Heterologous expression and characterization of a novel subtilisin-like protease from a thermophilic Thermus thermophilus HB8.

Subtilisins are a family of serine proteases used widely throughout the detergent, leather and food industries, with the identification and development of new enzymes holding much potential value. Thermus thermophilus HB8 was examined for serine proteases and found TTHA0724 gene. Sequence analysis of this putative serine protease placed it within the subtilisin family. To obtain active T. thermophilus HB8 subtilisins, three genes encoding prepro-subtilisin, pro-subtilisin and mature-subtilisin were cloned and expressed in Escherichia coli Transetta (DE3). Although direct expression of the mature-subtilisin gene was found to produce inactive inclusion bodies, expression of the pro-subtilisin gene resulted in active mature-subtilisin, indicating that the pro-sequence of translated pro-subtilisin underwent autoproteolysis. The resulting mature-subtilisin exhibited maximal activity between 65 and 85 °C at pH 7.5. The mature-subtilisin showed good stability, maintaining 50% activity after 48 h at 75 °C and >78% activity across the pH range 5.0-9.5. Furthermore, the mature-subtilisin demonstrated broad substrate specificity, with no requirement for the presence of metal ions which are essential for other subtilisin enzymes. Despite this Cu2+ was able to increase enzyme activity, while Ca2+ partially inhibited the activity. These properties suggest that T. thermophilus HB8 mature-subtilisin has potential value in its application in many industries.

Co-utilization of glucose and xylose by evolved Thermus thermophilus LC113 strain elucidated by (13)C metabolic flux analysis and whole genome sequencing.

We evolved Thermus thermophilus to efficiently co-utilize glucose and xylose, the two most abundant sugars in lignocellulosic biomass, at high temperatures without carbon catabolite repression. To generate the strain, T. thermophilus HB8 was first evolved on glucose to improve its growth characteristics, followed by evolution on xylose. The resulting strain, T. thermophilus LC113, was characterized in growth studies, by whole genome sequencing, and (13)C-metabolic flux analysis ((13)C-MFA) with [1,6-(13)C]glucose, [5-(13)C]xylose, and [1,6-(13)C]glucose+[5-(13)C]xylose as isotopic tracers. Compared to the starting strain, the evolved strain had an increased growth rate (~2-fold), increased biomass yield, increased tolerance to high temperatures up to 90°C, and gained the ability to grow on xylose in minimal medium. At the optimal growth temperature of 81°C, the maximum growth rate on glucose and xylose was 0.44 and 0.46h(-1), respectively. In medium containing glucose and xylose the strain efficiently co-utilized the two sugars. (13)C-MFA results provided insights into the metabolism of T. thermophilus LC113 that allows efficient co-utilization of glucose and xylose. Specifically, (13)C-MFA revealed that metabolic fluxes in the upper part of metabolism adjust flexibly to sugar availability, while fluxes in the lower part of metabolism remain relatively constant. Whole genome sequence analysis revealed two large structural changes that can help explain the physiology of the evolved strain: a duplication of a chromosome region that contains many sugar transporters, and a 5x multiplication of a region on the pVV8 plasmid that contains xylose isomerase and xylulokinase genes, the first two enzymes of xylose catabolism. Taken together, (13)C-MFA and genome sequence analysis provided complementary insights into the physiology of the evolved strain.

The regulatory mechanism underlying light-inducible production of carotenoids in nonphototrophic bacteria.

Light is a ubiquitous environmental factor serving as an energy source and external stimulus. Here, I review the conserved molecular mechanism of light-inducible production of carotenoids in three nonphototrophic bacteria: Streptomyces coelicolor A3(2), Thermus thermophilus HB27, and Bacillus megaterium QM B1551. A MerR family transcriptional regulator, LitR, commonly plays a central role in their light-inducible carotenoid production. Genetic and biochemical studies on LitR proteins revealed a conserved function: LitR in complex with adenosyl B12 (AdoB12) has a light-sensitive DNA-binding activity and thus suppresses the expression of the Crt biosynthesis gene cluster. The in vitro DNA-binding and transcription assays showed that the LitR-AdoB12 complex serves as a repressor allowing transcription initiation by RNA polymerase in response to illumination. The existence of novel light-inducible genes and the unique role of the megaplasmid were revealed by the transcriptomic analysis of T. thermophilus. The findings suggest that LitR is a general regulator responsible for the light-inducible carotenoid production in the phylogenetically divergent nonphototrophic bacteria, and that LitR performs diverse physiological functions in bacteria.

Identification of signature proteins that are distinctive of the Deinococcus-Thermus phylum.

The members of the Deinococcus-Thermus phylum, which include many species that are resistant to extreme radiation, as well as several thermophiles, have been recognized solely on the basis of their branching patterns in 16S rRNA and other phylogenetic trees. No biochemical or physiological characteristic is currently known that is unique to this group of species. To identify genes/proteins that are exclusive of this group of species, systematic protein basic local alignment tool (Blastp) searches were carried out on each open reading frame (ORF) in the genome of Deinococcus radiodurans. These studies identified 65 proteins that were only found in all three sequenced Deinococcus-Thermus genomes (viz. D. radiodurans, D. geothermalis and Thermus thermophilus), but not in any other bacteria. In addition, these studies also identified 206 proteins that are exclusively found in the two Deinocococci species, and 399 proteins that are unique to D. radiodurans. The identified proteins, which represent a genetic repertoire distinctive to the Deinococcus-Thermus group, or to Deinococci species, provide novel molecular markers for their identification and characterization. The cellular functions of most of these proteins are not known and their studies should prove useful in identifying novel biochemical and physiological characteristics that are exclusive of these groups of bacteria and also those responsible for the extreme radiation resistance of Deinococci.

Expression and characterization of recombinant thermostable alkaline phosphatase from a novel thermophilic bacterium Thermus thermophilus XM.

A gene (tap) encoding a thermostable alkaline phosphatase from the thermophilic bacterium Thermus thermophilus XM was cloned and sequenced. It is 1506 bp long and encodes a protein of 501 amino acid residues with a calculated molecular mass of 54.7 kDa. Comparison of the deduced amino acid sequence with other alkaline phosphatases showed that the regions in the vicinity of the phosphorylation site and metal binding sites are highly conserved. The recombinant thermostable alkaline phosphatase was expressed as a His6-tagged fusion protein in Escherichia coli and its enzymatic properties were characterized after purification. The pH and temperature optima for the recombinant thermostable alkaline phosphatases activity were pH 12 and 75 degrees C. As expected, the enzyme displayed high thermostability, retaining more than 50% activity after incubating for 6 h at 80 degrees C. Its catalytic function was accelerated in the presence of 0.1 mM Co2+, Fe2+, Mg2+, or Mn2+ but was strongly inhibited by 2.0 mM Fe2+. Under optimal conditions, the Michaelis constant (K(m)) for cleavage of p-nitrophenyl-phosphate was 0.034 mM. Although it has much in common with other alkaline phosphatases, the recombinant thermostable alkaline phosphatase possesses some unique features, such as high optimal pH and good thermostability.

Identification of signature proteins that are distinctive of the Deinococcus-Thermus phylum

The members of the Deinococcus-Thermus phylum, which include many species that are resistant to extreme radiation, as well as several thermophiles, have been recognized solely on the basis of their branching patterns in 16S rRNA and other phylogenetic trees. No biochemical or physiological characteristic is currently known that is unique to this group of species. To identify genes/proteins that are exclusive of this group of species, systematic protein basic local alignment tool (Blastp) searches were carried out on each open reading frame (ORF) in the genome of Deinococcus radiodurans. These studies identified 65 proteins that were only found in all three sequenced Deinococcus-Thermus genomes (viz. D. radiodurans, D. geothermalis and Thermus thermophilus), but not in any other bacteria. In addition, these studies also identified 206 proteins that are exclusively found in the two Deinocococci species, and 399 proteins that are unique to D. radiodurans. The identified proteins, which represent a genetic repertoire distinctive to the Deinococcus-Thermus group, or to Deinococci species, provide novel molecular markers for their identification and characterization. The cellular functions of most of these proteins are not known and their studies should prove useful in identifying novel biochemical and physiological characteristics that are exclusive of these groups of bacteria and also those responsible for the extreme radiation resistance of Deinococci (AU)

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Estimation of phylogeny and invariant sites under the general Markov model of nucleotide sequence evolution.

The models of nucleotide substitution used by most maximum likelihood-based methods assume that the evolutionary process is stationary, reversible, and homogeneous. We present an extension of the Barry and Hartigan model, which can be used to estimate parameters by maximum likelihood (ML) when the data contain invariant sites and there are violations of the assumptions of stationarity, reversibility, and homogeneity. Unlike most ML methods for estimating invariant sites, we estimate the nucleotide composition of invariant sites separately from that of variable sites. We analyze a bacterial data set where problems due to lack of stationarity and homogeneity have been previously well noted and use the parametric bootstrap to show that the data are consistent with our general Markov model. We also show that estimates of invariant sites obtained using our method are fairly accurate when applied to data simulated under the general Markov model.

Polyphasic analysis of Thermus isolates from geothermal areas in Iceland.

Genetic relationships and diversity of 101 Thermus isolates from different geothermal regions in Iceland were investigated by using multilocus enzyme electrophoresis (MLEE) and small subunit ribosomal rRNA (SSU rRNA) sequence analysis. Ten polymorphic enzymes were used and seven distinct and genetically highly divergent lineages of Thermus were observed. Six of seven lineages could be assigned to species whose names have been validated. The most diverse lineage was Thermus scotoductus. In contrast to the other lineages, this lineage was divided into very distinct genetic sublineages that may represent subspecies with different habitat preferences. The least diverse lineage was Thermus brockianus. Phenotypic and physiological analysis was carried out on a subset of the isolates. No relationship was found between growth on specific single carbon source to the grouping obtained by the isoenzyme analysis. The response to various salts was distinguishing in a few cases. No relationship was found between temperature at the isolation site and the different lineages, but pH indicated a relation to specific lineages.

Crystal structure of elongation factor P from Thermus thermophilus HB8.

Translation elongation factor P (EF-P) stimulates ribosomal peptidyltransferase activity. EF-P is conserved in bacteria and is essential for cell viability. Eukarya and Archaea have an EF-P homologue, eukaryotic initiation factor 5A (eIF-5A). In the present study, we determined the crystal structure of EF-P from Thermus thermophilus HB8 at a 1.65-A resolution. EF-P consists of three beta-barrel domains (I, II, and III), whereas eIF-5A has only two domains (N and C domains). Domain I of EF-P is topologically the same as the N domain of eIF-5A. On the other hand, EF-P domains II and III share the same topology as that of the eIF-5A C domain, indicating that domains II and III arose by duplication. Intriguingly, the N-terminal half of domain II and the C-terminal half of domain III of EF-P have sequence homologies to the N- and C-terminal halves, respectively, of the eIF-5A C domain. The three domains of EF-P are arranged in an "L" shape, with 65- and 53-A-long arms at an angle of 95 degrees, which is reminiscent of tRNA. Furthermore, most of the EF-P protein surface is negatively charged. Therefore, EF-P mimics the tRNA shape but uses domain topologies different from those of the known tRNA-mimicry translation factors. Domain I of EF-P has a conserved positive charge at its tip, like the eIF-5A N domain.

Isolation of a new thermohalophilic Thermus thermophilus strain from hot spring, able to grow on a renewable source of polysaccharide.

A thermohalophilic strain, Samu-Sal, isolated from hot springs of the Mount Grillo (Baia, Naples, Italy) at a depth of 60 m, according to its genotypic analyses is related to Thermus genus and should be classified as a new strain of Thermus thermophilus. Strain Samu-SA1 grew using, as sole carbon source, a polysaccharide extracted from waste industrial tomato process with a yield of 3.5 g l(-1). Strain Samu-SA1 synthesized several alpha- and beta-glycosidases.

Isolation and characterization of highly thermophilic xylanolytic Thermus thermophilus strains from hot composts.

This is the first detailed report of xylanolytic activity in Thermus strains. Two highly thermophilic xylanolytic bacteria, very closely related to non-xylanolytic T. thermophilus strains, have been isolated from the hottest zones of compost piles. Strain X6 was investigated in more detail. The growth rate (optical density monitoring) on xylan was 0.404.h-1 at 75 degrees C. Maximal growth temperature was 81 degrees C. Xylanase activity was mainly cell-bound, but was solubilized into the medium by sonication. It was induced by xylan or xylose in the culture medium. The temperature and pH optima of the xylanases were determined to be around 100 degrees C and pH 6, respectively. Xylanase activity was fairly thermostable; only 39% of activity was lost after an incubation period of 48 h at 90 degrees C in the absence of substrate. Xylanolytic T. thermophilus strains could contribute to the degradation of hemicellulose during the thermogenic phase of industrial composting.

Isolation and characterization of Thermus thermophilus Gy1211 from a deep-sea hydrothermal vent.

We examined a single, non-spore-forming, aerobic, thermophilic strain that was isolated from a deep-sea hydrothermal vent in the Guaymas Basin at a depth of 2000 m and initially placed in a phenetic group with Thermus scotoductus (X-1). We identified this deep-sea isolate as a new strain belonging to Thermus thermophilus using several parameters. DNA-DNA hybridization under stringent conditions showed 74% similarity between the deep-sea isolate and T. thermophilus HB-8T (T = type strain). Phenotypic characteristics, such as the utilization of carbon sources, hydrolysis of different compounds, and antibiotic sensitivity were identical in the two strains. The polar lipids composition showed that strain Gy1211 belonged to the genus Thermus. The fatty acids composition indicated that this strain was related to the marine T. thermophilus strain isolated from the Azores. The new isolate T. thermophilus strain Gy1211 grew optimally at 75 degrees C, pH 8.0; and 2% NaCl. A hydrostatic pressure of 20 MPa, similar to the in situ hydrostatic pressure of the deep-sea vent from which the strain was isolated, had no effect on growth. Strain HB-8T, however, showed slower growth under these conditions.

Cloning and sequence analysis of the structural gene for the bc1-type Rieske iron-sulfur protein from Thermus thermophilus HB8.

The structural gene encoding the Rieske iron-sulfur protein from Thermus thermophilus HB8 has been cloned and sequenced. The gene encodes a protein of 209 amino acids that begins with a hydrophilic N-terminus followed by a stretch of 21 hydrophobic amino acids that could serve as a transmembrane helix. The remainder of the protein has a hydrophobicity pattern typical of a water-soluble protein. A phylogenetic analysis of 26 Rieske proteins that are part of bc1 or b6f complexes shows that they fall into three major groups: eubacterial and mitochondrial, cyanobacterial and plastid, and five highly divergent outliers, including that of Thermus. Although the overall homology with other Rieske proteins is very low, the C-terminal half of the Thermus protein contains the signature sequence CTHLGC-(13X)-CPCH that most likely provides the ligands of the [2Fe-2S] cluster. It is proposed that this region of the protein represents a small domain that folds independently and that the encoding DNA sequence may have been transferred during evolution to several unrelated genes to provide the cluster attachment site to proteins of different origin. The role of individual residues in this domain of the Thermus protein is discussed vis-a-vis the three-dimensional structure of the bovine protein (Iwata et al., 1996 Structure 4, 567-579).

Mapping of 61 genes on the refined physical map of the chromosome of Thermus thermophilus HB27 and comparison of genome organization with that of T. thermophilus HB8.

We have constructed refined physical maps of the chromosome (1 center dot 82 Mb) and the large plasmid pTT27 (250 kb) of Thermus thermophilus HB27. A total of 49 cleavage sites with five restriction enzymes, EcoRI, SspI, MunI, EcoRV and ClaI, were determined on the maps. The location of 61 genes was determined by using as probes 64 genes cloned from T. thermophilus or other Thermus strains. Comparison of the genomic organization of the chromosomes of T. thermophilus HB27 and HB8 revealed that they were basically identical, but some genes were located in different regions. Among 32 genes whose locations were determined on both the HB27 and the HB8 chromosomes, the copy number of rpsL-rpsG-fus-tufA, the locations of glyS, pol, and one copy of nusG-rplK-rplA were different. The IS1000 sequence was located only in one region on the HB27 chromosome. In contrast, IS1000 sequences were scattered over four regions on the chromosome of HB8. As each region in which glyS, pol, or one copy of nusG-rplK-rplA are present also contained IS1000 in HB8, it is suggested that IS1000 may play an important role in genomic rearrangements in Thermus strains.

DNA relatedness of Thermus strains, description of Thermus brockianus sp. nov., and proposal to reestablish Thermus thermophilus (Oshima and Imahori)

Aerobic, thermophilic, gram-negative bacteria obtained from Yellowstone National Park that were placed in the genus Thermus on the basis of phenotypic data were examined by chemotaxonomic techniques to determine their peptidoglycan compositions, their respiratory quinones, their mean DNA base compositions, and their levels of DNA-DNA homology as determined by both the filter hybridization and reassociation rate methods. These isolates from hot springs included Thermus aquaticus strains and strains of a new genospecies. We propose the name Thermus brockianus for this new genospecies; strain YS38 is the type strain of this taxon. A collection of 10 strains, including the type strain of "Thermus thermophilus", which were isolated from widely separated geothermal sites, exhibited high levels of DNA-DNA homology with each other and had similar physiological properties. Therefore, we propose that the species Thermus thermophilus (Oshima and Imahori) should be reestablished, with strain HB8 as the type strain.