Complete genome sequence of the thermophilic bacterium Geobacillus thermoleovorans CCB_US3_UF5

Geobacillus thermoleovorans CCB_US3_UF5 is a thermophilic bacterium isolated from a hot spring in Malaysia. Here, we report the complete genome of G. thermoleovorans CCB_US3_UF5, which shows high similarity to the genome of Geobacillus kaustophilus HTA 426 in terms of synteny and orthologous genes.     

A new Thermus sp. class-IIS enzyme sub-family: isolation of a 'twin' endonuclease TspDTI with a novel specificity 5'-ATGAA(N(11/9))-3', related to TspGWI,TaqII and Tth111II

The TspDTI restriction endonuclease, which shows a novel recognition specificity 5'-ATGAA(N(11/9))-3', was isolated from Thermus sp. DT. TspDTI appears to be a 'twin' of restriction endonuclease TspGWI from Thermus sp. GW, as we have previously reported. TspGWI was isolated from the same location as TspDTI, it recognizes a related sequence 5'-ACGGA(N(11/9))-3' and has conserved cleavage positions. Both enzymes resemble two other class-IIS endonucleases from Thermus sp.: TaqII and Tth111II. N-terminal amino acid sequences of TspGWI tryptic peptides exhibit 88.9-100% similarity to the TaqII sequence. All four enzymes were purified to homogeneity; their polypeptide sizes (114.5-122 kDa) make them the largest class-IIS restriction endonucleases known to date. The existence of a Thermus sp. sub-family of class-IIS restriction endonucleases of a common origin is herein proposed.     

TspGWI, a thermophilic class-IIS restriction endonuclease from Thermus sp., recognizes novel asymmetric sequence 5'-ACGGA(N11/9)-3'

A novel prototype class-IIS restriction endonuclease, TspGWI, was isolated from the thermophilic bacterium Thermus sp. GW. The recognition sequence and cleavage positions have been established: TspGWI recognizes the non-palindromic 5-bp sequence 5'-ACGGA-3' and cleaves the DNA 11 and 9 nt downstream in the top and bottom strand, respectively. In addition, an accompanying endonuclease, TspGWII, an isoschizomer of Pst I, was found in Thermus sp. GW cells.     

Polyamine distributions in thermophilic eubacteria belonging to Thermus and Acidothermus

Triamines such as norspermidine, spermidine, and homospermidine and tetraamines such as norspermine, spermine, thermospermine, and aminopropylhomospermidine were found to be distributed ubiquitously in the eight extremely thermophilic (growing at 70 degrees C) Thermus species tested. Three linear pentaamine (caldopentamine, homocaldopentamine, and thermopentamine), two linear hexaamines (caldohexamine and homocaldohexamine), two tertiary branched tetraamines (N4-aminopropylnorspermidine and N4-aminopropyl-spermidine), and quaternary branched pentaamines such as N4-bis(aminopropyl)norspermidine and N4-bis(aminopropyl)spermidine were detected in T. thermophilus HB8, T. filiformis Wai33 A1, T. flavus AT-62, and T. caldophilus GK24. The linear hexaamines and branched polyamines were absent in T. aquaticus YT-1, T. sp. X-1, T. sp. T2, and T. sp. T351, in which linear pentaamines were minor components. Moderately thermophilic Thermus ruber and Thermus sp. K-2 contained putrescine, spermidine, norspermidine, homospermidine, spermine, norspermine, thermospermine, and aminopropylhomospermidine. No pentaamines, hexaamines, or branched polyamines were found in these two moderately thermophilic Thermus species. On the other hand, moderately thermophilic, acidophilic Acidothermus cellulolyticus was devoid of all the polyamines.     

Development of Thermus-Escherichia shuttle vectors and their use for expression of the Clostridium thermocellum celA gene in Thermus thermophilus.

We describe the self-selection of replication origins of undescribed cryptic plasmids from Thermus aquaticus Y-VII-51B (ATCC 25105) and a Thermus sp. strain (ATCC 27737) by random insertion of a thermostable kanamycin adenyltransferase cartridge. Once selected, these autonomous replication origins were cloned into the Escherichia coli vector pUC9 or pUC19. The bifunctional plasmids were analyzed for their sizes, relationships, and properties as shuttle vectors for Thermus-Escherichia cloning. Seven different vectors with diverse kanamycin resistance levels, stabilities, transformation efficiencies, and copy numbers were obtained. As a general rule, those from T. aquaticus (pLU1 to pLU4) were more stable than those from the Thermus sp. (pMY1 to pMY3). To probe their usefulness, we used one of the plasmids (pMY1) to clone in E. coli a modified form of the cellulase gene (celA) from Clostridium thermocellum in which the native signal peptide was replaced in vitro by that from the S-layer gene of T. thermophilus HB8. The hybrid product was expressed and exported by E. coli. When the gene was transferred by transformation into T. thermophilus, the cellulase protein was also expressed and secreted at 70 degrees C.     

Cloning and expression of the thermostable pullulanase gene from Thermus sp. strain AMD-33 in Escherichia coli

Abstract The gene coding for a thermostable pullulanase from a thermophile, Thermus sp. strain AMD-33, was cloned in Escherichia coli using pDR540 as a vector. A restriction map was determined for the plasmid pTPS131 which contained the fragment carrying the pullulanase gene. DNA-DNA hybridisation analysis showed that the DNA fragment contained the gene from Thermus sp. strain AMD-33. The strain of E. coli harbouring the plasmid pTPS131 produced most of the pullulanase protein cellularly, whereas Thermus sp. strain AMD-33 produced pullulanase extracellularly. Comparative studies of the enzyme from the thermophile and the plasmid-encoded enzyme in E. coli demonstrated that the optimum temperature and pH of the enzymes were closely similar.     

Quantification of intra- and extra-cellular thermophilic lipase/esterase production by Thermus sp.

Four Thermus strains produced lipolytic activity when grown in liquid medium for 30 h at 70 degrees C. The highest total lipase/esterase activity (57 U l(-1)) was in Thermus aquaticus YT-1, followed by Thermus thermophilus HB27 and HB8 (33 and 25 U l(-1), respectively), and finally by Thermus sp. (16 U l(-1)). Extra-cellular activity was detected in T. aquaticus YT-1 and T. thermophilus HB27 (33 and 17 U l(-1)). All enzymes were stable at 80 degrees C over 30 min, and their activity towards fatty acid esters increased as substrate chain-length diminished (i.e. hydrolysis rate was up to 6-fold higher on p-nitrophenyl caproate than on laurate).     

Production of galactooligosaccharides from lactose using a beta-glucosidase from Thermus sp. Z-1

A thermostable beta-glucosidase from Thermus sp. Z-1 that not only hydrolyzes beta-glucosides but also beta-galactosides was shown to efficiently produce oligosaccharides during hydrolysis of lactose. The yield of oligosaccharides was more than 40% for 0.88 M lactose solution at 70 degrees C at pH 7.0. The major product was a trisaccharide, 3'-galactosyllactose, formed by a galactosyltransfer reaction.     

Inducible and constitutive expression using new plasmid and integrative expression vectors for Thermus sp.

AIMS: To develop molecular tools and examine inducible and constitutive gene expression in Thermus thermophilus. METHODS AND RESULTS: Two plasmid promoter probe vectors and an integrative promoter probe vector were constructed using a promoterless thermostable kanamycin nucleotidyltransferase (KmR) cassette. Three expression vectors were constructed based on a constitutive promoter J17, that functions in both Thermus and Escherichia coli. An inducible expression vector was constructed using the heat-shock inducible promoter (70 to 85 degrees C) from the dnaK gene of T. flavus, and the malate dehydrogenase gene (mdh) from T. flavus was cloned and expressed in both E. coli and T. thermophilus HB27. CONCLUSION: This report describes the construction and use of improved promoter probe and expression vectors for use in Thermus species. The mdh gene can be used as a high temperature (85 degrees C) reporter gene for Thermus sp. The dnaK promoter is thermo-inducible. Significance and Impact of the Study: The expression vectors and molecular tools described here are significant improvements over previously reported vectors for Thermus sp. The mdh gene and the thermo-inducible dnaK promoter will facilitate high temperature studies employing Thermus species.     

Purification and characterization of the recombinant Thermus sp. strain T2 alpha-galactosidase expressed in Escherichia coli

The nucleotide sequence of the Thermus sp. strain T2 DNA coding for a thermostable alpha-galactosidase was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 474 amino acids (M(r), 53,514). The observed homology between the deduced amino acid sequences of the enzyme and alpha-galactosidase from Thermus brockianus was over 70%. Thermus sp. strain T2 alpha-galactosidase was expressed in its active form in Escherichia coli and purified. Native polyacrylamide gel electrophoresis and gel filtration chromatography data suggest that the enzyme is octameric. The enzyme was most active at 75 degrees C for p-nitrophenyl-alpha-D-galactopyranoside hydrolysis, and it retained 50% of its initial activity after 1 h of incubation at 70 degrees C. The enzyme was extremely stable over a broad range of pH (pH 6 to 13) after treatment at 40 degrees C for 1 h. The enzyme acted on the terminal alpha-galactosyl residue, not on the side chain residue, of the galactomanno-oligosaccharides as well as those of yeasts and Mortierella vinacea alpha-galactosidase I. The enzyme has only one Cys residue in the molecule. para-Chloromercuribenzoic acid completely inhibited the enzyme but did not affect the mutant enzyme which contained Ala instead of Cys, indicating that this Cys residue is not responsible for its catalytic function.     

Pyrococcus prefoldin stabilizes protein-folding intermediates and transfers them to chaperonins for correct folding

A molecular chaperone prefoldin/GimC from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 was characterized. Pyrococcus prefoldin protected porcine heart citrate synthase from thermal aggregation whereas each subunit alone afforded little protection. It also arrested the spontaneous refolding of acid-denatured green fluorescent protein and then transferred it not only to a group II chaperonin from the hyperthermophilic archaeum Thermococcus sp. strain KS-1, but also to a group I chaperonin from the thermophilic bacterium Thermus thermophilus HB8 for subsequent ATP dependent refolding.     

嗜热菌Thermus sp.YBJ-1的分离和淀粉酶基因的克隆

从西藏热泉水样分离得到一株嗜热菌(YBJ-1),其16S rDNA(1511bp)序列与栖热菌(Thermus scotoductus ITI-252T)的同源性为98％。通过PCR技术将Thermus sp．YBJ-1的淀粉酶基因(amyT)全长开放阅读框克隆到T载体。分析表明,amyT的ORF全长为1767bp,编码588个氨基酸。推导的氨基酸序列与嗜热脂肪芽孢杆菌的阿尔法环糊精酶(Bacillus stearothermophilus alpha-eyclodextrinase)和栖热菌Thermus sp．IM6501的麦芽糖淀粉酶(Thermus sp．IM6501 mahogenic amylase)分别有99％和96％的同源性,与嗜热脂肪芽孢杆菌的新普鲁兰酶(neopullulanase)的同源性为81％。     

Phosphoenolpyruvate carboxylase of Thermus sp.: role of a divergent glycine-rich region

In the phosphoenolpyruvate carboxylases (PEPCs) of mesophiles, a glycine(Gly)-rich region is well conserved and involved in catalytic activity, whereas the sequence of the corresponding region is considerably divergent in PEPC of Thermus sp. In this study, the Gly-rich region of Thermus PEPC was converted to that of Escherichia coli PEPC. The resulting mutant enzyme was as heat stable as the wild-type. However, its optimum temperature was markedly decreased. Thus, the divergent Gly-rich region of Thermus PEPC contributes to its catalytic activity but not to stability at high temperature.     

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.     

Thermophilic biodegradation of BTEX by two Thermus Species

Two thermophilic bacteria, Thermus aquaticus ATCC 25104 and Thermus species ATCC 27978, were investigated for their abilities to degrade BTEX (benzene, toluene, ethylbenzene, and xylenes). Thermus aquaticus and the Thermus sp. were grown in a nominal medium at 70 degrees C and 60 degrees C, respectively, and resting cell suspensions were used to study BTEX biodegradation at the same corresponding temperatures. The degradation of BTEX by these cell suspensions was measured in sealed serum bottles against controls that also displayed significant abiotic removals of BTEX under such high-temperature conditions. For T. aquaticus at a suspension density of only 1.3 x 10(7) cells/mL and an aqueous total BTEX concentration of 2.04 mg/L (0.022 mM), benzene, toluene, ethylbenzene, m-xylene, and an unresolved mixture of o-and p-xylenes were biodegraded by 10, 12, 18, 20, and 20%, respectively, after 45 days of incubation at 70 degrees C. For the Thermus sp. at a suspension density of 1.1 x 10(7) cells/mL and an aqueous total BTEX concentration of 6.98 mg/L (0.079 mM), benzene, toluene, ethylbenzene, m-xylene, and the unresolved mixture of o-and p-xylenes were biodegraded by 40, 35, 32, 33, and 33%, respectively, after 45 days of incubation at 60 degrees C. Raising the BTEX concentrations lowered the extents of biodegradation. The biodegradations of both benzene and toluene were enhanced when T. aquaticus and the Thermus sp. were pregrown on catechol and o-cresol, respectively, as carbon sources. Use of [U-(14)C]benzene and [ring-(14)C]toluene verified that a small fraction of these two compounds was metabolized within 7 days to water-soluble products and CO(2) by these nongrowing cell suspensions. Our investigation also revealed that the nominal medium can be simplified by eliminating the yeast extract and using a higher tryptone concentration (0.2%) without affecting the growth and BTEX degrading activities of these cells. (c) 1995 John Wiley & Sons, Inc.     

Description of five novel thermophilic species of the genus Thermus: Thermus hydrothermalis sp. nov., Thermus neutrinimicus sp. nov., Thermus thalpophilus sp. nov., Thermus albus sp. nov., and Thermus altitudinis sp. nov., isolated from hot spring sediments

Biological denitrification is a significant process in nitrogen biogeochemical cycle of terrestrial geothermal environments, and Thermus species have been shown to be crucial heterotrophic denitrifier in hydrothermal system. Five Gram-stain negative, aerobic and rod-shaped thermophilic bacterial strains were isolated from hot spring sediments in Tibet, China. Phylogenetic analysis based on 16S rRNA gene and whole genome sequences indicated that these isolates should be assigned to the genus Thermus and were most closely related to Thermus caldifontis YIM 73026^T, and Thermus brockianus YS38^T. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the five strains and the type strains of the genus Thermus were lower than the threshold values (95% and 70%, respectively) recommended for bacterial species, which clearly distinguished the five isolates from other species of the genus Thermus and indicated that they represent independent species. Colonies are circular, convex, non-transparent. Cell growth occurred at 37–80 °C (optimum, 60–65 °C), pH 6.0–8.0 (optimum, pH 7.0) and with 0–2.0% (w/v) NaCl (optimum, 0–0.5%). Denitrification genes (narG, nirK, nirS, and norB genes) detected in their genomes indicated their potential function in nitrogen metabolism. The obtained results combined with those of morphological, physiological, and chemotaxonomic characteristics, including the menaquinones, polar lipids, and cellular fatty acids showed that the isolates are proposed as representing five novel species of the genus Thermus, which are proposed as Thermus hydrothermalis sp. nov. SYSU G00291^T, Thermus neutrinimicus sp. nov. SYSU G00388^T, Thermus thalpophilus sp. nov. SYSU G00506^T, Thermus albus sp. nov. SYSU G00608^T, Thermus altitudinis sp. nov. SYSU G00630^T.     

Dissimilatory reduction of Fe(III) and other electron acceptors by a Thermus isolate

A thermophilic bacterium that can use O2, NO3-, Fe(III), and S0 as terminal electron acceptors for growth was isolated from groundwater sampled at a 3.2-km depth in a South African gold mine. This organism, designated SA-01, clustered most closely with members of the genus Thermus, as determined by 16S rRNA gene (rDNA) sequence analysis. The 16S rDNA sequence of SA-01 was >98% similar to that of Thermus strain NMX2 A.1, which was previously isolated by other investigators from a thermal spring in New Mexico. Strain NMX2 A.1 was also able to reduce Fe(III) and other electron acceptors. Neither SA-01 nor NMX2 A.1 grew fermentatively, i.e., addition of an external electron acceptor was required for anaerobic growth. Thermus strain SA-01 reduced soluble Fe(III) complexed with citrate or nitrilotriacetic acid (NTA); however, it could reduce only relatively small quantities (0.5 mM) of hydrous ferric oxide except when the humic acid analog 2,6-anthraquinone disulfonate was added as an electron shuttle, in which case 10 mM Fe(III) was reduced. Fe(III)-NTA was reduced quantitatively to Fe(II); reduction of Fe(III)-NTA was coupled to the oxidation of lactate and supported growth through three consecutive transfers. Suspensions of Thermus strain SA-01 cells also reduced Mn(IV), Co(III)-EDTA, Cr(VI), and U(VI). Mn(IV)-oxide was reduced in the presence of either lactate or H2. Both strains were also able to mineralize NTA to CO2 and to couple its oxidation to Fe(III) reduction and growth. The optimum temperature for growth and Fe(III) reduction by Thermus strains SA-01 and NMX2 A.1 is approximately 65 degrees C; their optimum pH is 6.5 to 7.0. This is the first report of a Thermus sp. being able to couple the oxidation of organic compounds to the reduction of Fe, Mn, or S.     

Sequence analysis and characterizations of two novel plasmids isolated from Thermus sp. 4C

Two novel plasmids, named pS4C and pL4C, were isolated from the thermophilic bacterium Thermus sp. 4C. The pS4C with a length of 5015bp and 58.25% of G+C content, contains 9 putative open reading frames (ORFs). The larger plasmid, pL4C, consisting of 21,248bp, has a G+C content of 68.60% and 34 putative ORFs. Both plasmids encode their own replication protein. The ORF 22 of pL4C and the ORF 4 of pS4C encode proteins with high sequence similarities to integrase (97%) and transposase (97%), respectively, which are both involved in DNA rearrangement and exchange. Furthermore, sequence analysis of pL4C also showed that several plasmid-encoded genes may be involved in DNA modification and repair, such as DNA G:T-mismatch repair endonuclease and micrococcal nuclease-like protein. These proteins may be involved in raising the repair efficiency and other minor editing needs. Interestingly, the elimination of plasmids significantly lowered the growth temperature of Thermus sp. 4C. Few reports dealing with the DNA repair enzymes on the plasmid from Thermus strains were published so far.     

Isolation and characterization of a mixotrophic sulfur-oxidizing Thermus scotoductus

Thermophilic, faculatatively mixotrophic sulfur-oxidizing bacteria were isolated from a sulfide-rich, neutral hot spring in Iceland. The strain, IT-7254, used thiosulfate and elemental sulfur as electron donors, oxygen and nitrate as electron acceptors, and acetate and other organic compounds as carbon sources. After a few days of growth in the presence of thiosulfate, this strain formed sulfur globules. Comparison of intracellular enzymes and heme proteins of heterotrophically and mixotrophically grown cells showed some differences. The new isolate belonged to Thermus scotoductus because the small subunit (SSU) rRNA gene sequence analysis showed 98.6% sequence similarity and 84% DNA:DNA reassociation to Thermus scotoductus NMX2 A. 1. It is also close to Thermus antranikianii HN3-7, with 98.3% and 79% SSU rRNA sequence similarity and DNA:DNA reassociation, respectively. It was also found that both Thermus NMX2 A.1 and T. antranikianii HN3-7 were able to oxidize thiosulfate but that the T. scotoductus type strain SE-1 was not. This is the first report of Thermus strains that are capable of mixotrophic growth with sulfur oxidation.