Protein expression in Escherichia coli minicells containing recombinant plasmids specifying trimethoprim-resistant dihydrofolate reductases.

Deoxyribonucleic acid fragments containing the structural genes for several trimethoprim-resistant dihydrofolate reductases from naturally occurring plasmids were inserted into small cloning vehicles. The genetic expression of these hybrid plasmids was studied in purified Escherichia coli minicells. The type I dihydrofolate reductase, encoded by plasmid R483 and residing within transposon 7 (Tn7), had a subunit molecular weight of 18,000. The type II dihydrofolate reductase, specified by plasmid R67, had a subunit molecular weight of 9,000. These two enzymes were antigenically distinct in that anti-type II dihydrofolate reductase (R67) antibody did not cross-react with the type I (R483) protein. The trimethoprim-resistant reductase specified by plasmid R388 had a subunit molecular weight of about 10,500 and was immunologically related to the type II (R67) enzyme. A 9,000 subunit of the dihydrofolate encoded by the transposition element Tn402 was also antigenically related to the R67 reductase.     

Protein expression in E. coli minicells by recombinant plasmids.

The polypeptides synthesized in E. coli minicells from recombinant plasmids containing DNA fragments from cauliflower mosaic virus, Drosophila melanogaster, and mouse mitochondria were examined. Molecularly cloned fragments of cauliflower mosaic virus DNA directed the synthesis of high levels of three polypeptides, which were synthesized entirely from within the cloned virus DNA fragments independent of their insertion into the plasmid vehicles. Several fragments of D. melanogaster DNA were capable of initiating polypeptide synthesis; however, termination of these polypeptides was dependent upon the insertion into the plasmid vehicle. The majority of D. melanogaster DNA fragments examined did not direct the detectable synthesis of any polypeptides. Insertion of DNA into the Eco RI site of ColE1 and pSC101 plasmids resulted in the altered expression of plasmid-encoded polypeptides. In the case of ColE1, this site of insertion lies within the colicin E1 structural gene, and insertion of foreign DNA into the site results in the synthesis of an inactive truncated colicin E1 molecule. It is probable that the Eco RI site in pSC101 lies within the structural gene for a polypeptide involved in tetracycline resistance, and insertion of DNA into this site may also result in the synthesis of a truncated or elongated polypeptide.     

Intermediates of replication of NR1 plasmid DNA in Escherichia coli mini-cells

The pulse label of NRL plasmid-containing mini-cells has been shown to be localized mainly in DNA with a floating density in the CsCl-EtBr gradient different from the floating density of supercoil and open circle DNAs. During the chase of the pulse label, the DNA is transfered from the fraction with the intermediate floating density varying between the values for the supercoil and open circle DNA fractions to the fraction located below supercoil DNA in the equilibrium gradient and further to the open circle fraction. Electron microscopic analysis of the material with a higher floating density as compared to supercoil DNA has demonstrated the presence of "heavy" intermediates--covalently closed loosely supercoiled molecules. It is also supported by the sedimentation pattern of the characterized fraction in neutral and alkaline saccharose gradients. Molecules located in the CsCl-EtBr gradient between supercoil and open circle DNAs have the sedimentation constant characteristic for the elongation intermediates. It is suggested that NRL DNA molecules in E. coli mini-cells pass through all the basic stages of replication which results in the formation of open circle DNA or supercoil relaxation complexes.     

Loss of RecA function affects the ability of Escherichia coli to maintain recombinant plasmids containing a Ter site.

In the Escherichia coli chromosome, DNA replication forks arrested by a Tus-Ter complex or by DNA damage are reinitiated through pathways that involve RecA and numerous other recombination functions. To examine the role of recombination in the processing of replication forks arrested by a Tus-Ter complex, the requirements for recombination-associated gene products were assessed in cells carrying Ter plasmids, i.e., plasmids that contain a Ter site oriented to block DNA replication. Of the E. coli recombination functions tested, only loss of recA conferred an observable phenotype on cells containing a Ter plasmid, which was inefficient transformation and reduced ability to maintain a Ter plasmid when Tus was expressed. Given the current understanding of replication reinitiation, the simplest explanation for the restriction of Ter plasmid maintenance was a reduced ability to restart plasmid replication in a recA tus(+) background. However, we were unable to detect a difference in the efficiency of replication arrest by Tus in recA-proficient and recA-deficient cells, which suggests that the inability to restart arrested replication forks is not the cause of the restriction on growth, but is due to an additional function provided by RecA. Other explanations for restriction of Ter plasmid maintenance were examined, including plasmid multimerization, plasmid rearrangements, and copy number differences. The most likely cause of the restriction on Ter plasmid maintenance was a reduced copy number in recA cells that was detected when the copy number was measured in relation to an external control. Possibly, loss of RecA function leads to improper processing of replication forks arrested at a Ter site, leading to the generation of degradation-prone substrates.     

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.     

Growth inhibition of Escherichia coli by E colicin plasmids

Plasmids were isolated from E colicinogenic strains and transformed into prototrophic Escherichia coli K 12 strain DB364. Screening of E colicinogenic transformants for growth on defined medium revealed an apparent amino acid auxotrophy mediated by E4 and, to a lesser extent, E7 colicin plasmids. The auxotrophy was further investigated in E4 colicinogenic strains. From such auxotrophic transformants, denoted Pmi+ (plasmid-mediated inhibition of growth), Pmi- variants were obtained at a frequency of 3 X 10(-4) per bacterium. Plasmid loss was not detected among Pmi- clones. Isolation of E4 colicin plasmids from Pmi- clones and retransformation of strain DB364 with these plasmids showed that 40% of the plasmids were unable to inhibit growth of DB364 and were inferred to have alterations in an E4 colicin plasmid gene termed pmi. All such plasmids were indistinguishable from native E4 colicin plasmids, with respect to colicin immunity, colicin production and excretion, and sensitivity to lysis by mitomycin C. Experiments examining the nutritional basis of the plasmid-mediated auxotrophy indicated that at least seven amino acids, isoleucine, leucine, valine, arginine, methionine, serine and glycine, were involved in the auxotrophy. However, supplementation with only these seven amino acids did not completely restore growth. Assays of the activities of enzymes involved in amino acid biosynthesis in colicinogenic and non-colicinogenic strains under repressing and derepressing growth conditions suggested that E4 colicin plasmids did not repress synthesis of the implicated amino acids.     

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.     

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.     

Binding of NR1 plasmid DNA to membrane during replication in Escherichia coli mini-cells

Studying the replication of NR1 plasmid in E. coli mini-cells it was shown that the character of bond between plasmid DNA and membrane depends on the stage of replication cycle of the plasmid. On initiation the DNA-membrane complex is sensitive to the action of ionic force. In the process of elongation the bond of DNA molecules with the membrane is unstable if exists at all, and can be broken even by the nonionic detergent. At the final stage of replication the newly synthesized molecules form a complex with the membrane structures which is unstable in the presence of 0,5 M NaCl. The destruction of the complex followed by the open cycle of plasmid DNA coming out of it takes place under the action of ionic detergent.     

R46-derived recombinant plasmids affecting DNA repair and mutation in E. coli

Summary Recombinant plasmids which render their host less mutable and more sensitive to some DNA-damaging agents have been isolated from the N-group plasmid R46. These plasmids have been physically mapped and found to originate from the region of R46 that has been deleted in pKM101. This deleted region is well removed from the muc region of R46 and pKM101 which is responsible for the mutator effects of these plasmids.The effect of these anti-mutagenic plasmids on the ability of pKM101 to complement umuC mutations has been examined, and they have been found to inhibit the complementation of such strains. We propose that these plasmids may code for a negative control function acting on the muc gene.     

The structures and fidelity of replication of mouse mitochondrial DNA-pSC101 EcoRI recombinant plasmids grown in E. coli K12

Recombinant DNAs containing the E. coli plasmid pSC101 and mouse cell (LA9) mitochondrial DNA (mtDNA) were formed in vitro via ligation of DNA fragments from limit EcoRI endonuclease digests and were used to transform E. coli K12. Four structurally different recombinant plasmid DNAs from transformed clones were characterized. Two of these were analyzed extensively and the mtDNA portions compared with mtDNA from LA9 cells. No differences were detected in the physical or chemical properties examined, except that the E. coli mtDNA lacked the alkali lability characteristic of animal mtDNAs.Heteroduplexes between the LA9 portions of the recombinant plasmids and LA9 mtDNA were analyzed by absorbance melting. The melting temperatures were indistinguishable from reannealed LA9 mtDNA homoduplexes, indicating that single-base replication errors occur at a frequency of fewer than 1 nucleotide in 300. Electron microscopic analyses of plasmid-LA9 mtDNA heteroduplexes and a comparison of agarose gel electrophoresis of restriction endonuclease fragments also indicated no differences. These results were independent of the order or the relative orientation of the pSC101 and mtDNA fragments.A third EcoRI fragment in LA9 mtDNA, not found in an earlier study (Brown and Vinograd, 1974), has been positioned in the LA9, EcoRI map. This fragment contains 165±10 nucleotide pairs.     

Stability of Escherichia coli strains harboring recombinant plasmids for L-threonine production

Escherichia coli recombinant strains bearing the thr operon have been previously selected for threonine production and phenotypically classified according to antibiotic resistance properties (Nudel et al. 1987).Further analysis of those strains permitted the isolation and restriction mapping of two different plasmids of 13 kb and 18.6 kb. The smaller one, which expressed tetracycline resistance gave better results on threonine accumulation but it was rather unstable when grown without antibiotic pressure. Therefore, other hosts were transformed with those plasmids to improve stability.A threonine-auxotrophic strain was a better host for plasmid maintenance and expression of thr operon. Host influence in plasmid-mediated threonine production was studied in terms of specific yields (the ratios of threonine accumulated to biomass values) and of plasmid maintenance (percent of Ap^rTc^r clones after cultivation in non selective media).We also determined that semisynthetic media of defined composition were better than rich media for threonine expression, due to feed-back controls exerted by undesired catabolites accumulated in complex media.     

Identification of the Escherichia coli tonB gene product in minicells containing tonB hybrid plasmids.

Sodium dodecyl sulfate/polyacrylamide gel analysis of proteins encoded by a series of tonB⁺ plasmids in minicells has identified the ton B gene product as a protein with an apparent molecular weight of 36,000. A parallel analysis of seven ton B mutations which have been genetically crossed onto a tonB⁺ plasmid supports this identification; the 36,000 M_r protein is absent from the set of proteins encoded by each tonB^? plasmid. Four of the tonB mutations are apparently IS1 insertions. The locations of these insertions within tonB have been determined by restriction endonuclease mapping. Correlation of these IS1 insertion sites with the molecular weights of prematurely terminated tonB polypeptides, suggests that tonB is transcribed in the direction opposite to that of the nearby tryptophan operon. In addition, a protein encoded by one of the inverted repeat sequences of the transposable element Tn5 has been tentatively identified.     

ColE1 hybrid plasmids containing Escherichia coli genes involved in the biosynthesis of glutamate and glutamine

The Clarke-Carbon bank of Escherichia coli strains carrying ColE1 hybrid plasmids was screened for complementation of gdh, gltB, and glnA mutations affecting nitrogen metabolism in E. coli. Plasmids which complemented each one of these mutations were isolated. In every case, the plasmids conferred to otherwise mutant cells the capacity to synthesize the corresponding wild-type enzymes: glutamate dehydrogenase, glutamate synthase, and glutamine synthetase (GS), respectively. For three representative plasmids, endonuclease restriction maps were constructed. One of the plasmids, pACR1, which complemented glnA mutations, including the glnA21::Tn5 insertion, was deemed to carry the glnA⁺ allele. GS synthesis by pACR1 $\text{glnA}{}^{\mathrm{+}}\text{glnA20}$ heterozygous merodiploids was subjected to repression by growth on 15 mm NH₄⁺ and had a twofold high derepressed level than wild-type (glnA⁺) haploid cells when grown on 0.5 mm NH₄⁺ or on glutamate as only nitrogen sources. The presence of glutamine as sole nitrogen source promoted repressed GS synthesis in the $\text{glnA}{}^{\mathrm{+}}\text{glnA20}$ merodiploids. By contrast, glutamine allowed almost fully derepressed synthesis of GS in glnA⁺ haploid cells.