Mechanism of Genetic Recombination During Bacterial Conjugation of Escherzchza Coli K-12

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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 recombinants (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 incorporation of the labeled donor DNA fragment. This method allows to detect the inactivation 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 counterselection 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, resuspended 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 recombinants 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 transferred 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 conjugation 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 postconjugation 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 mononuclear 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 multinuclear cells grown on 20 pg P/ml. Specific activity of PS2 in both cases is 16mC/mg. tion owing to external &radiation (FUERSTand STENT1956). Then the samples were frozen in liquid nitrogen. At increasing time intervals, samples were thawed and their titer was determined on AP agar. Curve 1 (Figure 2) represents the kinetics of cells suicide by P3*decay. This is a typical one-hit curve indicating that most of the cell population is mononuclear (MCFALLet al. 1958). The plateau on the kinetic curve after a long time of “radioactive suicide” is due to a small part of the cell population which did not undergo inactivation (DRISKELL-ZAMENHOFand ADELBERG1963). For comparison curve 2 is drawn on the same figure. It demonstrates the multihit kinetics of suicide, measured for cells grown in conditions of optimal phosphate content. The third experiment was a control for the formation of spontaneous Smr mutants among receipient cells. At the chosen streptomycin concentration (250 pg/ml) the background of spontaneous mutants was practically equal to zero. Now we shall describe our main experiment. Bacteria were grown from beginning to end on media containing no more than 2.5 pg P/ml for ensuring their mononuclearity. This enabled us to shorten the time of phenotypic expression for the recessive marker, because there was no need for segregation of fertilized and nonfertilized nuclei. P10 Sm’ cells labeled with PS2were conjugated with nonradioactive cells PA 260-1 T6’ in minimal medium supplied with P32. Immediately before conjugation the auxotrophic recipient cells were starved for amino acids during 2.5 hours. According to MAALOE(1961), in these conditions the chromosomes are able only to complete their replication cycle but do not initiate a new one. During the conjugation period the amino acid starvation of F- cells continued. This prevented the F- cells from assimilating radioactive phosphate during the conjugation period. After the conjugation lasted for 70 min at 37”C, phage T6 and DNase were added to the suspension. Then the cells were centrifuged to get rid of the radioactive medium, washed and resuspended in fresh nonradioactive medium supplied RECOMBINATION DURING CONJUGATION 121 with nutrient additions. The incubation of the cell suspension at 37°C continued for a period necessary for the phenotypic expression of Sm’ recombinants. At definite time periods, aliquots of the cell suspension were taken, diluted with the protective medium, divided into samples and frozen in liquid nitrogen. After P??decay, the samples were thawed and plated for Sm‘ recombinants. The results of this analysis are shown on Figure 3. Curves 4,3 and 2 demonstrate the kinetics of “radioactive suicide” of Sm‘ recombinants taken after 270,340 and 41 0 minutes respectively from the start of conjugation. Such long times of segregation of Sm‘ recombinants depend on the slow growth of the culture on the medium used (the generation-time in 2.5 pg-Tris-H medium is about 90 min) and the delay due to the replacement of the medium after conjugation. In their initial part, the curves fall sharply and are nearly parallel to each other. Then they come to a plateau, each on a different level. The fall of the recombinant titer is 5,4.3 and 3.7 times respectively. In the same time the titer of F- cells (curve 1) determined for samples taken after 70 min of conjugation practically remains constant (its fall did not exceed 1.2 times). Consequently, the decay of the recombinants carrying the Smr marker cannot be caused by the assimilation of radioactive phosphate by recipient cells. It is due to the disintegration of radioactive phosphorous in labeled donor DNA fragments incorporated into the genome of the recombinants.

cl25 [ FRACTION DF P” ATOMS DECAYED FIGURE3.-Kinetics of “radioactive suicide” of F- cells and Smr recombinants segregated at different moments of the postconjugation period. Curve 1: Change in the amount of viable F- cells subjected to conjugation in radioactive medium after amino acid starvation; Curve 2: after 410 min; Curve 3: after 340 min; Curve 4: after 270 min. Time is measured from the start of conjugation. 122 S. E. BRESLER AND V. A. LANZOV

DISCUSSION The phenotypic expression of a recessive character is connected with the dis- appearance of diploidy for this character. Hence, the segregating Sm‘ cells are real recombinants. The decay of recombinants due to “radioactive suicide” is a direct evidence of the incorporation of labeled donor DNA into the recombinant genome. Kinetics curves 2, 3 and 4 (Figure 3) follow the equation n = N1eLkt i- N,, where NI is the number of Sm‘ recombinants which contain P3?labeled fragments of donor DNA and undergo decay, and N, is the number of Sm‘ recombinants which are stable. In the beginning, the phenomenon of P3,decay is manifested in its pure form. We observed an exponential fall of curves 4,3 and 2. When N1etbecomes lower than N,, the curves form a plateau. The ratios N,/Nl for curves 4, 3 and 2 are 20, 23 and 28% respectively. The presence of some stable recombinants is due to a fraction of cells which underwent two divisions in the postrecombination period; this causes the formation of nonradioactive nuclei. A relatively slow increase of the percentage of stable recombinant cells with time is easily explained if we take into consideration that the multiplication of nonradioactive recombinants occurs on a background of the intensively growing population of new segregating radioactive Sm‘ recombinants. In conclusion we shall briefly discuss an apparent contradiction between our data and the results of JACOBand WOLLMAN(1961 ) . In their experiment prototrophic Hfr’s labeled with P3?were crossed on nonradioactive medium with auxotrophic nonradioactive F- cells. The zygotes formed were frozen at various stages of their development. The influence of P32decay on the recombinant yield was studied. When the samples were frozen immediately after the formation of zygotes, a sharp exponential decrease in the amount of recombinants was observed. On the other hand, when samples were taken after 1 hour or more, the influence of P3*disintegration was weakened and after 120 min became negligible. Thus the conclusion could be drawn that in 2 hours the genetic information contained in the labeled fragment of the donor DNA is transferred to structures which do not contain radioactive P3?.It is easy to se? that the system used by these authors in quantitatively very different from ours. First, in their case the donor DNA fragment transferred during conjugation contained only one P32labeled strand of DNA; secondly, the genetic character for which the selection was realized was a dominant one. Hence, one semiconservative replication of the merozygote even without cell division would be sufficient for the formation of a structure insensitive to P32 disintegration. On a selective medium this cell would behave as a prototroph indistinguishable from a true recombinant. Naturally, 2 hours are sufficient for the formation of such structures, while in our case we needed the segregation of true recombinants, and not less than two divisions, to arrive at a structure containing no radioactive phosphorus. Besides, one should bear in mind that in our experiment the time scale was much longer, since the culture was placed into a medium with limiting phosphate. Consequently, the difference in “stabilization times” in our experiment and that of JACOBand WOLLMANis quite natural. RECOMBINATION DURING CONJUGATION 123

SUMMARY The kinetics of “radioactive suicide” of bacterial recombinants yielded by conjugation of very hot P32-labelledHfr cells with cold F- cells was measured. A recessive genetic marker (Smr) was chosen for the purpose of discriminating true recombinants from heterozygotes. The experimental data presented here enable us to state that the transfer of genetic information from the donor cell to the recombinant is due to the physical incorporation of a fragment of donor DNA into the chromosome of the recombinant. This conclusion is in agreement with the mechanism of genetic recombination by breakage and reunion.

LITERATURE CITED

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