Mol Gen Genet (1987) 210:288-293 MGG © Springer-Verlag 1987 Transductional analysis of chromosome replication time Mark C. Hanks* and Millicent Masters Department of Molecular Biology, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK Summary. Following transduction of exponentially growing cultures of Escherichia coli with phage P 1, cells with recom- binant phenotype begin to increase in number after an ini- tial lag of about one generation time. We show that trans- ductants for markers located at different positions on the chromosome begin to increase at different times, in reverse order to that in which they are replicated. The period over which this happens is equal in duration to the time taken to replicate the chromosome and we have used this relation- ship to calculate the C-period of E. coli K12 growing at 30 ° C. We exclude transduction-induced filamentation as the cause of the initial lag and suggest that the lag may result from the way in which donor DNA is inherited. Key words: Transduction- DNA replication - Recombina- tion - Bacteriophage P 1 Introduction Following the transduction of enteric bacteria with general- ized transducing phage there is a lag of several generation times before cells inheriting the donor markers increase in number. This has been noted for Salmonella typhimurium transduced with P22 (Ebel-Tsipsis et al. 1972), Escherichia coli transduced with P1 (Helling 1967; Sandri and Berger 1980) and Klebsiella aerogenes, also transduced with PI (Bender and Sambucetti 1983). Bender and Sambucetti (1983) transduced exponentially growing cultures of Kleb- siella and observed a delay of 6-8 generations before trans- ductants increased in number. Since filaments (long cells) appeared to be selectively enriched for the transduced phe- notype these authors hypothesized that the transductional process causes filamentation and that transductants fail to increase in number until these filaments recover the ability to divide. The prospect of substantially enriching transduced pop- ulations for transductants by simple size selection led us to analyse in some detail the behaviour of E. coli cells imme- diately following transduction with P 1. Although we were easily able to separate the small number of filaments in our transduced populations from the normal-sized cells, we did not find the filaments to be significantly enriched for transductants. This rules out a delay in division as the cause * Present address: Poultry Research Unit, Roslin, Midlothian, UK Offprint requests to: M. Masters of the lag in increase of transductants. Our observation that the length of lag is marker dependent, varying with the position of the selected marker vis-a-vis the chromosom- al origin of replication, leads us to conclude that transduc- tants fail to increase in number immediately because of the time required to complete the segregation process. Anal- ysis of the marker-dependent differences in length of this segregation lag has enabled us to estimate the time required for chromosome replication (the C-time; Cooper and Hehn- stetter 1968) by E. coli growing at 30 ° C in broth. Numbers of transductants do not begin to increase until two generations after mixing phage and cells. This can most easily be explained on the hypothesis that transductional recombination, like post-conjugational recombination, usu- ally proceeds via single-strand replacement of recipient with donor DNA (Siddiqi and Fox 1973) rather than by double- stranded replacement as has been suggested on the basis of physical evidence (Ebel-Tsipsis et al. 1972; Sandri and Berger 1980). Materials and methods Bacteria, phage and strain constructions. Transductions for the purpose of strain construction were carried out with Plkc using lysates prepared on the wild-type strain W3110 as described by Masters (1970, 1977). Transductional recipi- ents were MM 303 (zx trp-tonB argH1 ilv 142 metB 1 pyrE41 lacY1 xyl-7 thi-1 rpsL tnaA1 tsx uhp2) and MM327 (MM303 purF lysA pyrE+). MM327 was constructed by transducing a thyA derivative of MM323 (Masters 1977) to lysA thyA +. Media were as previously described. Transduction of exponential cultures. For segregation experi- ments, cultures were grown exponentially in L broth to an optical density at 540 nm of 0.3. Then 20 ml of cells were centrifuged (15 min at room temperature) and resuspended in 1 ml of P 1 transducing lysate with a titre of 2 × 10 l° pfu/ ml (m.o.i. = 5) containing Ca + + at 2.5 mM. After 10 min adsorption at 37 ° C the mixture was diluted into 20 ml of prewarmed L broth containing 10 mM sodium citrate (to chelate the Ca + +) and grown with shaking. Optical density was maintained below 0.3 by dilution once per generation. Samples were withdrawn and plated on selective plates or diluted and plated for viable counts. Enrichment of filamentous cells. Ceils were grown and trans- duced as above. Samples were withdrawn and filtered under