Recombination-Deficient Mutants of an Extreme Thermophile, Thermus thermophilus

Recombination-deficient strains of the extreme thermophile Thermus thermophilus have been prepared from a leucine-isoleucine mutant strain (NM6). The availability of such recombination-deficient thermophilic bacterial strains may provide especially good hosts for work with plasmid vectors.     

Physical characterization of a plasmid (pTT1) isolated from Thermus thermophilus

A small circular supercoiled DNA molecule species with a molecular weight of about 5.4 × 10⁶ has been isolated from the extreme thermophile Thermus thermophilus HB8. This plasmid (pTT1) has a G plus C content of 68%, similar to that of the host chromosome. The superhelix density is the same as that of bacteriophage PM2 DNA. A physical map of the plasmid has been obtained using restriction endonucleases.     

Enigmas of biosyntheses of unusual polyamines in an extreme thermophile,Thermus thermophilus

Thermus thermophilus, an extreme thermophile belonging to Domain Bacteria, produces unusual polyamines in addition to standard polyamines. To understand mechanisms of changes of polyamine compositions of the thermophile upon change of growth conditions such as environmental temperature, metabolic pathways of polyamine biosyntheses of T. thermophilus have been studied and a new polyamine metabolic pathway was proposed. However, many enigmas remain to be solved in future studies. In this paper, biosyntheses of two non-standard polyamines, thermospermine and sym-homospermidine which are also produced and play important roles in plant cells, of the extreme thermophile are discussed in relation to the biosynthetic reactions in plants.     

Cloning and expression of the leucine gene from Thermus thermophilus in Escherichia coli

A pBR322-T.leu hybrid plasmid was constructed which contains a 3.75 Md HindIII-fragment derived from Thermus thermophilus HB27 chromosomal DNA. In the Escherichia coli host, this plasmid coded for the β-IPM dehy drogenase (product of leuB) activity, the optimal temperature of which was 80°C, suggesting that information on the thermostability of the enzyme lies in its structural gene. 10-day propagation of E. coli [pBR322-T.leu] at 37°C decreased the temperature optimum from 80°C to 75°C. This change, which was found to depend on the plasmid but not on the host cells, might be due to selection of some mutation at the non-restrictive temperature of 37°C. Our results suggest that the 3.75 Md HindIII-fragment of pBR322-T.leu carries a promoter of the thermophile, which could function in E. coli.     

Gene expression of Bacillus subtilis subtilisin E in Thermus thermophilus

The Bacillus subtilis subtilisin E gene was cloned into an expression vector of the extreme thermophile, Thermus thermophilus. Active subtilisin E was produced in E. coli, indicating that the Thermus promoter functions in E. coli. When the plasmid was further introduced into T. thermophilus, the subtilisin E gene was expressed and the gene product accumulated as an inactive pro-form, because the autoprocessing of the wild-type enzyme to the active-form did not occur at 50°C or above. Received 17 March 1999/ Accepted in revised form 28 June 1999     

A two-host fosmid system for functional screening of (meta)genomic libraries from extreme thermophiles

A new cloning system is described, which allows the construction of large-insert fosmid libraries in Escherichia coli and the transfer of the recombinant libraries to the extreme thermophile Thermus thermophilus via natural transformation. Libraries are established in the thermophilic host by site-specific chromosomal insertion of the recombinant fosmids via single crossover or double crossover recombination at the T. thermophilus pyr locus. Comparative screening of a fosmid library constructed from genomic DNA from the thermophilic spirochaete, Spirochaeta thermophila, for clones expressing thermoactive xylanase activity revealed that 50% of the fosmids that conferred xylanase activity upon the corresponding T. thermophilus transformants did not give rise to xylanase-positive E. coli clones, indicating that significantly more S. thermophila genes are functionally expressed in T. thermophilus than in E. coli. The novel T. thermophilus host/vector system may be of value for the construction and functional screening of recombinant DNA libraries from individual thermophilic or extremely thermophilic organisms as well as from complex metagenomes isolated from thermophilic microbial communities.     

Purification,catalytic properties and thermostability of 3-isopropylmalate dehydrogenase from Escherichia coli

3-isopropylmalate dehydrogenase (IPMDH) from Escherichia coli was overexpressed, purified and crystallized. The enzyme was characterized and compared to its thermophilic counterpart from Thermus thermophilus strain HB8. As in the thermophile enzyme, the activity of E. coli IPMDH was dependent on the divalent cations, Mg²⁺ or Mn²⁺, with Mn²⁺ being the preferred cation. Activity was also strongly influenced by KCl: 0.3 M were necessary for the optimal activity. At 40°C the K_m of E. coli IPMDH was 105 μM for IPM and 321 μM for NAD, the k_cat was 69 s⁻¹. The half denaturationn temperature was 64°C, which was 20°C lower than that of the thermophile enzyme.     

Production and Extracellular Secretion of Aqualysin I (a Thermophilic Subtilisin-Type Protease) in a Host-Vector System for Thermus thermophilus

Aqualysin I is synthesized as a large precursor, processed, and secreted into the culture medium by Thermus aquaticus YT-1. An expression plasmid for the aqualysin I gene in T. thermophilus HB27 was constructed. T. thermophilus cells harboring the recombinant plasmid produced correctly processed aqualysin I, and the mature enzyme was secreted into the culture medium.     

The organization of the leuC,leuD and leuB genes of the extreme thermophile Thermus thermophilus

3-Isopropylmalate dehydrogenase is encoded by leuB gene while leuC and leuB genes encode the large and small subunits of isopropylmalate isomerase in leucine biosynthetic pathway, respectively. Organization of the leuB, leuC and leuD genes of an extreme thermophile, Thermus thermophilus, was investigated by sequence analysis. Location of the genes was also tested by complementation analysis of leu deficiency of the thermophile and Escherichia coli. The order was the leuC, leuD, and leuB genes and, in contrast to a previous report, they did not overlap with each other. Sequence analysis of the leuC and leuD genes suggested that cysteine residues for iron–sulfur binding and other amino acid residues involved in isomerase activity, which have been inferred from analysis of a related protein, aconitase, were highly conserved.     

Pilin-Like Proteins in the Extremely Thermophilic Bacterium Thermus thermophilus HB27: Implication in Competence for Natural Transformation and Links to Type IV Pilus Biogenesis

The extreme thermophile Thermus thermophilus HB27 exhibits high frequencies of natural transformation. Although we recently reported identification of the first competence genes in Thermus, the molecular basis of DNA uptake is unknown. A pilus-like structure is assumed to be involved. Twelve genes encoding prepilin-like proteins were identified in three loci in the genome of T. thermophilus. Mutational analyses, described in this paper, revealed that one locus, which contains four genes that encode prepilin-like proteins (pilA1 to pilA4), is essential for natural transformation. Additionally, comZ, a new competence gene with no similarity to known genes, was identified. Analysis of the piliation phenotype revealed wild-type piliation of a pilA1-pilA3Δkat mutant and a comZ mutant, whereas a pilA4 mutant was found to be completely devoid of pilus structures. These findings, together with the significant similarity of PilA4 to prepilins, led to the conclusion that the T. thermophilus pilus structures are type IV pili. Furthermore, the loss of the transformation and piliation phenotype in the pilA4 mutant suggests that type IV pili are implicated in natural transformation of T. thermophilus HB27.     

Assessment of the tolerance of Lactococcus lactis cells at elevated temperatures

When Lactococcus lactis strains were exposed directly to the lethal temperature of 50 C for 30 ;min, 0.1–31% of the cells survived. However, when pre-exposed to 40 °C, prior to exposure at 50 °C, 4–61% of the cells survived. A plasmid carrying a unique heat shock gene from the thermophile Streptococcus thermophilus was cloned into L. ;lactis. When the transformed cells were cultivated at 30 °C the introduction of the plasmid had no obvious effect on the growth of L. ;lactis. However, when the temperature was abruptly shifted from 30 °C to 42 °C at mid-growth phase the growth decreased by 50%.     

Reversible thermal unfolding of thermostable phosphoglycerate kinase. Thermostability associated with mean zero enthalpy change.

Reversible thermal denaturation of phosphoglycerate kinases (E.C. 2.7.2.3) from an extremely thermophilic bacterium Thermus thermophilus and from yeast were studied by measuring their circular dichroism and fluorescence intensity. The thermal denaturation in the presence of guanidine hydrochloride was completely reversible. The thermodynamic parameters for the reaction were calculated based on a two-state mechanism. The free energy changes in denaturation at 25 °C in the absence of denaturant were estimated to be 11.87 ± 0.21 kcal/mol for T. thermophilus phosphoglycerate kinase and 5.33 ± 0.13 kcal/mol for that of yeast. It was found that the van't Hoff plot of the equilibrium constant for the denaturation reaction was almost independent of temperature in the temperature range 0 to 60 °C for T. thermophilus phosphoglycerate kinase, while that of yeast phosphoglycerate kinase was strongly temperature-dependent as reported for other thermolabile proteins. The enthalpy change in denaturation varies from 0.03 to 6.2 kcal/mol (0 to 60 °C) for T. thermophilus phosphoglycerate kinase and from ?27 to 31 kcal/mol (10 to 35 °C) for yeast enzyme. The entropy change in denaturation varies from ?3.9 to 21 entropy units for T. thermophilus phosphoglycerate kinase and ?96 to 104 entropys unit (10 to 35 °C) for yeast enzyme. The heat capacity change in denaturation is between 1.4 and 63 cal/deg. mol for the thermophile enzyme and between 1530 and 1750 cal/deg. mol for yeast enzyme at 20 °C. The observations that the enthalpy changes as well as the heat capacity changes in denaturation of the thermophilic enzyme were negligibly small suggest an explanation for the unusual stability to heat of T. thermophilus phosphoglycerate kinase.We also propose three possible mechanisms for the thermostability of proteins in general.     

Studies on Isoleucyl-tRNA Synthetase of an Extreme Thermophile,Thermus thermophilus HB8

Isoleucyl-tRNA synthetase (IRS) was partially purified from an extreme thermophile, T. thermophilus HB 8. The molecular weight (11.5 ×10⁴) and some kinetic constants were obtained and compared with IRS from other sources.

The present IRS catalyzed both isoleucine dependent and valine dependent ATP-PPi exchange reactions (optimum at around 80°C) but not valyl-tRNA formation. The optimum temperature for isoleucyl-tRNA formation was 62°C with E. coli tRNA and 75°C with T. thermophilus tRNA.

The enzyme showed a remarkable thermostability. The addition of E. coli or T. thermophilus tRNA enhanced the thermostability of the enzyme, which was shown to be fully active up to 77°C. When E. coli tRNA was used, the loading activity decreased in parallel to the unfolding of the substrate tRNA molecule. From these results the relation is discussed between tRNA conformation and function.     

Toxicity of indoxyl derivative accumulation in bacteria and its use as a new counterselection principle

In this work we describe the conditional toxic effect of the expression of enzymes that cleave 5-bromo-4-chloro-3-indolyl (BCI) substrates and its use as a new counterselection principle useful for the generation of clean and unmarked mutations in the genomes of bacteria. The application of this principle was demonstrated in the thermophile Thermus thermophilus HB27 and in a mesophile for which currently no counterselection markers are available, Micrococcus luteus ATCC 27141. For T. thermophilus, the indigogenic substrate BCI-β-glucoside was used in combination with the T. thermophilus β-glucosidase gene (bgl). For M. luteus, a combination of BCI-β-galactoside and the E. coli lacZ gene was implemented. We observed a strong growth-inhibiting effect when the strains were grown on agar plates containing the appropriate BCI substrates, the inhibition being proportional to the substrate concentration and the level of bgl/lacZ expression. The growth inhibition apparently depends on intracellular BCI substrate cleavage and accumulation of toxic indoxyl precipitates. The bgl and lacZ genes were used as counterselection markers for the rapid generation of scar-less chromosomal deletions in T. thermophilus HB27 (both in a Δbgl and in a wild type background) and in M. luteus ATCC 27141.     

Overproduction of carotenoids in Thermus thermophilus

Phytoene synthase encoded by the crtB gene is one of the rate-limiting enzymes for carotenoid production in Thermus thermophilus. We introduced a multicopy recombinant plasmid, pCOP1, in which the Thermus crtB gene was cloned, into carotenoid overproducing mutants of T. thermophilus. The overproducing mutants carrying a pCOP1 produced about twenty times as much carotenoids as the parental strain did.     

Sequencing and analysis of the Thermus thermophilus ribosomal protein gene cluster equivalent to the spectinomycin operon

To assess the organization of the Thermus thermophilus ribosomal protein genes, a fragment of DNA containing the complete S10 region and ten ribosomal protein genes of the spc region was cloned, using an oligonucleotide coding for the N-terminal amino acid (aa) sequence of T. thermophilus S8 protein as hybridization probe. The nucleotide sequence of a 4290 bp region between the rps17 and rpl15 genes was determined. Comparative analysis of this gene cluster showed that the gene arrangement (S17, L14, L24, L5, S14, S8, L6, L18, S5, L30 and L15) is identical to that of eubacteria. However, T. thermophilus ribosomal protein genes corresponding to the Escherichia coli S10 and spc operons are not resolved into two clusters: the stop codon of the rps17 gene (the last gene of the S10 operon in E. coli) and the start codon of the rpl14 gene (the first gene of the spc operon in E. coli) overlap. Most genes, except the rps14-rps8 intergenic spacer (69 bp), are separated by very short (only 3–7 bp) spacer regions or partially overlapped. The deduced aa sequences of T. thermophilus proteins share about 51–100% identities with the sequences of homologous proteins from thermophile Thermus aquaticus and Thermotoga maritima and 27–70% identities with the sequences of their mesophile counterparts.     

Conjugative plasmid transfer from <Emphasis Type="Italic">Escherichia coli</Emphasis> is a versatile approach for genetic transformation of thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> and <Emphasis Type="Italic">Geobacillus</Emphasis> species

We previously demonstrated efficient transformation of the thermophile Geobacillus kaustophilus HTA426 using conjugative plasmid transfer from Escherichia coli BR408. To evaluate the versatility of this approach to thermophile transformation, this study examined genetic transformation of various thermophilic Bacillus and Geobacillus spp. using conjugative plasmid transfer from E. coli strains. E. coli BR408 successfully transferred the E. coli–Geobacillus shuttle plasmid pUCG18T to 16 of 18 thermophiles with transformation efficiencies between 4.1 × 10^?7 and 3.8 × 10^?2/recipient. Other E. coli strains that are different from E. coli BR408 in intracellular DNA methylation also generated transformants from 9 to 15 of the 18 thermophiles, including one that E. coli BR408 could not transform, although the transformation efficiencies of these strains were generally lower than those of E. coli BR408. The conjugation was performed by simple incubation of an E. coli donor and a thermophile recipient without optimization of experimental conditions. Moreover, thermophile transformants were distinguished from abundant E. coli donor only by high temperature incubation. These observations suggest that conjugative plasmid transfer, particularly using E. coli BR408, is a facile and versatile approach for plasmid introduction into thermophilic Bacillus and Geobacillus spp., and potentially a variety of other thermophiles.     

Application of the shsp Gene, Encoding a Small Heat Shock Protein, as a Food-Grade Selection Marker for Lactic Acid Bacteria

Plasmid pSt04 of Streptococcus thermophilus contains a gene encoding a protein with homology to small heat shock proteins (A. Geis, H. A. M. El Demerdash, and K. J. Heller, Plasmid 50:53-69, 2003). Strains cured from the shsp plasmids showed significantly reduced heat and acid resistance and a lower maximal growth temperature. Transformation of the cloned shsp gene into S. thermophilus St11 lacking a plasmid encoding shsp resulted in increased resistance to incubation at 60°C or pH 3.5 and in the ability to grow at 52°C. A food-grade cloning system for S. thermophilus, based on the plasmid-encoded shsp gene as a selection marker, was developed. This approach allowed selection after transfer of native and recombinant shsp plasmids into different S. thermophilus and Lactococcus lactis strains. Using a recombinant plasmid carrying an erythromycin resistance (Em^r) gene in addition to shsp, we demonstrated that both markers are equally efficient in selecting for plasmid-bearing cells. The average transformation rates in S. thermophilus (when we were selecting for heat resistance) were determined to be 2.4 × 10⁴ and 1.0 × 10⁴ CFU/0.5 μg of DNA, with standard deviations of 0.54 × 10⁴ and 0.32 × 10⁴, for shsp and Em^r selection, respectively. When we selected for pH resistance, the average transformation rates were determined to be 2.25 × 10⁴ and 3.8 × 10³ CFU/0.5 μg of DNA, with standard deviations of 0.63 × 10⁴ and 3.48 × 10³, for shsp and Em^r selection, respectively. The applicability of shsp as a selection marker was further demonstrated by constructing S. thermophilus plasmid pHRM1 carrying the shsp gene as a selection marker and the restriction-modification genes of another S. thermophilus plasmid as a functional trait.     

Studies on rDNA from the extreme thermophilic eubacterium Thermus thermophilus HB8: The 23 S rDNA region D

23 S ribosomal ribonucleic acid gene from the extreme thermophile eubacterium Thermus thermophilus HB8 has been cloned in pBR322, and the nucleotide sequence of region D has been determined, which encompasses 873 nucleotides at the 3′-end of the RNA. We compare the primary and secondary structure of this region with the respective part of the 23 S rRNA from Escherichia coli and Bacillus stearothermophilus. A high level of structural conservation can be observed, throughout the RNA domain, albeit the usage of G/C basepairs is substantial even in comparison with another thermophilic eubacterium B. stearothermophilus. It is surprising that, in contrast to the usage of ^3′U-G^5′, the occurrence of ^3′G-U^5′ is comparable in E. coli as well as in B. stearothermophilus and T. thermophilus. Furthermore, it is most remarkable that the use of ^3′A-U^5′ and ^3′U-A^5′ is, compared to E. coli, only slightly reduced in B. stearothermophilus, but drastically decreased in T. thermophilus.     

Occurrence of sym-homospermidine in extremely thermophilic bacteria

Triamines produced by an extreme thermophile, Thermus thermophilus, were isolated and their chemical structures were determined. It was found that two novel triamines, norspermidine (1,7-diamino-4-azaheptane, NH₂(CH₂)₃· NH(CH₂)₃NH₂) and sym-homospermidine (1,9-diamino-5-azanonane, NH₂(CH₂)₄NH· (CH₂)₄NH₂) are present in the thermophile cells in addition to spermidine (1,8-diamino-4-azaoctane, NH₂(CH₂)₃NH(CH₂)₄NH₂).