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. In Escherichia coli, expression of succinate dehydrogenase was necessary for OAT1-associated growth defects to occur, and ablating OAT1 caused a significant increase in acetate and other metabolites associated with anaerobic respiration. OAT1 increased the succinate dehydrogenase reaction rate by 35% in in vitro assays with physiological concentrations of both succinate and malate. Our results suggest that OAT1 is a universal metabolite repair enzyme that is required to maximize aerobic respiration efficiency by preventing succinate dehydrogenase inhibition.

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 H2 as an electron donor to reduce CO2 to CH4, but in the absence of H2 many can also use formate. Formate dehydrogenase (Fdh) is essential for formate oxidation, where it transfers electrons for the reduction of coenzyme F420 or to a flavin-based electron bifurcating reaction catalyzed by heterodisulfide reductase (Hdr), the terminal reaction of methanogenesis. Furthermore, methanogens that use formate encode at least two isoforms of Fdh in their genomes, but how these different isoforms participate in methanogenesis is unknown. Using Methanococcus maripaludis, we undertook a biochemical characterization of both Fdh isoforms involved in methanogenesis. Both Fdh1 and Fdh2 interacted with Hdr to catalyze the flavin-based electron bifurcating reaction, and both reduced F420 at similar rates. F420 reduction preceded flavin-based electron bifurcation activity for both enzymes. In a Δfdh1 mutant background, a suppressor mutation was required for Fdh2 activity. Genome sequencing revealed that this mutation resulted in the loss of a specific molybdopterin transferase (moeA), allowing for Fdh2-dependent growth, and the metal content of the proteins suggested that isoforms are dependent on either molybdenum or tungsten for activity. These data suggest that both isoforms of Fdh are functionally redundant, but their activities in vivo may be limited by gene regulation or metal availability under different growth conditions. Together these results expand our understanding of formate oxidation and the role of Fdh in methanogenesis.

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. Here, we applied multi-omics analysis to explore molecular alterations in the bladder and their sensitivity to inosine following SCI. Canonical pathways regulated by SCI included those associated with protein synthesis, neuroplasticity, wound healing, and neurotransmitter degradation. Upstream regulator analysis identified MYC as a key regulator, whereas causal network analysis predicted multiple regulators of DNA damage response signaling following injury, including PARP-1. Staining for both DNA damage (γH2AX) and PARP activity (poly-ADP-ribose) markers in the bladder was increased following SCI, and attenuated in inosine-treated tissues. Proteomics analysis suggested that SCI induced changes in protein synthesis-, neuroplasticity-, and oxidative stress-associated pathways, a subset of which were shown in transcriptomics data to be inosine-sensitive. These findings provide novel insights into the molecular landscape of the bladder following SCI, and highlight a potential role for PARP inhibition to treat neurogenic bladder dysfunction.

Model Organisms To Study Methanogenesis, a Uniquely Archaeal Metabolism.

Methanogenic archaea are the only organisms that produce CH4 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 Archaea. Here, we discuss the importance of model organisms in the history of methanogen research, including their role in the discovery of the archaea and in the biochemical and genetic characterization of methanogenesis. We also discuss outstanding questions in the field and newly emerging model systems that will expand our understanding of this uniquely archaeal metabolism.

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

Background: 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. Materials and methods: Unilateral urinary diversion with stented BLSF conduits was executed in five adult female, Yucatan mini-swine over a 3 month period. Longitudinal imaging analyses including ultrasonography, retrograde ureteropyelography and video-endoscopy were carried out monthly. Histological, immunohistochemical (IHC), and histomorphometric assessments were performed on neoconduits at harvest. Results: All animals survived until scheduled euthanasia and displayed moderate hydronephrosis (Grades 1-3) in reconstructed collecting systems over the course of the study period. Stented BLSF constructs supported formation of vascularized, retroperitoneal tubes capable of facilitating external urinary drainage. By 3 months post-operative, neoconduits contained α-smooth muscle actin+ and SM22α+ smooth muscle as well as uroplakin 3A+ and pan-cytokeratin + urothelium. However, the degree of tissue regeneration in neotissues was significantly lower in comparison to ureteral controls as determined by histomorphometry. In addition, neoconduit stenting was necessary to prevent stomal occlusion. Conclusion: BLSF biomaterials represent emerging platforms for urinary conduit construction and may offer a functional replacement for conventional urinary diversion techniques following further optimization of mechanical properties and regenerative responses.

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. Among these are genes encoding components of the type IV-like pilus, transcription/translation associated genes, genes encoding putative membrane bound transport proteins, and genes of unknown function. Interestingly, similar genes were identified regardless of whether replicating or integrating plasmids were provided as a substrate for transformation. Using allelic replacement mutagenesis, we confirmed that several genes identified in these screens are essential for transformation. Finally, we identified a homolog of a membrane bound substrate transporter in Methanoculleus thermophilus and verified its importance for transformation using allelic replacement mutagenesis, suggesting a conserved mechanism for DNA transfer in multiple archaea. These data represent an initial characterization of the genes important for transformation which will inform efforts to understand gene flow in natural populations. Additionally, knowledge of the genes necessary for natural transformation may assist in identifying signatures of transformation machinery in archaeal genomes and aid the establishment of new model genetic systems for studying archaea.

Interspecies Formate Exchange Drives Syntrophic Growth of Syntrophotalea carbinolica and Methanococcus maripaludis.

The complete remineralization of organic matter in anoxic environments relies on communities of microorganisms that ferment organic acids and alcohols to CH4. This is accomplished through syntrophic association of H2 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 H2 or formate exchanged is limited. Here, we establish a model partnership between Syntrophotalea carbinolica and Methanococcus maripaludis. Through sequencing a transposon mutant library of M. maripaludis grown with ethanol oxidizing S. carbinolica, we found that genes encoding the F420-dependent formate dehydrogenase (Fdh) and F420-dependent methylene-tetrahydromethanopterin dehydrogenase (Mtd) are important for growth. Competitive growth of M. maripaludis mutants defective in either H2 or formate metabolism verified that, across multiple substrates, interspecies formate exchange was dominant in these communities. Agitation of these cultures to facilitate diffusive loss of H2 to the culture headspace resulted in an even greater competitive advantage for M. maripaludis strains capable of oxidizing formate. Finally, we verified that an M. maripaludis Δmtd mutant had a defect during syntrophic growth. Together, these results highlight the importance of formate exchange for the growth of methanogens under syntrophic conditions. IMPORTANCE In the environment, methane is typically generated by fermentative bacteria and methanogenic archaea working together in a process called syntrophy. Efficient exchange of small molecules like H2 or formate is essential for growth of both organisms. However, difficulties in determining the relative contribution of these intermediates to methanogenesis often hamper efforts to understand syntrophic interactions. Here, we establish a model syntrophic coculture composed of S. carbinolica and the genetically tractable methanogen M. maripaludis. Using mutant strains of M. maripaludis that are defective for either H2 or formate metabolism, we determined that interspecies formate exchange drives syntrophic growth of these organisms. Together, these results advance our understanding of the degradation of organic matter in anoxic environments.

The Function of Two Radical-SAM Enzymes, HcgA and HcgG, in the Biosynthesis of the [Fe]-Hydrogenase Cofactor.

In the biosynthesis of the iron-guanylylpyridinol (FeGP) cofactor, 6-carboxymethyl-5-methyl-4-hydroxy-2-pyridinol (1) is 3-methylated to form 2, then 4-guanylylated to form 3, and converted into the full cofactor. HcgA-G proteins catalyze the biosynthetic reactions. Herein, we report the function of two radical S-adenosyl methionine enzymes, HcgA and HcgG, as uncovered by in vitro complementation experiments and the use of purified enzymes. In vitro biosynthesis using the cell extract from the Methanococcus maripaludis ΔhcgA strain was complemented with HcgA or precursors 1, 2 or 3. The results suggested that HcgA catalyzes the biosynthetic reaction that forms 1. We demonstrated the formation of 1 by HcgA using the 3 kDa cell extract filtrate as the substrate. Biosynthesis in the ΔhcgG system was recovered by HcgG but not by 3, which indicated that HcgG catalyzes the reactions after the biosynthesis of 3. The data indicated that HcgG contributes to the formation of CO and completes biosynthesis of the FeGP cofactor.

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. Derivatives of FAST were successfully applied for protein abundance analysis, subcellular localization analysis, and determination of protein-protein interactions. FAST fusions to both formate dehydrogenase (Fdh) and F420-reducing hydrogenase (Fru) displayed increased fluorescence in cells grown on formate-containing medium, consistent with previous studies suggesting the increased abundance of these proteins in the absence of H2. Additionally, FAST fusions to both Fru and the ATPase associated with the archaellum (FlaI) showed a membrane localization in single cells observed using anoxic fluorescence microscopy. Finally, a split reporter translationally fused to the alpha and beta subunits of Fdh reconstituted a functionally fluorescent molecule in vivo via bimolecular fluorescence complementation. Together, these observations demonstrate the utility of FAST as a tool for studying members of the methanogenic archaea. IMPORTANCE Methanogenic archaea are important members of anaerobic microbial communities where they catalyze essential reactions in the degradation of organic matter. Developing additional tools for studying the cell biology of these organisms is essential to understanding them at a mechanistic level. Here, we show that FAST, in combination with the fluorogenic ligand HMBR, can be used to monitor protein dynamics in live cells of M. maripaludis. The application of FAST holds promise for future studies focused on the metabolism and physiology of methanogenic archaea.

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

In the FeGP cofactor of [Fe]-hydrogenase, low-spin FeII 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. Here, we report an in vitro biosynthesis assay of the FeGP cofactor using the cell extract of the ΔhcgBΔhcgC strain of Methanococcus maripaludis, which does not biosynthesize 3. We chemically synthesized pyridinol precursors 1 and 2, and detected the production of the FeGP cofactor from 1, 2 and 3. These results indicated that 1, 2 and 3 are the precursors of the FeGP cofactor, and the carboxy group of 3 is converted to the acyl ligand.

A Genetic Study of Nif-Associated Genes in a Hyperthermophilic Methanogen.

Methanocaldococcus sp. strain FS406-22, a hyperthermophilic methanogen, fixes nitrogen with a minimal set of known nif genes. Only four structural nif genes, nifH, nifD, nifK, and nifE, are present in a cluster, and a nifB homolog is present elsewhere in the genome. nifN, essential for the final synthesis of the iron-molybdenum cofactor of nitrogenase in well-characterized diazotrophs, is absent from FS406-22. In addition, FS406-22 encodes four novel hypothetical proteins, and a ferredoxin, in the nif cluster. Here, we develop a set of genetic tools for FS406-22 and test the functionality of genes in the nif cluster by making markerless in-frame deletion mutations. Deletion of the gene for one hypothetical protein, designated Hp4, delayed the initiation of diazotrophic growth and decreased the growth rate, an effect we confirmed by genetic complementation. NifE also appeared to play a role in diazotrophic growth, and the encoding of Hp4 and NifE in a single operon suggested they may work together in some way in the synthesis of the nitrogenase cofactor. No role could be discerned for any of the other hypothetical proteins, nor for the ferredoxin, despite the presence of these genes in a variety of related organisms. Possible pathways and evolutionary scenarios for the synthesis of the nitrogenase cofactor in an organism that lacks nifN are discussed. IMPORTANCEMethanocaldococcus has been considered a model genus, but genetic tools have not been forthcoming until recently. Here, we develop and illustrate the utility of positive selection with either of two selective agents (simvastatin and neomycin), negative selection, generation of markerless in-frame deletion mutations, and genetic complementation. These genetic tools should be useful for a variety of related species. We address the question of the minimal set of nif genes, which has implications for how nitrogen fixation evolved.

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. Moreover, biofilms develop dynamically over time. Here, we describe a cost-effective coverslip holder, printed with a three-dimensional (3D) printer, that facilitates surface adhesion assays under a broad range of standing and shaking culture conditions. This multipanel adhesion (mPAD) mount further allows cultures to be sampled at multiple time points, ensuring consistency and comparability between samples and enabling analyses of the dynamics of biofilm formation. As a proof of principle, using the mPAD mount for shaking, oxic cultures, we confirm previous flow chamber experiments showing that the Pseudomonas aeruginosa wild-type strain and a phenazine deletion mutant (Δphz) strain form biofilms with similar structure but reduced density in the mutant strain. Extending this analysis to anoxic conditions, we reveal that microcolony formation and biofilm formation can only be observed under shaking conditions and are decreased in the Δphz mutant compared to wild-type cultures, indicating that phenazines are crucial for the formation of biofilms if oxygen as an electron acceptor is unavailable. Furthermore, while the model archaeon Haloferax volcanii does not require archaella for surface attachment under static conditions, we demonstrate that an H. volcanii mutant that lacks archaella is impaired in early stages of biofilm formation under shaking conditions. IMPORTANCE Due to the versatility of the mPAD mount, we anticipate that it will aid the analysis of biofilm formation in a broad range of bacteria and archaea. Thereby, it contributes to answering critical biological questions about the regulatory and structural components of biofilm formation and understanding this process in a wide array of environmental, biotechnological, and medical contexts.

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 H2, 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. AglB glycosylates several cellular structures, such as pili, archaella, and the cell surface layer (S-layer). We show that the S-layer of strain JJ, but not strain S2, is a glycoprotein, that only strain JJ was capable of growth on surfaces, and that deletion of aglB blocked S-layer glycosylation and abolished surface-associated growth. A strain JJ mutant lacking structural components of the type IV-like pilus did not have a growth defect under any conditions tested, while a mutant lacking the preflagellin peptidase (ΔflaK) was defective for surface growth only when formate was provided as the sole electron donor. Finally, for strains that are capable of Fe0 oxidation, we show that deletion of aglB decreases the rate of anaerobic Fe0 oxidation, presumably due to decreased association of biomass with the Fe0 surface. Together, these data provide an initial characterization of surface-associated growth in a member of the methanogenic archaea. IMPORTANCE Methanogenic archaea are responsible for producing the majority of methane on Earth and catalyze the terminal reactions in the degradation of organic matter in anoxic environments. Methanogens often grow as biofilms associated with surfaces or partner organisms; however, the molecular details of surface-associated growth remain uncharacterized. We have found evidence that glycosylation of the cell surface layer is essential for growth of M. maripaludis on surfaces and can enhance rates of anaerobic iron corrosion. These results provide insight into the physiology of surface-associated methanogenic organisms and highlight the importance of surface association for anaerobic iron corrosion.

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. Pharmacologic inhibitor and rescue experiments revealed that epithelialization of neotissues is significantly dependent in part on pro-survival stimuli capable of suppressing caspase activity in epithelial progenitors via activation of hepatocyte growth factor receptor (c-MET), tropomyosin receptor kinase A (TrkA), phosphoinositide 3-kinase (PI3K), and protein kinase B (Akt) signaling mechanisms. These data highlight the molecular machinery involved in esophageal epithelial regeneration following surgical repair with acellular implants.

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. Adult Yucatan mini-swine (n = 5) were subjected to esophageal reconstruction with tubular BLSF grafts (2 cm in length) in combination with transient esophageal stenting for 2 months followed by a 1-month period, where the graft site was unstented. All animals receiving BLSF grafts survived and were capable of solid food consumption, however strictures were noted at graft regions in 60% of the experimental cohort between 2 and 3 months postop and required balloon dilation. In addition, fluoroscopic analysis showed peristaltic function in only 1/5 neotissues. Following swine harvest at 3 months, ex vivo tissue bath evaluations revealed that neoconduits exhibited contractile responses to carbachol, electric field stimulation, and KCl, whereas sodium nitroprusside and isoproterenol induced relaxation effects. Histological (Masson's Trichrome) and immunohistochemical analyses of regenerated tissue conduits showed a stratified, squamous epithelium expressing pan-cytokeratins buttressed by a vascularized lamina propria containing a smooth muscle-rich muscularis mucosa surrounded by a muscularis externa. Neuronal density, characterized by the presence of synaptophysin-positive boutons, was significantly lower in neotissues in comparison to nonsurgical controls. BLSF scaffolds represent a promising platform for the repair of tubular esophageal defects, however improvements in scaffold design are needed to reduce the rate of complications and improve the extent of constructive tissue remodeling. Impact statement The search for a superior "off-the-shelf" scaffold capable of repairing tubularesophageal defects as well as overcoming limitations associated with conventional autologous gastrointestinal segments remains elusive. The purpose of this study was to investigate the performance of an acellular, bilayer silk fibroin graft (BLSF) for tubular esophagoplasty in a porcine model. Our results demonstrated that BLSF scaffolds supported the formation of tubular neotissues with innervated, vascularized epithelial and muscular components capable of contractile and relaxation responses. BLSF scaffolds represent a promising platform for esophageal tissue engineering.

Formate-Dependent Heterodisulfide Reduction in a Methanomicrobiales Archaeon.

Hydrogenotrophic methanogens produce CH4 using H2 as an electron donor to reduce CO2 In the absence of H2, many are able to use formate or alcohols as alternate electron donors. Methanogens from the order Methanomicrobiales are capable of growth with H2, 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 Methanomicrobiales, we undertook a genetic and biochemical study of heterodisulfide reductase (Hdr) in Methanoculleus thermophilus Hdr catalyzes an essential reaction by coupling the first and last steps of methanogenesis through flavin-based electron bifurcation. Hdr from M. thermophilus copurified with formate dehydrogenase (Fdh) and only displayed activity when formate was supplied as an electron donor. We found no evidence of an Hdr-associated hydrogenase, and H2 could not function as an electron donor, even with Hdr purified from cells grown on H2 We found that cells catalyze a formate hydrogenlyase activity that is likely essential for generating the formate needed for the Hdr reaction. Together, these results highlight the importance of formate as an electron donor for methanogenesis and suggest the ability to use formate is closely integrated into the methanogenic pathway in organisms from the order MethanomicrobialesIMPORTANCE Methanogens from the order Methanomicrobiales are thought to prefer H2 as an electron donor for growth. They are ubiquitous in anaerobic environments, such as in wastewater treatment facilities, anaerobic digesters, and the rumen, where they catalyze the terminal steps in the breakdown of organic matter. However, despite their importance, the metabolism of these organisms remains understudied. Using a genetic and biochemical approach, we show that formate metabolism is closely integrated into methanogenesis in Methanoculleus thermophilus This is due to a requirement for formate as the electron donor to heterodisulfide reductase (Hdr), an enzyme responsible for catalyzing essential reactions in methanogenesis by linking the initial CO2 fixing step to the exergonic terminal reaction of the pathway. These results suggest that hydrogen is not necessarily the preferred electron donor for all hydrogenotrophic methanogens and provide insight into the metabolism of methanogens from the order Methanomicrobiales.

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. We describe two naturally competent organisms, Methanococcus maripaludis and Methanoculleus thermophilus In M. maripaludis, replicative vectors were transferred with an average efficiency of 2.4 × 103 transformants µg-1 DNA. In M. thermophilus, integrative vectors were transferred with an average efficiency of 2.7 × 103 transformants µg-1 DNA. Additionally, natural transformation of M. thermophilus could be used to introduce chromosomal mutations. To our knowledge, this is the first demonstration of a method to introduce targeted mutations in a member of the order Methanomicrobiales For both organisms, mutants lacking structural components of the type IV-like pilus filament were defective for DNA uptake, demonstrating the importance of pili for natural transformation. Interestingly, competence could be induced in a noncompetent strain of M. maripaludis by expressing pilin genes from a replicative vector. These results expand the known natural competence pili to include examples from the archaeal domain and highlight the importance of pili for DNA uptake in diverse microbial organisms.IMPORTANCE Microbial organisms adapt and evolve by acquiring new genetic material through horizontal gene transfer. One way that this occurs is natural transformation, the direct uptake and genomic incorporation of environmental DNA by competent organisms. Archaea represent up to a third of the biodiversity on Earth, yet little is known about transformation in these organisms. Here, we provide the first characterization of a component of the archaeal DNA uptake machinery. We show that the type IV-like pilus is essential for natural transformation in two archaeal species. This suggests that pili are important for transformation across the tree of life and further expands our understanding of gene flow in archaea.

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. SUBJECTS AND METHODS: Tracheal reconstruction of adult Sprague-Dawley rats was performed with BLSF (n = 38) or SIS (n = 32) matrices for up to 3 months of implantation. Functional evaluations of repaired conduits as well as histologic, immunohistochemical, and histomorphometric analyses of neotissues were assessed. RESULTS: Prior to scheduled euthanasia, survival rates of rats receiving BLSF or SIS grafts were ≥94%, with no clinical signs of airway obstruction observed over the course of the study. Micro-computed tomography analysis revealed that the mean percentage of stenosis was <20% in both implant groups. BLSF and SIS grafts supported formation of pseudostratified ciliated columnar epithelium by 1 week postoperatively; however, each matrix failed to promote de novo chondrogenesis by 3 months following repair. CONCLUSIONS: BLSF scaffolds can be used for reconstruction of rat tracheal patch defects with functional outcomes comparable to those of SIS matrices.