MECHANISM OF GENETIC RECOMBINATION DURING BACTERIAL CONJUGATION OF ESCHERZCHZA COLI K-12. 11. INCORPORATION OF THE DONOR DNA FRAGMENT INTO THE RECOMBINANT CHROMOSOME S. E. BRESLER AND V. A. LANZOV Institute of High Molecular Weight Compounds, Academy of Sciences of USSR, Leningrad Received November 30, 1966 INVESTIGATION of genetic recombination in bacteriophages and bacteria (KELLENBERGER.ZICHICHI and WEIGLE1961 ; MESELSONand WEIGLE1961 ; BODMERand GANESAN1964; FOXand ALLEN1965; OPPENHEIMand RILEY1966) has already shown that of the two recombination mechanisms, ILcopychoice” and “breakage-reunion”, the latter is the more plausible. Nevertheless, the model of “copy choice” acquired wide popularity. Therefore it is important to perform new experiments giving direct proofs of physical integration of parental material into recombinants. One experiment of this type fulfilled with a conjugation system is that of SIDDIQI( 1963). Using radioactively labeled F- cells and selecting true recombi- nants (according to a recessive marker T6‘) SIDDIQIsucceeded in demonstrating that the main part of the recipient cell genome (+ 80%) is inherited by the recombinant. Our aim was to design a complementary experiment to show that a part of the labeled donor genome can be detected in the cells which are true recombinants after conjugation. Since it was expected that the recombinant inherits only a small fragment of the donor DNA, the method has to be sufficiently sensitive. The experimental system satisfying these conditions is as follows: 1. The experiment is performed using a population of mainly mononuclear E. coti cells. The possibility of obtaining such a population was shown in the papers of WITKIN( 1951) and MCFALL,PARDEE and STENT(1958). 2. According to the conjugation model proposed by JACOBand BRENNER (1963) and experimentally confirmed by BLINKOVA,BRESLER and LANZOV(1965) and GROSSand CARO(1966), one of the two donor DNA strands transferred during conjugation is synthetized in the course of transfer. In order to ensure that both transferred DNA strands are labeled with P32,radioactive Hfr cells were crossed with unlabeled F- cells in P32containing medium. 3. Selection of recombinants after conjugation was effected for the recessive character Sm‘ which has the lowest time of phenotypic expression comparing with other recessive markers. 4. The method of “radioactive suicide” was used for revealing the incorpora- tion of the labeled donor DNA fragment. This method allows to detect the inacti- vation of the recombinant (in this case Sm?)when a small donor DNA fragment labeled with “hot” P3::is incorporated. Genetics 56: 117-124 May 19Gi. 118 S. E. BRESLER AND V. 11. L4h-ZOV Data presented in this paper prove that the transfer of genetic information from the donor to the recombinant is mediated by physical incorporation of a donor DNA fragment into the recombinant chromosome. MATERIALS AND METHODS Bacteria. Two strains of E. coli were used in this work: PI0 Smr, an Hfr strain (order: 0-thi-Sm-ser/gly F) of genotype thr--leu-SmT; and PA260-1 T@, an F- strain of genotype ser/gly-his-Smg. Both initial strains P10 and PA260 were obtained from F. JACOB. Phage. Phage T6 suspension of high titre was prepared by the method of ADAMS(1950). After purification the phage was resusp-nded in 2.5 ml of Tris-H medium and stored. For counter- selection of Hfr the phage suspension was introduced into the conjugating mixture with a multi- plicity not less than 500 phage particles per Hfr cell. Media. AP is the aminopeptide maximal medium. AP-agar is the AP medium with 2% of agar. Tris-H is the complete synthetic medium containing the main components of H medium (STENTand FUERST1955). Its composition is: KC1 1.5g; NaCl5g; NH4C1 lg; MgS04.7H,0 0.25g; Ca(N0,) 0.01g; thiamine 0.003% ; glycerol 2.5g, Neopeptone (Difco) 0.5g; Casamino acids (Difco) 0.5g; Tris buffer 0.1 M; pH 7.4; bidistilled water to liJ'10 ml. Figures in front of the medium symbd mean that the indicated amounts of phosphorus are introduced as inorganic phosphate per 1 ml. This was due exclusively to the Casamino acids preparation. For obtaining Tris-H medium poor in inorganic phosphate we purified the Casamino acids especially by mag- nesium ammonium phosphate precipitation, The resulting phosphorus content in the media was measured by the method of CHENet al. (1956). Tris-H-C is the synthetic minimal medium withoJt Casamino acids used for conjugation. In contrast to previously described H media, the phosphorus concentration required in Tris-H-C (2.5 pg/ml) was obtained by adding solutions of the salts KH2P04and Na2HP04.This medium contained also 80 pg/ml of aspartic acid. Low end3genous concentration of inorganic phosphate in Tris-H media enabled us to use high specific activities of P32 (100mC/mg). Preparation of labeled Hfr's, unlabeled F- cells and conjugatim procedure in radioactive medium. Hfr: 0.3 ml of 2.5 pg-Tris-H medium (100mC/mg P) were inxulated with a 17-hour old colony of Hfr's. It was aerated by slow shaking for one night at 37°C. The overnight culture was inoculated at the volume ratio 1:20 into the same medium and aerated at 37°C. During the exponential phase (after 5 to 6 hours) the culture was harvested, thoroughly washed to remove traces of the medium, and then resuspended in Tris-H-C medium. F-: cells were inoculated from an overnight culture into 2.5 pg-Tris-H medium. The inoculation of F- cells was effected 2 to 2.5 hours before that of Hfr's. This enabled us to subject them to amino acid starvation for the same time interval. Therefore the F- cells practically did not synthesize DNA and did not incorporate Piz during conjugation. The F- cells were taken in the exponential phase, centrifuged, resus- pended in Tris H-C medium and starved with aeration for 2 hours. During this period the tur- bidity of the culturc determined spectrophotometrically increased 1.4 to 1.7 times. The cells were centrifuged once more and resuspended in Tris-H-C medium. Cultures prepared in this way were mixed for conjugation in concentrations 2.101 l/ml Hfr x 4.108 l/ml F-. The total volume of the conjugation suspension was 2 ml. In the moment of mixing, radioactive phosphate was introduced into the suspension; its final specific activity was 100mC/mg P. The mixture was aerated by slow rotational shaking at 37°C for 70 min. Then a calculated amount of phage T6 was added, and also desoxyribonuclease at 20 pg/ml for the cleavage of DNA liberated from lysed Hfr cells. The phage resistant F- cells were filtered through a Millipore filter, washed with warm 2.5 pg-Tris-H medium, resuspended in 10 ml of the same medium and aerated by slow shaking at 37°C for phenotypic expression of streptomycin resistance. At various times aliquots were extracted from the suspension and divided into many equal samples; one of the samples was used for determining the titer of the Smr recombinants, and all other samples were diluted in a glycerol-Casamino medium, frozen in liquid nitrogen, and stored for P3*decay. Selection of recombinants: For more complete counterselection of Hfr Smr cells we used RECOMBINATION DURING CONJUGATION 119 hesides phage T6 also selection of prototrophs for the markers thr-leu. Selective agar for recombi- nants selection was prepared according to JACOB and WOLLMAN'Sprescription (1961) and supplied with streptomycin (250 pg/ml) . "Radioactive suicide" was observed according to a procedure described by BLINKOVAet al. (1965). EXPERIMENTAL RESULTS Three preliminary experiments were performed. First the yield of Sm" and ser/gZy+ recombinants was measured as a function of the time of phenotypic expression. Both Sm' and ser/gZy+ markers are trans- ferred during conjugation with a 12 min interval. PI0 Sm' and PA260-1 T6' cultures were mixed in a Tris-H medium under standard conditions. The conju- gation was interrupted by adding T6 phage for a short period; thereafter the mixture was diluted 1:lO with fresh Tris-H medium and aerated at 37" for a long time. At definite time intervals aliquots of the suspension were taken and the titer of the Sm' and ser/gZy+ recombinants determined. The results are plotted in Figure 1. Curve 1 shows the amount of prototrophic recombinants (ser/gZy+) versus time elapsed from the start of conjugation. This is a typical curve of delayed multiplication of recombinants for a dominant marker in the postcon- jugation period. Curve 2 shows segregation kinetics of recombinants for a recessive character Sm'. Comparison of both curves indicates that Sm' segregation begins approximately 60 min before the division of ser/gZy+ recombinants. In the second experiment, we verified the efficiency of formation of mono- nuclear cells by means of growing the culture in limiting phosphate. PA260-1 T6' cells were grown on 2.5 pg-Tris-H medium in the presence of P3*(+ 16 mC/mg P) during 3 to 4 generations, i.e., a time sufficient for the establishment of equilibrium between the phosphate in bacteria and in the medium (HERSHEY et at. 1951) . Then cells were diluted in glycerol-Casamino medium, and the radio- activity was lowered to 4 pC/ml. This level is sufficient to exclude cell inactiva- x' , ~ 120 140 160 180 200 220 hme (min) FIGURE1.-Increase of the yield of Sm' (curve 2) and ser/gly+ (curve 1) recombinants in the postconjugation period. 120 S. E. BRESLER AND V. A. LANZOV FRACTION OF P“ ATOMS DECAYED FIGURE2.-Decay kinetics of mononuclear and multinuclear radioactive cells. - Curve 1: Kinetics for mononuclear cells grown on medium with 2.5 ,pg P/ml. Curve 2: Kinetics for multi- nuclear cells grown on 20 pg P/ml.