Cloning and identification of the product of the dnaE gene of Escherichia coli

We successively subcloned the dnaE gene of Escherichia coli into pBR322, resulting in a plasmid that contains 4.6 kilobases of E. coli DNA. This plasmid can complement a dnaE temperature-sensitive mutation. A restriction map of the dnaE gene and the surrounding 10.7-kilobase region of the E. coli chromosome was determined. A unique HindIII restriction endonuclease site within the cloned segment of DNA was identified as a site required for expression of the dnaE gene. By using the maxicell plasmid-directed protein synthesizing system, we demonstrated that dnaE codes for the alpha subunit of DNA polymerase III.     

Effects of 3' end deletions from the Vibrio harveyi luxB gene on luciferase subunit folding and enzyme assembly: generation of temperature-sensitive polypeptide folding mutants

Ten recombinant plasmids have been constructed by deletion of specific regions from the plasmid pTB7 that carries the luxA and luxB genes, encoding the alpha and beta subunits of luciferase from Vibrio harveyi, such that luciferases with normal alpha subunits and variant beta subunits were produced in Escherichia coli cells carrying the recombinant plasmids. The original plasmid, which conferred bioluminescence (upon addition of exogenous aldehyde substrate) on E. coli carrying it, was constructed by insertion of a 4.0-kb HindIII fragment of V. harveyi DNA into the HindIII site of plasmid pBR322 [Baldwin, T.O., Berends, T., Bunch, T. A., Holzman, T. F., Rausch, S. K., Shamansky, L., Treat, M. L., & Ziegler, M. M. (1984) Biochemistry 23, 3663-3667]. Deletion mutants in the 3' region of luxB were divided into three groups: (A) those with deletions in the 3' untranslated region that left the coding sequences intact, (B) those that left the 3' untranslated sequences intact but deleted short stretches of the 3' coding region of the beta subunit, and (C) those for which the 3' deletions extended from the untranslated region into the coding sequences. Analysis of the expression of luciferase from these variant plasmids has demonstrated two points concerning the synthesis of luciferase subunits and the assembly of those subunits into active luciferase in E. coli. First, deletion of DNA sequences 3' to the translational open reading frame of the beta subunit that contain a potential stem and loop structure resulted in dramatic reduction in the level of accumulation of active luciferase in cells carrying the variant plasmids, even though the luxAB coding regions remained intact.     

Suppression of dnaE nonsense mutations by pcbA1.

DNA polymerase III has been recognized as the required replication enzyme in Escherichia coli. The synthesis subunit of DNA polymerase III holoenzyme (alpha subunit) is encoded by the dnaE gene. We have reported that E. coli cells can survive and grow in the absence of a functional dnaE gene product if DNA polymerase I and the pcbA1 mutation are present. Existing mutations in the dnaE gene have been conditionally defective thermolabile mutations. We report here construction of nonsense mutations in the dnaE gene by use of a temperature-sensitive suppressor mutation to permit survival at the permissive temperature (32 degrees C). Introduction of the pcbA1 mutation eliminated the temperature-sensitive phenotype. We confirmed by immunoblotting the lack of detectable alpha subunit at 43 degrees C.     

Nucleotide sequences of dnaE, the gene for the polymerase subunit of DNA polymerase III in Salmonella typhimurium, and a variant that facilitates growth in the absence of another polymerase subunit.

The dnaE gene of Salmonella typhimurium, like that of Escherichia coli, encodes the alpha subunit containing the polymerase activity of the principal replicative enzyme, DNA polymerase III. This gene, or one nearby, has been identified as the locus of suppressor mutations that promote growth by cells deleted for dnaQ, the gene for the editing subunit of this enzyme complex. Using a combination of nucleotide sequencing and marker rescue experiments, the alteration in one such suppressor was identified as a valine-to-glycine substitution at amino acid 832 of the 1,160-amino-acid alpha polypeptide. The alpha polypeptides of E. coli and S. typhimurium are identical in size and in 97% of their amino acid residues. Their identity includes the valine residue that was changed in the suppressor allele of S. typhimurium. We also localized a temperature-sensitive dnaE mutation to the 3' half of dnaE.     

The polymerase subunit of DNA polymerase III of Escherichia coli. I. Amplification of the dnaE gene product and polymerase activity of the alpha subunit

The Escherichia coli dnaE gene, which encodes the alpha subunit of DNA polymerase III (pol III) holoenzyme, has been cloned in a plasmid containing the PL promoter of phage lambda and thermally induced to overproduce the alpha subunit. In cells carrying this plasmid (pKH167), the alpha subunit was amplified, after heat induction, to a level of about 0.2% of the total cellular protein. Polymerase activity was assayed in three ways: (i) gap-filling by pol III holoenzyme and subassemblies of it, (ii) the extensive replication of a primed, single-stranded DNA circle only by pol III holoenzyme, and (iii) complementation of a crude, inactive pol III holoenzyme (temperature-sensitive dnaE mutant fraction) in replication of a primed, single-stranded DNA circle. Amplification of the alpha subunit raised the polymerase level 10-fold in assay (i), indicative of the dependence of pol III gap-filling activity on this polypeptide; pol III holoenzyme activity remained unaffected (assay (ii)), but the complementation activity was raised 5-fold (assay (iii)). Thus, the elevated alpha subunit (free or in a subassembly form) can substitute in vitro for a defective alpha subunit in pol III holoenzyme, but cannot increase the in vivo level of about eight pol III holoenzyme molecules per cell. This low level of pol III holoenzyme is fixed in wild type cells (bearing no plasmid) despite the presence of a 5-fold excess of the alpha subunit, as inferred from the various assays. These results suggest that the low level of pol III holoenzyme is determined by a factor or factors other than the level of the alpha subunit.     

Proton leakiness caused by cloned genes for the F0 sector of the proton-translocating ATPase of Escherichia coli: requirement for F1 genes.

To study expression of uncG, the gene coding for the gamma subunit of the Escherichia coli proton-translocating ATPase, deletions were made in the intergenic region between uncA, the gene coding for the alpha subunit, and uncG. Two deletions which fused uncA and uncG coded for alpha-gamma fusion polypeptides which were synthesized well both in vitro and in vivo, demonstrating that uncG expression is normally controlled by nucleotides in the intergenic region. Multicopy plasmids carrying these fusion genes and the genes for the other subunits of the ATPase had a harmful effect on the growth of E. coli. The effect was overcome by N,N'-dicyclohexylcarbodiimide, indicating that the cells probably leaked protons. The deleterious effect was eliminated by making a nonpolar deletion in the upstream F0 gene uncB, or by cloning each of the uncA-uncG fusion genes onto a separate plasmid, removed from the F0 genes, thus demonstrating that the fusion genes were not primarily responsible for the proton permeability. A plasmid which carried F0 genes and the gene for the delta subunit caused deleterious proton leakiness in unc+ cells but not in cells from which the unc operon was deleted. The proton leakiness caused by these different plasmids was therefore due to the production of a leaky F0 proton channel and required the presence of F1 genes. The results support a model for ATPase assembly in which F1 genes or polypeptides are involved in the formation or opening of the F0 proton channel.     

Nucleotide sequence of the genes for tryptophan synthase in Pseudomonas aeruginosa

We have determined the DNA sequence of the two adjacent genes for the alpha and beta chains of tryptophan synthase in Pseudomonas aeruginosa, along with 34 5'-flanking and 799 3'-flanking base pairs. The gene order is trpBA as predicted from earlier genetic studies, and the two cistrons overlap by 4 bp; a ribosome binding site for the second gene is evident in the coding sequence of the first gene. We have also determined the location of three large deletions eliminating portions of each gene. A detailed comparison of the deduced P. aeruginosa amino acid sequence with those published for E. coli, Bacillus subtilis, and Saccharomyces cerevisiae shows much similarity throughout the beta and most of the alpha subunit. Most of the residues implicated by chemical modification or mutation as being critical for enzymatic activity are conserved, along with many others, suggesting that three-dimensional structure has remained largely constant during evolution. We also report the construction of a recombinant plasmid that overproduces a slightly modified alpha subunit from P. aeruginosa that can form a functionally effective multimer with normal E. coli beta 2 subunit in vivo.      

A strong mutator effect caused by an amino acid change in the alpha subunit of DNA polymerase III of Escherichia coli

Most potent mutators heretofore detected in Escherichia coli are associated with defects in epsilon subunit of DNA polymerase III, encoded by the dnaQ gene. To elucidate the role of the alpha subunit, the catalytic subunit of the polymerase, in maintaining the high fidelity of DNA replication, we isolated a mutator mutant, the mutation (dnaE173) of which resides on the dnaE gene, encoding the alpha subunit. The dnaE173 mutant was unable to grow in salt-free L broth at temperatures exceeding 44.5 degrees C and exhibited an increased frequency of spontaneous mutations, 1,000 to 10,000-fold the wild type level, at permissive temperatures. The mutator effect of dnaE173 mutation is dominant over the wild type allele. These phenotypes are caused by a single base substitution, resulting in one amino acid change, Glu612 (GAA)----Lys(AAA), in the alpha subunit molecule. DNA polymerase III purified from the dnaE173 mutant contained both alpha and epsilon subunits, in a normal molar ratio. We found no differences between wild type and mutant polymerases in the Vmax, thermolabilities, and salt sensitivities. However, the apparent Km for the substrate nucleotide of the mutant polymerase was 1/6 of that determined with the wild type polymerase. Although the mutant polymerase retained a normal level of 3'----5' exonuclease activity, the proofreading capacity determined by "turnover assay" was significantly lower in the mutant polymerase, as compared with findings in the normal enzyme. It seems likely that the enhanced mutability in the dnaE173 strain results from, at least in part, a defect in the editing function of DNA polymerase III, and further suggests that a portion of the alpha subunit in which the amino acid change resides may be important for the proper setting of the two subunits at the replication fork so as to facilitate efficient editing during the DNA replication.     

DNA polymerase III holoenzyme of Escherichia coli. Purification and resolution into subunits.

DNA polymerase III holoenzyme has been purified from Escherichia coli HMS-83, using, as an assay, the conversion of coliphage G4 single-stranded DNA to the duplex replicative form. The holoenzyme consists of at least four different subunits: alpha, beta, gamma, and delta of 140,000, 40,000, 52,000, and 32,000 daltons, respectively. The alpha subunit is DNA polymerase III, the dnaE gene product. The holoenzyme has been resolved by phosphocellulose chromatography into an alpha - gamma - delta complex and a subunit beta (copolymerase III*); neither possesses detectable activity in the G4 system but together reconstitute holoenzyme-like activity. The alpha - gamma - delta complex has been further resolved to yield a gamma - delta complex which reconstitutes alpha - gamma - delta activity when added to DNA polymerase III. The gamma - delta complex contains a product of the dnaZ gene and has been purified from a strain which contains a ColE1-dnaZ hybrid plasmid.     

Alternate pathways of DNA replication in Escherichia coli

We have described the pcbA1 mutation which enables E. coli cells to replicate DNA in the absence of a functional dnaE gene product if DNA polymerase I (the polA gene product) is present. The pcbA1 mutation phenotypically suppresses multiple dnaEts and dnaEam alleles. The pcbA1/PolI replication pathway differs from normal in sensitivity to certain DNA-damaging agents such as methylmethane sulfonate (MMS) and a lack of damage-directed mutagenesis. We report here cloning of the pcbA1 gene in a multicopy plasmid. The pcbA1 mutation is detected only in cis; therefore, cloning necessitated gene eviction. The pcbA1 gene lies closely- linked to gyrB. We have demonstrated the physical presence of DNA polymerase I in the replicating holoenzyme complex by immunoblotting using dnaEam strains. We conclude that E. coli has two alternate replisome structures: REP-A, in which DNA polymerase I is the functional synthetic subunit; and REP-E, in which the alpha-subunit, product of the dnaE gene, is functional. To investigate further the role of individual DNA polymerases in replication, we have isolated the polB gene on multicopy plasmids.     

Isolation and characterization of the yeast 3-phosphoglycerokinase gene (PGK) by an immunological screening technique

An immunological screening technique has been used for the detection of a specific antigen-producing clone in a bank of bacterial colonies containing hybrid plasmids. This technique involves covalent attachment of antiserum to cyanogen bromide-activated paper discs, contact of this paper with lysed colonies on agar plates, and finally detection of the bound antigen with 125I-labeled antibody. Using this method, we have identified an Escherichia coli colony, containing a yeast DNA insert in plasmid ColE1, that produces antigen which combines with antibody directed against purified yeast 3-phosphoglycerate kinase. The hybrid plasmid (pYe57E2) obtained by this procedure has been shown by both biochemical and genetic methods to contain the structural gene PGK for yeast 3-phosphoglycerate kinase. The location of the PGK structural gene on pYe56E2 was determined by immunological screening of E. coli colonies bearing plasmids containing various reconstructions of the original yeast DNA insert. Examination of the expression of the cloned yeast PGK gene in both E. coli and yeast has shown that functional enzyme is synthesized from the cloned gene in yeast, but not in E. coli.     

Gene engineering by selectable intraplasmid recombination: construction of novel dihydrofolate reductase minigenes

An intraplasmid recombination system in Escherichia coli has been designed to make possible the engineering of various genes using methods that greatly reduce dependence on appropriately placed restriction enzyme sites. This system has been used to manipulate intervening sequences in dihydrofolate reductase minigenes and to vary the number of 48-bp repeats in the promoter region. In this method, the two fragments to be recombined are cloned into a plasmid separated by a fragment of DNA containing an expressible galactokinase-encoding gene (galK). Selection for loss of the galK gene, but for retention of the plasmid in E. coli, results in a plasmid in which the two fragments have undergone homologous recombination. Several new plasmids are reported here which contain an expressible galK gene flanked by multiple restriction sites. These plasmids should be useful in recombination and as convenient sources of a gene for which both positive and negative selections are available in E. coli.     

Specificity in the formation of delta tra F-prime plasmids.

Twenty-three independent delta tra F-prime plasmids from three different Escherichia coli K-12 sublines were isolated from Hfr strains whose points of origin coincided with the IS3 element alpha 3 beta 3 or alpha 4 beta 4 in the lac-purE region of the E. coli chromosome. Electrophoretic analysis of plasmid deoxyribonucleic acid digested with EcoRI and hybridization analysis of plasmid deoxyribonucleic acid digested with BglII revealed that at least 14 of these plasmids were formed by processes involving specific bacterial and F loci. Two of the specific bacterial loci involved in delta tra F-prime formation were located at approximately 3.3 and 11.7 min on the E. coli chromosomal map. Two of the delta tra F-prime plasmids contained bacterial deoxyribonucleic acid with circularization endpoints that mapped very near the termini of the IS2 element that is normally located between lac and proC.     

Cloning and sequence analysis of the Escherichia coli metH gene encoding cobalamin-dependent methionine synthase and isolation of a tryptic fragment containing the cobalamin-binding domain

A gene encoding cobalamin-dependent methionine synthase (EC 2.1.1.13) has been isolated from a plasmid library of Escherichia coli K-12 DNA by complementation to methionine prototrophy in an E. coli strain lacking both cobalamin-dependent and -independent methionine synthase activities (RK4536:metE, metHH). Maxicell expression of a series of plasmids containing deletions in the metH structural gene was employed to map the position and orientation of the gene on the cloned DNA fragment. A 6.3-kilobase EcoRI-SalI fragment containing the gene was cloned into the sequencing vector pGEM3B for double-stranded DNA sequencing; the MetH coding region consists of 3372 nucleotides. The enzyme was purified from an overproducing strain of E. coli harboring the recombinant plasmid, in which the level of methionine synthase was elevated 30- to 40-fold over wild-type E. coli. Recombinant enzyme is a protein of 123,640 molecular weight and has a turnover number of 1,450 min-1 in the standard assay. These values are to be compared with previously reported values of 133,000 for the molecular weight and 1,240-1,560 min-1 for the turnover number of the homogenous enzyme purified from a wild-type strain of E. coli B (Frasca, V., Banerjee, R. V., Dunham, W. R., Sands, R. H., and Matthews, R. G. (1988) Biochemistry 27, 8458-8465). Limited proteolysis of the native enzyme with trypsin resulted in loss of enzyme activity but retention of bound cobalamin on a peptide fragment of 28,000 molecular weight. This fragment has been shown to extend from residue 643 to residue 900 of the 1124-residue deduced amino acid sequence.     

Sequence of the small subunit of yeast carbamyl phosphate synthetase and identification of its catalytic domain

The yeast gene CPA1 coding for the small subunit of arginine-specific carbamyl phosphate synthetase has been cloned by complementation of a cpa 1 mutant with a plasmid library of total yeast chromosomal DNA. Two of the plasmids, pJL113/ST4 and pJL113/ST15, contain DNA inserts in opposite orientations with overlapping sequences of 2.6 kilobases. The nucleotide sequence of a 2.2-kilobase region of the DNA insert carrying the CPA1 gene has been determined. The CPA1 gene has been identified to be 1233 nucleotides long and to code for a polypeptide of 411 amino acids with a calculated molecular weight of 45,358. The amino acid sequence encoded in CPA1 is homologous to the recently determined sequence of the small subunit of Escherichia coli carbamyl phosphate synthetase (Piette, J., Nyunoya, H., Lusty, C.J., Cunin, R., Weyens, G., Crabeel, M., Charlier, D., Glandsdorff, N., and Pierard, A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4134-4138) over the entire length of the polypeptide chain. Comparison of the amino acid sequences of the small subunits of yeast and E. coli carbamyl phosphate synthetases to the sequences of Component II of anthranilate and p-aminobenzoate synthases suggests that these amidotransferases are evolutionarily related. The most highly conserved region of the yeast and E. coli enzymes includes a cysteine residue previously found to be at the active site of Pseudomonas putida anthranilate synthase Component II (Kawamura, M., Keim, P.S., Goto, Y., Zalkin, H., and Heinrikson, R.L. (1978) J. Biol. Chem. 253, 4659-4668). Based on the observed homologies in the primary sequences of the other amidotransferases examined, we propose a 13-amino acid long sequence to be part of the catalytic domain of this class of enzymes.     

The dnaE173 mutator mutation confers on the alpha subunit of Escherichia coli DNA polymerase III a capacity for highly processive DNA synthesis and stable binding to primer/template DNA

The strong mutator mutation dnaE173 which causes an amino-acid substitution in the alpha subunit of DNA polymerase III is unique in its ability to induce sequence-substitution mutations. We showed previously that multiple biochemical properties of DNA polymerase III holoenzyme of Escherichia coli are simultaneously affected by the dnaE173 mutation. These effects include a severely reduced proofreading capacity, an increased resistance to replication-pausing on the template DNA, a capability to readily promote strand-displacement DNA synthesis, a reduced rate of DNA chain elongation, and an ability to catalyze highly processive DNA synthesis in the absence of the beta-clamp subunit. Here we show that, in contrast to distributive DNA synthesis exhibited by wild-type alpha subunit, the dnaE173 mutant form of alpha subunit catalyzes highly processive DNA chain elongation without the aid of the beta-clamp. More surprisingly, the dnaE173 alpha subunit appeared to form a stable complex with primer/template DNA, while no such affinity was detected with wild-type alpha subunit. We consider that the highly increased affinity of alpha subunit for primer/template DNA is the basis for the pleiotropic effects of the dnaE173 mutation on DNA polymerase III, and provides a clue to the molecular mechanisms underlying sequence substitution mutagenesis.     

A novel shuttle vector for Streptomyces spp. and Escherichia coli as a tool in site-directed mutagenesis.

This paper describes the construction and utilization of a novel shuttle vector for Streptomyces spp. and Escherichia coli as a useful vector in site-directed mutagenesis. The shuttle vector pIAFS20 (6.7 kb) has the following features: a replicon for Streptomyces spp., isolated from plasmid pIJ702; the thiostrepton-resistance gene as a selective marker in Streptomyces; the ColE1 origin, allowing replication in E. coli; and the ampicillin-resistance gene as a selective marker in E. coli. Vector pIAFS20 also contains the phage f1 intergenic region, which permits production of single-stranded DNA in E. coli after superinfection with helper phage M13K07. Moreover, the lac promoter is located in front of the multiple cloning sites cassette, allowing eventual expression of the cloned genes in E. coli. After mutagenesis and screening of the mutants in E. coli, the plasmids can be readily used to transform Streptomyces spp. As a demonstration, a 3.2-kb DNA fragment containing the gene encoding the xylanase A from Streptomyces lividans 1326 was inserted into pIAFS20, and the promoter region of this gene served as a target for site-directed mutagenesis. The two deletions reported here confirm the efficiency of this new vector as a tool in mutagenesis.     

Cloning the KpnI restriction-modification system in Escherichia coli

The genes encoding the KpnI restriction and modification (R-M) system from Klebsiella pneumoniae, recognizing the sequence, 5'-GGTAC decreases C-3', were cloned and expressed in Escherichia coli. Although the restriction endonuclease (ENase)- and methyltransferase (MTase)-encoding genes were closely linked, initial attempts to clone both genes as a single DNA fragment in a plasmid vector resulted in deletions spanning all or part of the gene coding for the ENase. Initial protection of the E. coli host with MTase expressed on a plasmid was required to stabilize a compatible plasmid carrying both the ENase- and the MTase-encoding genes on a single DNA fragment. However, once established, the MTase activity can be supplied in cis to the kpnIR gene, without an extra copy of kpnIM. A chromosomal map was generated localizing the kpnIR and kpnIM genes on 1.7-kb and 3.5-kb fragments, respectively. A final E. coli strain was constructed, AH29, which contained two compatible plasmids: an inducible plasmid carrying the kpnIR gene which amplifies copy number at elevated temperatures and a pBR322 derivative expressing M.KpnI. This strain produces approx. 10 million units of R.KpnI/g of wet-weight cells, which is several 1000-fold higher than the level of R.KpnI produced by K. pneumoniae. In addition, DNA methylated with M.KpnI in vivo does not appear to be restricted by the mcrA, mcrB or mrr systems of E. coli.     

Deletion analysis of the cloned replication origin region from bacteriophage M13.

A cloned 270-nucleotide fragment from the origin region of the M13 duplex replicative form DNA confers an M13-dependent replication mechanism upon the plasmid vector pBR322. This M13 insert permits M13 helper-dependent replication of the hybrid plasmid in polA cells which are unable to replicate the pBR322 replicon alone. Using in vitro techniques, we have constructed several plasmids containing deletions in the M13 DNa insert. The endpoints of these deletions have been determined by DNA sequence analysis and correlated with the transformation and replication properties of each plasmid. Characterization of these deletion plasmids allows the following conclusions. (i) The initiation site for M13 viral strand replication is required for helper-dependent propagation of the chimeric plasmid. (ii) A DNA sequence in the M13 insert, localized between 89 and 129 nucleotides from the viral strand initiation site, is necessary for efficient transformation of polA cells. A chimeric plasmid containing the viral strand initiation site, but lacking this additional 40 nucleotide M13 sequence, transforms helper-infected cells at a frequency approximately 10(4)-fold less than that of plasmids containing this additional DNA segment. (iii) The entire M13 complementary strand origin can be deleted without affecting M13-dependent transformation by the hybrid plasmids. We propose a model in which replication of one strand of duplex chimera initiates by nicking at the gene II protein nicking site in the viral strand of the M13 insert, followed by asymmetric single-strand synthesis. Initiation of the complementary strand possibly occurs within plasmid sequences.