Two mechanisms necessary for the stable inheritance of plasmid RP4.

Plasmid RP4 is stably maintained in strains of Escherichia coli and other Gram-negative bacteria. Inactivation of the plasmid primase gene (pri) or removal of the PstIC fragment gave RP4 derivatives that are slightly unstably maintained in E. coli. Removal of the Tn 1 multimer resolution system (res and tnpR) did not lead to any detectable plasmid loss. Removal of all three of these regions, however, gave rise to pNJ5000 which is lost at high frequency. We have dissected the mechanisms causing this phenomenon. In contrast to RP4, pNJ5000 accumulates significantly as plasmid multimers in a Rec+ host; in a recA host, multimers are not seen and the plasmid is stably maintained. Multimers therefore appear to form by recA-mediated homologous recombination and cause plasmid instability, perhaps by interfering with partition. We demonstrate a mechanism provided by the PstIC fragment which acts on multimers analogously to the Tn1/3 resolution system on plasmid cointegrates, being effective only when cloned in cis. The loss of pri, on the other hand, can be complemented in trans. Our results are consistent with the view that primase prevents multimers forming (rather than resolving them once formed), perhaps by binding specifically to single-stranded regions of the plasmid and preventing homologous pairing.

Replication defective RP4 plasmids recovered after chromosomal integration.

pHH6000 is a composite replicon made by the in vitro ligation of the IncP plasmid RP4 to a fragment of bacteriophage lambda capable of autonomous replication. Derivatives were selected in which it had integrated into the Escherichia coli chromosome by homologous recombination with the resident lambda prophage, and plasmids were subsequently regenerated from the integrated molecules. Although of the same molecular size as pHH6000, all had altered properties: those recovered from the chromosome of cells simultaneously carrying a distinguishable autonomous IncP plasmid showed a 100- to 1000-fold reduction in their ability to become established in a lambda lysogen; those regenerated from cells with no autonomous IncP plasmid were no longer RP4 replicons, now being dependent on replication functions encoded by the lambda DNA they carry and therefore unable to form a plasmid in a lambda lysogen. This second class of plasmids still exhibited normal RP4 incompatibility and stability even though neither property is encoded by the lambda replicator DNA. It was concluded that expression of RP4 incompatibility and partitioning control do not require an intact RP4 replicon. The data also suggest that the presence in the chromosome of a normal RP4 molecule may be deleterious to the host, although the manner in which the integrated molecules were obtained allows other explanations. The composite plasmids replicating from cloned lambda genes should be useful in analysis of the regulated distribution of RP4 molecules at cell division.

Replication control of IncP plasmids.

Clones of Escherichia coli with a chromosomally integrated RP4-prime plasmid were isolated and characterized. Chromosome transfer was increased about 50-fold and the Hfr still carried an autonomous plasmid indistinguishable from the original RP4-prime. This could be eliminated by pRP64 or R751, two distinguishably marked incompatible plasmids, giving rise to strains which stably retained the resistance patterns of both plasmids and which continued to transfer the chromosome at enhanced levels. In both cases, however, the copy number of the autonomous plasmid was reduced by the presence of a chromosomal RP4 such that the total number of P plasmid genomes (integrated and autonomous) remained constant. The results are consistent with the idea that copy number is controlled by diffusible inhibitors or initiators of replication.

A broad-host-range cloning vector transposable to various replicons.

A system is described for the stable insertion of cloned DNA sequences into the chromosomes of Gram-negative bacteria. Two broad-host-range plasmids form the basis of the system: one (the "carrier") contains a transposable DNA sequence into which foreign DNA can be cloned; the second (the "helper") provides transposition functions in trans. Both plasmids can be readily transferred between Gram-negative bacteria by conjugation. Instability of the carrier allows enrichment for the products of transposition to the chromosome of the new host, following which the insertion can be stabilised by elimination of the helper. The system was successfully tested in Escherichia coli, Methylophilus methylotrophus and Pseudomonas aeruginosa, and the insertions were stable in each case (less than 0.02% loss per generation).

Conjugal transfer system of plasmid RP4: analysis by transposon 7 insertion.

We have begun an analysis in Escherichia coli of the conjugal transfer functions of the broad-host-range plasmid RP4. We have isolated 19 tra mutants of RP4, generated by insertion of transposon 7, and mapped their insertion sites by restriction endonuclease analysis. These sites fall into two separate regions on either side of the kanamycin resistance determinant. The transfer rates of the mutants range from 10% of that of RP4 to an undetectable level. Spot tests with the P-1 pilus-specific phages PRR1, Pf3, and PR4 and electron microscopic examination for pili have classified the mutants into several phenotypes consistent with their having normal, retracted, or no pili. Analysis of transient plasmid heterozygotes, created by P1 transduction, divided the tra mutants into a minimum of five complementation groups. Some of these groups contain more than one phenotypic class and may represent more than one gene because of the possible polar and deletion effects of Tn7 insertion.

Characterization of SmSu plasmids by restriction endonuclease cleavage and compatibility testing.

Twelve plasmids carrying genes for streptomycin and sulfonamide resistance were studied for the number and distribution of sites on the plasmid moleucles susceptible to cleavage by the restriction endonuclease EcoRI. Ten of the twelve were found to have a single cut site, one plasmid (R678) had three such sites, and plasmid PB165, which was isolated as three supercoiled deoxyribonucleic acid species with molecular weights 7.4 x 10(6), 14.7 x 10(6), and 21.4 x 10(6) was reduced to a single (linear) species of molecular weight 7.6 x 10(6) after cutting with EcoRI. We conclude that PB165 forms oligomers in Escherichia coli and that the number of copies of these per chromosome is more consistant and that the number of copies of these per chromosome is more consistent with a negative than a positive control mechanism for plasmid replication. Compatibility testing of a positive control mechanism for plasmid replication. Compatibility testing of these plasmids showed they all belong to the same incompatibility group, which we designate IncQ, suggesting that they may have come from a common ancestor.

Transposition of a deoxyribonucleic acid sequence encoding trimethoprim and streptomycin resistances from R483 to other replicons.

R483, a plasmid of the Ialpha incompatibility group, contained a deoxyribonucleic acid (DNA) sequence encoding resistance to trimethoprim (TpR) and streptomycin (SmR) that could be transposed to other replicons, i.e., to the Escherichia coli chromosome and to related and unrelated plasmids. Each transposition resulted in the acquisition by the recipient replicon of a segment of DNA of about 9 X 10(6) daltons, both resistance genes, but never the colicin Ia or pilus genes of R483. Transposition took place at a single chromosomal site between dnaA and ilv and did not suppress the DnaA phenotype, in contrast to integration of the whole R483 plasmid. The chromosome, having received the transposition, could secondarily act as a transposition donor to another plasmid. Such a plasmid was indistinguishable from one having received a direct transposition from R483. TpR SmR transposition was very site specific and did not require a functional recA+ gene. We postulate that the TpR SmR segment of R483 is a transposon (TnC) with specific boundary sequences.

Plasmid-determined beta-lactamase indistinguishable from the chromosomal beta-lactamase of Escherichia coli.

A plasmid, derived from a naturally occurring strain of Proteus mirabilis, conferred resistance to cephalosporins, apparently mediated by a beta-lactamase indistinguishable from that determined by the chromosomal gene of Escherichia coli K-12. There was evidence for a recombination event between the wild-type plasmid and a defective F factor (Fsp) in the Escherichia coli K-12 culture in which it was stored.

Compatibility properties of P1 and PhiAMP prophages.

Although phages P1 and PhiAmp are heteroimmune (Chesney and Scott, 1975 and Yarmolinsky, unpublished), their plasmid prophages are incompatible. Thus, the immunity and compatibility systems are two distinct regulators of phage replication. The two prophages, and plasmid P15B (Ikeda, Inzuka and Tomizawa, 1970) constitute a new compatibility group, designated Y.

Assay of deoxyribonucleic acid homology using a single-strand-specific nuclease at 75 C.

We investigated the conditions under which a crude preparation of endonuclease S1 gives maximal hydrolysis of denatured deoxyribonucleic acid (DNA) while giving minimal hydrolysis of native DNA. The hydrolysis was measured by filtering and determining the acid-insoluble reaction product using 3H-labeled substrates. We also investigated various parameters in making this measurement. Under appropriate conditions (in 1 mM ZnSO-4, 0.168 M NaCl at pH 4.8) denatured DNA is hydrolyzed within 3% of completion whereas native DNA is essentially unaffected. The reaction was applied to assay plasmid DNA homoand heteroduplexes for which the method proves to be simple, fast, and reproducible.

Comparison of the deoxyribonucleic acid molecular weights and homologies of plasmids conferring linked resistance to streptomycin and sulfonamides.

Bacterial strains showing linked resistance to streptomycin (Sm) and sulfonamides (Su) were chosen representing a wide taxonomic and geographical range. Their SmSu resistances were transferred to Escherichia coli K-12 and then plasmid deoxyribonucleic acid (DNA) was isolated by ethidium bromide CsCl centrifugation. The plasmid DNA was examined by electron microscopy and analyzed by sedimentation through 5 to 20% neutral sucrose gradients. Plasmid DNA from strains having transmissible SmSu resistance consisted of two or three molecular species, one of which had a molecular mass of about 5.7 Mdal (10(6) daltons), the others varying between 20 to 60 Mdal. By using transformation or F' mobilization, we isolated the SmSu-resistance determinant from any fellow resident plasmids in each strain and again isolated the plasmid DNA. Cosedimentation of each of these with a differently labeled reference plasmid DNA (R300B) showed 9 out of 12 of the plasmids to have a molecular mass not significantly different from the reference (5.7 Mdal); two others were 6.3 and 9.2 Mdal, but PB165 consisted of three plasmids of 7.4, 14.7, and 21.4 Mdal. Three separate isolations of the SmSu determinant from PB165 gave the same three plasmids, which we conclude may be monomer, dimer, and trimer, respectively. DNA-DNA hybridizations at 75 C demonstrated 80 to 93% homology between reference R300B DNA and each isolated SmSu plasmid DNA, except for the 9.2-Mdal plasmid which had 45% homology and PB165 which had 35%. All the SmSu plasmids were present as multiple copies (about 10) per chromosome. The conjugative plasmid of R300 (present as 1.3 copies per chromosome) has been shown to have negligible effect on the number of copies of its accompanying SmSu plasmid R300B. We conclude that the SmSu plasmids are closely related and probably have a common evolutionary origin.