A High-Transformation-Efficiency Cloning Vector for Thermus thermophilus

The cloning vector pMK18 was developed through the fusion of the minimal replicative region from an indigenous plasmid of Thermus sp. ATCC27737, a gene cassette encoding a thermostable resistance to kanamycin, and the replicative origin and multiple cloning site of pUC18. Plasmid pMK18 showed transformation efficiencies from 10⁸ to 10⁹ per microgram of plasmid in Thermus thermophilus HB8 and HB27, both by natural competence and by electroporation. We also show that T. thermophilus HB27 can take pMK18 modified by the Escherichia coli methylation system with the same efficiency as its own DNA. To demonstrate its usefulness as a cloning vector, a gene encoding the β-subunit of a thermostable nitrate reductase was directly cloned in T. thermophilus HB27 from a gene library. Its further transfer to E. coli also proved its utility as a shuttle vector.     

Production of polyhydroxyalkanoates from whey by Thermus thermophilus HB8

The thermophilic bacterium Thermus thermophilus HB8 is able to utilize lactose from whey-based media for the biosynthesis of polyhydroxyalkanoates (PHAs) under nitrogen limitation. T. thermophilus can utilize both, glucose and galactose, the products of lactose hydrolysis. When T. thermophilus HB8 was grown in culture media containing 24% (v/v) whey, PHA was accumulated up to 35% (w/w) of its biomass after 24 h of cultivation. The effect of initial phosphate concentration on the PHA production was also investigated. Using an initial phosphate concentration of 50 mM the PHA accumulation was enhanced. Analysis of the produced PHA from T. thermophilous HB8 grown in whey-based media revealed a novel heteropolymer consisting of the short chain length 3-hydroxyvalerate (3HV; 38 mol%) and the medium chain length, 3-hydroxyheptanoate (3HHp; 9.89 mol%), 3-hydroxynanoate (3HN; 16.59 mol%) and 3-hydroxyundecanoate (3HU; 35.42 mol%). Despite the low molecular weight of the produced PHA by T. thermophilus, whey could be an excellent substrate for the production of heteropolymers with unique properties.     

Molecular Analyses of the Natural Transformation Machinery and Identification of Pilus Structures in the Extremely Thermophilic Bacterium Thermus thermophilus Strain HB27

Thermus thermophilus HB27, an extremely thermophilic bacterium, exhibits high competence for natural transformation. To identify genes of the natural transformation machinery of T. thermophilus HB27, we performed homology searches in the partially completed T. thermophilus genomic sequence for conserved competence genes. These analyses resulted in the detection of 28 open reading frames (ORFs) exhibiting significant similarities to known competence proteins of gram-negative and gram-positive bacteria. Disruption of 15 selected potential competence genes led to the identification of 8 noncompetent mutants and one transformation-deficient mutant with a 100-fold reduced transformation frequency. One competence protein is similar to DprA of Haemophilus influenzae, seven are similar to type IV pilus proteins of Pseudomonas aeruginosa or Neisseria gonorrhoeae (PilM, PilN, PilO, PilQ, PilF, PilC, PilD), and another deduced protein (PilW) is similar to a protein of unknown function in Deinococcus radiodurans R1. Analysis of the piliation phenotype of T. thermophilus HB27 revealed the presence of single pilus structures on the surface of the wild-type cells, whereas the noncompetent pil mutants of Thermus, with the exception of the pilF mutant, were devoid of pilus structures. These results suggest that pili and natural transformation in T. thermophilus HB27 are functionally linked.     

Resistance of Thermus spp. to Potassium Tellurite

Two members of the genus Thermus were examined for their resistance to toxic inorganic compounds. They both proved to be fairly resistant to tellurite and selenite and to many other heavy metal salts. Cell extracts of Thermus thermophilus HB8 and of T. flavus AT-62 catalyze the reduction of K₂TeO₃ in a reaction which is dependent on NADH oxidation.     

Unmasking of an essential thiol during function of the membrane-bound enzyme II of the phosphoenolpyruvate β-glucoside phosphotransferase system of Escherichia coli

β-Glucoside transport by phosphoenolpyruvate-hexose phosphotransferase system in Escherichia coli is inactivated in vivo by thiol reagents. This inactivation is strongly enhanced by the presence of transported substrates. In a system reconstituted from soluble and membrane-bound components, only the particulate component, the membrane-bound enzyme II^bgl appeared as the target of N-ethylmaleimide inactivation. The same feature was found in the case of methyl-α-d-glucoside uptake via enzyme II^glc.It is shown that the sensitizing effect of substrates is specific and not generalized, methyl-α-d-glucoside only sensitizes enzyme II^bglc and $\text{p-}\text{nitrophenyl-β-}\text{d}\text{-glucoside}$ only sensitizes enzyme II^bgl towards N-ethylmaleimide inactivation.The inactivation of enzyme II^bgl by thiol reagents is also promoted in vivo by fluoride inhibition of phosphoenolpyruvate synthesis. In toluene-treated bacteria, the presence of phosphoenolpyruvate protects against inactivation by thiol reagents of $\text{p-}\text{nitrophenyl-β-}\text{d}\text{-glucoside}$ phosphorylation. Both results suggest that the inactivator resistent form of enzyme II^bgl is an energized form of the enzyme.     

Bioenergetics at extreme temperature: Thermus thermophilus ba3- and caa3-type cytochrome c oxidases

Seven years into the completion of the genome sequencing projects of the thermophilic bacterium Thermus thermophilus strains HB8 and HB27, many questions remain on its bioenergetic mechanisms. A key fact that is occasionally overlooked is that oxygen has a very limited solubility in water at high temperatures. The HB8 strain is a facultative anaerobe whereas its relative HB27 is strictly aerobic. This has been attributed to the absence of nitrate respiration genes from the HB27 genome that are carried on a mobilizable but highly-unstable plasmid. In T. thermophilus, the nitrate respiration complements the primary aerobic respiration. It is widely known that many organisms encode multiple biochemically-redundant components of the respiratory complexes. In this minireview, the presence of the two cytochrome c oxidases (CcO) in T. thermophilus, the ba₃- and caa₃-types, is outlined along with functional considerations. We argue for the distinct evolutionary histories of these two CcO including their respective genetic and molecular organizations, with the caa₃-oxidase subunits having been initially ‘fused’. Coupled with sequence analysis, the ba₃-oxidase crystal structure has provided evolutionary and functional information; for example, its subunit I is more closely related to archaeal sequences than bacterial and the substrate–enzyme interaction is hydrophobic as the elevated growth temperature weakens the electrostatic interactions common in mesophiles. Discussion on the role of cofactors in intra- and intermolecular electron transfer and proton pumping mechanism is also included. This article is part of a Special Issue entitled: Respiratory Oxidases.     

The role of the PHP domain associated with DNA polymerase X from Thermus thermophilus HB8 in base excision repair

Base excision repair (BER) is one of the most commonly used DNA repair pathways involved in genome stability. X-family DNA polymerases (PolXs) play critical roles in BER, especially in filling single-nucleotide gaps. In addition to a polymerase core domain, bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain with phosphoesterase activity which is also required for BER. However, the role of the PHP domain of PolX in bacterial BER remains unresolved. We found that the PHP domain of Thermus thermophilus HB8 PolX (ttPolX) functions as two types of phosphoesterase in BER, including a 3′-phosphatase and an apurinic/apyrimidinic (AP) endonuclease. Experiments using T. thermophilus HB8 cell lysates revealed that the majority of the 3′-phosphatase and AP endonuclease activities are attributable to the another phosphoesterase in T. thermophilus HB8, endonuclease IV (ttEndoIV). However, ttPolX possesses significant 3′-phosphatase activity in ΔttendoIV cell lysate, indicating possible complementation. Our experiments also reveal that there are only two enzymes that display the 3′-phosphatase activity in the T. thermophilus HB8 cell, ttPolX and ttEndoIV. Furthermore, phenotypic analysis of ΔttpolX, ΔttendoIV, and ΔttpolX/ΔttendoIV using hydrogen peroxide and sodium nitrite supports the hypothesis that ttPolX functions as a backup for ttEndoIV in BER.     

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.     

Development of a new host–vector system for colour selection of cloned DNA inserts using a newly designed β-galactosidase gene containing multiple cloning sites in Thermus thermophilus HB27

We constructed a new Thermus thermophilus cloning vector which enables the colour selection of cloned DNA inserts in the T. thermophilus HB27 host strain (β-gal⁻) on growth plates containing 3,4-cyclohexenoesculetin β-d-galactopyranoside (S-gal) in the medium. This vector harbors a modified β-galactosidase gene (TTP0042 of T. thermophilus HB27) with 12 unique restriction enzyme sites (Acc65I, AvrII, BlpI, BssHII, EcoRI, EcoRV, HindIII, NruI, SalI, SpeI, SphI and XbaI) as multiple cloning sites under the control of the T. thermophilus slpA promoter. This host–vector system facilitates cloning procedures in T. thermophilus HB27.

     

Role of galE on biofilm formation by Thermus spp.

Thermus thermophilus and Thermus aquaticus are thermophilic bacteria that are frequently found to attach to solid surfaces in hot springs to form biofilms. Uridine diphosphate (UDP)-galactose-4′-epimerase (GalE) is an enzyme that catalyzes the conversion of UDP-galactose to UDP-glucose, an important biochemical step in exopolysaccharide synthesis. We expressed GalE obtained from T. thermophilus HB8 in Escherichia coli and found that the enzyme is stable at 80 °C and can epimerize UDP-galactose to UDP-glucose and UDP-N-acetylgalactosamine (UDP-GalNAc) to UDP-N-acetylglucosamine (UDP-GlcNAc). Enzyme overexpression in T. thermophilus HB27 led to an increased capacity of biofilm production. Therefore, the galE gene is important to biofilm formation because of its involvement in epimerizing UDP-galactose and UDP-N-acetylgalactosamine for exopolysaccharide biosynthesis.     

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.     

Identification of new enzyme activities of several strains of Thermus species

New thermostable enzyme activities of seven Thermus strains were compared using the API ZYM system. All the strains exhibited high levels of - and -glycosidases, esterase (C₄) and esterase-lipase (C₈) activities intracellularly. Only T. thermophilus HB8 (ATCC 27634) showed -glucosidase and esterase activities in the supernatant. According to the intensity of -galactosidase activity, Thermus strains were divided in three groups. Group 0, which showed a weak -galactosidase activity, included Thermus spp. ATCC 31674 (T351) and 27978 (X-1) as well as T. thermophilus ATCC 27634 (HB8). Group I which consisted of T. aquaticus ATCC 25104 (YT-1), ATCC 25105 (Y-VII-51B) and Thermus sp. ATCC 27737 (T2), had a specific activity of approximately 40.0 U mg^–1 and galactose as inducer. T. aquaticus ATCC 31558 (group 2) was particularly effective for -galactosidase production (2840 U) with a specific activity of 98 U mg^–1. For each strain, galactose (0.5%) was a better inducer of -galactosidase production than lactose (1%). The detection of -galactosidase activity was dependent on the derivative chromogenic substrates used (naphthyl or nitrophenol coupled to sugar). Oligosaccharides were synthesized from cellobiose, lactulose, maltose or lactose as substrates at high temperature in some strains of Thermus.     

Recombinant phosphoribosyl pyrophosphate synthetases from <Emphasis Type="Italic">Thermus thermophilus</Emphasis> HB27: Isolation and properties

Two genes of T. thermophilus HB27, TT_C1184 and TT_C1274, encoding proteins with phosphoribosyl pyrophosphate synthetase activity were cloned in an expressing vector pET23d+. Escherichia coli strains overproducing two relevant proteins in soluble forms were obtained. The methods of isolation of thermophilic phosphoribosyl pyrophosphate synthetases Tth PRPPS1 and Tth PRPPS2 were developed. The activities of these enzymes were determined as a function of concentration of metal ions, inorganic phosphate, and temperature. The kinetic parameters for basic natural substrates were calculated; the substrate specificity for different carbohydrate 5-phosphates of D-series was studied. It was shown that the two proteins differ significantly in these characteristics. According to the results and comparison of amino acid sequences of new proteins with those of other phosphoribosyl pyrophosphate synthetases, both enzymes belong to class I PRPPS.     

Anaerobic Growth,a Property Horizontally Transferred by an Hfr-Like Mechanism among Extreme Thermophiles

Despite the fact that the extreme thermophilic bacteria belonging to the genus Thermus are classified as strict aerobes, we have shown that Thermus thermophilus HB8 (ATCC 27634) can grow anaerobically when nitrate is present in the growth medium. This strain-specific property is encoded by a respiratory nitrate reductase gene cluster (nar) whose expression is induced by anoxia and nitrate (S. Ramírez-Arcos, L. A. Fernández-Herrero, and J. Berenguer, Biochim. Biophys. Acta, 1396:215–1997). We show here that this nar operon can be transferred by conjugation to an aerobic Thermus strain, enabling it to grow under anaerobic conditions. We show that this transfer takes place through a DNase-insensitive mechanism which, as for the Hfr (high frequency of recombination) derivatives of Escherichia coli, can also mobilize other chromosomal markers in a time-dependent way. Three lines of evidence are presented to support a genetic linkage between nar and a conjugative plasmid integrated into the chromosome. First, the nar operon is absent from a plasmid-free derivative and from a closely related strain. Second, we have identified an origin for autonomous replication (oriV) overlapping the last gene of the nar cluster. Finally, the mating time required for the transfer of the nar operon is in good agreement with the time expected if the transfer origin (oriT) were located nearby and downstream of nar.

Most extreme thermophiles that live in geothermal environments are strict anaerobes (3, 11) as a consequence of the adaptation to the low solubility of oxygen at these temperatures. However, members of the genus Thermus constitute an exception to this general rule, being described taxonomically as strictly aerobic chemorganotrophs (2).However, we recently showed that one of the most thermophilic isolates of this genus, Thermus thermophilus HB8, was able to grow anaerobically when nitrate was present in the medium. Biochemical and genetic evidence demonstrated that this ability was related to the synthesis of a membrane-bound respiratory nitrate reductase complex whose protein components, the α (NarG; 136 kDa), β (NarH; 57 kDa), and γ (NarI; 28 kDa) subunits, were homologous (about 48 to 50% sequence identity) to those from mesophilic facultative anaerobes (e.g., Escherichia coli). The genes encoding these subunits were located within a single operon (nar) that was induced under low oxygen concentrations when nitrate was present (21). In contrast to those described for most nitrate reducers, the product of nitrate respiration was secreted to the growth medium through an unknown transporter.We also observed that even a closely related strain, such as T. thermophilus HB27, was unable to grow under such anaerobic conditions (21). Since the main difference between strains HB8 and HB27 of T. thermophilus is the absence of plasmids from the latter, the possibility that the nar operon could be encoded by a transferable genetic element, such as a plasmid, was considered.In this article, we analyze this possibility and demonstrate that the ability to grow by nitrate respiration can be transferred to the aerobic strain T. thermophilus HB27 by conjugation. We also relate this ability to the integration of a nar-carrying conjugative plasmid into the chromosome of T. thermophilus HB8. Moreover, we show that, as for the Hfr strains of E. coli, this integrated plasmid can also mobilize other chromosomal genes in a time-dependent way.     

Transferable Denitrification Capability of Thermus thermophilus

Laboratory-adapted strains of Thermus spp. have been shown to require oxygen for growth, including the model strains T. thermophilus HB27 and HB8. In contrast, many isolates of this species that have not been intensively grown under laboratory conditions keep the capability to grow anaerobically with one or more electron acceptors. The use of nitrogen oxides, especially nitrate, as electron acceptors is one of the most widespread capabilities among these facultative strains. In this process, nitrate is reduced to nitrite by a reductase (Nar) that also functions as electron transporter toward nitrite and nitric oxide reductases when nitrate is scarce, effectively replacing respiratory complex III. In many T. thermophilus denitrificant strains, most electrons for Nar are provided by a new class of NADH dehydrogenase (Nrc). The ability to reduce nitrite to NO and subsequently to N₂O by the corresponding Nir and Nor reductases is also strain specific. The genes encoding the capabilities for nitrate (nar) and nitrite (nir and nor) respiration are easily transferred between T. thermophilus strains by natural competence or by a conjugation-like process and may be easily lost upon continuous growth under aerobic conditions. The reason for this instability is apparently related to the fact that these metabolic capabilities are encoded in gene cluster islands, which are delimited by insertion sequences and integrated within highly variable regions of easily transferable extrachromosomal elements. Together with the chromosomal genes, these plasmid-associated genetic islands constitute the extended pangenome of T. thermophilus that provides this species with an enhanced capability to adapt to changing environments.     

Enzymatic properties and physiological function of glutamate racemase from Thermus thermophilus

d-Amino acids are physiologically important components of peptidoglycan in the bacterial cell wall, maintaining cell structure and aiding adaptation to environmental changes through peptidoglycan remodelling. Therefore, the biosynthesis of d-amino acids is essential for bacteria to adapt to different environmental conditions. The peptidoglycan of the extremely thermophilic bacterium Thermus thermophilus contains d-alanine (d-Ala) and d-glutamate (d-Glu), but its d-amino acid metabolism remains poorly understood. Here, we investigated the enzyme activity and function of the product of the TTHA1643 gene, which is annotated to be a Glu racemase in the T. thermophilus HB8 genome. Among 21 amino acids tested, TTHA1643 showed highly specific activity toward Glu as the substrate. The catalytic efficiency (k_cat/K_m) of TTHA1643 toward d- and l-Glu was comparable; however, the k_cat value was 18-fold higher for l-Glu than for d-Glu. Temperature and pH profiles showed that the racemase activity of TTHA1643 is high under physiological conditions for T. thermophilus growth. To assess physiological relevance, we constructed a TTHA1643-deficient strain (∆TTHA1643) by replacing the TTHA1643 gene with the thermostable hygromycin resistance gene. Growth of the ∆TTHA1643 strain in synthetic medium without d-Glu was clearly diminished relative to wild type, although the TTHA1643 deletion was not lethal, suggesting that alternative d-Glu biosynthetic pathways may exist. The deterioration in growth was restored by adding d-Glu to the culture medium, showing that d-Glu is required for normal growth of T. thermophilus. Collectively, our findings show that TTHA1643 is a Glu racemase and has the physiological function of d-Glu production in T. thermophilus.     

Type IV Pilus Biogenesis,Twitching Motility,and DNA Uptake in Thermus thermophilus: Discrete Roles of Antagonistic ATPases PilF,PilT1, and PilT2

Natural transformation has a large impact on lateral gene flow and has contributed significantly to the ecological diversification and adaptation of bacterial species. Thermus thermophilus HB27 has emerged as the leading model organism for studies of DNA transporters in thermophilic bacteria. Recently, we identified a zinc-binding polymerization nucleoside triphosphatase (NTPase), PilF, which is essential for the transport of DNA through the outer membrane. Here, we present genetic evidence that PilF is also essential for the biogenesis of pili. One of the most challenging questions was whether T. thermophilus has any depolymerization NTPase acting as a counterplayer of PilF. We identified two depolymerization NTPases, PilT1 (TTC1621) and PilT2 (TTC1415), both of which are required for type IV pilus (T4P)-mediated twitching motility and adhesion but dispensable for natural transformation. This suggests that T4P dynamics are not required for natural transformation. The latter finding is consistent with our suggestion that in T. thermophilus, T4P and natural transformation are linked but distinct systems.