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Mutation Research, 73 (1980) 209--213 © Elsevier/North-Holland Biomedical Press

Short Communicat ion

ANTIMUTATION EFFECT OF AN E. coli MEMBRANE FRACTION ON UV-MUTAGENESIS

DIANE HARPER, STEVE KRISTOFF and RICHARD BOCKRATH

Department of Microbiology and Immunology, and Medical Biophysics Program, Indiana University School of Medicine, Indianapolis, IN 46223 (U.S.A.)

(Received 2 January 1980) (Revision received 29 May 1980) (Accepted 30 May 1980)

Adler, Crow and Gill [1] have recently shown that addition of a particulate fraction of the cytoplasmic membrane of an E. coli B/r strain induced septation in cultures of irradiated Ion cells, when the cells were incubated with the mem- brane fraction anaerobically for at least 4 h after irradiation. The Ion genotype in E. coli will cause inhibition of septum formation upon exposure of the cell to radiation. The inhibition of septum formation leads to filamentous growth which is of ten lethal. Therefore, the induction of septation by the membrane fraction had the effect of increasing the survival of the irradiated cells.

We have studied the depression of mutagenesis that occurs when irradiated E. coli are plated at high densities [2]. The number of mutan t colonies indi- cated increases linearly with increasing plate density to about 10 s bacteria per plate. At higher plate densities, suppressor mutat ions are very sensitive to crowding depression of mutagenesis and backmutations are somewhat sensitive. We suggested that this crowding depression of mutagenesis resulted from an effect on the rec/lex response syste~m, which provides a pleiotropic response to genetic injury including: stimulated synthesis of the recA protein [8], enhanced survival and mutagenesis of infecting, irradiated phage [5], induction of endogenous prophage [6 ], inhibition of respiration [ 10], inhibition of DNA degradation [7], induced mutational repair of damage to cellular DNA [13], and filamentous growth in a lon background [12]. Since filamentous growth and mutational repair may be related through their dependency on the rec/lex gene system, we wondered if the particulate membrane fraction which induced septation in a lon strain would have an effect on mutagenesis.

Materials and methods

We used 2 strains of E. coli B/r: WU3610-11 and WU-11. Both contained an amber defect in the leucine metabolic pathway and an ochre defect in the tyro- sine pathway. They also contained a class 2 amber suppressor (supE) that sup-

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pressed the leucine requirement. In addition, WU-11 was uvrA and therefore lacked excision repair [2]. The particulate membrane fraction was provided by Dr. Howard Adler, Oak Ridge National Laboratory. It was prepared from an E. coli B/r strain disrupted by 2--3 passages through a French press (20 000 psi) and fractionated in 0.16 mM MgC12 as the supernatant of 3000 g for 5 min and the pellet of 226 000 g for 2 h.

Cells were grown in supplemented A-0 minimal salts medium [3] to an op- tical density of 0.20 measured at 450 nm. They were sedimented by centrifuga- tion and resuspended in A-0. They were then exposed to 254 nm light, 10 J /m 2 for the excision-proficient strain and 1.5 J /m 2 for the excision-deficient strain. After irradiation, 0.2-ml samples of the cells were put into prepared tubes held at 50°C that contained top agar and samples of the membrane fraction (extract). Each mixture was then immediately poured onto plates. Both the top agar and the medium in the plates were A-0 hardened with 1.5% agar and sup- plemented with glucose (4 mg/ml), leucine (20/~g/ml), and nutrient broth (200 pg/ml). This medium allowed selection for tyrosine revertants.

As soon as the plates hardened, half were placed in a 37 ° incubator and half were placed in a Gas-Pak which was placed in a 37 ° incubator. The Gas Pak (Baltimore Biological Laboratories) provided an anaerobic environment. The plates remained in anaerobic conditions for 4 h after which they were taken out of the Gas-Pak and placed back in the incubator. Both sets of plates were incubated for a total of 36--48 h after which revertant colonies were counted. Our early trial experiments included using pour plates or the spreading tech- nique on regular agar plates. We found the top agar method to be the most effective and it was used for the data reported here.

In all cases the number of spontaneous revertant colonies found on a plate was less than 10 and the data presented was not corrected for spontaneous mu- tants.

Results and discussion

We found that the yield of revertants to tyrosine p ro to t rophy was reduced when cells were plated with extract and kept in the anarobic environment (Ta- ble 1). No such depression of mutagenesis occurred when cells were plated with extract and kept in an aerobic environment. When cells were plated wi thout extract and placed in an aerobic environment there was a slight effect on mu- tagenesis. The data show an approximately 2-fold reduction in the number of

T A B L E 1

A N T I M U T A T I O N E F F E C T O F T H E P A R T I C U L A T E M E M B R A N E F R A C T I O N

M u t a n t y i e l d per pla te a

A e r o b i c A n a e r o b i c

W i t h o u t e x t r a c t 4 9 0 409 W i t h e x t r a c t 567 241

a N u m b e r s are ave rages o f 3 E x p t s .

T A B L E 2

D I F F E R E N T I A L A N T I M U T A T I O N E F F E C T

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M u t a n t y ie ld per p late a

C o n v e r t e d B a c k m u t a n t s suppressors

de n o v o suppressors

A. E x c i s i o n - p r o f i c i e n t ce l l s w i t h o u t e x t r a c t 324 63 18 w i t h ex trac t 188 60 21

B. E x c i s i o n - d e f i c i e n t cel ls w i t h o u t e x t r a c t 117 194 162 w i t h ex t rac t 59 126 83

a N u m b e r s are averages o f 3 Expts . using anaerobic incubat ion .

tyrosine revertants when cells were plated with the membrane fraction and incubated anaerobically.

By using ochre and amber defective T4 phage we were able to differentiate between backmutations, de novo ochre suppressor mutations, and ochre sup- pressor mutations that were converted from the original amber suppressor mu- tations [3] . These were determined by streaking 3 lanes of phage across a plate containing nutrient agar. Each lane contained a T4 phage with a different defect. The phage in lane 1 had a defect corrected by a class 1 or 2 amber sup- pressor. The phage in lane 2 contained a defect corrected by either a class 1 or 3, amber or ochre suppressor. The phage in lane 3 had a defect corrected by a class 1 or 2 ochre suppressor. Revertant colonies were picked from the assay plates, isolated and streaked across the phage lanes. Lysis resulted only when the bacteria contained the proper suppressor tRNA mutat ion to correct the defect in the phage gene. We were able to tell if the tyrosine revertants con- tained a backmutation, a class 2 de novo suppressor mutation, or a class 2 con- vetted suppressor mutat ion.

In excision-proficient cells a large effect by the membrane fraction was seen in converted suppressors, while little or no effect was seen in the backmutants or de novo suppressors (Table 2A). In the excision-repair-deficient strain we saw an effect on all 3 types of revertants. There was a 2-fold decrease in the number of suppressor mutants and a 1.5-fold reduction in the number of back-

T A B L E 3

E F F E C T OF M E M B R A N E P A R T I C U L A T E F R A C T I O N ON S U R V I V A L OF E X C I S I O N D E F I C I E N T C E L L S

Viable bac ter ia per plate a

A e r o b i c A n a e r o b i c

W i t h o u t e x t r a c t 136 1 4 0 With e x t r a c t 161 155

a N o r m a l dens i ty e u l t u ~ o f irradiated WU-11 is d i lu ted by a fac tor o f 1 0 6, p la ted w i t h or w i t h o u t the m e m b r a n e frac t ion , and i n c u b a t e d aerobica l ly or anaerobica l ly . N u m b e r s are averages o f c o l o n i e s per plate f r o m dupl ica te plat ings.

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mutants when these cells were incubated anaerobically with the membrane fraction after irradiation (Table 2B).

One way to explain the similar loss of all 3 types of mutants in the excision- deficient cells is that the membrane fraction in anaerobic conditions enhanced killing. Our data show that the extract in no way reduced survival of excision- deficient cells in either aerobic or anaerobic conditions leading us to conclude that the membrane fraction effected only the mechanism of mutat ion (Ta- ble 3).

The data presented for excision-proficient bacteria clearly show an antimuta- tion effect by the membrane fraction on converted suppressor mutat ion and apparently no effect on backmutat ion or de novo suppressor mutat ion. The lack of a noticeable effect on de novo suppressor mutat ion may be due to the fact that very low numbers of this type of mutant were obtained in the top- agar assay system used here. When the same bacterial strain was similarly treated and assayed in the convential manner by spreading, a large portion of the revertant population consisted of de novo suppressor mutat ions [3]. The very low numbers found in this study may be attributable to a mutat ion fre- quency decline-like mechanism operating because of transient temperature ele- vation [11]. Mutation-frequency decline strongly decreases the number of de novo suppressors and has little effect on backmutants or converted suppressors [3].

We studied mutagenesis of bacteria deficient in excision repair to avoid mu- tation-frequency decline and therefore to obtain more de novo suppressors. In this background more de novo suppressors were seen and the membrane frac- tion effected de novo suppressors, converted suppressors, and backmutants. The relative numbers of the 3 types of revertants were similar and the effect by the membrane fraction on each type was similar.

These results suggest that the membrane fraction preferentially causes a dis- ruption of the post-replication mutat ion process(es) distinguished by Nishioka and Doudney [9], through which presumably only suppressor mutations are developed in excision-proficient cells and all types of mutations are developed in excision<leficient cells. This route for mutagenesis seems to require induced levels of mutational repair [4] and therefore the results seen here may occur because the extract plus anaerobic incubation inhibits post-UV protein syn- thesis. We have no data regarding protein synthesis in cells during the assay with extract.

Acknowledgements

We thank Dr. Adler for sending us samples of the membrane fraction and for his comments and suggestions. This research was supported by Public Health Service Grant GM21788 from the National Institutes of Health.

References

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10 Swenson, P.A., and R.L. Schenley, Respirat ion, growth, and viability of repair-deficient mutants of Eseher ich ia col i after ul traviolet i rradiat ion, Int. J. Radiat . Biol., 25 (1974) 51--60.

11 Witkin, E.M., Time, temperature and protein synthesis: A s tudy of ultraviolet- induced muta t ion in bacteria, Cold Spr. Harb. Symp. Quant. Biol., 21 (1956) 123--240.

12 Witkin, E.M., The radiat ion sensit ivity o f E s c h e r i e h i a col i B: A hypothesis relat ing f i lament format ion and prophage induct ion, Proc. Natl. Acad. Sci. (U.S.A.), 57 (1987) 1275--1279.

13 Witkin, E.M., Ultraviolet mutagenesis and inducible DNA repair in Escher ich ia eol i , Bacteriol. Rev., 40 (1976) 87--103.