Analysis of mini-F plasmid replication by transposition mutagenesis

Derivatives of a mini-F plasmid in which Tn3 is inserted in F deoxyribonucleic acid were obtained, and the sites of insertion for 40 of the derivatives were mapped. Tn3 was found to insert at many sites within mini-F, but most insertions were within the 43.0- to 43.7-kilobase (kb), 44.2- to 44.7-kb, and 45.9- to 46.3-kb segments. Hence, these segments are unnecessary for mini-F replication. Most of the Tn3 derivatives were similar to their parent miniplasmid with respect to copy number, stability, and incompatibility. Insertions at 45.15 kb and near 46.0 kb caused a moderate disruption of copy number control, and insertion at 47.6 kb resulted in unstable maintenance. Deletion derivatives lacking deoxyribonucleic acid between 40.3 and 44.76 kb and between 45.92 and 49.4 kb were obtained. This observation suggests either that mini-F contains a third origin, in addition to those already reported to be at 42.6 and 44.4 kb, or that the reported position of the secondary origin, 44.4 kb, is incorrect and that this origin is between 44.76 and 45.92 kb.     

Prophage lambda induction caused by mini-F plasmid genes

Summary When bacterial cells harboring a temperaturesensitive replication plasmid, which carries the particular ccd segment of a mini-F plasmid, are transferred to 42°C, cell division is inhibited after incubation for an appropriate time. The inhibition occurs, when the copy number of the plasmid decreases to become critically low, about one per cell (Ogura and Hiraga 1983 b). In phage lysogens carrying this type of plasmid, the prophage is induced in a small portion of the cell population under the same conditions, in addition to the inhibition of cell division in most of cells. The prophage induction, but not the inhibition of normal cell division, depends on normal recA function. Both induction of prophage and inhibition of cell division are suppressed by the simultaneous presence of a replication proficient plasmid carrying the ccdA gene. We discuss molecular mechanisms of the ccd function that couples host cell division to plasmid proliferation and induces the prophage. Additionally, we propose a hypothesis that the ccd mechanism of F plasmid contributes to indirect induction of prophage by an F plasmid damaged by UV-irradiation and then introduced into a lysogen via conjugation.Abbreviations kb kilobase pairs. m.o.i., multiplicity of infection     

Amber-mutants of plasmid mini-F defective in replication

Summary Amber mutants of the mini-F plasmid pML31 were isolated with the mutagen hydroxylamine. Under non-permissive conditions amber mutants segregate and show no incorporation of label into supercoiled plasmid DNA in double-label experiments. Wild-type and one mutant of mini-F were integrated by recombinant DNA techniques into the single EcoRI site of plasmid vector pBR322. Plasmid specific proteins were labeled in minicells and analysed by SDS-PAGE. A 34,000 dalton molecular weight protein was identified to be missing in the amber mutant of plasmid mini-F.     

Additional genes essential for replication of the mini-F plasmid from origin I

Summary We isolated a series of Tn5-insertional mutants from the mini-F plasmid, which has a deletion in the origin II region and replicates exclusively from origin I, and found that the mutants that had Tn5 in either the F4 or the F5 gene were defective in their replication. It is concluded that, in addition to the F3 gene on which we have reported previously, both the F4 and the F5 genes are essential for the replication from origin I.     

Replication of mini-F plasmid in vitro promoted by purified E protein

Summary An in vitro system for replication of mini-F plasmid DNA was constructed. This system consists of an ammonium sulfate fraction II (Fuller et al. 1981) from Escherichia coli extract, exogeneously added purified E protein encoded by mini-F plasmid, and mini-F DNA in a closed circular form. Experiments with this system showed that the 217 bp DNA region which contains the A+T rich cluster and the four 19 bp direct repeats responsible for incB incompatibility is essential for mini-F DNA replication.     

An essential gene for replication of the mini-F plasmid from origin I

Summary We constructed a series of defective mini-F plasmids, which have deletion(s) in the replication origin I and/or origin II, and their derivatives, which do not produce F3 protein, by insertion of the XhoI fragment of Tn5 into the XhoI site at 41.0 F (kilobases on the coordinate map of F-plasmid). Using these mutant mini-F plasmids, we found that F3 protein is essential for the replication of mini-F from origin I, but not from origin II.     

Rapid microscale procedure for visualizing intracellular plasmid DNA by electron microscopy

A rapid microscale procedure is described that releases plasmid DNA in situ from the bacterial cell and that allows selective observation of the plasmid bound to cellular components. The released plasmid DNA was adsorbed preferentially on mica in a divalent cation-free medium then processed for electron microscopy. The plasmid DNAs studied were pAO3 (1683 base pairs (bp²)), λdv021 (3505 bp), pTSO118 (4000 bp), pAO65 (4786 bp), ColE1 (6500 bp), and RSF2124 (11,400 bp). These DNAs were seen as supercoiled circles or as relaxed circles of corresponding length. Occasionally an internal loop of replicating DNA was present. One micrometer of measured length corresponded to 3100 bp.     

Stabilization of anE. coli plasmid by a mini-F fragment of DNA

Summary In anEscherichia coli K-12 strain (trpA trpE tnaA) cultured in LB broth without selective pressure, a pBR322 derivative containing the gene for tryptophan synthase (pBR322-trpBA) was found to be unstable. After 70 cell-number doublings, only 50% of the host cells retained the gene for ampicillin resistance (Ap^r). Insertion of the mini-F fragment of F factor DNA into this plasmid could effectively reduce the plasmid loss. Partial derepression of the tryptophan promotor-operator by 3-indopleacrylic acid further decreased the stability of the pBR322-trpBA but not that of the mini-F inserted plasmid (pBR322F-trpBA) The vector pBR322F-trpBA could be maintained at high copy number in the culture after 100 generations of growth; the culture was able to overproduce tryptophan synthase in the presence of 3-indoleacrylic acid.l-Tryptophan was produced from indole andl-serine using andE. coli host transformed with.pBR322F-trpBA DNA. After 8 h of incubation, the expression level was approximately 180 g/l.     

Complementation of replication-deficient deletion derivatives of plasmid mini-F.

Deleted mini-F plasmids with defects in replication were constructed and tested to see whether they could be rescued through complementation by a helper plasmid. This allowed us to identify two genetic loci determining trans-acting functions required for stable maintenance of plasmid mini-F, one encoded by the PstI fragment from 45.7 to 47.3 F-coordinates (F) and the other most probably located in the region from 43.1 to 43.8 F. The smallest mini-F plasmid that could be established through complementation consists of the PstI fragment 44.0 to 45.7 F, encoding origin II and the incB locus.     

Study of the binding of RNA polymerase by a recombinant plasmid using electron microscopy

RNA-polymerase of E. coli was bound in vitro under physiological conditions to a recombinant plasmid pBR322 carrying two identical segments of bacteriophage T4 DNA, each containing a complete gene coding for T4 DNA ligase. After fixation of the complex with formaldehyde it was analysed by electron microscopy. A map of binding sites of the enzyme to DNA was obtained after a statistical assessment of micrographs. The inserted repeat revealed itself in the map as two regions of identical binding patterns, thus proving the adequacy of the preparation procedure. In pBR322 the strong binding sites correlate with the position of promoters. Also, apart from this, there are other strong binding sites within the T4 sequences which correlate strongly with the regions of abnormally high AT content. That means that under physiological conditions the RNA polymerase forms strong binding complexes with any AT rich DNA regions, as well as with real promoters.     

Behavior of sister copies of mini-F plasmid after synchronized plasmid replication in Escherichia coli cells

To clarify whether sister copies of mini-F plasmid are immediately separated from each other after replication, we analyzed the behavior of sister mini-F copies after synchronized replication of mini-F. Sister copies of mini-F were separated immediately or shortly after replication, in contrast to sister oriC copies of the Escherichia coli chromosome.     

Mutants of the mini-F plasmid pML31 thermosensitive in replication.

Hydroxylamine mutagenesis was used for the induction of thermosensitive replication mutants of the mini-F plasmid pML31. Replication mutants were characterized by studying the segregation kinetics and the incorporation of [3H]-thymidine into plasmid deoxyribonucleic acid at the nonpermissive temperature. Based on these experiments two types of mutants could be distinguished. Mutants of type I are fast segregating with the kinetics expected if plasmid replication was blocked immediately. Double-label experiments showed a rapid shut-off of replication in these mutants at 42 degrees C. Mutants of type II segregate slower, showing only a partial inhibition of plasmid deoxyribonucleic acid synthesis at the nonpermissive temperature. The label incorporated at 42 degrees C was predominantly found in open circular plasmid molecules.     

Analysis of human chromosomal variants by quantitative electron microscopy

Two human ring chromosomes 18 were isolated intact in a Langmuir trough, critical-point dried, and studied by quantitative electron microscopy. The portion of deleted genetic material is calculated from dry-mass determinations and is found to be comparable to the total genetic material present in the bacterium Salmonella typhimurium.Preliminary presentation of this study was made (Golomb and Bahr, 1971a).This study has been supported by American Cancer Society Grant No. P259-j.The opinions or assertions contained herein are the private views of authors and are not to be construed as official or as reflecting the views of the department of the Army or the Department of Defense.