Cloning, nucleotide sequence, and expression in Escherichia coli of the Bacillus stearothermophilus peroxidase gene (perA).

The gene encoding a thermostable peroxidase was cloned from the chromosomal DNA of Bacillus stearothermophilus IAM11001 in Escherichia coli. The nucleotide sequence of the 3.1-kilobase EcoRI fragment containing the peroxidase gene (perA) and its flanking region was determined. A 2,193-base-pair open reading frame encoding a peroxidase of 731 amino acid residues (Mr, 82,963) was observed. A Shine-Dalgarno sequence was found 9 base pairs upstream from the translational starting site. The deduced amino acid sequence coincides with those of the amino terminus and four peptides derived from the purified peroxidase of B. stearothermophilus IAM11001. E. coli harboring a recombinant plasmid containing perA produced a large amount of thermostable peroxidase which comigrated on polyacrylamide gel electrophoresis with the B. stearothermophilus peroxidase. The peroxidase of B. stearothermophilus showed 48% homology in the amino acid sequence to the catalase-peroxidase of E. coli.     

A common sequence motif, -E-G-Y-A-T-A-, identified within the primase domains of plasmid-encoded I- and P-type DNA primases and the alpha protein of the Escherichia coli satellite phage P4.

DNA primases encoded by the conjugative plasmids ColIb-P9 (IncI1), RP4, and R751 (IncP), and the protein of the Escherichia coli satellite phage P4 alpha were shown to contain a common amino acid sequence motif -E-G-Y-A-T-A-. The P4 alpha gene product, required for initiation of phage DNA replication, exhibits primase activity on single-stranded circular DNA templates. This priming activity resembles the enzymatic activity of DNA primases encoded by conjugative plasmids in terms of template utilization and the ability to synthesize primers that can be elongated by DNA polymerase III holoenzyme. The -E-G-Y-A-T-A- motif is part of an extended sequence region most conserved within the primase domains of the four enzymes. Single amino acid substitutions generated in the -E-G-Y-A-T-A- motif of the RP4 TraC2 and the P4 alpha protein affect priming activity, supporting the hypothesis that the conserved sequence motif is part of the active center for primase function. A mutation that eliminates priming activity causes P4 phage to grow poorly and to depend upon the host dnaG primase. Computer analysis identified two additional sequence motifs within the amino acid sequence of the P4 alpha protein: a potential zinc-finger motif and a "type A" nucleotide binding site, both strikingly similar to sequence motifs described in various DNA primases and helicases.     

Gene cloning, sequence analysis, purification, and characterization of a thermostable aminoacylase from Bacillus stearothermophilus.

A genomic DNA fragment encoding aminoacylase activity of the eubacterium Bacillus stearothermophilus was cloned into Escherichia coli. Transformants expressing aminoacylase activity were selected by their ability to complement E. coli mutants defective in acetylornithine deacetylase activity, the enzyme that converts N-acetylornithine to ornithine in the arginine biosynthetic pathway. The 2.3-kb cloned fragment has been entirely sequenced. Analysis of the sequence revealed two open reading frames, one of which encoded the aminoacylase. B. stearothermophilus aminoacylase, produced in E. coli, was purified to near homogeneity in three steps, one of which took advantage of the intrinsic thermostability of the enzyme. The enzyme exists as homotetramer of 43-kDa subunits as shown by cross-linking experiments. The deacetylating capacity of purified aminoacylase varies considerably depending on the nature of the amino acid residue in the substrate. The enzyme hydrolyzes N-acyl derivatives of aromatic amino acids most efficiently. Comparison of the predicted amino acid sequence of B. stearothermophilus aminoacylase with those of eubacterial acetylornithine deacylase, succinyldiaminopimelate desuccinylase, carboxypeptidase G2, and eukaryotic aminoacylase I suggests a common origin for these enzymes.     

Characterization of a novel DNA primase from the Salmonella typhimurium bacteriophage SP6

The gene for the DNA primase encoded by Salmonella typhimurium bacteriophage SP6 has been cloned and expressed in Escherichia coli and its 74-kDa protein product purified to homogeneity. The SP6 primase is a DNA-dependent RNA polymerase that synthesizes short oligoribonucleotides containing each of the four canonical ribonucleotides. GTP and CTP are both required for the initiation of oligoribonucleotide synthesis. In reactions containing only GTP and CTP, SP6 primase incorporates GTP at the 5'-end of oligoribonucleotides and CMP at the second position. On synthetic DNA templates, pppGpC dinucleotides are synthesized most rapidly in the presence of the sequence 5'-GCA-3'. This trinucleotide sequence, containing a cryptic dA at the 3'-end, differs from other known bacterial and phage primase recognition sites. SP6 primase shares some properties with the well-characterized E. colibacteriophage T7 primase. The T7 DNA polymerase can use oligoribonucleotides synthesized by SP6 primase as primers for DNA synthesis. However, oligoribonucleotide synthesis by SP6 primase is not stimulated by either the E. coli- or the T7-encoded ssDNA binding protein. An amino acid sequence alignment of the SP6 and T7 primases, which share only 22.4% amino acid identity, indicates amino acids likely critical for oligoribonucleotide synthesis as well as a putative Cys(3)His zinc finger motif that may be involved in DNA binding.     

On the DNA binding protein II from Bacillus stearothermophilus. II. The amino acid sequence and its relation to those of homologous proteins from other prokaryotes

The complete amino acid sequence of DNA binding protein II from Bacillus stearothermophilus has been determined. The protein contains 90 amino acid residues and has a calculated Mr of 9716. The sequence is compared to homologous molecules from Escherichia coli, Thermoplasma acidophilum, and Pseudomonas aeruginosa (where only a partial sequence is available). The B. stearothermophilus molecule has 58% and 59% residues identical with the two forms of the E. coli protein and 32% with the T. acidophilum protein. There are totally conserved residues at positions 46-48 and 61-65 with an intervening cluster of basic amino acids in all four proteins.     

Structure of the zinc-binding domain of Bacillus stearothermophilus DNA primase

BACKGROUND: DNA primases catalyse the synthesis of the short RNA primers that are required for DNA replication by DNA polymerases. Primases comprise three functional domains: a zinc-binding domain that is responsible for template recognition, a polymerase domain, and a domain that interacts with the replicative helicase, DnaB. RESULTS: We present the crystal structure of the zinc-binding domain of DNA primase from Bacillus stearothermophilus, determined at 1.7 A resolution. This is the first high-resolution structural information about any DNA primase. A model is discussed for the interaction of this domain with the single-stranded DNA template. CONCLUSIONS: The structure of the DNA primase zinc-binding domain confirms that the protein belongs to the zinc ribbon subfamily. Structural comparison with other nucleic acid binding proteins suggests that the beta sheet of primase is likely to be the DNA-binding surface, with conserved residues on this surface being involved in the binding and recognition of DNA.     

Nucleotide sequence, heterologous expression and novel purification of DNA ligase from Bacillus stearothermophilus(1).

The gene for DNA ligase (EC 6.5.1.2) from thermophilic bacterium Bacillus stearothermophilus NCA1503 has been cloned and the complete nucleotide sequence determined. The ligase gene encodes a protein 670 amino acids in length. The gene was overexpressed in Escherichia coli and the enzyme has been purified to homogeneity. Preliminary characterisation confirms that it is a thermostable, NAD(+)-dependent DNA ligase.     

Cloning and sequence analysis of the genes encoding the alpha and beta subunits of the E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

A 4175-bp EcoRI fragment of DNA that encodes the alpha and beta chains of the pyruvate dehydrogenase (lipoamide) component (E1) of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. Open reading frames (pdhA, pdhB) corresponding to the E1 alpha subunit (368 amino acids, Mr 41,312, without the initiating methionine residue) and E1 beta subunit (324 amino acids, Mr 35,306, without the initiating methionine residue) were identified and confirmed with the aid of amino acid sequences determined directly from the purified polypeptide chains. The E1 beta gene begins just 3 bp downstream from the E1 alpha stop codon. It is followed, after a longer gap of 73 bp, by the start of another but incomplete open reading frame that, on the basis of its known amino acid sequence, encodes the dihydrolipoyl acetyltransferase (E2) component of the complex. All three genes are preceded by potential ribosome-binding sites and the gene cluster is located immediately downstream from a region of DNA showing numerous possible promoter sequences. The E1 alpha and E1 beta subunits of the B. stearothermophilus pyruvate dehydrogenase complex exhibit substantial sequence similarity with the E1 alpha and E1 beta subunits of pyruvate and branched-chain 2-oxo-acid dehydrogenase complexes from mammalian mitochondria and Pseudomonas putida. In particular, the E1 alpha chain contains the highly conserved sequence motif that has been found in all enzymes utilizing thiamin diphosphate as cofactor.     

Mouse primase p49 subunit molecular cloning indicates conserved and divergent regions

Primase is a specialized RNA polymerase that synthesizes RNA primers for initiation of DNA synthesis. A full cDNA clone of the p49 subunit of mouse primase, a heterodimeric enzyme, has been isolated using a primase p49-specific polyclonal antibody to screen a lambda gt11 mouse cDNA expression library. The cDNA indicated the subunit is a 417-amino acid polypeptide with a calculated molecular mass of 49,295 daltons. The p49 mRNA is approximately 1500 nucleotides long with a 5'-untranslated region of 74 nucleotides and a 3'-untranslated region of 200 nucleotides. Comparison with a similar sized primase subunit from yeast showed highly conserved amino acid sequences in the N-terminal halves of the polypeptides and included a potential metal-binding domain suggesting the functional importance of this region for DNA binding. In contrast, the 3' portion of the cDNA has rapidly diverged in nucleotide sequence, as primase mRNA can be detected in mouse and rat cells with a 3' probe (including coding and noncoding) but not in RNA from hamster or human cells. A full-length cDNA probe detected mRNA from hamster and human cell lines, indicating a conserved 5' portion and divergent 3' region of the expressed gene. The rapid divergence may be related to the species-specific protein interactions found for the DNA polymerase alpha-primase complex. The mRNA is detected in proliferating but not in quiescent cells consistent with its function in DNA replication.     

Gene cloning and sequence determination of leucine dehydrogenase from Bacillus stearothermophilus and structural comparison with other NAD(P)+-dependent dehydrogenases

The gene for leucine dehydrogenase (EC 1.4.1.9) from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The selection for the cloned gene was based upon activity staining of the replica printed E. coli cells. A transformant showing high leucine dehydrogenase activity was found to carry an about 9 kilobase pair plasmid, which contained 4.6 kilobase pairs of B. stearothermophilus DNA. The nucleotide sequence including the 1287 base pair coding region of the leucine dehydrogenase gene was determined by the dideoxy chain termination method. The translated amino acid sequence was confirmed by automated Edman degradation of several peptide fragments produced from the purified enzyme by trypsin digestion. The polypeptide contained 429 amino acid residues corresponding to the subunit (Mr 49,000) of the hexameric enzyme. Comparison of the amino acid sequence of leucine dehydrogenase with those of other pyridine nucleotide dependent oxidoreductases registered in a protein data bank revealed significant sequence similarity, particularly between leucine and glutamate dehydrogenases, in the regions containing the coenzyme binding domain and certain specific residues with catalytic importance.     

Twinkle, the Mitochondrial Replicative DNA Helicase, Is Widespread in the Eukaryotic Radiation and May Also Be the Mitochondrial DNA Primase in Most Eukaryotes

Recently, the human protein responsible for replicative mtDNA helicase activity was identified and designated Twinkle. Twinkle has been implicated in autosomal dominant progressive external ophthalmoplegia (adPEO), a mitochondrial disorder characterized by mtDNA deletions. The Twinkle protein appears to have evolved from an ancestor shared with the bifunctional primase-helicase found in the T-odd bacteriophages. However, the question has been raised as to whether human Twinkle possesses primase activity, due to amino acid sequence divergence and absence of a zinc-finger motif thought to play an integral role in DNA binding. To date, a primase protein participating in mtDNA replication has not been identified in any eukaryote. Here we investigate the wider phylogenetic distribution of Twinkle by surveying and analyzing data from ongoing EST and genome sequencing projects. We identify Twinkle homologues in representatives from five of six major eukaryotic assemblages (“supergroups”) and present the sequence of the complete Twinkle gene from two members of Amoebozoa, a supergroup of amoeboid protists at the base of the opisthokont (fungal/metazoan) radiation. Notably, we identify conserved primase motifs including the zinc finger in all Twinkle sequences outside of Metazoa. Accordingly, we propose that Twinkle likely serves as the primase as well as the helicase for mtDNA replication in most eukaryotes whose genome encodes it, with the exception of Metazoa. [Reviewing Editior: Dr. Rüdiger Cerff]     

Thermostable alanine racemase from Bacillus stearothermophilus: DNA and protein sequence determination and secondary structure prediction

The nucleotide sequence of the alanine racemase (EC 5.1.1.1) gene from a thermophile, Bacillus stearothermophilus, was determined by the dideoxy chain termination method with universal and synthetic site-specific primers. The amino acid sequence of the enzyme predicted from the nucleotide sequence was confirmed by peptide sequence information derived from the N-terminal amino acid residues and several tryptic fragments. The alanine racemase gene consists of 1158 base pairs encoding a protein of 386 amino acid residues; the molecular weight of the apoenzyme is estimated as 43,341. The racemase gene of B. stearothermophilus has a closely similar size (1158 vs 1167 base pairs) to that of the gene of a mesophile, B. subtilis, but shows a higher preference for codons ending in G or C. A comparison of the amino acid sequence with those of Bacillus subtilis and Salmonella typhimurium dadB and alr enzymes revealed overall sequence homologies of 31-54%, including an identical octapeptide bearing the pyridoxal 5'-phosphate binding site. Although the residues common in the four racemases are not continuously arrayed, these constitute distinct domains and their hydropathy profiles are very similar. The secondary structure of B. stearothermophilus alanine racemase was predicted from the results obtained by theoretical analysis and circular dichroism measurement.     

Complete nucleotide sequence of a thermophilic alpha-amylase gene: homology between prokaryotic and eukaryotic alpha-amylases at the active sites

The nucleotide sequence of a thermophilic, liquefying alpha-amylase gene cloned from B. stearothermophilus was determined. The NH2-terminal amino acid sequence analysis of the B. stearothermophilus alpha-amylase confirmed that the reading frame of the gene consisted of 1,644 base pairs (548 amino acids). The B. stearothermophilus alpha-amylase had a signal sequence of 34 amino acids, which was cleaved at exactly the same site in E. coli. The mature enzyme contained two cysteine residues, which might play an important role in maintenance of a stable protein conformation. Comparison of the amino acid sequence inferred from the B. stearothermophilus alpha-amylase gene with those inferred from other bacterial liquefying alpha-amylase genes and with the amino acid sequences of eukaryotic alpha-amylases showed three homologous sequences in the enzymatically functional regions.     

Amino acid and nucleotide sequence homologies among E. coli RNA polymerase core enzyme subunits, DNA primase, elongation factor Tu, F1-ATPase alpha, ribosomal protein L3, DNA polymerase I, T7 phage DNA polymerase, and MS2 phage RNA replicase beta subunit

The 330 residue-long N-terminal domains (NTDs) of beta and beta' subunits of the Escherichia coli RNA polymerase (RPase) core enzyme were found to be significantly homologous to the entire length of its alpha subunit. The C-terminal domains (CTDs) of the RPase beta subunit and DNA primase (dnaG protein) were not only strongly homologous to each other but also considerably homologous to the RPase alpha, suggesting that an alpha subunit-like enzyme must have been commonly ancestral to core enzyme subunits and primase. The N-terminal region (NTR) of RPase alpha was also found to show significant homologies with NTRs of the E. coli EF-Tu and F1-ATPase alpha subunit, and a possible weak homology with ribosomal protein L3. A most important finding was that the C-terminal regions (CTRs) of DNA polymerase (DPase) I, T7 phage DPase and MS2 phage RNA replicase beta subunit are closely homologous with one another. These CTRs showed considerable homologies to RPase alpha NTD and RPase beta CTD. These conclusions are based on statistical evaluations of homologies in base and/or amino acid sequence alignments.     

Cloning, expression, and isolation of the mannitol transport protein from the thermophilic bacterium Bacillus stearothermophilus.

A mannitol phosphotransferase system (PTS) was identified in Bacillus stearothermophilus by in vitro complementation with Escherichia coli EI, HPr, and IIA(Mtl). Degenerate primers based on regions of high amino acid similarity in the E. coli and Staphylococcus carnosus EII(Mt1) were used to develop a digoxigenin-labeled probe by PCR. Using this probe, we isolated three overlapping DNA fragments totaling 7.2 kb which contain the genes mtlA, mtlR, mtlF, and mtlD, encoding the mannitol IICB,a regulator, IIA, and a mannitol-1-phosphate dehydrogenase, respectively. The mtl4 gene consists of 1,413 bp coding for a 471-amino-acid protein with a calculated mass of 50.1 kDa. The amino acid sequence shows high similarity with the sequence of IICB(Mtl) of S. carnosus and the IICB part of the IICBA(Mtl)s of E. coli and B. subtilis. The enzyme could be functionally expressed in E. coli by placing it behind the strong tac promoter. The rate of thermal inactivation at 60 degrees C of B. stearothermophilus HCB(Mt1) expressed in E. coli was two times lower than that of E. coli IICB(Mtl). IICB(Mtl) in B. stearothermophilus is maximally active at 85 degrees C and thus very thermostable. The enzyme was purified on Ni-nitrilotriacetic acid resin to greater than 95% purity after six histidines were fused to the C-terminal part of the transporter.     

Nucleotide sequence and linkage map position of the secX gene in maize chloroplast and evidence that it encodes a protein belonging to the 50S ribosomal subunit

The nucleotide sequence of the segment of maize chloroplast DNA lying between the map coordinate positions 32.59 and 32.98 Kb and containing the secX gene has been determined. The derived amino acid sequence of maize chloroplast secX is 95%, 87% and 62% identical to the corresponding derived amino acid sequences from two plant chloroplasts and Escherichia coli, respectively. It is also 70% identical to the experimentally determined amino acid sequence of a protein isolated from Bacillus stearothermophilus ribosomes. Separation of the 50S ribosomal subunit proteins of E. coli by reversed phase HPLC gave a peak which contained pure secX protein, as determined by N-terminal amino acid sequencing. Spinach chloroplast 50S subunit proteins separated by HPLC also gave a peak corresponding to pure secX protein. From these results we conclude that the secX gene in E. coli and in plant chloroplasts encodes a small (37-38 amino acid residues) ribosomal protein belonging to the 50S subunit. The same conclusion has been reached recently by A. Wada with respect to E. coli secX. In agreement with Wada, we name the secX protein L36. Its chloroplast gene is designated rpL36.