DNA:DNA hybridization and chemotaxonomic studies of Thermus scotoductus.

The species Thermus scotoductus was recently described as containing several non-pigmented isolates from Selfoss, Iceland, and the X-1 strain from the USA (Kristjansson et al., 1994). In this study, we performed DNA:DNA hybridizations and chemotaxonomic studies on several non-pigmented Thermus isolates from other geographical areas to assess their relationship to the strains originally assigned to this species. The results of DNA:DNA hybridizations showed that strains NH and Dl from London and strains Vl-7a and Vl-13 from Vizela, Portugal, belonged to T. scotoductus. T. scotoductus X-1 (ATCC 27978) was composed of two stable colony types, one of which had a major glycolipid different from the one present in the other colony type and from all other Thermus strains examined as well. The fatty acid composition of the isolates from Selfoss and London were practically identical. However, the fatty acid composition of strain X-1, the individual colony types of this strain and the Vizela strains were different from the Selfoss-London isolates and from each other. Another non-pigmented strain, designated SPS-11, belonged to a different DNA homology group.

Thermus silvanus sp. nov. and Thermus chliarophilus sp. nov., two new species related to thermus ruber but with lower growth temperatures.

Strains of Thermus silvanus sp. nov. and strains of Thermus chliarophilus sp. nov. were isolated from the hot spring at Vizela in northern Portugal and the hot spring at Alcafache in central Portugal, respectively. The strains of T. silvanus produce orange-red-pigmented colonies and have an optimum growth temperature of about 55 degrees C, while the strains of T. chliarophilus produce yellow-pigmented colonies and have an optimum growth temperature of about 50 degrees C. The strains of both species are catalase negative. These species can be distinguished from each other and from Thermus ruber by biochemical characteristics, fatty acid composition data, and 16S rRNA gene sequence data. Our phylogenetic analysis showed that strains VI-R2T (T = type strain) and ALT-8T belong to the T. ruber line of descent. The type strain of T. silvanus is strain VI-R2 (= DSM 9946), and the type strain of T. chliarophilus is strain ALT-8 (= DSM 9957).

Polar lipids and fatty acid composition of Thermus strains from New Zealand.

The polar lipids and fatty acid composition of Thermus aquaticus YT-1 and YS 041, T. filiformis Wai33 A1 and eighteen isolates from New Zealand, several of which are attributed to T. filiformis, were compared to complement the taxonomy of these organisms. The polar lipid patterns were essentially similar in all strains and consisted of one major phospholipid and one major glycolipid. The fatty acid analysis produced three basic groups corresponding to T. filiformis Wai33 A1, T. aquaticus and the third to the other New Zealand strains. The presence of hydroxy fatty acids is reported in Thermus spp. for the first time.

Phylogenetic diversity of the deinococci as determined by 16S ribosomal DNA sequence comparison.

16S ribosomal DNA (rDNA) sequences were determined for the five species of the genus Deinococcus (Deinococcus erythromyxa, Deinococcus proteolyticus, Deinococcus radiodurans, Deinococcus radiophilus, and Deinococcus radiopugnans) and the single species of the genus. Deinobacter (Deinobacter grandis). With the exception of Deinococcus erythromyxa, the deinococci form a coherent phylogenetic cluster which is related to the Thermus-Meiothermus lineage. An analysis of the 16S rDNA sequence of Deinococcus erythromyxa revealed that this organism is an actinomycete and a member of the genus Kocuria. Deinobacter grandis falls within the radiation of the genus Deinococcus and phylogenetically can be considered a member of this genus. The results of the phylogenetic analyses are consistent with chemotaxonomic data. On the basis of our data, Deinobacter grandis is transferred to the genus Deinococcus as Deinococcus grandis comb. nov., the description of the genus Deinococcus is emended accordingly, and Deinococcus erythromyxa is transferred to the genus Kocuria as Kocuria erythromyxa comb. nov. The description of the family Deinococcaceae is emended to include organisms with rod-shaped cells, and a set of 16S rDNA signature nucleotides is designated for this group. On the basis of the distinct phylogenetic position of the Deinococcus lineage and a set of 16S rDNA signature nucleotides, the order Deinococcales ord. nov. is described.

Thermonema rossianum sp. nov., a new thermophilic and slightly halophilic species from saline hot springs in Naples, Italy.

Six slightly halophilic, thermophilic bacterial strains were isolated from saline hot springs along the Bay of Naples, Italy. These strains produce bright yellow colonies and have a filamentous morphology and an optimum growth temperature of about 60 degrees C. Lipid composition and 16S ribosomal DNA sequence analyses showed that these strains belong to the genus Thermonema, a member of the cytophaga-flavobacter-bacteroides phylum. Growth was observed in medium containing 1 to 3% NaCl. The DNA G + C content was 50.9 mol%. DNA-DNA hybridization studies showed that these strains represent a new species of the genus Thermonema. We propose that strain NR-27T (T = type strain) and the other strains represent a new species, Thermonema rossianum. Strain NR-27 (= DSM 10300) is the type strain of this species.

Meiothermus cerbereus sp. nov., a new slightly thermophilic species with high levels of 3-hydroxy fatty acids.

Strains of Meiothermus cerbereus sp. nov. were isolated from the hot springs within the Geysir geothermal area of Iceland. The strains of Meiothermus cerbereus produce red-orange-pigmented colonies, have an optimum growth temperature of about 55 degrees C, and have higher levels of 3-OH fatty acids than the strains of the other species of the genus Meiothermus. These strains, unlike all other strains of the species of the genus Meiothermus examined previously, required cysteine, thiosulfate, or thioglycolate for growth in liquid Thermus medium, but not in the corresponding medium solidified with agar. Several strains belonging to Meiothermus silvanus, isolated from Geysir, also required reduced sulfur compounds for growth in liquid medium, leading to the hypothesis that this requirement is not a taxonomic characteristic of the new species. The new species represented by strains GY-1T and GY-5 can be distinguished from the other species of the genus Meiothermus by biochemical characteristics, fatty acid composition, DNA-DNA reassociation values, and 16S ribosomal DNA sequence. The type strain for Meiothermus cerbereus is GY-1 (= DSM 11376).

Determination of the structure of a novel glycolipid from Thermus aquaticus 15004 and demonstration that hydroxy fatty acids are amide linked to glycolipids in Thermus spp.

The compositions of the major glycolipids (GL-1) of five strains of Thermus aquaticus, the type strain of T. filiformis, T. oshimai SPS-11, and Thermnus sp. strain CG-2 were examined by gas chromatography, gas chromatography-mass spectroscopy, fast atom bombardment-mass spectroscopy, and chemical methods. The results showed that, with the exception of T. aquaticus 15004, the organisms each have a major glycolipid whose structure was established as diglycosyl-(N-acyl)glycosaminyl-glycosyl diacylglycerol. Glucosamine was present in GL-1 of T. oshimai SPS-11 and Thermus sp. strain CG-2, while galactosamine was present in the GL-1 of T. aquaticus and T. filiformis. The novel major glycolipid of T. aquaticus 15004 was identified as galactofuranosyl-(N-acetyl)galactosaminyl-(N-acyl)galactosaminyl-gluc - osyl diacylglycerol. The hydroxy fatty acids found in the T. aquaticus strains and in the type strain of T. filiformis were exclusively amide linked to the galactosamine of the major glycolipid. Ester-linked hydroxy fatty acids were not detected in the diacylglycerol moiety of GL-1 of these organisms. Hydroxy fatty acids were detected neither in the major glycolipid of T. oshimai SPS-11 and Thermnus sp. strain CG-2, in which glucosamine is present, nor in the major phospholipid of any of the strains examined.

Characterization of novel long-chain 1,2-diols in Thermus species and demonstration that Thermus strains contain both glycerol-linked and diol-linked glycolipids.

In this study, we purified and characterized tetra- and triglycosyl glycolipids (GL-1 and GL-2, respectively) from two different colonial forms of Thermus scotoductus X-1, from T. filiformis Tok4 A2, and from T. oshimai SPS-11. Acid hydrolysis of the purified glycolipids liberated, in addition to the expected long-chain fatty acids, two components which were identified by gas chromatography-mass spectrometry as 16-methylheptadecane-1,2-diol and 15-methylheptadecane-1,2-diol. Fast atom bombardment mass spectrometry of the intact glycolipids indicated that a major proportion consisted of components with glycan head groups linked to long-chain 1,2-diols rather than to glycerol, although in all cases glycerol-linked compounds containing similar glycan head groups were also present. As in other Thermus strains, the polar head group of GL-1 from T. filiformis Tok4 A2 and from T. scotoductus X-1 colony type t2 was a glucosylgalactosyl-(N-acyl)glucosaminylglucosyl moiety. However, GL-2 from T. scotoductus X-1 colony type t1 and from T. oshimai SPS-11 was a truncated analog which lacked the nonreducing terminal glucose. Long-chain 1,2-diols have been previously reported in the polar lipids of Thermomicrobium roseum and (possibly) Chloroflexus aurantiacus, but to our knowledge, this is the first report of their detection in other bacteria and the first account of the structural determination of long-chain diol-linked glycolipids.

Usefulness of fatty acid composition for differentiation of Legionella species.

Numerical analysis of fatty acid methyl ester (FAME) profiles of 199 isolates and 76 reference strains, belonging to all validly described species of the genus Legionella that can be cultured in laboratory media, was used to differentiate between the species of this genus. With the exception of the strains that autofluoresced red, it was possible to differentiate all the other Legionella species. The strains of the species L. bozemanii, L. dumoffii, L. feeleii, L. gormanii, L. maceachernii, L. micdadei, and L. quinlivanii did not form single clusters, showing some degree of variability in the fatty acid compositions. The strains of the blue-white autofluorescent species had very similar fatty acid compositions and were difficult to distinguish from each other. Nine isolates had fatty acid profiles unlike those of any of the validly described species and may represent different FAME groups of known species or undescribed Legionella species. The method used in this study was useful for screening and discriminating large number of isolates of Legionella species. Moreover, the results obtained can be included in a database of fatty acid profiles, leading to a more accurate automatic identification of Legionella isolates.

Tepidimonas ignava gen. nov., sp. nov., a new chemolithoheterotrophic and slightly thermophilic member of the beta-Proteobacteria.

A bacterial isolate with an optimum growth temperature of about 55 degrees C was recovered on a medium composed of one part Kligler's iron agar and four parts of Thermus Agar from the host spring at São Pedro do Sul in central Portugal. Phylogenetic analyses using the 16S rRNA gene sequence of strain SPS-1037T indicated that the new organism represented a new genus and species of beta-Proteobacteria. The major fatty acids of strain SPS-1037T are C16:0 and C17:0. Ubiquinone 8 is the major respiratory quinone, and the major polar lipids are phosphatidylethanolamine and phosphatidylglycerol. The new isolate is aerobic and chemolithoheterotrophic. Thiosulfate and tetrathionate were oxidized to sulfate. The growth yield of the organism was improved by the addition of thiosulfate to media containing organic carbon sources, but the organism did not grow autotrophically under the conditions examined. Heterotrophic growth of strain SPS-1037T occurs on amino acids and organic acids, but this organism does not assimilate carbohydrates. On the basis of the phylogenetic analyses, and physiological and biochemical characteristics, it is proposed that strain SPS-1037T represents a new genus and a new species for which the name Tepidimonas ignava is proposed.

Thermus igniterrae sp. nov. and Thermus antranikianii sp. nov., two new species from Iceland.

Several yellow-pigmented isolates, with optimum growth temperatures of about 65-70 degrees C, were recovered from hot springs in Iceland. Phylogenetic analysis of the 16S rDNA and DNA-DNA reassociation values showed that these organisms represented two new species of the genus Thermus. Strains RF-4T and HN1-8 had maximum temperatures for growth below 80 degrees C, while strains HN3-7T and HN2-7, unlike all other strains of the species of the genus Thermus except those belonging to Thermus thermophilus, grew at 80 degrees C. The new isolates from Iceland could not be distinguished easily from each other or from other strains of the species of the genus Thermus by biochemical characteristics; however, strains RF-4T and HN1-8 assimilated ribitol, a characteristic which was not detected in any of the other strains examined. Moreover, the species represented by strains RF-4T and HN1-8 and the species represented by strains HN3-7T and HN2-7 could be distinguished clearly from the other species of Thermus by their fatty acid composition. Strains RF-4T and HN1-8 have the highest combined levels of iso-15:0 and iso-17:0 and the lowest levels of iso-16:0 of any of the strains of the species of Thermus, while strains HN3-7T and HN2-7 are characterized by a very low iso-15:0/iso-17:0 ratio. On the basis of the phylogenetic analysis, DNA-DNA reassociation values, physiological and biochemical characteristics and fatty acid composition, the name Thermus igniterrae sp. nov. is proposed for the species represented by strains RF-4T and HN1-8 and the name Thermus antranikianii sp. nov. is proposed for the species represented by strains HN3-7T and HN2-7.

Characterization and radiation resistance of new isolates of Rubrobacter radiotolerans and Rubrobacter xylanophilus.

In this study we characterized new strains of the slightly thermophilic species Rubrobacter radiotolerans and the thermophilic species Rubrobacter xylanophilus, both of which were previously represented only by the type strains isolated, respectively, from Japan and the United Kingdom. The new isolates were recovered from two hot springs in central Portugal after gamma irradiation of water and biofilm samples. We assessed biochemical characteristics, performed DNA-DNA hybridization, and carried out 16S rDNA sequence analysis to demonstrate that the new Rubrobacter isolates belong to the species R. radiotolerans and R. xylanophilus. We also show for the first time that the strains of R. xylanophilus and other strains of R. radiotolerans are extremely gamma radiation resistant.

Deinococcus geothermalis sp. nov. and Deinococcus murrayi sp. nov., two extremely radiation-resistant and slightly thermophilic species from hot springs.

Strains of Deinococcus geothermalis sp. nov. were isolated from the hot spring and runoff at Agnano, Naples, Italy, and from the hot spring at São Pedro do Sul in central Portugal, while strains of Deinococcus murrayi sp. nov. were isolated from the hot springs at São Pedro do Sul, São Gemil, and Alcafache in central Portugal. The strains of D. geothermalis and D. murrayi produce orange-pigmented colonies and have an optimum growth temperature of about 45 to 50 degrees C. The type strains of the two new species are extremely gamma radiation resistant. The fatty acids of these new species are primarily branched-chain fatty acids. The two new species can be distinguished from each other by the lower pH range of D. geothermalis than of D. murrayi, by their fatty acid compositions, and by several biochemical parameters, including the ability of D. geothermalis to grow in minimal medium without yeast extract. 16S rRNA gene sequencing also showed that the isolates constitute two species and that these species are distinct from the other species of the genus Deinococcus. The type strain of D. geothermalis is AG-3a (= DSM 11300), and the type strain of D. murrayi is ALT-1b (= DSM 11303).

Alicyclobacillus hesperidum sp. nov. and a related genomic species from solfataric soils of São Miguel in the Azores.

Several acidophilic, slightly thermophilic or thermophilic Gram-positive isolates were recovered from solfataric soil at Furnas on the Island of São Miguel in the Azores. Phylogenetic analysis of the 16S rRNA gene sequence showed that these organisms represented two novel species of the genus Alicyclobacillus. Strains FR-11T and FR-1b had an optimum growth temperature of about 50 degrees C, whereas strains FR-3 and FR-6T had an optimum growth temperature of about 60 degrees C. Biochemical, physiological and chemotaxonomic characteristics did not distinguish isolates FR-3 and FR-6T from the type strain of Alicyclobacillus acidocaldarius; however, strains FR-11T and FR-1b could be easily distinguished from the type strain of Alicyclobacillus acidoterrestris by the carbon source assimilation pattern and the fatty acid composition. On the basis of the phylogenetic analysis, physiological and biochemical characteristics, and fatty acid composition the name Alicyclobacillus hesperidum is proposed for the species represented by strains FR-11T and FR-1b; a formal name for the new genomic species represented by strains FR-3 and FR-6T is not proposed at this time.