Cloning and nucleotide sequence of the aspartase gene of Escherichia coli W.

The aspA gene of Escherichia coli W which encodes aspartase was cloned into the plasmid vector pBR322. The nucleotide sequences of aspA and its flanking regions were determined. The aspA gene encodes a protein with a molecular weight of 52,224 consisted of 477 amino acid residues. The amino acid sequence of the protein predicted from the nucleotide sequence was consistent with those of the NH2- and COOH-terminal regions and also with the amino acid composition of the purified aspartase determined previously. Potential promoter and terminator sequences for aspA were also found in the determined sequence.     

Cloning, expression, and nucleotide sequence of a mutant glgC gene from Escherichia coli B.

A mutant glgC gene contained in a 10.9-kb PstI fragment was cloned from the Escherichia coli B strain SG5 via colony hybridization by using a wild-type glgC probe. The altered allosteric properties of the expressed ADPglucose synthetase were found to result from the conversion of proline to serine at amino acid residue 295.     

Cloning, nucleotide sequence, and expression of the Escherichia coli gene encoding carnitine dehydratase.

Carnitine dehydratase from Escherichia coli O44 K74 is an inducible enzyme detectable in cells grown anaerobically in the presence of L-(-)-carnitine or crotonobetaine. The purified enzyme catalyzes the dehydration of L-(-)-carnitine to crotonobetaine (H. Jung, K. Jung, and H.-P. Kleber, Biochim. Biophys. Acta 1003:270-276, 1989). The caiB gene, encoding carnitine dehydratase, was isolated by oligonucleotide screening from a genomic library of E. coli O44 K74. The caiB gene is 1,215 bp long, and it encodes a protein of 405 amino acids with a predicted M(r) of 45,074. The identity of the gene product was first assessed by its comigration in sodium dodecyl sulfate-polyacrylamide gels with the purified enzyme after overexpression in the pT7 system and by its enzymatic activity. Moreover, the N-terminal amino acid sequence of the purified protein was found to be identical to that predicted from the gene sequence. Northern (RNA) analysis showed that caiB is likely to be cotranscribed with at least one other gene. This other gene could be the gene encoding a 47-kDa protein, which was overexpressed upstream of caiB.     

Cloning, expression, and nucleotide sequence of the Escherichia coli K-12 ackA gene.

The Escherichia coli K-12 ackA gene, which encodes an acetate kinase, was cloned. The acetate kinase activities of ackA+ plasmid-containing strains were amplified 160- to 180-fold. The complete nucleotide sequence of the ackA gene was determined. It was deduced that the ackA gene coded for a protein of 400 amino acids with an Mr of 43,297. The ackA gene was found to be located about 15 kilobases upstream of the purF-folC-hisT region of the chromosome.     

Cloning and complete nucleotide sequence of the Escherichia coli glutamine permease operon (glnHPQ)

Summary The glutamine permease operon encoding the high-affinity transport system of glutamine in Escherichia coli could be cloned in one of the mini F plasmids, but not in pBR322 or pACYC184, by selection for restoration of the Gln⁺ phenotype, the ability to utilize glutamine as a sole carbon source. We determined the nucleotide sequence of the glutamine permease operon, which contains the structural gene of the periplasmic glutamine-binding protein (glnH), an indispensable component of the permease activity. The N-terminal amino acid sequence and the overall amino acid composition of the purified glutamine-binding protein were in good agreement with those predicted from the nucleotide sequence, if the N-terminal 22 amino acid residues were discounted. The latter comprised two Lys residues (nos. 2 and 6) followed by 16 hydrophobic amino acid residues and was assumed to be a signal peptide for transport into the periplasmic space. There were two additional reading frames (glnP and glnQ) downstream of glnH sharing a common promoter. It was concluded that the glnP and glnQ proteins as well as the glnH protein are essential for glutamine permease activity.     

Complete nucleotide sequence of the Escherichia coli recB gene.

The complete nucleotide sequence of the Escherichia coli recB gene which encodes a subunit of the ATP-dependent DNase, Exonuclease V, has been determined. The proposed coding region for the RecB protein is 3543 nucleotides long and would encode a polypeptide of 1180 amino acids with a calculated molecular weight of 133,973. The start of the recB coding sequence overlaps the 3' end of the upstream ptr gene, and the recB termination codon overlaps the initiation codon of the downstream recD gene, suggesting that these genes may form an operon. No sequences which reasonably fit the consensus for an E. coli promoter could be identified upstream of the proposed recB translational start. The predicted RecB amino acid sequence contains regions of homology with ATPases, DNA binding proteins and DNA repair enzymes.     

Cloning, nucleotide sequence, and expression in Escherichia coli of the phospholipase D gene from Corynebacterium pseudotuberculosis.

The phospholipase D (PLD) gene from Corynebacterium pseudotuberculosis has been cloned, sequenced, and expressed in Escherichia coli. Analysis of DNA sequence data reveals a major open reading frame encoding a 31.4-kilodalton protein, a size consistent with that estimated for the PLD protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of these data with the amino-terminal protein sequence indicates that the mature PLD protein is preceded by a 24-residue signal sequence. Expression of the PLD gene in E. coli is initiated from the corynebacterial promoter, and the resulting protein has sphingomyelinase activity. Primer extension mapping localized the 5' end of the PLD gene mRNA to a site 5 to 7 base pairs downstream of a region similar to the consensus sequence for E. coli promoters. Northern and Southern blot analyses suggest that the gene is transcribed from mRNA approximately 1.1 kilobases in length and that it is present in a single copy within the C. pseudotuberculosis genome.     

Nucleotide sequence of the secA gene and secA(Ts) mutations preventing protein export in Escherichia coli.

The DNA sequence of the secA gene, essential for protein export in Escherichia coli, was determined and found to encode a hydrophilic protein of 901 amino acid residues with a predicted molecular weight of 101,902, consistent with its previously determined size and subcellular location. Sequence analysis of 9 secA(Ts) mutations conferring general protein export and secA regulatory defects revealed that these mutations were clustered in three specific regions within the first 170 amino acid residues of the SecA protein and were the result of single amino acid changes predicted to be severely disruptive of protein structure and function. The DNA sequence immediately upstream of secA was shown to encode a previously inferred gene, gene X. Sequence analysis of a conditionally lethal amber mutation, am109, previously inferred to be located proximally in the secA gene, revealed that it was located distally in gene X and was conditionally lethal due to its polar effect on secA expression. This and additional evidence are presented indicating that gene X and secA are cotranscribed.     

Cloning, nucleotide sequence and expression in Escherichia coli of a lipase gene from Bacillus subtilis 168.

The gene coding for an extracellular lipase of Bacillus subtilis 168 was cloned and found to be expressed in Escherichia coli. Enzyme activity measurements showed no fatty acid chain length preference. A set of Tn5 insertions which inactivate the gene were localized and used to initiate its sequencing. The nucleotide sequence was determined on two independent clones expressed in E. coli. In one of these clones, the sequence revealed a frameshift, due to the presence of an additional adenine in the N-terminal region, which caused the interruption of the open reading frame, probably allowing translation to initiate at a second ATG codon. The sequence of the wild-type lip gene from B. subtilis was confirmed on the chromosomal fragment amplified by polymerase chain reaction (PCR). When compared to other lipases sequenced to date, the enzyme described here lacks the conserved pentapeptide Gly-X-Ser-X-Gly supposed to be essential for catalysis. However, alignments of several microbial lipase sequences suggest that the pentapeptide Ala-X-Ser-X-Gly present in the lipase B. subtilis may function as the catalytic site. Homologies were found in the N-terminal protein region with lipases from different Pseudomonas species. The predicted M(r) and isoelectric point for the mature protein are 19,348 and 9.7 respectively.     

Cloning, nucleotide sequence, and expression in Escherichia coli of the gene for poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis.

The extracellular poly(3-hydroxybutyrate) depolymerase gene from Alcaligenes faecalis T1 was cloned into Escherichia coli DH1 by using the plasmid pUC8. An A. faecalis T1 genomic library was prepared in E. coli from a partial Sau3AI digest and screened with antibody against the depolymerase. Of the 29 antibody-positive clones, 1 (pDP14), containing about 4 kilobase pairs of A. faecalis T1 DNA, caused expression of a high level of depolymerase activity in E. coli. The enzyme purified from E. coli was not significantly different from the depolymerase of A. faecalis in molecular weight, immunological properties, peptide map, specific activity, or substrate specificity. Most of the expressed enzyme was found to be localized in the periplasmic space of E. coli, although about 10% of the total activity was found in the culture medium. Results of a deletion experiment with pDP14 showed that a large SalI fragment of about 2 kilobase pairs was responsible for expression of the enzyme in E. coli. The nucleotide sequence of the large SalI fragment has been determined. Comparison of the deduced amino terminus with that obtained from sequence analysis of the purified protein indicated that poly(3-hydroxybutyrate) depolymerase exists as a 488-amino-acid precursor with a signal peptide of 27 amino acids.     

Cloning, expression in Escherichia coli and nucleotide sequence of a tetracycline-resistance gene from Streptomyces rimosus

Determinants of tetracycline resistance have been cloned from two different tetracycline-producing industrial strains of Streptomyces into Streptomyces lividans using the plasmid vector pUT206. Three plasmids, pUT250 and pUT260 with a 9.5 and a 7.5 kb insert respectively of Streptomyces rimosus DNA, and pUT270 with a 14.0 kb insert of Streptomyces aureofaciens DNA, conferring resistance to tetracycline, have been isolated. By in vitro sub-cloning, a similar fragment of 2.45 kb containing the tetracycline resistance gene (tet347) was further localized on these plasmids. The S. rimosus gene has been cloned into Escherichia coli and expressed under the control of lambda pL or Lpp promoters. Differential protein extraction of E. coli cells revealed the presence of an additional membrane-embedded protein in tetracycline-resistant cells. On the basis of available restriction endonuclease maps, the tet347 gene is probably identical to the tetB gene from S. rimosus recently identified by T. Ohnuki and co-workers as responsible for the reduced accumulation of tetracycline. The nucleotide sequence of a 2052 bp DNA fragment containing the TcR structural gene from S. rimosus has been determined. The amino acid sequence of the tet347 protein (Mr35818) deduced from the nucleotide sequence shows a limited but significant homology to other characterized tetracycline transport acting determinants from pathogenic bacteria.     

Cloning, expression, and nucleotide sequence of glgC gene from an allosteric mutant of Escherichia coli B.

The Escherichia coli B mutant strain CL1136 accumulates glycogen at a 3.4- to 4-fold greater rate than the parent E. coli B strain and contains an ADPglucose synthetase with altered kinetic and allosteric properties. The enzyme from CL1136 is less dependent on the allosteric activator, fructose 1,6-bisphosphate, for activity and less sensitive to inhibition by AMP than the parent strain enzyme. The structural gene, glgC, for the allosteric mutant enzyme was selected by colony hybridization and cloned into the bacterial plasmid pBR322 by insertion of the chromosomal DNA at the PstI site. One recombinant plasmid, designated pKG3, was isolated from the genomic library of CL1136 containing glgC. The cloned ADPglucose synthetase from the mutant CL1136 was expressed and characterized with respect to kinetic and allosteric properties and found to be identical to the enzyme purified from the CL1136 strain. The mutant glgC was then subcloned into pUC118/119 for dideoxy sequencing of both strands. The mutant glgC sequence was found to differ from the wild-type at the deduced amino acid residue 67 where a single point mutation resulted in a change from arginine to cysteine.     

Cloning, nucleotide sequence, and overexpression of the L-rhamnose isomerase gene from Pseudomonas stutzeri in Escherichia coli

The gene encoding L-rhamnose isomerase (L-RhI) from Pseudomonas stutzeri was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the L-RhI gene revealed an open reading frame of 1,290 bp coding for a protein of 430 amino acid residues with a predicted molecular mass of 46,946 Da. A comparison of the deduced amino acid sequence with sequences in relevant databases indicated that no significant homology has previously been identified. An amino acid sequence alignment, however, suggested that the residues involved in the active site of L-RhI from E. coli are conserved in that from P. stutzeri. The L-RhI gene was then overexpressed in E. coli cells under the control of the T5 promoter. The recombinant clone, E. coli JM109, produced significant levels of L-RhI activity, with a specific activity of 140 U/mg and a volumetric yield of 20,000 U of soluble enzyme per liter of medium. This reflected a 20-fold increase in the volumetric yield compared to the value for the intrinsic yield. The recombinant L-RhI protein was purified to apparent homogeneity on the basis of three-step chromatography. The purified recombinant enzyme showed a single band with an estimated molecular weight of 42,000 in a sodium dodecyl sulfate-polyacrylamide gel. The overall enzymatic properties of the purified recombinant L-RhI protein were the same as those of the authentic one, as the optimal activity was measured at 60 degrees C within a broad pH range from 5.0 to 11.0, with an optimum at pH 9.0.     

Complete nucleotide sequence of the Escherichia coli gdhA gene

The DNA sequence of the gdhA gene of Escherichia coli K12, which encodes the 447 amino acid polypeptide subunit of NADP-specific glutamate dehydrogenase, is presented. The deduced protein sequence is strongly homologous to the corresponding enzyme of the eukaryotic fungus Neurospora crassa. The upstream DNA sequence includes several overlapping promoter consensus sequences. The downstream DNA sequence contains inverted repeats, predicted as forming long stable stem-loop structures in RNA, homologous to those found in several enterobacterial intergenic regions.     

Chromosomal location and nucleotide sequence of the Escherichia coli dapA gene.

Restriction fragments hybridizing to phage HP1c1 DNA were identified in digests of DNA from lysogenic strains of Haemophilus influenzae. The results showed that the cohesive ends of the mature phage DNA were joined in lysogens and that the phage genome was covalently linked to the host DNA, indicating that lysogeny involves recombination between specific sites on the phage and host chromosomes. The site on the phage chromosome at which this recombination occurred was between 110 and 750 base pairs of the left end on the mature phage genome.     

The nucleotide sequence of the Escherichia coli rts gene

The nucleotide sequence of rts, an essential Escherichia coli gene, has been determined. Transformation of an rts mutant with the plasmid, pJAF1, containing the rts gene resulted in rescue of the defect. The transformation experiments indicate that the rts gene is distinct from the flanking birA, tRNA and tufB genes.