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A strictly thermophilic anaerobe, designated strain VF08T, was isolated from a water sample collected from a Great Artesian Basin bore (registered bore number 22981) situated at Mitchell, QLD, Australia. Cells of isolate VF08Twere slightly curved, non-sporulating rods (1.5–3.5×0.4–0.8 μm), which stained Gram-negative but possessed a Gram-positive cell-wall ultrastructure. The strain grew optimally in tryptone-yeast extract-glucose (TYEG) medium at 55 °C (temperature growth range between 37 and 60 °C) and a pH of 7 (pH growth range, 6.0–9.0). Yeast extract or tryptone was required for growth on glucose, fructose, xylose, maltose, sucrose, raffinose, cellobiose, ribose, pyruvate, tryptone, peptone, Casamino acids, amyl media and serine, but could also support growth as the sole carbon source. End products from glucose fermentation were acetate, ethanol, CO2and H2. The strain reduced vanadium(V), but not iron(III), manganese(IV), elemental sulfur, sulfate, thiosulfate, sulfite, nitrate or nitrite in the presence of 0.2 % yeast extract, peptone, tryptone, glucose, sucrose and Casamino acids, but an increase in the growth rate or cell yield was not observed. Growth was inhibited by chloramphenicol, streptomycin, tetracycline, penicillin, ampicillin and ≥2 % NaCl (w/v). The G+C content of the DNA was 38.4±0.8 mol% as determined by the thermal denaturation (Tm) method. 16S rRNA gene sequence analysis revealed that isolate VF08Twas a member of the genusCaloramatorwithCaloramator australicusandCaloramator fervidus(formerlyClostridium fervidus) being the closest relatives with similarity values of 85.0 and 86.1 %, respectively, when helix 6 nucleotides were included in the analysis, and 95.2 % and 94 %, respectively, when these nucleotides were masked from the analysis. Further analysis revealed that strain VF08Tformed an individual cluster (cluster II) within the genusCaloramatorand could be distinguished from other species within the genusCaloramator(clusters I, III and IV) on the basis of signature nucleotides and differences in phenotypic traits. These data suggest that strain VF08Tis a novel species of the genusCaloramator, for which the nameCaloramator mitchellensissp. nov. is proposed. The type strain is VF08T(=JCM 15828T=KCTC 5735T). An emended description of the genusCaloramatoris also provided.

The V-type ATPase of the thermophile Caloramator fervidus is an ATP-driven Na+ pump. The nucleotide sequence of the ntpFIKECGABD operon containing the structural genes coding for the nine subunits of the enzyme complex was determined. The identity of the proteins in two pairs of subunits (D, E and F, G) that have very similar mobilities on SDS-PAGE of the purified complex (24.3 and 22.7 kDa, and 12.3 and 11.6 kDa) was established by tryptic digestion of the protein bands followed by mass spectrometric analysis of the peptides.

A strictly anaerobic, thermophilic bacterium, designated strain RC3T, was isolated from microbial mats colonizing thermal waters of a run-off channel formed by free-flowing waters from a bore well (registered no. 17263) of the Great Artesian Basin, Australia. The slightly curved rods (2.5–4.2×0.8–1.0 μm) of strain RC3T stained Gram-positive and grew optimally in tryptone-yeast extract-glucose medium at 60 °C (range 45–70 °C) and pH 7 (range pH 5–9). Strain RC3T grew poorly on yeast extract (0.2 %) but did not grow on tryptone (0.2 %) as a sole carbon source; yeast extract was required for growth on other energy sources, which included glucose, fructose, galactose, xylose, maltose, sucrose, raffinose, mannose, cellobiose, cellulose, starch, amylopectin, xylan, peptone, amyl media (Research Achievement), threonine and pyruvate but did not include arabinose, ribose, lactose, CM-cellulose, myo-inositol, mannitol, chitin, casein, formate, acetate, succinate, propionate, lactate, benzoate, glycerol, ethanol, Casamino acids, arginine, alanine, serine, glycine, glutamine, leucine, isoleucine, methionine or aspartate. The end products of glucose fermentation were ethanol and acetate. In the presence of 0.2 % yeast extract, iron(III), manganese(IV) and elemental sulfur were reduced but not sulfate, sulfite, thiosulfate, nitrate or nitrite. Iron(III) was also reduced in the presence of peptone, tryptone, amyl media, threonine and glycerol but not chitin, xylan, pectin, starch, pyruvate, acetate, benzoate, lactate, propionate, succinate, inositol, ethanol, mannitol, arginine, glutamine or serine. Strain RC3T was not able to utilize molecular hydrogen and/or carbon dioxide in the presence or absence of iron(III). In the presence of iron(III) and glycerol, increased concentrations of Fe(II) corresponded to increased cell numbers, demonstrating that strain RC3T was able to conserve energy to support growth from the reduction of Fe(III) to Fe(II). Chloramphenicol, streptomycin, tetracycline, penicillin and ampicillin and NaCl concentrations greater than 2 % inhibited growth. The G+C content of the DNA was 34±1 mol% as determined by the thermal denaturation (T m) method. 16S rRNA gene sequence analysis indicated that strain RC3T was affiliated to Caloramator fervidus (95.8 % similarity to the type strain) and to other Caloramator species (average similarity of 91.6 %) within the phylum Firmicutes. On the basis of phylogenetic and phenotypic characteristics, it is proposed that strain RC3T should be classified in the genus Caloramator as a representative of a novel species, Caloramator australicus sp. nov. The type strain is RC3T (=JCM 1508T =KCTC 5601T).

The Na+-pumping V-ATPase complex of the thermophilic bacterium Caloramator fervidus was purified and dissociated under controlled conditions. The structure of purified V-1-ATPase subcomplexes differing in subunit composition was analyzed by electron microscopy and single particle analysis of 50 000 projections. Difference mapping of subcomplex projections revealed the presence and position of two subunits in the central stalk. A density with an elongated shape similar to the gamma subunit of F-ATPases is partly located within V-1 and corresponds, most likely, to subunit E. Subunit E is connected to the membrane-bound part V-0 via subunit C, a spherical density that is connected to the center of V-0. The presence of subunit C makes the central stalk substantially longer in comparison to the F-ATPases, in which the subunit connects directly to F-0.