E. S. Anderson

A simple method for the preparation of large quantities of pure plasmid DNA.

Polyethylene glycol quantitatively precipitates plasmid DNA of molecular weight 6-123-10-6, from cleared lysates of plasmid-carrying bacterial strains, After resuspension and density-gradient centrifugation of the precipitated DNA, it is unchanged in length and in transformation efficiency for Escherichia coli K12. Plasmid DNA can be easily prepared in large quantities by including a polyethylene glycol precipitation step in standard plasmid isolation procedures.

Mutagenesis of plasmid DNA with hydroxylamine: Isolation of mutants of multi-copy plasmids

SummaryAn investigation of in vitro mutagenesis of plasmid DNA with hydroxylamine is described. The treated plasmid DNA was used to transform Escherichia coli K12. Mutants of the plasmid NTP3, which codes for resistance to ampicillin and sulphonamides, were isolated and characterised. They were classified according to the reduction in level of their β-lactamase activity. Hydroxylamine-induced mutants of NTP14 were also isolated. This plasmid codes for ampicillin resistance, synthesis of colicin E1, and the EcoRI restriction and modification enzymes. One class of mutants is lethal to the host strain at temperatures above 33° C, but carrier strains grow well at 28° C. There is evidence that these mutants code for a temperature-sensitive EcoRI modification activity: the lethal effect probably results from the cleavage of the host-cell DNA by the restriction enzyme at non-permissive temperatures. The possible genetic uses of the mutant plasmids for the production of hybrid plasmids in the bacterial cell are discussed.

R factor compatibility groups

SummaryEight R factors are described which fall into four compatibility groups, distinct from previously described F-like and I-like groups. E. coli K12 carrying one of these factors, TP114, supports multiplication of the I-specific phage If1. However, TP114 is fully compatible with the I-like factor T-Δ and with all the other R factors described in this paper. Interactions between TP114 and T-Δ are described. Another R factor, TP122, inhibits F-fertility and is therefore fi+, although strains carrying it do not support multiplication of the F-specific phage μ2. TP122 is compatible with the F-like R factor R1, but incompatible with three other factors, all of which fall into the N group. Two further factors, TP116 and TP117, are incompatible with each other and constitute a new group, designated Group H. The final factor, TP113, is compatible with all the R factors with which it has been tested, so that it represents yet a further group. A second member of this group has recently been identified.

Molecular studies of an fi+ plasmid from strains of Salmonella typhimurium

SummaryPlasmid DNA has been isolated from five fi+ strains of Salmonella typhimurium of independent origin, including type 36 and LT2. The mean contour length of the plasmids was between 27.3 and 29.3 μm. A variant line of S. typhimurium type 36 which was fi- yeilded no plasmid DNA. These results support the hypothesis that the fi+ property of S. typhimurium is coded by a plasmid. In S. typhimurium 36 this plasmid, designated MP1036, also appears to code for restriction of non-donor-specific phages. Molecular studies indicate that superinfection of S. typhimurium 36 with the kanamycin resistance determinant K, which results in loss of the fi+ property, is correlated with loss of MP1036. Reassociation experiments demonstrate a high degree of homology between the DNA of all five S. typhimurium plasmids, and between MP1036 and K. MP1036 has some homology with F and F-like R factors, but not with plasmids of other compatibility groups. A recombinant between an ampicillin resistance determinant and MP1036 is autotransferable at low frequency. The significance of these findings is discussed.

Genetic and molecular characterisation of some non-transferring plasmids.

SummaryCompatibility and molecular studies were performed on a number of non auto-transferring plasmids for drug resistance and colicinogeny. The ampicillin (A) and streptomycin-sulphonamide (SSu) resistance determinants of Salmonella typhimurium type 29 are compatible with each other, and thus represent different compatibility groups. The laboratory-made resistance determinant ASu is incompatible with SSu and was used for compatibility studies with other determinants. Nineteen of 26 wild streptomycin-sulphonamide resistant strains of salmonellae and Escherichia coli carried non-transferring SSu determinants incompatible with ASu, and therefore probably phylogenetically related to the SSu determinant of type 29. A wild tetracycline resistance determinant (T) and the non-transferring colicinogeny determinants E1, E2 and E3 were compatible with each other and with A and SSu. A tentative classification can thus be suggested for these non-transferring plasmids which places A, SSu and its homologues, T, ColE1, ColE2 and ColE3 in separate compatibility groups. Molecular studies of ten of the plasmids showed that they consisted of covalentlyclosed circular DNA molecules with mean contour lengths between 2.22 and 4.53 μm. All were present in multiple copies per chromosome in E. coli K12.

The Molecular Relatedness of R Factors in Enterobacteria of Human and Animal Origin

The molecular length and DNA homology of R factors isolated from enterobacteria of human and animal origin have been examined. DNA from plasmids of the same compatibility group, whether of human or animal origin is indistinguishable, after allowance has been made for the regions coding for different antibiotic resistances. These results indicate that there is a common pool of R factors in man and ainmals.

Application of Agarose Gel Electrophoresis to the Characterization of Plasmid DNA in Drug-resistant Enterobacteria

A simple gel electrophoresis method has been described for the detection of plasmid DNA in bacteria (Meyers et al., 1976). We investigated further the problems encountered in using this method for the analysis of plasmids in wild enterobacterial strains. The migration of open circular and linear plasmid DNA was examined, since these forms sometimes caused difficulty in the interpretation of the plasmid content of uncharacterized strains. Electrophoresis at different agarose concentrations was employed to resolve clearly plasmid DNA from the chromosomal DNA fragments in the crude preparations. Dissociation of some plasmids occurs in Salmonella typhimurium, and this was detected by electrophoresis. The technique was applied to the study of drug-resistant strains of S. typhimurium phage type 208 from several Middle Eastern countries. The cultures carry a drug resistance plasmid of the FIme compatibility group, and at least two other plasmids which were detected and identified by gel electrophoresis. The studies supported and extended the genetic findings and provided information on the distribution of particular plasmids.

Characterisation of plasmids coding for the restriction endonuclease EcoRI

SummaryThe properties of two plasmids coding for the EcoRI restriction and modification enzymes are described. Both plasmids are non auto-transferring (NTP) but can be mobilised by transfer factors. Strains carrying NTP13 produce colicin E1 and the EcoRI enzymes. This plasmid has a molecular weight of 6x106 daltons and is present as approximately 12 copies per chromosome. The second plasmid, NTP14, was detected after mobilisation of the EcoRI plasmid with the R factor R1–19. NTP14 codes for ampicillin resistance, synthesis of the EcoRI enzymes and colicin E1. The molecular weight of NTP14 is 10.7x106 daltons and there are about 14 copies per chromosome. DNA-DNA reassociation experiments were performed to determine the interrelationships of NTP13, NTP14, ColE1 and the R factor R1–19. NTP13 and NTP14 continue to replicate when cellular protein synthesis is inhibited by the addition of chloramphenicol.

Transduction of resistance determinants and R factors of the Δ transfer systems by phage P1kc

SummaryTransduction by P1ke shows that Δ-mediated R factors fall into two groups: those in which the resistance and the Δ transfer factor are transduced as a single unit; and those in which the resistance determinant is transduced independently of the transfer factor. The first group is exemplified by the T-Δ R factor, which is transferable after transduction. An example of the second group is the SSu,Δ R factor, in which the SSu determinant is transduced independently to recipient cells. The SSu resistance is therefore not transferable until Δ is introduced into these recipients. These observations support the postulate, originally based on conjugational observations, that R factors are of two classes. In Class 1 the resistance determinant and the transfer factor form a single covalently bonded complex which is transferred intact to recipient cells; T-Δ belongs to this class. In Class 2 the resistance determinant and the transfer factor are separate plasmids. Although the transfer factor is necessary for transfer of the determinant in this class, independence of the plasmids is maintained in new hosts, and the nature of the association between the respective plasmids during transfer requires clarification.

Molecular studies of F1me resistance plasmids, particularly in epidemic Salmonella typhimurium

SummaryThe molecular sizes of F1me resistance plasmids from strains of Salmonella typhimurium, S. wien and S. typhi were within the range 87.9–102.6×106 daltons. DNA reassociation studies indicated that the plasmids from these hosts had at least 80% of their nucleotide sequences in common. A high proportion of F1me plasmids cannot mediate their own transfer. The non-autotransferring property of such plasmids is the result of DNA deletion; a non-autotransferring F1me plasmid was about 10×106 daltons shorter than autotransferring representatives of the group, and its DNA showed 100% homology with them. Plasmids of the F1me group are incompatible with the F factor and with F1R factors. F1me plasmids are incompatible with the fi+ MP10 plasmid of S. typhimurium, whereas F and F1 factors are compatible with MP10 (Anderson et al., 1977). There was no significant DNA homology between members of the F1me group and MP10, and these plasmids may share only a small region of DNA responsible for their incompatibility. The F1me R factors examined had 29–37% DNA homology with the F factor, and 50–58% homology with the F1 resistance plasmid, R162. Molecular examination therefore supports the genetic differentiation of members of the F1me group from other F-like plasmids. Both types of investigation can thus be used in epidemiological studies of bacterial strains carrying resistance or other plasmids.