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.     

EcoRI restriction endonuclease map of the composite R plasmid NR1.

A physical map of the composite R plasmid NR1 has been constructed using specific cleavage of deoxyribonucleic acid (DNA) by the restriction endonuclease EcoR-. Digestion of composite NR1 DNA by EcoRI yields thirteen fragments. The six largest fragments (designated A to F) are from the resistance transfer factor component that harbors the tetracycline resistance genes (RTF-TC). The seven smallest fragments (designated G to M) are from the r-determinants component that harbors the chloramphenicol (CM), streptomycin-spectinomycin (SM/SP), and sulfonamide (SA) resistance genes. The largest fragment of several RTF-TC segregants of NR1 that have deleted the r-determinants component is 0.8 X 10(6) daltons larger than fragment A of composite NR1. Only a part of fragment H of the r-determinants component is amplified in transitioned NR1 DNA in Proteus mirabilis, which consists of multiple, tandem sequences of r-determinants attached to a single copy of the RTF-TC component. Both of these changes can be explained by the locations of the excision sites at the RTF-TC: r-determinants junctions that are involved in the dissociation and reassociation of the RTF-TC and r-determinants components. The thirteen fragments of composite NR1 DNA produced by EcoRI have been ordered using partial digestion techniques. The order of the fragments is: A-D-C-E-F-B-H-I-L-K-G-M-J. The approximate locations of the TC, CM, SM/SP, and SA resistance genes on the EcoRI map were determined by analyzing several deletion mutants of NR1.     

Generation of miniplasmids from copy number mutants of the R plasmid NR1.

Small, closed circular deoxyribonucleic acid molecules, called miniplasmids, were observed in Escherichia coli harboring copy number mutants of the R plasmid NR1 after growth in medium containing tetracycline. The level of tetracycline resistance conferred by the copy mutant plasmids was lower (3 to 6 microgram/ml) than that conferred by NR1 (100 MICROGRAM/ML). The presence of the miniplasmid enhanced the level of tetracycline resistance conferred by the copy mutant. Miniplasmids of molecular weights 4 X 10(6) to 13 X 10(6) were found. They carried no antibiotic resistance markers and could be eliminated by growth in the presence of chloramphenicol and/or streptomycin-spectinomycin. Studies with the restriction endonucleases EcoRI and Sal I indicated that the miniplasmids are derived from the region of the copy mutant plasmids that contains the origin for replication of the resistance transfer factor. There were approximately 12 copies of the miniplasmid per chromosome, compared with 3 and 6 copies of the copy mutants of NR1. The miniplasmids appeared to be incompatible with the copy mutant plasmids.     

Frequency of replication from alternative origins in the composite R plasmid NR1.

The relative frequency of initiation of DNA replication within the RTF-Tc and r-determinant components of the composite drug resistance plasmid NR1 in Proteus mirabilis was evaluated. Using fractionated radioactively labeled plasmid DNA, analytical procedures that distinguished between the two components of the composite plasmid were carried out. A mixture of uniformly 14C-labeled and 3H-pulse-labeled plasmid DNA (pulse-labeled origin[s] of replication) was used in each of three experiments. First, shear products of the DNA were analyzed using CsCl density gradient centrifugation. Second, fragmented DNA was hybridized to nonradioactive RTF-Tc and r-determinant DNAs immobilized on nitrocellulose filters. Third, the radioactive plasmid DNAs immobilized on nitrocellulose filters. Third, the radioactive plasmid DNA was digested with restriction enzyme (EcoRI), producing a set of RTF-Tc and r-determinant fragments with differing 3H/14C isotpe ratios. The three experiments suggested that under the conditions used to accumulate replicating plasmid DNA molecules (DNA substrate limitation), the r-determinant origin of replication was preferentially utilized in the composite plasmid.     

Repetition of tetracycline resistance determinant genes on R plasmid pRSD1 in Escherichia coli.

Summary The 30 megadalton (Mdal)-conjgaative, fi^- plasmid pRSD1 determines inducible tetracycline resistance (Tc) in Escherichia coli. As shown by restriction analysis, a 3.5 Mdal-EcoRI fragment of pRSD1 spliced into the small plasmid pRSD2124 comprises the entire Tc determinant (tet) region. A restriction map of pRSD1 is presented which includes the location of the tet region and of an underwound loop not related to Tc (Burkardt et al., 1978). Selective amplification of tet genes is demonstrated by three lines of evidence. (i) The resistance level of cell harbouring pRSD1 increases approximately tenfold by induction with 10g/ml of tetracycline. Further groth in the presence of 100 g/ml of the drug (tet-racycline stress) selects for cells with even higher resistance levels (about 300 g/ml) in rec ⁺ cells. In a recA strain, a smaller proportion of cells attains these high resistance levels suggesting the involvement of host recombination. (ii) Electron micrographs of pRSD1-DNA isolated from tetracycline-stressed cells reveal a heterogeneous population of circular DNA molecules ranging between 1.7 and 21.6 m. The distribution of contour lengths shows a discrete pattern ascribed to the presence of autonomous single-and multiple-copy Tc determinants and to intact plasmids containing zero to six tet regions in tandem repeats. (iii) This interpretation is supported by heteroduplex and restriction analyses which demonstrate the presence of multiple copies of the 3.5 Mdal-element encompassing the tet region in pRSD1 molecules selected by tetracycline stress. It has been concluded that gene amplification leading to tandem repetition of the tet region ensues in pRSD1. Such plasmids confer increased tetracycline resistance and can, therefore, be selected by high doses of the drug.     

The incompatibility product of IncFII R plasmid NR1 controls gene expression in the plasmid replication region.

The incompatibility properties of IncFII R plasmid NR1 were compared with those of two of its copy number mutants, pRR12 and pRR21. pRR12 produced an altered incompatibility product and also had an altered incompatibility target site. The target site appeared to be located within the incompatibility gene, which is located more than 1,200 base pairs from the plasmid origin of replication. The incompatibility properties of pRR21 were indistinguishable from those of NR1. Lambda phages have been constructed which contain the incompatibility region of NR1 or of one of its copy mutants fused to the lacZ gene. In lysogens constructed with these phages, beta-galactosidase was produced under the control of a promoter located within the plasmid incompatibility region. Lysogens containing prophages with the incompatibility regions from pRR12 and pRR21 produced higher levels of beta-galactosidase than did lysogens containing prophages with the incompatibility region from the wild-type NR1. The introduction into these inc-lac lysogens of pBR322 plasmids carrying the incompatibility regions of the wild-type or mutant plasmids resulted in decreased levels of beta-galactosidase production. For a given lysogen, the decrease was greater when the pBR322 derivative expressed a stronger incompatibility toward the plasmid from which the fragment in the prophage was derived. This suggested that the incompatibility product acts on its target to repress gene expression in the plasmid replication region.     

Cloning of replication, incompatibility, and stability functions of R plasmid NR1.

The region of R plasmid NR1 that is capable of mediating autonomous replication was cloned by using EcoRI, SalI, and PstI restriction endonucleases. The only EcoRI fragment capable of mediating autonomous replication in either a pol+ or a polA host was fragment B. SalI fragment E joined in native orientation with the part of SalI fragment C that overlapped with EcoRI fragment B, and also two contiguous PstI fragments of sizes 1.6 and 1.1 kilobases from EcoRI fragment B-mediated autonomous replication. When these individual SalI fragments were cloned onto plasmid pBR313 or the individual PstI fragments were cloned onto plasmid pBR322, none of these single fragments could rescue the replication of the ColE1-like vectors in a polA host, even in the presence of a compatible "helper" plasmid derived from a copy mutant of NR1. In contrast to the results reported for closely related R plasmid R6, EcoRI fragment A of NR1 could not rescue the replication of ColE1 derivative RSF2124 in a polA(Am) mutant or in a polA(Ts) mutant at the restrictive temperature. Although capable of autonomous replication, EcoRI fragment B of NR1 (or smaller replicator fragments cloned from it by using other restriction enzymes) was not stably inherited in the absence of selection for the recombinant plasmid. When EcoRI fragment B was ligated to EcoRI fragment A of NR1, the recombinant plasmid was stable. Thus, EcoRI fragment A contained a stability (stb) function. The stb function did not act in trans since EcoRI fragment B was not stably inherited when a ColE1 derivative (RSF2124) ligated to EcoRI fragment A was present in the same cell. A cointegrate plasmid consisting of EcoRI fragment B of NR1 ligated to RSF2124 was also not stably inherited, whereas only EcoRI fragment B was unstable when both RSF2124 and EcoRI fragment B coexisted as autonomous plasmids in the same cell. The incompatibility gene of NR1 was shown to be located within the region of overlap between SalI fragment E and the PstI 1.1-kilobase fragment. A copy mutant of NR1 (called pRR12) was found to have greatly reduced incompatibility with NR1; this Inc- phenotype is cis dominant.     

Autoregulation of the stability operon of IncFII plasmid NR1.

The stb locus of IncFII plasmid NR1, which mediates stable inheritance of the plasmid, is composed of an essential cis-acting DNA site located upstream from two tandem genes that encode essential stability proteins. The two tandem genes, stbA and stbB, are transcribed as an operon from promoter PAB. Using PAB-lacZ gene fusions, it was found that the stb operon is autoregulated. A low-copy-number stb+ plasmid introduced into the same cell with the PAB-lacZ fusion plasmid repressed beta-galactosidase activity about 5-fold, whereas a high-copy-number stb+ plasmid repressed beta-galactosidase about 15-fold. The details of autoregulation were analyzed by varying the concentrations of StbA and StbB to examine their effects on expression from the PAB-lacZ fusion plasmid. StbB protein by itself had autorepressor activity. Although StbA protein by itself had no detectable repressor activity, plasmids that encoded both stbA and stbB repressed more effectively than did those that encoded stbB alone. Plasmids with a mutation in stbA had reduced repressor activity. One mutation in stbB that inactivated the stability function also reduced, but did not eliminate, repressor activity. Repressor activity of the mutant StbB protein was effectively enhanced by stbA. These results indicate that StbB serves two functions, one for stable inheritance and one for autoregulation of the stb operon, both of which may be influenced by StbA protein.     

Transition of deletion mutants of the composite resistance plasmid NR1 in Escherichia coli and Salmonella typhimurium.

Derivatives of the composite R plasmid NR1 from which a portion of the resistance determinants (r-determinants) component had been deleted were found to undergo amplification of the remaining r-determinants region in Escherichia coli and Salmonella typhimurium. The wild-type NR1 plasmid does not amplify in these genera, although all of these plasmids undergo amplification in Proteus mirabilis. The deletion mutants retained the mercuric ion resistance operon (mer) but conferred a much lower level of sulfonamide resistance than NR1. The remaining r-determinants region, which is bounded by direct repeats of the insertion element IS1, formed multiple tandem duplications in E. coli, S. typhimurium, and P. mirabilis after subculturing the host cells in medium containing high concentrations of sulfonamide. Gene amplification was characterized by restriction endonuclease analysis, analytical buoyant density centrifugation, DNA-DNA hybridization, and sedimentation in sucrose gradients. The tandem repeats remained attached to the resistance transfer factor component of the plasmid in at least part of the plasmid population; autonomous tandem repeats of r-determinants were probably also present. Amplification did not occur in host recA mutants. Amplified strains subcultured in drug-free medium lost the amplified r-determinants. By using a strain temperature sensitive for the recA gene, it was possible to obtain gene amplification at the permissive temperature. Loss of r-determinants took place at the permissive temperature, but not at the nonpermissive temperature. The termini of the deletions of several independent mutants which conferred low sulfonamide resistance were found to be located within the adjacent streptomycin-spectinomycin resistance gene.     

oriT sequence of the antibiotic resistance plasmid R100.

We present the nucleotide sequence of the oriT region from plasmid R100. Comparison to other IncF plasmids revealed homology around the proposed nick sites as well as conservation of inverted repeated sequences in the nonhomologous region. Three areas showed strong homology (eight of nine nucleotides) to the consensus sequence for binding of integration host factor, suggesting a role for this DNA-binding protein in nicking at oriT.     

Effects of chloramphenicol and rifampicin on the replication of R plasmid NR1 deoxyribonucleic acid in Escherichia coli.

The effects of inhibition of protein and ribonucleic acid (RNA) synthesis on the replication of the plasmids NR1 and F′lac in Escherichia coli were studied. When protein synthesis is inhibited, there is approximately a 25% increase in R plasmid deoxyribonucleic acid (DNA), but this newly synthesized DNA is not recoverable in the covalently closed circular (CCC) form until protein synthesis is allowed to resume. When RNA synthesis is inhibited, there is also approximately a 20% increase in R plasmid DNA, but this DNA is immediately recoverable in the CCC form. F′lac DNA, unlike R plasmid DNA, can continue to replicate for at least a generation time in the absence of protein synthesis, and this F′lac DNA is immediately recoverable in the CCC form.     

Cloning the origin of transfer region of the resistance plasmid R1

The insertion of a 7.7-kb EcoRI fragment of the resistance plasmid R1 into pBR325 yielded a plasmid which is mobilizable by pDB12, a multicopy derivative of R1drd-19 lacking most of the resistance determinants. The vector alone was not mobilizable in this system. From this observation we conclude that we have cloned the origin of transfer (oriT) of R1. After inserting a 5.3-kb PvuII-EcoRI fragment of the 7.7-kb region into pUC9 the DNA was cleaved randomly with DNaseI and BamHI linkers were attached to the ends. A subsequent BamHI digestion and electrophoretic separation of the resulting DNA molecules by their size allowed us to generate an ordered series of stepwise shortened plasmids. Plasmids with a deletion of approximately 3400 bp could no longer be mobilized. Since the next larger plasmid with 284 additional base pairs could be mobilized, we are able to confine the oriT location within this extra nucleotide stretch. The DNA sequence of this region was determined. Dominant features within the DNA region are a high AT content and five inverted repeats, which might function as recognition or substrate sites for proteins of the conjugational transfer system.