Methanogenium,a new genus of marine methanogenic bacteria,and characterization ofMethanogenium cariaci sp. nov. andMethanogenium marisnigri sp. nov.

A new genus of marine methanogenic bacteria and two species within this genus are described.Methanogenium is the proposed genus andMethanogenium cariaci the type species. Cells of the type species are Gram-negative, peritrichously flagellated, irregular cocci with a periodic wall surface pattern. Colonies formed by these bacteria are yellow, circular and umbonate with entire edges. The DNA base composition is 52 mol% guanine plus cytosine. Formate or hydrogen and carbon dioxide serve as substrates for growth. Cells ofMethanogenium marisnigri are of similar shape but smaller diameter thanM. cariaci. The colonies ofM. marisnigri are convex, and the DNA base composition is 61 mol % G+C. Formate or hydrogen and carbon dioxide are growth substrates. Sodium chloride is required for growth of both methanogens.Abbreviations SDS sodium dodecylsulfate - PIPES piperazine-N,N-bis (2 ethanesulfonic acid) - HS-CoM coenzyme M, 2-mercaptoethanesulfonic acid     

Methanogenium marinum sp. nov., a H2-using methanogen from Skan Bay,Alaska, and kinetics of H2 utilization

A methanogen, strain AK-1, was isolated from permanently cold marine sediments, 38- to 45-cm below the sediment surface at Skan Bay, Alaska. The cells were highly irregular, nonmotile coccoids (diameter, 1 to 1.2 μm), occurring singly. Cells grew by reducing CO₂ with H₂ or formate as electron donor. Growth on formate was much slower than that on H₂. Acetate, methanol, ethanol, 1- or 2-propanol, 1- or 2-butanol and trimethylamine were not catabolized. The cells required acetate, thiamine, riboflavin, a high concentration of vitamin B₁₂, and peptones for growth; yeast extract stimulated growth but was not required. The cells grew fastest at 25 °C (range 5 °C to 25 °C), at a pH of 6.0 – 6.6 (growth range, pH 5.5 – 7.5), and at a salinity of 0.25 – 1.25 M Na⁺. Cells of this and other H₂-using methanogens from saline environments metabolized H₂ to a very low threshold pressure (less than 1 Pa) that was dependent on the methane partial pressure. We propose that the threshold pressure may be limited by the energetics of catabolism. The sequence of the 16S rDNA gene of strain AK-1 was most similar (98%) to the sequences of Methanogenium cariaci JR-1 and Methanogenium frigidum Ace-2. DNA–DNA hybridization between strain AK-1 and these two strains showed only 34.9% similarity to strain JR-1 and 56.5% similarity to strain Ace-2. These analyses indicated strain AK-1 should be classified as a new species within the genus Methanogenium. Phenotypic differences between strain AK-1 and these strains (including growth temperature, salinity range, pH range, and nutrient requirements) support this. Therefore, a new species, Methanogenium marinum, is proposed with strain AK-1 as type strain. This revised version was published online in August 2006 with corrections to the Cover Date.     

Methanogenium liminatans spec. nov., a new coccoid,mesophilic methanogen able to oxidize secondary alcohols

A new mesophilic, coccoid methanogen, assigned as Methanogenium liminatans spec. nov. strain DSM 4140, was isolated from effluent of a reactor for the anaerobic treatment of industrial waste water. Cells of M. Liminatans formed irregular cocci, about 1.5 m in size, and occurred singly. The cell envelope was an S-layer with hexagonally arranged glycoprotein subunits (M_r=118000). The center-to-center spacings were 15.4 nm. The polar lipid pattern was similar to that of Methanogenium tationis, the polyamine content similar to that found in several Methanogenium species. Strain DSM 4140 grew with H₂/CO₂, formate, 2-propanol/CO₂, 2-butanol/CO₂ and cyclopentanol/CO₂. For growth with the different substrates acetate was required as an additional carbon source. Growth on H₂/CO₂ was stimulated by the addition of tungstate. The optimal concentration was 1–2 M Na₂WO₄. ¹⁸⁵WO _inf4 ^sup2- was incorporated into cells. Growth was not influenced by 0–600 mM NaCl, but no growth occurred in the presence of 800 mM NaCl. Increasing concentrations of KCl up to 100 mM were slightly inhibitory for growth. The optimal growth temperature was around 40°C. The G+C content of the DNA was 59.3 mol% (T_m) or 60.5 mol% (HPLC).Abbreviations HPLC = high performance liquid chromatography - PAS = periodic acid-Schiff reagent - sADH = secondary alcohol dehydrogenase - F₄₂₀ = 7,8-didemethyl-8-hydroxy-5-deazariboflavin     

Classification of secondary alcohol-utilizing methanogens including a new thermophilic isolate

A thermophilic coccoid methanogenic bacterium, strain TCI, that grew optimally around 55° C was isolated with 2-propanol as hydrogen donor for methanogenesis from CO₂. H₂, formate or 2-butanol were used in addition. Each secondary alcohol was oxidized to its ketone. Growth occurred in defined freshwater as well as salt (2% NaCl, w/v) medium. Acetate was required as carbon source, and 4-aminobenzoate and biotin as growth factors. A need for molybdate or alternatively tungstate was shown.Strain TCI was further characterized together with two formerly isolated mesophilic secondary alcohol-utilizing methanogens, the coccoid strain CV and the spirilloid strain SK. The guanine plus cytosine content of the DNA of the three strains was 55,47, and 39 mol%, respectively. Determination of the molecular weights of the methylreductase subunits and sequencing of ribosomal 16S RNA of strains TCI and CV revealed close relationships to the genus Methanogenium. The new isolate TCI is classified as a strain of the existing species, Methanogenium thermophilum (thermophilicum). For strain CV, that uses ethanol or 1-propanol in addition, a classification as new species, Methanogenium organophilum, is proposed. Strain SK is affiliated with the existing species, Methanospirillum hungatei. The ability to use secondary alcohols was also tested with described species of methanogens. Growth with secondary alcohols was observed with Methanogenium marisnigri, Methanospirillum hungatei strain GP1 and Methanobacterium bryantii, but not with Methanospirillum strains JF1 and M1h, Methanosarcina barkeri, Methanococcus species or thermophilic strains or species other than the new isolate TCI.     

Isolation and characterization of a new thermophilic and autotrophic methane producing bacterium:Methanobacterium thermoaggregans spec. nov.

Methanobacterium thermoaggregans is a new thermophilic autotrophic rod-shaped methane producing bacterium. The organism likes to form aggregates during growth and utilizes only H₂ and CO₂ as substrates. Growth optimum is at 65°C with a doubling time of 3.5 h. Optimal growth occurs at pH-values between 7 and 7.5. The addition of yeast extract to the mineral salt medium stimulates growth. The DNA base composition is 42 mol% G+C. The organism was isolated from mud taken from a cattle pasture. Because of its optimal growth temperature and its tendency to form aggregates the nameMethanobacterium thermoaggregans is suggested.Abbreviations G+C Guanine+cytosine     

Methanobacterium thermoalcaliphilum spec. nov., a new moderately alkaliphilic and thermophilic autotrophic methanogen

An autotrophic moderately alkaliphilic and thermophilic nonmotile rod-shaped methanogen was isolated from a biogas plant. The isolate grows only on H₂+CO₂ and requires yeast extract. Growth optimum is at 60°C with a generation time of 4 h. In the absence of substrates complete lysis occurs. The pH range for growth is 7.5–8.5. Growth was also observed at pH values above 9.0. The DNA base composition is 38.8 mol% G+C. According to its physiological properties the nameMethanobacterium thermoalcaliphilum is proposed.Abbreviations G+C Guanine+cytosine     

Nucleotide distribution in bacterial DNA's differing in G + C content

Summary The DNA's ofMicrococcus lysodeikticus andClostridium perfringens were fragmented to about 7 000 nucleotide pairs long by shear and fractionated with respect to buoyant density of mercury complexes in Cs₂SO₄. The distribution of G + C content in both DNA's was characteristically asymmetric. InM. lysodeikticus DNA, low G + C fragments were more numerous than high G + C fragments, whereas inC. perfringens DNA, high G + C fragments were more numerous than low G + C fragments. The G + C content of fragments ofM. lysodeikticus DNA varied from 70 to 77%, with a mean and standard deviation of 73.7 ± 1.92% G + C and that ofC. perfringens DNA varied from 27 to 34%, with a mean and standard deviation of 29.8 ± 1.34% G + C. The standard deviation was smaller than that ofEscherichia coli DNA fragments of similar size. Biological meanings of relatively low heterogeneity in nucleotide composition inM. lysodeikticus andC. perfringens are discussed.     

Thiobacillus plumbophilus spec. nov., a novel galena and hydrogen oxidizer

From an uranium mine three strains of rodshaped, mesophilic, chemolithoautotrophic bacteria were isolated. They grow by oxidation of H₂S, galena (PbS) and H₂. Anglesite (PbSO₄) is formed from galena. No ferrous iron is oxidized by the isolates. They grow between pH 4 and 6.5 at temperatures of about 9 to 41°C (optimum around 27°C). The G+C content of the DNA is around 66 mol %. Based on their ability to oxidize sulfur compounds, the new organisms belong to the genus Thiobacillus. No significant homology with Thiobacillus ferrooxidans and Thiobacillus cuprinus was detected by DNA-DNA hybridization. Therefore the new isolates represent a new species within the genus Thiobacillus. Based on the unusual growth on galena, we name the new species Thiobacillus plumbophilus (type strain Gro 7; DSM 6690).     

Isolation and characterization of Methanoplanus endosymbiosus sp. nov., an endosymbiont of the marine sapropelic ciliate Metopus contortus quennerstedt

Epifluorescence microscopy revealed the presence of a methanogenic bacterium as an endosymbiont in the sapropelic marine ciliate Metopus contortus. The in situ methanogenic activity of the symbiont could be demonstrated. The isolated endosymbiont was an irregular, disc-shaped bacterium with a diameter of 1.6–3.4 m. It had a generation time of 7 or 12 hours on growth on H₂/CO₂ or formate, respectively. The temperature range for growth was between 16 and 36°C with an optimum at 32°C. The optimal pH range for growth was 6.8 to 7.3. Salts, with an optimum concentration of 0.25 M, and tungsten were required for growth. The mol% G+C was 38.7%. The cell envelope consisted of proteins and a glycoprotein with an apparent molecular weight of 110,000. Morphology, antigenic relationship and the G+C content established the isolate MC1 as a new species of the genus Methanoplanus, and the name Methanoplanus endosymbiosus is proposed.Abbreviations G+C Guanine+cytosine - SDS sodium dodecylsulfate - PIPES piperazine-N,N-bis (2-ethane) sulfonic acid     

Salinicoccus salsiraiae sp. nov.: a new moderately halophilic gram-positive bacterium isolated from salted skate

Two moderately halophilic low G + C Gram-positive bacteria were isolated from a sample of salted skate (Class Chondrychthyes, Genus Raja). Phylogenetic analysis of the 16S rRNA gene sequence of strains RH1^T and RH4 showed that these organisms represented a novel species of the genus Salinicoccus. The new isolates formed pink–red colonies and flocculated in liquid media, with optimum growth in media containing 4% NaCl and pH of about 8.0. These organisms are aerobic but reduce nitrate to nitrite under anaerobic conditions. Acid is produced from several carbohydrates. Oxidase and catalase were detected. Menaquinone 6 was the major respiratory quinone. The major fatty acids of strains RH1^T and RH4 were 15:0 anteiso and 15:0 iso. The G + C contents of DNA were 46.2 and 46.0 mol%, respectively. The peptidoglycan was of A3alpha L-Lys-Gly_5–6 type. On the basis of the phylogenetic analyses, physiological and biochemical characteristics, we suggest that strain RH1^T (=LMG 22840 = CIP 108576) represents a new species of the genus Salinicoccus, for which we propose the name Salinicoccus salsiraiae.     

An extremely thermophilic Methanococcus from a deep sea hydrothermal vent and its plasmid

An extremely thermophilic methanogen was isolated from a hydrothermal vent core sample from Guaymas Basin, Gulf of California, at a depth of 2003 m. The isolate, designated strain AG86, was a coccoid autotroph using H₂-CO₂ as energy and carbon source with a growth temperature range of 48 to 92°C, optimum, 85°C. AG86 required NaCl and Mg²⁺ and trace amounts of selenite and tungstate. Vitamins were not required. However, yeast extract, Casamino acids and Trypticase stimulated growth significantly. When grown in the presence of these stimulants and at the optimal growth temperature and pH 6.5, the minimum doubling time was 20 min. Cells were fragile and readily lysed by detergents. The mol% G+C was 33%. These results and partial 16S rRNA sequencing indicated that AG86 belonged to the genus Methanococcus and closely resembled Methanococcus jannaschii. Tests for extrachromosomal DNA revealed a plasmid in AG86 and two plasmids in M. jannaschii. Different patterns were obtained from restriction endonuclease digestion of the three plasmids, and no homology was observed with DNA-DNA hybridization.Abbreviations CCC DNA covalently close circular DNA - DM defined marine medium - G+C Guanine plus cytosine - MPN most probable number     

Characterization of a new mesophilic,secondary alcohol-utilizing methanogen,Methanobacterium palustre spec. nov. from a peat bog

The isolation and characterization of a new methanogen from a peat bog, Methanobacterium palustre spec. nov., strain F, is described. Strain F grew on H₂/CO₂ and formate in complex medium. It also grew autotrophically on H₂/CO₂. Furthermore, growth on 2-propanol/CO₂ was observed. Methane was formed from CO₂ by oxidation of 2-propanol to acetone or 2-butanol to 2-butanone, but growth on 2-butanol plus CO₂ apparently was too little to be measurable. Similarly, Methanobacterium bryantii M. o. H. and M. o. H. G formed acetone and 2-butanone from 2-propanol and 2-butanol, but no growth was measurable.On the basis of morphological and biochemical features strain F could be excluded from the genus Methanobrevibacter. Due to its cell morphology, lipid composition and polyamine pattern it belonged to the genus Methanobacterium. From known members of this genus strain F could be distinguished either by a different G+C content of the DNA, low DNA-DNA homology with reference strains, lacking serological reactions with anti-S probes and differences in the substrate spectrum.An alcohol dehydrogenase activity, specific for secondary alcohols and its substrate specificity was determined in crude extracts of strain F. NADP⁺ was the only electron carrier that was utilized. No reaction was found with NAD⁺, F₄₂₀, FMN and FAD.Abbreviations NAD⁺ nicotinamide adenine dinucleotide - NADH₂ reduced form of NAD⁺ - NADP⁺ nicotinamide adenine dinucleotide phosphate - NADPH₂ reduced form of NADP⁺ - FMN flavin adenine mononucleotide - FAD flavin adenine dinucleotide - ADH alcohol dehydrogenase - F₄₂₀ 8-hydroxy-7,8-didemethyl-5-deazaflavin - SSC standard saline citrate (0.15 M NaCl, 0.015 M trisodium citrate, pH 7.5)     

Psychrotrophic,lactic acid-producing bacteria from anoxic waters in Ace Lake,Antarctica; Carnobacterium funditum sp. nov. and Carnobacterium alterfunditum sp. nov.

Heterofermentative, lactic acid-producing, gram-positive, motile bacteria were isolated from the waters of Ace Lake, Antarctica. All strains produced virtually only l(+)lactic acid from d(+)glucose. d(–)ribose was fermented to lactic, acetic, and formic acids, and ethanol. Cell walls contained meso-diaminopimaleic acid. The strains did not grow at 30°C and were psychrotrophic. Whole cells contained 18:1cis 9 as a major component of their fatty acids. At 20°C, the strains grew better anaerobically than aerobically and all strains lacked catalase, oxidase and respiratory lipoquinones. DNA that coded for most of the 16S rRNA gene of one of the strains was amplified by the polymerase chain reaction and sequenced. The strain was phylogenetically most closely related to Carnobacterium mobile (K_nuc=0.0214). The isolates separated into two phenotypes. DNA/DNA homology studies determined on a representative from each phenotype showed low homology between the phenotypes (38±8%), and with Carnobacterium mobile (26±2%, 34±2%). Carnobacterium funditum sp. nov. produced acid from mannitol, trehalose, but not amygdalin. The G+C content of the DNA was 32–34%, and the Type strain is DSM 5970 (=ACAM 312). Carnobacterium alterfunditum sp. nov. produced acid weakly from amygdalin but not from mannitol or trehalose. The G+C content was 33–34%, and the Type strain is DSM 5972 (=ACAM 313).     

Growth and pigment production byArthrobacter pyridinolis n. sp.

A new bacterium capable of growing on 2-hydroxypyridine as sole source of carbon and nitrogen was isolated from soil. During its growth on solid medium, approximately 50% of this substrate was converted to a brilliant blue crystalline pigment which was deposited extracellularly in the colony mass. The pigment was identical to that produced byArthrobacter crystallopoietes during its growth on 2-hydroxypyridine. The new isolate exhibited the typical cycle of morphogenesis characteristic of the genusArthrobacter. The organism is different from all other reported species ofArthrobacter. It is proposed that the organism be namedArthrobacter pyridinolis n. sp.List of Abbreviations MSP mineral salts phosphate basal culture medium containing 2-hydroxypyridine, yeast extract and trace salts - 2-HP 2-hydroxypyridine - PFU plaque forming units - G+C guanine+cytosine - T _m midpoint of thermal denaturation     

DNA of Akodon (Rodentia,Cricetidae). I. Biophysical characterization

Buoyant density in CsCl, melting temperature, and G + C base content of the DNA from four species of Akodon (Rodentia, Cricetidae) were determined. The buoyant density values of 1.699–1.701 g/cm³ were in accordance with the data reported for other cricetids. No satellite bands were seen in neutral CsCl. The T _m values determined in 1 × SSC ranged from 86.2 to 87.0 C, which corresponds to G + C contents of 41.2–43.2%. There was good agreement in DNA base composition of the four species, although values were slightly higher in A. obscurus, suggesting a certain degree of interspecies variability.This study was supported by grants from Comisión de Investigaciones Científicas de la Provincia de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, and Organización de Estados Americanos.     

Clostridium mayombei sp. nov., an H2/CO2 acetogenic bacterium from the gut of the African soil-feeding termite,Cubitermes speciosus

Clostridium mayombei sp. nov., a previously undescribed H₂-oxidizing CO₂-reducing acetogenic bacterium, was isolated from gut contents of the African soilfeeding termite, Cubitermes speciosus. Cells were anaerobic, Gram positive, catalase and oxidase negative, endospore-forming motile rods which measured 1×2 – 6 m and which had a DNA base composition of 25.6 mol% G+C (strain SFC-5). Optimum conditions for growth on H₂+CO₂ were at 33°C and pH 7.3, and under these conditions cells produced acetate according to the equation: 4 H₂+2 CO₂CH₃COOH+2 H₂O. Other substrates supporting good growth included carbohydrates (e.g. glucose, xylose, starch), sugar alcohols, and organic and amino acids, and with these substrates acetate was almost always the principle fermentation product. Comparative analysis of 16S rRNA nucleotide sequences confirmed that C. mayombei was closely related to various members of the genus Clostridium. However, morphological and physiological differences between C. mayombei and other homoacetogenic clostridia were deemed significant enough to warrant creation of a new taxon. Results are discussed in light of the diversity of H₂/CO₂ acetogens recently isolated from various termites, and in terms of the relative importance of H₂/CO₂ acetogenesis to termite nutrition.     

Isolation and characterization of Thermanaerothrix daxensis gen. nov., sp. nov., a thermophilic anaerobic bacterium pertaining to the phylum "Chloroflexi", isolated from a deep hot aquifer in the Aquitaine Basin

A new strictly anaerobic thermophilic multicellular filamentous bacterium (0.2–0.3 μm × >100 μm), designated GNS-1^T, was isolated from a deep hot aquifer in France. It was non-motile, and stained Gram-negative. Optimal growth was observed at 65 °C, pH 7.0, and 2 g L⁻¹ of NaCl. Strain GNS-1^T was chemoorganotrophic fermenting ribose, glucose, galactose, arabinose, fructose, mannose, maltose, sucrose, xylose, raffinose, pyruvate, and xylan. Yeast extract was required for growth. The end products of glucose fermentation were lactate, acetate, CO₂, and H₂. The G + C content of the DNA was 57.6 mol%. Its closest phylogenetic relative was Bellilinea caldifistulae with 92.5% similarity. Based on phylogenetic, genotypic and phenotypic characteristics, strain GNS-1^T (DSM 23592^T, JCM 16980^T) is proposed to be assigned to a novel species of a novel genus within the class Anaerolineae (subphylum I), phylum “Chloroflexi”, Thermanaerothrix daxensis gen. nov., sp. nov. The GenBank accession number is HM596746.     

Eubacterium aggreganssp. nov., a New Homoacetogenic Bacterium from Olive Mill Wastewater Treatment Digestor

A strictly anaerobic, homoacetogenic, Gram-positive, non spore-forming bacterium, designated strain SR12^T(T=type strain), was isolated from an anaerobic methanogenic digestor fed with olive mill wastewater. Yeast extract was required for growth but could also be used as sole carbon and energy source. Strain SR12^Tutilized a few carbohydrates (glucose, fructose and sucrose), organic compounds (lactate, crotonate, formate and betaine), alcohols (methanol), the methoxyl group of some methoxylated aromatic compounds, and H₂+CO₂. The end-products of carbohydrate fermentation were acetate, formate, butyrate, H₂and CO₂. End-products from lactate and methoxylated aromatic compounds were acetate and butyrate. Strain SR12^Twas non-motile, formed aggregates, had a G+C content of 55 mol % and grew optimally at 35°C and pH 7.2 on a medium containing glucose. Phylogenetically, strain SR12^Twas related toEubacterium barkeri, E. callanderi, andE. limosumwithE. barkerias the closest relative (similarity of 98%) with which it bears little phenotypic similarity or DNA homology (60%). On the basis of its phenotypic, genotypic, and phylogenetic characteristics, we propose to designate strain SR12^TasEubacterium aggreganssp. nov. The type strain is SR12^T(=DSM 12183).     

Characterization of Desulfovibrio fructosovorans sp. nov.

Desulfovibrio strain JJ isolated from estuarine sediment differed from all other described Desulfovibrio species by the ability to degrade fructose. The oxidation was incomplete, leading to acetate production. Fructose, malate and fumarate were fermented mainly to succinate and acetate in the absence of an external electron acceptor. The pH and temperature optima for growth were 7.0 and 35° C respectively. Strain JJ was motile by means of a single polar flagellum. The DNA base composition was 64.13% G+C. Cytochrome c ₃ and desulfoviridin were present. These characteristics established the isolate as a new species of the genus Desulfovibrio, and the name Desulfovibrio fructosovorans is proposed.     

Acetonema longum gen.nov.sp.nov., an H2/CO2 acetogenic bacterium from the termite,Pterotermes occidentis

A previously undescribed, H₂-oxidizing CO₂-reducing acetogenic bacterium was isolated from gut contents of the wood-feeding termite, Pterotermes occidentis. Cells of representative strain APO-1 were strictly anaerobic, Gram-negative, endospore-forming motile rods which measured 0.30–0.40×6–60 m. Cells were catalase positive, oxidase negative, and had 51.5 mol percent G+C in their DNA. Optimum conditions for growth on H₂+CO₂ were at 30–33°C and pH (initial) 7.8, and under these conditions cells formed acetate according to the equation: 4 H₂+2 CO₂CH₃COOH+2 H₂O. Other energy sources supporting good growth of strain APO-1 included glucose, ribose, and various organic acids. Acetate and butyrate were major fermentation products from most organic compounds tested, however propionate, succinate, and 1,2-propanediol were also formed from some substrates. Based on comparative analysis of 16S rRNA nucleotide sequences, strain APO-1 was related to, but distinct from, members of the genus Sporomusa. Moreover, physiological and morphological differences between strain APO-1 and the six known species of Sporomusa were significant. Consequently, it is proposed herewith that a new genus, Acetonema, be established with strain APO-1 as the type strain of the new species, Acetonema longum. A. longum may contribute to the nutrition of P. occidentis by forming acetate, propionate and butyrate, compounds which are important carbon and energy sources for termites.