Plasmid co-integrates of prophage lambda and R factor R100

Abstract

Single and tandem insertions of prophage X into R100 have been isolated. Insertions into the transfer genes, insertions into the transfer control gene finO, and insertions into regions that result in no detectable phenotypic change were found. From the last type, deletion mutants were isolated which established the sequence of antibiotic resistance genes as tet-cml-fus-str-sul-mer in R100. Highfrequency transducing phage preparations Xmer, Xsul str, and Xsul str cml were also isolated from this type. R factor R100 is a conjugative plasmid existing as a covalently closed circular deoxyribonucleic acid (DNA) molecule of 60 x 10 to 70 x 106 molecular weight in Escherichia coli K-12. The functions that this plasmid determines include: (i) the ability to replicate in a controlled fashion in the host cell, together with the incompatibility of this replication with that of related plasmids in the same cell; (ii) resistances to tetracycline, chloramphenicol, streptomycin (and spectinomycin, using the same adenylase [3,9]), sulfonamides, mercuric ions, and fusidic acid (5); (iii) a transfer system that allows it to transfer its DNA by conjugation to a suitable recipient cell; and (iv) a two-stage system that controls expression of the transfer genes (7).As a long-term goal, we are interested in elucidating the detailed genetic and physical structure of R100, including in particular the mechanisms whereby expression of the transfer genes is controlled. As a step towards that end, we first isolated a number of "co-integrates" of R100 and X by using the techniques for X insertion into "unusual" attachment sites pioneered by Shimada et al. (16). These X insertions are frequently strongly polar and therefore help to identify operons. Second, from insertions of a XcI857 phage, deletion mutants can be selected as survivors to high temperatures. These can be used to locate the genes responsible for the various R factor functions, and. they are also useful in the characterization of operons and the genes controlling them. Third, it is possible to isolate transducing phages from such insertions, which should allow gene amplification where it is required to purify a gene product, and serve as a source of DNA for measuring transcription of discrete regions of R100 using hybridization techniques. This report presents the methods used to isolate R100 (X) co-integrates and summarizes results showing the approximate locations of the prophage insertions. Deletion mutants from two integrates are described that have allowed a genetic map of the antibiotic resistance genes of R100 to be constructed. MATERIALS AND METHODSBacterial strains. The host strain for R100 or R100-1 in which the X insertions were generated was ED2149. This is a derivative of ED395 that is T66 and carries a deletion covering nia, gal, att,, and bio (ED395 is a LacAu,24-derivative of W3110). The att, deletion was transduced with P1 grown on CT1O (obtained from C. Town) into a (XcI857 susJ6 xis-i) + lysogen ofthe parental attx+ strain, since all spontaneous gal att, bio ...

Phenotypic reversion of an IS1-mediated deletion mutation: a combined role for point mutations and deletions in transposon evolution.

“…We have analysed the plasmid pEDR104 by restriction cleavage and Southern hybridisation ( Figure 1) and have constructed the physical map shown in Figure 2. As predicted by Dempsey and Willetts (1976), most of the r-determinant has been deleted. The deletion was mediated by ISIb and pEDR104 still carries two copies of ISI.…”

“…The plasmid pEDR104 was isolated as a temperatureresistant revertant of a strain harbouring RIOO::XcI857susS7b5l5b519 (Dempsey and Willetts, 1976).…”

“…The RTF part flanked by two ISi elements contains the Tcr transposon TnlO, Tra genes, and Rep region. The arrow marked X on the Tn2670 map shows the integration site of the X genome in the RIOO::X cointegrate which is the precursor of pEDR104 (Dempsey and Willetts, 1976;Dempsey et al, 1978). In pEDR104, an ISlb-mediated deletion has removed most of the r-determinant together with the integrated X DNA and produced Tn267OA104.…”

“…The R plasmid pEDR104 was kindly provided by W.B. Dempsey (Dempsey and Willetts, 1976). Phage Xrl4::ISl (Hirsch et al, 1972) and plasmid pBR325 (Bolivar, 1978;Prentki et al, 1981) were used as hybridisation probes for S1 and the cat gene, respectively.…”

“…In the course of these studies we found that the cat gene product also confers resistance to fusidic acid (Far) (Marcoli et al, 1980;Volker et al, 1982). We were, therefore, intrigued by a report that another deletion derivative of TN2670 on a R100 derivative, called pEDR104, provided resistance to Cm but not to Fa (Dempsey and Willetts, 1976). We analysed strains containing this plasmid and have cleared up the contradiction.…”