Mapping of the drug resistance genes carried by the r-determinant of the R100.1 plasmid

Summary We have cloned the EcoRI fragments of pLC1, a circular DNA element found in an Escherichia coli dnaA _ts strain integratively suppressed by R100.1 (Chandler et al., 1977a), using the plasmid vector pCR1. All the resistance genes known to be present on the r-determinant of R100.1 were found to be present on pLC1. The isolation of pCR1 derivatives carrying various EcoRI fragments of either pLC1 or R100.1 has allowed a more precise mapping of the position of the resistance genes on the R100.1 molecule.     

Dissection of the r-determinant of the plasmid R100.1: the sequence at the extremities of Tn21.

We have sequenced the extremities of the transposon Tn21, isolated from the r-determinant of the multiple antibiotic resistance plasmid R100.1, and show that it is a member of the Tn3 family of elements.     

Production of extrachromosomal r-determinant circles from integrated R100.1: Involvement of the E. coli recombination system

Summary The drug resistance plasmid R100.1 can integrate into the E. coli chromosome at several sites on the plasmid. Many of the resulting Hfr strains continuously produce extrachromosomal circular forms of the r-determinant. These r-det plasmids seem incapable of stable autonomous replication. We show that their presence in the cell requires the continuous activity of functional recA and recC genes but does not require the lexA function.The production of r-det circular forms is correlated with an increased copy number of r-det sequences, relative to RTF sequences. This copy number increase is, however, also found in a recA ^- backgound where no circular forms of r-det are found. These results show that a specific replication of r-det sequences, not present in the wild-type R100.1 plasmid, occurs in these R-Hfr strains. They suggest that a rec promoted recombination, posterior to the specific replication event, is needed for the production of circular r-det forms.     

The repA2 gene of the plasmid R100.1 encodes a represser of plasmid replication

We have constructed two miniplasmids, derived from the resistance plasmid R100.1. In one of these plasmids 400 bp of R100.1 DNA have been replaced by DNA from the transposon Tn1000 (gamma-delta). This substitution removes the amino-terminal end of the repA2 coding sequence of R100.1 and results in an increased copy number of the plasmid carrying the substitution. The copy number of the substituted plasmid is reduced to normal levels in the presence of R100.1. The repA2 gene thus encodes a trans-acting repressor function involved in the control of plasmid replication.     

The construction and replication properties of hybrid plasmids composed of the r-determinant of R100.1 and the plasmids pCRI or pSC201

Summary We have cloned the entire r-determinant of the antibiotic resistance plasmid R100.1 on the plasmid vectors pCR1 and pSC201. We find that the hybrid plasmids segregate from cultures in which replication of the vector is blocked. This suggests that the r-det is not capable of autonomous replication.     

Localization of Kpnl restriction sites in the r-determinant of plasmid NR1

Restriction KpnI sites on a molecule of the plasmid NR1 were localized. The position of KpnA (58,0 MD) and KpnB (1,0 MD) fragments on a physical map of the NR1 molecule was shown using endonucleases EcoRI and SaII. Both restriction sites are situated on a r-determinant of the EcoJ fragment of the plasmid NR1.     

Deletions in the r-determinant mer region of plasmid R100-1 selected for loss of mercury hypersensitivy.

A mutant of plasmid R100-1, which conferred cellular hypersensitivity to Hg2+ because of the insertion of Tn801 (TnA) into the gene determining synthesis of mercuric reductase enzyme, allowed further mutational events to be selected which resulted in either reversion to Hg2+ resistance (characteristic plasmid R100-1) or sensitivity at a level characteristic of plasmidless strains. Restriction endonuclease EcoRI and BamHI analysis showed that reversion to resistance resulted from loss of TnA from the R100-mer:Tn801 plasmid, whereas the change from hypersensitivity to sensitivity to Hg2+ usually resulted from deletion of part or all of Tn801 plus plasmid deoxyribonucleic acid sequences corresponding to the operator-proximal end of the mer operon.     

Suppression of induction of SOS functions in an Escherichia coli tif-1 mutant by plasmid R100.1.

The tif-1 mutation in the recA gene of Escherichia coli caused, at 40 degrees C, lethal cell filamentation, induction of the recA protein, mutagenesis, and, in lambda lysogens, prophage induction. The presence of plasmid R100.1 in tif-1 strains suppressed tif-mediated cell filamentation and killing, recA protein induction, and prophage induction in lysogens. It also reduced mutagenesis in a tif-1 sfiA11(R100.1) strain. Plasmids F'lac, P1, and pMB9, in contrast, had little or no effect on tif-mediated induction of lambda. The presence of R100.1 did not inhibit the induction of the recA protein or of lambda by ultraviolet irradiation or mitomycin C treatment of tif-1(R100.1) or tif-1(lambda)(R100.1) strains.     

Tn10 mediated integration of the plasmid R100.1 into the bacterial chromosome: Inverse transposition

Summary Upon integration into the bacterial chromosome the drug resistance plasmid R100.1 often loses its tetracycline resistance character. We have analyzed an Hfr strain formed by such an integration and an R-prime plasmid derived from it. We find that integration took place within the Tn10 transposon, that the two IS10 sequences were retained, but that at least 80% of the transposon segment located between them, and carrying the tetracycline resistance genes, had been lost. We suggest that integration of R100.1 was mediated by an inverse transposition using the IS10 sequences.     

DNA inversion mediated by the r-determinant of plasmid NR1: evidence for the intramolecular replicative transposition of a 23 kb IS1-flanked transposon?

Summary The r-determinant (r-det) of the R plasmid NR1-Basel is a 23 kb, IS1-flanked transposon, called Tn2671, which has been shown to transpose to the genome of bacteriophage P7. Among the derivatives of phage P7::r-det we found one which carried two copies of the r-det as inverted repeats and which also contained the P7 genome segment between them in inverted orientation. Its generation is best explained by assuming that the entire 23 kb Tn2671 transposon has undergone intramolecular replicative transposition.     

Recombination between two IS/s flanking the r-determinant of R100-1: involvement of dor and recA gene functions in Salmonella typhimurium

Drug resistance genes of the r-determinant component of a composite R plasmid R100-1 were frequently lost in Salmonella typhimurium. Various deletion mutants were analyzed by restriction endonuclease cleavage, Southern blotting, and hybridization techniques. The loss of the r-determinant was found to be the result of a reciprocal recombination between the two IS/s flanking the r-determinant. This recombination depended upon both dor and recA gene functions.     

A mutant of the plasmid R100.1 capable of producing autonomous circular forms of its resistance determinant

We have isolated a derivative of plasmid R100.1, following integration into and excision from the Escherichia coli chromosome, which exhibits the ability to produce circular copies of its resistance-determinant (r-det) region. The plasmid also shows unusually frequent loss of the r-det resistance genes. Both these phenomena are inheritable plasmid characteristics and are dependent on the host recombination system. The production of r-det can be repressed in trans by the presence in the same cell of a second plasmid closely related to R100.1.     

On the role of IS1 in the formation of hybrids between the bacteriophage P1 and the R plasmid NR1

Summary The genomes of bacteriophage P1 derivatives carrying drug resistance genes derived from an R plasmid NR1 were analysed by restriction cleavage and be DNA-DNA hybridization. Two representatives of a class of oversized P1CmSmSu phages were identified as P1 carrying the entire r-determinant of NR1 together with its two flanking, directly repeated IS1. In one case the r-determinant insertion is carried at the site of the residential IS1 of P1, in the other case it is transposed into another region of the P1 genome. Models postulate that the first type resulted from reciprocal recombination within IS1 elements and that the formation of the second type of P1-R hybrid depended both on IS1 mediated transposition and reciprocal recombination. Plaque forming P1Cm or P1CmSm phages are explained as IS1 mediated deletion derivatives of P1CmSmSu, although an alternative model postulates that sometimes P1Cm phages might result from two consecutive transposition events of only one IS1 without involving reciprocal recombination. Secondary P1 derivatives carrying only one IS1 at the site of the original r-determinant or of Cm insertions into P1 must have been produced by reciprocal recombination between the two IS1 flanking the insertions. An implication from this study, that any genetic material carried adjacent to an IS1 element may undergo passive transposition, is discussed.     

Involvement of transposable elements in the formation of hybrid phages between bacteriophage P1 and the R plasmid NR1

Homologous recombination between IS1 elements present on both replicons, P1 and NR1, resulted in P1-NR1 cointegrates and P1-RTF and P1-r-det phages. Cointegration between P1 and NR1-B, and NR1 derivative with multiple DNA rearrangements including insertion of the transposable element γδ, was also mediated by reciprocal recombination in IS1 sequences. However, all 4 hybrids studied carried deletions promoted by γδ residing on NR1-B. Further IS1-mediated deletions on the hybrid genomes resulted in plaque-forming P1Cm phages.     

Intra- and intermolecular recombination of test plasmids in K12 Escherichia coli cells carrying an RTF derivative of the R1drd-19 plasmid

The RTF derivative of the plasmid R1drd-19 was found to stimulate recombination of the tester plasmids in a recB mutant of Escherichia coli K12. The frequency of intramolecular recombination is increased 3.5 and 20-fold, as compared to the one in rec+ and rec- strains, respectively. The frequency of interplasmid recombination is enhanced 4 and 9-fold, respectively. Considerable heterogeneity of the recombination products of the tester plasmid intramolecular recombination in recB-/RTFR1-19 strain has been revealed. It is hypothesized that a "recombinase" encoded by Rldrd-19 plasmid determines a new minor pathway in recB- (Rec P) which differs in activity and, perhaps substrate specificity from the main Rec BCD pathway.     

Formation of an IS1-induced deletion in pNt6::IS1 plasmid

The expression of oLpLN region of the plasmid pNT6 causes the high instability of the plasmid. Mutations in the promoter pL region and lesions in the structural part of the N gene result in the stable inheritance of the plasmid. The plasmids pNT6::IS1 containing the IS1-element inserted into the different loci of oLpLN region restore the high instability of the plasmid inheritance in the strain 4830 coding for oLpLN. The plasmids pIG3 and pIG4 of the series pNT6: :IS1 permit one to obtain the collection of random deletions in the cloned fragments induced by IS1-element.     

Origin and direction of replication of the drug resistance plasmid R100.1 and of a resistance transfer factor derivative in synchronized cultures.

The origin and direction of replication of the resistance plasmid R100.1 and its resistance transfer factor derivative, pAR132, were studied by electron microscopy autoradiography of partially denatured molecules and partial denaturation mapping of replicative intermediates. Results of these studies indicate the existence of an origin of replication at 8.8 kilobases on the R100 map. Replication from this origin in cultures synchronized for initiation of replication is predominantly unidirectional in a single direction.