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.     

Cloned DNA fragment specifying major outer membrane protein a in Escherichia coli K-12.

Plasmid pMC44 is a recombinant plasmid that contains a 2-megadalton EcoRI fragment of Escherichia coli K-12 DNA joined to the cloning vehicle, pSC101. The polypeptides specified by plasmid pMC44 were identified and compared with those specified by pSC101 to determine those that are unique to pMC44. Three polypeptides specified by plasmid pMC44 were localized in the cell envelope fraction of minicells: a Sarkosyl-insoluble outer membrane polypeptide (designated M2), specified by the cloned 2-megadalton DNA fragment, and two Sarkosyl-soluble membrane polypeptides specified by the cloning plasmid pSC101. Bacteria containing plasmid pMC44 synthesized quantities of M2 approximately equal to the most abundant E. coli K-12 outer membrane protein. Evidence is presented that outer membrane polypeptide M2, specified by the recombinant plasmid pMC44, is the normal E. coli outer membrane protein designated protein a by Lugtenberg and 3b by Schnaitman.     

Effect of nalidixic acid and novobiocin on pBR322 genetic expression in Escherichia coli minicells.

The effects of two deoxyribonucleic acid (DNA) gyrase inhibitors, nalidixic acid and novobiocin, on the gene expression of plasmid pBR322 in Escherichia coli minicells were studied. Quantitative estimates of the synthesis of pBR322-coded polypeptides in novobiocin-treated minicells showed that the synthesis of a polypeptide of molecular weight of 34,000 (the tetracycline resistance protein) was reduced to 11 to 20% of control levels, whereas the amount of a polypeptide of 30,500 (the beta-lactamase precursor) was increased to as much as 200%. Nalidixic acid affected the synthesis of the tetracycline resistance protein similarly to novobiocin, although to a lesser extent. The effects of nalidixic acid were not observed in a nalidixic-resistant mutant; those induced by novobiocin were only partially suppressed in a novobiocin-resistant mutant. The synthesis of one of the inducible tetracycline-resistant proteins (34,000) coded by plasmid pSC101 was also reduced in nalidixic acid- and novobiocin-treated minicells. These results suggest that the gyrase inhibitors modified the interaction of ribonucleic acid polymerase with some promoters, either by decreasing the supercoiling density of plasmid DNA or by altering the association constant of the gyrase to specific DNA sites.     

Identification by deletion analysis of an inducible protein required for plasmid pSC101-mediated tetracycline resistance.

Plasmids containing small deletions within a tetracycline (Tc) resistance gene(s) of plasmid pHA121 were isolated. Plasmid pHA121 was formed by ligating the EcoRI-digested Tc resistance plasmid pSC101 and similarly digested mini-ColE1 plasmid pHA105. The DNA deletion plasmids were constructed by digesting plasmid pHA121 DNA with the restriction endonucleases BamH1 and Sal1 and, in addition, λ exonuclease. Two plasmids, designated pJT131 and pJT133, had small deletions of approximately 0.64 to 0.8 kb and a comparison of the radioactive polypeptides synthesized in plasmid-containing minicells revealed that a 34-kdal polypeptide was not specified by either pJT131 or pJT133. We conclude that the 34-kdal polypeptide is required for the expression of Tc resistance and that its structural gene probably maps in the deleted region of pSC101 DNA.     

Sequence organization and expression of a yeast plasmid DNA.

Saccharomyces cerevisiae strain A364A D5 contains circular double-stranded DNA molecules of 6230 +/- 30 base pairs (2mu DNA) which are present in 68 copies per cell and make up 2.4% of the haploid genome. About 0.4% of non-poly A containing yeast RNA hybridizes to the yeast DNA circles. When denatured and then self-annealed, the DNA molecules assume a characteristic "dumbbell" shape in the electron microscope indicating that each circle possesses a non-tandem inverted repeat sequence of 630 +/- 10 base pairs. Eco-RI digestion of purified 2mu DNA yields 4 fragments on an agarose gel whose combined molecular mass is twice that of the monomer circle, suggesting that there are 2 populations of circles, each of the same molecular weight. Representatives of each population have been separated by cloning in Escherichia coli via the bacterial plasmid pSC101. Heteroduplex analysis of the cloned circles show that the 2 different populations arise because of intramolecular recombination between the inverted repeat sequences. Acrylamide gel patterns of polypeptides synthesized in bacterial mini-cells containing the hybrid plasmids between 2mu DNA and pSC101 are significantly different than the pattern obtained from mini-cells containing pSC101 alone.     

Analysis of the promoter-distal region of the tra operon of the F sex factor of Escherichia coli K-12 encoded by EcoRI restriction fragments f17, f19, and f2.

The promoter-distal region of the tra operon of the F sex factor Escherichia coli K-12 was analyzed, using the chimeric plasmid pRS31, which contains the F EcoRI restriction fragments f17, f19, and f2 cloned into the EcoRI site of pSC101. A series of deletion plasmids of pRS31, extending increasing distances from a site in f17 through f19 and ending in f2, were isolated. These plasmids were examined by heteroduplex analysis with the parent DNA, and a restriction map of this region of DNA was constructed. A series of Tn5 insertion derivatives of pRS31 were also isolated and mapped, using both heteroduplex analysis and restriction mapping. Both the insertion and deletion mutants were tested in minicells for the synthesis of radioactively labeled proteins. This allowed the identification of the individual gene products and mapping of the genes. The result is a saturated physical map of this region of DNA from fragment f17 through to the IS3 insertion sequence near the promoter-distal end of f2.     

Translation in Escherichia coli mini-cells containing hamster mitochondrial DNA-Co1E1 - Ampr recombinant plasmids.

Hamster mitochondrial DNA is cleaved into two fragments (4.2 and 11.4 kilobase pairs of DNA (kb)) by the restriction enzyme, Eco RI. Recombinant DNA molecules formed in vitro between an Escherichia coli plasmid, Co1E1 - Ampr, and Eco RI-digested hamster mitochondrial DNA were transformed into E. coli K12. The translation products of the parent plasmid, Co1E1 - Ampr, and recombinant plasmid DNAs containing (i) the 4.2 kb mitochondrial DNA fragment and (ii) the 11.4 kb fragment were characterized on sodium dodecyl sulfate-polyacrylamide gels using bacterial mini-cell lysates. The Co1E1 - Ampr plasmid specifies at least six polypeptides whose structural genes comprise 56% of the plasmid DNA. Insertion of hamster mitochondrial DNA at the Eco RI site of the plasmid alters the relative rate of synthesis of these six polypeptides and induces the occurrence of a new band on sodium dodecyl sulfate-polyacrylamide gels which is probably not specified by the inserted mitochondrial DNA sequences.     

Polypeptides encoded by the mer operon.

HgCl2-induced polypeptides synthesized by Escherichia coli minicells containing recombinant or natural HgR plasmids were labeled with [35S]methionine and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All plasmids examined encoded two heavily labeled, HgCl2-inducible polypeptides of 69,000 and 12,000 daltons. Most plasmids also encoded two additional HgCl2-inducible proteins in the 14,000- to 17,000-dalton range. Antiserum prepared against a purified mercuric ion reductase reacts with the 69,000-dalton polypeptide and a minor 66,000-dalton protein seen in several different HgR minicells. Recombinant plasmids constructed from portions of mer DNA from the IncFII plasmid NR1 were also analyzed in the minicell system. Five HgCl2-inducible polypeptides (69,000, 66,000, 15,100, 14,000, and 12,000 daltons) were synthesized in minicells carrying pRR130, a recombinant derivative containing the EcoRI-H and EcoRI-I restriction fragments of NR1. The EcoRI-H fragment of NR1 encodes the three small mer proteins of 15,100, 14,000, and 12,000 daltons and the amino-terminal 40,000 daltons of the mercuric ion reductase monomer.     

Plasmid segregation into minicells is associated with membrane attachment and independent of plasmid replication.

The role of plasmid replication in the segregation of plasmids into Escherichia coli minicells was investigated with temperature-sensitive replication mutants derived from E. coli plasmids ColE1 and pSC101. For as long as six generations of growth, at permissive or nonpermissive temperatures (when greater than 80% of plasmid replication was inhibited), the same amount of previously 3H-labeled plasmid DNA segregated into minicells. Density gradient separations of wild-type and temperature-sensitive plasmid DNA from both replicons segregated into the minicells showed that about 20 to 25% was stably associated with the minicell membrane at both temperatures. Electron microscopy showed this DNA to consist of circular plasmid molecules attached to the minicell membrane. These combined findings suggest that segregation of plasmids into minicells and their association with the minicell membrane are interrelated and independent of plasmid replication.     

Molecular and genetic studies of an R factor system consisting of independent transfer and drug resistance plasmids

Certain genetic, structural, and biochemical properties of a class 2 R-factor system consisting of the conjugally proficient transfer plasmid I and the naturally occurring non-conjugative tetracycline (Tc) resistance plasmid 219 are reported. I and 219 exist as separate plasmid deoxyribonucleic acid (DNA) species in both Escherichia coli and Salmonella panama, having molecular weights of 42 x 10(6) and 5.8 x 10(6), respectively. The buoyant densities of I and 219 are 1.702 and 1.710 g/cm(3), respectively, in neutral cesium chloride. Although the Tc resistance plasmid is not transmissible in a normal conjugal mating, it is mobilized in a three-component mating by plasmid I and by certain other conjugative plasmids of the fi(+) or fi(-) phenotype. Mobilization does not appear to involve intermolecular recombination between plasmids, and no covalent linkage of resistance markers and fertility functions is observed. Transformation of CaCl(2)-treated E. coli by plasmid DNA is shown to be a useful procedure for studying the biological properties of different plasmid molecular species that have been fractionated in vitro, and for selectively inserting non-self-transmissible plasmids into specific bacterial strains. The effects of tetracycline on the rate of protein synthesis carried out by plasmid 219 were studied by using isolated E. coli minicells into which this plasmid had segregated. Consistent with the results of earlier investigations showing the inducibility of plasmid-mediated Tc resistance in E. coli, the antibiotic was observed to stimulate protein synthesis in minicells carrying the plasmid 219 and totally inhibit (3)H-leucine incorporation by minicells lacking the Tc resistance marker. Five discrete polypeptide species were synthesized by minicells carrying plasmid 219; exposure of minicells or parent bacteria to Tc resulted in specific and reproducible changes in polypeptide synthesis patterns.     

hisT is part of a multigene operon in Escherichia coli K-12.

The Escherichia coli K-12 hisT gene has been cloned, and its organization and expression have been analyzed on multicopy plasmids. The hisT gene, which encodes tRNA pseudouridine synthase I (PSUI), was isolated on a Clarke-Carbon plasmid known to contain the purF gene. The presence of the hisT gene on this plasmid was suggested by its ability to restore both production of PSUI enzymatic activity and suppression of amber mutations in a hisT mutant strain. A 2.3-kilobase HindIII-ClaI restriction fragment containing the hisT gene was subcloned into plasmid pBR322, and the resulting plasmid (designated psi 300) was mapped with restriction enzymes. Complementation analysis with different kinds of hisT mutations and tRNA structural analysis confirmed that plasmid psi 300 contained the hisT structural gene. Enzyme assays showed that plasmid psi 300 overproduced PSUI activity by ca. 20-fold compared with the wild-type level. Subclones containing restriction fragments from plasmid psi 300 inserted downstream from the lac promoter established that the hisT gene is oriented from the HindIII site toward the ClaI site. Other subclones and derivatives of plasmid psi 300 containing insertion or deletion mutations were constructed and assayed for production of PSUI activity and production of proteins in minicells. These experiments showed that: (i) the proximal 1.3-kilobase HindIII-BssHII restriction fragment contains a promoter for the hisT gene and encodes a 45,000-dalton polypeptide that is not PSUI; (ii) the distal 1.0-kilobase BssHII-ClaI restriction fragment encodes the 31,000-dalton PSUI polypeptide; (iii) the 45,000-dalton polypeptide is synthesized in an approximately eightfold excess compared with PSUI; and (iv) synthesis of the two polypeptides is coupled, suggesting that the two genes are part of an operon. Insertion of mini-Mu d1 (lac Km) phage into plasmid psi 300 confirmed that the hisT gene is the downstream gene in the operon.     

Identification of the Escherichia coli K-12 cpxA locus as a single gene: construction and analysis of biologically-active cpxA gene fusions

In the accompanying communication we showed that a 2 kb EcoRI-BamHI restriction fragment from the pfkA-rha interval of the Escherichia coli K-12 chromosome fully complemented a chromosomal cpxA mutation when the fragment was cloned in pBR325. The same fragment cloned in pBR322 lacked any complementing activity. We show here that minicells containing the pBR325 derivative (pRA310) synthesized a 33 kDa polypeptide, designated phi 33, that was not synthesized in minicells containing the pBR322 derivative (pRA311) or either of the parent plasmids. Synthesis of the phi 33 polypeptide did not occur in minicells containing Tn5 insertion alleles of pRA310 that inactivated its cpxA complementing activity. These insertions mapped within the vector cat (chloramphenicol acetyltransferase gene) sequence immediately adjacent to the EcoRI site of pRA310 and within the 700-800 bp of the cloned EcoRI-BamHI fragment immediately adjacent to the EcoRI site. Tn5 insertions located within the fragment but closer to the BamHI terminus affected neither the cpxA complementing activity of pRA310 nor synthesis of the phi 33 polypeptide in minicells. Plasmid pRA311 could be converted to a plasmid with cpxA complementing activity by cloning into its EcoRI site a restriction fragment containing a hybrid trp-lacUV5 promoter, the lacZ ribosome binding site, and the first eight lacZ codons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic mapping of the F plasmid gene that promotes degradation of stable ribonucleic acid in Escherichia coli.

F+ Escherichi coli cells that contain an srnA mutant allele degrade their stable ribonucleic acid (RNA) extensively after RNA synthesis is blocked at 42 degrees C. The relevant gene promoting degradation of stable RNA, srnB+, or its promoter was mapped between 1.7 and 2.8 kilobases on the F plasmid by using deleted F' plasmids and chimeric plasmids composed of pSC101 and fragments of F plasmid.     

On the nature of tetracycline resistance controlled by the plasmid pSC101.

In vitro enzymatic alteration of plasmid phenotype and in vitro construction of recombinant plasmids containing genetic information derived from the plasmid pSC101 have been used to investigate the mechanism of function of tetracycline resistance determined by the plasmid pSC101. The resistance has been shown to be inducible and involves the increased synthesis of membrane-associated polypeptides of 34,000, 26,000 and 14,000 daltons that are encoded for by the plasmid. The 34,000 dalton polypeptide along with another plasmid-encoded polypeptide of 18,000 daltons function in an ATP-independent manner to prevent the accumulation of tetracycline by the cell. These polypeptides are sufficient for resistance. A second component of plasmid-determined resistance involves the 14,000 dalton polypeptide and reduces the initial adsorption of tetracycline by sensitive cells, but is not alone sufficient for the generation of resistance. The role of the 26,000 dalton polypeptide in tetracycline resistance has not been identified.     

Plasmid pSC101 replication mutants generated by insertion of the transposon Tn1000

A derivative of pSC101, pLC709, was constructed by ligation of the HincII-A fragment of pSC101 to the mini-colEI plasmid pVH51 and to a DNA fragment encoding resistance to the antibiotics streptomycin and spectinomycin. Insertions of the transposon Tn1000 (gamma-delta) into the pSC101 replication region of pLC709 were isolated following cotransfer of the plasmid with the sex factor F. The sites of insertion of the transposon were determined by restriction enzyme analysis and the replication and incompatibility properties of the insertion plasmids and DNA fragments cloned from them were analysed. The insertion mutations defined a locus, inc, of approximately 200 base-pairs that is responsible for pSC101-specific incompatibility. Two mutations adjacent to this region inactivate pSC101 replication but can be complemented in trans by a wild-type pSC101 plasmid, and thus define a trans-acting replication function, rep. The inc locus is within a larger region of some 450 base-pairs that is essential for pSC101 replication and that includes the origin of replication. This 450 base-pair segment can replicate in the presence of a helper plasmid that supplies the rep function in trans.     

Molecular cloning of an Escherichia coli plasmid determinant than encodes for the production of heat-stable enterotoxin.

A conjugative plasmid, ESF0041 was isolated from an enterotoxigenic strain of Escherichia coli from calves. ESF0041 was found to be 65 x 10(6) daltons in mass of a member of the F incompatibility complex. Acquisition of ESF0041 by E. coli K-12 was invariably associated with the capacity to produce heat-stable (ST) enterotoxin. ESF0041 and pSC101 deoxyribonucleic acids were cleaved with EcoRI, and the fragments were ligated with polynucleotide ligase. Transformation of E. coli K-12 with the ligation mixture led to the isolation of an ST+ clone. Further analysis of the plasmid deoxyribonucleic acid from this clone showed that a structural gene(s) associated with ST biosynthesis had been isolated as a 5.7 x 10(6)-dalton ESF0041 fragment in pSC101. In turn, 5.7 x 10(6)-dalton fragment was ligated to a multicopy COLE1 derivative, RSF2124, so that toxin synthesis was amplified about threefold.