Long-distance electron transfer in a filamentous Gram-positive bacterium.

Long-distance extracellular electron transfer has been observed in Gram-negative bacteria and plays roles in both natural and engineering processes. The electron transfer can be mediated by conductive protein appendages (in short unicellular bacteria such as Geobacter species) or by conductive cell envelopes (in filamentous multicellular cable bacteria). Here we show that Lysinibacillus varians GY32, a filamentous unicellular Gram-positive bacterium, is capable of bidirectional extracellular electron transfer. In microbial fuel cells, L. varians can form centimetre-range conductive cellular networks and, when grown on graphite electrodes, the cells can reach a remarkable length of 1.08 mm. Atomic force microscopy and microelectrode analyses suggest that the conductivity is linked to pili-like protein appendages. Our results show that long-distance electron transfer is not limited to Gram-negative bacteria.

Ecological distribution of extremely thermophilic bacteria belonging to the genus Calditerricola using the novel enrichment MPN-PCR method.

A unique compost called Satsuma soil is produced from sewage sludge by a hyperthermal composting process in Kagoshima City, Japan. The composting process is carried out at a controlled temperature of at least 80°C and the resulting compost might be useful for recycling sustainable agricultural products. The extremely thermophilic bacterial genus Calditerricola was initially isolated from the high-temperature compost. Likewise, the bacteria were previously isolated from material sludge. It is believed that bacteria in this genus might be involved in the hyperthermal composting process. Calditerricola bacteria are distributed not only in compost, but also in all of its material sludge, and are more abundant in material sludge than in compost. Moreover, based on investigations of samples near geothermal areas in high temperature conditions, such as volcanoes, Calditerricola was presumed to originate in the volcanic ash of Mt. Sakurajima in Kagoshima City, Japan. However, its precise origin and ecology are unclear. Thus, in this study, a new molecular biological method called enrichment most probable number (MPN)-PCR (eMPN-PCR) was established and used to quantitatively investigate the population and distribution of the extreme thermophile Calditerricola in environmental samples using genus-specific PCR primers. The eMPN-PCR method was an effective quantitative detection method with high sensitivity, yielding MPN estimates that were highly correlated with colony forming unit (CFU) estimates but a low detection threshold value.

Jilinibacillus soli gen. nov., sp. nov., a novel member of the family Bacillaceae.

A Gram-positive, aerobic, rod-shaped, motile, endospore-forming bacterium, designated strain A12(T), was isolated from a saline and alkali soil samples in Baicheng City, western of Jilin Province, China. Growth occurred in 15-45 °C (optimum, 30 °C) and at pH 7.0-11.5 (optimum, pH 9.0) and in the presence of 0-10 % (w/v) NaCl [optimum, 1-3 % (w/v) NaCl]. Meso-DAP was present in the peptidoglycan. The predominant menaquinone was MK-7. The major polar lipid profile was phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidyl inositol-methyl and phosphotidylinositol dimannosid. The major fatty acid (>10 % of total fatty acids) was anteiso-C15:0. DNA G + C content was 36.2 mol %. The level of 16S rRNA gene sequence similarity between strain A12(T) and other recognized species of the family was below 95.6 %. Phylogenetic analysis based on 16S rRNA gene sequence data indicated that the strain A12(T) fell with the family Bacillaceae and formed a distinct taxon. Based on physiological, chemotaxonomic and phylogenetic analyses, strain A12(T) was considered to represent a novel species of a new genus, for which the name Jilinibacillus soli gen. nov., sp. nov. was proposed. The type strain of Jilinibacillus soli was A12(T) (=GIMN1.014(T) = CCTCC M2011164(T) = KCTC 33417(T)).

Characterisation and anti-biofilm activity of extracellular polymeric substances from Oceanobacillus iheyensis.

Oceanobacillus iheyensis BK6, isolated from a marine natural biofilm, produced approximately 40 0mg L(-1) extracellular polymeric substances (EPS). FTIR analysis of the EPS revealed different functional groups (halide groups, uronic acid and saccharides). The GCMS showed that the extracellular polysacchrides comprised of mannose (47.78%), glucose (29.71%) and arabinose (22.46%). The molecular mass of the EPS was about 2.14 × 10(6)Da. It was thermally stable and showed pseudoplastic rheology and emulsifying activity (66.47%). The EPS exhibited antibiofilm activity against a pathogenic strain of Staphylococcus aureus. This is the first report on the characterisation of EPS from the genus Oceanobacillus. The high viscosity, emulsifying properties and antibiofilm activity of EPS make it suitable for potential pharmaceutical and industrial applications.

Genome sequence of Lysinibacillus boronitolerans F1182, isolated from a traditional Korean fermented soybean product.

Lysinibacillus is a Gram-positive, rod-shaped, and round-spore-forming bacterial genus of the family Bacillaceae. We analyzed the genome sequence of Lysinibacillus boronitolerans F1182, isolated from a traditional Korean fermented soybean product. The genome sequence contained 4.46 Mbp with a G+C content of 37.5%. This is the first report of an L. boronitolerans genome.

Isolation of a Lysinibacillus fusiformis strain with tetrodotoxin-producing ability from puffer fish Fugu obscurus and the characterization of this strain.

Six dominant strains were isolated from the livers of the puffer fish Fugu obscurus and were screened for their TTX production-ability. Electrospray ionization-mass spectrometry (ESI-MS) analyses revealed that strain B-1 produced TTX and related substances; mouse bioassay revealed that 23.9 mouse units (MU) of toxins were present in 200ml of broth medium. On the basis of the physiological and biochemical characteristics of this strain and the results of 16S rRNA analysis, strain B-1 was identified as Lysinibacillus fusiformis.

Lentibacillus persicus sp. nov., a moderately halophilic species isolated from a saline lake.

A Gram-staining-positive, moderately halophilic bacterium, designated strain Amb31(T), was isolated from water of the hypersaline lake Aran-Bidgol in Iran and characterized taxonomically using a polyphasic approach. Cells were rods, motile and able to produce ellipsoidal endospores at a central position in swollen sporangia. Strain Amb31(T) was facultatively anaerobic and catalase- and oxidase-positive. The strain grew in a complex medium supplemented with 3-25 % (w/v) NaCl (optimum 7.5-10 %). Optimal growth was at 30-35 degrees C and pH 7.5. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that strain Amb31(T) belonged to the genus Lentibacillus; it exhibited 16S rRNA gene sequence similarity values of 96.8 and 96.4 % to Lentibacillus salicampi SF-20(T) and Lentibacillus salinarum AHS-1(T), respectively, and values of 95.9-94.7 % to the type strains of other recognized species of Lentibacillus. The cell-wall peptidoglycan of strain Amb31(T) was based on meso-diaminopimelic acid and MK-7 was the respiratory isoprenoid quinone. The major fatty acids were anteiso-C(15 : 0) (44.7 %), iso-C(16 : 0) (21.4 %) and anteiso-C(17 : 0) (15.9 %) and the polar lipid pattern consisted of phosphatidylglycerol, diphosphatidylglycerol, five phospholipids and a glycolipid. The DNA G+C content was 44.1 mol%. All these features confirmed the placement of strain Amb31(T) within the genus Lentibacillus and the strain could be clearly differentiated from strains of the other species of Lentibacillus on the basis of several phenotypic, genotypic and chemotaxonomic features. DNA-DNA relatedness with the type strain of the most closely related strain, L. salicampi DSM 16425(T), was 28 %. Therefore, strain Amb31(T) represents a novel species of the genus Lentibacillus, for which the name Lentibacillus persicus sp. nov. is proposed. The type strain is Amb31(T) (=CCM 7683(T) =CECT 7524(T) =DSM 22530(T) =LMG 25304(T)).

Halobacillus profundi sp. nov. and Halobacillus kuroshimensis sp. nov., moderately halophilic bacteria isolated from a deep-sea methane cold seep.

Two Gram-positive, rod-shaped, moderately halophilic bacteria were isolated from a deep-sea carbonate rock at a methane cold seep in Kuroshima Knoll, Japan. These bacteria, strains IS-Hb4(T) and IS-Hb7(T), were spore-forming and non-motile. They were able to grow at temperatures as low as 9 degrees C and hydrostatic pressures up to 30 MPa. Based on high sequence similarity of their 16S rRNA genes to those of type strains of the genus Halobacillus, from 96.4 % (strain IS-Hb7(T) to Halobacillus halophilus NCIMB 9251(T)) to 99.4 % (strain IS-Hb4(T) to Halobacillus dabanensis D-8(T)), the strains were shown to belong to this genus. DNA-DNA relatedness values of 49.5 % and 1.0-33.0 %, respectively, were determined between strains IS-Hb4(T) and IS-Hb7(T) and between these strains and other Halobacillus type strains. Both strains showed the major menaquinone MK7 and L-orn-D-Asp cell-wall peptidoglycan type. Straight-chain C(16 : 0), unsaturated C(16 : 1)omega7c alcohol and C(18 : 1)omega7c and cyclopropane C(19 : 0) cyc fatty acids were predominant in both strains. The DNA G+C contents of IS-Hb4(T) and IS-Hb7(T) were respectively 43.3 and 42.1 mol%. Physiological and biochemical analyses combined with DNA-DNA hybridization results allowed us to place strains IS-Hb4(T) (=JCM 14154(T)=DSM 18394(T)) and IS-Hb7(T) (=JCM 14155(T)=DSM 18393(T)) in the genus Halobacillus as the respective type strains of the novel species Halobacillus profundi sp. nov. and Halobacillus kuroshimensis sp. nov.

Oceanobacillus chironomi sp. nov., a halotolerant and facultatively alkaliphilic species isolated from a chironomid egg mass.

Chironomids (Diptera; Chironomidae) are the most abundant insects in freshwater aquatic habitats. Females of the genus Chironomus lay egg masses containing hundreds of eggs embedded in a gelatinous matrix. A bacterial strain, designated T3944D(T), was isolated from a chironomid egg mass sampled from a waste-stabilization pond in northern Israel and was found to be Gram-positive, motile by peritrichous flagella, endospore-forming, halotolerant and facultatively alkaliphilic. Comparative 16S rRNA gene sequence analysis showed that strain T3944D(T) belonged to the genus Oceanobacillus, exhibiting the highest levels of similarity with the sequences of Oceanobacillus oncorhynchi subsp. incaldanensis DSM 16557(T) (94.9 %), Oceanobacillus oncorhynchi subsp. oncorhynchi JCM 12661(T) (94.8 %), Oceanobacillus iheyensis JCM 11309(T) (94.7 %) and Oceanobacillus picturae LMG 19416 (94.5 %). Strain T3944D(T) grew optimally at 1-3 % NaCl, pH 8.5 and 37 degrees C. The major cellular fatty acids were anteiso-C(15 : 0) (60.0 %) and anteiso-C(17 : 0) (12.9 %) and the DNA G+C content was 38.1 mol%. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain T3944D(T) represents a novel species in the genus Oceanobacillus, for which the name Oceanobacillus chironomi sp. nov. is proposed. The type strain is T3944D(T) (=LMG 23627(T)=DSM 18262(T)).

Exiguobacterium profundum sp. nov., a moderately thermophilic, lactic acid-producing bacterium isolated from a deep-sea hydrothermal vent.

A facultatively anaerobic, halotolerant, moderately thermophilic and non-sporulating bacterium, designated strain 10C(T), was isolated from deep-sea hydrothermal vent samples collected on the 13 degrees N East Pacific Rise at a depth of approximately 2600 m. Cells of strain 10C(T) were Gram-positive, motile rods, and grew optimally at 45 degrees C (range 12-49 degrees C), pH 7.0 (range pH 5.5-9.5) and 0-2 % NaCl (range 0-11 %). (+)-L-Lactate was the main organic acid detected from carbohydrate fermentation with traces of formate, acetate and ethanol. Strain 10C(T) was catalase-positive, oxidase-negative and reduced nitrate to nitrite under anaerobic conditions. The DNA G+C content was 50.4 mol%. Its closest phylogenetic relatives were Exiguobacterium aestuarii TF-16(T) and Exiguobacterium marinum TF-80(T) (16S rRNA gene sequence similarity >99 %). However, strain 10C(T) differed genotypically from these two Exiguobacterium species as indicated by DNA-DNA relatedness data. Therefore, on the basis of phenotypic, genotypic and phylogenetic characteristics, strain 10C(T) is considered to represent a novel species of the genus Exiguobacterium, for which the name Exiguobacterium profundum sp. nov. is proposed. The type strain is 10C(T) (=CCUG 50949(T)=DSM 17289(T)).

Oceanobacillus profundus sp. nov., isolated from a deep-sea sediment core.

A halotolerant, facultatively alkaliphilic bacterium, designated CL-MP28T, was isolated from the surface of a sediment core sample collected at a depth of 2247 m in the Ulleung Basin of the East Sea, Korea. Phylogenetic analysis of the 16S rRNA gene sequence of strain CL-MP28T revealed an affiliation with the genus Oceanobacillus. The sequence similarities between the isolate and type strains of members of the genus Oceanobacillus were in the range 95.0-96.0 %, indicating that strain CL-MP28T represented a novel species within the genus. The strain was found to be Gram-positive, rod-shaped and motile by means of peritrichous flagella and was shown to produce ellipsoidal spores. The strain was strictly aerobic and able to grow with NaCl at concentrations in the range 0-14 % (w/v) at pH 7.5. The strain grew at temperatures of 15-42 degrees C and at pH 6.5-9.5. The major fatty acids were anteiso-C(15 : 0) (64.9 %), anteiso-C(17 : 0) (11.9 %) and iso-C(16 : 0) (7.7 %). The major isoprenoid quinone was MK-7. The DNA G+C content was 40.2 mol%. According to the 16S rRNA gene sequence, DNA-DNA relatedness and physiological data and the fatty acid composition, CL-MP28T could be assigned to the genus Oceanobacillus, but is distinguishable from the recognized species of this genus. Strain CL-MP28T therefore represents a novel species within the genus Oceanobacillus, for which the name Oceanobacillus profundus sp. nov. is proposed. The type strain is CL-MP28T (=KCCM 42318T=DSM 18246T).

Isolation and characterization of a Geobacillus thermoleovorans strain from an ultra-deep South African gold mine.

A thermophilic facultative bacterial isolate was recovered from 3.2km depth in a gold mine in South Africa. This isolate, designated GE-7, was cultivated from pH 8.0, 50 degrees C water from a dripping fracture near the top of an exploration tunnel. GE-7 grows optimally at 65 degrees C and pH 6.5 on a wide range of carbon substrates including cellobiose, hydrocarbons and lactate. In addition to O(2), GE-7 also utilizes nitrate as an electron acceptor. GE-7 is a long rod-shaped bacterium (4-6microm longx0.5microm wide) with terminal endospores and flagella. Phylogenetic analysis of GE-7 16S rDNA sequence revealed high sequence similarity with G. thermoleovorans DSM 5366(T) (99.6%), however, certain phenotypic characteristics of GE-7 were distinct from this and other previously described strains of G. thermoleovorans.

Vulcanibacillus modesticaldus gen. nov., sp. nov., a strictly anaerobic, nitrate-reducing bacterium from deep-sea hydrothermal vents.

A novel anaerobic, moderately thermophilic, spore-forming bacterium, designated strain BRT, was isolated from deep-sea hydrothermal core samples collected at the Rainbow vent field on the Mid-Atlantic Ridge (36 degrees 14' N 33 degrees 54' W). The cells were found to be rod-shaped, non-motile, Gram-positive and spore-forming. The organism grew in the temperature range 37-60 degrees C, with an optimum at 55 degrees C, and at pH values in the range 6-8.5, with an optimum around pH 7. NaCl concentrations for growth were in the range 10-40 g l(-1), with an optimum at 20-30 g l(-1). Strain BRT grew chemo-organoheterotrophically with carbohydrates, proteinaceous substrates and organic acids with nitrate as electron acceptor. The novel isolate was not able to ferment. The G+C content of the genomic DNA was 34.5 mol%. Phylogenetic analysis of the 16S rRNA gene sequence placed strain BRT in the Bacillaceae within the class 'Bacilli'. On the basis of the phenotypic and phylogenetic data, this isolate should be described as a member of a novel genus, for which the name Vulcanibacillus gen. nov. is proposed. The type species is Vulcanibacillus modesticaldus sp. nov., with the type strain BRT (=DSM 14931T=JCM 12998T).

Characterization of Exiguobacterium isolates from the Siberian permafrost. Description of Exiguobacterium sibiricum sp. nov.

Three Gram-positive bacterial strains, 7-3, 255-15 and 190-11, previously isolated from Siberian permafrost, were characterized and taxonomically classified. These microorganisms are rod-shaped, facultative aerobic, motile with peritrichous flagella and their growth ranges are from -2.5 to 40 degrees C. The chemotaxonomic markers indicated that the three strains belong to the genus Exiguobacterium. Their peptidoglycan type was A3alpha L-Lys-Gly. The predominant menaquinone detected in all three strains was MK7. The polar lipids present were phosphatidyl-glycerol, diphosphatidyl-glycerol and phosphatidyl-ethanolamine. The major fatty acids were iso-C13:0, anteiso-C13:0, iso-C15:0, C16:0 and iso-C17:0. Phylogenetic analysis based on 16S rRNA and six diverse genes, gyrB (gyrase subunit B), rpoB (DNA-directed RNA polymerase beta subunit), recA (homologous recombination), csp (cold shock protein), hsp70 (ClassI-heat shock protein-chaperonin) and citC (isocitrate dehydrogenase), indicated that the strains were closely related to Exiguobacterium undae (DSM 14481(T)) and Exiguobacterium antarcticum (DSM 14480(T)). On the basis of the phenotypic characteristics, phylogenetic data and DNA-DNA reassociation data, strain 190-11 was classified as E. undae, while the other two isolates, 7-3 and 255-15, comprise a novel species, for which the name Exiguobacterium sibiricum sp. nov. is proposed.

Characterization of potential stress responses in ancient Siberian permafrost psychroactive bacteria.

Past studies of cold-acclimated bacteria have focused primarily on organisms not capable of sub-zero growth. Siberian permafrost isolates Exiguobacterium sp. 255-15 and Psychrobacter sp. 273-4, which grow at subzero temperatures, were used to study cold-acclimated physiology. Changes in membrane composition and exopolysaccharides were defined as a function of growth at 24, 4 and -2.5 degrees C in the presence and absence of 5% NaCl. As expected, there was a decrease in fatty acid saturation and chain length at the colder temperatures and a further decrease in the degree of saturation at higher osmolarity. A shift in carbon source utilization and antibiotic resistance occurred at 4 versus 24 degrees C growth, perhaps due to changes in the membrane transport. Some carbon substrates were used uniquely at 4 degrees C and, in general, increased antibiotic sensitivity was observed at 4 degrees C. All the permafrost strains tested were resistant to long-term freezing (1 year) and were not particularly unique in their UVC tolerance. Most of the tested isolates had moderate ice nucleation activity, and particularly interesting was the fact that the Gram-positive Exiguobacterium showed some soluble ice nucleation activity. In general the features measured suggest that the Siberian organisms have adapted to the conditions of long-term freezing at least for the temperatures of the Kolyma region which are -10 to -12 degrees C where intracellular water is likely not frozen.

Anoxybacillus kamchatkensis sp. nov., a novel thermophilic facultative aerobic bacterium with a broad pH optimum from the Geyser valley, Kamchatka.

A facultative aerobic, moderately thermophilic, spore forming bacterium, strain JW/VK-KG4 was isolated from an enrichment culture obtained from the Geyser valley, a geo-thermally heated environment located in the Kamchatka peninsula (Far East region of Russia). The cells were rod shaped, motile, peritrichous flagellated stained Gram positive and had a Gram positive type cell wall. Aerobically, the strain utilized a range of carbohydrates including glucose, fructose, trehalose, proteinuous substrates, and pectin as well. Anaerobically, only carbohydrates are utilized. When growing on carbohydrates, the strain required yeast extract and vitamin B(12). Anaerobically, glucose was fermented to lactate as main product and acetate, formate, ethanol as minor products. Aerobically, even in well-aerated cultures (agitated at 500 rpm), glucose oxidation was incomplete and lactate and acetate were found in culture supernatants as by-products. Optimal growth of the isolate was observed at pH(25 C) 6.8-8.5 and 60 degrees C. The doubling times on glucose at optimal growth conditions were 34 min (aerobically) and 40 min (anaerobically). The G+C content was 42.3 mol% as determined by T(m) assay. Sequence analysis of the 16S rRNA gene indicated an affiliation of strain JW/VK-KG4 with Anoxybacillus species. Based on its morphology, physiology, phylogenetic relationship and its low DNA-DNA homology with validly published species of Anoxybacillus, it is proposed that strain JW/VK-KG4 represents a new species in the genus Anoxybacillus as A. kamchatkensis sp. nov. The type strain for the novel species is JW/VK-KG4(T) (=DSM 14988, =ATCC BAA-549). The GenBank accession number for the 16S rDNA sequence is AF510985.

Pontibacillus marinus sp. nov., a moderately halophilic bacterium from a solar saltern, and emended description of the genus Pontibacillus.

A moderately halophilic, Gram-positive, rod-shaped bacterium (BH030004(T)) was isolated from a solar saltern in Korea. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain BH030004(T) belonged to the genus Pontibacillus. Chemotaxonomic data (DNA G + C content, 42 mol%; major isoprenoid quinone, MK-7; cell-wall type, A1gamma-type meso-diaminopimelic acid; major fatty acids, iso-C(15 : 0) and anteiso-C(15 : 0)) also supported the affiliation of the isolate to the genus Pontibacillus. Although the 16S rRNA gene sequence similarity between strain BH030004(T) and Pontibacillus chungwhensis DSM 16287(T) was relatively high (99.1 %), physiological properties and DNA-DNA hybridization (about 7 % DNA-DNA relatedness) allowed genotypic and phenotypic differentiation of strain BH030004(T) from the type strain of P. chungwhensis. Therefore, strain BH030004(T) represents a novel species of the genus Pontibacillus, for which the name Pontibacillus marinus sp. nov. is proposed. The type strain is BH030004(T) (=KCTC 3917(T) = DSM 16465(T)).

Lentibacillus salarius sp. nov., isolated from saline sediment in China, and emended description of the genus Lentibacillus.

A Gram-positive, spore-forming and moderately halophilic bacterium, strain BH139(T), was isolated from saline sediment of Xinjiang Province, China. Cells of strain BH139(T) were motile, flagellated rods. The organism grew optimally at 30-35 degrees C in the presence 12-14 % (w/v) NaCl. The major fatty acids were branched saturated fatty acids such as iso-C(16 : 0), anteiso-C(15 : 0), iso-C(15 : 0), iso-C(14 : 0) and anteiso-C(17 : 0). The G + C content of the genomic DNA was about 43 mol% and the predominant isoprenoid quinone was MK-7. Comparative 16S rRNA gene sequence analyses showed that strain BH139(T) was most closely related to Lentibacillus salicampi KCCM 41560(T) (96.9 % 16S rRNA gene sequence similarity) and formed a distinct phyletic line from that species. On the basis of physiological and molecular properties, the isolate represents a novel species within the genus Lentibacillus, for which the name Lentibacillus salarius sp. nov. is proposed. The type strain is BH139(T) (= KCTC 3911(T) = DSM 16459(T)).

Geobacillus jurassicus sp. nov., a new thermophilic bacterium isolated from a high-temperature petroleum reservoir, and the validation of the Geobacillus species.

Four thermophilic, spore-forming bacterial strains, DS1(T), DS2, 46 and 49, were isolated from the high-temperature Dagang oilfield, located in China. The strains were identified by using the polyphasic taxonomy approach. These were aerobic, gram-positive, rod-shaped, moderately thermophilic (with an optimum growth temperature of 60-65 degrees C), chemoorganotrophic bacteria capable of growing on various sugars, carboxylic acids and crude oil. Two strains, DS1(T) and DS2, were capable of growing on individual saturated hydrocarbons. The G + C content of the DNA of strains DS1(T) and DS2 was 54.5 and 53.8 mol%, respectively. The phylogenetic analysis of the 16S rDNA of strains DS1(T) and DS2 showed that they form a separate cluster within the genus Geobacillus. The cellular fatty acids of the isolates were dominated by iso-15:0, iso-16:0 and iso-17:0 acids, which are the typical fatty acids of bacteria from the genus Geobacillus. The DNA-DNA hybridization study and the comparative analysis of the morphological and chemotaxonomic characteristics of strains DS1(T) and DS2 showed that they differ from the previously described Geobacillus species and belong to a new species, which was called Geobacillus jurassicus. DS1(T) (=VKM B2301(T), = DSM 15726(T)) is the type strain of this species. According to both DNA-DNA reassociation studies and 16S rDNA sequence analysis, two other strains, 46 and 49, were assigned to the species G. stearothermophilus. In this paper, we provide evidence that the new combinations G. stearothermophilus, G. thermoleovorans, G. kaustophilus, G. thermoglucosidasius and G. thermodenitrificans may be considered to be valid.

Geobacillus lituanicus sp. nov.

Obligately thermophilic, aerobic, proteolytic, endospore-forming strain N-3(T) was isolated from a high-temperature oilfield in Lithuania. 16S rRNA gene sequence analysis placed this strain in genetic group 5 of the endospore formers. Geobacillus thermoleovorans appeared to be the closest phylogenetic neighbour (99.4 % sequence similarity). The G+C content of strain N-3(T) was 52.5 mol% and matched the range established for the genus Geobacillus. Studies of DNA-DNA relatedness and morphological and physiological analyses enabled strain N-3(T) to be described as a member of the genus Geobacillus, but could not assign this strain to any other known species of this genus. Results of this polyphasic study allowed characterization of strain N-3(T) as a novel species in the genus Geobacillus - Geobacillus lituanicus sp. nov. This species can be distinguished from G. thermoleovorans and Geobacillus stearothermophilus on the basis of 16S rRNA gene PCR-RFLP assays with the restriction endonucleases AluI, HaeIII and TaqI. The type strain of the novel species is N-3(T) (=DSM 15325(T)=VKM B-2294(T)).