Molecular cloning and characterization of the aroD gene encoding 3-dehydroquinase from Salmonella typhi

The aroD gene from Salmonella typhi, encoding 5-dehydroquinate hydrolyase (3-dehydroquinase), has been cloned into Escherichia coli and the DNA sequence determined. The aroD gene was isolated from a cosmid gene bank by complementation of an S. typhimurium aroD mutant. Analysis of the DNA sequence revealed the presence of an open reading frame capable of encoding a protein of 252 amino acids with a calculated Mr of 27706. Comparison of the deduced S. typhi 3-dehydroquinase protein sequence with that elucidated for E. coli revealed 69% homology. Alignment of the S. typhi sequence and equivalent Aspergillus nidulans and Saccharomyces cerevisiae sequences showed that homology was lower, at 24%, but still significant. Use of a minicell expression system demonstrated that a polyclonal antibody raised against E. coli 3-dehydroquinase cross-related with its S. typhi counterpart.     

Cloning of dapD, aroD and asd of Leptospira interrogans serovar icterohaemorrhagiae, and nucleotide sequence of the asd gene.

Metabolites such as diaminopimelate and some aromatic derivatives, not synthesized in mammalian cells, are essential for growth of bacteria. As a first step towards the design of a new human live vaccine that uses attenuated strains of Leptospira interrogans, the asd, aroD and dapD genes, encoding aspartate beta-semialdehyde dehydrogenase, 3-dehydroquinase and tetrahydrodipicolinate N-succinyltransferase, respectively, were cloned by complementation of Escherichia coli mutants. The complete nucleotide sequence of the asd gene was determined and found to contain an open reading frame capable of encoding a protein of 349 amino acids with a calculated Mr of 38,007. Comparison of this deduced L. interrogans aspartate beta-semialdehyde dehydrogenase amino acid sequence with those of the same enzyme from Saccharomyces cerevisiae and Corynebacterium glutamicum revealed 46% and 36% identity, respectively. By contrast, the identity between the L. interrogans enzyme and the Streptococcus mutans or E. coli enzymes was less than 31%. Highly conserved sequences within aspartate semialdehyde dehydrogenase from the five organisms were observed at the amino and carboxyl termini, and around the cysteine of the active site.     

Cloning, expression, sequence analysis and biochemical characterization of an autolytic amidase of Bacillus subtilis 168 trpC2.

By use of a functional assay for activity, an autolysin structural gene was cloned on a 3 kb EcoRI DNA fragment from a lambda gt11 expression library of Bacillus subtilis 168 trpC2 genomic DNA. Sequencing of the fragment showed five open reading frames, the central one of which encoded the lytic enzyme as found by subclone activity mapping and its homology to a recently sequenced autolysin gene. The protein had a deduced sequence of 272 amino acids and a molecular mass of 29957 Da. When expressed in Escherichia coli DH5 alpha, the protein was processed to a 21 kDa form, as estimated by renaturing SDS-PAGE. The autolysin was an N-acetylmuramyl-L-alanine amidase and its activity was MgCl2-dependent (20 mM optimum) and LiCl-sensitive. The enzyme could bind to and hydrolyse a wide range of peptidoglycan substrates isolated from Gram-positive bacteria; the binding was also MgCl2-dependent. Initial mapping experiments located the autolysin gene near aroD on the B. subtilis 168 chromosome.     

Cloning of a sequence of Aquaspirillum magnetotacticum that complements the aroD gene of Escherichia coli

A 2 kb DNA fragment isolated from a cosmid library of Aquaspirillum magnetotacticum strain MS-1 complements the aromatic-metabolite requirements and iron-uptake deficiencies of Escherichia coli and Salmonella typhimurium strains that lack a functional aroD (biosynthetic dehydrodquinase) sequence. All recombinant cosmids selected for their aroD complementation property carry this sequence. No DNA sequence homology has, however, been detected by Southern hybridization between the cloned fragment and the aroD gene of E. coli or the qa2 (catabolic dehydroquinase) gene of Neurospora crassa.     

Biosynthesis of bacterial glycogen. Primary structure of Escherichia coli 1,4-alpha-D-glucan:1,4-alpha-D-glucan 6-alpha-D-(1, 4-alpha-D-glucano)-transferase as deduced from the nucleotide sequence of the glg B gene

The nucleotide sequence of the glg B gene, coding for branching enzyme (EC 2.4.1.18), was elucidated. It consists of 2181 base pairs specifying a protein of 727 amino acids. The deduced amino acid sequence was consistent with the amino acid analysis that was obtained with the pure protein as well as with the molecular weight determined from sodium dodecyl sulfate-gel electrophoresis. The deduced amino acid sequence was also consistent with the amino-terminal amino acid sequence and the amino acid sequence analysis of various peptides obtained from CNBr degradation of purified branching enzyme.     

Nucleotide sequence of the G6-amylase gene from alkalophilic Bacillus sp. H-167

The nucleotide sequence of the G6-amylase gene from alkalophilic Bacillus sp. H-167 was determined. The open reading frame of the gene consisted of 2865 base pairs, encoding 955 amino acids. The NH2-terminal amino acid sequence analysis of the G6-amylase indicated that the enzyme had a single peptide of 33 amino acid residues and the mature enzyme was composed of 922 amino acids, giving a molecular mass of 102,598. Identity of the NH2-terminal amino acid sequences among each component of the multiform G6-amylase suggested the proteolytic processing of the COOH-terminal side of the enzyme. The DNA sequence and the deduced amino acid sequence of the G6-amylase gene showed no homology with those of other bacterial alpha-amylases although the consensus amino acid sequences of the active center were well conserved.     

The primary structure of phosphoenolpyruvate carboxylase of Escherichia coli. Nucleotide sequence of the ppc gene and deduced amino acid sequence

The nucleotide sequence of the ppc gene, the structural gene for phosphoenolpyruvate carboxylase [EC 4.1.1.31], of Escherichia coli K-12 was determined. The gene codes for a polypeptide comprising 883 amino acid residues with a calculated molecular weight of 99,061. The amino acid sequence deduced from the nucleotide sequence was entirely consistent with the protein chemical data obtained with the purified enzyme, including the NH2- and COOH-terminal sequences and amino acid composition. The coding region is preceded by two putative ribosome binding sites, and is followed closely by a good representative of rho-independent terminator. The codon usage in the ppc gene suggests a moderate expression of the gene. The secondary structure of the enzyme was predicted from the deduced amino acid sequence.     

Nucleotide sequence of the G6-amylase gene from alkalophilic Bacillus sp. H-167

The nucleotide sequence of the G₆-amylase gene from alkalophilic Bacillus sp. H-167 was determined. The open reading frame of the gene consisted of 2865 base pairs, encoding 955 amino acids. The NH₂-terminal amino acid sequence analysis of the G₆-amylase indicated that the enzyme had a single peptide of 33 amino acid residues and the mature enzyme was composed of 922 amino acids, giving a molecular mass of 102598. Identity of the NH₂-terminal amino acid sequences among each component of the multiform G₆-amylase suggested the proteolytic processing of the COOH-terminal side of the enzyme. The DNA sequence and the deduced amino acid sequence of the G₆-amylase gene showed no homology with those of other bacterial α-amylases although the consensus amino acid sequences of the active center were well conserved.     

Purification, characterization, and molecular cloning of a chitinase from the seeds of Benincasa hispida

A chitinase was purified from the seeds of Benincasa hispida, a medicinal plant also called white gourd, and a member of the Cucurbitaceae family. Purification was done by using a procedure consisting of only two fractionation steps: an acid denaturation step followed by ion-exchange chromatography. The sequence of the N-terminal forty amino acid residues was analyzed and the sequence indicated that the enzyme is a class III chitinase. The enzyme, which is a basic chitinase, is one of at least five chitinases detected in the seed extract of B. hispida. Like other class III chitinases, this enzyme also has lysozyme activity. A genomic clone of the gene encoding the enzyme was isolated and sequenced. The gene has the potential to encode a protein of 301 amino acid residues. The deduced amino acid sequence of the protein, as expected from the N-terminal amino acid sequence, shares high degrees of similarity with other class III chitinases.     

The Escherichia coli pldC gene encoding lysophospholipase L(1) is identical to the apeA and tesA genes encoding protease I and thioesterase I, respectively.

We deduced the amino acid sequence of Escherichia coli lysophospholipase L(1) by determining the nucleotide sequence of the pldC gene encoding this enzyme. The translated protein was found to contain 208 amino acid residues with a hydrophobic leader sequence of 26 amino acid residues. The molecular weight of the purified enzyme (20,500) was in good agreement with the predicted size (20,399) of the processed protein. A search involving a data bank showed that the nucleotide sequence of the pldC gene was identical to those of the apeA and tesA genes encoding protease I and thioesterase I, respectively. Consistent with the identity of the pldC gene with these two genes, the enzyme purified from E. coli overexpressing the pldC gene showed both protease I and thioesterase I activities.     

Cloning and sequence analysis of the gene encoding Lactococcus lactis malolactic enzyme: Relationships with malic enzymes

Abstract Malolactic enzyme is the key enzyme in the degradation of L-malic acid by lactic acid bacteria. Using degenerated primers designed from the first 20 N-terminal amino acid sequence of lactococcal malolactic enzyme, a 60-bp DNA fragment containing part of the mleS gene was amplified from Lactococcus lactis in a polymerase chain reaction. This specific probe was used to isolate two contiguous fragments covering the gene as a whole. The 1.9-kb region sequenced contains an open reading frame of 1623 bp, coding a putative protein of 540 amino acids. The deduced amino acid sequence reveals that lactococcal putative protein (Mlep) is highly homologous to the malic enzyme of other organisms. Expression of the mleS gene in Escherichia coli results in malolactic activity.     

Cloning, sequencing, and expression of the meso-diaminopimelate dehydrogenase gene from Bacillus sphaericus

The gene encoding the meso-diaminopimelate dehydrogenase of Bacillus sphaericus was cloned into E. coli cells and its complete DNA sequence was determined. The meso-diaminopimelate dehydrogenase gene consisted of 978 nucleotides and encoded 326 amino acid residues corresponding to the subunit of the dimeric enzyme. The amino acid sequence deduced from the nucleotide sequence of the enzyme gene of B. sphaericus showed 50% identity with those of the enzymes from Corynebacterium glutamicum and Brevibacterium flavum. The enzyme gene from B. sphaericus was highly expressed in E. coli cells. We purified the enzyme to homogeneity from a transformant with 76% recovery. The N-terminal amino acid of both the enzyme from B. sphaericus and the transformant were serine, indicating that the N-terminal methionine is removed by post-translational modification in B. sphaericus and E. coli cells.     

Dihydrofolate reductase of the extremely halophilic archaebacterium Halobacterium volcanii. The enzyme and its coding gene

Halobacterium volcanii mutants that are resistant to the dihydrofolate reductase inhibitor trimethoprim contain DNA sequence amplifications. This paper describes the cloning and nucleic acid sequencing of the amplified DNA sequence of the H. volcanii mutant WR215. This sequence contains an open reading frame that codes for an amino acid sequence that is homologous to the amino acid sequences of dihydrofolate reductases from different sources. As a result of the gene amplification, the trimethoprim-resistant mutant overproduces dihydrofolate reductase. This enzyme was purified to homogeneity using ammonium sulfate-mediated chromatographies. It is shown that the enzyme comprises 5% of the cell protein. The amino acid sequence of the first 15 amino acids of the enzyme fits the coding sequence of the gene. Preliminary biochemical characterization shows that the enzyme is unstable at salt concentrations lower than 2 M and that its activity increases with increase in the KCl or NaCl concentrations.     

Isolation and sequencing of a genomic clone encoding aspartic proteinase of Rhizopus niveus.

A gene encoding Rhizopus niveus aspartic proteinase was isolated from an R. niveus genomic library by using oligonucleotides probes corresponding to its partial amino acid sequence, and its nucleotide sequence was determined. By comparing its deduced amino acid sequence with the amino acid sequence of rhizopuspepsin (5, 26), we concluded that the R. niveus aspartic proteinase gene has an intron within its coding region and that it has a preproenzyme sequence of 66 amino acids upstream of the mature enzyme of 323 amino acids.     

The overexpression, purification and complete amino acid sequence of chorismate synthase from Escherichia coli K12 and its comparison with the enzyme from Neurospora crassa.

The enzyme chorismate synthase was purified in milligram quantities from an overproducing strain of Escherichia coli. The amino acid sequence was deduced from the nucleotide sequence of the aroC gene and confirmed by determining the N-terminal amino acid sequence of the purified enzyme. The complete polypeptide chain consists of 357 amino acid residues and has a calculated subunit Mr of 38,183. Cross-linking and gel-filtration experiments show that the enzyme is tetrameric. An improved purification of chorismate synthase from Neurospora crassa is also described. Cross-linking and gel-filtration experiments on the N. crassa enzyme show that it is also tetrameric with a subunit Mr of 50,000. It is proposed that the subunits of the N. crassa enzyme are larger because they contain a diaphorase domain that is absent from the E. coli enzyme.     

Bacillus licheniformis APase I gene promoter: a strong well-regulated promoter in B. subtilis

The 5' regulatory region and the portion of the structural gene coding for the amino-terminal sequence of alkaline phosphatase I (APase I) were isolated from Bacillus licheniformis MC14 using a synthetic oligodeoxynucleotide deduced from the amino acid sequence of the enzyme. The DNA sequence analysis of this region revealed an open reading frame of 129 amino acids containing the amino-terminal sequence of the mature APase protein. The protein sequence was preceded by a putative signal sequence of 32 amino acid residues. The predicted amino acid sequence of the partial APase clone as well as the experimentally determined amino acid sequence of the enzyme indicated that B. licheniformis APase retains the important features conserved among other APases of Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, and various human tissues. Heterologous expression studies of the promoter using a fusion with the lacZ gene indicated that it functions as a very strong inducible promoter in B. subtilis that is tightly regulated by phosphate concentration.     

Cloning, sequencing and expression of a sialidase gene from Clostridium sordellii G12

A 4.3 kb XbaI restriction fragment of DNA from Clostridium sordellii G12 hybridized with a synthetic oligonucleotide representing the N-terminus of the sialidase protein secreted by C. sordellii. This cloned fragment was shown to encode only part of the sialidase protein. The sialidase gene of C. sordellii was completed by a 0.7 kb RsaI restriction fragment overlapping one end of the XbaI fragment. After combining the two fragments and transformation of Escherichia coli, a clone that expressed sialidase was obtained. The nucleotide sequence of the sialidase gene of C. sordellii G12 was determined. The sequence of the 18 N-terminal amino acids of the purified extracellular enzyme perfectly matched the predicted amino acid sequence near the beginning of the structural gene. The amino acid sequence derived from the complete gene corresponds to a protein with a molecular mass of 44,735 Da. Upstream from the putative ATG initiation codon, ribosomal-binding site and promoter-like consensus sequences were found. The encoded protein has a leader sequence of 27 amino acids. The enzyme expressed in E. coli has similar properties to the enzyme isolated from C. sordellii, except for small differences in size and isoelectric point. Significant homology (70%) was found with a sialidase gene from C. perfringens.