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.     

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.     

Pyrimidine biosynthesis genes (pyrE and pyrF) of an extreme thermophile, Thermus thermophilus.

We have isolated uracil auxotrophic mutants of an extreme thermophile, Thermus thermophilus. A part of the pyrimidine biosynthetic operon including genes for orotate phosphoribosyltransferase (pyrE) and for orotidine-5'-monophosphate decarboxylase (pyrF) was cloned and sequenced. The pyrE gene can be a bidirectional marker for the gene manipulation system of the thermophile.     

Thermoadaptation of alpha-galactosidase AgaB1 in Thermus thermophilus

The evolutionary potential of a thermostable alpha-galactosidase, with regard to improved catalytic activity at high temperatures, was investigated by employing an in vivo selection system based on thermophilic bacteria. For this purpose, hybrid alpha-galactosidase genes of agaA and agaB from Bacillus stearothermophilus KVE39, designated agaA1 and agaB1, were cloned into an autonomously replicating Thermus vector and introduced into Thermus thermophilus OF1053GD (DeltaagaT) by transformation. This selector strain is unable to metabolize melibiose (alpha-galactoside) without recombinant alpha-galactosidases, because the native alpha-galactosidase gene, agaT, has been deleted. Growth conditions were established under which the strain was able to utilize melibiose as a single carbohydrate source when harboring a plasmid-encoded agaA1 gene but unable when harboring a plasmid-encoded agaB1 gene. With incubation of the agaB1 plasmid-harboring strain under selective pressure at a restrictive temperature (67 degrees C) in a minimal melibiose medium, spontaneous mutants as well as N-methyl-N'-nitro-N-nitrosoguanidine-induced mutants able to grow on the selective medium were isolated. The mutant alpha-galactosidase genes were amplified by PCR, cloned in Escherichia coli, and sequenced. A single-base substitution that replaces glutamic acid residue 355 with glycine or valine was found in the mutant agaB1 genes. The mutant enzymes displayed the optimum hydrolyzing activity at higher temperatures together with improved catalytic capacity compared to the wild-type enzyme and furthermore showed an enhanced thermal stability. To our knowledge, this is the first report of an in vivo evolution of glycoside-hydrolyzing enzyme and selection within a thermophilic host cell.     

Phylogenetic analysis of several Thermus strains from Rehai of Tengchong, Yunnan, China

Several Thermus strains were isolated from 10 hot springs of the Rehai geothermal area in Tengchong, Yunnan province. The diversity of Thermus strains was examined by sequencing the 16S rRNA genes and comparing their sequences. Phylogenetic analysis showed that the 16S rDNA sequences from the Rehai geothermal isolates form four branches in the phylogenetic tree and had greater than 95.9% similarity in the phylogroup. Secondary structure comparison also indicated that the 16S rRNA from the Rehai geothermal isolates have unique secondary structure characteristics in helix 6, helix 9, and helix 10 (reference to Escherichia coli). This research is the first attempt to reveal the diversity of Thermus strains that are distributed in the Rehai geothermal area.     

Ribotyping of 16S and 23S rRNA genes and organization of rrn operons in members of the bacterial genera Gemmata,Planctomyces, Thermotoga,Thermus, and Verrucomicrobium

The number of organization of rrn genes of two members of the order Planctomycetales, Planctomyces limnophilus and Gemmata obscuriglobus, as well as three species from other bacterial phyla, namely Thermotoga maritima, Thermus aquaticus and Verrucomicrobium spinosum were examined by Southern blot hybridization analysis of restricted DNA with labeled 16S- and 23S rRNAs. Ribotyping analysis revealed that two species contain unlinked 16S- and 23S rRNA genes. Planctomyces limnophilus possessed two unlinked rrn genes which were separated from each other by at least 4.3 kb, and Thermus aquaticus had to unlinked 16S and 23S rRNA genes, separated from each other by at least 2.5 kb. Gemmata obscuriglobus exhibited five genes for which the organization could as yet not be determined because of the complex hybridization patterns. In the other two species, rrn genes clustered in operons. Thermotoga maritima had a single gene for each rRNA species which were separated by not more than 1.5 kb, while Verrucomicrobium spinosum had four copies of probably linked 16S and 23S rRNA genes with a maximal distance between 16S and 23S rRNA genes of 1.3 kb.     

Ribosomal protein S1 from Thermus thermophilus: its detection,identification and overproduction

Ribosomal protein S1 has been identified in Thermus thermophilus ribosomes. The gene of ribosomal protein S1 from Thermus thermophilus has been cloned and overexpressed in Escherichia coli. A procedure for purification of the protein has been developed.     

A plasmid vector for an extreme thermophile, Thermus thermophilus

The host-vector system for an extreme thermophile, Thermus thermophilus HB27, was developed. The host strain has a mutation in tryptophan synthetase gene (trpB), and the mutation was determined to be a missense mutation by DNA sequence analysis. A Thermus-E. coli shuttle vector pYK109 was constructed. pYK109 consists of Thermus cryptic plasmid pTT8, tryptophan synthetase gene (trpB) of Thermus T2 and E. coli plasmid vector pUC13. pYK109 transformed T. thermophilus HB27 trpB5 to Trp+ at a frequency of 10(6) transformants per microgram DNA.     

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.     

Sequence and identification of the nucleotide binding site for the elongation factor Tu from Thermus thermophilus HB8.

Two structural genes for the Thermus thermophilus elongation factor Tu (tuf) were identified by cross-hybridization with the tufA gene from E. coli. The sequence of one of these tuf genes, localized on a 6.6 kb Bam HI fragment, was determined and confirmed by partial protein sequencing of an authentic elongation factor Tu from T. thermophilus HB8. Expression of this tuf gene in E. coli minicells provided a low amount of immuno-precipitable thermophilic EF-Tu. Affinity labeling of the T. thermophilus EF-Tu and sequence comparison with homologous proteins from other organisms were used to identify the guanosine-nucleotide binding domain.     

The genome sequence of the extreme thermophile Thermus thermophilus

Thermus thermophilus HB27 is an extremely thermophilic, halotolerant bacterium, which was originally isolated from a natural thermal environment in Japan. This organism has considerable biotechnological potential; many thermostable proteins isolated from members of the genus Thermus are indispensable in research and in industrial applications. We present here the complete genome sequence of T. thermophilus HB27, the first for the genus Thermus. The genome consists of a 1,894,877 base pair chromosome and a 232,605 base pair megaplasmid, designated pTT27. The 2,218 identified putative genes were compared to those of the closest relative sequenced so far, the mesophilic bacterium Deinococcus radiodurans. Both organisms share a similar set of proteins, although their genomes lack extensive synteny. Many new genes of potential interest for biotechnological applications were found in T. thermophilus HB27. Candidates include various proteases and key enzymes of other fundamental biological processes such as DNA replication, DNA repair and RNA maturation.     

Thermus thermophilus bacteriophage phiYS40 genome and proteomic characterization of virions

We determined the sequence of the 152,372 bp genome of phiYS40, a lytic tailed bacteriophage of Thermus thermophilus. The genome contains 170 putative open reading frames and three tRNA genes. Functions for 25% of phiYS40 gene products were predicted on the basis of similarity to proteins of known function from diverse phages and bacteria. phiYS40 encodes a cluster of proteins involved in nucleotide salvage, such as flavin-dependent thymidylate synthase, thymidylate kinase, ribonucleotide reductase, and deoxycytidylate deaminase, and in DNA replication, such as DNA primase, helicase, type A DNA polymerase, and predicted terminal protein involved in initiation of DNA synthesis. The structural genes of phiYS40, most of which have no similarity to sequences in public databases, were identified by mass spectrometric analysis of purified virions. Various phiYS40 proteins have different phylogenetic neighbors, including myovirus, podovirus, and siphovirus gene products, bacterial genes and, in one case, a dUTPase from a eukaryotic virus. phiYS40 has apparently arisen through multiple acts of recombination between different phage genomes as well as through acquisition of bacterial genes.     

Comparative analysis of ribosomal protein L5 sequences from bacteria of the genus Thermus.

The genes for the ribosomal 5S rRNA binding protein L5 have been cloned from three extremely thermophilic eubacteria, Thermus flavus, Thermus thermophilus HB8 and Thermus aquaticus (Jahn et al, submitted). Genes for protein L5 from the three Thermus strains display 95% G/C in third positions of codons. Amino acid sequences deduced from the DNA sequence were shown to be identical for T flavus and T thermophilus, although the corresponding DNA sequences differed by two T to C transitions in the T thermophilus gene. Protein L5 sequences from T flavus and T thermophilus are 95% homologous to L5 from T aquaticus and 56.5% homologous to the corresponding E coli sequence. The lowest degrees of homology were found between the T flavus/T thermophilus L5 proteins and those of yeast L16 (27.5%), Halobacterium marismortui (34.0%) and Methanococcus vannielii (36.6%). From sequence comparison it becomes clear that thermostability of Thermus L5 proteins is achieved by an increase in hydrophobic interactions and/or by restriction of steric flexibility due to the introduction of amino acids with branched aliphatic side chains such as leucine. Alignment of the nine protein sequences equivalent to Thermus L5 proteins led to identification of a conserved internal segment, rich in acidic amino acids, which shows homology to subsequences of E coli L18 and L25. The occurrence of conserved sequence elements in 5S rRNA binding proteins and ribosomal proteins in general is discussed in terms of evolution and function.     

Genomic restriction map of the extremely thermophilic bacterium Thermus thermophilus HB8.

A physical map of the chromosome of the extremely thermophilic eubacterium Thermus thermophilus HB8 has been constructed by using pulsed-field gel electrophoresis techniques. A total of 26 cleavage sites for the rarely cutting restriction endonucleases HpaI, MunI, and NdeI were located on the genome. On the basis of the sizes of the restriction fragments generated, the genome size was estimated to be 1.74 Mbp, which is significantly smaller than the chromosomes of Escherichia coli and other mesophiles. Partial digestion experiments revealed the order of the six HpaI bands on the chromosome. Hybridization of isolated restriction fragments to pulsed-field gel-separated restriction digestions confirmed the deduced order of the HpaI fragments and allowed ordering and alignment of the NdeI and MunI fragments. In addition, 16 genes or gene clusters cloned from several different Thermus strains were located on the T. thermophilus HB8 chromosomal map by hybridization of gene probes to pulsed-field gel-resolved restriction digestions.     

Disruption of Thermus thermophilus genes by homologous recombination using a thermostable kanamycin-resistant marker

Genes of an extremely thermophilic bacterium, Thermus thermophilus, were disrupted by homologous recombination using a recently developed, thermostable kanamycin-resistant marker. First, the trpE gene was disrupted with various constructions of DNA. The transformation efficiency was exponentially increased as the length of the homologous regions flanking the marker gene increased above the minimum length (200-300 bp). We then disrupted five genes of the nucleotide excision repair system and examined their phenotypes. The convenience and high reliability of this method should prompt its application to the high-throughput systematic disruption of the genes of this thermophilic bacterium.     

Development of a gene expression vector for Thermus thermophilus based on the promoter of the respiratory nitrate reductase

A specific expression system for Thermus spp. is described. Plasmid pMKE1 contains replicative origins for Escherichia coli and Thermus spp., a selection gene encoding a thermostable resistance to kanamycin, and a 720 bp DNA region containing the promoter (Pnar), and the regulatory sequences of the respiratory nitrate reductase operon of Thermus thermophilus HB8. Two genes, encoding a thermophilic beta-galactosidase and an alkaline phosphatase were cloned in pMKE1 as cytoplasmic and periplasmic reporters, respectively. The expression of the reporters was specifically induced by the combined action of nitrate and anoxia in facultative anaerobic derivatives of T. thermophilus HB27 to which the gene cluster for nitrate respiration was transferred by conjugation. Overexpressions in the range of approximately 200-fold were obtained for the cytoplasmic reporter, whereas that of the periplasmic reporter was limited to approximately 20-fold, with respect to their intrinsic respective activities.     

Intra- and extracellular beta-galactosidases from Bifidobacterium bifidum and B. infantis: molecular cloning, heterologous expression, and comparative characterization

Three beta-galactosidase genes from Bifidobacterium bifidum DSM20215 and one beta-galactosidase gene from Bifidobacterium infantis DSM20088 were isolated and characterized. The three B. bifidum beta-galactosidases exhibited a low degree of amino acid sequence similarity to each other and to previously published beta-galactosidases classified as family 2 glycosyl hydrolases. Likewise, the B. infantis beta-galactosidase was distantly related to enzymes classified as family 42 glycosyl hydrolases. One of the enzymes from B. bifidum, termed BIF3, is most probably an extracellular enzyme, since it contained a signal sequence which was cleaved off during heterologous expression of the enzyme in Escherichia coli. Other exceptional features of the BIF3 beta-galactosidase were (i) the monomeric structure of the active enzyme, comprising 1,752 amino acid residues (188 kDa) and (ii) the molecular organization into an N-terminal beta-galactosidase domain and a C-terminal galactose binding domain. The other two B. bifidum beta-galactosidases and the enzyme from B. infantis were multimeric, intracellular enzymes with molecular masses similar to typical family 2 and family 42 glycosyl hydrolases, respectively. Despite the differences in size, molecular composition, and amino acid sequence, all four beta-galactosidases were highly specific for hydrolysis of beta-D-galactosidic linkages, and all four enzymes were able to transgalactosylate with lactose as a substrate.     

Xylose (glucose) isomerase gene from the thermophile Thermus thermophilus: cloning, sequencing, and comparison with other thermostable xylose isomerases.

The xylose isomerase gene from the thermophile Thermus thermophilus was cloned by using a fragment of the Streptomyces griseofuscus gene as a probe. The complete nucleotide sequence of the gene was determined. T. thermophilus is the most thermophilic organism from which a xylose isomerase gene has been cloned and characterized. The gene codes for a polypeptide of 387 amino acids with a molecular weight of 44,000. The Thermus xylose isomerase is considerably more thermostable than other described xylose isomerases. Production of the enzyme in Escherichia coli, by using the tac promoter, increases the xylose isomerase yield 45-fold compared with production in T. thermophilus. Moreover, the enzyme from E. coli can be purified 20-fold by simply heating the cell extract at 85 degrees C for 10 min. The characteristics of the enzyme made in E. coli are the same as those of enzyme made in T. thermophilus. Comparison of the Thermus xylose isomerase amino acid sequence with xylose isomerase sequences from other organisms showed that amino acids involved in substrate binding and isomerization are well conserved. Analysis of amino acid substitutions that distinguish the Thermus xylose isomerase from other thermostable xylose isomerases suggests that the further increase in thermostability in T. thermophilus is due to substitution of amino acids which react during irreversible inactivation and results also from increased hydrophobicity.