Delivery for Microbial Electrosynthesis via 3D-Printed Cathode},

author = {Frauke Kracke and Jörg S. Deutzmann and Buddhinie S. Jayathilake and Simon H. Pang and Swetha Chandrasekaran and Sarah E. Baker and Alfred M. Spormann},

url = {https://www.frontiersin.org/articles/10.3389/fmicb.2021.696473/full},

doi = {10.3389/fmicb.2021.696473},

year  = {2021},

date = {2021-08-03},

urldate = {2021-08-03},

journal = {Frontiers in Microbiology},

volume = {12},

number = {696473},

abstract = {The efficient delivery of electrochemically in situ produced H2 can be a key advantage of microbial electrosynthesis over traditional gas fermentation. However, the technical details of how to supply large amounts of electric current per volume in a biocompatible manner remain unresolved. Here, we explored for the first time the flexibility of complex 3D-printed custom electrodes to fine tune H2 delivery during microbial electrosynthesis. Using a model system for H2-mediated electromethanogenesis comprised of 3D fabricated carbon aerogel cathodes plated with nickel-molybdenum and Methanococcus maripaludis, we showed that novel 3D-printed cathodes facilitated sustained and efficient electromethanogenesis from electricity and CO2 at an unprecedented volumetric production rate of 2.2 LCH4 /Lcatholyte/day and at a coulombic efficiency of 99%. Importantly, our experiments revealed that the efficiency of this process strongly depends on the current density. At identical total current supplied, larger surface area cathodes enabled higher methane production and minimized escape of H2. Specifically, low current density (<1 mA/cm2) enabled by high surface area cathodes was found to be critical for fast start-up times of the microbial culture, stable steady state performance, and high coulombic efficiencies. Our data demonstrate that 3D-printing of electrodes presents a promising design tool to mitigate effects of bubble formation and local pH gradients within the boundary layer and, thus, resolve key critical limitations for in situ electron delivery in microbial electrosynthesis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Ruth2021,

title = {Enzyme Electrochemistry for Industrial Energy Applications—A Perspective on Future Areas of Focus},

author = {Ruth, John C. and Spormann, Alfred},

url = {https://pubs.acs.org/doi/10.1021/acscatal.1c00708},

doi = {10.1021/acscatal.1c00708},

year  = {2021},

date = {2021-04-30},

journal = {ACS Catalysis},

volume = {11},

pages = {5951–5967},

abstract = {While enzymes catalyze reactions with high selectivity and specificity at ambient temperature and pressure, electroactive enzymes retain such remarkable catalytic properties for catalyzing redox reactions on the basis of direct or mediated electron transfer with an electrode. They offer the possibility for alternative, environmentally friendly production for a variety of industrially relevant chemicals, such as hydrogen gas, ammonia, and methanol. This Perspective summarizes the recent progress in electrochemistry involving hydrogenases, nitrogenases, and enzymes involved in the chemistry of one-carbon compounds. We discuss the current challenges of achieving high catalytic rates and stability on the basis of two promising improvements to existing enzymatic electrochemical systems—redox polymers and gas-diffusion electrodes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Muller2021,

title = {An alternative resource allocation strategy in the chemolithoautotrophic archaeon Methanococcus maripaludis},

author = {Muller, Albert and Gu, Wenyu and Patsalo, Vadim and Deutzmann, Jörg and Williamson, James R. and Spormann, Alfred},

url = {https://www.pnas.org/content/118/16/e2025854118.short},

doi = {10.1073/pnas.2025854118},

year  = {2021},

date = {2021-04-20},

journal = {Proceedings of the National Acadamy of Science},

volume = {118},

number = {16},

abstract = {Most microorganisms in nature spend the majority of time in a state of slow or zero growth and slow metabolism under limited energy or nutrient flux rather than growing at maximum rates. Yet, most of our knowledge has been derived from studies on fast-growing bacteria. Here, we systematically characterized the physiology of the methanogenic archaeon Methanococcus maripaludis during slow growth. M. maripaludis was grown in continuous culture under energy (formate)-limiting conditions at different dilution rates ranging from 0.09 to 0.002 h−1, the latter corresponding to 1% of its maximum growth rate under laboratory conditions (0.23 h−1). While the specific rate of methanogenesis correlated with growth rate as expected, the fraction of cellular energy used for maintenance increased and the maintenance energy per biomass decreased at slower growth. Notably, proteome allocation between catabolic and anabolic pathways was invariant with growth rate. Unexpectedly, cells maintained their maximum methanogenesis capacity over a wide range of growth rates, except for the lowest rates tested. Cell size, cellular DNA, RNA, and protein content as well as ribosome numbers also were largely invariant with growth rate. A reduced protein synthesis rate during slow growth was achieved by a reduction in ribosome activity rather than via the number of cellular ribosomes. Our data revealed a resource allocation strategy of a methanogenic archaeon during energy limitation that is fundamentally different from commonly studied versatile chemoheterotrophic bacteria such as E. coli.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{McCully2020,

title = {Direct cathodic electron uptake coupled to sulfate reduction by Desulfovibrio ferrophilus IS5 biofilms},

author = {McCully, Ali and Spormann, Alfred},

url = {https://sfamjournals.onlinelibrary.wiley.com/doi/abs/10.1111/1462-2920.15235},

doi = {10.1111/1462-2920.15235},

year  = {2020},

date = {2020-09-17},

abstract = {Direct electron uptake is emerging as a key process for electron transfer in anaerobic microbial communities, both between species and from extracellular sources, such as zero‐valent iron (Fe0) or cathodic surfaces. In this study, we investigated cathodic electron uptake by Fe0‐corroding Desulfovibrio ferrophilus IS5 and showed that electron uptake is dependent on direct cell contact via a biofilm on the cathode surface rather than through secreted intermediates. Induction of cathodic electron uptake by lactate‐starved D. ferrophilus IS5 cells resulted in the expression of all components necessary for electron uptake; however, protein synthesis was required for full biofilm formation. Notably, proteinase K treatment uncoupled electron uptake from biofilm formation, likely through proteolytic degradation of proteinaceous components of the electron uptake machinery. We also showed that cathodic electron uptake is dependent on SO42‐ reduction. The insensitivity of Fe0 corrosion to proteinase K treatment suggests that electron uptake from a cathode might involve different mechanism(s) than those involved in Fe0 corrosion.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Kracke2020,

title = {In situ electrochemical H2 production for efficient and stable power-to-gas electromethanogenesis},

author = {Kracke, Frauke and Deutzmann, Jörg and Gu, Wenyu and Spormann, Alfred},

url = {https://pubs.rsc.org/en/content/articlehtml/2020/gc/d0gc01894e},

doi = {10.1039/D0GC01894E},

year  = {2020},

date = {2020-09-02},

journal = {Green Chemistry},

volume = {22},

pages = {6194-6203},

abstract = {Bioelectrochemical power-to-gas presents a promising technology for long-term storage of excess renewable energy in the form of methane. The transition of the technology from laboratory to applied scale is currently challenged by low volumetric production rates, energy losses at the cathode, as well as the unknown physiology of the microbes in an electrochemical reactor. Here, we introduce a stable electromethanogenesis system based on efficient in situ hydrogen production by non-precious-metal catalysts and effective hydrogen uptake by the methanogenic microorganisms. Using NiMo-cathodes and pure cultures of hydrogenotrophic Methanococcus maripaludis, our system achieved an unprecedented volumetric methane production rate from CO2 of 1.4 L methane per L per day. The system performed stably for over 4 weeks with columbic efficiencies steadily above 90%. A physiological analysis of cells in the electromethanogenic reactor revealed robustly-growing cells with nearly identical protein expression patterns to gas-fed controls. Local pH fluctuations at the surface of cathode and cation exchange membrane resulted in a small but noticeable fraction of cell lysis. Our data collectively indicate that physiologically uncompromised cells of a pure methanogenic culture can perform methanogenesis robustly at high specific rate in a biocompatible electromethanogenic reactor using inexpensive, earth-abundant cathode materials.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Ruth2020,

title = {Enhanced Electrosynthetic Hydrogen Evolution by Hydrogenases Embedded in a Redox‐Active Hydrogel},

author = {Ruth, John C. and Milton, Ross D. and Gu, Wenyu and Spormann, Alfred},

url = {https://onlinelibrary.wiley.com/doi/10.1002/chem.202000750},

doi = {10.1002/chem.202000750},

year  = {2020},

date = {2020-02-19},

journal = {Chemistry - A European Journal},

abstract = {Molecular hydrogen is a major high‐energy carrier for future energy technologies, if produced from renewable electrical energy. Hydrogenase enzymes offer a pathway for bioelectrochemically producing hydrogen that is advantageous over traditional platforms for hydrogen production because of low overpotentials and ambient operating temperature and pressure. However, electron delivery from the electrode surface to the enzyme’s active site is often rate‐limiting. Here, we show three different hydrogenases from Clostridium pasteurianum and Methanococcus maripaludis , when immobilized at a cathode in a cobaltocene‐functionalized polyallylamine (Cc‐PAA) redox polymer, mediate rapid and efficient hydrogen evolution. We further show that Cc‐PAA‐mediated hydrogenases can operate at high faradaic efficiency (80‐100%) and low overpotential (‐0.578 to ‐0.593 V vs. SHE). Specific activities of these hydrogenases in the electrosynthetic Cc‐PAA assay were comparable to their respective activities in traditional methyl viologen assays, indicating that Cc‐PAA mediates electron transfer at high rates, to most of the embedded enzymes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Yee2020,

title = {Cultivating electroactive microbes—from field to bench},

author = {Yee, Mon Oo and Deutzmann, Jörg and Spormann, Alfred and Rotaru, Amelia-Elena},

url = {https://iopscience.iop.org/article/10.1088/1361-6528/ab6ab5/meta},

doi = {10.1088/1361-6528/ab6ab5},

year  = {2020},

date = {2020-02-13},

journal = {Nanotechnology},

volume = {31},

number = {17},

abstract = {Electromicrobiology is an emerging field investigating and exploiting the interaction of microorganisms with insoluble electron donors or acceptors. Some of the most recently categorized electroactive microorganisms became of interest to sustainable bioengineering practices. However, laboratories worldwide typically maintain electroactive microorganisms on soluble substrates, which often leads to a decrease or loss of the ability to effectively exchange electrons with solid electrode surfaces. In order to develop future sustainable technologies, we cannot rely solely on existing lab-isolates. Therefore, we must develop isolation strategies for environmental strains with electroactive properties superior to strains in culture collections. In this article, we provide an overview of the studies that isolated or enriched electroactive microorganisms from the environment using an anode as the sole electron acceptor (electricity-generating microorganisms) or a cathode as the sole electron donor (electricity-consuming microorganisms). Next, we recommend a selective strategy for the isolation of electroactive microorganisms. Furthermore, we provide a practical guide for setting up electrochemical reactors and highlight crucial electrochemical techniques to determine electroactivity and the mode of electron transfer in novel organisms.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Grembi2019,

title = {High-throughput multi-parallel enteropathogen quantification via nano-liter qPCR},

author = {Grembi, Jessica A. and Mayer-Blackwell, Koshlan and Luby, Stephen and Spormann, Alfred},

doi = {10.1101/746446},

year  = {2019},

date = {2019-08-24},

abstract = {Quantitative molecular diagnostic methods, such as qPCR, can effectively detect pathogen-specific nucleic acid sequences. However, costs associated with multi-pathogen quantitative molecular diagnostics hinder their widespread use. Nano-liter qPCR (nL-qPCR) is a miniaturized tool for quantification of multiple targets in large numbers of samples based on assay parallelization on a single chip, with potentially significant cost-savings due to rapid throughput and reduced reagent volumes. We evaluated a suite of novel and published assays to detect 17 enteric pathogens using a commercially available nL-qPCR technology. Assay efficiencies ranged from 88-98% (mean 91%) and were reproducible across four operators at two separate facilities. When applied to complex fecal material, assays were sensitive and selective (99.8% of DNA amplified were genes from the target organism). Detection limits were 1-2 orders of magnitude higher for nL-qPCR than an existing enteric TaqMan Array Card (TAC), due to nanofluidic volumes. Compared to the TAC, nL-qPCR displayed 97% (95% CI 0.96, 0.98) negative percent agreement and 63% (95% CI 0.60, 0.66) overall positive percent agreement. Positive percent agreement was 90% for target concentrations above the nL-qPCR detection limits. nL-qPCR assays showed an underestimation bias of 0.34 log10 copies/gram of stool [IQR -0.41, -0.28] compared with the enteric TAC. Higher detection limits, inherent to nL-qPCR, do not hinder detection of clinically relevant pathogen concentrations. With 12 times higher throughput for a sixth of the per-sample cost of the enteric TAC, the nL-qPCR chip described here is a viable alternative for enteropathogen quantification for studies where other technologies are cost-prohibitive.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Kracke2019,

title = {Robust and biocompatible catalysts for efficient hydrogen-driven microbial electrosynthesis},

author = {Kracke, Frauke and Wong, Andrew Barnabas and Maegaard, Karen and Deutzmann, Jörg and Hubert, McKenzie A. and Hahn, Christopher and Jaramillo, Thomas F. and Spormann, Alfred},

url = {https://www.nature.com/articles/s42004-019-0145-0},

doi = {/10.1038/s42004-019-0145-0},

year  = {2019},

date = {2019-04-12},

journal = {Nature Communications Chemistry},

volume = {2},

number = {45},

abstract = {CO2 reduction by combined electro- and bio-catalytic reactions is a promising technology platform for sustainable production of chemicals from CO2 and electricity. While heterogeneous electrocatalysts can reduce CO2 to a variety of organic compounds at relatively high reaction rates, these catalysts have limitations achieving high selectivity for any single product beyond CO. Conversely, microbial CO2 reduction pathways proceed at high selectivity; however, the rates at bio-cathodes using direct electron supply via electricity are commonly limiting. Here we demonstrate the use of non-precious metal cathodes that produce hydrogen in situ to support microbial CO2 reduction to C1 and C2 compounds. CoP, MoS2 and NiMo cathodes perform durable hydrogen evolution under biologically relevant conditions, and the integrated system achieves coulombic efficiencies close to 100% without accumulating hydrogen. Moreover, the one-reactor hybrid platform is successfully used for efficient acetate production from electricity and CO2 by microbes previously reported to be inactive in bioelectrochemical systems.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Milton2018,

title = {Methanococcus maripaludis employs three functional heterodisulfide reductase complexes for flavin-based electron bifurcation using hydrogen and formate},

author = {Milton, Ross D. and Ruth, John C. and Deutzmann, Jörg and Spormann, Alfred},

doi = {10.1021/acs.biochem.8b00662},

year  = {2018},

date = {2018-07-16},

journal = {Biochemistry},

abstract = {Hydrogenotrophic methanogens oxidize molecular hydrogen to reduce carbon dioxide to methane. In methanogens without cytochromes, the initial endergonic reduction of CO2 to formylmethanofuran with H2-derived electrons is coupled to the exergonic reduction of a heterodisulfide of coenzymes B and M, by flavin-based electron bifurcation (FBEB). In Methanococcus maripaludis, FBEB is performed by a heterodisulfide reductase (Hdr) enzyme complex that involves hydrogenase (Vhu), although formate dehydrogenase (Fdh) has been proposed as an alternative to Vhu. We have identified and purified three Hdr complexes of M. maripaludis, where homodimeric Hdr complexes containing (Vhu)2 or (Fdh)2 were found, in addition to a heterocomplex that contains both Vhu and Fdh. Formate was found in in vitro assays using purified Hdr complex to act directly as the electron donor for FBEB via the associated Fdh. Furthermore, while ferredoxin was slowly reduced to 30% (-360 mV vs. SHE) by H2 and formate (0.8 atm and 30 mM, according to thermodynamics), the addition of CoB-S-S-CoM as the high potential electron acceptor (E0’ = -140 mV vs. SHE; to induce FBEB) resulted in the rapid and more complete reduction of Fd to 94% (-455 mV vs. SHE).},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Karim2018,

title = {Fine-tuned Protein Production in Methanosarcina acetivorans C2A},

author = {Karim, Ann A. and Gestaut, Daniel R. and Fincker, Maeva and Ruth, John C. and Holmes, Eric and Sheu, Wayne and Spormann, Alfred},

doi = {10.1021/acssynbio.8b00062},

year  = {2018},

date = {2018-06-19},

journal = {ACS Synthetic Biology},

abstract = {Methanogenic archaea can be integrated into a sustainable, carbon-neutral cycle for producing organic chemicals from C1 compounds if the rate, yield, and titer of product synthesis can be improved using metabolic engineering. However, metabolic engineering techniques are limited in methanogens by insufficient methods for controlling cellular protein levels. We conducted a systematic approach to tune protein levels in Methanosarcina acetivorans C2A, a model methanogen, by regulating transcription and translation initiation. Rationally designed core promoter and ribosome binding site mutations in M. acetivorans C2A resulted in a predicable change in protein levels over a 60 fold range. The overall range of protein levels was increased an additional 3 fold by introducing the 5′ untranslated region of the mcrB transcript. This work demonstrates a wide range of precisely controlled protein levels in M. acetivorans C2A, which will help facilitate systematic metabolic engineering efforts in methanogens.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Lienemann2018,

title = {Mediator-free enzymatic electrosynthesis of formate by the Methanococcus maripaludis heterodisulfide reductase supercomplex},

author = {Lienemann, Michael and Deutzmann, Jörg and Milton, Ross and Sahin, Merve and Spormann, Alfred},

url = {https://doi.org/10.1016/j.biortech.2018.01.036},

year  = {2018},

date = {2018-01-09},

journal = {Bioresource Technology},

volume = {254},

pages = {278-283},

abstract = {Electrosynthesis of formate is a promising technology to convert CO2 and electricity from renewable sources into a biocompatible, soluble, non-flammable, and easily storable compound. In the model methanogen Methanococcus maripaludis, uptake of cathodic electrons was shown to proceed indirectly via formation of formate or H2 by undefined, cell-derived enzymes. Here, we identified that the multi-enzyme heterodisulfide reductase supercomplex (Hdr-SC) of M. maripaludis is capable of direct electron uptake and catalyzes rapid H2 and formate formation in electrochemical reactors (−800 mV vs Ag/AgCl) and in Fe(0) corrosion assays. In Fe(0) corrosion assays and electrochemical reactors, purified Hdr-SC primarily catalyzed CO2 reduction to formate with a coulombic efficiency of 90% in the electrochemical cells for 5 days. Thus, this report identified the first enzyme that stably catalyzes the mediator-free electrochemical reduction of CO2 to formate, which can serve as the basis of an enzyme electrode for sustained electrochemical production of formate.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Sewell2017,

title = {Homoacetogenesis in Deep-Sea Chloroflexi, as Inferred by Single-Cell Genomics, Provides a Link to Reductive Dehalogenation in Terrestrial Dehalococcoidetes},

author = {Sewell, Holly and Kaster, Anne-Kristen and Spormann, Alfred},

url = {https://mbio.asm.org/content/8/6/e02022-17.short},

doi = {10.1128/mBio.02022-17},

year  = {2017},

date = {2017-12-29},

journal = {mBio},

volume = {8},

number = {6},

pages = {e02022-17},

abstract = {The deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylum Chloroflexi. In this report, we investigated genomes of single cells obtained from deep-sea sediments of the Peruvian Margin, which are enriched in such Chloroflexi. 16S rRNA gene sequence analysis placed two of these single-cell-derived genomes (DscP3 and Dsc4) in a clade of subphylum I Chloroflexi which were previously recovered from deep-sea sediment in the Okinawa Trough and a third (DscP2-2) as a member of the previously reported DscP2 population from Peruvian Margin site 1230. The presence of genes encoding enzymes of a complete Wood-Ljungdahl pathway, glycolysis/gluconeogenesis, a Rhodobacter nitrogen fixation (Rnf) complex, glyosyltransferases, and formate dehydrogenases in the single-cell genomes of DscP3 and Dsc4 and the presence of an NADH-dependent reduced ferredoxin:NADP oxidoreductase (Nfn) and Rnf in the genome of DscP2-2 imply a homoacetogenic lifestyle of these abundant marine Chloroflexi. We also report here the first complete pathway for anaerobic benzoate oxidation to acetyl coenzyme A (CoA) in the phylum Chloroflexi (DscP3 and Dsc4), including a class I benzoyl-CoA reductase. Of remarkable evolutionary significance, we discovered a gene encoding a formate dehydrogenase (FdnI) with reciprocal closest identity to the formate dehydrogenase-like protein (complex iron-sulfur molybdoenzyme [CISM], DET0187) of terrestrial Dehalococcoides/Dehalogenimonas spp. This formate dehydrogenase-like protein has been shown to lack formate dehydrogenase activity in Dehalococcoides/Dehalogenimonas spp. and is instead hypothesized to couple HupL hydrogenase to a reductive dehalogenase in the catabolic reductive dehalogenation pathway. This finding of a close functional homologue provides an important missing link for understanding the origin and the metabolic core of terrestrial Dehalococcoides/Dehalogenimonas spp. and of reductive dehalogenation, as well as the biology of abundant deep-sea Chloroflexi.



IMPORTANCE The deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylum Chloroflexi. In this report, we investigated genomes of single cells obtained from deep-sea sediments and provide evidence for a homacetogenic lifestyle of these abundant marine Chloroflexi. Moreover, genome signature and key metabolic genes indicate an evolutionary relationship between these deep-sea sediment microbes and terrestrial, reductively dehalogenating Dehalococcoides.},

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pubstate = {published},

tppubtype = {article}

}

@article{Fincker2017,

title = {Biochemistry of Catabolic Reductive Dehalogenation},

author = {Fincker, Maeva and Spormann, Alfred},

url = {http://www.annualreviews.org/doi/abs/10.1146/annurev-biochem-061516-044829},

doi = {10.1146/annurev-biochem-061516-044829},

year  = {2017},

date = {2017-06-01},

journal = {Annual Review of Biochemistry},

volume = {86},

pages = {357-386},

abstract = {A wide range of phylogenetically diverse microorganisms couple the reductive dehalogenation of organohalides to energy conservation. Key enzymes of such anaerobic catabolic pathways are corrinoid and Fe–S cluster–containing, membrane-associated reductive dehalogenases. These enzymes catalyze the reductive elimination of a halide and constitute the terminal reductases of a short electron transfer chain. Enzymatic and physiological studies revealed the existence of quinone-dependent and quinone-independent reductive dehalogenases that are distinguishable at the amino acid sequence level, implying different modes of energy conservation in the respective microorganisms. In this review, we summarize current knowledge about catabolic reductive dehalogenases and the electron transfer chain they are part of. We review reaction mechanisms and the role of the corrinoid and Fe–S cluster cofactors and discuss physiological implications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Deutzmann2017,

title = {Enhanced microbial electrosynthesis by using defined co-cultures},

author = {Deutzmann, Jörg and Spormann, Alfred},

url = {https://www.nature.com/articles/ismej2016149.pdf},

doi = {10.1038/ismej.2016.149},

year  = {2017},

date = {2017-03-01},

journal = {The ISME Journal},

volume = {11},

pages = {704-714},

abstract = {Microbial uptake of free cathodic electrons presents a poorly understood aspect of microbial physiology. Uptake of cathodic electrons is particularly important in microbial electrosynthesis of sustainable fuel and chemical precursors using only CO2 and electricity as carbon, electron and energy source. Typically, large overpotentials (200 to 400 mV) were reported to be required for cathodic electron uptake during electrosynthesis of, for example, methane and acetate, or low electrosynthesis rates were observed. To address these limitations and to explore conceptual alternatives, we studied defined co-cultures metabolizing cathodic electrons. The Fe(0)-corroding strain IS4 was used to catalyze the electron uptake reaction from the cathode forming molecular hydrogen as intermediate, and Methanococcus maripaludis and Acetobacterium woodii were used as model microorganisms for hydrogenotrophic synthesis of methane and acetate, respectively. The IS4-M. maripaludis co-cultures achieved electromethanogenesis rates of 0.1–0.14 μmol cm−2 h−1 at −400 mV vs standard hydrogen electrode and 0.6–0.9 μmol cm−2 h−1 at −500 mV. Co-cultures of strain IS4 and A. woodii formed acetate at rates of 0.21–0.23 μmol cm−2 h−1 at −400 mV and 0.57–0.74 μmol cm−2 h−1 at −500 mV. These data show that defined co-cultures coupling cathodic electron uptake with synthesis reactions via interspecies hydrogen transfer may lay the foundation for an engineering strategy for microbial electrosynthesis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Mayer-Blackwell2016b,

title = {Survival of Vinyl Chloride Respiring Dehalococcoides mccartyi under Long-Term Electron Donor Limitation},

author = {Mayer-Blackwell, Koshlan and Azizian, Mohammad and Green, Jennifer and Spormann, Alfred and Semprini, Lewis},

url = {https://pubs.acs.org/doi/abs/10.1021/acs.est.6b05050},

doi = {10.1021/acs.est.6b05050},

year  = {2016},

date = {2016-12-21},

journal = {Environmental Science and Technology},

volume = {51},

number = {3},

pages = {1635-1642},

abstract = {In anoxic groundwater aquifers, the long-term survival of Dehalococcoides mccartyi populations expressing the gene vcrA (or bvcA) encoding reductive vinyl chloride dehalogenases are important to achieve complete dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE) to nonchlorinated ethene. The absence or inactivity of vcrA-containing Dehalococcoides results in the accumulation of the harmful chlorinated intermediates dichloroethene (DCE) and vinyl chloride (VC). Although vcrA-containing Dehalococcoides subpopulations depend on synergistic interaction with other organohalide-respiring populations generating their metabolic electron acceptors (DCE and VC), their survival requires successful competition for electron donor within the entire organohalide-respiring microbial community. To understand this dualism of synergy and competition under growth conditions relevant in contaminated aquifers, we investigated Dehalococcoides-level population structure when subjected to a change in the ratio of electron donor to chlorinated electron acceptor in continuously stirred tank reactors (CSTRs) operated over 7 years. When the electron donor formate was supplied in stoichiometric excess to TCE, both tceA-containing and vcrA-containing Dehalococcoides populations persisted, and near-complete dechlorination to ethene was stably maintained. When the electron donor formate was supplied at substoichiometric concentrations, the interactions between tceA-containing and vcrA-containing populations shifted toward direct competition for the same limiting catabolic electron donor substrate with subsequent niche exclusion of the vcrA-containing population. After more than 2000 days of operation under electron donor limitation, increasing the electron donor to TCE ratio facilitated a recovery of the vcrA-containing Dehalococoides population to its original frequency. We demonstrate that electron donor scarcity alone, in the absence of competing metabolic processes or inhibitory dechlorination intermediate products, is sufficient to alter the Dehalococcoides population structure. These results underscore the importance of electron donor and chloroethene stoichiometry in maintaining balanced functional performance within consortia composed of multiple D. mccartyi subpopulations, even when other competing electron acceptor processes are absent.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Mayer-Blackwell2016,

title = {1,2-Dichloroethane Exposure Alters the Population Structure, Metabolism, and Kinetics of a Trichloroethene-Dechlorinating Dehalococcoides mccartyi Consortium},

author = {Mayer-Blackwell, Koshlan and Fincker, Maeva and Molenda, Olivia and Callahan, Benjamin and Sewell, Holly and Holmes, Susan and Edwards, Elizabeth and Spormann, Alfred},

url = {http://pubs.acs.org/doi/abs/10.1021/acs.est.6b02957},

doi = {10.1021/acs.est.6b02957},

year  = {2016},

date = {2016-11-03},

journal = {Environmental Science & Technology},

volume = {50},

number = {22},

pages = {12187-12196},

abstract = {Bioremediation of groundwater contaminated with chlorinated aliphatic hydrocarbons such as perchloroethene and trichloroethene can result in the accumulation of the undesirable intermediate vinyl chloride. Such accumulation can either be due to the absence of specific vinyl chloride respiring Dehalococcoides mccartyi or to the inhibition of such strains by the metabolism of other microorganisms. The fitness of vinyl chloride respiring Dehalococcoides mccartyi subpopulations is particularly uncertain in the presence of chloroethene/chloroethane cocontaminant mixtures, which are commonly found in contaminated groundwater. Therefore, we investigated the structure of Dehalococcoides populations in a continuously fed reactor system under changing chloroethene/ethane influent conditions. We observed that increasing the influent ratio of 1,2-dichloroethane to trichloroethene was associated with ecological selection of a tceA-containing Dehalococcoides population relative to a vcrA-containing Dehalococcoides population. Although both vinyl chloride and 1,2-dichloroethane could be simultaneously transformed to ethene, prolonged exposure to 1,2-dichloroethane diminished the vinyl chloride transforming capacity of the culture. Kinetic tests revealed that dechlorination of 1,2-dichloroethane by the consortium was strongly inhibited by cis-dichloroethene but not vinyl chloride. Native polyacrylamide gel electrophoresis and mass spectrometry revealed that a trichloroethene reductive dehalogenase (TceA) homologue was the most consistently expressed of four detectable reductive dehalogenases during 1,2-dichloroethane exposure, suggesting that it catalyzes the reductive dihaloelimination of 1,2-dichloroethane to ethene.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Nazik2015,

title = {Effects of Iron Chelators on the Formation and Development of Aspergillus fumigatus Biofilm},

author = {Nazik, Hasan and Penner, John and Ferreira, Jose and Haagensen, Janus A.J. and Cohen, Kevin and Spormann, Alfred and Martinez, Marife and Chen, Vicky and Hsu, Joe and Clemons, Karl and Stephens, David},

url = {http://aac.asm.org/content/59/10/6514.short},

doi = {10.1128/AAC.01684-15},

year  = {2015},

date = {2015-10-01},

journal = {Antimicrobial Agents and Chemotherapy},

volume = {59},

number = {10},

pages = {6514-6520},

abstract = {Iron acquisition is crucial for the growth of Aspergillus fumigatus. A. fumigatus biofilm formation occurs in vitro and in vivo and is associated with physiological changes. In this study, we assessed the effects of Fe chelators on biofilm formation and development. Deferiprone (DFP), deferasirox (DFS), and deferoxamine (DFM) were tested for MIC against a reference isolate via a broth macrodilution method. The metabolic effects (assessed by XTT [2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt]) on biofilm formation by conidia were studied upon exposure to DFP, DFM, DFP plus FeCl3, or FeCl3 alone. A preformed biofilm was exposed to DFP with or without FeCl3. The DFP and DFS MIC50 against planktonic A. fumigatus was 1,250 μM, and XTT gave the same result. DFM showed no planktonic inhibition at concentrations of ≤2,500 μM. By XTT testing, DFM concentrations of <1,250 μM had no effect, whereas 2,500 μM increased biofilms forming in A. fumigatus or preformed biofilms (P < 0.01). DFP at 156 to 2,500 μM inhibited biofilm formation (P < 0.01 to 0.001) in a dose-responsive manner. Biofilm formation with 625 μM DFP plus any concentration of FeCl3 was lower than that in the controls (P < 0.05 to 0.001). FeCl3 at ≥625 μM reversed the DFP inhibitory effect (P < 0.05 to 0.01), but the reversal was incomplete compared to the controls (P < 0.05 to 0.01). For preformed biofilms, DFP in the range of ≥625 to 1,250 μM was inhibitory compared to the controls (P < 0.01 to 0.001). FeCl3 at ≥625 μM overcame inhibition by 625 μM DFP (P < 0.001). FeCl3 alone at ≥156 μM stimulated biofilm formation (P < 0.05 to 0.001). Preformed A. fumigatus biofilm increased with 2,500 μM FeCl3 only (P < 0.05). In a strain survey, various susceptibilities of biofilms of A. fumigatus clinical isolates to DFP were noted. In conclusion, iron stimulates biofilm formation and preformed biofilms. Chelators can inhibit or enhance biofilms. Chelation may be a potential therapy for A. fumigatus, but we show here that chelators must be chosen carefully. Individual isolate susceptibility assessments may be needed.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Ferreira2015,

title = {Inhibition of Aspergillus fumigatus and Its Biofilm by Pseudomonas aeruginosa Is Dependent on the Source, Phenotype and Growth Conditions of the Bacterium},

author = {Ferreira, Jose and Penner, John and Moss, Richard and Haagensen, Janus A.J. and Clemons, Karl and Spormann, Alfred and Nazik, Hasan and Cohen, Kevin and Banaei, Niaz and Carolino, Elisabete and Stephens, David},

url = {http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0134692},

doi = {10.1371/journal.pone.0134692},

year  = {2015},

date = {2015-08-07},

journal = {PLOS One},

abstract = {Aspergillus fumigatus (Af) and Pseudomonas aeruginosa (Pa) are leading fungal and bacterial pathogens, respectively, in many clinical situations. Relevant to this, their interface and co-existence has been studied. In some experiments in vitro, Pa products have been defined that are inhibitory to Af. In some clinical situations, both can be biofilm producers, and biofilm could alter their physiology and affect their interaction. That may be most relevant to airways in cystic fibrosis (CF), where both are often prominent residents.



We have studied clinical Pa isolates from several sources for their effects on Af, including testing involving their biofilms. We show that the described inhibition of Af is related to the source and phenotype of the Pa isolate. Pa cells inhibited the growth and formation of Af biofilm from conidia, with CF isolates more inhibitory than non-CF isolates, and non-mucoid CF isolates most inhibitory. Inhibition did not require live Pa contact, as culture filtrates were also inhibitory, and again non-mucoid>mucoid CF>non-CF. Preformed Af biofilm was more resistant to Pa, and inhibition that occurred could be reproduced with filtrates. Inhibition of Af biofilm appears also dependent on bacterial growth conditions; filtrates from Pa grown as biofilm were more inhibitory than from Pa grown planktonically. The differences in Pa shown from these different sources are consistent with the extensive evolutionary Pa changes that have been described in association with chronic residence in CF airways, and may reflect adaptive changes to life in a polymicrobial environment.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Haagensen2015,

title = {New In Vitro Model To Study the Effect of Human Simulated Antibiotic Concentrations on Bacterial Biofilms},

author = {Haagensen, Janus A.J. and Verotta, Davide and Huang, Liusheng and Spormann, Alfred},

url = {http://aac.asm.org/content/59/7/4074.short},

doi = {10.1128/AAC.05037-14},

year  = {2015},

date = {2015-07-01},

journal = {Antimicrobial Agents and Chemotherapy},

volume = {59},

number = {7},

pages = {4074-4081},

abstract = {A new in vitro pharmacokinetic/pharmacodynamic simulator for bacterial biofilms utilizing flow cell technology and confocal laser scanning microscopy is described. The device has the ability to simulate the changing antibiotic concentrations in humans associated with intravenous dosing on bacterial biofilms grown under continuous culture conditions. The free drug concentrations of a single 2-g meropenem intravenous bolus dose and first-order elimination utilizing a half-life of 0.895 h (elimination rate constant, 0.776 h−1) were simulated. The antibacterial activity of meropenem against biofilms of Pseudomonas aeruginosa PAO1 and three clinical strains isolated from patients with cystic fibrosis was investigated. Additionally, the effect of meropenem on PAO1 biofilms cultured for 24 h versus that on biofilms cultured for 72 h was examined. Using confocal laser scanning microscopy, rapid biofilm killing was observed in the first hour of the dosing interval for all biofilms. However, for PAO1 biofilms cultured for 72 h, only bacterial subpopulations at the periphery of the biofilm were affected, with subpopulations at the substratum remaining viable, even at the conclusion of the dosing interval. The described model is a novel method to investigate antimicrobial killing of bacterial biofilms using human simulated concentrations.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Deutzmann2015,

title = {Extracellular Enzymes Facilitate Electron Uptake in Biocorrosion and Bioelectrosynthesis},

author = {Deutzmann, Jörg and Sahin, Merve and Spormann, Alfred},

editor = {Harwood, Caroline},

url = {http://mbio.asm.org/content/6/2/e00496-15.short},

doi = {10.1128/mBio.00496-15},

year  = {2015},

date = {2015-04-21},

journal = {mBio},

volume = {6},

number = {2},

abstract = {Direct, mediator-free transfer of electrons between a microbial cell and a solid phase in its surrounding environment has been suggested to be a widespread and ecologically significant process. The high rates of microbial electron uptake observed during microbially influenced corrosion of iron [Fe(0)] and during microbial electrosynthesis have been considered support for a direct electron uptake in these microbial processes. However, the underlying molecular mechanisms of direct electron uptake are unknown. We investigated the electron uptake characteristics of the Fe(0)-corroding and electromethanogenic archaeon Methanococcus maripaludis and discovered that free, surface-associated redox enzymes, such as hydrogenases and presumably formate dehydrogenases, are sufficient to mediate an apparent direct electron uptake. In genetic and biochemical experiments, we showed that these enzymes, which are released from cells during routine culturing, catalyze the formation of H2 or formate when sorbed to an appropriate redox-active surface. These low-molecular-weight products are rapidly consumed by M. maripaludis cells when present, thereby preventing their accumulation to any appreciable or even detectable level. Rates of H2 and formate formation by cell-free spent culture medium were sufficient to explain the observed rates of methane formation from Fe(0) and cathode-derived electrons by wild-type M. maripaludis as well as by a mutant strain carrying deletions in all catabolic hydrogenases. Our data collectively show that cell-derived free enzymes can mimic direct extracellular electron transfer during Fe(0) corrosion and microbial electrosynthesis and may represent an ecologically important but so far overlooked mechanism in biological electron transfer.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{A2015,

title = {Biochemical and EPR-spectroscopic Investigation into Heterologously Expressed Vinyl Chloride Reductive Dehalogenase (VcrA) from Dehalococcoides mccartyi Strain VS.},

author = {Parthasarathy A and Stich TA and Lohner ST and Lesnefsky A and Britt RD and Spormann, Alfred},

url = {http://pubs.acs.org/doi/abs/10.1021/ja511653d},

doi = {10.1021/ja511653d},

year  = {2015},

date = {2015-03-18},

journal = {J Am Chem Soc.},

volume = {137},

number = {10},

pages = {3525-3532},

abstract = {Reductive dehalogenases play a critical role in the microbial detoxification of aquifers contaminated with chloroethenes and chlorethanes by catalyzing the reductive elimination of a halogen. We report here the first heterologous production of vinyl chloride reductase VcrA from Dehalococcoides mccartyi strain VS. Heterologously expressed VcrA was reconstituted to its active form by addition of hydroxocobalamin/adenosylcobalamin, Fe3+, and sulfide in the presence of mercaptoethanol. The kinetic properties of reconstituted VcrA catalyzing vinyl chloride reduction with Ti(III)-citrate as reductant and methyl viologen as mediator were similar to those obtained previously for VcrA as isolated from D. mccartyi strain VS. VcrA was also found to catalyze a novel reaction, the environmentally important dihaloelimination of 1,2-dichloroethane to ethene. Electron paramagnetic resonance (EPR) spectroscopic studies with reconstituted VcrA in the presence of mercaptoethanol revealed the presence of Cob(II)alamin. Addition of Ti(III)-citrate resulted in the appearance of a new signal characteristic of a reduced [4Fe–4S] cluster and the disappearance of the Cob(II)alamin signal. UV–vis absorption spectroscopy of Ti(III)citrate-treated samples revealed the formation of two new absorption maxima characteristic of Cob(I)alamin. No evidence for the presence of a [3Fe–4S] cluster was found. We postulate that during the reaction cycle of VcrA, a reduced [4Fe–4S] cluster reduces Co(II) to Co(I) of the enzyme-bound cobalamin. Vinyl chloride reduction to ethene would be initiated when Cob(I)alamin transfers an electron to the substrate, generating a vinyl radical as a potential reaction intermediate.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Woebken2014,

title = {Revisiting N2 fixation in Guerrero Negro intertidal microbial mats with a functional single-cell approach},

author = {Woebken, Dagmar and Burow, Luke and Behnam, Faris and Mayali, Xavier and Schintlmeister, Arno and Fleming, Erich and Prufert-Bebout, Leslie and Singer, Stephen and Cortes, Alejandro and Hoehler, Tori abd Pett-Ridge, Jennifer and Spormann, Alfred and Wagner, Michael and Weber, Peter and Bebout, Brad},

url = {https://www.nature.com/ismej/journal/v9/n2/abs/ismej2014144a.html},

doi = {10.1038/ismej.2014.144},

year  = {2014},

date = {2014-10-10},

journal = {The ISME Journal},

volume = {9},

pages = {485-496},

abstract = {Photosynthetic microbial mats are complex, stratified ecosystems in which high rates of primary production create a demand for nitrogen, met partially by N2 fixation. Dinitrogenase reductase (nifH) genes and transcripts from Cyanobacteria and heterotrophic bacteria (for example, Deltaproteobacteria) were detected in these mats, yet their contribution to N2 fixation is poorly understood. We used a combined approach of manipulation experiments with inhibitors, nifH sequencing and single-cell isotope analysis to investigate the active diazotrophic community in intertidal microbial mats at Laguna Ojo de Liebre near Guerrero Negro, Mexico. Acetylene reduction assays with specific metabolic inhibitors suggested that both sulfate reducers and members of the Cyanobacteria contributed to N2 fixation, whereas 15N2 tracer experiments at the bulk level only supported a contribution of Cyanobacteria. Cyanobacterial and nifH Cluster III (including deltaproteobacterial sulfate reducers) sequences dominated the nifH gene pool, whereas the nifH transcript pool was dominated by sequences related to Lyngbya spp. Single-cell isotope analysis of 15N2-incubated mat samples via high-resolution secondary ion mass spectrometry (NanoSIMS) revealed that Cyanobacteria were enriched in 15N, with the highest enrichment being detected in Lyngbya spp. filaments (on average 4.4 at% 15N), whereas the Deltaproteobacteria (identified by CARD-FISH) were not significantly enriched. We investigated the potential dilution effect from CARD-FISH on the isotopic composition and concluded that the dilution bias was not substantial enough to influence our conclusions. Our combined data provide evidence that members of the Cyanobacteria, especially Lyngbya spp., actively contributed to N2 fixation in the intertidal mats, whereas support for significant N2 fixation activity of the targeted deltaproteobacterial sulfate reducers could not be found.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Seedorf2014,

title = {Bacteria from Diverse Habitats Colonize and Compete in the Mouse Gut},

author = {Seedorf, Henning and Griffin, Nicholas and Ridaura, Vanessa and Reyes, Alejandro and Cheng, Jiye and Rey, Federico and Smith, Michelle and Simon, Gabriel and Scheffrahn, Rudolf and Woebken, Dagmar and Spormann, Alfred and Van Treuren, William and Ursell, Luke and Pirrung, Megan and Robbins-Pianka, Adam and Cantarel, Brandi and Lombard, Vincent and Henrissat, Bernard and Knight, Rob and Gordon, Jeffrey},

url = {http://www.sciencedirect.com/science/article/pii/S0092867414011568},

doi = {10.1016/j.cell.2014.09.008},

year  = {2014},

date = {2014-10-09},

journal = {Cell},

volume = {159},

number = {2},

pages = {253-266},

abstract = {To study how microbes establish themselves in a mammalian gut environment, we colonized germ-free mice with microbial communities from human, zebrafish, and termite guts, human skin and tongue, soil, and estuarine microbial mats. Bacteria from these foreign environments colonized and persisted in the mouse gut; their capacity to metabolize dietary and host carbohydrates and bile acids correlated with colonization success. Cohousing mice harboring these xenomicrobiota or a mouse cecal microbiota, along with germ-free “bystanders,” revealed the success of particular bacterial taxa in invading guts with established communities and empty gut habitats. Unanticipated patterns of ecological succession were observed; for example, a soil-derived bacterium dominated even in the presence of bacteria from other gut communities (zebrafish and termite), and human-derived bacteria colonized germ-free bystander mice before mouse-derived organisms. This approach can be generalized to address a variety of mechanistic questions about succession, including succession in the context of microbiota-directed therapeutics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Mayer-Blackwell2014,

title = {Nanoliter qPCR Platform for Highly Parallel, Quantitative Assessment of Reductive Dehalogenase Genes and Populations of Dehalogenating Microorganisms in Complex Environments},

author = {Mayer-Blackwell, Koshlan and Azizian, Mohammad and Machak, Christina and Vitale, Elena and Carpani, Giovanna and de Ferra, Francesca and Semprini, Lewis and Spormann, Alfred},

url = {http://pubs.acs.org/doi/abs/10.1021/es500918w},

doi = {10.1021/es500918w},

year  = {2014},

date = {2014-07-21},

journal = {Environmental Science & Technology},

volume = {48},

number = {16},

pages = {9659-9667},

abstract = {Idiosyncratic combinations of reductive dehalogenase (rdh) genes are a distinguishing genomic feature of closely related organohalogen-respiring bacteria. This feature can be used to deconvolute the population structure of organohalogen-respiring bacteria in complex environments and to identify relevant subpopulations, which is important for tracking interspecies dynamics needed for successful site remediation. Here we report the development of a nanoliter qPCR platform to identify organohalogen-respiring bacteria and populations by quantifying major orthologous reductive dehalogenase gene groups. The qPCR assays can be operated in parallel within a 5184-well nanoliter qPCR (nL-qPCR) chip at a single annealing temperature and buffer condition. We developed a robust bioinformatics approach to select from thousands of computationally proposed primer pairs those that are specific to individual rdh gene groups and compatible with a single amplification condition. We validated hundreds of the most selective qPCR assays and examined their performance in a trichloroethene-degrading bioreactor, revealing population structures as well as their unexpected shifts in abundance and community dynamics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Kaster2014,

title = {Single cell genomic study of Dehalococcoidetes species from deep-sea sediments of the Peruvian Margin},

author = {Kaster, Anne-Kristen and Mayer-Blackwell, Koshlan and Pasarelli, Ben and Spormann, Alfred},

url = {https://www.nature.com/ismej/journal/v8/n9/abs/ismej201424a.html},

doi = {10.1038/ismej.2014.24},

year  = {2014},

date = {2014-03-06},

journal = {The ISME Journal},

volume = {8},

pages = {1831-1842},

abstract = {The phylum Chloroflexi is one of the most frequently detected phyla in the subseafloor of the Pacific Ocean margins. Dehalogenating Chloroflexi (Dehalococcoidetes) was originally discovered as the key microorganisms mediating reductive dehalogenation via their key enzymes reductive dehalogenases (Rdh) as sole mode of energy conservation in terrestrial environments. The frequent detection of Dehalococcoidetes-related 16S rRNA and rdh genes in the marine subsurface implies a role for dissimilatory dehalorespiration in this environment; however, the two genes have never been linked to each other. To provide fundamental insights into the metabolism, genomic population structure and evolution of marine subsurface Dehalococcoidetes sp., we analyzed a non-contaminated deep-sea sediment core sample from the Peruvian Margin Ocean Drilling Program (ODP) site 1230, collected 7.3 m below the seafloor by a single cell genomic approach. We present for the first time single cell genomic data on three deep-sea Chloroflexi (Dsc) single cells from a marine subsurface environment. Two of the single cells were considered to be part of a local Dehalococcoidetes population and assembled together into a 1.38-Mb genome, which appears to be at least 85% complete. Despite a high degree of sequence-level similarity between the shared proteins in the Dsc and terrestrial Dehalococcoidetes, no evidence for catabolic reductive dehalogenation was found in Dsc. The genome content is however consistent with a strictly anaerobic organotrophic or lithotrophic lifestyle.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Lee2014,

title = {Fermentation couples Chloroflexi and sulfate-reducing bacteria to Cyanobacteria in hypersaline microbial mats.},

author = {Lee, JZ and Burow, LC and Woebken, Dagmar and Everroad, RC and Kubo, MD and Spormann, Alfred and Weber, Peter and Pett-Ridge, Jennifer and Bebout, Brad and Hoehler, Tori},

url = {https://www.ncbi.nlm.nih.gov/m/pubmed/24616716/},

doi = {10.3389/fmicb.2014.00061},

year  = {2014},

date = {2014-02-26},

journal = {Frontiers in Microbiology},

abstract = {Past studies of hydrogen cycling in hypersaline microbial mats have shown an active nighttime cycle, with production largely from Cyanobacteria and consumption from sulfate-reducing bacteria (SRB). However, the mechanisms and magnitude of hydrogen cycling have not been extensively studied. Two mats types near Guerrero Negro, Mexico-permanently submerged Microcoleus microbial mat (GN-S), and intertidal Lyngbya microbial mat (GN-I)-were used in microcosm diel manipulation experiments with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), molybdate, ammonium addition, and physical disruption to understand the processes responsible for hydrogen cycling between mat microbes. Across microcosms, H2 production occurred under dark anoxic conditions with simultaneous production of a suite of organic acids. H2 production was not significantly affected by inhibition of nitrogen fixation, but rather appears to result from constitutive fermentation of photosynthetic storage products by oxygenic phototrophs. Comparison to accumulated glycogen and to CO2 flux indicated that, in the GN-I mat, fermentation released almost all of the carbon fixed via photosynthesis during the preceding day, primarily as organic acids. Across mats, although oxygenic and anoxygenic phototrophs were detected, cyanobacterial [NiFe]-hydrogenase transcripts predominated. Molybdate inhibition experiments indicated that SRBs from a wide distribution of DsrA phylotypes were responsible for H2 consumption. Incubation with (13)C-acetate and NanoSIMS (secondary ion mass-spectrometry) indicated higher uptake in both Chloroflexi and SRBs relative to other filamentous bacteria. These manipulations and diel incubations confirm that Cyanobacteria were the main fermenters in Guerrero Negro mats and that the net flux of nighttime fermentation byproducts (not only hydrogen) was largely regulated by the interplay between Cyanobacteria, SRBs, and Chloroflexi.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Marshall2014,

title = {Inferring community dynamics of organohalide-respiring bacteria in chemostats by covariance of rdhA gene abundance},

author = {Marshall, Ian and Azizian, Mohammad and Semprini, Lewis and Spormann, Alfred},

url = {https://academic.oup.com/femsec/article/87/2/428/481878},

doi = {10.1111/1574-6941.12235},

year  = {2014},

date = {2014-02-01},

journal = {FEMS Microbiology Ecology},

volume = {87},

number = {2},

pages = {428-440},

abstract = {We have developed a novel approach to identifying and quantifying closely related organohalide-respiring bacteria. Our approach made use of the unique genomic associations of specific reductive dehalogenase subunit A encoding genes (rdhA) that exist in known strains of Dehalococcoides mccartyi and Desulfitobacterium and the distinguishing covariance pattern of observed rdhA genes to assign genes to unknown strains. To test this approach, we operated five anaerobic reductively dechlorinating chemostats for 3–4 years with tetrachloroethene and trichloroethene as terminal electron acceptors and lactate/formate as electron donors. The presence and abundance of rdhA genes were determined comprehensively at the community level using a custom-developed Reductive Dehalogenase Chip (RDH Chip) DNA microarray and used to define putative strains of Dehalococcoides mccartyi and Desulfitobacterium sp. This monitoring revealed that stable chemical performance of chemostats was reflected by a stable community of reductively dechlorinating bacteria. However, perturbations introduced by, for example, electron donor limitation or addition of the competing electron acceptor sulfate led to overall changes in the chemostat performance, including incomplete reduction in the chloroethene substrates, and in the population composition of reductively dehalogenating bacteria. Interestingly, there was a high diversity of operationally defined D. mccartyi strains between the chemostats with almost all strains unique to their specific chemostats in spite of similar selective pressure and similar inocula shared between chemostats.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Jew2013,

title = {Microbially enhanced dissolution of HgS in an acid mine drainage system in the California Coast Range},

author = {Jew, A.D. and Behrens, S.F. and Rytuba, J.J. and Kappler, A. and Spormann, Alfred and Brown Jr., G.E.},

url = {http://onlinelibrary.wiley.com/doi/10.1111/gbi.12066/full},

doi = {10.1111/gbi.12066},

year  = {2013},

date = {2013-11-13},

journal = {Geobiology},

volume = {12},

number = {1},

pages = {20-33},

abstract = {Mercury sulfides (cinnabar and metacinnabar) are the main ores of Hg and are relatively stable under oxic conditions (Ksp = 10−54 and 10−52, respectively). However, until now their stability in the presence of micro-organisms inhabiting acid mine drainage (AMD) systems was unknown. We tested the effects of the AMD microbial community from the inoperative Hg mine at New Idria, CA, present in sediments of an AMD settling pond adjacent to the main waste pile and in a microbial biofilm on the surface of this pond, on the solubility of crystalline HgS. A 16S rRNA gene clone library revealed that the AMD microbial community was dominated by Fe-oxidizing (orders Ferritrophicales and Gallionellas) and S-oxidizing bacteria (Thiomonas sp.), with smaller amounts (≤6%) being comprised of the orders Xanthomondales and Rhodospirillales. Though the order Ferritrophicales dominate the 16S rRNA clones (>60%), qPCR results of the microbial community indicate that the Thiomonas sp. represents ~55% of the total micro-organisms in the top 1 cm of the AMD microbial community. Although supersaturated with respect to cinnabar and metacinnabar, microcosms inoculated with the AMD microbial community were capable of releasing significantly more Hg into solution compared to inactivated or abiotic controls. Four different Hg-containing materials were tested for bacterially enhanced HgS dissolution: pure cinnabar, pure metacinnabar, mine tailings, and calcine material (processed ore). In the microcosm with metacinnabar, the presence of the AMD microbial community resulted in an increase of dissolved Hg concentrations up to 500 μg L-1 during the first 30 days of incubation. In abiotic control microcosms, dissolved Hg concentrations did not increase above 100 ng L−1. When Hg concentrations were below 50 μg L-1, the Fe-oxidizing bacteria in the AMD microbial community were still capable of oxidizing Fe(II) to Fe(III) in the AMD solution, whereas concentrations above 50 μg L−1 resulted in inhibition of microbial iron oxidation. Our experiments show that the AMD microbial community contributes to the dissolution of mercury sulfide minerals. These findings have major implications for risk assessment and future management of inoperative Hg mines worldwide.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Lohner2013,

title = {Identification of a reductive tetrachloroethene dehalogenase in Shewanella sediminis},

author = {Lohner, ST and Spormann, Alfred},

year  = {2013},

date = {2013-10-01},

journal = {The Royal Society of Biological Sciences},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Chao2013,

title = {PdeB, a Cyclic Di-GMP-Specific Phosphodiesterase That Regulates Shewanella oneidensis MR-1 Motility and Biofilm Formation},

author = {Chao, Lily and Rakshe, Shauna and Leff, Maija and Spormann, Alfred},

url = {http://jb.asm.org/content/195/17/3827.short},

doi = {10.1128/JB.00498-13},

year  = {2013},

date = {2013-09-01},

journal = {Journal of Bacteriology},

volume = {195},

number = {17},

pages = {3827-3833},

abstract = {Shewanella oneidensis MR-1, a gammaproteobacterium with respiratory versatility, forms biofilms on mineral surfaces through a process controlled by the cyclic dinucleotide messenger c-di-GMP. Cellular concentrations of c-di-GMP are maintained by proteins containing GGDEF and EAL domains, which encode diguanylate cyclases for c-di-GMP synthesis and phosphodiesterases for c-di-GMP hydrolysis, respectively. The S. oneidensis MR-1 genome encodes several GGDEF and EAL domain proteins (50 and 31, respectively), with a significant fraction (∼10) predicted to be multidomain (e.g., GGDEF-EAL) enzymes containing an additional Per-Arnt-Sim (PAS) sensor domain. However, the biochemical activities and physiological functions of these multidomain enzymes remain largely unknown. Here, we present genetic and biochemical analyses of a predicted PAS-GGDEF-EAL domain-containing protein, SO0437, here named PdeB. A pdeB deletion mutant exhibited decreased swimming motility and increased biofilm formation under rich growth medium conditions, which was consistent with an increase in intracellular c-di-GMP. A mutation inactivating the EAL domain also produced similar swimming and biofilm phenotypes, indicating that the increase in c-di-GMP was likely due to a loss in phosphodiesterase activity. Therefore, we also examined the enzymatic activity of purified PdeB and found that the protein exhibited phosphodiesterase activity via the EAL domain. No diguanylate cyclase activity was observed. In addition to the motility and biofilm phenotypes, transcriptional profiling by DNA microarray analysis of biofilms of pdeB (in-frame deletion and EAL) mutant cells revealed that expression of genes involved in sulfate uptake and assimilation were repressed. Addition of sulfate to the growth medium resulted in significantly less motile pdeB mutants. Together, these results indicate a link between c-di-GMP metabolism, S. oneidensis MR-1 biofilm development, and sulfate uptake/assimilation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Marcobal2013,

title = {A metabolomic view of how the human gut microbiota impacts the host metabolome using humanized and gnotobiotic mice},

author = {Marcobal, A and Kashyap, P and Nelson, T and Aronov, P and Donia, M and Spormann, Alfred and Fischbach, M and Sonnenburg, J},

url = {https://www.nature.com/ismej/journal/v7/n10/abs/ismej201389a.html},

doi = {10.1038/ismej.2013.89},

year  = {2013},

date = {2013-06-06},

journal = {The ISME Journal},

volume = {7},

pages = {1933-1943},

abstract = {Defining the functional status of host-associated microbial ecosystems has proven challenging owing to the vast number of predicted genes within the microbiome and relatively poor understanding of community dynamics and community–host interaction. Metabolomic approaches, in which a large number of small molecule metabolites can be defined in a biological sample, offer a promising avenue to ‘fingerprint’ microbiota functional status. Here, we examined the effects of the human gut microbiota on the fecal and urinary metabolome of a humanized (HUM) mouse using an optimized ultra performance liquid chromatography–mass spectrometry-based method. Differences between HUM and conventional mouse urine and fecal metabolomic profiles support host-specific aspects of the microbiota’s metabolomic contribution, consistent with distinct microbial compositions. Comparison of microbiota composition and metabolome of mice humanized with different human donors revealed that the vast majority of metabolomic features observed in donor samples are produced in the corresponding HUM mice, and individual-specific features suggest ‘personalized’ aspects of functionality can be reconstituted in mice. Feeding the mice a defined, custom diet resulted in modification of the metabolite signatures, illustrating that host diet provides an avenue for altering gut microbiota functionality, which in turn can be monitored via metabolomics. Using a defined model microbiota consisting of one or two species, we show that simplified communities can drive major changes in the host metabolomic profile. Our results demonstrate that metabolomics constitutes a powerful avenue for functional characterization of the intestinal microbiota and its interaction with the host.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Muller2013,

title = {The mxd operon in Shewanella oneidensis MR-1 is induced in response to starvation and regulated by ArcS/ArcA and BarA/UvrY},

author = {Muller, Jana and Shukla, Soni and Jost, Kathinka and Spormann, Alfred},

url = {https://bmcmicrobiol.biomedcentral.com/articles/10.1186/1471-2180-13-119},

doi = {10.1186/1471-2180-13-119},

year  = {2013},

date = {2013-05-27},

journal = {BMC Microbiology},

volume = {13},

number = {119},

abstract = {Background

S. oneidensis MR-1 is a dissimilatory metal-reducing bacterium. Under anoxic conditions S. oneidensis MR-1 attaches to and uses insoluble minerals such as Fe(III) and Mn(IV) oxides as electron acceptors. In the laboratory, S. oneidensis MR-1 forms biofilms under hydrodynamic flow conditions on a borosilicate glass surface; formation of biofilms was previously found to be dependent on the mxd gene cluster (mxdABCD).



Results

This study revealed environmental and genetic factors regulating expression of the mxd genes in S. oneidensis MR-1. Physiological experiments conducted with a S. oneidensis MR-1 strain carrying a transcriptional lacZ fusion to the mxd promoter identified electron donor starvation as a key factor inducing mxd gene expression. Tn5 mutagenesis identified the ArcS/ArcA two-component signaling system as a repressor of mxd expression in S. oneidensis MR-1 under planktonic conditions. Biofilms of ∆arcS and ∆arcA strains carrying a transcriptional gfp -reporter fused to the mxd promoter revealed a reduced mxd expression, suggesting that ArcS/ArcA are necessary for activation of mxd expression under biofilm conditions. Biofilms of ∆arcS and ∆arcA mutants were unable to form a compact three-dimensional structure consistent with a low level of mxd expression. In addition, BarA/UvrY was identified as a major regulator of mxd expression under planktonic conditions. Interestingly, biofilms of ∆barA and ∆uvrY mutants were able to form three-dimensional structures that were, however, less compact compared to wild type biofilms.



Conclusions

We have shown here that the mxd genes in S. oneidensis MR-1 are controlled transcriptionally in response to carbon starvation and by the ArcS/ArcA and the BarA/UvrY signaling system. BarA might function as a sensor to assess the metabolic state of the cell, including carbon starvation, leading to expression of the mxd operon and therefore control biofilm formation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Loffler2013,

title = {Dehalococcoides mccartyi gen. nov., sp. nov., obligately organohalide-respiring anaerobic bacteria relevant to halogen cycling and bioremediation, belong to a novel bacterial class, Dehalococcoidia classis nov., order Dehalococcoidales ord. nov. and family Dehalococcoidaceae fam. nov., within the phylum Chloroflexi},

author = {Loffler, Frank and Yan, Jun and Ritalahti, Kirsti and Adrian, Lorenz and Edwards, Elizabeth and Konstantinidis, Konstantinos and Muller, Jochen and Fullerton, Heather and Zinder, Stephen and Spormann, Alfred},

url = {http://ijs.microbiologyresearch.org/content/journal/ijsem/10.1099/ijs.0.034926-0},

doi = {10.1099/ijs.0.034926-0},

year  = {2013},

date = {2013-02-01},

journal = {International Journal of Systematic and Evolutionary Microbiology},

volume = {63},

pages = {625-635},

abstract = {Six obligately anaerobic bacterial isolates (195T, CBDB1, BAV1, VS, FL2 and GT) with strictly organohalide-respiring metabolisms were obtained from chlorinated solvent-contaminated aquifers, contaminated and uncontaminated river sediments or anoxic digester sludge. Cells were non-motile with a disc-shaped morphology, 0.3–1 µm in diameter and 0.1–0.2 µm thick, and characteristic indentations on opposite flat sides of the cell. Growth occurred in completely synthetic, reduced medium amended with a haloorganic electron acceptor (mostly chlorinated but also some brominated compounds), hydrogen as electron donor, acetate as carbon source, and vitamins. No other growth-supporting redox couples were identified. Aqueous hydrogen consumption threshold concentrations were <1 nM. Growth ceased when vitamin B12 was omitted from the medium. Addition of sterile cell-free supernatant of Dehalococcoides-containing enrichment cultures enhanced dechlorination and growth of strains 195 and FL2, suggesting the existence of so-far unidentified stimulants. Dechlorination occurred between pH 6.5 and 8.0 and over a temperature range of 15–35 °C, with an optimum growth temperature between 25 and 30 °C. The major phospholipid fatty acids were 14 : 0 (15.7 mol%), br15 : 0 (6.2 mol%), 16 : 0 (22.7 mol%), 10-methyl 16 : 0 (25.8 mol%) and 18 : 0 (16.6 mol%). Unusual furan fatty acids including 9-(5-pentyl-2-furyl)-nonanoate and 8-(5-hexyl-2-furyl)-octanoate were detected in strains FL2, BAV1 and GT, but not in strains 195T and CBDB1. The 16S rRNA gene sequences of the six isolates shared more than 98 % identity, and phylogenetic analysis revealed an affiliation with the phylum Chloroflexi and more than 10 % sequence divergence from other described isolates. The genome sizes and G+C contents ranged from 1.34 to 1.47 Mbp and 47 to 48.9 mol% G+C, respectively. Based on 16S rRNA gene sequence comparisons, genome-wide average nucleotide identity and phenotypic characteristics, the organohalide-respiring isolates represent a new genus and species, for which the name Dehalococcoides mccartyi gen. nov., sp. nov. is proposed. Isolates BAV1 ( = ATCC BAA-2100  = JCM 16839  = KCTC 5957), FL2 ( = ATCC BAA-2098  = DSM 23585  = JCM 16840  = KCTC 5959), GT ( = ATCC BAA-2099  = JCM 16841  = KCTC 5958), CBDB1, 195T ( = ATCC BAA-2266T  = KCTC 15142T) and VS are considered strains of Dehalococcoides mccartyi, with strain 195T as the type strain. The new class Dehalococcoidia classis nov., order Dehalococcoidales ord. nov. and family Dehalococcoidaceae fam. nov. are described to accommodate the new taxon.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Berggren2013,

title = {Effects of Sulfate Reduction on the Bacterial Community and Kinetic Parameters of a Dechlorinating Culture under Chemostat Growth Conditions},

author = {Berggren, Dusty and Marshall, Ian and Azizian, Mohammad and Spormann, Alfred and Semprini, Lewis},

url = {http://pubs.acs.org/doi/abs/10.1021/es304244z},

doi = {10.1021/es304244z},

year  = {2013},

date = {2013-01-14},

journal = {Environmental Science & Technology},

volume = {47},

number = {4},

pages = {1879-1886},

abstract = {Results are presented from a chemostat study where the reductive dehalogenation of PCE was evaluated in the absence and presence of sulfate. Two chemostats inoculated with the Point Mugu culture, which contains strains of Dehalococcoides mccartyi, were operated at a 50 day HRT and fed PCE (1.12 mM) and lactate (4.3 mM). The control chemostat (PM-5L, no sulfate), achieved pseudo-steady-state transformation of PCE to ethene (98%) and VC (2%) at 2.4 nM of H2. Batch kinetic tests with chemostat harvested cells showed the maximum rate (kmaxX) value for each dehalogenation step remained fairly constant, while hupL clone library analyses showed maintenance of a diverse D. mccartyi community. Sulfate (1 mM) was introduced to the second chemostat, PM-2L. Effective sulfate reduction was achieved 110 days later, resulting in 600 μM of total sulfide. PCE dechlorination efficiency decreased following complete sulfate reduction, yielding ethene (25%), VC (67%), and cis-DCE (8%). VC dechlorination was most affected, with kmaxX values decreasing by a factor of 50. The decrease was associated with the enrichment of the Cornell group of D. mccartyi and decline of the Pinellas group. Long-term exposure to sulfides and/or competition for H2 may have been responsible for the community shift.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Burow2012,

title = {Anoxic carbon flux in photosynthetic microbial mats as revealed by metatranscriptomics},

author = {Burow, Luke and Woebken, Dagmar and Marshall, Ian and Lindquist, Erika and Bebout, Brad and Prufert-Bebout, Leslie and Hoehler, Tori and Tringe, Susannah and Pett-Ridge, Jennifer and Weber, Peter and Spormann, Alfred and Singer, Stephen},

url = {https://www.nature.com/ismej/journal/v7/n4/abs/ismej2012150a.html},

doi = {10.1038/ismej.2012.150},

year  = {2012},

date = {2012-11-29},

journal = {The ISME Journal},

volume = {7},

pages = {817-829},

abstract = {Photosynthetic microbial mats possess extraordinary phylogenetic and functional diversity that makes linking specific pathways with individual microbial populations a daunting task. Close metabolic and spatial relationships between Cyanobacteria and Chloroflexi have previously been observed in diverse microbial mats. Here, we report that an expressed metabolic pathway for the anoxic catabolism of photosynthate involving Cyanobacteria and Chloroflexi in microbial mats can be reconstructed through metatranscriptomic sequencing of mats collected at Elkhorn Slough, Monterey Bay, CA, USA. In this reconstruction, Microcoleus spp., the most abundant cyanobacterial group in the mats, ferment photosynthate to organic acids, CO2 and H2 through multiple pathways, and an uncultivated lineage of the Chloroflexi take up these organic acids to store carbon as polyhydroxyalkanoates. The metabolic reconstruction is consistent with metabolite measurements and single cell microbial imaging with fluorescence in situ hybridization and NanoSIMS.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Marshall2012,

title = {A Single-Cell Genome for Thiovulum sp.},

author = {Marshall, Ian and Blainey, Paul and Spormann, Alfred and Quake, Stephen},

url = {http://aem.asm.org/content/78/24/8555.short},

doi = {10.1128/AEM.02314-12},

year  = {2012},

date = {2012-09-28},

journal = {Applied and Environmental Microbiology},

volume = {78},

number = {24},

pages = {8555-8563},

abstract = {We determined a significant fraction of the genome sequence of a representative of Thiovulum, the uncultivated genus of colorless sulfur Epsilonproteobacteria, by analyzing the genome sequences of four individual cells collected from phototrophic mats from Elkhorn Slough, California. These cells were isolated utilizing a microfluidic laser-tweezing system, and their genomes were amplified by multiple-displacement amplification prior to sequencing. Thiovulum is a gradient bacterium found at oxic-anoxic marine interfaces and noted for its distinctive morphology and rapid swimming motility. The genomic sequences of the four individual cells were assembled into a composite genome consisting of 221 contigs covering 2.083 Mb including 2,162 genes. This single-cell genome represents a genomic view of the physiological capabilities of isolated Thiovulum cells. Thiovulum is the second-fastest bacterium ever observed, swimming at 615 μm/s, and this genome shows that this rapid swimming motility is a result of a standard flagellar machinery that has been extensively characterized in other bacteria. This suggests that standard flagella are capable of propelling bacterial cells at speeds much faster than typically thought. Analysis of the genome suggests that naturally occurring Thiovulum populations are more diverse than previously recognized and that studies performed in the past probably address a wide range of unrecognized genotypic and phenotypic diversities of Thiovulum. The genome presented in this article provides a basis for future isolation-independent studies of Thiovulum, where single-cell and metagenomic tools can be used to differentiate between different Thiovulum genotypes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Woebken2012,

title = {Identification of a novel cyanobacterial group as active diazotrophs in a coastal microbial mat using NanoSIMS analysis},

author = {Woebken, Dagmar and Burow, Luke and Prufert-Bebout, Leslie and Bebout, Brad and Hoehler, Tori and Pett-Ridge, Jennifer and Spormann, Alfred and Weber, Peter and Singer, Stephen},

url = {https://www.nature.com/ismej/journal/v6/n7/abs/ismej2011200a.html},

doi = {10.1038/ismej.2011.200},

year  = {2012},

date = {2012-01-12},

journal = {The ISME Journal},

volume = {6},

pages = {1427-1439},

abstract = {N2 fixation is a key process in photosynthetic microbial mats to support the nitrogen demands associated with primary production. Despite its importance, groups that actively fix N2 and contribute to the input of organic N in these ecosystems still remain largely unclear. To investigate the active diazotrophic community in microbial mats from the Elkhorn Slough estuary, Monterey Bay, CA, USA, we conducted an extensive combined approach, including biogeochemical, molecular and high-resolution secondary ion mass spectrometry (NanoSIMS) analyses. Detailed analysis of dinitrogenase reductase (nifH) transcript clone libraries from mat samples that fixed N2 at night indicated that cyanobacterial nifH transcripts were abundant and formed a novel monophyletic lineage. Independent NanoSIMS analysis of 15N2-incubated samples revealed significant incorporation of 15N into small, non-heterocystous cyanobacterial filaments. Mat-derived enrichment cultures yielded a unicyanobacterial culture with similar filaments (named Elkhorn Slough Filamentous Cyanobacterium-1 (ESFC-1)) that contained nifH gene sequences grouping with the novel cyanobacterial lineage identified in the transcript clone libraries, displaying up to 100% amino-acid sequence identity. The 16S rRNA gene sequence recovered from this enrichment allowed for the identification of related sequences from Elkhorn Slough mats and revealed great sequence diversity in this cluster. Furthermore, by combining 15N2 tracer experiments, fluorescence in situ hybridization and NanoSIMS, in situ N2 fixation activity by the novel ESFC-1 group was demonstrated, suggesting that this group may be the most active cyanobacterial diazotroph in the Elkhorn Slough mat. Pyrotag sequences affiliated with ESFC-1 were recovered from mat samples throughout 2009, demonstrating the prevalence of this group. This work illustrates that combining standard and single-cell analyses can link phylogeny and function to identify previously unknown key functional groups in complex ecosystems.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Burow2011,

title = {Hydrogen production in photosynthetic microbial mats in the Elkhorn Slough estuary, Monterey Bay},

author = {Burow, Luke and Woebken, Dagmar and Bebout, Brad and McMurdie, Paul and Singer, Stephen and Pett-Ridge, Jennifer and Prufert-Bebout, Leslie and Spormann, Alfred and Weber, Peter and Hoehler, Tori},

url = {https://www.nature.com/ismej/journal/v6/n4/abs/ismej2011142a.html},

doi = {10.1038/ismej.2011.142},

year  = {2011},

date = {2011-10-20},

journal = {The ISME Journal},

volume = {6},

pages = {863-874},

abstract = {Hydrogen (H2) release from photosynthetic microbial mats has contributed to the chemical evolution of Earth and could potentially be a source of renewable H2 in the future. However, the taxonomy of H2-producing microorganisms (hydrogenogens) in these mats has not been previously determined. With combined biogeochemical and molecular studies of microbial mats collected from Elkhorn Slough, Monterey Bay, California, we characterized the mechanisms of H2 production and identified a dominant hydrogenogen. Net production of H2 was observed within the upper photosynthetic layer (0–2 mm) of the mats under dark and anoxic conditions. Pyrosequencing of rRNA gene libraries generated from this layer demonstrated the presence of 64 phyla, with Bacteriodetes, Cyanobacteria and Proteobacteria dominating the sequences. Sequencing of rRNA transcripts obtained from this layer demonstrated that Cyanobacteria dominated rRNA transcript pyrotag libraries. An OTU affiliated to Microcoleus spp. was the most abundant OTU in both rRNA gene and transcript libraries. Depriving mats of sunlight resulted in an order of magnitude decrease in subsequent nighttime H2 production, suggesting that newly fixed carbon is critical to H2 production. Suppression of nitrogen (N2)-fixation in the mats did not suppress H2 production, which indicates that co-metabolic production of H2 during N2-fixation is not an important contributor to H2 production. Concomitant production of organic acids is consistent with fermentation of recently produced photosynthate as the dominant mode of H2 production. Analysis of rRNA % transcript:% gene ratios and H2-evolving bidirectional [NiFe] hydrogenase % transcript:% gene ratios indicated that Microcoelus spp. are dominant hydrogenogens in the Elkhorn Slough mats.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Marshall2011,

title = {The Hydrogenase Chip: a tiling oligonucleotide DNA microarray technique for characterizing hydrogen-producing and -consuming microbes in microbial communities},

author = {Marshall, Ian and Berggren, Dusty and Azizian, Mohammad and Burow, Luke and Semprini, Lewis and Spormann, Alfred},

url = {https://www.nature.com/ismej/journal/v6/n4/abs/ismej2011136a.html},

doi = {10.1038/ismej.2011.136},

year  = {2011},

date = {2011-10-13},

journal = {The ISME Journal},

volume = {6},

pages = {814-826},

abstract = {We developed a broad-ranging method for identifying key hydrogen-producing and consuming microorganisms through analysis of hydrogenase gene content and expression in complex anaerobic microbial communities. The method is based on a tiling hydrogenase gene oligonucleotide DNA microarray (Hydrogenase Chip), which implements a high number of probes per gene by tiling probe sequences across genes of interest at 1.67 × –2 × coverage. This design favors the avoidance of false positive gene identification in samples of DNA or RNA extracted from complex microbial communities. We applied this technique to interrogate interspecies hydrogen transfer in complex communities in (i) lab-scale reductive dehalogenating microcosms enabling us to delineate key H2-consuming microorganisms, and (ii) hydrogen-generating microbial mats where we found evidence for significant H2 production by cyanobacteria. Independent quantitative PCR analysis on selected hydrogenase genes showed that this Hydrogenase Chip technique is semiquantitative. We also determined that as microbial community complexity increases, specificity must be traded for sensitivity in analyzing data from tiling DNA microarrays.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{McMurdie2011,

title = {Site-Specific Mobilization of Vinyl Chloride Respiration Islands by a Mechanism Common in Dehalococcoides},

author = {McMurdie, Paul and Hug, Laura and Edwards, Elizabeth and Holmes, Susan and Spormann, Alfred},

url = {https://bmcgenomics.biomedcentral.com/articles/10.1186/1471-2164-12-287},

doi = {10.1186/1471-2164-12-287},

year  = {2011},

date = {2011-06-02},

journal = {BMC Genomics},

volume = {12},

number = {287},

abstract = {Background

Vinyl chloride is a widespread groundwater pollutant and Group 1 carcinogen. A previous comparative genomic analysis revealed that the vinyl chloride reductase operon, vcrABC, of Dehalococcoides sp. strain VS is embedded in a horizontally-acquired genomic island that integrated at the single-copy tmRNA gene, ssrA.



Results

We targeted conserved positions in available genomic islands to amplify and sequence four additional vcrABC -containing genomic islands from previously-unsequenced vinyl chloride respiring Dehalococcoides enrichments. We identified a total of 31 ssrA-specific genomic islands from Dehalococcoides genomic data, accounting for 47 reductive dehalogenase homologous genes and many other non-core genes. Sixteen of these genomic islands contain a syntenic module of integration-associated genes located adjacent to the predicted site of integration, and among these islands, eight contain vcrABC as genetic 'cargo'. These eight vcrABC -containing genomic islands are syntenic across their ~12 kbp length, but have two phylogenetically discordant segments that unambiguously differentiate the integration module from the vcrABC cargo. Using available Dehalococcoides phylogenomic data we estimate that these ssrA-specific genomic islands are at least as old as the Dehalococcoides group itself, which in turn is much older than human civilization.



Conclusions

The vcrABC -containing genomic islands are a recently-acquired subset of a diverse collection of ssrA-specific mobile elements that are a major contributor to strain-level diversity in Dehalococcoides, and may have been throughout its evolution. The high similarity between vcrABC sequences is quantitatively consistent with recent horizontal acquisition driven by ~100 years of industrial pollution with chlorinated ethenes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Saville2011,

title = {Energy-Dependent Stability of Shewanella oneidensis MR-1 Biofilms},

author = {Saville, Renee and Rakshe, Shauna and Haagensen, Janus A.J. and Shukla, Soni and Spormann, Alfred},

url = {http://jb.asm.org/content/193/13/3257.short},

doi = {10.1128/JB.00251-11},

year  = {2011},

date = {2011-05-13},

journal = {Journal of Bacteriology},

volume = {193},

number = {13},

pages = {3257-3264},

abstract = {Stability and resistance to dissolution are key features of microbial biofilms. How these macroscopic properties are determined by the physiological state of individual biofilm cells in their local physical-chemical and cellular environment is largely unknown. In order to obtain molecular and energetic insight into biofilm stability, we investigated whether maintenance of biofilm stability is an energy-dependent process and whether transcription and/or translation is required for biofilm dissolution. We found that in 12-hour-old Shewanella oneidensis MR-1 biofilms, a reduction in cellular ATP concentration, induced either by oxygen deprivation or by addition of the inhibitor of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone (CCCP), dinitrophenol (DNP), or CN−, resulted in massive dissolution. In 60-hour-old biofilms, the extent of uncoupler-induced cell loss was strongly attenuated, indicating that the integrity of older biofilms is maintained by means other than those operating in younger biofilms. In experiments with 12-hour-old biofilms, the transcriptional and translational inhibitors rifampin, tetracycline, and erythromycin were found to be ineffective in preventing energy starvation-induced detachment, suggesting that neither transcription nor translation is required for this process. Biofilms of Vibrio cholerae were also induced to dissolve upon CCCP addition to an extent similar to that in S. oneidensis. However, Pseudomonas aeruginosa and P. putida biofilms remained insensitive to CCCP addition. Collectively, our data show that metabolic energy is directly or indirectly required for maintaining cell attachment, and this may represent a common but not ubiquitous mechanism for stability of microbial biofilms.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Kapoor2011,

title = {Antimicrobial Peptoids Are Effective against Pseudomonas aeruginosa Biofilms},

author = {Kapoor, Rinki and Wadman, Mayken and Dohm, Michelle and Czyzewski, Ann and Spormann, Alfred and Barron, Annelise},

url = {http://aac.asm.org/content/55/6/3054.short},

doi = {10.1128/AAC.01516-10},

year  = {2011},

date = {2011-03-21},

journal = {Antimicrobial Agents and Chemotherapy},

volume = {55},

number = {6},

pages = {3054-3057},

abstract = {The resistance of biofilms to conventional antibiotics complicates the treatment of chronic cystic fibrosis (CF). We investigated the effects of peptoids, peptides, and conventional antibiotics on the biomass and cell viability within Pseudomonas aeruginosa biofilms. At their MICs, peptoids 1 and 1-C134mer caused maximum reductions in biomass and cell viability, respectively. These results suggest that peptoids of this class could be worth exploring for the treatment of pulmonary infections occurring in CF patients.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Cordova2011,

title = {Partial Functional Replacement of CymA by SirCD in Shewanella oneidensis MR-1},

author = {Cordova, Carmen and Schicklberger, Marcus and Yu, Yang and Spormann, Alfred},

url = {http://jb.asm.org/content/193/9/2312.short},

doi = {10.1128/JB.01355-10},

year  = {2011},

date = {2011-03-04},

journal = {Journal of Bacteriology},

volume = {193},

number = {9},

pages = {2312-2321},

abstract = {The gammaproteobacterium Shewanella oneidensis MR-1 utilizes a complex electron transfer network composed primarily of c-type cytochromes to respire under anoxic conditions a variety of compounds, including fumarate, nitrate, and dimethyl sulfoxide (DMSO), in addition to the minerals Fe(III) and Mn(IV). Central to several respiratory pathways is CymA, a cytoplasmic membrane-bound tetraheme c-type cytochrome that functions as the major hydroquinone dehydrogenase. To investigate functional redundancy and plasticity in S. oneidensis MR-1 electron transport, we isolated ΔcymA suppressor mutants and characterized one biochemically and genetically. Interestingly, in the characterized ΔcymA suppressor mutant, respiration of fumarate, ferric citrate, and DMSO was restored but that of nitrate was not. The suppression was found to be due to transcriptional activation of sirC and sirD, encoding a periplasmic iron sulfur protein and an integral membrane hydroquinone dehydrogenase, respectively. Biochemical in vitro reconstitution experiments confirmed electron transport between formate and fumarate via fumarate reductase by suppressor membrane fractions. The suppression was found to be caused by insertion of an ISSod1 element upstream of the sirCD transcriptional start site, generating a novel, constitutively active hybrid promoter. This work revealed that adaptation of an alternative electron transfer pathway from quinol to terminal oxidoreductases independent of CymA occurs rapidly in S. oneidensis MR-1.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Rakshe2011,

title = {Indirect Modulation of the Intracellular c-Di-GMP Level in Shewanella oneidensis MR-1 by MxdA},

author = {Rakshe, Shauna and Leff, Maija and Spormann, Alfred},

url = {http://aem.asm.org/content/77/6/2196.short},

doi = {10.1128/AEM.01985-10},

year  = {2011},

date = {2011-01-28},

journal = {Applied and Environmental Microbiology},

volume = {77},

number = {6},

pages = {2196-2198},

abstract = {The GGDEF domain protein MxdA, which is important for biofilm formation in Shewanella oneidensis MR-1, was hypothesized to possess diguanylate cyclase activity. Here, we demonstrate that while MxdA controls the cellular level of c-di-GMP in S. oneidensis, it modulates the c-di-GMP pool indirectly.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Nelson2010,

title = {PhyloChip microarray analysis reveals altered gastrointestinal microbial communities in a rat model of colonic hypersensitivity},

author = {Nelson, T and Holmes, Susan and Alekseyenko, A and Shenoy, M and Desantis, T and Wu, C and Andersen, G and Winston, J and Sonnenburg, J and Pasricha, P and Spormann, Alfred},

url = {http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2982.2010.01637.x/full},

doi = {10.1111/j.1365-2982.2010.01637.x},

year  = {2010},

date = {2010-12-03},

journal = {Neurogastroenterology & Motility},

volume = {23},

number = {2},

pages = {169-e42},

abstract = {Background  Irritable bowel syndrome (IBS) is a chronic, episodic gastrointestinal disorder that is prevalent in a significant fraction of western human populations; and changes in the microbiota of the large bowel have been implicated in the pathology of the disease.



Methods  Using a novel comprehensive, high-density DNA microarray (PhyloChip) we performed a phylogenetic analysis of the microbial community of the large bowel in a rat model in which intracolonic acetic acid in neonates was used to induce long lasting colonic hypersensitivity and decreased stool water content and frequency, representing the equivalent of human constipation-predominant IBS.



Key Results  Our results revealed a significantly increased compositional difference in the microbial communities in rats with neonatal irritation as compared with controls. Even more striking was the dramatic change in the ratio of Firmicutes relative to Bacteroidetes, where neonatally irritated rats were enriched more with Bacteroidetes and also contained a different composition of species within this phylum. Our study also revealed differences at the level of bacterial families and species.



Conclusions & Inferences  The PhyloChip is a useful and convenient method to study enteric microflora. Further, this rat model system may be a useful experimental platform to study the causes and consequences of changes in microbial community composition associated with IBS.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}