Mol Gen Genet (1983) 192:282-284

© Springer-Verlag 1983

Short Communication

Evidence that the phr + Gene Enhances the Ultraviolet Resistance of Escherichia coli recA Strains in the Dark

Kazuo Yamamoto 1, Yoshisada Fujiwara 1, and Hideo Shinagawa 2 1 Department of Radiation Biophysics, Kobe University School of Medicine, Kobe 650, Japan 2 Department of Experimental Chemotherapy, Research Institute for Microbial Diseases, Osaka University, Suita 565, Japan

Summary. An Escherichia coli recA phr + purA strain was more resistant to ultraviolet radiation than its isogenic de- rivative recA phr + purA + in the absence of photoreactivat- ing light, whereas their nearly isogenic derivative recA phr showed most UV-induced lethality. The amounts of photo- reactivating enzyme (PRE) per cell in the recA phr ÷ purA was higher than in the recA phr + purA +. The recA phr is defective for photoreactivation. Thus, in the recA strain, UV resistance in the dark increased in proportion to the amounts of PRE per cell, suggesting that PRE participates in the process of dark repair of UV-damaged DNA.

Previously we have found that an Escherichia coli recA strain carrying many copies of the phr + gene on multicopy plasmids is more resistant in the dark to ultraviolet (UV) radiation than that without the phr+-plasmid (Yamamoto et al. 1983). From these results, it is reasonable to assume that a recA strain with the phr + gene is more resistant in the dark to UV radiation than that without the phr + gene. The present experiments were designed to test this assumption. In order to compare the dark repair capacity among isogenic recA strains with different amounts of pho- toreactivating enzyme (PRE) activity, a purA mutation was introduced in our strains, since it was reported (Nishioka and Harm 1972) that purA mutations enhance the activity of PRE. E. eoli K12 ES4 (phr + gal purA), obtained from Dr. B.J. Bachmann, was used as a recipient strain to con- struct desired strains by transduction with P1 lysates pre- pared from E. eoIi B Hs30 (phr gal + uvrB purA +) (Kondo et al. 1970). The KY12 (phr + gal purA +) strain was ob- tained as a purA + transductant, and KYI3 (phr gal + purA) strain as a gal + phr transductant. The phr locus is closely linked to the gal locus and they are co-transducible with PI at an average frequency of 30% (Youngs and Smith 1978). Next we attempted to introduce a recA mutation into above-mentioned strain from strain JC10240 (Hfr recA56 srlR::TnlO) (Csonka and Clark 1980) by transduc- tion with P1 but failed to obtain transductants due to low titer PI lysates. To construct a recA56 srlR::TnlO strain which gives high titer P1 lysates, JC10240 was mated with

Offprint requests to: Kazuo Yamamoto

ABl157 (Howard-Flanders et al. 1966) and the exconju- gants were selected for the characteristics of streptomycin resistance, tetracycline resistance (TC), and UV sensitivity. One of the exconjugants was named KY9 (recA56 srlR: :Tnl0) and used as a donor for the reeA56 transduc- tion with PI. ES4 (phr+purA), KY12 (phr + purA +) and KY13 (phr purA) were then infected with P1 lysates ob- tained from KY9 and selected for Tc r colonies. The trans- ductants were further checked for UV sensitivity and one of the TC transductants which were UV sensitive was iso- lated as a recA56 transductant for each strain. The strains thus constructed were designated as KY115 (recA56 purA), KY125 (recA56) and KY135 (reeA56phrpurA). The proce- dure described above will guarantee that they are nearly isogenic.

The UV sensitivities of the strains were compared. As shown in Fig. la, KYI35 was the most sensitive to UV radiation, KY125 moderately sensitive and KY115 the least sensitive. On the other hand, three recA + strain ES4, KY12 and KY13 which correspond to KYI15, KY125 and KY135, respectively, were equally resistant to UV (data not shown).

The amounts of PRE per cell were estimated from kinet- ics of survival increment after flash-photoreactivation, "dose decrement" method (Harm 1969). In these recA56 strains, however, we encountered difficulties to obtain con- stant "dose decrement". The idea of "dose decrement" is based on the fact that PRE does work only in the presence of PR light. However, in a recA strain, PRE has repair effect in the absence of PR light (Yamamoto et al. 1983). "Dose decrement" is affected by flash light in one hand, and by dark repair of PRE during incubation in the plates. This may be the reason why, in the recA strains, we were unable to obtain constant "dose decrement". We, there- fore, used isogenic uvrBpurA and its uvrBpurA + derivatives of ES4, since PRE has been found to have no dark repair effects in a uvrB strain (Yamamoto et al. 1983). KY14 (purA uvrB phr +) was constructed by introducing gal + uvrB from Hs30 by PI transduction into ES4. We next introduced purA + by P1 transduction to KY14, named KY16 (purA + uvrB phr +). "Dose decrement" of KY14 was 1.4 J/m 2, and KY16 0.4 J/m 2. Since a UV dose of 1 J/m z produces 65 dimers per E. coli chromosome, we calculated the amounts of PRE per cell as shown in Table 1. These values, shown

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Fig. l a, b. UV sensitivity of isogenic recA56 strains with different amounts of PRE per cell. The bacteria were grown to stationary phase in Luria broth, washed in 0.067 M phosphate buffer by centrifugation, resuspended to 1 x 10 7 cells/ml, irradiated with 254 nm UV at a dose rate of 0.1 J/me/s, and serially diluted in phosphate buffer, a E. coli K12 strains. [] KYl15 (recA56 purA), Lx KY125 (recA56), o KY135 (recA56 phr purA). The bacteria were spread on four Luria plates per one point and incubated overnight at 37 ° C. Each point represents an average of 3-4 independent experiments with standard error shown, b E. coli B strains, z~ H/r30Rrec (recA56), o H/r30rec (recA56 phr). Experiments were carried out three times and were highly reproducible. The results represent the mean value of two plates of one representative experiment done in parallel. All the experiments a and b were done under yellow light

Table 1. The amounts of PRE per cell

Strains a Relevant genotype Amounts of PRE per cell b

KY 14 uvrB purA 90 KY16 uvrB purA + 26

" Bacterial strains have been described in the text. KY115 (recA56 purA) in Fig. 1 a is isogenic to KY14, and KY125 (recA56purA +) to KY16. The mutant genotype of parental strain ES4 is purA, lac, gal, xyl, mtl, tonA, tsx, supE44

b The amounts of PRE per cell were calculated according to the method of Harm (1969). The principle of which has been de- scribed by Sancar and Rupert (1978). We used Hakuba Otto DC-20N electric flash unit as a PR light in the form of single flash

in Table 1, are almost equal to those reported by Harm (1969). Now KY14 is isogenic to KY115, and K Y I 6 to KY125. This may indicate that KYl15 possesses higher amounts of PRE than KY125. Although the strains used for measuring UV sensitivity and for calculating "dose de- crement" are different, there was a good correlation be- tween the UV sensitivity and the amounts of PRE per cell among these three strains; the least sensitive strain (KY115) possesses higher amounts of PRE per cell than moderately sensitive strain (KY125), and the most sensitive strain (KY135) is defective for PR.

We also constructed an isogenic recA56 srIR:: Tnl 0 pair from a previously established E. coli B strain H/r30 (phr) and its phr + derivatives H/r30R (phr +) (Kondo et al. 1970) and they were designated as H/r30rec (recA56 phr) and H/ r30Rrec (recA56 phr+). The UV sensitivity of these strains

is shown in Fig. 1 b. Again the phr + strain was more resis- tant than the phr strain in the recA background, although H/r30 and H/r30R are known to be equally resistant to UV in the dark.

It is known that PRE has PR capacity (Rupert 1975). Furthermore, combining the present studies with our pre- vious studies (indicating phr-plasmid mediated rescue of UV sensitivity in recA strains (Yamamoto et al. 1983)), we may conclude that PRE partly facilitates dark repair in the recA strain.

Acknowledgements. The authors express their gratitude to Dr. B.J. Bachmann, AJ. Clark and S. Kondo for strains, to Dr. S. Kondo for comments and suggestions during the preparation of the manu- script, and to Dr. A. Nakata and P.P. Mehta for suggestions. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.

References

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Kondo S, Ichikawa H, Iwo K, Kato T (1970) Base-change muta- genesis and prophage induction in strains of Escherichia eoli with different DNA repair capacities. Genetics 66:187-217

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Rupert CS (1975) Enzymatic photoreactivation: Overview. In: Hanawalt PC, Setlow RB (eds) Molecular mechanisms for re- pair of DNA, part A. Plenum Press, New York, pp 73-87

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Communicated by M. Takanami

Received June 14, t983