Increased sensitivity of Escherichia coli K12 to certain mutagens as a consequence of a mutation leading to phage U3 resistance

Mutation Research, 104 (1982)55-60

55

Elsevier Biomedical Press

Increased sensitivity of Escherichia coli K12 to certain mutagens as a consequence of a mutation leading to phage U3 resistance J a r g e n Ellenberger* Zentrallaboratorium far Mutagenit#tsprafung der Deutschen Forschungsgemeinschaft, Freiburg i. Br. (Federal Republic of Germany)

(Accepted 26 November 1981)

In the development of bacterial indicators for assessing potential mutagenicity of chemicals, emphasis has been placed, above all, on an enhancement of mutagen sensitivity of the tester strains next to the search for suitable genetic end-points. Such enhancement has been achieved by the introduction of mutations or genetic factors that alter the DNA-repair capacity of the cell (e.g. Ames et al., 1973; Green and Muriel, 1976; MacPhee, 1973; McCann et al., 1975; Mohn et al., 1980), and by decreasing the permeability barrier of the cell wall. The latter increases penetration into the bacterial cell of substances of large molecular size. Ethylenediaminetetraacetate-treated E. coli show an increase in general permeability (Leive, 1968). After introduction of specific mutations, e.g. envA and rfa, strains of S. typhimurium and E. coli become more sensitive to some chemical agents than the corresponding wild-types (Aline and Reznikoff, 1975; Ames et al., 1973; Moreau et al., 1976; Normark et al., 1969). Both in E. coli and in S. typhimurium a correlation between bacteriophage resistance and alterations in bacterial cell wall composition has been described. (For literature see Hancock and Reeves, 1976.) Watson and Paigen (1971) isolated an E. coli bacteriophage that can only adsorb if galactose is present in the cell wall. This phage U3 specifically lyses cells with the complete K12 core (Schmidt, 1973). Mutants resistant to phage U3 have a defective cell wall and should show increased permeability to large chemicals. In the course of the present study the question of whether or not U3 resistance would enhance the sensitivity of tester strain 343/113 of E. coli K I2, previously used for detecting chemical mutagens (see Mohn and Ellenberger, 1977, 1980), was examined. To isolate U3-resistant (U3 r) mutants, 0.1-ml aliquots of a culture of

*Present address: Department of Experimental Pathology and Toxicology, Boehringer Ingelheim KG, 6507 lngelheim (Federal Republic of Germany). 0165 7992/82/0000-0000/$02.75 © Elsevier BiomedicalPress

56

strain 343/113 growing logarithmically were added to 2.5-ml aliquots of nutrient broth containing soft agar, and the mixture was poured over nutrient-broth agar plates. Aliquots of a U3 suspension (about 1 x 10 l° plaque-forming particles per ml; originally obtained from H. Saedler) were dropped onto the plates, and these were then incubated for 24 h at 37 °C. Colonies grown within the plaques were isolated and restreaked on nutrient-broth agar to produce single colonies. Purification of the isolated clones was performed by repeated restreaking of individual colonies. For the assay of U3 resistance, cultures growing logarithmically were cross-streaked on nutrient-broth agar against a U3 suspension. The absence of lysis at the crossing indicated resistance. A total of 201 U3 r derivatives of strain 343/113, arising spontaneously, were isolated in this manner. 9 of these derivatives showed greater sensitivity to different substances than strain 343/113, and 1 strain (referred to as 343/113, U3 r in this study) was chosen for further trials. The data presented in Table 1 demonstrate the greatly enhanced sensitivity of this strain towards crystal violet, sodium deoxycholate and rifampicin. No enhanced sensitivity for a number of alkylating agents (e.g. nitrosoguanidine) was apparent on introduction of the U3 r mutation. Neither the toxic nor the mutagenic efficiency was increased (data not shown). In contrast to the parent strain, the new tester strain was mutagenized by lower concentrations of some heterocyclic aromatic compounds. Fig. 1 demonstrates this by the entozon example. For a further comparison of the strain sensitivities, investigations using treat-andplate tests were performed. As part of a study of the photomutagenic activity of heterocyclic aromatic compounds (to be published), neutral red was chosen, here, as model substance. Additionally, the uvrB derivative of strain 343/113 (isolated by B.W. Glickman and G.R. Mohn after co-transduction with bio- and selection via chlorate resistance) was included in the trial. In combination with white light, neutral red was mutagenic in all 3 E. coli strains (Fig. 2). Both with strain 343/113

TABLE 1 SENSITIVITIES OF T H E E. coli STRAINS 343/113 A N D 343/113,U3 r FOR C R Y S T A L VIOLET, SODIUM D E O X Y C H O L A T E A N D R I F A M P I C I N Substance

Crystal violet (0.5 m g / m l ) Sodium deoxycholate (200 m g / m l ) Rifampicin (0.2 m g / m l )

Diameter of zone of growth inhibition (mm) 343/113

343/113,U3 r

0 0 0

21 14 10

A bacterial culture (0.1 ml containing about 2 × 10 s washed cells per ml) was poured into 3 ml of complete soft agar on the surface of complete growth medium contained in petri dishes. Subsequently, 10 ul of an aqueous solution of the given chemical were added in the middle of the plates. After the plates had been incubated for 20 h at 37 °C in the dark, the zone of growth inhibition was measured on the spot of application of the chemical.

57

Fig. 1. Comparison of the mutagenic effect of entozon, 9-(3-diethylamino-2-hydroxypropylamino)-6,7dimethoxy-3-nitroacridine dihydrochloride, in the nad113 system of E. coli 343/113 (A) and 343/113,U3 r (B). A bacterial culture (0.1 ml, containing about 2 × 108 washed cells per ml) was spread over the surface of selective medium. Subsequently, 50 #1 of an aqueous solution of the substance under test (0.5 mg/ml) were added in the middle of the plates. After 2 days of incubation at 37 °C in the dark, strain 343/113 showed mutation induction on the spot of mutagen. Strain 343/113,U3 r responded more sensitively: the high concentration of the mutagen at the point ef application inhibited the bacteria, and a zone of mutant colonies appeared at a range of lower concentration around the spot.

a n d with its u v r B derivative the i n d u c t i o n o f m u t a n t s reached s a t u r a t i o n at concentrations a b o v e a b o u t 40 #M. A d o u b l i n g o f the exposure c o n c e n t r a t i o n from 50 to 100 ~M, with a c o n s t a n t m o d e r a t e l y toxic effect of the agent, did n o t increase the yield o f m u t a n t s . C o m p a r e d with this, a c o n t i n u o u s d o s e - e f f e c t relationship in the c o n c e n t r a t i o n range up to 70 #M resulted for strain 343/113,U3 r. P r o n o u n c e d cell growth i n h i b i t i o n on the selective m e d i u m at higher c o n c e n t r a t i o n s did not allow accurate c o l o n y c o u n t i n g at 100 #M. Even with the lower c o n c e n t r a t i o n s the n u m b e r of revertants lay a b o v e the values f o u n d for the 2 other strains*. These results with n e u t r a l red permit the following i n t e r p r e t a t i o n . During t r e a t m e n t only a certain (limited) q u a n t i t y o f the test substance could penetrate into those bacterial cells with a n intact, i.e. n o r m a l l y permeable, celt wall a n d display there its m u t a g e n i c effect. Owing to the lack of excision repair, more p r e m u t a t i o n s m a n i f e s t e d themselves in strain 343/113, u r v B t h a n in the repair-proficient strain 343/113. As the cell wall had become more permeable in c o n n e c t i o n with U3

* The U3r property did not increase the photosensitivity of the tester strain. In the dark no induction of arg + mutations by neutral red were observed (data not shown).

58

100L o o

o 3431113

• -

-

3431113,uvrB

•

\° 50 >

~

o

~

=

3431113,U3r

ffl

i

0

i

50

500-

100

D yo

3431113'u3r

/

O

400-

"E 300

i

3

3431113.uvrB

"

•

o

o343/113

E

~

2OO

='/ ° •~/ ~ ° '

~oo 7 i

i

100

50 Conc. {p,MI

Fig. 2. Survival and induction of a r g + mutations in strain E . c o l i 343/113 and in its u v r B and U3 r derivatives after treatment with neutral red. a, Survival of colony-forming ability, b, Mutation induction. Growing cells were treated for 120 min at 37 °C under irradiation with white light. As source of light two Osram-L 65W/25 fluorescent lamps (effective intensity of light, 4000 lux) were used at a distance of 30 cm. Ventilation protected the incubation mixtures from additional heat caused by the intense illumination. For a detailed description of the testing procedure see Mohn and EUenberger (1977). The points represent the mean values of 2 separate Expts.

r e s i s t a n c e , m o r e m u t a g e n i c p r i n c i p l e s p e n e t r a t e d i n t o t h e U 3 r t e s t cells a n d r e a c h e d the

target

material.

This

explanation

molecules of alkylating mutagens, same

arg

÷

is s u p p o r t e d

by the

as a l r e a d y m e n t i o n e d

finding

that

small

above, examined in the

t e s t s y s t e m , w e r e a s e f f e c t i v e i n s t r a i n 3 4 3 / 1 1 3 , U 3 r as i n s t r a i n 3 4 3 / 1 1 3 .

59 TABLE 2 MUTAGENICITY OF 0.5 mM 8-METHOXYPSORALEN IN THE DARK Each value represents the mean from 3 plates in 1 Expt. Strain

Survivors (07o)

Number of induced arg + revertants per plate

343/113 343/113,U3 r

69 42 54 64

122 174 771 143

343/l13,uvrB

343/113(pKMI01)

Growing cells were treated for 120 min at 37 °C in the dark in a rotary shaker as described previously by Mohn and Ellenberger (1977). To avoid photodynamic effects, preparation of incubation mixtures, dilution of treated bacteria, and plating on the media were carried out under yellow light. The following spontaneous mutation frequencies per plate were observed: 343/113, 47; 343/113,U3 r, 56; 343/l13,uvrB, 157; 343/lI3(pKM101), 61. Strain 343/I13(pKMI01) is a resistance-plasmid-bearing derivative (Mohn et al., 1980).

This is so b e c a u s e it c a n n o t be expected that a n o r m a l cell wall represents a n o t i c e a b l e o b s t a c l e for such substances with a relatively small m o l e c u f a r configuration. T h e finding that 8 - m e t h o x y p s o r a l e n was not n o t i c e a b l y m o r e effective in strain 3 4 3 / 1 1 3 , U 3 r t h a n in strain 343/113 ( T a b l e 2) m a y be f u r t h e r p r o o f that no p r o n o u n c e d p e n e t r a t i o n p r o b l e m s exist for this s u b s t a n c e a n d the m u t a g e n i c i t y f o u n d is p r i m a r i l y d e p e n d e n t on each c h o s e n test system (cf. C l a r k e a n d W a d e , 1975; Bridges a n d M o t t e r s h e a d , 1977). M o r e o v e r , as d e m o n s t r a t e d with the u v r B derivative in the p r e s e n t e x a m p l e , the state o f D N A - r e p a i r synthesis influences the m u t a g e n i c activity o f 8 - m e t h o x y p s o r a l e n . T h e s p o n t a n e o u s reversion frequencies d e t e r m i n e d for the various m u t a t i o n systems o f strain 343/113 (cf. M o h n a n d Ellenberger, 1977) are not c h a n g e d by the U3 r p r o p e r t y . So f a r as o b s e r v e d u p to now, the U3 r p r o p e r t y does n o t influence the m u t a g e n specificity o f the i n d i v i d u a l test systems. The g a l - p h e n o t y p e a n d the reversion to gal ÷ is n o r m a l in the U3-reststant strain. This indicate6 that the m u t a t i o n leading to resistance did not occur in the galE gene. W i t h E. coli 343/113,U3 r a tester strain is a v a i l a b l e that, p r o b a b l y owing to a defective a n d t h e r e f o r e p e r m e a b l e cell wall, m a y be used successfully for verifying an otherwise w e a k or u n c e r t a i n m u t a g e n i c i t y o f substances o f large m o l e c u l a r size.

References Aline Jr., R.F., and W.S. Reznikoff (1975) Bacteriophage Mu-l-induced permeability mutants in Escherichia coil K-12, J. Bacteriol., 124, 578-581.

60 Ames, B.N., F.D. Eee and W.E. Durston (1973) An improved bacterial test system for the detection and classification of mutagens and carcinogens, Proc. Natl. Acad. Sci. (U.S.A.), 70, 782-786. Bridges, B.A., and R.P. Mottershead (1977) Frameshift mutagenesis in bacteria by 8-methoxypsoralen (rnethoxalen) in the dark, Mutation Res., 44, 305-312. Clarke, C.H., and M.J. Wade (1975) Evidence that caffeine, 8-methoxypsoralen and steroidal diamines are frameshift mutagens for E. coli K-12, Mutation Res., 28, 123-125. Green, M.H.E., and W.J. Murie[ (1976) Mutagen testing using Trp + reversion in Escherichia coli, Mutation Res., 38, 3-32. Hancock, R.E.W., and P. Reeves (1976) Eipopolysaccharide-deficient, bacteriophage-resistant mutants of Escherichia coli KI2, J. Bacteriol., 127, 98-108. Leive, L. (1968) Studies on the permeability change produced in coliform bacteria by EDTA, J. Biol. Chem., 243, 2373-2380. MacPhee, D.G. (1973) Salmonella typhimurium hisG46 (R-Utrecht): Possible use in screening mutagens and carcinogens, Appl. Microbiol., 26, 1004-1005. McCann, J., N.E. Spingarn, J. Kobori and B.N. Ames (1975) Detection of carcinogens as mutagens: Bacterial tester strains with R factor plasmids, Proc. Natl. Acad. Sci. (U.S.A.), 72, 979-983. Mohn, G.R., and J. Ellenberger (1977) The use of Escherichia eoli K12/343/113(k) as a multi-purpose indicator strain in various mutagenicity testing procedures, in: B.J. Kilbey et al. (Eds.), Handbook of Mutagenicity Test Procedures, Elsevier, Amsterdam, pp. 95-118. Mohn, G.R., and J. Ellenberger (1980) Appreciation of the value of different bacterial test systems for detecting and for ranking chemical mutagens, Arch. Toxicol., 46, 45-60. Mohn, G.R., N. Guijt and B.W. Glickman (1980) Influence of DNA adenine methylation dam mutation and of plasmid pKM 101 on the spontaneous and induced mutability of certain genes in Escherichia coli K12, Mutation Res., 74, 255-265. Moreau, P., A. Bailone and R. Devoret (1976) Prophage k induction in Escherichia coli KI2 envA uvrB: A highly sensitive test for potential carcinogens, Proc. Natl. Acad. Sci. (U.S.A.), 73, 3700-3704. Normark, S., H.G. Boman and E. Matsson (1969) Mutant of Escherichia coli with anomalous cell division and ability to decrease episomally and chromosomally mediated resistance to ampicillin and several other antibiotics, J. Bacteriol., 97, 1334-1342. Schmidt, G. (1973) Genetical studies on the lipopolysaccharide structure of Escherichia coli K12, J. Gen. Microbiol., 77, 151-160. Watson, G., and K. Paigen (1971) isolation and characterization of an Escherichia coli bacteriophage requiring cell wall galactose, J. Virol., 8, 669-674.