Mitochondrial Dysfunction: At the Nexus between Alcohol-Associated Immunometabolic Dysregulation and Tissue Injury.

Alcohol misuse, directly or indirectly as a result of its metabolism, negatively impacts most tissues, including four with critical roles in energy metabolism regulation: the liver, pancreas, adipose, and skeletal muscle. Mitochondria have long been studied for their biosynthetic roles, such as ATP synthesis and initiation of apoptosis. However, current research has provided evidence that mitochondria participate in myriad cellular processes, including immune activation, nutrient sensing in pancreatic ß-cells, and skeletal muscle stem and progenitor cell differentiation. The literature indicates that alcohol impairs mitochondrial respiratory capacity, promoting reactive oxygen species (ROS) generation and disrupting mitochondrial dynamics, leading to dysfunctional mitochondria accumulation. As discussed in this review, mitochondrial dyshomeostasis emerges at a nexus between alcohol-disrupted cellular energy metabolism and tissue injury. Here, we highlight this link and focus on alcohol-mediated disruption of immunometabolism, which refers to two distinct, yet interrelated processes. Extrinsic immunometabolism involves processes whereby immune cells and their products influence cellular and/or tissue metabolism. Intrinsic immunometabolism describes immune cell fuel utilization and bioenergetics that affect intracellular processes. Alcohol-induced mitochondrial dysregulation negatively impacts immunometabolism in immune cells, contributing to tissue injury. This review will present the current state of literature, describing alcohol-mediated metabolic and immunometabolic dysregulation from a mitochondrial perspective.

A review of alcohol-pathogen interactions: New insights into combined disease pathomechanisms.

Progression of chronic infections to end-stage diseases and poor treatment results are frequently associated with alcohol abuse. Alcohol metabolism suppresses innate and adaptive immunity leading to increased viral load and its spread. In case of hepatotropic infections, viruses accelerate alcohol-induced hepatitis and liver fibrosis, thereby promoting end-stage outcomes, including cirrhosis and hepatocellular carcinoma (HCC). In this review, we concentrate on several unexplored aspects of these phenomena, which illustrate the combined effects of viral/bacterial infections and alcohol in disease development. We review alcohol-induced alterations implicated in immunometabolism as a central mechanism impacting metabolic homeostasis and viral pathogenesis in Simian immunodeficiency virus/human immunodeficiency virus infection. Furthermore, in hepatocytes, both HIV infection and alcohol activate oxidative stress to cause lysosomal dysfunction and leakage and apoptotic cell death, thereby increasing hepatotoxicity. In addition, we discuss the mechanisms of hepatocellular carcinoma and tumor signaling in hepatitis C virus infection. Finally, we analyze studies that review and describe the immune derangements in hepatotropic viral infections focusing on the development of novel targets and strategies to restore effective immunocompetency in alcohol-associated liver disease. In conclusion, alcohol exacerbates the pathogenesis of viral infections, contributing to a chronic course and poor outcomes, but the mechanisms behind these events are virus specific and depend on virus-alcohol interactions, which differ among the various infections.

Intact functional fourteen-subunit respiratory membrane-bound [NiFe]-hydrogenase complex of the hyperthermophilic archaeon Pyrococcus furiosus.

The archaeon Pyrococcus furiosus grows optimally at 100 °C by converting carbohydrates to acetate, CO2, and H2, obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is encoded by a 14-gene operon with both hydrogenase and Na(+)/H(+) antiporter modules. Herein a His-tagged MBH was expressed in P. furiosus and the detergent-solubilized complex purified under anaerobic conditions by affinity chromatography. Purified MBH contains all 14 subunits by electrophoretic analysis (13 subunits were also identified by mass spectrometry) and had a measured iron:nickel ratio of 15:1, resembling the predicted value of 13:1. The as-purified enzyme exhibited a rhombic EPR signal characteristic of the ready nickel-boron state. The purified and membrane-bound forms of MBH both preferentially evolved H2 with the physiological donor (reduced ferredoxin) as well as with standard dyes. The O2 sensitivities of the two forms were similar (half-lives of ∼ 15 h in air), but the purified enzyme was more thermolabile (half-lives at 90 °C of 1 and 25 h, respectively). Structural analysis of purified MBH by small angle x-ray scattering indicated a Z-shaped structure with a mass of 310 kDa, resembling the predicted value (298 kDa). The angle x-ray scattering analyses reinforce and extend the conserved sequence relationships of group 4 enzymes and complex I (NADH quinone oxidoreductase). This is the first report on the properties of a solubilized form of an intact respiratory MBH complex that is proposed to evolve H2 and pump Na(+) ions.

Proton-coupled electron transfer dynamics in the catalytic mechanism of a [NiFe]-hydrogenase.

The movement of protons and electrons is common to the synthesis of all chemical fuels such as H2. Hydrogenases, which catalyze the reversible reduction of protons, necessitate transport and reactivity between protons and electrons, but a detailed mechanism has thus far been elusive. Here, we use a phototriggered chemical potential jump method to rapidly initiate the proton reduction activity of a [NiFe] hydrogenase. Coupling the photochemical initiation approach to nanosecond transient infrared and visible absorbance spectroscopy afforded direct observation of interfacial electron transfer and active site chemistry. Tuning of intramolecular proton transport by pH and isotopic substitution revealed distinct concerted and stepwise proton-coupled electron transfer mechanisms in catalysis. The observed heterogeneity in the two sequential proton-associated reduction processes suggests a highly engineered protein environment modulating catalysis and implicates three new reaction intermediates; Nia-I, Nia-D, and Nia-SR(-). The results establish an elementary mechanistic understanding of catalysis in a [NiFe] hydrogenase with implications in enzymatic proton-coupled electron transfer and biomimetic catalyst design.

Production and Application of a Soluble Hydrogenase from Pyrococcus furiosus.

Hydrogen gas is a potential renewable alternative energy carrier that could be used in the future to help supplement humanity's growing energy needs. Unfortunately, current industrial methods for hydrogen production are expensive or environmentally unfriendly. In recent years research has focused on biological mechanisms for hydrogen production and specifically on hydrogenases, the enzyme responsible for catalyzing the reduction of protons to generate hydrogen. In particular, a better understanding of this enzyme might allow us to generate hydrogen that does not use expensive metals, such as platinum, as catalysts. The soluble hydrogenase I (SHI) from the hyperthermophile Pyrococcus furiosus, a member of the euryarchaeota, has been studied extensively and used in various biotechnological applications. This review summarizes the strategies used in engineering and characterizing three different forms of SHI and the properties of the recombinant enzymes. SHI has also been used in in vitro systems for hydrogen production and NADPH generation and these systems are also discussed.

High yield purification of a tagged cytoplasmic [NiFe]-hydrogenase and a catalytically-active nickel-free intermediate form.

The cytoplasmic [NiFe]-hydrogenase I (SHI) of the hyperthermophile Pyrococcus furiosus evolves hydrogen gas (H2) from NADPH. It has been previously used for biohydrogen production from sugars using a mixture of enzymes in an in vitro cell-free synthetic pathway. The theoretical yield (12 H2/glucose) is three times greater than microbial fermentation (4 H2/glucose), making the in vitro approach very promising for large scale biohydrogen production. Further development of this process at an industrial scale is limited by the availability of the H2-producing SHI. To overcome the obstacles of the complex biosynthetic and maturation pathway for the [NiFe] site of SHI, the four gene operon encoding the enzyme was overexpressed in P. furiosus and included a polyhistidine affinity tag. The one-step purification resulted in a 50-fold increase in yield compared to the four-step purification procedure for the native enzyme. A trimeric form was also identified that lacked the [NiFe]-catalytic subunit but catalyzed NADPH oxidation with a specific activity similar to that of the tetrameric form. The presence of an active trimeric intermediate confirms the proposed maturation pathway where, in the terminal step, the NiFe-containing catalytic subunit assembles with NADPH-oxidizing trimeric form to give the active holoenzyme.

Mannosylglycerate and di-myo-inositol phosphate have interchangeable roles during adaptation of Pyrococcus furiosus to heat stress.

Marine hyperthermophiles accumulate small organic compounds, known as compatible solutes, in response to supraoptimal temperatures or salinities. Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at temperatures near 100°C. This organism accumulates mannosylglycerate (MG) and di-myo-inositol phosphate (DIP) in response to osmotic and heat stress, respectively. It has been assumed that MG and DIP are involved in cell protection; however, firm evidence for the roles of these solutes in stress adaptation is still missing, largely due to the lack of genetic tools to produce suitable mutants of hyperthermophiles. Recently, such tools were developed for P. furiosus, making this organism an ideal target for that purpose. In this work, genes coding for the synthases in the biosynthetic pathways of MG and DIP were deleted by double-crossover homologous recombination. The growth profiles and solute patterns of the two mutants and the parent strain were investigated under optimal growth conditions and also at supraoptimal temperatures and NaCl concentrations. DIP was a suitable replacement for MG during heat stress, but substitution of MG for DIP and aspartate led to less efficient growth under conditions of osmotic stress. The results suggest that the cascade of molecular events leading to MG synthesis is tuned for osmotic adjustment, while the machinery for induction of DIP synthesis responds to either stress agent. MG protects cells against heat as effectively as DIP, despite the finding that the amount of DIP consistently increases in response to heat stress in the nine (hyper)thermophiles examined thus far.

Engineering hyperthermophilic archaeon Pyrococcus furiosus to overproduce its cytoplasmic [NiFe]-hydrogenase.

The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H(2) production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production.

Minoxidil weakens newly synthesized collagen in fibrotic synoviocytes from osteoarthritis patients.

PURPOSE: Synovial fibrosis (SFb) formation and turnover attributable to knee osteoarthritis (KOA) can impart painful stiffness and persist following arthroplasty. To supplement joint conditioning aimed at maximizing peri-operative function, we evaluated the antifibrotic effect of Minoxidil (MXD) on formation of pyridinoline (Pyd) cross-links catalyzed by Plod2-encoded lysyl hydroxylase (LH)2b that strengthen newly synthesized type-I collagen (COL1) in fibroblastic synovial cells (FSCs) from KOA patients. MXD was predicted to decrease Pyd without significant alterations to Col1a1 transcription by FSCs stimulated with transforming growth factor (TGF)ß1. METHODS: Synovium from 10 KOA patients grouped by SFb severity was preserved for picrosirius and LH2b histology or culture. Protein and RNA were purified from fibrotic FSCs after 8 days with or without 0.5 µM MXD and/or 4 ng/mL of TGFß1. COL1 and Pyd protein concentrations from ELISA and expression of Col1a1, Acta2, and Plod2 genes by qPCR were compared by parametric tests with α = 0.05. RESULTS: Histological LH2b expression corresponded to SFb severity. MXD attenuated COL1 output in KOA FSCs but only in the absence of TGFß1 and consistently decreased Pyd under all conditions with significant downregulation of Plod2 but minimal alterations to Col1a1 and Acta2 transcripts. CONCLUSIONS: MXD is an attractive candidate for local antifibrotic pharmacotherapy for SFb by compromising the integrity of newly formed fibrous deposits by FSCs during KOA and following arthroplasty. Targeted antifibrotic supplementation could improve physical therapy and arthroscopic lysis strategies aimed at breaking down joint scarring. However, the effect of MXD on other joint-specific TGFß1-mediated processes or non-fibrotic components requires further investigation.

Chronic Binge Alcohol and Ovarian Hormone Loss Dysregulate Circulating Immune Cell SIV Co-Receptor Expression and Mitochondrial Homeostasis in SIV-Infected Rhesus Macaques.

Effective antiretroviral therapy (ART) has transitioned HIV to a chronic disease, with more than 50% of people living with HIV (PLWH) being over the age of 50. HIV targets activated CD4+ T cells expressing HIV-specific co-receptors (CCR5 and CXCR4). Previously, we reported that chronic binge alcohol (CBA)-administered male rhesus macaques had a higher percentage of gut CD4+ T cells expressing simian immunodeficiency virus (SIV) co-receptor CXCR4. Evidence also suggests that gonadal hormone loss increased activated peripheral T cells. Further, mitochondrial function is critical for HIV replication and alcohol dysregulates mitochondrial homeostasis. Hence, we tested the hypothesis that CBA and ovariectomy (OVX) increase circulating activated CD4+ T cells expressing SIV co-receptors and dysregulate mitochondrial homeostasis in SIV-infected female rhesus macaques. Results showed that at the study end-point, CBA/SHAM animals had increased peripheral CD4+ T cell SIV co-receptor expression, and a lower CD4+ T cell count compared to CBA/OVX animals. CBA and OVX animals had altered peripheral immune cell gene expression important for maintaining mitochondrial homeostasis. These results provide insights into how at-risk alcohol use could potentially impact viral expression in cellular reservoirs, particularly in SIV-infected ovariectomized rhesus macaques.

Alcohol Impairs Immunometabolism and Promotes Naïve T Cell Differentiation to Pro-Inflammatory Th1 CD4+ T Cells.

CD4+ T cell differentiation to pro-inflammatory and immunosuppressive subsets depends on immunometabolism. Pro-inflammatory CD4+ subsets rely on glycolysis, while immunosuppressive Treg cells require functional mitochondria for their differentiation and function. Previous pre-clinical studies have shown that ethanol (EtOH) administration increases pro-inflammatory CD4+ T cell subsets; whether this shift in immunophenotype is linked to alterations in CD4+ T cell metabolism had not been previously examined. The objective of this study was to determine whether ethanol alters CD4+ immunometabolism, and whether this affects CD4+ T cell differentiation. Naïve human CD4+ T cells were plated on anti-CD3 coated plates with soluble anti-CD28, and differentiated with IL-12 in the presence of ethanol (0 and 50 mM) for 3 days. Both Tbet-expressing (Th1) and FOXP3-expressing (Treg) CD4+ T cells increased after differentiation. Ethanol dysregulated CD4+ T cell differentiation by increasing Th1 and decreasing Treg CD4+ T cell subsets. Ethanol increased glycolysis and impaired oxidative phosphorylation in differentiated CD4+ T cells. Moreover, the glycolytic inhibitor 2-deoxyglucose (2-DG) prevented the ethanol-mediated increase in Tbet-expressing CD4+ T cells but did not attenuate the decrease in FOXP3 expression in differentiated CD4+ T cells. Ethanol increased Treg mitochondrial volume and altered expression of genes implicated in mitophagy and autophagosome formation (PINK1 and ATG7). These results suggest that ethanol impairs CD4+ T cell immunometabolism and disrupts mitochondrial repair processes as it promotes CD4+ T cell differentiation to a pro-inflammatory phenotype.

A Novel FACS-Based Workflow for Simultaneous Assessment of RedOx Status, Cellular Phenotype, and Mitochondrial Genome Stability.

Intracellular reduction-oxidation (RedOx) status mediates a myriad of critical biological processes. Importantly, RedOx status regulates the differentiation of hematopoietic stem and progenitor cells (HSPCs), mesenchymal stromal cells (MSCs) and maturation of CD8+ T Lymphocytes. In most cells, mitochondria are the greatest contributors of intracellular reactive oxygen species (ROS). Excess ROS leads to mitochondrial DNA (mtDNA) damage and protein depletion. We have developed a fluorescence-activated cell sorting (FACS)-based protocol to simultaneously analyze RedOx status and mtDNA integrity. This simultaneous analysis includes measurements of ROS (reduced glutathione (GSH)), ATP5H (nuclear encoded protein), MTCO1 (mitochondrial DNA encoded protein), and cell surface markers to allow discrimination of different cell populations. Using the ratio of MTCO1 to ATP5H median fluorescence intensity (MFI), we can gain an understanding of mtDNA genomic stability, since MTCO1 levels are decreased when mtDNA becomes significantly damaged. Furthermore, this workflow can be optimized for sorting cells, using any of the above parameters, allowing for downstream quantification of mtDNA genome copies/nucleus by quantitative PCR (qPCR). This unique methodology can be used to enhance analyses of the impacts of pharmacological interventions, as well as physiological and pathophysiological processes on RedOx status along with mitochondrial dynamics in most cell types.

Engineering the respiratory membrane-bound hydrogenase of the hyperthermophilic archaeon Pyrococcus furiosus and characterization of the catalytically active cytoplasmic subcomplex.

The archaeon Pyrococcus furiosus grows optimally at 100°C by converting carbohydrates to acetate, carbon dioxide and hydrogen gas (H2), obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is classified as a Group 4 hydrogenase and is encoded by a 14-gene operon that contains hydrogenase and Na(+)/H(+) antiporter modules. Herein a His-tagged 4-subunit cytoplasmic subcomplex of MBH (C-MBH) was engineered and expressed in P. furiosus by differential transcription of the MBH operon. It was purified under anaerobic conditions by affinity chromatography without detergent. Purified C-MBH had a Fe : Ni ratio of 14 : 1, similar to the predicted value of 13 : 1. The O2 sensitivities of C-MBH and the 14-subunit membrane-bound version were similar (half-lives of ∼15 h in air), but C-MBH was more thermolabile (half-lives at 90°C of 8 and 25 h, respectively). C-MBH evolved H2 with the physiological electron donor, reduced ferredoxin, optimally at 60°C. This is the first report of the engineering and characterization of a soluble catalytically active subcomplex of a membrane-bound respiratory hydrogenase.

Biological conversion of carbon dioxide and hydrogen into liquid fuels and industrial chemicals.

Non-photosynthetic routes for biological fixation of carbon dioxide into valuable industrial chemical precursors and fuels are moving from concept to reality. The development of 'electrofuel'-producing microorganisms leverages techniques in synthetic biology, genetic and metabolic engineering, as well as systems-level multi-omic analysis, directed evolution, and in silico modeling. Electrofuel processes are being developed for a range of microorganisms and energy sources (e.g. hydrogen, formate, electricity) to produce a variety of target molecules (e.g. alcohols, terpenes, alkenes). This review examines the current landscape of electrofuel projects with a focus on hydrogen-utilizing organisms covering the biochemistry of hydrogenases and carbonic anhydrases, kinetic and energetic analyses of the known carbon fixation pathways, and the state of genetic systems for current and prospective electrofuel-producing microorganisms.

Homologous expression of a subcomplex of Pyrococcus furiosus hydrogenase that interacts with pyruvate ferredoxin oxidoreductase.

Hydrogen gas is an attractive alternative fuel as it is carbon neutral and has higher energy content per unit mass than fossil fuels. The biological enzyme responsible for utilizing molecular hydrogen is hydrogenase, a heteromeric metalloenzyme requiring a complex maturation process to assemble its O(2)-sensitive dinuclear-catalytic site containing nickel and iron atoms. To facilitate their utility in applied processes, it is essential that tools are available to engineer hydrogenases to tailor catalytic activity and electron carrier specificity, and decrease oxygen sensitivity using standard molecular biology techniques. As a model system we are using hydrogen-producing Pyrococcus furiosus, which grows optimally at 100°C. We have taken advantage of a recently developed genetic system that allows markerless chromosomal integrations via homologous recombination. We have combined a new gene marker system with a highly-expressed constitutive promoter to enable high-level homologous expression of an engineered form of the cytoplasmic NADP-dependent hydrogenase (SHI) of P. furiosus. In a step towards obtaining 'minimal' hydrogenases, we have successfully produced the heterodimeric form of SHI that contains only two of the four subunits found in the native heterotetrameric enzyme. The heterodimeric form is highly active (150 units mg(-1) in H(2) production using the artificial electron donor methyl viologen) and thermostable (t(1/2) ∼0.5 hour at 90°C). Moreover, the heterodimer does not use NADPH and instead can directly utilize reductant supplied by pyruvate ferredoxin oxidoreductase from P. furiosus. The SHI heterodimer and POR therefore represent a two-enzyme system that oxidizes pyruvate and produces H(2) in vitro without the need for an intermediate electron carrier.

Heterologous expression and maturation of an NADP-dependent [NiFe]-hydrogenase: a key enzyme in biofuel production.

Hydrogen gas is a major biofuel and is metabolized by a wide range of microorganisms. Microbial hydrogen production is catalyzed by hydrogenase, an extremely complex, air-sensitive enzyme that utilizes a binuclear nickel-iron [NiFe] catalytic site. Production and engineering of recombinant [NiFe]-hydrogenases in a genetically-tractable organism, as with metalloprotein complexes in general, has met with limited success due to the elaborate maturation process that is required, primarily in the absence of oxygen, to assemble the catalytic center and functional enzyme. We report here the successful production in Escherichia coli of the recombinant form of a cytoplasmic, NADP-dependent hydrogenase from Pyrococcus furiosus, an anaerobic hyperthermophile. This was achieved using novel expression vectors for the co-expression of thirteen P. furiosus genes (four structural genes encoding the hydrogenase and nine encoding maturation proteins). Remarkably, the native E. coli maturation machinery will also generate a functional hydrogenase when provided with only the genes encoding the hydrogenase subunits and a single protease from P. furiosus. Another novel feature is that their expression was induced by anaerobic conditions, whereby E. coli was grown aerobically and production of recombinant hydrogenase was achieved by simply changing the gas feed from air to an inert gas (N2). The recombinant enzyme was purified and shown to be functionally similar to the native enzyme purified from P. furiosus. The methodology to generate this key hydrogen-producing enzyme has dramatic implications for the production of hydrogen and NADPH as vehicles for energy storage and transport, for engineering hydrogenase to optimize production and catalysis, as well as for the general production of complex, oxygen-sensitive metalloproteins.

Description of four novel psychrophilic, ionizing radiation-sensitive Deinococcus species from alpine environments.

Five psychrophilic bacterial strains were isolated from soil samples collected above the treeline of alpine environments. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these organisms represent four novel species of the genus Deinococcus; levels of sequence similarity to the type strains of recognized Deinococcus species were in the range 89.3-94.7 %. Strains PO-04-20-132T, PO-04-20-144, PO-04-19-125T, ME-04-01-32T and ME-04-04-52T grew aerobically, with optimum growth at 10 degrees C and at pH 6-9. The major respiratory menaquinone was MK-8. The fatty acid profiles of strains PO-04-20-132T, PO-04-20-144, PO-04-19-125T and ME-04-01-32T were dominated by 16 : 1omega7c, 17 : 0 iso and 15 : 1omega6c, whereas 16 : 1omega7c, 17 : 0 cyclo and 16 : 0 predominated in strain ME-04-04-52T. The DNA G+C contents of strains PO-04-20-132T, PO-04-19-125T, ME-04-01-32T and ME-04-04-52T were 63.2, 63.1, 65.9 and 62.6 mol%, respectively. Strains PO-04-20-132T, PO-04-19-125T, ME-04-01-32T and ME-04-04-52T had gamma radiation D10 (dose required to reduce the bacterial population by 10-fold) values of < or =4 kGy. These four strains showed sensitivity to UV radiation and extended desiccation as compared with Deinococcus radiodurans. On the basis of the phylogenetic analyses, and chemotaxonomic and phenotypic data, it is proposed that strains PO-04-20-132T (=LMG 24019T=NRRL B-41950T; Deinococcus radiomollis sp. nov.), PO-04-19-125T (=LMG 24282T=NRRL B-41949T; Deinococcus claudionis sp. nov.), ME-04-01-32T (=LMG 24022T=NRRL B-41947T; Deinococcus altitudinis sp. nov.) and ME-04-04-52T (=LMG 24283T=NRRL B-41948T; Deinococcus alpinitundrae sp. nov.) represent the type strains of four novel species of the genus Deinococcus.