Microbiology (1 9961, 142, 2399-2406 Printed in Great Britain 1 School of Biological Sciences, University of Birmingham, Edgbaston, Birmingham B15 ZlT, UK M i cr o bi o logy, S r i Padmavathi Mahila Viswavidyalayem, Tirupati- 517 507 (AP) India 2 Department of Curing of F-like plasmid TP181 by plumbagin is due to interference with both replication and maintenance functions V. V. Lakshmi1#2 and Christopher M. Thomas' Author for correspondence: Christopher M. Thomas. Tel: $44 121 424 5903. Fax: +44 121 414 i925. e-mail : C M.Thoinas@ bham.ac.uk F-like plasmid TP181 is particularly susceptible to curing by the naphthoquinone derivative plumbagin, which may attack DNA gyrase. TPI81 should provide particular insight into the basis of plasmid elimination, which has application in a number of contexts. Curing was found to be optimal at pH 7.2. An EcoRl fragment containing the RFlA replicon of TP181 was joined to a KmR determinant (giving miniTPl81). MiniTPl81 had the same increased susceptibility to curing by plumbagin when compared to miniF as TP181 had relative to F. Plumbagin interfered with replication of miniTPl81, depressing its copy number and increasing the rate of segregation. Plumbagin also blocked the lethal effect of the ccd locus after rifampicin treatment, which mimics production of plasmid-free segregants, so that more of these plasmid- free cells would survive. Restriction mapping and DNA sequence analysis indicated that the ccd locus of TP181 is almost identical to that of F but that TP181 lacks the repC gene present in F which is needed to activate replication from oriW. Thus the sensitivity of TP181 may be due t o its dependence on both a replicon which is hypersensitive to perturbation of supercoiling by plumbagin and a host-killing system which is blocked by plumbagin. Keywords : plasmid curing, plumbagin, rifampicin, ccd locus INTRODUCTION Bacterial plasmids are extrachromosomal DNA elements which are normally inherited stably as the bacterial host grows and divides. Many plasmids are self-transmissible and if they integrate into the chromosome they can also promote conjugative transfer of chromosomal genes. Many phenotypic traits are encoded by plasmid-borne genes. These include properties such as resistance to antibiotics and heavy metals { Stanisich, 1 988). Resistance to antibiotics is once again a major clinical problem and strategies are needed to help combat the spread of antibiotic resistance which is mediated by plasmids. One strategy to minimize plasmid transmission of anti- biotic resistance is to eliminate the plasmids. This process is known as 'curing' and many compounds have been shown to be capable of causing this effect (Caro eb d., 1984; Trevors, 1386). Some of these compounds may The GenBank accession number for the sequence of the TP181 ccd region is U51588. cause curing by a rather nonspecific process - they damage and stress the cells so that aberrant cells emerge. Other agents seem to act much more selectively. Acridine orange is an example of this sort. It causes elimination of the sex factor F at concentrations where it has little effect on the bacterial host (Hohn & Korn, 1969). Mutations in the copB locus gave resistance to this curing effect and it was suggested that this region might contain the target fur acridine orange (LVechsler & Kline, 1980). However, this resistance was probably not very specific and could have arisen simply from the elevated copy number of the mutants, which would decrease the segregation rate even if replication inhibition were observed. Resistance was also found to arise in some temperature-stable revertants of repts mutants, suggesting that acridine orange sen- sitivity is a property of the basic replicon (Lane, 1381). The molecular basis for the sensitivity has not been further defined. However, it is remarkable that any single agent could cause efficient plasmid loss since detailed analysis of F and other plasmids has revealed multiple plasmid strategies for survival. F and F-like plasmids appear to have two or 0002-0673 0 1996 SGM 2399