Cloning and expression of Acinetobacter calcoaceticus catechol 1,2-dioxygenase structural gene catA in Escherichia coli.

Catechol 1,2-dioxygenase (EC 1.13.1.1), the product of the catA gene, catalyzes the first step in catechol utilization via the beta-ketoadipate pathway. Enzymes mediating subsequent steps in the pathway are encoded by the catBCDE genes which are carried on a 5-kilobase-pair (kbp) EcoRI restriction fragment isolated from Acinetobacter calcoaceticus. This DNA was used as a probe to identify Escherichia coli colonies carrying recombinant pUC19 plasmids with overlapping sequences. Repetition of the procedure yielded an A. calcoaceticus 6.7-kbp EcoRI restriction fragment which contained the catA gene and bordered the original 5-kbp EcoRI restriction fragment. When the catA-containing fragment was placed under the control of the lac promoter on pUC19 and induced with isopropylthiogalactopyranoside, catechol dioxygenase was formed in E. coli at twice the level found in fully induced cultures of A. calcoaceticus. A. calcoaceticus strains with mutations in the catA gene were transformed to wild type by DNA from lysates of E. coli strains carrying the catA gene on recombinant plasmids. Thus, A. calcoaceticus strains with a mutated gene can be used in a transformation assay to identify E. coli clones in which at least part of the wild-type gene is present but not necessarily expressed.     

Acinetobacter calcoaceticus encoded mutarotase: nucleotide sequence analysis of the gene and characterization of its secretion in Escherichia coli.

The nucleotide sequence of the mutarotase gene from Acinetobacter calcoaceticus has been determined. It reveals an open reading frame of 381 amino acids. The codon usage of A. calcoaceticus for this gene is similar to E. coli except for the amino acids Leu, Ala, Glu, and Arg where major differences exist. This did not interfere drastically with high level expression in E. coli. The regulatory sequences for the initiation of translation are similar to the ones described for E. coli. The N-terminal 20 amino acids, which are not found in the mature enzyme, show homology to signal sequences of exported proteins. In A. calcoaceticus and E. coli mutarotase is specifically secreted into the periplasmic space. Processing of the signal sequence occurs at identical sites in both organisms. The mature mutarotase consists of 361 amino acids and has a calculated molecular weight of 38457 Da. Expression of mutarotase at a high level in a recombinant E. coli destabilizes the outer membrane. This results in coordinated leakage of mutarotase and beta-lactamase into the culture broth.     

Cloning and expression of Acinetobacter calcoaceticus catBCDE genes in Pseudomonas putida and Escherichia coli.

This report describes the isolation and preliminary characterization of a 5.0-kilobase-pair (kbp) EcoRI DNA restriction fragment carrying the catBCDE genes from Acinetobacter calcoaceticus. The respective genes encode enzymes that catalyze four consecutive reactions in the catechol branch of the beta-ketoadipate pathway: catB, muconate lactonizing enzyme (EC 5.5.1.1); catC, muconolactone isomerase (EC 5.3.3.4); catD, beta-ketoadipate enol-lactone hydrolase (EC 3.1.1.24); and catE, beta-ketoadipate succinyl-coenzyme A transferase (EC 2.8.3.6). In A. calcoaceticus, pcaDE genes encode products with the same enzyme activities as those encoded by the respective catDE genes. In Pseudomonas putida, the requirements for both catDE and pcaDE genes are met by a single set of genes, designated pcaDE. A P. putida mutant with a dysfunctional pcaE gene was used to select a recombinant pKT230 plasmid carrying the 5.0-kbp EcoRI restriction fragment containing the A. calcoaceticus catE structural gene. The recombinant plasmid, pAN1, complemented P. putida mutants with lesions in catB, catC, pcaD, and pcaE genes; the complemented activities were expressed constitutively in the recombinant P. putida strains. After introduction into Escherichia coli, the pAN1 plasmid expressed the activities constitutively but at much lower levels that those found in the P. putida transformants or in fully induced cultures of A. calcoaceticus or P. putida. When placed under the control of a lac promoter on a recombinant pUC13 plasmid in E. coli, the A. calcoaceticus restriction fragment expressed catBCDE activities at levels severalfold higher than those found in fully induced cultures of A. calcoaceticus. Thus there is no translational barrier to expression of the A. calcoaceticus genes at high levels in E. coli. The genetic origin of the cloned catBCDE genes was demonstrated by the fact that the 5.0-kbp EcoRI restriction fragment hybridized with a corresponding fragment from wild-type A. calcoaceticus DNA. This fragment was missing in DNA from an A. calcoaceticus mutant in which the cat genes had been removed by deletion. The properties of the cloned fragment demonstrate physical linkage of the catBCDE genes and suggest that they are coordinately transcribed.     

High level expression of Acinetobacter calcoaceticus mutarotase in Escherichia coli is achieved by improving the translational control sequence and removal of the signal sequence

Summary The expression of enzymatically active Acinetobacter calcoaceticus encoded mutarotase in Escherichia coli is increased 10-fold upon fusion of the mro reading frame to an efficient ribosome binding sequence and deletion of the signal sequence. This was demonstrated by fusing the mro gene to the highly expressed tetR gene under control of the strong phage promoter P _L(Oehmichen et al. 1984 EMBO J. 3:539–543). Deleting the leader sequence yielded cytoplasmic expression of 5×10⁴ u of active mutarotase while constructions bearing the leader sequence expressed only 2×10³ u active enzyme. It was shown by protein and Western blot analysis that the amounts of protein expressed are nearly the same with and without signal sequence. However, under the conditions of P _Linduction the major portion of the wild type mutarotase expressed at high level remains insoluble and is not processed nor active while at low level of expression processing is complete to yield active mutarotase. The implications of these results on production of pharmaceutically interesting proteins in E. coli are discussed.     

Cloning and expression of the transhydrogenase gene of Escherichia coli.

Based on the rationale that Escherichia coli cells harboring plasmids containing the pnt gene would contain elevated levels of enzyme, we have isolated three clones bearing the transhydrogenase gene from the Clarke and Carbon colony bank. The three plasmids were subjected to restriction endonuclease analysis. A 10.4-kilobase restriction fragment which overlapped all three plasmids was cloned into the PstI site of plasmid pUC13. Examination of several deletion derivatives of the resulting plasmid and subsequent treatment with exonuclease BAL 31 revealed that enhanced transhydrogenase expression was localized within a 3.05-kilobase segment. This segment was located at 35.4 min in the E. coli genome. Plasmid pDC21 conferred on its host 70-fold overproduction of transhydrogenase. The protein products of plasmids carrying the pnt gene were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes from cells containing the plasmids. Two polypeptides of molecular weights 50,000 and 47,000 were coded by the 3.05-kilobase fragment of pDC11. Both polypeptides were required for expression of transhydrogenase activity.     

Cloning and expression of the metE gene in Escherichia coli

A lambda-transducing phage was isolated that contains the metE gene. This gene codes for N5-methyl-H4-folate:homocysteine methyltransferase (EC 2.1.1.14), an enzyme that catalyzes the terminal reaction in methionine biosynthesis. A 9.1-kb EcoR1 fragment of this phage, containing the metE gene, was then cloned into pBR325. This plasmid, pJ19, was used to transform Escherichia coli strain 2276, a metE mutant, and restore the MetE+ phenotype. Although the transformed cells produced large amounts of the metE protein in vivo, in vitro studies using pJ19 as template showed low synthesis of the metE protein.     

Cloning and expression of the Escherichia coli K-12 sad gene.

The Escherichia coli K-12 sad gene, which encodes an NAD-dependent succinic semialdehyde dehydrogenase, was cloned into a high-copy-number vector. Minicells carrying a sad+ plasmid produced a 55,000-dalton peptide, the probable sad gene product.     

Acinetobacter calcoaceticus genes involved in biosynthesis of the coenzyme pyrrolo-quinoline-quinone: nucleotide sequence and expression in Escherichia coli K-12.

Synthesis of the coenzyme pyrrolo-quinoline-quinone (PQQ) from Acinetobacter calcoaceticus requires the products of at least four different genes. In this paper we present the nucleotide sequence of a 5,085-base-pair DNA fragment containing these four genes. Within the DNA fragment three reading frames are present, coding for proteins of Mr 10,800, 29,700, and 43,600 and corresponding to three of the PQQ genes. In the DNA region where the fourth PQQ gene was mapped the largest possible reading frame encodes for a polypeptide of only 24 amino acids. Still, the expression of this region is essential for the biosynthesis of PQQ. A possible role for this DNA region is discussed. Sandwiched between two PQQ genes an additional reading frame is present, coding for a protein of Mr 33,600. This gene, which is probably transcribed in the same operon as three of the PQQ genes, seems not required for PQQ synthesis. Expression of the PQQ genes in Acinetobacter lwoffi and Escherichia coli K-12 led to the synthesis of the coenzyme in these organisms.     

Cloning and expression of the pepD gene of Escherichia coli

Peptidase D of Escherichia coli, cleaving the unusual dipeptide carnosine, was found to be encoded by the ColE1 hybrid plasmid pLC44-11. From this plasmid the pepD gene was subcloned into small vectors. As shown by successive reduction of the flanking sequences of genomic DNA, the order of genes in the region at 6 min of the E. coli K12 map is phoE, pepD, in the clockwise orientation. Insertional inactivation of the pepD gene and expression of recombinant plasmids in maxicells allowed the identification of the pepD product as a 52 kDa protein. Comparison with the 100 kDa protein molecular mass determined by gel filtration suggests that active peptidase D is probably a dimer.     

Cloning and expression of the pneumococcal neuraminidase gene in Escherichia coli

A gene bank of Sau3AI-generated Streptococcus pneumoniae DNA fragments was constructed in Escherichia coli K-12 by cloning into the BamHI site of the cosmid vector pHC79. One clone capable of cleaving the fluorogenic neuraminidase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetyl-neuraminic acid was isolated. This activity was inhibited by treatment with a mouse antiserum raised against purified pneumococcal neuraminidase. The recombinant plasmid purified from this clone (designated pJCP301) contained approximately 3.0 kb of pneumococcal DNA. Western-blot analysis indicated that E. coli K-12[pJCP301] produced a 98-kDa polypeptide which reacted with antineuraminidase serum.     

Cloning and expression of the pneumococcal autolysin gene in Escherichia coli

Summary A 7.5 kb BclI-fragment of Streptococcs pneumoniae DNA has been cloned in Escherichia coli HB101 using pBR322 as a vector. The new plasmid (pGL30) of 12.0 kb expresses a protein that has been characterized by biochemical, immunological and genetic methods as the inactive form (E-form) of the pneumococcal N-acetyl-muramyl-l-alanyl amidase (EC 3.5.1.28). Our results demonstrate that the E-form is the primary product of the lyt gene of S. pneumoniae. The inactive E-form can be converted to the active C-form in vitro by incubation of the E-form enzyme with choline-containing pneumococcal cell walls at low temperature in a similar way to enzyme production in the homologous system. The production of this protein in E. coli HB101 was 500-fold higher than in the homologous host. E. coli CSR603 containing pGL30 and labeled with [³⁵S]methionine synthesized a 35 kd protein. pGL30 can transform at high frequency an autolysin-defective mutant of S. pneumoniae to the lyt⁺ phenotype.     

Cloning and expression in Escherichia coli of the gene encoding a novel L-2,4-diaminobutyrate decarboxylase of Acinetobacter baumannii

Abstract The gene encoding L-2,4-diaminobutyrate decarboxylase (DABA DC) was cloned from Acinetobacter baumannii ATCC 19606. The gene was evidently under the control of its own promoter. Interestingly, the host carrying this clone also produced an appreciable amount of 1,3-diaminopropane. Restriction mapping and subsequent subcloning of the cloned insert localized the DABA DC gene within a 2.45-kb SphI/Eco RI fragment. For endogenous production of DAP, a 1.75-kb Eco RI/ Pst I region downstream from the DABA DC gene was further required. Southern blot hybridization revealed some heterogeneity in the DABA DC genes among other Acinetobacter species.     

Cloning and constitutive expression of the N-acetylneuraminate lyase gene of Escherichia coli.

The N-acetylneuraminate (NANA) lyase (EC 4.1.3.3) gene from Escherichia coli was self-cloned in E. coli. Transformants were selected by complementation of a NANA lyase-deficient E. coli strain. One clone was found to produce NANA lyase, and it contained a recombinant plasmid, pNAL1, with a 9.0-kilobase HindIII insert. The cloning of the NANA lyase gene resulted in the change from inducible to constitutive production of the enzyme. The level of expression of the NANA lyase gene in E. coli(pNAL1) clones was two- to three-fold higher than that in the fully induced wild-type strains.     

Cloning and expression in Escherichia coli of the dnaK gene of Zymomonas mobilis.

The DnaK protein of Zymomonas mobilis (DnaKz) was identified and found to be 80% identical to the DnaK protein of Escherichia coli on the basis of the sequence of the N-terminal 21 amino acids. The dnaKz gene was cloned and found to be expressed in a thermosensitive dnaK mutant of Escherichia coli. Expression of the foreign gene restored a thermoresistant phenotype but failed to modulate the heat shock response in E. coli.     

Cloning and expression of the Staphylococcus aureus protein A gene in Escherichia coli.

A gene bank of Staphylococcus aureus strain Cowan I was established using an E. coli HB101/pBR327 host-vector system. Recombinants expressing staphylococcal protein A (SPA) were detected using an IgG-binding assay. A 3.2 Kb DNA fragment directing the synthesis of SPA in E. coli was identified. SPA produced by E. coli was characterised in minicells and by Western blotting and double diffusion experiments.     

Cloning and expression of the yeast galactokinase gene in an Escherichia coli plasmid.

This report describes the construction and isolation of a plasmid, derived from pBR322, which carries a BglII restriction fragment of DNA containing the galactokinase gene from Saccharomyces cerevisiae. This was accomplished by the following procedure: (1) Purified galactokinase mRNA, labelled with 125I, was hybridized to BglII digests of yeast DNA employing Southern's filter transfer technique to identify a restriction fragment containing the galactokinase gene. (2) This fragment was partially purified by agarose gel electrophoresis, ligated into the BamHI site of pBR322 and transformed into Escherichia coli to generate a clone bank containing the galactokinase gene. (3) This bank was screened by in situ colony hybridization with galactokinase mRNA resulting in the identification of a plasmid carrying this gene. This plasmid DNA hybridized with the galactokinase mRNA to the same extent in the presence of absence of a large excess of unlabelled mRNA from cells that were not induced for galactokinase synthesis, while the same amount of unlabelled galactose-induced mRNA reduced the hybridization by 95%. When this plasmid was introduced into an E. coli strain deleted for the galactose operon it caused the synthesis of low levels of yeast galactokinase activity.