Metagenome-assembled genomes of freshwater sp. G-191 and sp. enriched from Cedar Swamp, Woods Hole, MA.

are facultative denitrifying anaerobes capable of using one-carbon compounds as a sole carbon source. sp. G-191 was enriched from Cedar Swamp, Woods Hole, Massachusetts, using a selective medium for methanol-utilizing bacteria.

An Extracellular, Ca-Activated Nuclease (EcnA) Mediates Transformation in a Naturally Competent Archaeon.

Transformation, the uptake of DNA directly from the environment, is a major driver of gene flow in microbial populations. In bacteria, DNA uptake requires a nuclease that processes dsDNA to ssDNA, which is subsequently transferred into the cell and incorporated into the genome. However, the process of DNA uptake in archaea is still unknown.

High-throughput genetics enables identification of nutrient utilization and accessory energy metabolism genes in a model methanogen.

Archaea are widespread in the environment and play fundamental roles in diverse ecosystems; however, characterization of their unique biology requires advanced tools. This is particularly challenging when characterizing gene function. Here, we generate randomly barcoded transposon libraries in the model methanogenic archaeon and use high-throughput growth methods to conduct fitness assays (RB-TnSeq) across over 100 unique growth conditions.

A universal metabolite repair enzyme removes a strong inhibitor of the TCA cycle.

A prevalent side-reaction of succinate dehydrogenase oxidizes malate to enol-oxaloacetate (OAA), a metabolically inactive form of OAA that is a strong inhibitor of succinate dehydrogenase. We purified from cow heart mitochondria an enzyme (OAT1) with OAA tautomerase (OAT) activity that converts enol-OAA to the physiological keto-OAA form, and determined that it belongs to the highly conserved and previously uncharacterized Fumarylacetoacetate_hydrolase_domain-containing protein family. From all three domains of life, heterologously expressed proteins were shown to have strong OAT activity, and ablating the OAT1 homolog caused significant growth defects.

Integrated omics analysis unveils a DNA damage response to neurogenic injury.

Spinal cord injury (SCI) evokes profound bladder dysfunction. Current treatments are limited by a lack of molecular data to inform novel therapeutic avenues. Previously, we showed systemic inosine treatment improved bladder function following SCI in rats.

Functionally redundant formate dehydrogenases enable formate-dependent growth in Methanococcus maripaludis.

Methanogens are essential for the complete remineralization of organic matter in anoxic environments. Most cultured methanogens are hydrogenotrophic, using H as an electron donor to reduce CO to CH, but in the absence of H many can also use formate. Formate dehydrogenase (Fdh) is essential for formate oxidation, where it transfers electrons for the reduction of coenzyme F or to a flavin-based electron bifurcating reaction catalyzed by heterodisulfide reductase (Hdr), the terminal reaction of methanogenesis.

Model Organisms To Study Methanogenesis, a Uniquely Archaeal Metabolism.

Methanogenic archaea are the only organisms that produce CH as part of their energy-generating metabolism. They are ubiquitous in oxidant-depleted, anoxic environments such as aquatic sediments, anaerobic digesters, inundated agricultural fields, the rumen of cattle, and the hindgut of termites, where they catalyze the terminal reactions in the degradation of organic matter. Methanogenesis is the only metabolism that is restricted to members of the domain .

Random transposon mutagenesis identifies genes essential for transformation in Methanococcus maripaludis.

Natural transformation, the process whereby a cell acquires DNA directly from the environment, is an important driver of evolution in microbial populations, yet the mechanism of DNA uptake is only characterized in bacteria. To expand our understanding of natural transformation in archaea, we undertook a genetic approach to identify a catalog of genes necessary for transformation in Methanococcus maripaludis. Using an optimized method to generate random transposon mutants, we screened 6144 mutant strains for defects in natural transformation and identified 25 transformation-associated candidate genes.

Evaluation of silk fibroin-based urinary conduits in a porcine model of urinary diversion.

The primary strategy for urinary diversion in radical cystectomy patients involves incorporation of autologous gastrointestinal conduits into the urinary tract which leads to deleterious consequences including chronic infections and metabolic abnormalities. This report investigates the efficacy of an acellular, tubular bi-layer silk fibroin (BLSF) graft to function as an alternative urinary conduit in a porcine model of urinary diversion. Unilateral urinary diversion with stented BLSF conduits was executed in five adult female, Yucatan mini-swine over a 3 month period.

Interspecies Formate Exchange Drives Syntrophic Growth of and Methanococcus maripaludis.

The complete remineralization of organic matter in anoxic environments relies on communities of microorganisms that ferment organic acids and alcohols to CH. This is accomplished through syntrophic association of H or formate producing bacteria and methanogenic archaea, where exchange of these intermediates enables growth of both organisms. While these communities are essential to Earth's carbon cycle, our understanding of the dynamics of H or formate exchanged is limited.

The Fluorescence-Activating and Absorption-Shifting Tag (FAST) Enables Live-Cell Fluorescence Imaging of Methanococcus maripaludis.

Live-cell fluorescence imaging of methanogenic archaea has been limited due to the strictly anoxic conditions required for growth and issues with autofluorescence associated with electron carriers in central metabolism. Here, we show that the fluorescence-activating and absorption-shifting tag (FAST) complexed with the fluorogenic ligand 4-hydroxy-3-methylbenzylidene-rhodanine (HMBR) overcomes these issues and displays robust fluorescence in Methanococcus maripaludis. We also describe a mechanism to visualize cells under anoxic conditions using a fluorescence microscope.

In Vitro Biosynthesis of the [Fe]-Hydrogenase Cofactor Verifies the Proposed Biosynthetic Precursors.

In the FeGP cofactor of [Fe]-hydrogenase, low-spin Fe is in complex with two CO ligands and a pyridinol derivative; the latter ligates the iron with a 6-acylmethyl substituent and the pyridinol nitrogen. A guanylylpyridinol derivative, 6-carboxymethyl-3,5-dimethyl-4-guanylyl-2-pyridinol (3), is produced by the decomposition of the FeGP cofactor under irradiation with UV-A/blue light and is also postulated to be a precursor of FeGP cofactor biosynthesis. HcgC and HcgB catalyze consecutive biosynthesis steps leading to 3.

Advanced Understanding of Prokaryotic Biofilm Formation through Use of a Cost-Effective and Versatile Multipanel Adhesion (mPAD) Mount.

Most microorganisms exist in biofilms, which comprise aggregates of cells surrounded by an extracellular matrix that provides protection from external stresses. Based on the conditions under which they form, biofilm structures vary in significant ways. For instance, biofilms that develop when microbes are incubated under static conditions differ from those formed when microbes encounter the shear forces of a flowing liquid.

Complete Genome Sequence of the Secondary Alcohol-Utilizing Methanogen Methanospirillum hungatei Strain GP1.

We report the complete genome sequence of Methanospirillum hungatei strain GP1 (DSM 1101). Strain GP1 oxidizes H, formate, and secondary alcohols as the substrates for methanogenesis. Members of the genus are model organisms used to study syntrophic growth with bacterial partners, but secondary alcohol metabolism remains poorly studied.

The Oligosaccharyltransferase AglB Supports Surface-Associated Growth and Iron Oxidation in Methanococcus maripaludis.

Most microbial organisms grow as surface-attached communities known as biofilms. However, the mechanisms whereby methanogenic archaea grow attached to surfaces have remained understudied. Here, we show that the oligosaccharyltransferase AglB is essential for growth of Methanococcus maripaludis strain JJ on glass or metal surfaces.

Molecular mechanisms of esophageal epithelial regeneration following repair of surgical defects with acellular silk fibroin grafts.

Constructive remodeling of focal esophageal defects with biodegradable acellular grafts relies on the ability of host progenitor cell populations to repopulate implant regions and facilitate growth of de novo functional tissue. Intrinsic molecular mechanisms governing esophageal repair processes following biomaterial-based, surgical reconstruction is largely unknown. In the present study, we utilized mass spectrometry-based quantitative proteomics and in silico pathway evaluations to identify signaling cascades which were significantly activated during neoepithelial formation in a Sprague Dawley rat model of onlay esophagoplasty with acellular silk fibroin scaffolds.

Formate-Dependent Heterodisulfide Reduction in a Archaeon.

Hydrogenotrophic methanogens produce CH using H as an electron donor to reduce CO In the absence of H, many are able to use formate or alcohols as alternate electron donors. Methanogens from the order are capable of growth with H, but many lack genes encoding hydrogenases that are typically found in other hydrogenotrophic methanogens. In an effort to better understand electron flow in methanogens from the , we undertook a genetic and biochemical study of heterodisulfide reductase (Hdr) in Hdr catalyzes an essential reaction by coupling the first and last steps of methanogenesis through flavin-based electron bifurcation.

Type IV-Like Pili Facilitate Transformation in Naturally Competent Archaea.

Naturally competent organisms are capable of DNA uptake directly from the environment through the process of transformation. Despite the importance of transformation to microbial evolution, DNA uptake remains poorly characterized outside of the bacterial domain. Here, we identify the pilus as a necessary component of the transformation machinery in archaea.

Evaluation of Bilayer Silk Fibroin Grafts for Tubular Esophagoplasty in a Porcine Defect Model.

Surgical reconstruction of tubular esophageal defects with autologous gastrointestinal segments is the gold standard treatment to replace damaged or diseased esophageal tissues. Unfortunately, this approach is associated with adverse complications, including dysphagia, donor-site morbidity, and in some cases patient death. Bilayer silk fibroin (BLSF) scaffolds were investigated as alternative, acellular grafts for tubular esophagoplasty in a porcine defect model for 3 months of implantation.

Evaluation of Acellular Bilayer Silk Fibroin Grafts for Onlay Tracheoplasty in a Rat Defect Model.

Objective: To assess the efficacy of acellular bilayer silk fibroin (BLSF) grafts to repair full-thickness tracheal defects and to compare the performance with conventional porcine small intestinal submucosa (SIS) implants.

Study Design: A prospective controlled animal trial in a rat model of onlay tracheoplasty.

Setting: Pediatric medical center.

Augmentation Cystoplasty of Diseased Porcine Bladders with Bi-Layer Silk Fibroin Grafts.

The search for an ideal "off-the-shelf" biomaterial for augmentation cystoplasty remains elusive and current scaffold configurations are hampered by mechanical and biocompatibility restrictions. In addition, preclinical evaluations of potential scaffold designs for bladder repair are limited by the lack of tractable large animal models of obstructive bladder disease that can mimic clinical pathology. The results of this study describe a novel, minimally invasive, porcine model of partial bladder outlet obstruction that simulates clinically relevant phenotypes.

Repair of injured urethras with silk fibroin scaffolds in a rabbit model of onlay urethroplasty.

Background: Preclinical validation of scaffold-based technologies in animal models of urethral disease is desired to assess wound healing efficacy in scenarios that mimic the target patient population. This study investigates the feasibility of bilayer silk fibroin (BLSF) scaffolds for the repair of previously damaged urethras in a rabbit model of onlay urethroplasty.

Materials And Methods: A focal, partial thickness urethral injury was created in adult male rabbits (n = 12) via electrocoagulation and then onlay urethroplasty with 50 mm BLSF grafts was carried out 2 wk after injury.