A simple method for constructing recA strains in Escherichia coli

We isolated mutations in cys genes located in close vicinity of recA gene in Escherichia coli, by insertion of Tnl, Tn5 or TnlO. We deviced a simple method constructing recA strains using these mutations. The cys mutations were transduced by Pike into an appropriate recipient selecting for drug resistance of the mutations. The resultant Cys transductants were crossed with an Hfr RecA~ strain, KL16-99, and Cyst recombinants were picked up. Among them, small colony f ormers were Recstrains. In Escherichia coli genetics, there are many instances in which construction of recA strains is desired. One conventional technique to attain this is isolation of a Thyauxotrophy by selection with trimethoprim (Stacey and Simson 1965) followed by isolation of Thy+ ReCArecombinants of the strain by a cross with an Hfr recA strain such as KL16-99 (Bachmann 1972). Recently, an Hfr strain convenient for transferring a recA mutation to a recipient had been constructed. The strain carries two linked mutations, recA and srl :: Tn10. Using the strain as a donor, the recA mutation could be transferred either by conjugation or P1-mediated transduction to recipients selecting resistance to tetracycline (Tc) (Csonka and Clark 1980). However, the method is not applicable in recipients already resistant to Tc, or in those which can not express TnlO encoded Tc resistance. It was reported that the level of Tc resistance was very low in strains carrying an amber suppressor (Zupancic et al. 1980) . And transduction by Pike of TnlO insertions into such strains conferring resistance to low concentration of Tc was very tricky (Harayama, unpublished). Therefore, we intended to devise another method to construct recA strains in E. coli. We isolated mutations in the cyst-C cluster located at 59 min on recent EK coli linkage map (Bachmann and Low 1980), by insertion mutagenesis of either Tnl, Tn5 or TnlO. Insertion mutagenesis of Tn5 using Abb kan-1 (Berg et al. 1975), of TnlO using A55 (Kleckner et al. 1978), or of Tnl using a temperature-sensitive mutant of RP4 (Harayama et al. 1980), had been de190 S. HARAYAMA and T. IINo scribed (Harayama et al. 1979, 1980, 1981). Out of each 2000 to 3000 colonies mutagenised either by one of these transposons, we found 4, 4 and 1 mutants defective in cyst-C cluster by Tnl, Tn5 and TnlO insertions, respectively. We used three strains, TH1099 (cys :: Tnl), TH579 (cys :: Tn5) and TH517 (cys :: T nlO), in the following experiments. Pike lysates of these three strains were prepared on L broth (LB) (Miller 1972) plates containing 10-2M CaC12 and 10-3M L-cysteine and drug resistances of of the strains (30 ;ug/ml of ampicillin (Ap) in the case of Tn1, 40 ig/ml of kanamycin (Km) in the case of Tn5 and 30 ig/ml of Tc in the case of TnlO) were transduced into EJ502 (Fhis rpsL). All Ap resistant transductants (12/12), about the half of Km resistant transductants (79/150) and all Tc resistant transductants (40/40) by Pike propagated from T111099, TH517 and TH579, respectively, were Cys-. Translocation of transposon from the initial insertion site to elsewhere, through phage-mediated transduction, had been observed previously (Kleckner et al. 1978; Harayama et a1.1979; Biek and Roth 1980) . These Cystransductants were mated with an Hf r recA strain, KL1699, in LB at 37°C for 30 minutes at a ratio of 1 donor to 5 recipients. Then, each mating mixture was washed two times with 0.8% NaCI and cells were spread, after serial dilutions, on glucose minimal (M9) plates (Miller 1972) containing 10-3M L-histidine and 100 ig/ml streptomycin, and the plates were incubated for 3 days at 37°C. About one third of colonies developed on the plates were small in size. They were picked up and streaked on the minimal plates for single colony isolation. Each 10 clones obtained by cross of EJ502 cys :: Tnl X KL16-99, EJ502 cys :: Tn5 X KL16-99 and EJ502 cys :: T nl0 X KL16-99, were examined for their histidine requirement and for their UV sensitivity (recA strain is hypersensitive to UV). All the clones were UV' and His-. In a method described by Csonka and Clark (1980), a recA mutation could be transferred with srl :: T n l 0 either by Hf r cross or by P1-mediated transduction. Since the mutations in the cyst-C cluster are located more than 1 min apart from recA (Bachman and Low 1980), P1-mediated transduction of recA with cysJ-C may be more difficult than the co-transduction of recA with srl :: T n l 0. In summary, using transposon insertion mutations in the cysJ-C cluster, recA strains could be constructed by two steps: 1) transfer of a cys mutation by Pike to an appropriate recipient selecting for drug resistance; 2) isolation of a small Cyst colony arisen by conjugation of the Cysrecipient with an Hfr strain, KL16-99. Since three drug resistance, namely, Ap, Km and Tc, are available in the present method, this can be used in a variety of circumstances. The work was supported by Grants in Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. Construction of recA strains in E. coli 191