Nitrosoguanidine assay of episomal integration in Escherichia coli

JOURNAL OF BACTERIOLOGY, Nov. 1973, p. 527-530 Copyright i 1973 American Society for Microbiology Vol. 116, No. 2 Printed in U.S.A. Nitrosoguanidine Assay of Episomal Integration in Escherichia coli CRISTINA P. LLOVERES AND E. CERDA-OLMEDO Departamento de Genetica, Facultad de Ciencias, Universidad de Sevilla, Spain Received for publication 10 July 1973 Mutations affecting utilization of lactose and resistance to the male-specific phages fl, f2, and Q8 tend to occur simultaneously more often than expected by chance in Hfr strains whose origin of transfer is close to the genes for lactose utilization, but not in F+ strains. Strains derived from the Hfr, but exhibiting poor ability to transfer early chromosomal genes, may or may not show this comutation phenomenon. These results support the concept that the F factor is integrated into the Hfr chromosome during vegetative growth, but is autonomous in the F+ strains and could serve as an assay for episomal localization. N-methyl-N'-nitro-N-nitrosoguanidine is an effective mutagen in Escherichia,coli (2, 11) and in many other organisms. The inactivation of the F factor by nitrosoguanidine, as we will abbreviate its name, has been described (13), and many mutations induced by this agent have been used in the genetic analysis of F functions (review in reference 1). Although all genes are susceptible to mutation by nitrosoguanidine, the mutations in any particular cell are not randomly distributed over the chromosome but crowded together in small regions, extending over less than 2 min of the E. coli chromosome (6), and placed at the positions occupied by the replication points at the time of the treatment (5). Genetic sites closer than 2 min exhibit comutation, that is, they tend to mutate simultaneously more often than expected by chance. Comutation may be a useful tool in some problems of genetic mapping, such as the recognition of the integrated and autonomous states of episomes. We have used the F factor as a model system, defining it as an episome conferring sensitivity to male-specific phages, such as the ribonucleic acid (RNA) phages Q,B and f2 and the deoxyribonucleic acid (DNA) phage fl. MATERIALS AND METHODS Bacteria and phages. X15 is a prototrophic F+ strain of Escherichia coli, derived from the F+ strain W1485; X493 is a prototrophic Hfr strain, derived from X15 and transferring its chromosome in the order O-proB-proA-leu-thr-... -lac-F. Both were received from R. Curtiss (3). DF73 is an Fstrain carrying the markers strA, thyA, thr, leu, thi, pro, arg, lac, gal, ara, xyl, mtl, derived from the strain AB1157 and received from K. Brooks. The wild-type RNA phages Qf and f2 were received from A. J. Clark, and the wild-type DNA phage fl was from N. D. Zinder. Culture conditions. Bacteria were grown at 37 C in tris(hydroxymethyl)aminomethane (Tris)-salts minimal medium (10), with D-glucose at 2 g/liter as carbon and energy source. Solid media were prepared in the same way with 25 g of agar (Difco) per liter. When appropriate, amino acids and thymine were added at 20 gg/ml, thiamine at 1 zg/ml, and streptomycin at 100 ug/ml, and ,-lactose was substituted for glucose at 2 g/liter. Nitrosoguanidine treatment. Cells from exponentially growing cultures were treated with 100 yg of nitrosoguanidine (Serva, Heidelberg, Germany) per ml of Tris-maleate buffer, pH 7.5, at 37 C for 30 min (4); they were washed twice by centrifugation both before and after the treatment. Test for phage resistance. Phage stocks were produced in broth-grown E. coli strain X493. A drop of phage stock, containing over 109 plaque-forming units of the indicated phage or phage mixture, was extended in a diametrical band on an agar plate with a wire loop. Bacteria from single colonies were streaked across the phage-containing band with a sterile toothpick. A colony was considered resistant when bacterial growth was the same on both sides of the phage-containing band, as observed after overnight incubation at 37 C. RESULTS New strains. Two Lacderivatives of the Hfr strain x493, unable to utilize lactose, were isolated after nitrosoguanidine treatment and called SE12 and SE13. To this end, exponentially-growing cells of strain x493 were treated with nitrosoguanidine, allowed to grow in glucose liquid medium for 3 h, and plated on glucose solid medium; individual colonies were then tested for growth on lactose solid medium. The conjugational ability and the order of gene 527 LLOVERES AND CERDA-OLMEDO transfer were checked in these strains by interrupted mating (9) with DF73, and they were found to be the same as in the original strain. Two Lacderivatives of the F+ strain X15 were isolated by the same procedure as for the Hfr, and called SE20 and SE21. They were checked to be still F+ by observing the appearance of resistance to the male-specific phages fl, f2, and QB after prolonged incubation with acridine orange (7). Strains exhibiting poor conjugational ability were isolated from the LacHfr. Bacteria from SE12 colonies were spotted onto plates covered with some 109 cells of strain DF73 and containing adequate supplements for the selection of Leu+ Thr+ Str+ recombinants (that is, containing streptomycin, thiamine, thymine, proline, and arginine). In most cases a thick growth of recombinants appeared, but some colonies, constituting about 1% of the total, did not produce any recombinants. After reisolation and retesting, four of them were called SE18, SE19, SE22, and SE23. The same procedure was applied to the strain SE13, and strains SE15, SE16, and SE17 were isolated. These strains were all found to be sensitive to male-specific phages. Comutation. The lac mutations in strains SE12, SE13, SE20, and SE21 were obtained independently. The survival of these strains after nitrosoguanidine treatment and the frequencies of Lac+ revertants are indicated in Table 1, which gives the results of typical experiments in which bacterial samples, taken just before or just after the treatment with nitrosoguanidine, were plated on glucose and factose media. Higher survival rates can be obtained by decreasing the pH at the time of the treatment to 5.5. It can be noted that nitrosoguanidine usually increases the frequency of revertants more than 1,000-fold. The strains derived from SE12 and SE13 behave like their parents in these respects. When untreated samples were plated on either glucose or lactose, the resulting colonies were always phage sensitive. Not a single phage-resistant colony was found after testing a total of 8,000 colonies belonging to all the strains obtained in the previous section. After the mutagenic treatment, a sizable proportion of phage-resistant colonies was found both among the general population (glucose plates) and among the Lac+ revertants (lactose plates). Table 2 gives the incidence of resistance to phages QB and f2, and Table 3 gives the incidence of resistance to phage fl. The colonies resistant to fl were found to be resistant to Q, and f2, but the reverse was often untrue. As a consequence, the proportions in Table 2 tend to be higher than those in Table 3. TABLE 1. Survival and reversion to Lac+ after nitrosoguanidine treatment Frequency of Lac+ revertants Strain S rv ing (per colony-forming unit) Surviving frcin Before the After the treatment treatment SE12 (Hfr) 0.15 2.2 x 10' 2.7 x 10-' SE13 (Hfr) 0.050 7.5 x 10-7 9.4 x 10-4 SE20 (F+) 0.096 < 10-8 4.1 x 10-4 SE21 (F+) 0.072 < 10-8 2.8 x 10In the Hfr strains the mutation to resistance to male-specific phages was always more frequent among the Lac+ revertants than among the general population; thus, both kinds of mutations tended to occur simultaneously in the same cells. No such association was found in the F+ strains. where both kinds of mutations occurred as independent events. One of the seven strains isolated from the Hfr because of their poor ability to transfer early chromosomal genes showed comutation between phage resistance and Lac+ reversion, but the others did not. DISCUSSION The two kinds of mutations investigated here were chosen because one is presumed to depend on chromosomal genes and the other is presumed to depend on F-factor genes. The experimental result is that in the Hfr the two kinds of mutations occur simultaneously more often than expected by chance after nitrosoguanidine treatment, whereas this does not happen in F+ strains. This result indicates that the genes responsible for the two mutations replicate at about the same time in the Hfr but not in the F+. This is in excellent agreement with the hypothesis (8) that the F factor of the Hfr is integrated with the chromosome and replicates as a part of it, even during vegetative growth unrelated to conjugation. The strains isolated from the Hfr because of their poor ability to transfer early chromosomal genes may be expected a priori to fall into two groups, one lacking comutation between episomal and chromosomal genes (presumably due to detachment from the chromosome of an F factor that does not carry the relevant chromosomal genes) and another exhibiting comutation (detachment of an F factor carrying the relevant chromosomal genes, or mutational loss of conjugational ability without detachment or acquisition of resistance to male-specific phages). Six strains of the first group and one of the second group were isolated. Guerola et al. (6) defined the comutation index as the frequency of double mutants rela528 J. BACTERIOL. EPISOMAL INTEGRATION IN E. COLI TABLE 2. Resistance to phages QpS and f2 after nitrosoguanidine treatment Plated on glucose Plated on lactose Strain Statistical Colonies Percent Colonies Percent significance" tested resistant tested resistant Hfr strains SE12 800 4.6 800 9.2 P < 0.001 SE13 1825 4.8 1825 7.9 P < 0.001 F+ strains SE20 700 5.0 800 5.1 NS SE21 400 4.7 300 5.3 NS Conjugation-defective strains derived from Hfr SE15 400 3.5 400 12.5 P < 0.001 SE16 400 4.7 400 5.0 NS SE17 400 5.2 400 6.7 NS SE18 400 4.0 400 4.0 NS SE19 400 5.0 400 7.0 NS SE22 500 5.0 500 5.6 NS SE23 400 5.0 400 5.4 NS a The probability of a chance deviation equal to or larger than the experimental one Was calculated after the arCsin transformation and the t test (12). All "not significant" (NS) cases gave P > 0.2. TABLz 3. Resistance to phage fl after nitrosoguanidine treatment Strain Hfr strains SE12 SE13 F+ strains SE21 Conjugation-defective strains derived from Hfr SE17 SE18 Plated on glucose Colonies tested 500 375 500 500 500 Percent resistant 3.2 2.1 2.6 2.4 1.4 Plated on lactose Colonies tested 500 375 400 500 500 Percent resistant 8.6 7.2 3.0 2.0 1.4 tive to the product of the frequencies of the single mutants; they found an index of 15 for genes placed 1 min away in the chromosome map. The comutation index for Lac+ reversion and fl resistance is about 3 in the Hfr strains. By comparison with Guerola's results, the relevant genes may be placed some 1.5 min away in the Hfr chromosome. Comutation is clearer and the index is slightly higher when fl is used to assay for resistance than when Qfi and f2 are used. On the other hand, bacteria resistant to fl are usually resistant to Qfi and f2, but not vice versa. This may be due to mutations conferring resistance to Q8 and f2, but not fl, located in chromosomal genes far from lac and the F factor, and is probably related to the different participation of bacterial genes in the development of the two types of phage. Comutation studies fashioned in different ways may be used to test for the integration of an episome into a chromosome. Such a test is independent of whether the episome confers conjugational ability, of whether a genetic system has been developed in the bacterial species under study, and of whether the episome carries any indispensable genes (and thus cannot be lost upon incubation with acridine dyes). For example, a mutational change in the episome would be selected, either in the forward or Statistical significance P < 0.001 P < 0.001 NS NS NS VOL. 116, 1973 529 530 LLOVERES AND reverse direction, after treatment with nitrosoguanidine, and the resulting mutants would be assayed for the presence of chromosomal mutations. Auxotrophic mutations are convenient to assay and are usually spread over the chromosome. If the episomal mutation is frequently associated with a particular growth requirement, the implication would be that the episome is close to the genes responsible for that kind of auxotrophy. In fact comutation only means that the two genetic structures replicate simultaneously. Comutation might be found between genes in two independent replicons if there is a precise synchronization in their replication. Autonomous episomes are known to replicate at certain times of the cell cycle (14, 15), but there is no indication that the synchronization is good enough to show appreciable comutation with chromosomal genes. Reversion to wild-type phenotype may sometimes be due more to intergenic suppression than to intragenic reversion and the comutation results would then be misinterpreted. In our experience such high relative incidence of intergenic suppression is uncommon and errors can be prevented by the study of different original mutations of the same type. ACKNOWLEDGMENTS We thank K. Brooks, A. J. Clark, R. Curtiss Ill, and N. D. Zinder for the original strains of bacteria and phages, and J. Yebra for expert technical help. LITERATURE CITED 1. Achtman, M. 1973. Genetics of the F sex factor in .ERDA-OLMEDO J. BACTERIOL. Enterobacteriaceae. Curr. Top. Microbiol. Immunol. 60:79-123. 2. Adelberg, E. A., M. Mandel, and G. C. C. Chen. 1965. Optimal conditions for mutagenesis by N-methyl-N'- nitro-N-nitrosoguanidine in Escherichia coli K12. Biochem. Biophys. Res. Commun. 18:788-795. 3. Berg, C. M., and R. Curtiss. 1967. Transposition derivatives of an Hfr strain of Escherichia coli K-12. Genetics 56:503-525. 4. Cerda-Olmedo, E., and P. C. Hanawalt. 1968. Diazomethane as the active agent in nitrosoguanidine mutagenesis and lethality. Mol. Gen. Genet. 101:191-202. 5. Cerda-Olmedo, E., P. C. Hanawalt, and N. Guerola. 1968. Mutagenesis of the replication point by nitrosoguanidine: Map and pattern of replication of the Escherichia coli chromosome. J. Mol. Biol. 33:705-719. 6. Guerola, N., J. L. Ingraham, and E. Cerda-Olmedo. 1971. Induction of closely linked multiple mutations by nitrosoguanidine. Nature (London) 230:122-125. 7. Hirota, Y. 1960. The effect of acridine dyes on mating type factors in Escherichia coli. Proc. Nat. Acad. Sci. U.S.A. 46:57-64. 8. Jacob, F., and E. L. Wollman. 1957. Analyse des groupes de liaison genetique de differentes souches donatrices d'Escherichia coli K12. C. R. Acad. Sci. 245:1840-1843. 9. Low, B., and T. H. Wood. 1965. A quick and efficient method for interruption of bacterial conjugation. Genet. Res. 6:300-303. 10. Maaloe, O., and P. C. Hanawalt. 1961. Thymine deficiency and the normal DNA replication cycle. J. Mol. Biol. 3:144-155. 11. Mandell, J. D., and J. Greenberg. 1960. A new chemical mutagen for bacteria: 1-methyl-3-nitro-1-nitrosoguanidine. Biochem. Biophys. Res. Commun. 3:575-578. 12. Sokal, R. R., and F. J. Rohlf. 1969. Biometry, p. 607. W. H. Freeman and Co, San Francisco. 13. Takahashi, I., and R. A. Barnard. 1967. Effect of Nmethyl-N'-nitro-N-nitrosoguanidine on the F factor of Escherichia coli. Mutat. Res. 4:111-117. 14. Yabe, Y., and S. Mitsuhashi. 1971. Replication and transfer of the R factor in a synchronized culture of Escherichia coli. Jap. J. Microbiol. 15:21-27. 15. Zeuthen, J., and M. L. Pato. 1971. Replication of the F'lac sex factor in the cell cycle of Escherichia coli. Mol. Gen. Genet. 111:242-255. 10 c