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.     

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 In one of these plasmids 400 bp of R100.1 DNA have been replaced by DNA from the transposon Tn1000 (gamma-delta).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.     

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.     

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.     

Relationships between autonomous and integrated forms of tetracycline resistance plasmid in Staphylococcus aureus.

Recombinant plasmids carrying apparently the complete genome of a small staphylococcal plasmid, pT181, or of its temperature-sensitive replication mutant, pSA0301, were isolated and characterized; in these recombinants, pT181 or pSA0301 were considered as “integrated” into the other plasmid, inasmuch as they seem to have a subsidiary role in the replication of the respective recombinant plasmids. Using these recombinants, the incompatibility relationships between integrated and autonomous forms of the same plasmid were studied. The results obtained showed that, although integrated plasmids express their incompatibility toward autonomous ones, they are not susceptible to the incompatibility manifested by an autonomous or another integrated plasmid. No differences were observed between pT181 and pSA0301 in their response to the incompatibility manifested by recombinant plasmids. The expression of the incompatibility of an integrated plasmid did not require the function of the repC gene, involved in plasmid autonomous replication. Moreover, the pT181 repC⁺ gene seems not to be expressed when pT181 is integrated into another plasmid in that the integrated form does not complement autonomous pSA0301 for replication at nonpermissive temperature.     

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.     

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.     

Involvement of IS1 in the dissociation of the r-determinant and RTF components of the plasmid R100.1

Summary Detailed mapping localized the PHO 1 mutation between the OLI 2 and OLI 4 loci on mitochondrial DNA of Saccharomyces cerevisiae.In its mitochondrially integrated form, the PHO 1-ATPase³ was difficult to identify either immunologically or by specific inhibitors like oligomycin and DCCD. Solubilization by Triton X-100 allowed unambiguuous identification of this enzyme as an authentic mitochondrial ATPase. However, Triton extraction produced a 2 to 3 fold enhancement of the PHO 1-ATPase activity which also became drastically cold-sensitive. The wild type ATPase was neither activated nor made cold-labile by solubilization, and retained full sensitivity to oligomycin and DCCD.Sucrose gradient analysis of the Triton-extracted ATPase from wild type, PHO 1 mutant and rho ^- strains showed a density difference between the solubilized PHO 1-and wild type ATPase, and similarity between solubilized PHO 1-and rho ^- ATPase (F₁).Whole cells of the PHO 1 mutant present considerably increased respiration rates.Comparison of oligomycin-sensitivity in whole cells, coupled isolated mitochondria and membrane-bound ATPase indicates a contrast between oligomycin-resistance of the ATPase and oligomycin-sensitivity of in vivo or in vitro coupling systems, which might characterize the products of this region of mitochondrial DNA.     

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.     

Deletion mutant analysis of the Staphylococcus aureus plasmid pI258 mercury-resistance determinant

Deletion mutant analysis of the mercury-resistant determinant (mer operon) from the Staphylococcus aureus plasmid pI258 was used to verify the location of the merA and merB genes and to show the existence of mercuric ion transport gene(s). ORF5 was confirmed to be a transport gene and has an amino acid product sequence homologous to the merT gene products from several gram-negative bacteria and a Bacillus species. Deletion analysis established that inactivation of merA on a broad-spectrum mer resistance determinant resulted in a mercury-hypersensitive phenotype. Gene dosage had no apparent effect on the level of resistance conferred by the intact mer operon or on the expression of an inducible phenotype, except that when the intact pI258 mer operon was on a high copy number plasmid, uninduced cells possessed a volatilization rate that was at most only 3.5-fold less than that observed for induced cells. There was no need for mercury ion transport proteins for full resistance when the mer operon was expressed in a high copy number plasmid.     

Host components required for the replication of the resistance plasmid R124 and a copy mutant derivative

The replication of R124, and a copy mutant derivative of it, was measured with respect to dependence on the host DnaA, DnaB, DnaC, DnaE, DnaG, and PolA gene products. Both plasmids replicated under conditions where the DnaA gene product was inactivated or where the polymerising activity of the PolA gene product was reduced. In contrast, neither plasmid replicated to any appreciable extent, if the DnaB, DnaC, DnaE or DnaG gene products were inactivated. R124 integratively suppressed the lesion of the dnaA mutant but the copy mutant derivative had only a very weak suppressing effect. Neither plasmid suppressed the lesions of any of the other dna mutants.     

Further characterization of the R plasmid Rts1 and its mutant pTW2: replication and incompatibility of the plasmid.

Incompatibility of the R plasmid Rts1 and its replication mutant pTW2 was studied in recA host cells of Escherichia coli. When the R plasmid R401, belonging to the same incompatibility group as Rts1, was used as a test plasmid, R401 was eliminated preferentially from (Rts-R401)+ cells irrespective of the direction of transfer. In contrast, pTW2 and R401 were mutually excluded. The decreased incompatibility of pTW2 was confirmed by a direct incompatibility test in which a derivative of Rts1 expelled pTW2 exclusively. Alkaline sucrose gradients of pTW2 and Rts1 DNA indicated that approximately one-fourth of the Rts1 genome was deleted in pTW2. In addition, both the various temperature-dependent properties of Rts1 and the inhibitory effect on phage T4 development were also lost in pTW2. A possible mechanism that regulates the stringent replication of Rts1 is discussed.     

Single-stranded circular DNA generated from broad host range plasmid R1162 and its derivatives

Extraction of R1162 plasmid DNA with the alkaline lysis method yields considerable amounts of single-stranded circular plasmid DNA. Destabilization of plasmid DNA is stimulated by the R1162 mob region in cis. The formation of single-stranded circular DNA is initiated at a specific site on the plasmid, presumably the origin of transfer (oriT).     

Nucleotide sequence of the R26 chloramphenicol resistance determinant and identification of its gene product

The cml gene of plasmid R26 is carried on a 1.9-kb HindIII fragment and specifies low-level, inducible resistance to chloramphenicol (Cm). In this paper we report the identification of its product as an approx. 31 kDa protein in minicell experiments, and the determination of the nucleotide sequence of cml, which indicates that the gene product is a relatively hydrophobic protein of Mr 33,800. The protein has no detectable homology to other characterised chloramphenicol-resistance (CmR) proteins, nor any to the membrane-associated tetracycline-resistance (TcR) proteins. The presumptive ribosome-binding site (RBS) of cml mRNA is within a region showing potential for secondary structure.     

Characteristics of the deletion mutant of plasmid R6K]

Deletion plasmide R6Kdelta with the mol wt of 17.2.10(6) dalton isolated from the E. coli chi 925 (R6K) is described. This plasmide expresses no resistance to streptomycin, is replicated in the E. coli K12 under relaxed control and is resistant to the treatment with the eliminating agents. Analysis of plasmide DNA with the aid of electrophoresis in agarose gel demonstrated that R6K delta has one site attacked by restriction endonucleases Eco. RI and Bam HI. These data were confirmed by the determination of the transforming activity of the corresponding DNA restrictors. It is supposed that the isolated plasmide was identical with plasmide RSF1040. A possibility of using R6K delta as a genetic vector for obtaining recombination DNA molecules in vitro is discussed.