SUPPRESSION OF THE FORMATION OF POLYGENOTYPIC RECOMBINANT COLONIES BY A maf MUTATION IN MATING WITH HfrH

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1 SUPPRESSION OF THE FORMATION OF POLYGENOTYPIC RECOMBINANT COLONIES BY A maf MUTATION IN MATING WITH HfrH JONATHAN T. OU AND LI-ME1 KUO The Instituie for Cancer Research, Fox Chase Cancer Cenier, Philadelphia, Pennsylvania Manuscript received June 7, 1979 ABSTRACT W3011, a Cavalli-type Hfr (HfrC), was mated with KY9474, maf-1, which cannot maintain F or like plasmids, and with OU9474, Maf+, a spontaneous revertant of KY9474. The recombinant colonies obtained were 100% monogenotypic from KY9474 and 90% monogenotypic from OU9474. On the other hand, in matings with OU11, a Hayes-type Hfr (HfrH), and these two strains, recombinant colonies derived from KY9474 showed only 22% polygenotypic recombinant colonies; whereas, those derived from OU9474 showed a high production rate (57%) of polygenotypic recombinant colonies. Among the polygenotypic recombinant colonies derived from KY9474 maf-1, 50% contained three or more recombinant types. These were probably derived from a small fraction of Maf+ revertants in the KY9474 population, as suggested by the results of mating this strain with M80, an F strain that contains an amber mutation in trah. These results support the hypothesis that the donor DNA fragments derived from an HfrH can undergo a limited replication in the recipient to produce polygenotypic recombinant colonies, whereas those derived from HfrC cannot. OME 20 years ago in pedigree analyses, LEDERBERG (1957) showed that single exconjugants produce mainly single-type (monogenotypic) recombinants, using a Cavalli type Hfr (HfrC), whereas ANDERSON (1958) and his co-worker (ANDERSON and MAZB 1957), using a Hayes-type Hfr (HfrH), demonstrated that exconjugants often produce multiple recombinant types (polygenotypic). Since then, a number of investigators have confirmed these observations (WOOD 1967; BRESLER, LANZOV and BLINKOVA 1967; LOTAN, YAGIL and BRACHA 1972; BRESLER, LANZOV and MANUKIAN 1973; NAJER and KUNICKI-GOLDFINGER 1974; Ou 1975; Ou and ANDERSON 1976), and have concluded that the difference is associated with the type of Hfr used (WOOD 1967; LOTAN, YAGIL and BRACHA 1972; Ou 1975; Ou and ANDERSON 1976) rather than with the strains used. Furthermore, a number of investigators suggested that the ability of HfrH to produce polygenotypic recombinant colonies is likely to be due to the ability of the transferred donor DNA to replicate a few times in the host before integration, whereas the transferred DNA derived from HfrC cannot (LOTAN, YAGIL and BRACHA 1972; Ou 1975; Ou and ANDERSON 1976). We further proposed that the transferred DNA derived from HfrH may carry machinery for its own replica- Genetirs 93: October, 1979

2 346 J. T. OU AND L-M. KUO tion, whereas that derived from HfrC does not (Ou 1975; Ou and ANDERSON 1976). Utilizing a maf (maintenance of an autonomous F factor) mutant strain (WADA and YURA 1971; WADA, HIRAGA and YURA 1976) that inhibits the replication of F plasmids in mating experiments, we have obtained further supporting evidence that the formation of polygenotypic recombinant colonies from HfrH is indeed most likely due to the ability of the transferred DNA fragments to replicate in the recipient. MATERIALS AND METHODS Bacterial strains: Bacterial strains used are listed in Table 1. All are derivatives of Escherichia coli K12. Media: All bacteria were routinely grown and mated in L-broth containing per liter: 10 g Tryptane, 5 g yeast extract, 10 g NaC1, and 1 g glucose. Agar plates used were L-agar, McConkey agar (Difco), and minimum medium consisting of M9 medium supplemented with appropriate nutrients and drugs. The drugs used were streptomycin-sulfate (Str) 100 pg per ml, nalidixic acid (Nal) 30 pg per ml, and phenethyl alcohol (Pea) 0.23% (v/v). Mating procedures: Exponentially growing (- 4 x 108 bacteria per ml) donor and recipient bacteria were mixed at one-to-one ratio and incubated for 90 min with gentle shaking at 37". The mixtures were then mechanically blended (Low and WOOD 1965), diluted and plated on selective plates: arginine or leucine for HfrC mating and tryptophan for HfrH mating. In each case, streptomycin was used to counter-select against the donors. Determination of genetic constitution: Each entire colony was taken, resuspended in 1 ml of broth, and plated on L-agar plates with appropriate dilution. After 20 to 24 hr incubation, 52 secondary colonies were picked from each and transferred to a new L-agar plate to form a grid. The grid plate was again incubated for 20 to 24 hr and replica-plated onto desired selective plates for determination of genetic constitution. The replica plates were read after 20 to 24 hr incubation. All incubations were carried out at 37". TABLE 1 Escherichia coli K12 strains Strain ou11 W3011 M80 KY9474 KY9475 OU9474 OU41113 Sex Hfr Hfr F' Genotype' Hayes type, thi rel-1 A-; injects chromosome with the order thr maf leu proa lac.... Cavalli type, ilu metb1 rela1 galk2 tona57 spot1 A-; injects chromosome with the order zsx lac proa leu maf thr.... JCFL80 plasmid with trah8o (amber) in JC6255, which is lac trp supd maf-1 A(trp-tonB) proa ilu arg leu lac tm rpsl pea supe Same as KY9474 except maf+ leu+ Spontaneous Maf + revertant of KY9474 lacy1 or 24 galk2 arghl meta28 tsx-6 thi-1 Mu+ nala92 Xyl-5 or Xyl-7 tfr-3 supe44 deo? Spontaneous Nalr mutant of OU41112, which is a derivative of NH4111 by curing F'lac+ (F42) with acridine orange Source This laboratory E. LEDERBERG N. WILLETTS T. YURA T. YURA This laboratory This laboratory * See BACHMAN, Low and TAYLOR (1976) for gene symbols.

3 maf GENE AND POLYGENOTYPIC RECOMBINANTS 347 RESULTS Formation of polygenotypic recombinant colonies in HfrH X matings: OU11, an HfrH strain, bacteria were separately mated to KY9474 (maf-l) and OU9474, a spontaneous Maff revertant of KY9474, and Trp+Str recombinants were selected. The purity of these recombinant colonies was then determined by testing the recombinants for four unselected markers: Lac+, Pro+, Pea and Leu+. We have not tested these recombinants for the Maf marker. As shown in Table 2, only eight in 36, or 22%, of recombinant colonies from OU11 X KY9474 were of polygenotypic types, whereas 17 of 30, or 57%, recombinant colonies from OUll X OU9474 were polygenotypic. Although HfrH injects maf+ early (- 7 min), it has been shown to be recessive (WADA, HIRAGA and YURA 1976) and thus maff from the donor DNA probably did not cause the formation of polygenotypic recombinant colonies by allowing the donor DNA fragments to replicate. The x2-test showed p = with 1 d.f., indicating a significant difference between the matings. Examination of recombinant types showed that 14 of 16 possible genetic constitutions were found. When those eight polygenotypic colonies produced in OU11 x KY9474 matings were examined, four (or 50%) of them contained more than three recombinant types, suggesting that they occurred by the bacterial host allowing replication of the donor DNA fragment. One possibility is that these recombinant colonies are actually produced by minority Maf + revertants that are always present in the population of KY9474 bacteria, despite our use of freshly isolated strains. Proportion of Maff revertants in KY9474 population: To test the above possibility, we set out to measure the proportion of Maf+ revertants in the KY9474 population. In order to select these revertants, we used an F lac+ strain because maf-l- bacteria can not maintain F Zacf. Therefore, only Maf+ revertants are able to maintain F Zac+ and can form stable F lac+ transconjugants. Furthermore, Maf+ revertants will be a minority in the maf-l- population, so that the frequency of F Zac+ transconjugant formation in a mating between F lacf donors and maf-1- recipients should be low compared to that in a mating between F Zac+ donors and maff recipients, where most of the transferred F Zac+ would be stably maintained in the recipient. The maf-i- bacteria containing an F plasmid, although unable to maintain it, can transfer the F plasmid to other maf-i- bacteria in the population (WADA, TABLE 2 The frequencies of the formation of polygenotypic recombinant colonies in OUll matings with KY9474 and OE9474 Recipient No. of monogenotypic No. of olygenotypic strams Trp+ colonies (percent) ~rp+ coponies (percent) KY9474 maf-i 28 (78) OU9474 Maff 13 (43) Total 41 Total 8 (22) (59) x2 test (SIMPSON, ROE and LEWONTIN 1960) showed x2= 6.851, P = with 1 d.f

4 348 J. T. OU AND L-M. KUO HIRAGA and YURA 1976). Thus, if a Traf F plasmid is used, transconjugant colonies would appear in a maf-1- population because of the secondary F plasmid transfer within the colonies. To prevent this secondary transfer, the result of which may complicate the measurement, we chose a donor strain containing an amber tra- (transfer) mutation so that the F plasmid cannot be expressed in the recipient, which is Su-, and thus the transconjugants will be Tra-. For these reasons, M80 (supd,tra+), an F Zac+ strain containing the JCFL80 plasmid, which has an amber mutation in trcrh, (ACHTMAN, WILLETTS and CLARK 1971) was chosen. This donor strain was mated with KY9474 maf-l, OU9474 Maf+, and OU41113 Maf+, and the frequency of Lac+ transconjugant formation and the stability of Lac+ transconjugants determined. Among these three strains, KY9474 and OU9474 did not suppress the trah80 amber mutation although they contain supe, and thus the transconjugants derived from these strains were Tra-. On the other hand, OU41113 did suppress the trah amber mutation, and the transconjugants obtained were Tra+. Matings were carried out using an excess of M80 (1.5 to one recipient) to insure that every bacterium was mated. As shown in Table 3, compared to Maf+ strains the Maf- KY9474 strain showed much lower mating efficiency: x per bacterium compared to x per bacterium in the case of the Maf+ strains. Furthermore, most or all of the transconjugants obtained were stable. This result indicates a high reversion rate of KY9474 maf-l to become Maf + : at least 1.3 X per bacterium. There are two situations where a transconjugant will be stable: (1) the plasmid replicates in the host, and (2) the bacterial chromosomal segment in the plasmid (lac+ in this case) either recombines with the host chromosome or the entire TABLE 3 Efjiciency of transconjugant formation and stability of transconjugants obtained in 90 minute matings of F M80 to four strains strains Transconjugants KY9474 Lac+Slrr (maf-i-) KY9475 LacfStrr (mnf-i+) OU94.74 Lac+Strr (Maff) OU41113 Lac+NaP (Maf + ) No. of stabla transconjugants/ Eificiency of mating* No. of transconjugants tested.;- Expr. 1 Expt. 2 Expt. 3 Expt. 1 Expt.2 Expt x x x1G-2 17/20 8/8 19/20 310x /10 11 ox /10 10/ X 1 o- 10/10 * No. of transconjugant colonies per bacterium at the time of Mixing. It can he greater than one because there was bacterial growth in 90 minutes. t Transconjugants were inoculated in L-broth and allowed to grow for at least 20 generations. The cultures were plated on McConkey agar to determine the relative number of Lac+ and Lac- colonies. An arbitrary criterion was used to determine the stability of transconjugants: when the ratio of Lac+ to Lac- colonies was one or larger, the transconjugants were regarded as stable; otherwise, they were considered unstable. As it turned out, most of the cases showed a clear-cut result of producing a great majority of either Lac+ or Lac- colonies.

5 maf GENE AND POLYGENOTYPIC RECOMBINANTS 349 plasmid becomes integrated into the host chromosome. Further tests showed that all transconjugants examined were resistant to $11 phage, a female-specific phage, and the lac+ marker was readily curable by treatment with agents such as acridine orange. These test results suggest that the stable transconjugants contain autonomous plasmids, and thus indicate that they were derived from Maf + revertants in the population. It appears that the polygenotypic recombinant colonies observed in OU11 x KY9474 matings could have been derived from the Maf + revertants in the population of KY9474. Formation of polygenotypic recombinant colonies in HfrC X mdings: The above results suggest that the production of polygenotypic colonies in OU11 X OU9474 matings was due to the replication of donor DNA in OU9474 bacteria. One question remains, however: the polygenotypic colonies obtained in the OU11 x OU9474 mating might be due to the inherent nature of OU9474; that is to say, when mated, OU9474 bacteria might produce polygenotypic colonies regardless of Hfr strains. To check this, we have mated W3011, a Cavali-type Hfr well-known for not producing polygenotypic colonies, with the two (OU9474 and KY9474) strains. Argf [Strr] or Leu+ [Sti] recombinants were selected, and unselected markers Leu+, Peas, Pro+ and Lac+ were checked. The results showed that all 20 recombinant colonies derived from KY9474 were monogenotypic and only two digenotypic recombinant colonies (containing two recombinant types each) were found in 20 recombinant colonies tested from the mating with OU9474. These results suggest that the HfrH strain is responsible for the production of the polygenotypic colonies described above. DISCUSSION The different behavior of Hayes-type Hfr and Cavalli-type Hfr in forming recombinants has long been observed and studied extensively. Based on a series of experiments, we proposed (Ou 1975; Ou and ANDERSON 1976) that the transferred DNA donated by HfrH bacteria could undergo a few rounds of replication before integration into the host chromosome, thereby producing a number of different recombinant types from a single exconjugant. On the other hand, the transferred DNA from HfrC bacteria could not replicate; therefore, generally only one recombinant type would be produced from a single exconjugant. Thus, if one somehow inhibited the replication of the HfrH-derived donor DNA in the host, one should be able to reduce the number of polygenotypic recombinant colonies. W e found earlier that simply by raising the ph of the medium in which mating with HfrH is performed one can reduce substantially the number of polygenotypic recombinant colonies (OU 1975). The replication of the donor DNA fragment appears to be controlled by the DNA fragment itself and not by the host DNA synthesizing machinery, or the HfrC-derived DNA fragment should be able to replicate and produce polygenotypic recombinant colonies. That is to say, the replication is likely to be carried out by the replication system of the F plasmid. The maf- mutation inhibits the replication of F plasmids, but not recombination (WADA and YURA 1971; WADA, HIRAGA and YURA 1976). Thus, bacteria containing a maf mutation should

6 350 J. T. OU AND L-M. KUO show a reduced number of polygenotypic recombinants when mated with HfrH. As presented above, this is what we found: the frequency of polygenotypic recombinant colonies was reduced about one-half in the HfrH X maf-1- compared to that in HfrH x Maff. The frequency (22%) of polygenotypic recombinant formation in the HfrH x maf-1 mating is similar to that found in our previous experiments in which we showed inhibition of the formation of polygenotypic recombinant formation by raising the ph of the medium to 7.6 (Ou 1976). However, somewhat unexpected in the present experiment is that only four out of eight (or 50%) polygenotypic colonies were digenotypic (contained two recombinant types) with three out of four containing exclusive recombinant types (no overlapping of unselected donor markers in recombinants; see Ou 1975), and among the rest, three were trigenotypic and one tetragenotypic; whereas, in the experiments done at ph 7.6 mentioned above (Ou 1975), most recombinant colonies were digenotypic with only 1.2% containing three recombinants. The reason for this is probably the following. The maf-1 mutation of strain KY9474 appears to be rather unstable and reverts back to Maf+ with a high frequency (OU9474 was thus obtained). Therefore, in any Mal? population, there would always be a small fraction of bacteria reverting back to Maff. These Maf+ revertants would have an advantage in forming recombinants once they received the donor DNA because of their ability to maintain and replicate the F plasmid. The results of F x matings supprt this possibility. Thus, the actual frequency of polygenotypic recombinants is probably much lower than the 22% obtained in the mating between OU11 and KY9474 here. The question of the inherent nature of OU9474 to produce polygenotypic recombinant colonies has been resolved by mating with an HfrC donor. AS shown above, this cross produced predominantly monogenotypic recombinant colonies, as expected. Thus, the results reported here further support the hypothesis that the donor DNA fragment derived from HfrH most likely contains genes required for replication. These genes are responsible for the formation of polygenotypic recombinant colonies. Apparently, HfrC-derived DNA fragments lack the ability to form multiple copies in recipient bacteria. We thank BARBARA ELBERTSON and EILEEN HANIGAN for their skillful technical assistance. We are grateful to E. LEDERBERG, N. WILLETTS and T. YURA for their generosity in supplying us with the E. coli strains used in this investigation. Special thanks to T. ANDERSON for his interest and constant encouragement. This investigation was supported by Public Health Service grants RR and CA to The Institute for Cancer Research, and by an appropriation from the Commonwealth of Pennsylvania. LITERATURE CITED ACHTMAN, im., N. WILLETTS and A. J. CLARK, 1971 Beginning a genetic analysis of conjugational transfer determined by the F factor in Escherichia coli by isolation and characterization of transfer-deficient mutants. J. Bacteriol. 106: ANDERSON, T. F., 1958 Recombination and segregation in Escherichia coli. Cold Spring Harbor Symp. Quant. Biol. 23:

7 maf GENE AND POLYGENOTYPIC RECOMBINANTS 35 1 ANDERSON, T. F. and R. MAzB, 1957 Analyse de la descendance de zygot6s formes par conjugaison chez Escherichia coli K12. Ann. Inst. Pasteur 93: BACHMAN, B. J., K. B. Low and A. L. TAYLOR, 1976 Recalibrated linkage map of Escherichia coli K12. Bacteriol. Rev. 40: RRESLER, S. E., V. A. LANZOV and A. BLINKOVA, 1967 Mechanism of genetic recombinations during bacterial conjugation of Escherichia coli K12. I. Heterogeneity of the progeny of conjugated cells. Genetics 56: BRESLER, S. E., V. A. LANZOV and L. R. MANUKIAN, 1973 Mechanism of genetic recombination during bacterial conjugation of E. coli K12. IV. Heterogeneity of progeny exconjugants. Role of donor and recipient strains. Molec. Gen. Genet. 123: LEDERBERG, J., 1957 Sibling recombinants in zygote pedigrees of Escherichia coli. Proc. Natl. Acad. Sci. US. 43: LOTAN, D., E. YAGIL and M. BRACHA, 1972 Bacterial conjugation: An analysis of mixed recombinant clones. Genetics 72: Low, B. and T. H. WOOD, 1965 A quick and efficient method for interruption of bacterial conjugation. Genet. Res. 4: NAJER, A. and W. J. H. KUNICKI-GOLDFINGER, 1974 Mechanism of conjugation and recombination in bacteria. XIII. Heterogenetic recombinant clones and the fate of transferred fragment of Hfr chromosome. Acta. Microbiol. Polon. Ser. A6 (23): Ou, J. T., 1975 Recombinant clone heterogeneity in Escherichia coli conjugation: Effect of ph and partially replicated recipient deoxyribonucleic acid. Genetics 80: Ou, J. T. and T. F. ANDERSON, 1976 F plasmids from HfrH and HfrC in reca- Escherichia coli. Genetics 83 : SIMPSON, C. G., A. ROE and R. C. LEWONTIN, 1960 Quantitative Zoology. Harcourt, Brace and Company, New York. WADA, C. and T. YURA, 1971 Phenethyl alcohol resistance in Escherichia coli. 11. Replication of F factor in the resistant strain C600. Genetics 69 : WADA, C., S. HIRAGA and T. YURA, 1976 A mutant of Escherichia coli incapable of supportive replication of like plasmids. J. Mol. Biol. 108: WOOD, T. H., 1967 Genetic recombination in Escherichia coli. Clone heterogeneity and the kinetics of segregation. Science 157: Corresponding editor: I. P. CRAWFORD