A novel virus family, the Rudiviridae: Structure, virus-host interactions and genome variability of the sulfolobus viruses SIRV1 and SIRV2.

The unenveloped, stiff-rod-shaped, linear double-stranded DNA viruses SIRV1 and SIRV2 from Icelandic Sulfolobus isolates form a novel virus family, the Rudiviridae. The sizes of the genomes are 32. 3 kbp for SIRV1 and 35.8 kbp for SIRV2. The virions consist of a tube-like superhelix formed by the DNA and a single basic 15.8-kD DNA-binding protein. The tube carries a plug and three tail fibers at each end. One turn of the DNA-protein superhelix measures 4.3 nm and comprises 16.5 turns of B DNA. The linear DNA molecules appear to have covalently closed hairpin ends. The viruses are not lytic and are present in their original hosts in carrier states. Both viruses are quite stable in these carrier states. In several laboratory hosts SIRV2 was invariant, but SIRV1 formed many different variants that completely replaced the wild-type virus. Some of these variants were still variable, whereas others were stable. Up to 10% nucleotide substitution was found between corresponding genome fragments of three variants. Some variants showed deletions. Wild-type SIRV1, but not SIRV2, induces an SOS-like response in Sulfolobus. We propose that wild-type SIRV1 is unable to propagate in some hosts but surmounts this host range barrier by inducing a host response effecting extensive variation of the viral genome.

Cloning and sequence analysis of the DNA ligase-encoding gene of Rhodothermus marinus, and overproduction, purification and characterization of two thermophilic DNA ligases.

In this paper we describe the cloning and sequence analysis of a gene encoding DNA ligase (Lig; EC 6.5.1.2) from the thermophilic bacterium Rhodothermus marinus (Rm). We also describe the overexpression of the Lig-encoding genes of Rm and the thermophile, Thermus scotoductus (Ts), in Escherichia coli, and the purification and characterization of the overproduced Lig. The Rm lig gene encodes a protein of 712 amino acids (aa) with a calculated molecular mass of 79,487 Da. Comparison with published sequences of bacterial Lig revealed significant homology between the NAD(+)-utilizing Lig, and alignment of their aa sequences revealed several blocks of conserved residues. Both of the purified Lig exhibit nick-closing activity over a wide range of temperatures. Under our assay conditions the Rm Lig was active at 5-75 degrees C with apparent optimal activity above 55 degrees C. The Ts enzyme showed activity at 15-75 degrees C with optimal activity above 65 degrees C. The half-life of the Lig at 91 degrees C was estimated to be 7 min for the Rm Lig and 26 min for the Ts Lig.

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.

Cloning, sequence analysis and overexpression of a rhodothermus marinus gene encoding a thermostable thymidine kinase.

Thymidine kinase type II is an important part of the pyrimidine salvage pathway. The thymidine kinase gene from the thermophilic eubacterium Rhodothermus marinus was cloned, sequenced and overexpressed. The gene is 639 bp and encodes a protein of 213 amino acids with a calculated molecular mass of 23.6 kDa. It shows homology to other thymidine kinase proteins from eukaryotic and prokaryotic organisms. The recombinant protein is inhibited by dNTPs but not by dNDPs. It is a tetramer in its native state. Its optimum temperature of activity is 65 degrees C and it has a half life of 15 min at 90 degrees C. This is the first thymidine kinase to be described from a thermophilic bacterium.

Species Composition of Cultivated and Noncultivated Bacteria from Short Filaments in an Icelandic Hot Spring at 88 degrees C.

Samples of short pink-grayish filaments were collected from a hot spring in the Hengill area in southwestern Iceland at 85-88 degrees C, pH 6.9 and 1.7 mg/L sulfide. The species composition was studied by cloning and sequencing small subunit rRNA genes obtained by PCR amplifications from mat DNA. Using 98% sequence similarity as a cutoff value, a total of 5 bacterial operational taxonomic units (OTUs) and 6 archaeal OTUs were detected among 68 bacterial clones and 97 archaeal clones. Database matching showed that 80.5% of the archaeal sequences were 99% similar to Pyrobaculum islandicum and 14.5% were closest to the Korarchaeota clone sequence SRI306. About 87% of the bacterial sequences had the closest database match (99%) to the clone sequence SRI48 but were also found to be 99% identical with hydrogen-oxidizing strains previously isolated in this laboratory from hot springs in the same region. Out of 7 Thermus sequences, 4 were 100% identical to T. scotoductus NMX2 A.1 but 3 represented a new uncultivated Thermus species. Four different media, varying in organic nutrients and phosphate composition were used to isolate 81 aerobic thermophilic heterotrophs. Four isolates were Bacillus spp; but out of 77 Thermus isolates, 42 belonged to T. scotoductus and 35 to T. brockianus. T. scotoductus seemed to be preferably isolated on media low in nutrients and phosphate, whereas for T. brockianus it was the opposite. The T. scotoductus clones and isolates had 99-100% sequence similarity to each other. No T. brockianus sequences were found in the bacterial clone library.

F-and V-ATPases in the genus Thermus and related species.

The discovery of a V-type ATPase in the gram-negative bacterium Thermus thermophilus HB8 (YOKOYAMA et al., J. Biol. Chem. 265, 21946, 1990) was unexpected, since only eukaryotic endomembranes and archaea were thought to contain this enzyme complex, and horizontal gene transfer was suggested to explain the finding. We examined membrane-associated ATPases from representatives of several groups of the genus Thermus. The enzymes were extracted with chloroform and purified by ion exchange chromatography or native gel electrophoresis. One novel Islandic isolate, T. scotoductus SE-1, as well as strain T. filiformis from New Zealand, possessed F-ATPases, as judged by the typical five subunit composition of the F1-moiety, sensitivity to azide, insensitivity to nitrate and a strong crossreaction with antibodies against the F1-ATPase from E. coli. In addition, N-terminal amino acid sequencing of the beta subunit from T. scotoductus SE-1 confirmed its homology with beta subunits from known F-ATPases. In contrast, the same extraction procedure released a V-ATPase from the membranes of T. thermophilus HB27 and T. aquaticus YT-1. The related species Meiothermus (formerly Thermus) chliarophilus ALT-8 also possessed a V-ATPase. All V-ATPases examined in this study contained larger major subunits than F-ATPases, crossreacted with antiserum against subunit A of the V-ATPase from the archaeon Halobacterium saccharovorum, and the N-terminal sequences of their major subunits were homologous to those of other V-ATPases. Sequences of the 16S rRNA gene clearly placed T. scotoductus SE-1, along with other non-pigmented Thermus strains, as a distinct species close to T. aquaticus. Our results suggested that at least two members of the genus, T. scotoductus SE-1 and T. filiformis, contain an F-ATPase, whereas several others possess a V-ATPase. These data could indicate a greater diversity of the genus Thermus than was previously thought. Alternatively, the genus may consist of species where horizontal gene transfer has occurred and others, where it has not.

Why do sulfate-reducing bacteria outcompete methanogenic bacteria for substrates?

The apparent Ks values for H2 of several phylogenetically distant strains of both methanogenic bacteria and sulfate-reducing bacteria were measured. The sulfate reducers had Ks values of about 2 µM whereas the Ks values of the methanogens were 6-20 µM. This indicates that probably all sulfate-reducing bacteria have a higher substrate affinity for H2 than the methanogenic bacteria. Difference in substrate affinity can thus account for the inhibition of methanogenesis from H2 and CO2 in sulfate-rich ecosystems (mainly saltwater marshes), where the H2 concentration is well below 5 µM. Possible explanations for this general phenomenon are discussed.

Extremely halotolerant bacteria characteristic of fully cured and dried cod.

Gram-positive cocci were isolated in high numbers from salted codfish during processing. They were found to be the main bacterial type in fully cured and dried salted cod. Phenotypic characterization of 37 strains showed them to belong to the novobiocin resistant staphylococci, most likely Staphylococcus arlettae or xylosus. Based on sequencing of 16S rDNA and comparison of 700 bases it was concluded that they should be assigned to the species Staphylococcus arlettae. They were found to be extremely halotolerant, growing well at salt concentrations from 0.06 M NaCl, and even displaying clear growth at 4.5 M NaCl. Likewise, the strains grew over a wide temperature range, from 8 to 45 degrees C. Optimal growth conditions were found to be at 0.4-0.6 M NaCl and 30-32 degrees C. This is all in accordance with findings for related staphylococci that have been isolated from other heavily salted meat or fish products.

A new ecological adaptation to high sulfide by a Hydrogenobacter sp. growing on sulfur compounds but not on hydrogen.

Thermophilic bacteria were isolated from a sulfide-rich, neutral hot spring in Iceland on gelrite minimal medium with 16 mM thiosulfate. The isolates were aerobic, obligate chemolithoautotrophs and used thiosulfate and sulfur as electron donors, producing sulfate from both substrates. No growth was observed with hydrogen as the sole electron donor, and no hydrogenase activity was detected. The cells were gram-negative and usually single, 4-5 microm long and 0.7 microm in diameter and formed sulfur globules after a few days of incubation. By SSU rRNA sequence comparisons, the bacterium was placed in the genus Hydrogenobacter with the closest relative to be Calderobacterium hydrogenophilum with 98.3% sequence similarity. This novel bacterium shows an ecological adaptation to high sulfide springs and is differentiated from its closest known relatives by lack of H2 oxidation, deposition of sulfur and lower growth temperature.

Ecology and habitats of extremophiles.

This review describes the main natural extreme environments, characterized by high temperature, high and low pH and high salinity, that can be colonized by microorganisms. The environments covered are: freshwater alkaline hot springs; acidic solfatara fields; anaerobic geothermal mud and soils; acidic sulphur and pyrite areas; carbonate springs and alkaline soil; and soda and highly saline lakes. The community structure, in terms of available energy sources and representative autotrophic and heterotrophic microorganisms, is discussed for each type of habitat.

Distribution of Thermus spp. in Icelandic Hot Springs and a Thermal Gradient.

The growth range in nature of bacteria belonging to the genus Thermus was investigated by sampling 55 different hot springs in Iceland. The springs ranged in temperature from 32 to 99 degrees C, and in pH from 2.1 to 10.1. Viable counts of Thermus spp. ranging from 10 to 10 CFU/100 ml of spring water were found in 27 of the springs sampled. The temperature range for these bacteria was found to be 55 to 85 degrees C, and the pH range was from about 6.5 to above 10. Thermus spp. were found in springs containing up to 1 mM dissolved sulfide and having conductivity up to 2,000 muS/cm. The distribution of Thermus spp. in a hot spring thermal gradient was also investigated and found to agree well with the overall distribution in individual springs.

Substrate binding site for nitrate reductase of Escherichia coli is on the inner aspect of the membrane.

Escherichia coli grown anaerobically on nitrate exhibited the same transport barrier to reduction of chlorate, relative to nitrate, as that exhibited by Paracoccus denitrificans. This establishes that the nitrate binding site of nitrate reductase (EC 1.7.99.4) in E. coli must also lie on the cell side of the nitrate transporter which is associated with the plasma membrane. Because nitrate reductase is membrane bound, the nitrate binding site is thus located on the inner aspect of the membrane. Nitrate pulse studies on E. coli in the absence of valinomycin showed a small transient alkalinization (leads to H+/NO3- congruent to --0.07) which did not occur with oxygen pulses. By analogy with P. denitrificans, the alkaline transient is interpreted to arise from proton-linked nitrate uptake which is closely followed by nitrite efflux. The result is consistent with internal reduction of nitrate, whereas external reduction would be expected to give leads to H+/NO3-ratios approaching --2.

Silicibacter lacuscaerulensis gen. nov., sp. nov., a mesophilic moderately halophilic bacterium characteristic of the Blue Lagoon geothermal lake in Iceland.

Mesophilic, moderately halophilic bacteria were isolated from a silica-rich geothermal lake, the Blue Lagoon in Iceland. The isolates are strictly aerobic, but reduce nitrate to nitrite, and are oxidase- and catalase-positive. The nonsporeforming and nonmotile Gram negative rods are 0.6-0.8 microm in diameter and variable in length (9-18 microm), and contain gas vacuoles. The GC content in their DNA is 66.15%. The minimum, optimum, and maximum temperatures for growth are 22 degrees C, 45 degrees C, and 50 degrees C, respectively. The isolates do not grow without added salt in the medium and can grow at up to 7% NaCl (w/v). The optimal salinity for growth is 3.5%-4% NaCl. The pH range for growth is 6.5-8.5, with the optimal pH at 7.0. At optimal conditions the bacterium has a doubling time of 80 min. The main cytochrome is a membrane-bound cytochrome c with an alpha-peak at 549nm. Sequencing of 16S rRNA from the type strain ITI-1157 revealed it to be a proteobacterium of the alpha-subclass with the closest relatives being Roseobacter litoralis and Paracoccuss kocuri. The new isolates do not contain bacteriochlorophyll a and are considered to represent a new genus and a new species, Silicibacter lacuscaerulensis.

First practical assay for soluble nitrous oxide reductase of denitrifying bacteria and a partial kinetic characterization.

Lysis of spheroplasts made from denitrification-adapted Paracoccus denitrificans released a soluble nitrous oxide reductase which was assayed spectrophotometrically under anaerobic conditions by following the oxidation of methyl or benzyl viologen cation radical upon reduction of N2O to N2. Other classes of reductants so far tested, including dithionite, could not substitute for viologen dyes. Viologen dyes, therefore, afford the first practical assay for this previously elusive enzyme of denitrifying bacteria. The assay is specifically an in vitro assay, because the dyes cannot couple with intracellular nitrous oxide reductase. The enzyme exhibited simple saturation kinetics with respect to both N2O and reduced viologen dye. The Km for N2O was about 5 microM at 22 degrees C and pH 7.1 and the apparent Km for reduced benzyl and methyl viologen was 0.9 and 0.5 microM, respectively. Both dyes afforded the same Vmax value. Oxidized viologen dyes were not inhibiting to 1 mM nor was N2 to 1 atm. In fresh lysates, Vmax was about 1.2 mumol of N2O X min-1 x mg of protein-1 or about twice that for intact cells or spheroplasts utilizing yeast extract or lactate. Enzyme activity was observed to be labile in crude preparations under anaerobic conditions. Nitrous oxide reductase was inhibited by acetylene, CO, azide, and cyanide with Ki values of 28, 3.5, 0.35, and 0.045 microM, respectively. All showed noncompetitive inhibition with respect to N2O.

Isolation of Halotolerant Thermus spp. from Submarine Hot Springs in Iceland.

Thermophilic, aerobic bacteria of the genus Thermus were isolated from submarine alkaline hot springs in Iceland. Five submarine hot springs were sampled, and all had viable counts of Thermus spp. of about 10 CFU/ml. All submarine strains grew in the presence of NaCl at 3% or higher, but no strains from terrestrial hot springs would grow at concentrations higher than 1% NaCl. The growth rate of submarine Thermus strains was not stimulated by NaCl and was reduced at NaCl concentrations higher than 1%. The pattern of growth of these isolates on single carbon sources was similar to that of terrestrial isolates.

Influence of pH and Temperature on Enumeration of Cellulose- and Hemicellulose-Degrading Thermophilic Anaerobes in Neutral and Alkaline Icelandic Hot Springs.

Cellulose- and hemicellulose-degrading thermophilic anaerobes were enumerated in biomat samples of various temperatures from two different hot springs in the Hverageroi area of Iceland: one spring had a pH near 7, the second had a pH near 9. The most-probable-number technique was used for enumeration of bacteria in the samples, with media at many different temperatures (37 to 90 degrees C) and two pH values (7 and 9). There were generally more xylan-degrading then cellulose-utilizing organisms in both environments. There was no growth at 80 degrees C in the neutral spring or at 37 degrees C in the alkaline spring. However, there were large numbers of both types of organisms in the alkaline spring at 80 degrees C and in the neutral spring at 37 degrees C. No cultures grew from the most-probable-number tubes inoculated with the Hverageroi samples and incubated at 90 degrees C or with media at pH 9. However, xylan-degrading cultures at 70 degrees C were enriched at pH 9 with samples from some other Icelandic hot springs.

Production of Rhizobium Inoculants for Lupinus nootkatensis on Nutrient-Supplemented Pumice.

The use of the legume Lupinus nootkatensis as a pioneer plant to fight soil erosion and to reclaim eroded soils in Iceland has been under development for a few years. Production of a robust, low-cost bacterial inoculant was therefore a prerequisite for the extended use of this plant. Volcanic pumice is a naturally expanded mineral which is available in vast amounts in Iceland. It was tested as a carrier for solid fermentation of Rhizobium lupini. Nutrient-supplemented pumice containing a small percentage of peat and diatomaceous earth and kept in sterile plastic bags promoted good growth of the bacteria. Viable-colony counts remained stable at 10 to 10/g for at least 35 weeks when the carrier was stored at 22 degrees C. The pumice-based inoculant had good storage and handling properties and could be mixed directly with the seeds during the sowing process. When seeds of L. nootkatensis were sown manually into nutrient-poor eroded sandy soils, about 56% of the first-year plants were successfully nodulated.

Sulfolobicins, specific proteinaceous toxins produced by strains of the extremely thermophilic archaeal genus Sulfolobus.

Several novel strains of "Sulfolobus islandicus" produced proteinaceous toxins, termed sulfolobicins, which killed cells of other strains of the same species, as well as of Sulfolobus solfataricus P1 and Sulfolobus shibatae B12, but not of the producer strains and of Sulfolobus acidocaldarius DSM639. The sulfolobicin purified from the strain HEN2/2 had a molecular mass of about 20 kDa. It was found to be associated with the producer cells as well as with cell-derived S-layer-coated spherical membrane vesicles 90 to 180 nm in diameter and was not released from the cells in soluble form.

Genetic diversity analysis of Rhodothermus reflects geographical origin of the isolates.

The genetic diversity and relationships of 81 Rhodothermus isolates from different geothermal environments in Iceland were examined by analysis of electrophoretically demonstrable allelic variation of 13 genes encoding enzymes. All the enzymes were polymorphic. A total of 71 distinctive multilocus genotypes (electrophoretic types, ETs) were identified. The mean genetic diversity per locus (H1) was 0.586. The relatively high genetic variance observed within Rhodothermus isolates from different locations is most likely the result of genetic changes occurring independently in the locations studied. A high Gst value (0.284) indicates that a considerable part of the variance observed is due to differences between locations. Cluster analysis revealed two major groups of ET clusters diverging at a genetic distance of 0.75, reflecting strongly the geographic origin of isolates. Estimation of the association index (I(A)) indicates that Rhodothermus marinus is a clonal species in which recombination events occur rarely. Partial or whole sequencing of the 16S rRNA genes of Rhodothermus isolates grouping at genetic distance of 0.40 confirmed that all the isolates belonged to the species Rhodothermus marinus. The results of this study confirm that, despite phylogenetic and phenotypic similarity, genetic diversity within Rhodothermus marinus is quite high.

Cloning and sequencing of a Rhodothermus marinus gene, bglA, coding for a thermostable beta-glucanase and its expression in Escherichia coli.

A gene library of the thermophilic eubacterium, Rhodothermus marinus, strain 21, was prepared in pUC18 and used to transform Escherichia coli. Of 5400 transformants, two produced halos on lichenan plates after Congo-red staining. Restriction mapping showed that the two clones shared an overlapping 1200-bp DNA fragment, which was used for DNA sequencing. Five potential methionine (Met) translational-initiation codons were identified. A putative signal peptide of 30 amino acids was identified with a hydrophobic core of nine hydrophobic amino acids. The molecular mass of the mature enzyme was estimated to be 29.7 kDa. A comparison of the primary protein sequence of beta-glucanase of Rhodothermus marinus with other glycosyl hydrolases showed 38.5% identity to the C-terminal part of the beta-1,3-glucanase of Bacillus circulans and limited identity to bacterial endo-beta-1,3-1,4-glucanases. The amino acid sequence showed high similarity to regions surrounding the catalytic Glu residue of bacterial beta-glucanases. A gene fragment of 889 bp containing the catalytic domain was overexpressed in E. coli using the pET23, T7-phage RNA polymerase system. The enzyme showed activity on lichenan, beta-glucan and laminarin but not on CMC cellulose or xylan. The expressed enzyme was purified by heat treatment of the host. The enzyme had a temperature and pH optima of 85 degrees C and pH 7.0, respectively, and was shown to retain full activity after incubation for 16 h at 80 degrees C and have a half life of 3 h at 85 degrees C.