Methods to Identify and Analyze Vesicle-Protected DNA Transfer.

Conjugation, transformation, and transduction constitute the three classical mechanisms involved in horizontal gene transfer (HGT) among prokaryotes. In addition, alternative HGT mechanisms exist in groups of organisms. Among them, the use of DNA-containing membrane vesicles as shuttle elements for HGT has been described for a number of microorganisms, including both thermophiles and mesophiles. Here we describe the methods followed to detect, purify, and analyze these vesicles.

Complete Genome Sequence of Thermus aquaticus Y51MC23.

Thermus aquaticus Y51MC23 was isolated from a boiling spring in the Lower Geyser Basin of Yellowstone National Park. Remarkably, this T. aquaticus strain is able to grow anaerobically and produces multiple morphological forms. Y51MC23 is a Gram-negative, rod-shaped organism that grows well between 50°C and 80°C with maximum growth rate at 65°C to 70°C. Growth studies suggest that Y51MC23 primarily scavenges protein from the environment, supported by the high number of secreted and intracellular proteases and peptidases as well as transporter systems for amino acids and peptides. The genome was assembled de novo using a 350 bp fragment library (paired end sequencing) and an 8 kb long span mate pair library. A closed and finished genome was obtained consisting of a single chromosome of 2.15 Mb and four plasmids of 11, 14, 70, and 79 kb. Unlike other Thermus species, functions usually found on megaplasmids were identified on the chromosome. The Y51MC23 genome contains two full and two partial prophage as well as numerous CRISPR loci. The high identity and synteny between Y51MC23 prophage 2 and that of Thermus sp. 2.9 is interesting, given the 8,800 km separation of the two hot springs from which they were isolated. The anaerobic lifestyle of Y51MC23 is complex, with multiple morphologies present in cultures. The use of fluorescence microscopy reveals new details about these unusual morphological features, including the presence of multiple types of large and small spheres, often forming a confluent layer of spheres. Many of the spheres appear to be formed not from cell envelope or outer membrane components as previously believed, but from a remodeled peptidoglycan cell wall. These complex morphological forms may serve multiple functions in the survival of the organism, including food and nucleic acid storage as well as colony attachment and organization.

Differences in Physical and Biochemical Properties of Thermus scotoductus SA-01 Cultured with Dielectric or Convection Heating.

A thermophile, Thermus scotoductus SA-01, was cultured within a constant-temperature (65°C) microwave (MW) digester to determine if MW-specific effects influenced the growth and physiology of the organism. As a control, T. scotoductus cells were also cultured using convection heating at the same temperature as the MW studies. Cell growth was analyzed by optical density (OD) measurements, and cell morphologies were characterized using electron microscopy imaging (scanning electron microscopy [SEM] and transmission electron microscopy [TEM]), dynamic light scattering (DLS), and atomic force microscopy (AFM). Biophysical properties (i.e., turgor pressure) were also calculated with AFM, and biochemical compositions (i.e., proteins, nucleic acids, fatty acids) were analyzed by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. Gas chromatography-mass spectrometry (GC-MS) was used to analyze the fatty acid methyl esters extracted from cell membranes. Here we report successful cultivation of a thermophile with only dielectric heating. Under the MW conditions for growth, cell walls remained intact and there were no indications of membrane damage or cell leakage. Results from these studies also demonstrated that T. scotoductus cells grown with MW heating exhibited accelerated growth rates in addition to altered cell morphologies and biochemical compositions compared with oven-grown cells.

[Isolation and characterization of a Thermus bacteriophage lytic from Tengchong Rehai Hot Spring lytic].

OBJECTIVE: Genus Thermus represents an ancient descendant within the domain of Bacteria. This research was focused on the isolation and characterization of Thermus bacteriophages from Tengchong Reihai hot spring. METHODS: Bacteriophage was isolated from Tengchong Rehai hot springs by double-layer plate method, and further characterized by morphology, temperature, pH and organic solvent effect on phage production, DNA restriction endonuclease digestion and protein composition analysis. RESULTS: One lytic bacteriophage was isolated from Tengchong hot spring. It's host strain Thermus sp. TC10 belonged to genus Thermus (16S rRNA gene accession number GU119889). This phage has a hexagonal head (67 nm in diameter) and an extremely long tail (837 nm in length and 10 nm in width). The optimum temperature and pH value for production of virons were about 65 degrees C and 7.6, respectively. The phage was not sensitive to chloroform. The differences between this phage and the other two Thermus Siphoviridae phages P23-45 and P74-26, which were isolated form Russia's Kamchatka peninsula, demostrated it was a novel bacteriaphage and was denoted as TTSP10 (Tengchong Thermus Siphoviridae phage).

A ring-like nucleoid is not necessary for radioresistance in the Deinococcaceae.

BACKGROUND: Transmission electron microscopy images of Deinococcus radiodurans R1 suggest that the nucleoid of this species exists as a "ring-like" body, and have led to speculation that this structure contributes to the radioresistance of the species. Since extreme radioresistance is characteristic of six other species of Deinococcus, we have attempted to correlate nucleoid morphology and radioresistance by determining whether the genomic DNA of each of these species exhibit similar structures. RESULTS: The nucleoid morphologies of seven recognized species of Deinococcus, the radioresistant bacterium Rubrobacter radiotolerans, and the more radiosensitive deinococcal relative Thermus aquaticus were evaluated using epifluorescence and deconvolution techniques. Although the nucleoids of Deinococcus murrayi, Deinococcus proteolyticus, Deinococcus radiophilus, and Deinococcus grandis have structures similar to D. radiodurans, the majority of nucleoids found in Deinococcus radiopugnans and Deinococcus geothermalis lack any specific organization. The nucleoid of R. radiotolerans consists of multiple highly condensed spheres of DNA scattered throughout the cell. The genomic DNA of Thermus aquaticus is uniformly distributed throughout the cell. CONCLUSION: There is no obvious relationship between the shape of a species' nucleoid and extreme radioresistance. However, the genomes of all extremely radioresistance species examined are highly condensed relative to more radiosensitive species. Whether DNA in this tightly packed configuration contributes to the radioresistance of these bacteria remains unknown, but this common structural feature appears to limit diffusion of fragments generated post-irradiation even in cells incapable of repairing strand breaks.

Cell wall structural divergence among Thermus spp.

The fine structure of sacculi from Thermus thermophilus HB27, T. aquaticus YT-1 and Thermus ATCC27737 has been worked out by HPLC analysis and mass spectrometry techniques. The three microorganisms have a murein composition of the rare A3beta chemotype, but showed substantial differences in muropeptide composition. Phenylacetylated muropeptides,previously described in T. thermophilus HB8, were detected exclusively in T. thermophilus HB27. Murein from T. aquaticusYT-1 was devoid of D-Ala-D-Ala terminated muropeptides, which were, in contrast, abundant in T. thermophilus HB27 and Thermus ATCC27737. The significance of these findings is discussed.

Evidence for association of bacteriocinogenic activity with membrane vesicles of Thermus rubens.

Thermus rubens has previously been reported to exhibit bacteriocin-like antagonism against other members of the genus Thermus when assayed via the deferred antagonism technique. Bacteriocin activity was not detectable in various age batch culture supernatants assayed by simple spotting procedures. Successful cell-free isolation was accomplished by way of a procedure employing French press disruption of T. rubens cells. Ultrafiltration and molecular exclusion chromatographic studies ascribed a high molecular weight to the agent which was found to be completely precipitable by ammonium sulphate. Activity was totally sedimentable upon ultracentrifugation. Transmission electron microscopic examination of column fractions containing high specific activity revealed numerous circular, apparently trilaminar, structures indicative of membrane vesicles.

Electron image analysis of ribosomal subunits from Thermus aquaticus.

Electron micrographs of ribosomal subunits from the thermophilic bacterium Thermus aquaticus were analysed using multivariate statistical analysis and characteristic views constructed to reproducible spatial resolutions ranging from 1.9 to 3.6 nm. These views were comparable to morphological classes of Escherichia coli ribosomal subunits, albeit with differences in fine features also found in archaebacterial ribosomes.

Filament formation in Thermus species in the presence of some D-amino acids or glycine.

A number of strains of Thermus spp. changed morphology from rods of about 6 to 8 microns long to multicellular filaments (unsheathed trichomes) up to many hundreds of micrometres long with the addition of glycine or certain D-amino acids to the growth medium. Associated with this change was the formation of braided trichomes and occasionally true knots. Filament formation was reversible by the removal of the causal agent, but only if growth was possible. Electron microscopy suggested that the wall structure was not changed, but only that cells did not separate due to the continuous nature of the outer membrane layer. The filaments were thus multicellular. The constituent cells were similar in length to the normal rod-shaped cells. Filament formation by Thermus spp. may have applications in industrial scale culture of these extracellular enzyme-producing thermophilic bacteria.

Characterization and preliminary crystallographic studies on large ribosomal subunits from Thermus thermophilus.

Diffracting crystals, suitable for X-ray crystallographic analysis, have been obtained from large (50 S) ribosomal subunits from Thermus thermophilus. These crystals, with P4(1)2(1)2 symmetry and a unit cell of 495 A x 495 A x 196 A, reach typically a size of 0.15 mm x 0.25 mm x 0.35 mm. Using synchrotron radiation at cryo-temperature, these crystals diffract X-rays to better than 9 A resolution, and do not show any measurable decay after a few days of irradiation. They complete a series of crystals, grown by us, from ribosomal particles of the same source, including a 30 S subunits, 70 S ribosomes and complexes of the latter with: (1) an oligomer of 35 uridine residues and (2) the same oligonucleotide together with approximately two Phe-tRNA(Phe) molecules. Crystallographic analysis of the various members of this series should provide information for investigating the conformational changes that take place upon the association of ribosomes from their subunits as well as upon binding of non-ribosomal components that participate in protein biosynthesis.

Studies on the NADH-menaquinone oxidoreductase segment of the respiratory chain in Thermus thermophilus HB-8.

Five distinct low potential iron-sulfur clusters have been identified potentiometrically in the membrane particles from Thermus thermophilus HB-8. Three of these clusters (designated as [N-1H]T, [N-2H]T, and [N-3]T) exhibit the following midpoint redox potentials and g values (Em8.0 = -274 mV, gx,y,z = 1.93, 1.94, 2.02), (Em8.0 = -304 mV, gx,y,z = 1.89, 1.95, 2.04), and (Em8.0 = -289 mV, gx,y,z = 1.80, 1.83, 2.06), respectively. These clusters, one binuclear and two tetranuclear, have been shown to be components of the energy coupled NADH-menaquinone oxidoreductase complex (NADH dh I). They are reducible by NADH in the piericidin A-inhibited aerobic membrane particles as well as in the purified NADH dh I complex. Two additional very low potential iron-sulfur clusters (one binuclear, [N-1L]T, and one tetranuclear, [N-2L]T) were observed in membrane particles. These clusters possess the following physiochemical properties (Em8.0 = -418 mV, gx,y,z = 1.93, 19.5, 2.02) and (Em8.0 = -437 mV, gx,y,z = 1.89, 1.95, 2.04), respectively. No high potential tetranuclear cluster equivalent to the mitochondrial iron-sulfur cluster [N-2]B was found in this bacterial system. In membrane particles isolated from T. thermophilus HB-8 cells, four different semiquinone species have been identified based on their redox midpoint potentials [Em9(Q/QH2) = 40, -100, -160, -300 mV] and sensitivity to the quinone analogue inhibitor, 2-heptyl-4-hydroxy quinoline-N-oxide. Of these semiquinone species the -100 mV component has been suggested to be part of the NADH dehydrogenase. Piericidin A sensitive delta psi formation has been demonstrated to be coupled to the NADH-MQ1 oxidoreductase in membrane vesicles of T. thermophilus HB-8.

Three-dimensional crystals of ribosomes and their subunits from eu- and archaebacteria.

Ordered three-dimensional crystals of 70S ribosomes as well as of 30S and 50S ribosomal subunits from various bacteria (E. coli, Bacillus stearothermophilus, Thermus thermophilus and Halobacterium marismortui) have been grown by vapour diffusion in hanging drops using mono- and polyalcohols. A new compact crystal form of 50S subunits has been obtained, and it is suitable for crystallographic studies at medium resolution. In addition, from one crystal form large crystals could be grown in X-ray capillaries. In all cases the crystals were obtained from functionally active ribosomal particles, and the particles from dissolved crystals retained their integrity and biological activity.

Outer and inner membrane preparations of extreme thermophile and their physico-chemical properties.

Outer and inner (cytoplasmic) membranes were partially purified from the gram negative extremely thermophilic bacteria, Thermus thermophilus HB-8 by sucrose density gradient centrifugation. In spite of our efforts to separate them, the inner membrane fraction contained some outer membrane components as determined by enzyme assay and electrophoresis. When studied by 5DS spin labeling, the outer membranes showed a larger 2T11 value (lower fluidity) than the inner membranes, although the fatty acid compositions were similar. The inner membranes of the cells cultured at higher temperature showed a larger 2T11 value than the cells cultured at lower temperature. A similar phenomenon was observed with the TEMPO parameter of liposomal membranes. The upper break point (Th) of the inner membranes observed by spin labeling was slightly lower than the culture temperature of the cells, and the lower break point (T1) corresponded well to the lowest temperature limit of growth. The calorimetric heating curve of the inner membranes had a broader temperature range of transition than that of the liposomal membranes. The transition temperature observed by calorimetry seems to reflect the melting properties of the membrane lipids, while fatty acid spin probe probably reports the local environment of the membrane, which is more directly related to its biological function.

[Thermophilic bacteria, Thermus ruber, that produce a bright orange pigment]. / Termofil'nye bakterii Thermus ruber, produtsiruiushchie iarko-oranzhevyi pigment.

A non-sporeforming gram-negative bacterium isolated from soils of Africa was shown to belong to the species Thermus ruber in its morphologo-cytological and physiologobiochemical properties. It differed from the Thermus ruber species described elsewhere (Loginova et al., 1975; Loginova and Egorova, 1975) only on colour: the former contained a bright-red pigment while the latter produced bright-orange pigments.

[Speroplast behavior in cultures of Thermus ruber]. / Povedenie sferoplastov v kul'turakh Thermus ruber.

Spheroplasts spontaneously originating in cultures of Thermus ruber, which are cultivated on potato-peptone media, are capable of growth and multiplication. They may reach 10 mc in diameter. Spheroplasts multiply by budding which can be preceded with inner division or budding; as a result 2--9 and more protoplast bodies are formed under the "envelope" of a spheroplast. Self-reproduction of spheroplasts is most active in a semiliquid potato-meat-peptone medium containing 0.2--0.3 per cent of agar. Microcolonies, which are clusters of spheroplasts in the process of budding, are formed at 45 degrees C in this medium during 1--2 days. Reversion of spheroplasts to rod-like cells occurs if L-lysine and/or DL-alpha-aline are added to the potato-peptone medium. Spheroplasts spontaneously produced by Thermus ruber are similar to the unstable type of L-form in their fine organization and behaviour in cultures.