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.     

Identification, subcellular localization and functional interactions of PilMNOWQ and PilA4 involved in transformation competency and pilus biogenesis in the thermophilic bacterium Thermus thermophilus HB27

The natural transformation system of the thermophilic bacterium Thermus thermophilus HB27 comprises at least 16 distinct competence proteins encoded by seven distinct loci. In this article, we present for the first time biochemical analyses of the Thermus thermophilus competence proteins PilMNOWQ and PilA4, and demonstrate that the pilMNOWQ genes are each essential for natural transformation. We identified three different forms of PilA4, one with an apparent molecular mass of 14 kDa, which correlates with that of the deduced protein, an 18-kDa form and a 23-kDa form; the last was found to be glycosylated. We demonstrate that PilM, PilN and PilO are located in the inner membrane, whereas PilW, PilQ and PilA4 are located in the inner and outer membranes. These data show that PilMNOWQ and PilA4 are components of a DNA translocator structure that spans the inner and outer membranes. We further show that PilA4 and PilQ both copurify with pilus structures. Possible functions of PilQ and PilA4 in DNA translocation and in pilus biogenesis are discussed. Comparative mutant studies revealed that mutations in either pilW or pilQ significantly affect the location of the other protein in the outer membrane. Furthermore, no PilA4 was present in the outer membranes of these mutants. From these findings, we conclude that the abilities of PilW, PilQ and PilA4 to stably localize or accumulate in the outer membrane fraction are strongly dependent on one another, which is in accord with an outer membrane DNA translocator complex comprising PilW, PilQ, and PilA4.     

Characterization of DNA transport in the thermophilic bacterium Thermus thermophilus HB27

Horizontal gene transfer has been a major force for genome plasticity over evolutionary history, and is largely responsible for fitness-enhancing traits, including antibiotic resistance and virulence factors. In particular, for adaptation of prokaryotes to extreme environments, lateral gene transfer seems to have played a crucial role. Recently, by performing a genome-wide mutagenesis approach with Thermus thermophilus HB27, we identified the first genes in a thermophilic bacterium for the uptake of free DNA, a process called natural transformation. Here, we present the first data on the biochemistry and bioenergetics of the DNA transport process in this thermophile. We report that linear and circular plasmid DNA are equally well taken up with a high maximal velocity of 1.5 microg DNA.(mg protein)(-1).min(-1), demonstrating an extremely efficient binding and uptake rate of 40 kb.s(-1).cell(-1). Uncouplers and ATPase inhibitors immediately inhibited DNA uptake, providing clear evidence that DNA translocation in HB27 is an energy-dependent process. DNA uptake studies with genomic DNA of Bacteria, Archaea and Eukarya revealed that Thermus thermophilus HB27 takes up DNA from members of all three domains of life. We propose that the extraordinary broad substrate specificity of the highly efficient Thermus thermophilus HB27 DNA uptake system may contribute significantly to thermoadaptation of Thermus thermophilus HB27 and to interdomain DNA transfer in hot environments.     

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.     

Characterization of a thermostable DNA photolyase from an extremely thermophilic bacterium, Thermus thermophilus HB27.

The photolyase gene from Thermus thermophilus was cloned and sequenced. The characteristic absorption and fluorescence spectra of the purified T. thermophilus photolyase suggested that the protein has flavin adenine dinucleotide as a chromophore. The second chromophore binding site was not conserved in T. thermophilus photolyase. The purified enzyme showed light-dependent photoreactivation activity in vitro at 35 and 65 degrees C and was stable when subjected to heat and acidic pH.     

Glutamine:phenylpyruvate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8

A subfamily I aminotransferase gene homologue containing an open reading frame encoding 381 amino acid residues (Mr=42,271) has been identified in the process of the genome project of an extremely thermophilic bacterium, Thermus thermophilus HB8. Alignment of the predicted amino acid sequence using FASTA shows that this protein is a member of aminotransferase subfamily Igamma. The protein shows around 40% identity with both T. thermophilus aspartate aminotransferase [EC 2.6.1.1] and mammalian glutamine:phenylpyruvate aminotransferase [EC 2.6.1.64]. The recombinant protein expressed in Escherichia coli is a homodimer with a subunit molecular weight of 42,000, has one pyridoxal 5'-phosphate per subunit, and is highly active toward glutamine, methionine, aromatic amino acids, and corresponding keto acids, but has no preference for alanine and dicarboxylic amino acids. These substrate specificities are similar to those described for mammalian glutamine: phenylpyruvate aminotransferase. This is the first enzyme reported so far that has the glutamine aminotransferase activity in non-eukaryotic cells. As the presence of aromatic amino acid:2-oxoglutarate aminotransferase [EC 2.6.1.57] has not been reported in T. thermophilus, this enzyme is expected to catalyze the last transamination step of phenylalanine and tyrosine biosynthesis. It may also be involved in the methionine regeneration pathway associated with polyamine biosynthesis. The enzyme shows a strikingly high pKa value (9.3) of the coenzyme Schiff base in comparison with other subfamily I aminotransferases. The origin of this unique pKa value and the substrate specificity is discussed based on the previous crystallographic data of T. thermophilus and E. coli aspartate aminotransferases.     

The high-affinity maltose/trehalose ABC transporter in the extremely thermophilic bacterium Thermus thermophilus HB27 also recognizes sucrose and palatinose

We have studied the transport of trehalose and maltose in the thernophilic bacterium Thermus thermophilus HB27, which grows optimally in the range of 70 to 75 degrees C. The K(m) values at 70 degrees C were 109 nM for trehalose and 114 nM for maltose; also, a high K(m) (424 nM) was found for the uptake of sucrose. Competition studies showed that a single transporter recognizes trehalose, maltose, and sucrose, while d-galactose, d-fucose, l-rhamnose, l-arabinose, and d-mannose were not competitive inhibitors. In the recently published genome of T. thermophilus HB27, two gene clusters designated malEFG1 (TTC1627 to -1629) and malEFG2 (TTC1288 to -1286) and two monocistronic genes designated malK1 (TTC0211) and malK2 (TTC0611) are annotated as trehalose/maltose and maltose/maltodextrin transport systems, respectively. To find out whether any of these systems is responsible for the transport of trehalose, the malE1 and malE2 genes, lacking the sequence encoding the signal peptides, were expressed in Escherichia coli. The binding activity of pure recombinant proteins was analyzed by equilibrium dialysis. MalE1 was able to bind maltose, trehalose, and sucrose but not glucose or maltotetraose (K(d) values of 103, 67, and 401 nM, respectively). Mutants with disruptions in either malF1 or malK1 were unable to grow on maltose, trehalose, sucrose, or palatinose, whereas mutants with disruption in malK2 or malF2 showed no growth defect on any of these sugars. Therefore, malEFG1 encodes the binding protein and the two transmembrane subunits of the trehalose/maltose/sucrose/palatinose ABC transporter, and malK1 encodes the ATP-binding subunit of this transporter. Despite the presence of an efficient transporter for trehalose, this compound was not used by HB27 for osmoprotection. MalE1 and MalE2 exhibited extremely high thermal stability: melting temperatures of 90 degrees C for MalE1 and 105 degrees C for MalE2 in the presence of 2.3 M guanidinium chloride. The latter protein did not bind any of the sugars examined and is not implicated in a maltose/maltodextrin transport system. This work demonstrates that malEFG1 and malK1 constitute the high-affinity ABC transport system of T. thermophilus HB27 for trehalose, maltose, sucrose, and palatinose.     

Characterization of homoisocitrate dehydrogenase involved in lysine biosynthesis of an extremely thermophilic bacterium,Thermus thermophilus HB27, and evolutionary implication of beta-decarboxylating dehydrogenase

Although the presence of an enzyme that catalyzes beta-decarboxylating dehydrogenation of homoisocitrate to synthesize 2-oxoadipate has been postulated in the lysine biosynthesis pathway through alpha-aminoadipate (AAA), the enzyme has not yet been analyzed at all, because no gene encoding the enzyme has been identified until recently. A gene encoding a protein with a significant amino acid sequence identity to both isocitrate dehydrogenase and 3-isopropylmalate dehydrogenase was cloned from Thermus thermophilus HB27. The gene product produced in recombinant Escherichia coli cells demonstrated homoisocitrate dehydrogenase (HICDH) activity. A knockout mutant of the gene showed an AAA-auxotrophic phenotype, indicating that the gene product is involved in lysine biosynthesis through AAA. We therefore named this gene hicdh. HICDH, the gene product, did not catalyze the conversion of 3-isopropylmalate to 2-oxoisocaproate, a leucine biosynthetic reaction, but it did recognize isocitrate, a related compound in the tricarboxylic acid cycle, as well as homoisocitrate as a substrate. It is of interest that HICDH catalyzes the reaction with isocitrate about 20 times more efficiently than the reaction with the putative native substrate, homoisocitrate. The broad specificity and possible dual function suggest that this enzyme represents a key link in the evolution of the pathways utilizing citrate derivatives. Site-directed mutagenesis study reveals that replacement of Arg(85) with Val in HICDH causes complete loss of activity with isocitrate but significant activity with 3-isopropylmalate and retains activity with homoisocitrate. These results indicate that Arg(85) is a key residue for both substrate specificity and evolution of beta-decarboxylating dehydrogenases.     

Structure of the PilM-PilN inner membrane type IV pilus biogenesis complex from Thermus thermophilus

Type IV pili are surface-exposed filaments, which extend from a variety of bacterial pathogens and play a major role in pathogenesis, motility, and DNA uptake. Here, we present the crystal structure of a complex between a cytoplasmic component of the type IV pilus biogenesis system from Thermus thermophilus, PilM, in complex with a peptide derived from the cytoplasmic portion of the inner membrane protein PilN. PilM also binds ATP, and its structure is most similar to the actin-like protein FtsA. PilN binds in a narrow channel between the 1A and 1C subdomains in PilM; the binding site is well conserved in other gram-negative bacteria, notably Neisseria meningitidis, Pseudomonas aeruginosa, and Vibrio cholerae. We find no evidence for the catalysis of ATP hydrolysis by PilM; fluorescence data indicate that the protein is likely to be saturated by ATP at physiological concentrations. In addition, binding of the PilN peptide appears to influence the environment of the ATP binding site. This is the first reported structure of a complex between two type IV pilus biogenesis proteins. We propose a model in which PilM binds ATP and then PilN as one of the first steps in the formation of the inner membrane platform of the type IV pilus biogenesis complex.     

Thermostable repair enzyme for oxidative DNA damage from extremely thermophilic bacterium, Thermus thermophilus HB8.

The mutM (fpg) gene, which encodes a DNA glycosylase that excises an oxidatively damaged form of guanine, was cloned from an extremely thermophilic bacterium, Thermus thermophilus HB8. Its nucleotide sequence encoded a 266 amino acid protein with a molecular mass of approximately 30 kDa. Its predicted amino acid sequence showed 42% identity with the Escherichia coli protein. The amino acid residues Cys, Asn, Gln and Met, known to be chemically unstable at high temperatures, were decreased in number in T.thermophilus MutM protein compared to those of the E.coli one, whereas the number of Pro residues, considered to increase protein stability, was increased. The T.thermophilus mutM gene complemented the mutability of the E.coli mutM mutY double mutant, suggesting that T. thermophilus MutM protein was active in E.coli. The T.thermophilus MutM protein was overproduced in E.coli and then purified to homogeneity. Size-exclusion chromatography indicated that T. thermophilus MutM protein exists as a more compact monomer than the E.coli MutM protein in solution. Circular dichroism measurements indicated that the alpha-helical content of the protein was approximately 30%. Thermus thermophilus MutM protein was stable up to 75 degrees C at neutral pH, and between pH 5 and 11 and in the presence of up to 4 M urea at 25 degrees C. Denaturation analysis of T.thermophilus MutM protein in the presence of urea suggested that the protein had at least two domains, with estimated stabilities of 8.6 and 16.2 kcal/mol-1, respectively. Thermus thermophilus MutM protein showed 8-oxoguanine DNA glycosylase activity in vitro at both low and high temperatures.     

Occurrence of transsulfuration in synthesis of L-homocysteine in an extremely thermophilic bacterium, Thermus thermophilus HB8

A cell extract of an extremely thermophilic bacterium, Thermus thermophilus HB8, cultured in a synthetic medium catalyzed cystathionine gamma-synthesis with O-acetyl-L-homoserine and L-cysteine as substrates but not beta-synthesis with DL-homocysteine and L-serine (or O-acetyl-L-serine). The amounts of synthesized enzymes metabolizing sulfur-containing amino acids were estimated by determining their catalytic activities in cell extracts. The syntheses of cystathionine beta-lyase (EC 4.4.1.8) and O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8) were markedly repressed by L-methionine supplemented to the medium. L-Cysteine and glutathione, both at 0.5 mM, added to the medium as the sole sulfur source repressed the synthesis of O-acetylserine sulfhydrylase by 55 and 73%, respectively, confirming that this enzyme functions as a cysteine synthase. Methionine employed at 1 to 5 mM in the same way derepressed the synthesis of O-acetylserine sulfhydrylase 2.1- to 2.5-fold. A method for assaying a low concentration of sulfide (0.01 to 0.05 mM) liberated from homocysteine by determining cysteine synthesized with it in the presence of excess amounts of O-acetylserine and a purified preparation of the sulfhydrylase was established. The extract of cells catalyzed the homocysteine gamma-lyase reaction, with a specific activity of 5 to 7 nmol/min/mg of protein, but not the methionine gamma-lyase reaction. These results suggested that cysteine was also synthesized under the conditions employed by the catalysis of O-acetylserine sulfhydrylase using sulfur of homocysteine derived from methionine. Methionine inhibited O-acetylserine sulfhydrylase markedly. The effects of sulfur sources added to the medium on the synthesis of O-acetylhomoserine sulfhydrylase and the inhibition of the enzyme activity by methionine were mostly understood by assuming that the organism has two proteins having O-acetylhomoserine sulfhydrylase activity, one of which is cystathionine gamma-synthase. Although it has been reported that homocysteine is directly synthesized in T. thermophilus HB27 by the catalysis of O-acetylhomoserine sulfhydrylase on the basis of genetic studies (T. Kosuge, D. Gao, and T. Hoshino, J. Biosci. Bioeng. 90:271-279, 2000), the results obtained in this study for the behaviors of related enzymes indicate that sulfur is first incorporated into cysteine and then transferred to homocysteine via cystathionine in T. thermophilus HB8.     

Lysophosphatidic acid acyltransferase from the thermophilic bacterium Thermus thermophilus HB8 displays substrate promiscuity

ABSTRACT

Lysophosphatidic acid acyltransferase is a phospholipid biosynthetic enzyme that introduces a fatty acyl group into the sn-2 position of phospholipids. Its substrate selectivity is physiologically important in defining the physicochemical properties of lipid membranes and modulating membrane protein function. However, it remains unclear how these enzymes recognize various fatty acids. Successful purification of bacterial lysophosphatidic acid acyltransferases (PlsCs) was recently reported and has paved a path for the detailed analysis of their reaction mechanisms. Here, we purified and characterized PlsC from the thermophilic bacterium Thermus thermophilus HB8. This integral membrane protein remained active even after solubilization and purification and showed reactivity toward saturated, unsaturated, and methyl-branched fatty acids, although branched-chain acyl groups are the major constituent of phospholipids of this bacterium. Multiple sequence alignment revealed the N-terminal end of the enzyme to be shorter than that of PlsCs with defined substrate selectivity, suggesting that the shortened N-terminus confers substrate promiscuity.     

Structure and function of PilQ, a secretin of the DNA transporter from the thermophilic bacterium Thermus thermophilus HB27

Secretins are a family of large bacterial outer membrane protein complexes mediating the transport of complex structures, such as type IV pili, DNA and filamentous phage, or various proteins, such as extracellular enzymes and pathogenicity determinants. PilQ of the thermophilic bacterium Thermus thermophilus HB27 is a member of the secretin family required for natural transformation. Here we report the isolation, structural, and functional analyses of a unique PilQ from T. thermophilus. Native PAGE, gel filtration chromatography, and electrophoretic mobility shift analyses indicated that PilQ forms a macromolecular homopolymeric complex that binds dsDNA. Electron microscopy showed that the PilQ complex is 15 nm wide and 34 nm long and consists of an extraordinary stable cone and cup structure and five ring structures with a large central channel. Moreover, the electron microscopic images together with secondary structure analyses combined with structural data of type II protein secretion system and type III protein secretion system secretins suggest that the individual rings are formed by conserved domains of alternating α-helices and β-sheets. The unprecedented length of the PilQ complex correlated well with the distance between the inner and outer membrane of T. thermophilus. Indeed, PilQ was found immunologically in both membranes, indicating that the PilQ complex spans the entire cell periphery of T. thermophilus. This is consistent with the hypothesis that PilQ accommodates a PilA4 comprising pseudopilus mediating DNA transport across the outer membrane and periplasmic space in a single-step process.     

Functional dissection of structural regions of the Thermus thermophilus competence protein PilW: Implication in secretin complex stability,natural transformation and pilus functions

Uptake of DNA from the environment into the bacterial cytoplasm is mediated by a macromolecular transport machinery that is similar in structure and function to type IV pili (T4P) and, indeed, DNA translocator and T4P share common components. One is the secretin PilQ which is assembled into homopolymeric complexes forming highly dynamic outer membrane (OM) channels mediating pilus extrusion and DNA uptake. How PilQ interacts with the motor is still enigmatic. Here, we have used biochemical and genetic techniques to study the interaction of PilQ with PilW, a unique protein which is essential for natural transformation and T4P extrusion of T. thermophilus. PilQ and PilW form high molecular mass complexes in the OM of T. thermophilus. When pilW was deleted, PilQ complexes were no longer observed but only PilQ monomers, accompanied by a loss of DNA uptake as well as a loss of T4P and twitching motility. Piliation of a ΔpilT2/ΔpilW double mutant suggests that PilW is important for stable assembly of PilQ complexes. To analyze the role of different regions of PilW, partial deletions (pilW_?2–40, pilW_?50–150, pilW_?163–216 and pilW_?216–292) were generated and the effect on DNA uptake, PilQ complex formation and T4P functions such as twitching motility, biofilm formation and cell-cell interaction was studied. These studies revealed that a central disordered region in PilW is required for pilus dynamics. We propose that PilW is part of a protein network that connects the transport ATPase to drive different functions of the DNA translocator and T4P.     

Gene cloning and overexpression of a geranylgeranyl diphosphate synthase of an extremely thermophilic bacterium, Thermus thermophilus

A geranylgeranyl diphosphate (GGPP) synthase gene of an extremely thermophilic bacterium, Thermus thermophilus, was cloned and sequenced. T. thermophilus GGPP synthase, overexpressed in Escherichia coli cells as a glutathione S-transferase fusion protein, was purified and characterized. The fusion protein, retaining thermostability, formed a homodimer, and showed higher specific activity than did a partially purified thermostable enzyme previously reported. Optimal reaction conditions and kinetic parameters were also examined. The deduced amino acid sequence indicated that T. thermophilus GGPP synthase was excluded from the group of bacterial type GGPP synthases and lacked the insertion amino acid residues in the first aspartate-rich motif as do archaeal and eukaryotic short-chain prenyltransferases.     

Characterization of chromosomal and megaplasmid partitioning loci in Thermus thermophilus HB27

Background

In low-copy-number plasmids, the partitioning loci (par) act to ensure proper plasmid segregation and copy number maintenance in the daughter cells. In many bacterial species, par gene homologues are encoded on the chromosome, but their function is much less understood. In the two-replicon, polyploid genome of the hyperthermophilic bacterium Thermus thermophilus, both the chromosome and the megaplasmid encode par gene homologues (parABc and parABm, respectively). The mode of partitioning of the two replicons and the role of the two Par systems in the replication, segregation and maintenance of the genome copies are completely unknown in this organism.

Results

We generated a series of chromosomal and megaplasmid par mutants and sGFP reporter strains and analyzed them with respect to DNA segregation defects, genome copy number and replication origin localization. We show that the two ParB proteins specifically bind their cognate centromere-like sequences parS, and that both ParB-parS complexes localize at the cell poles. Deletion of the chromosomal parAB genes did not apparently affect the cell growth, the frequency of cells with aberrant nucleoids, or the chromosome and megaplasmid replication. In contrast, deletion of the megaplasmid parAB operon or of the parB gene was not possible, indicating essentiality of the megaplasmid-encoded Par system. A mutant expressing lower amounts of ParABm showed growth defects, a high frequency of cells with irregular nucleoids and a loss of a large portion of the megaplasmid. The truncated megaplasmid could not be partitioned appropriately, as interlinked megaplasmid molecules (catenenes) could be detected, and the ParBm-parSm complexes in this mutant lost their polar localization.

Conclusions

We show that in T. thermophilus the chromosomal par locus is not required for either the chromosomal or megaplasmid bulk DNA replication and segregation. In contrast, the megaplasmid Par system of T. thermophilus is needed for the proper replication and segregation of the megaplasmid, and is essential for its maintenance. The two Par sets in T. thermophilus appear to function in a replicon-specific manner. To our knowledge, this is the first analysis of Par systems in a polyploid bacterium.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1523-3) contains supplementary material, which is available to authorized users.     

Occurrence of phosphenolpyruvate carboxylase in the extremely thermophilic bacterium Thermus aquaticus.

In the extreme thermophile Thermus aquaticus, phosphoenolpyruvate carboxylase catalyzes carbon dioxide fixation on the C3 metabolite phosphoenolpyruvate, producing oxaloacetate. In a moderately thermophilic Bacillus species this function is fulfilled by pyruvate carboyxlase. Like several of its mesophilic counterparts, the Thermus enzyme exhibits a requirement for acetyl coenzyme A.