Thermus brevis sp. nov., a moderately thermophilic bacterium isolated from a hot spring microbial mat.

Three closely related, facultative anaerobic, Gram-stain-negative, twitching motile, short rod-shaped, non-endospore-forming, moderately thermophilic bacteria, designated strains SYSU G05001T, SYSU G05003 and SYSU G05004, were isolated from a hot spring microbial mat, collected from Rehai National Park, Tengchong, Yunnan Province, south-western China. The results of phylogenetic analysis based on the 16S rRNA gene sequences indicated that these three strains were closely related to Thermus scotoductus SE-1T (97.97, 98.18, 97.90 % sequence similarity). Whole genome sequencing and polyphasic taxonomic approach were used to determine the genomic profile and taxonomic status of the novel strain SYSU G05001T. Cell growth occurred at 37-80 °C (optimum, 55 °C), pH 6.0-8.0 (optimum, pH 7.0) and with 0-3.0 % (w/v) NaCl (optimum, 1%). Thiosulfate enhanced cell growth. MK-8 was the predominant menaquinone. The major cellular fatty acids included iso-C15 : 0, iso-C17 : 0 and anteiso-C15 : 0. The major polar lipids were consisted of aminophospholipid, glycolipid and phospholipids. The whole genome of strain SYSU G05001T consisted of 2.55 Mbp and the DNA G+C content was 64.94 mol%. The average nucleotide identity (≤94.95 %) and digital DNA-DNA hybridization (≤62.3 %) values between strain SYSU G05001T and other members of the genus Thermus were all lower than the threshold values recommended for distinguishing novel prokaryotic species. On the basis of the presented polyphasic evidence and genotypic data, it is proposed that strain SYSU G05001T (=KCTC 82627T=MCCC 1K06118T) represents a novel species of the genus Thermus, for which the name Thermus brevis sp. nov. is proposed.

The integrity of the G2421-C2395 base pair in the ribosomal E-site is crucial for protein synthesis.

During the elongation cycle of protein biosynthesis, tRNAs traverse through the ribosome by consecutive binding to the 3 ribosomal binding sites (A-, P-, and E- sites). While the ribosomal A- and P-sites have been functionally well characterized in the past, the contribution of the E-site to protein biosynthesis is still poorly understood in molecular terms. Previous studies suggested an important functional interaction of the terminal residue A76 of E-tRNA with the nucleobase of the universally conserved 23S rRNA residue C2394. Using an atomic mutagenesis approach to introduce non-natural nucleoside analogs into the 23S rRNA, we could show that removal of the nucleobase or the ribose 2'-OH at C2394 had no effect on protein synthesis. On the other hand, our data disclose the importance of the highly conserved E-site base pair G2421-C2395 for effective translation. Ribosomes with a disrupted G2421-C2395 base pair are defective in tRNA binding to the E-site. This results in an impaired translation of genuine mRNAs, while homo-polymeric templates are not affected. Cumulatively our data emphasize the importance of E-site tRNA occupancy and in particular the intactness of the 23S rRNA base pair G2421-C2395 for productive protein biosynthesis.

MreB and MreC act as the geometric moderators of the cell wall synthetic machinery in Thermus thermophiles.

How cell morphology is maintained in thermophilic bacteria is unknown. In this study, the functions and mechanisms of the potential cell shape determinants (e.g. MreB, MreC, MreD and RodA homologues) of the model extremely thermophilic bacterium Thermus thermophilus were initially analyzed. Deletion of mreC, mreD or rodA only resulted in heterozygous mutants indicating that these genes are all essential. In the MreB-inhibited (by A22) strain and the heterozygous mreC, mreD or rodA mutant, cell morphologies were drastically changed, and enlarged spherical cells were eventually dead indicating that they are vital for cell shape maintenance. When fused to sGFP, MreB, MreC, MreD, RodA, and the enzymes involved in peptidoglycan synthesis (e.g. PBP2 and MurG) exhibited similar subcellular localization pattern, appearing as patches, or bands slightly angled to the cell length. The localizations and functions of all the 6 proteins required a natural peptidoglycan synthesis pattern, additionally those of MreD, RodA and MurG were dependent on MreB polymerization. Consistently, through comprehensive bacterial two-hybrid analyses, it was revealed that MreB could interact with itself, MreC, MreD, RodA and MurG, and MreC could associate with PBP2. In conclusion, in T. thermophilus, MreB, MreC, MreD, RodA and the peptidoglycan synthesis enzymes probably form a network of interactions centered with MreB and bridged with MreC, thereby maintaining cell morphology.

UV-induced crosslinks in the 16S rRNAs of Escherichia coli, Bacillus subtilis and Thermus aquaticus and their implications for ribosome structure and photochemistry.

Sixteen long-range crosslinks are induced in Escherichia coli 16S rRNA by far-UV irradiation. Crosslinking patterns in two other organisms, Bacillus subtilis and Thermus aquaticus, were investigated to determine if the number and location of crosslinks in E.coli occur because of unusually photoreactive nucleotides at particular locations in the rRNA sequence. Thirteen long-range crosslinks in B.subtilis and 15 long-range crosslinks in T.aquaticus were detected by gel electrophoresis and 10 crosslinks in each organism were identified completely by reverse transcription analysis. Of the 10 identified crosslinks in B.subtilis, eight correspond exactly to E.coli crosslinks and two crosslinks are formed close to sites of crosslinks in E.coli. Of the 10 identified crosslinks in T.aquaticus, five correspond exactly to E.coli crosslinks, three are formed close to E.coli crosslinking sites, one crosslink corresponds to a UV laser irradiation-induced crosslink in E.coli and the last is not seen in E.coli. The overall similarity of crosslink positions in the three organisms suggests that the crosslinks arise from tertiary interactions that are highly conserved but with differences in detail in some regions.

Production of thermostable lipolytic activity by thermus species.

A quantitative screening for intra- and extracellular lipolytic activity was performed in submerged cultures of four Thermus strains using two different media (named T or D medium). Major differences in the extracellular lipolytic activity were observed in T medium, the highest values being for Thermus thermophilus HB27 and Thermus aquaticus YT1 strains (18 and 33 U/L, respectively). Two enzymes with lipase/esterase activity were identified in the four Thermus strains by zymogram analysis, with molecular weights of 34 and 62 kDa. No kinetic typification of the enzymes as primary metabolites was possible for any of the Thermus strains, because of the lack of a good fitting of the experimental lipolytic activity production rates to the Luedecking and Piret model. However, a linear relationship was found between the absolute values of biomass and total lipase/esterase activity (sum of intracellular and extracellular). For T. thermophilus HB27, an increase in the aeration rate caused the increase in the production of biomass and, particularly, intracellular lipolytic activity but the extracellular lipolytic activity was not affected except for the series with the strongest oxygen limitation. Transmission electronic microscopy revealed that T. thermophilus HB27 formed rotund bodies surrounded by a common membrane in cultures in the early stationary phase. The results suggest the occurrence of a specific mechanism of lipase/esterase secretion that might be due to the different composition and permeability of the cell membranes and those surrounding the rotund bodies.

Reduction of U(VI) by the deep subsurface bacterium, Thermus scotoductus SA-01, and the involvement of the ABC transporter protein.

In this study we investigated the effect of uranium on the growth of the bacterium Thermus scotoductus strain SA-01 as well as the whole cell U(VI) reduction capabilities of the organism. Also, site-directed mutagenesis confirmed the identity of a protein capable of a possible alternative mechanism of U(VI) reduction. SA-01 can grow aerobically in up to 1.25 mM uranium and has the capability to reduce low levels of U(VI) in under 20 h. TEM analysis performed on cells exposed to uranium showed extracellular and membrane-bound accumulation of uranium. The reductase-like protein was surprisingly identified as a peptide ABC transporter, peptide-binding protein. This study showcases the concept of protein promiscuity, where this protein with a distinct function in situ can also have the unintended function of a reactant for the reduction of U(VI).

Strategies for improving extracellular lipolytic enzyme production by Thermus thermophilus HB27.

In Thermus thermophilus HB27 cultures the localisation of lipolytic activity is extracellular, intracellular and membrane bound, with low percentage for the former. Therefore, the extracellular secretion must be increased in order to simplify the downstream process and to reduce the economic cost. This study focuses on the design of an innovative operational strategy to increase extracellular lipolytic enzyme production by T. thermophilus HB27 at bioreactor scale. In order to favour its secretion, the effect of several operational variables was evaluated. Among them, the presence of oils in the culture medium leads to improvements in growth and lipolytic enzyme activity. Sunflower oil is the most efficient inducer showing better results when added after 10h of growth. On the other hand, although surfactants lead to an almost complete inhibition of growth and lipolytic enzyme production, their addition along the culture could affect the location of the enzyme. Thus, by addition of surfactants at the stationary phase, a release of intracellular and membrane enzyme which increases the extracellular enzyme proportion is detected. Based on these results, strategies with successive addition of oil and surfactant in several culture phases in shake flask are developed and verified in a laboratory scale stirred tank bioreactor.

[Coccoid cells and spheroplasts in cultures of the genus Thermus]. / Kokkovidnye kletki i sferoplasty kul'tur roda Thermus.

The formation of capable of self-reproduction coccoid cells, and of spheroplasts, was studied in the cultures of Thermus ruber and pigmentless Thermus sp. by light and electron microscopy. The primary origination of cocci might be due to non-uniform or multiple cell division. Spheroplasts can be produced either from coccoid or from rod-like cells as a result of autolysis of the glycopeptide layer of the cell wall. The spheroplasts of the cultures belonging to the Thermus genus are characterized by osmotic stability and a peculiar appearance due to a strongly developed periplasmic space and the eccentric arrangement of the protoplast in it.

A comparative analysis of extreme thermophilic bacteria belonging to the genus Thermus.

Several extreme thermophilic Gram negative bacteria found in a thermally polluted river in Belgium have been compared with Thermus strains isolated from widely distant geographical areas. This analysis has become possible after the design of a new culture medium (162). All strains examined (including the isolate successively denominated Flavobacterium thermophilum and Thermus thermophilus) were found to be morphologically identical with strain YT-1 of Thermus aquaticus. The cells are immotile, rod-like, strictly aerobic, catalase and oxidase positive. They produce amylase, hydrolyze gelatin and are confirmed to be highly sensitive towards penicillin. The nutritional pattern of all strains has been analysed extensively, by testing a broad spectrum of possible substrates. The strains display a uniform response to the microbiological tests applied and most probably belong to the same species: Thermus aquaticus.

Plasmid-associated aggregation in Thermus thermophilus HB8.

Thermus thermophilus HB8, a moderate thermophile, exhibits visible aggregation when growing on a rich broth. Strain HB8 also contains two cryptic plasmids. We isolated cured strains from HB8 and observed that loss of the 47-MDa plasmid was correlated with loss of aggregation. An enrichment procedure was developed for aggregating cells and used to demonstrate that aggregation was restored upon transformation of a cured strain with plasmid DNA. The aggregation phenotype of transformed cells was variably stable; most did not retain either the plasmid or the phenotype for prolonged periods of growth. Hybridization experiments using a partial sequence from the 47-MDa plasmid suggested the presence of a repeated DNA sequence on this plasmid and on the chromosome. This is the first report of a phenotype associated with a plasmid from a Thermus strain.

[Cytological characteristics of the thermophilic bacterium Thermus ruber]. / Tsitologicheskaia kharakteristika termofil'noi bakterii Thermus ruber.

The obligate thermophilic bacterium Thermus ruber 12b was examined using optical and electron microscopy. Its cells were shown to be polymorphous. In a liquid medium, the cells associate yielding complex spherical bodies having a surface layer in common and complexes consisting of two or more cells but without a common envelope. A large nucleoid is located in the central part of the cells; it contains polyphosphate granules. Fat inclusions and analogues of mitochondria with dehydrogenase activity typical of them can be detected at the periphery. The cell wall consists of a folded outer membrane, an underlying fibrillar material, and a thin rigid layer. Peculiar adhesion zones appear between the outer membrane and the rigid layer in places where folds are formed. An electron-dense substance is deposited on the surface of the three-layer cytoplasmic membrane.

[Experimental induction of rotund bodies in Thermus equaticus]. / Experimentelle Induktion von "rotund bodies" bei Thermus aquaticus.

With several isolates of T. aquaticus rotund bodies (rbs) have been artificially induced. The most active agents were lysozyme, pronase (not trypsin), cellulase (not alpha-amylase or beta-glucosidase), penicillin and some mineral salts. Two modifications of rbs were observed: the vesticular type arising from single cells or filaments and the aggregate-rbs resulting from secondary associations of several cells or filaments. In both cases the primary event is a partial detachment of the outer layer of cell envelopes followed by its swelling to form a hyaline bleb. Within these osmotically stable blebs cells and/or filaments involved in the ribs production become irreversibly trapped. Appearance of filaments and rbs in stationary phase cultures are discussed as a consequence of unbalanced growth culminating in abnormal autolytic reactions.

Crude-oil-degrading thermophilic bacterium isolated from an oil field.

Thermophilic bacterium strain C2, which has the ability to transform crude oils, was isolated from the reservoir of the Shengli oil field in East China. The Gram-negative, rod-shaped, nonmotile cells were grown at a high temperature, up to 83 degrees C, in the neutral to alkaline pH range. Depending on the culture conditions, the organism occurred as single rods or as filamentous aggregates. Strain C2 was grown chemoorganotrophically and produced metabolites, such as volatile fatty acids, 1,2-benzenedicarboxylic acid, bis(2-ethylhexyl)ester, dibutyl phthalate, and di-n-octyl phthalate. It could metabolize different organic substrates (acetate, D-glucose, fructose, glycerol, maltose, pyruvate, starch, sucrose, xylose, hexadecane). The G+C content (68 mol%) and the 16S rRNA sequence of strain C2 indicated that the isolate belonged to the genus Thermus. The strain affected different crude oils and changed their physical and chemical properties. The biochemical interactions between crude oils and strain C2 follow distinct trends characterized by a group of chemical markers (saturates, aromatics, resins, asphaltenes). Those trends show an increase in saturates and a decrease in aromatics, resins, and asphaltenes. The bioconversion of crude oils leads to an enrichment in lighter hydrocarbons and an overall redistribution of these hydrocarbons.