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.     

Compact reduced thioredoxin structure from the thermophilic bacteria Thermus thermophilus

The X-ray crystallographic structure of a thioredoxin from Thermus thermophilus was solved to 1.8 A resolution by molecular replacement. The crystals' space group was C2 with cell dimensions of a = 40.91, b = 95.44, c = 56.68 A, beta =91.41 degrees, with two molecules in the asymmetric unit. Unlike the reported thioredoxin structures, the biological unit of T. thermophilus thioredoxin is a dimer both in solution and in the crystal. The fold conforms to the "thioredoxin fold" that is common over a class of nine protein families including thioredoxin; however, the folded portion of this protein is much more compact than other thioredoxins previously solved by X-ray crystallography being reduced by one alpha-helix and one beta-strand. As with other thioredoxins, the active site is highly conserved even though the variation in sequence can be quite large. The T. thermophilus thioredoxin has some variability at the active site, especially compared with previously solved structures from bacterial sources.     

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.     

Identification of a crystalline surface layer on the cell envelope of the thermophilic eubacterium Thermus thermophilus

Abstract Analysis by electron microscopy of cells of Thermus thermophilus revealed the presence of a crystalline layer on the cell surface with the characteristic appearance of an S-layer. The layer is apparently built up by a single protein with a M _r of 100 000 in a hexagonal array. The unit cell dimension of the S-layer is 24 ± 2 nm.     

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.     

Mutagenesis of uracil-DNA glycosylase deficient mutants of the extremely thermophilic eubacterium Thermus thermophilus

Thermus thermophilus is an extremely thermophilic, aerobic, and gram-negative eubacterium that grows optimally at 70-75 degrees C, pH 7.5. In extremely high temperature environment, DNA damages in cells occur at a much higher frequency in thermophiles than mesophiles such as E. coli. When temperature rises, the deamination of cytosine residues in double-strand DNA is expected to increase greatly. T. thermophilus HB27 has two putative uracil-DNA glycosylase genes (udgA and udgB). Expression level of udgA gene was 2-3 times higher than that of udgB at 70, 74, and 78 degrees C when it was monitored by beta-glucosidase reporter assay. We developed hisD(3110), hisD(3113), hisD(3115), and hisD(174) marker allele that can specifically detect G:C-->A:T, C:G-->A:T, T:A-->A:T, and A:T-->G:C base-substitutions, respectively, by His(+) reverse mutations. We then disrupted udgA and udgB by thermostable kanamycin-resistant gene (htk) or pyrE gene insertion in each hisD background, and their spontaneous His(+) reversion frequencies were compared. A udgA,B double mutant showed a pronounced increase in G:C-->A:T reversion frequency compared with each single udg mutant, udgA or udgB. Estimated mutation rates of the udgA,B mutant cultured at 60, 70, and 78 degrees C were about 2, 12, and 117 His(+)/10(8)/generation, respectively. At 70 degrees C culture, increased ratio of the mutation rate compared with the udg(+) strain was 12-fold in udgA, 3-fold in udgB, and 56-fold in udgA,B mutant. On the other hand, no difference was observed in other mutations of C:G-->A:T, T:A-->A:T, and A:T-->G:C between udgA,B double mutant and the parent udg(+) strain. The present results indicated that gene products of udgB as well as udgA functioned in vivo to remove uracil in DNA and prevent G:C-->A:T transition mutations.     

A chaperonin from a thermophilic bacterium, Thermus thermophilus, that controls refoldings of several thermophilic enzymes.

A chaperonin has been purified from a thermophilic bacterium, Thermus thermophilus. It consists of two kinds of proteins with approximate Mr 58,000 and 10,000 and shows a 7-fold rotational symmetry from the top view and a "football"-like shape from the side view under the electron microscopic view. Its weak ATPase activity is inhibited by sulfite and activated by bicarbonate. ATP causes change of its mobility in nondenaturating polyacrylamide gel electrophoresis. The T. thermophilus chaperonin can promote in vitro refolding of several guanidine HCl-denatured enzymes from thermophilic bacteria. At high temperatures above 60 degrees C, where the native enzymes are stable but their spontaneous refoldings upon dilution of guanidine HCl fail, the chaperonin induces productive refolding in an ATP-dependent manner. No or very poor refolding is induced when the chaperonin is added to the solution aged after dilution. An excess amount of the chaperonin is inhibitory for refolding. At middle temperatures (30-50 degrees C), where spontaneous refoldings of the enzymes occur, the chaperonin arrests refolding in the absence of ATP and refolding is induced when ATP is supplemented. At temperatures below 20 degrees C, where spontaneous refoldings also occur, the chaperonin arrests the refolding but ATP does not induce refolding.     

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.     

Cysteine synthase of an extremely thermophilic bacterium,Thermus thermophilus HB8

O-Acetyl-L-serine sulfhydrylase (EC 4.2.99.8) was first purified from an extremely thermophilic bacterium, Thermus thermophilus HB8, in order to ascertain that it is responsible for the cysteine synthesis in this organism cultured with either sulfate or methionine given as a sole sulfur source. Polyacrylamide gel electrophoreses both with and without SDS found high purity of the enzyme preparations finally obtained, through ammonium sulfate fractionation, ion exchange chromatography, gel filtration, and hydrophobic chromatography (or affinity chromatography). The enzyme activity formed only one elution curve in each of the four different chromatographies, strongly suggesting the presence of only one enzyme species in this organism. Molecular masses of 34,000 and 68,000 were estimated for dissociated subunit and the native enzyme, respectively, suggesting a homodimeric structure. The enzyme was stable at 70 degrees C at pH 7.8 for 60 min, and more than 90% of the activity was retained after incubation of its solution at 80 degrees C with 10 mm dithiothreitol. The enzyme was also quite stable at pH 8-12 (50 degrees C, 30 min). It had an apparent Km of 4.8 mM for O-acetyl-L-serine (with 1 mM sulfide) and a Vmax of 435 micromol/min/mg of protein. The apparent Km for sulfide was approximately 50 microM (with 20 mM acetylserine), suggesting that the enzyme can react with sulfide liberated very slowly from methionine. The absorption spectrum of the holo-enzyme and inhibition of the activity by carbonyl reagents suggested the presence of pyridoxal 5'-phosphate as a cofactor. The apo-enzyme showed an apparent Km of 29 microM for the cofactor at pH 8. Monoiodoacetic acid (1 mM) almost completely inactivated the enzyme. The meaning of a very high enzyme content in the cell is discussed.     

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.     

Crystal structure of TTC0263, a thermophilic TPR protein from Thermus thermophilus HB27

The hypothetical protein TTC0263 of Thermus thermophilus HB27 is a thermophilic tetratricopeptide repeat (TPR)-containing protein. In the present study, the TPR region (residues 26-230) was resolved at 2.5 A with R-factors of R/Rfree = 23.6%/28.6%. TTC0263 consists of 11 helices that form five TPR units. Uniquely, it contains one atypical "extended" TPR (eTPR) unit. This comprises extended helical residues near the loop region of TTC0263, such that the helical length of eTPR is longer than that of the canonical TPR sequence. In addition, the hybrid TPR domain of TTC0263 possesses oligomer-forming characteristics. TPR domains are generally involved in forming multi-subunit complexes by interacting with each other or with other subunit proteins. The dynamic structure of TTC0263 described here goes some way to explaining how TPR domains mediate the formation of multi-subunit complexes.     

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 ℃. As expected, the enzyme displayed high thermostability, retaining more than 50% activity after incubating for 6 h at 80 ℃. Its catalytic function was accelerated in the presence of 0.1 mM Co^2＋, Fe^2＋, Mg^2＋, or Mn^2＋ but was strongly inhibited by 2.0 mM Fe^2＋. Under optimal conditions, the Michaelis constant （Kin） 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.     

Creation of genetic information by DNA polymerase of the thermophilic bacterium Thermus thermophilus.

Genetic information encoded in a template of a genome is replicated in a complementary way by DNA polymerase or RNA polymerase with high fidelity; no creation of information occurs in this reaction unless an error occurs. We report here that DNA polymerase of the thermophilic bacterium Thermus thermophilus can synthesize up to 200 kb linear double-stranded DNA in vitro in the complete absence of added primer and template DNAs, indicating that genetic information is actively created by protein. This ab initio DNA synthesis occurs at 74 degrees C and requires magnesium ion. There is a lag time of approximately 1 h and then the reaction proceeds linearly. The synthesized DNAs have a variety of sequences; they are mostly tandem repetitive sequences, e.g. (CATGTATA) n , (TGTATGTATACATACATA) n and (TATACGTA) n . Some degenerate sequences of these basic repeat units are also found. The similar repetitive sequences are found in many natural genes. These results, together with similar results found using DNA polymerase of archaeon Thermococcus litoralis , suggest that creative, non-replicative synthesis of DNA by protein was a driving force for diversification of genetic information at a certain stage of the evolution of life on the early earth.     

The structures of glycolipids isolated from the highly thermophilic bacterium Thermus thermophilus Samu-SA1

Thermophiles constitute a class of microorganisms able to grow at extremely elevated temperatures. Some of these species are classified as Gram-negative bacteria, because of the presence of an outer membrane in the cell envelope, which is located on the top of a thick murein layer. Unlike typical Gram-negative bacteria, the outer membranes of Thermus species are not composed of lipopolysaccharides but of peculiar glycolipids (GL), whose structures seem to be strictly involved in the adaptation to high temperatures. In this work, the complete structures of the major GL components from the cell envelope of the thermophilic bacterium Thermus thermophilus Samu-SA1 are presented. Protocols conventionally adopted for Gram-negative bacteria were used, and, for the first time, GL from Thermus were analyzed in their native form. Two GL and one phosphoglycolipid (PGL) were detected and characterized. The two GL, analyzed by nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR) mass spectrometry, possessed the same tetrasaccharide structure linked to a glycerol unit or, alternatively, to a long-chain diol. Moreover, a PGL from Thermus was characterized for the first time, in which N-glyceroyl-heptadecaneamine was present. These molecules are chemically related to other GL from thermophile bacteria, in which they play a crucial role in the adaptation of cell membranes to heat.     

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.     

The contribution of the zinc-finger motif to the function of Thermus thermophilus ribosomal protein S14

In the crystal structure of the 30S ribosomal subunit from Thermus thermophilus, cysteine 24 of ribosomal protein S14 (TthS14) occupies the first position in a CXXC-X12-CXXC motif that coordinates a zinc ion. The structural and functional importance of cysteine 24, which is widely conserved from bacteria to humans, was studied by its replacement with serine and by incorporating the resulting mutant into Escherichia coli ribosomes. The capability of such modified ribosomes in binding tRNA at the P and A-sites was equal to that obtained with ribosomes incorporating wild-type TthS14. In fact, both chimeric ribosomal species exhibited 20% lower tRNA affinity compared with native E. coli ribosomes. In addition, replacement of the native E. coli S14 by wild-type, and particularly by mutant TthS14, resulted in reduced capability of the 30S subunit for association with 50S subunits. Nevertheless, ribosomes from transformed cells sedimented normally and had a full complement of proteins. Unexpectedly, the peptidyl transferase activity in the chimeric ribosomes bearing mutant TthS14 was much lower than that measured in ribosomes incorporating wild-type TthS14. The catalytic center of the ribosome is located within the 50S subunit and, therefore, it is unlikely to be directly affected by changes in the structure of S14. More probably, the perturbing effects of S14 mutation on the catalytic center seem to be propagated by adjacent intersubunit bridges or the P-site tRNA molecule, resulting in weak donor-substrate reactivity. This hypothesis was verified by molecular dynamics simulation analysis.     

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.