M. S. Da Costa

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).

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

Abstract 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.

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.

Rubrobacter xylanophilus sp. nov., a new thermophilic species isolated from a thermally polluted effluent

One strain of a thermophilic, slightly halotolerant bacterium was isolated from a thermally polluted industrial runoff near Salisbury, United Kingdom. This organism, strain PRD-1T (T = type strain), for which we propose the name Rubrobacter xylanophilus sp. nov., produces short gram-positive rods and coccoid cells and forms pink colonies. The optimum growth temperature is approximately 60°C. Unusual internal branched-chain fatty acids (namely, 12-methylhexadecanoic acid and 14-methyloctadecanoic acid) make up the major acyl chains of the lipids. The results of our 16S rRNA sequence comparisons showed that strain PRD-1T is related to Rubrobacter radiotolerans and that these two organisms form a deep evolutionary line of descent within the gram-positive Bacteria.

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.

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 Kliglers 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.

Extracellular α-amylase from Thermus filiformis Ork A2: purification and biochemical characterization

Abstract An extracellular α-amylase produced by the thermophilic bacterium Thermus filiformis Ork A2 was purified from cell-free culture supernatant by ion exchange chromatography. The molecular mass was estimated to be 60 000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was rich in both basic and hydrophobic amino acids, presenting the following NH2-terminal amino acid sequence: Thr-Ala-Asp-Leu-Ile-Val-Lys-Ile-Asn-Phe. Amylolytic activity on soluble starch was optimal at pH 5.5–6.0 and 95°C, and the enzyme was stable in the pH range of 4.0–8.0. Calcium enhanced thermostability at temperatures above 80°C, increasing the half-life of activity to more than 8 h at 85°C, 80 min at 90°C, and 19 min at 95°C. Ethylenediaminetetraacetic acid (EDTA) inhibited amylase activity, the inhibition being reversed by the addition of calcium or strontium ions. The α-amylase was also inhibited by copper and mercuric ions, and p-chloromercuribenzoic acid, the latter being reversed in the presence of dithiothreitol. Dithiothreitol and β-mercaptoethanol activated the enzyme. The α-amylase exhibited Michaelis-Menten kinetics for starch, with a Km of 5.0 mg·ml−1 and kcat/Km of 5.2 × 105 ml·mg−1 s−1. Similar values were obtained for amylose, amylopectin, and glycogen. The hydrolysis pattern was similar for maltooligosaccharides and polysaccharides, with maltose being the major hydrolysis product. Glucose and maltotriose were generated as secondary products, although glucose was produced in high levels after a 6-h digestion. To our knowledge this is the first report of the characterization of an α-amylase from a strain of the genus Thermus.

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.

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).

A hot water supply as the source of Legionella pneumophila in incubators of a neonatology unit

The humidification trays of five of seven incubators in a neonatology unit of a hospital were found to be colonized with Legionella pneumophila, serogroup 1. Bacteriological analysis of the water in the humidification trays showed very large numbers of heterotrophic bacteria, one of which also contained Pseudomonas aeruginosa. Two hot water systems supply the neonatology unit, either of which is used to add water to the humidification trays; one system (A) is maintained at about 60 degrees C, while the other system (B) is maintained at 45 degrees C. The latter was also found to be colonized with L. pneumophila, Sg1. Monoclonal antibody (Mab) subgrouping of the isolates, indicated that system B was the source of colonization of the humidification trays of the incubators.