ColE1 hybrid plasmids for Escherichia coli genes of glycolysis and the hexose monophosphate shunt.

The Clarke-Carbon clone bank carrying ColE1-Escherichia coli DNA has been screened by conjugation for complementation of glycolysis and hexose monophosphate shunt mutations. Plasmids were identified for phosphofructokinase (pfkA), triose phosphate isomerase (tpi), phosphoglucose isomerase (pgi), glucose-6-phosphate dehydrogenase (zwf), gluconate-6-phosphate dehydrogenase (gnd), enolase (eno), phosphoglycerate kinase (pgk), and fructose-1,6-P2 aldolase (fda). Enzyme levels for the plasmid-carried gene ranged, for the various plasmids, from 4- to 25-fold the normal level.     

Regulation of the L-arabinose operon in strains of Escherichia coli containing ColE1-ara hybrid plasmids.

Summary Hybrid plasmids were constructed from fragments of F'ara episomes formed by the restriction endonuclease EcoRI and a linear form of the plasmid ColE1 created by cleavage with EcoRI. Hybrid plasmids were constructed containing the entire ara region or the ara region with various parts deleted. E. coli K12 host strains were constructed which contained different deletions of the ara region. The hybrid plasmids were transferred to those strains whose ara deletion complemented that of the plasmid. The initial differential rates of synthesis of L-arabinose isomerase, the product of the araA gene, were determined for the Ara⁺, plasmid containing strains. These studies demonstrated that strains containing (araO1BA)718 produce elevated levels of araC protein, suggesting the araC promoter has been altered by this deletion. Evidence is also presented which suggests that araC protein activates the ara-BAD operon to higher levels when it is present in cis rather than trans. Amplification of the products of the cloned genes is observed when compared to haploid levels in some cases.     

Synthetic ColE1 plasmids carrying genes for penicillin-binding proteins in Escherichia coli

Clarke and Carbon's collection of 2000 Escherichia coli strains which harbor ColE1 plasmids carrying small random segments of the E. coli chromosome was screened for the correction of mutational defects in penicillin-binding proteins (PBPs): ponA (PBP-1a), ponB (PBP-1b), dacB (PBP-4), and pfv (PBP-5). We found plasmids carrying chromosomal segments containing ponA⁺-aroB⁺ (pLC29-47), ponB⁺-tonA⁺ (pLC4-43, pLC4-44, and pLC19-19), and argG⁺-dacB⁺ (pLC10-46 and pLC18-38). Characters of these plasmids were analyzed. Two other plasmids (pLC26-6 and pLC4-14) previously found to correct ftsI mutation (Y. Nishimura, Y. Takeda, A. Nishimura, H. Suzuki, M. Inouye, and Y. Hirota (1977)Plasmid1, 67–77) were also investigated further. Restriction maps of chromosomal DNAs carried by pLC29-47, pLC4-44, pLC19-19, pLC18-38, pLC26-6, and pLC4-14 were constructed. The regions of ponB-tonA on pLC4-44 and pLC19-19, and of leuA-ftsI-murE and F on pLC26-6 were located on the restriction maps. Although both pLC26-6 and pLC4-14 corrected a thermosensitive mutation, ftsI, which causes a defect in cell division due to abnormal PBP-3, only pLC26-6 led to restoration of PBP-3 production by an ftsI mutant, while pLC4-14 did not. Restriction and heteroduplex analyses of pLC26-6 and pLC4-14 have shown the absence of nucleotide sequence homology between them. The plasmids, pLC29-47 carrying ponA⁺ and pLC4-43, pLC4-44, and pLC19-19 carrying ponB⁺ led the host cell to overproduce the respective PBP.     

Synthetic ColE1 plasmids carrying genes for cell division in Escherichia coli.

Clarke and Carbon's collection of 2000 E. coli strains, which harbor ColE1 plasmids carrying small random segments of the E. coli chromosome, was screened for the correction of thermosensitive defects in the processes of cell division and in the synthesis of murein-lipoprotein. The genetic defects examined in this screening were those in partition of daughter nuclei (par), cleavage of cells (fts), determination of a cell shape (rod), and synthesis of murein-lipoprotein (lpo). We found plasmids carrying E. coli chromosomal segments containing ftsB⁺, ftsE⁺,ftsI⁺,ftsM⁺, and parA⁺. However, none was found to transfer ftsA⁺, ftsC⁺, ftsF⁺, ftsG⁺, ftsJ⁺, ftsK⁺, ftsL⁺, parB⁺, rod⁺, and lpo⁺. One of the donor strains transferring a gene that corrected thermosensitive cell cleavage in the ftsI^? mutant overproduced the penicillin-binding protein 3 by ca. 10-fold.     

Glutamine synthetase cascade: enrichment of uridylyltransferase in Escherichia coli carrying hybrid ColE1 plasmids

Colchicine-binding properties of the total cytoplasmic pool of tubulin from rat liver were evaluated in tubulin-stabilizing (TS) supernates. Microtubules were separated from free tubulin using a microtubule-stabilizing solution (MTS) and ultracentrifugation. [³H]Colchicine-binding properties of microtubule-derived tubulin were investigated in supernates prepared after resuspension of MTS pellets in TS. In TS buffer at 37 °C the colchicine-binding activity of the total cytoplasmic pool of tubulin decayed with $\text{T}\text{1}\text{2}$ of 3.39 h. Resuspended pellet tubulin decayed much more rapidly under the same conditions with a $\text{T}\text{1}\text{2}$ of 0.72 h. This rapid time decay of microtubule-derived tubulin was found to be at least partially attributable to prior microtubule-stabilizing solution exposure. Since tartrate has been reported to increase the rate of colchicine binding to tubulin, sodium tartrate (150 mm) was added to our colchicine-binding system. This addition increased the detectable [³H]colchicine binding by 10% in the total cytoplasmic preparation and by 85% in the resuspended pellet preparation. Addition of tartrate (150 mm) also resulted in a 105% increase in the $\text{T}\text{1}\text{2}$ for total cytoplasmic tubulin and a 412% increase for microtubule derived tubulin. Total cytoplasmic supernates of liver bound [³H]colchicine linearly over a wide range of tissue concentrations. However, resuspended microtubule-stabilizing solution pellet supernates in tubulin-stabilizing solution showed some increase in colchicine binding per tissue weight in the more dilute samples. Our data which demonstrate differences in colchicine-binding properties for total cytoplasmic and microtubule-derived pools of tubulin suggest that present assays for hepatic tubulin polymerization which assume identical binding properties should be interpreted with caution.     

Two-step method for curing Escherichia coli of ColE1-derived plasmids

To cure Escherichia coli for plasmids derived from the ColE1 replicon advantage is taken of the fact that maintenance of this replicon requires a wild-type allele of polA, encoding DNA polymerase I. Curing is achieved by cotransduction of a mutant polA allele with metE::Tn10, fadAB::Tn10 or other transposon insertions near polA. Reciprocal transduction to Met(+) Pol(+) or to Fad(+) Pol(+) ensures reestablishment of the original genotype except for loss of the plasmid. A set of useful bacterial strains is provided.     

Stability of ColE1-like and pBR322-like plasmids in Escherichia coli

The average copy number, the level of ampicillin resistance conferred by one plasmid, and the degree of plasmid multimerization were determined for several ColE1-like and pBR322-like plasmids. From the results obtained, the variance of the units of partition corresponding to each plasmid studied was calculated. Experimentally determined plasmid stability was compared with that calculated using the variance of the units of partition and the ratio between the generation times of plasmid-free and of plasmid-carrying cells, assuming that the units of partition are distributed randomly between daughter cells. Stability of the pBR322-like plasmids present mainly as monomers in the bacterial host was consistent with random partitioning, whereas pBR322-like plasmids, present mainly as dimers, and the ColE1-like plasmid showed greater stability than that predicted with random partitioning at cell division.     

Homology between Escherichia coli plasmids ColE1 and p15A.

The location and extent of the homology between plasmids ColE1 and p15A were determined by analysis of heteroduplexes formed between them as well as with a related plasmid, pBR322, and by hybridization of radioactive deoxyribonucleic acids to restriction fragments of p15A and ColE1. The homology between the plasmids contained the entire region of ColE1 required for its replication as well as an additional 400 base pairs downstream from the origin of replication. This region on p15A, which was 980 +/- 43 base pairs, started at 0.1 of the molecular length from one end formed by cleavage with the restriction endonuclease BglI and extended to 0.54 of the molecular length from the same end. Restriction cleavage maps for the enzymes BglI, HpaI, HaeII, HaeIII, and HincII are also presented.     

Synthesis of mot and che gene products of Escherichia coli programmed by hybrid ColE1 plasmids in minicells.

Hybrid Escherichia coli ColE1 plasmids carrying the genes for motility (mot) and chemotaxis (che) were transferred to a minicell-producing strain. The mot and che genes on the hybrid plasmid directed protein synthesis in minicells. Polypeptides synthesized in minicells were identical to the products of the motA, motB, cheA, cheW, cheM, cheX, cheB, cheY, and cheZ genes previously identified by using hybrid lambda and ultraviolet-irradiated host cells (Silverman and Simon, J. Bacteriol. 130:1317-1325, 1977), thus confirming these gene product assignments. The products of some che genes (cheA and cheM) appeared as more than one band on polyacrylamide gel electrophoresis, but analysis of partial peptide digests of these polypeptides suggested that the multiple forms were coded for by a single gene. Measurement of the physical length of the hybrid plasmids allowed an estimate of the amount of coding capacity of the cloned deoxyribonucleic acid, which was devoted to the synthesis of the mot and che gene products. These estimates were also consistent with the hypothesis that the multiple polypeptides corresponding to cheA and cheM were the products of single genes.     

In situ detection of Escherichia coli cells containing ColE1-related plasmids by hybridization to regulatory RNA II

A method is described for the in situ detection of individual whole fixed cells of Escherichia coli containing ColE1-related plasmids. It makes use of fluorescence in situ hybridization (FISH) and the regulatory RNA II as a target molecule for both, Cy3- and HRP-labeled olinucleotide probes. Various methods for signal amplification were compared. Probes targeting the regulatory RNA I did not result in the in situ detection of plasmid-bearing cells.     

Construction and characterization of hybrid plasmids containing the Escherichia coli nrd region.

Recombinant plasmids containing all or part of the genetic region of Escherichia coli coding for the two subunits of ribonucleoside diphosphate reductase (proteins B1 and B2) were constructed with the aid of the multicopy plasmid pBR322. Two of these plasmids (pPS1 and pPS2) appeared to carry both a regulator and the complete structural information for the enzyme and, after transformation of E. coli, directed a 10- to 20-fold overproduction of both proteins B1 and B2. The other plasmids (pPS101 and pPS201) carried structural information for only protein B2. Cells carrying pPS1 and pPS2 showed a 5- to 500-fold increased resistance against the drug hydroxyurea. This establishes that in E. coli the inhibition of deoxyribonucleic acid synthesis by hydroxyurea is fully explained by its action on ribonucleotide reductase.     

Citrate synthase activity in Escherichia coli harbouring hybrid plasmids containing the gltA gene

A hybrid plasmid, pDB2, was constructed by ligating a 3.24 kb EcoRI/HindIII fragment of the Escherichia coli chromosome into pBR322. This was used to transform a gltA mutant which was devoid of citrate synthase activity. The resultant strain expressed very high citrate synthase activity and this enabled a simplified purification of the homogeneous enzyme in high yield. The subunit Mr was estimated as 47000-49000 by SDS gel electrophoresis, which closely resembles the eukaryotic form of the enzyme. Evidence for some conservation of sequence between the two proteins was revealed in the acid cleavage pattern at aspartyl-prolyl residues. In addition to coding for the structural gene for citrate synthase, the 3.24 kb EcoRI/HindIII fragment also retained the genetic structure necessary for control of enzyme synthesis since the expression of enzyme activity in the strain harbouring pDB2 was still subject to glucose repression.     

Recombinant plasmids with genes for the biosynthesis of alkaline phosphatase of Escherichia coli

Summary Restriction maps of several recombinant plasmids representing a section of the E. coli K12 chromosome 35,000 bp in size with the genes phoA, proC and phoB were prepared. The orientation of phoA and the exact position of its N-terminal end on this map were determined by identifying a subfragment which carried the phoA promoter and by determining the nucleotide sequence of a 160 bp portion of this subfragment comprising the codons for the N-terminal end of pre-alkaline phosphatase. From this DNA sequence the leader sequence of alkaline phosphatase which consists of 21 amino acids was derived.     

Isolation and study of hybrid plasmids carrying Escherichia coli threonine operon genes

A fragment of Escherichia coli chromosome containing the intact threonine operon or its distinct genes has been cloned on the pBR322 plasmid. This fragment has been mapped using some restriction endonucleases. Cloning results in an increased level of appropriate enzyme activity in cells containing hybrid plasmids. Those carrying the complete threonine operon are capable of accumulating threonine up to 5 g/l in culture medium during 48 h. When multi-copy plasmids are used for gene cloning, interpretation of experiments aimed at transformation of auxotrophic bacterial strains, might be complicated. For example, transformation of appropriate threonine auxotrophs by a hybrid plasmid carrying mutation in the threonine gene, might result in prototrophic phenotype. It is possible that the great amount of mutant enzyme molecules compensated their low activity. On the contrary, the presence of a gene within the plasmid, as shown by restriction and biochemical analysis, did not always ensure the growth on a minimal medium of auxotrophs transformed by this plasmid.     

Dual-origin plasmids containing an amplifiable ColE1 ori; temperature-controlled expression of cloned genes

Dual-origin plasmids comprising an inducible ColE1-derived origin of replication controlled by the λ p_R promoter, the c1857 temperature-sensitive represser gene and the pSC101 origin of replication and its associated par sequence, were constructed. Such plasmids carrying cloned genes were stably maintained at four copies per chromosome, and were readily amplifiable by thermal induction. Cloned gene expression increased with copy number, and accumulation values of > 20% total cellular protein were detected. These vectors should prove useful for the production of foreign protein on a large scale, since they provide for stable plasmid maintenance during the growth phase, and high-level gene expression without plasmid loss during the production phase.     

Hybrid plasmids containing the araBAD genes of Escherichia coli B/r.

The DNA fragments generated by restriction endonuclease BamI which contain the araCBAD genes from E.coli B/r have been cloned. The DNA fragments containing ara genes were idenified by a compairson of the BamI fragments of lambdah80dara phages containing different ara deletion mutations. The ara genes were cloned into the plasmid pBR317, a derivative of ColE1. The cloned DNA fragments were analyzed by digestion with pairs of restriction endonucleases to determine the molecular weight of the chimeras and to identify the cloned ara DNA fragments. The cloned ara fragments were also identified by genetic complementation and recombination tests.