Lysyl-tRNA synthetase of Bacillus stearothermophilus molecular cloning and expression of the gene

The gene of the lysyl-tRNA synthetase of Bacillus stearothermophilus NCA1503 was cloned and sequenced. The gene consists of 1485 bp nucleotides commencing with an ATG start codon and ending with a TAA stop codon, and encodes a polypeptide of 493 amino acids. The recombinant enzymes were expressed in E. coli using an expression plasmid containing the T7 RNA polymerase/promoter.     

The araBAD operon of Salmonella typhimurium LT2. II. Nucleotide sequence of araA and primary structure of its product, L-arabinose isomerase

The nucleotide sequence of gene araA of Salmonella typhimurium LT2 has been determined. The gene encodes an L-arabinose isomerase (EC 5.3.1.4) of 500 amino acid residues with a calculated Mr of 55814. The ATG start codon of araA is 10 bp distal to the TAA termination codon of araB. A presumed ribosome-binding site (RBS) "TAAGGA" 7 bp from the ATG codon overlaps the stop codon of araB. L-Arabinose isomerase was purified and the amino acid composition is in agreement with that predicted from the DNA sequence. The NH2-terminus of the protein is modified as the sequence cannot be analyzed by the automated Edman degradation. Amino acid composition analyses of both NH2-terminal and C-terminal cyanogen bromide (CNBr) cleaved peptides and partial amino acid sequence of the C-terminal peptide are consistent with the deduced amino acid sequence.     

Sequence and analysis of the DNA encoding protective antigen of Bacillus anthracis

The nucleotide sequence of the protective antigen (PA) gene from Bacillus anthracis and the 5' and 3' flanking sequences were determined. PA is one of three proteins comprising anthrax toxin; and its nucleotide sequence is the first to be reported from B. anthracis. The open reading frame (ORF) is 2319 bp long, of which 2205 bp encode the 735 amino acids of the secreted protein. This region is preceded by 29 codons, which appear to encode a signal peptide having characteristics in common with those of other secreted proteins. A consensus TATAAT sequence was located at the putative -10 promoter site. A Shine-Dalgarno site similar to that found in genes of other Bacillus sp. was located 7 bp upstream from the ATG start codon. The codon usage for the PA gene reflected its high A + T (69%) base composition and differed from those of genes for bacterial proteins from most other sequences examined. The TAA translation stop codon was followed by an inverted repeat forming a potential termination signal. In addition, a 192-codon ORF of unknown significance, theoretically encoding a 21.6-kDa protein, preceded the 5' end of the PA gene.     

Primary structure of the oligo-1,6-glucosidase of Bacillus cereus ATCC7064 deduced from the nucleotide sequence of the cloned gene

The gene coding for Bacillus cereus ATCC7064 (mesophile) oligo-1,6-glucosidase was cloned within a 2.8-kb SalI-EcoRI fragment of DNA, using the plasmid pUC19 as a vector and Escherichia coli C600 as a host. E. coli C600 bearing the hybrid plasmid pBCE4 accumulated oligo-1,6-glucosidase in the cytoplasm. The cloned enzyme coincided absolutely with B. cereus oligo-1,6-glucosidase in its Mr (65,000), in its electrophoretic behavior on a polyacrylamide gel with or without sodium dodecyl sulfate, in its isoelectric point (4.5), in the temperature dependence of its stability and activity, and in its antigenic determinants. The nucleotide sequence of B. cereus oligo-1,6-glucosidase gene and its flanking regions was determined with both complementary strands of DNA (each 2838 nucleotides). The gene consisted of an open reading frame of 1674 bp commencing with a ATG start codon and followed by a TAA stop codon. The amino acid sequence deduced from the nucleotide sequence predicted a protein of 558 amino acid residues with a Mr of 66,010. The amino acid composition and Mr were comparable with those of B. cereus oligo-1,6-glucosidase. The predicted N-terminal sequence of 10 amino acid residues agreed completely with that of the cloned ligo-1,6-glucosidase. The deduced amino acid sequence of B. cereus oligo-1,6-glucosidase was 72% and 42% similar to those from Bacillus thermoglucosidasius KP1006 (DSM2542, obligate thermophile) oligo-1,6-glucosidase and from Saccharomyces carlsbergensis CB11 alpha-glucosidase, respectively. Predictions of protein secondary structures along with amino acid sequence alignments demonstrated that B. cereus oligo-1,6-glucosidase may take the similar (alpha/beta)8-barrel super-secondary structure, a barrel of eight parallel beta-strands surrounded by eight alpha-helices, in its N-terminal active site domain as S. carlsbergensis alpha-glucosidase and Aspergillus oryzae alpha-amylase.     

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.     

Biosynthesis of bacterial glycogen: primary structure of Salmonella typhimurium ADPglucose synthetase as deduced from the nucleotide sequence of the glgC gene.

The nucleotide sequence of a 1.4-kilobase-pair fragment containing the Salmonella typhimurium LT2 glgC gene coding for ADPglucose synthetase was determined. The glgC structural gene contains 1,293 base pairs, having a coding capacity of 431 amino acids. The amino acid sequence deduced from the nucleotide sequence shows that the molecular weight of ADPglucose synthetase is 45,580. Previous results of the total amino acid composition analysis and amino acid sequencing (M. Lehmann and J. Preiss, J. Bacteriol. 143:120-127, 1980) of the first 27 amino acids from the N terminus agree with that deduced from nucleotide sequencing data. Comparison of the Escherichia coli K-12 and S. typhimurium LT2 ADPglucose synthetase shows that there is 80% homology in their nucleotide sequence and 90% homology in their deduced amino acid sequence. Moreover, the amino acid residues of the putative allosteric sites for the physiological activator fructose bisphosphate (amino acid residue 39) and inhibitor AMP (amino acid residue 114) are identical between the two enzymes. There is also extensive homology in the putative ADPglucose binding site. In both E. coli K-12 and S. typhimurium LT2, the first base of the translational start ATG of glgA overlaps with the third base TAA stop codon of the glgC gene.     

Structure of the beta-1,3-1,4-glucanase gene of Bacillus macerans: homologies to other beta-glucanases

Summary The nucleotide sequence of an 852 base pair (bp) DNA fragment containing the entire gene coding for thermostable beta- 1,3-1,4-glucanase ofBacillus macerans has been determined. ThebglM gene comprises an open reading frame (ORF) of 711 by (237 codons) starting with ATG at position 93 and extending to the translational stop codon TAA at position 804. The deduced amino acid sequence of the mature protein shows 70% homology to published sequences of mesophilic beta- 1,3-1,4-glucanases fromB. subtilis andB. amyloliquefaciens. The sequence coding for mature beta-glucanase is preceded by a putative signal peptide of 25 amino acid residues, and a sequence resembling a ribosome-binding site (GGAGG) before the initiation codon. By contrast with the processed protein, the N-terminal amino acid sequence constituting the putative leader peptide bears no or only weak homology to signal peptides of mesophilicBacillus endo-beta-glucanases. TheB. macerans signal peptide appears to be functional in exporting the enzyme to the periplasm inE. coli. More than 50% of the whole glucanase activity was localized in the periplasmic space and in the supernatant. Whereas homology to endo-1,4-beta-glucanases is completely lacking, a weak amino acid homology between the sequence surrounding the active site of phage T4 lysozyme and a sequence spanning residues 126 through 161 ofB. macerans endo-beta-glucanase could be identified.     

Nucleotide sequence of the celC gene encoding endoglucanase C of Clostridium thermocellum

The nucleotide sequence of the cellulase gene celC, encoding endoglucanase C of Clostridium thermocellum, has been determined. The coding region of 1032 bp was identified by comparison with the N-terminal amino acid (aa) sequence of endoglucanase C purified from Escherichia coli. The ATG start codon is preceded by an AGGAGG sequence typical of ribosome-binding sites in Gram-positive bacteria. The derived amino acid sequence corresponds to a protein of Mr 40,439. Amino acid analysis and apparent Mr of endoglucanase C are consistent with the amino acid sequence as derived from the DNA sequencing data. A proposed N-terminal 21-aa residue leader (signal) sequence differs from other prokaryotic signal peptides and is non-functional in E. coli. Most of the protein bears no resemblance to the endoglucanases A, B, and D of the same organism. However, a short region of homology between endoglucanases A and C was identified, which is similar to the established active sites of lysozymes and to related sequences of fungal cellulases.     

Identification of an alternative translation initiation site for the Pantoea ananatis lycopene cyclase (crtY) gene in E. coli and its evolutionary conservation

Previous sequence analyses of the lycopene cyclase gene (crt Y) from Pantoea ananatis revealed that translation of its protein product in Escherichia coli began at the ATG start codon. We found, however, that this enzyme could also be produced in E. coli without the ATG start codon present. Results of experiments using crt Y mutants revealed that a GTG (Val) sequence, located in-frame and 24 bp downstream of the ATG, could act as a potential start codon. Additionally, a point-mutated GTA (Val), replaced from alternative GTG start codon, also displayed its potential as a start codon although the strength as a translation initiation codon was considerably weak. This finding suggests that non-ATG codons, especially one base pairing with the anticodon (3'-UAC-5') in fMet-tRNA, might be also able to function as start codon in translation process. Furthermore, amino acid sequence alignment of lycopene cyclases from different sources suggested that a Val residue located within the N-terminus of these enzymes might be used as an alternative translation initiation site. In particular, presence of a conserved Asp, located in-frame and 12 bp upstream of potential start codon, supports this assumption in view of the fact that Asp (GAT or GAC) can function as part of the Shine-Dalgano sequence (AGGAGG).     

Characterization of the ksgA gene of Escherichia coli determining kasugamycin sensitivity

In the plasmid pUC8ksgA7, the coding region of the ksgA gene is preceded by the lac promoter (Plac) and a small open reading frame (ORF). This ORF of 15 codons is composed of nucleotides derived from the lacZ gene, a multiple cloning site and the ksgA gene itself. The reading frame begins with the ATG initiation codon of lacZ and ends a few nucleotides beyond the ATG start codon of ksgA. The ksgA gene is not preceded by a Shine-Dalgarno (SD) signal. Cells transformed with pUC8ksgA7 produce active methylase, the product of the ksgA gene. Introduction of an in-phase TAA stop codon in the small ORF abolishes methylase production in transformed cells. On the plasmid pUC8ksgA5, which contains the entire ksgA region, the promoter of the ksgA gene was found to reside in a 380 base pair Bgl1-Pvu2 restriction fragment, partly overlapping the ksgA gene, by two independent methods. Cloning of this fragment in front of the galK gene in plasmid pKO1 stimulates galactokinase activity in transformants and its insertion into the expression vector pKL203 makes beta-galactosidase synthesis independent of the presence of Plac. The sequence of the Bgl1-Pvu2 fragment was determined and a putative promoter sequence identified. An SD signal could not be distinguished at a proper distance upstream from the ksgA start codon. Instead, an ORF of 13 codons starting with ATG in tandem with an SD signal and ending 4 codons ahead of the ksgA gene was identified. This suggests that translation of the ORF is required for expression of the ksgA gene.(ABSTRACT TRUNCATED AT 250 WORDS)