Isolation and characterization of an isogenic set of Salmonella typhimurium strains analogous to the...

Mutation Research, 224 (1989) 453-464 453 Elsevier

MUTGEN 01487

Isolation and characterization of an isogenic set of Salmonella typhimurium strains analogous to the "Ames" tester strains

D a v i d J. P o p k i n , V a l e r i e M. D a v i s a n d M i c h a e l J. P r i v a l

Genetic Toxiocology Branch, Food and Drug Administration, Washington, DC 20204 (U.S.A.)

(Received 21 November 1988) (Revision received 15 May 1989)

(Accepted 6 June 1989)

Keywords: Isogenic Salmonella typhimurium tester strains; Ames test

Summary

The standard Ames tester strains of Salmonella typhimurium contain a number of genetic differences at loci other than his. The fact that these strains contain independently isolated uvrB-bio-gal deletions and rfa mutations implies that these are likely to vary from strain to strain. Since the strains were isolated from different parental stocks of S. typhimurium LT-2, they differ in their ability to metabolize arabinose. Other, unknown differences may exist because the isolation of some of the strains involved ultraviolet and chemical mutagenesis. We have isolated a set of isogenic S. typhimurium strains that contain the relevant genetic markers of the standard Ames tester strains. These strains all contain the same uvrB-bio-gal deletion and the same rfa mutation; they differ only in the nature of their his mutations and in the presence or absence of the plasmid pKM101. We have assessed the responsiveness of these strains to a number of mutagens and conclude that their mutagenic specificities are the same as those of the corresponding Ames strains: TA98, TA100, TA1535, TA1537 and TA1538. Therefore, the specificity of the standard Ames strains with respect to these mutagens is a result solely of the differences in the nature of their his mutations and the effects of pKM101.

The most widely used mutagenicity test is the assay using the histidine-requiring strains of Salmonella typhimurium developed in the labora- tory of Dr. Bruce N. Ames (Ames et al., 1975; Maron and Ames, 1983). The array of bacterial strains responding to a particular mutagen can indicate whether the mutagen causes frameshift or base-pair substitution mutations. Thus, one of the

Correspondence: Dr. Michael J. Prival, Genetic Toxicology Branch, HFF-166, Food and Drug Administration, 200 C Street S.W., Washington, DC 20204 (U.S.A.).

advantages of this method is that it provides infor- mation about the mechanism of mutagenicity.

Many investigators who work with the Ames S. typhimurium tester strains have assumed that they differ only in the nature of o their his mutations, in the presence or absence of the pKM101 plasmid, or in other defined ways specified in their known genotypes (Ames et al., 1975). If this were the case, the various tester strains could be referred to as "isogenic". However, because of the manner in which the tester strains were derived, they are not isogenic.

There are several reasons for the differences between the standard Ames tester strains. First,

0165-1218/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

454

they were isolated from different stocks of S. typhimurium LT-2. Busch et al. (1986) reported that the base-pair substitution strains TA1535 and TA100 are capable of utilizing arabinose as the sole source of carbon and energy, whereas the standard frameshift tester strains, TA1537, TA1538 and TA98, cannot grow on this sugar. Hartman (1987) explained this difference by pointing out that the hisC3076 and hisD3052 frameshift mutations were isolated in an S. typhimurium strain carrying the stable ara-9 mu- tation (Oeschger and Hartman, 1970), whereas the base-pair substitution mutation hisG46 was iso- lated in an ara ÷ strain (Hartman et al., 1960; see Fig. 1). The ara-9 strain itself was isolated follow- ing ultraviolet mutagenesis of a leu-39 mutant of LT-2; the leu-39 allele was then removed by trans- duction with an unspecified phage (Margolin, 1963). Both the hisC3076 and hisD3052 strains were isolated from the ara-9 strain after treatment with the frameshift mutagen ICR364-OH (Oeschger and Hartman, 1970), which, like the ultraviolet treatment used to obtain the ara-9 al-

lele, could have introduced any number of un- known mutations into these strains. The standard Ames tester strains were derived from the result- ing mutants without transfer of the his mutations to a common genetic background.

The development of the standard tester strains from the hisC3076, hisD3052 and hisG46 mutants involved the isolation of derivative strains contain- ing deletions that covered the uvrB and gal re- gions. Each of these deletions was independently isolated and has a different numerical designation, as shown in Table 1 and Fig. 1. None of these deletions is, therefore, likely to be the same as any of the others. It is not known precisely how large each of these deletions is, or how they differ. Although there is no evidence that the differences among the deletions in the current Ames tester strains are of any significance, it is relevant to note that strain TA97 as originally isolated (Levin et al., 1982) has growth problems (low levels of viability of overnight cultures, thin background lawns and pin-point colonies on mutagenesis plates) which Levin and Ames attribute to the

(leu-39) LT2

(hisG46)

hisG46

J (chl- 1005~ TA1530

I (rfa- 1001)

pKMI011

(ara-9)* leu-39 ~, leu-39ara-9

u+) ara-9

(h isC3076)/~D30521 * *

hisC3076 hisD3052 '-

(ch1-1007) I (chl-lO08) TA1532 TA1964

~ KM101)

TP15 TP17

(P1'TA1530)~ /(Pl'TA1532) (h isG46)~/(hisC3076)

(chl-2001) (rfa2001) ~ ( + pKM101) . TP164 " ; ( ~ " ~

(Pl"TP15~ / \ (Pl'TP1776 ~

~ KM101)

Fig. 1. Genealogy of Ames and isogenic tester strains. Ames tester strains in boxes; isogertic tester strains in ovals. Complete genotypes of all strains shown in Table 1. The allele or plasmid added at each step is shown in parentheses. * Indicates mutagenesis with ultraviolet light; ** indicates treatment with frameshift mutagen ICR364-OH. (TD) indicates that the leu + allele was introduced by transduction (phage unspecified); (P1) indicates that allele was introduced by transduction with bacteriophage PlcmCl-100. The donor strain for the PlcmCI-100 transductions is shown after the "'PI.". The allele transferred in each transduction is also indicated. All alleles not obtained by an indicated mutagenesis or transduction were derived by spontaneous mutation. (+pKM101) indicates that plasmid pKM101 was introduced by conjugation. Figure modeled after that published by

Busch et al. (1986).

TABLE 1

GENOTYPES OF S. typhimurium STRAINS SHOWN IN Fig. 1

455

Mutant Genotype a Derivation

Ames tester strains TA98 TA100 TA1535 TA1537 TA1538

ara-9, hisD3052, chl-lO08, rfa-lOO4/pKMl01 hisG46, chl-lO05, rfa-lOO1/pKMl01 hisG46, ch1-1005, rfa-lO01 ara-9, hisC3076, chl-1007, rfa-lO03 ara-9, hisD3052, chl-lO08, rfa-lO04

Mutants used in derioation hisC3076 hisD3052 hisG46 LT-2 leu-39 leu-39 ara-9 ara-9 TA1530 TA1532 TA1964 TA2000

lsogenic tester strains TP135 TP137 TP138 TP198 TP200

of Ames tester strains ara-9, hisC3076 ara-9, hisD3052 hisG46 Wild-type leu-39 leu-39, ara-9 ara-9 hisG46, chl-1005 ara-9, hisC3076, chl-1007 ara-9, hisD3052, chl-lO08 purF145/pKMlO1

ara-9, hisG46, chl-2001, rfa-2001 ara-9, hisC3076, chl-2001, rfa-2001 ara-9, hisD3052, chl-2001, rfa-2001 ara-9, hisD3052, chl-2001, rfa-2OOl /pKMlO1 ara-9, hisG46, chl-2001, rfa-2OO1/pKMlO1

Mutants used in derivation of isogenic tester strains SB3507/pKM101 trpB223/pKMlO1 TP15 ara-9, hisG46, chl-2001, rfa-2001 TP17 ara-9, hisC3076, chl-2001, rfa-2001 TP164 ara-9, hisD3052, chl-2001

Mating TA2000 --, TA1538 Mating TA2000 ~ TA1535 Spontaneous mutant of TA1530 Spontaneous mutant of TA1532 Spontaneous mutant of TA1964

ICR364-OH mutagenesis of ara-9 ICR364-OH mutagenesis of ara-9 Spontaneous mutant of LT-2

Spontaneous mutant of LT-2 Ultraviolet mutagenesis of leu-39 Transduction of leu-39 ara-9 Spontaneous mutant of hisG46 Spontaneous mutant of hisC3076 Spontaneous mutant of hisD3052 Mating hisG46/pKM101 ---, purF145

P1 transduction TP15 ---, TP138 P1 transduction TP17--, TP138 Spontaneous mutant of TP164 Mating SB3507/pKM101 ---, TP138 Mating SB3507/pKM101 ~ TP135

Mating TA98 ~ SB3507 P1 transduction TA1530 ---, TP138 P1 transduction TA1532 -- TP138 Spontaneous mutant of hisD3052

" chl mutations are deletions extending through the uvrB, bio and gal genes. References for strains in Table 1: All "TA" strains except TA2000: Busch et al. (1986); Ames et al. (1975). Strain TA2000: McCann et al. (1975b). Strains hisC3076 and hisD3052: Oesehger and Hartman (1970). Strain hisG46: Hartman et al. (1960). Strains leu-39, leu-39 ara-9, and ara-9: Margolin (1963). Strain SP3507/pKM101: Cebula (1986). All "TP" strains: this publication.

length of the uvrB-bio-gal deletion in this strain. As a result, a newer strain, designated TA97a, carrying a shorter deletion, is currently used and distributed by Ames's laboratory.

After the uvrB-bio-gal deletions were isolated, the next step in the construction of the Ames tester strains was the isolation of an rfa mutant from each strain. No data are available from which to ascertain whether the rfa mutations in the 3 different strains (TA1535, TA1537 and TA1538) are the same or different. They are most likely different since they were isolated indepen-

dently, as indicated by their different numerical designations (Table 1, Fig. 1).

The resulting 3 strains, TA1535, TA1537 and TA1538, carry the hisG46, hisC3076 and hisD3052 mutations, respectively. The plasmid pKM101 was introduced by conjugation into strains TA1535 and TA1538 to produce strains TA100 and TA98.

Thus, the 3 basic tester strains TA1535, TA1537 and TA1538 are not isogenic for 3 reasons: (1) They were isolated from different S. typhimurium stock cultures with known differences at the ara-9 locus; (2) their uorB-bio-gal deletions and rfa

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mutations were independently isolated; and (3) strains TA1537 and TA1538 were derived from strains treated with ultraviolet light and a chem- ical mutagen. In view of the differences among strains, the question arises as to whether the strain-specific responses seen with many mutagens are due solely to their known genetic differences.

The purpose of this work was to isolate strains of S. typhimurium that are isogenic, i.e., they are genetically different only at the his locus and with respect to the presence or absence of the pKM101 plasmid. We have also evaluated the responses of these isogenic strains to a number of mutagens to confirm that the differences in response among the various strains are due solely to the different his mutations that they contain and to the pres- ence or absence of the plasmid pKM101.

Materials and methods

Media and chemicals LB medium contains 10 g of tryptone, 5 g of

yeast extract, and 10 g of NaC1 per liter. LBC is LB medium containing 2.5 mM CaC12. Base agar media were prepared from these media by adding 15 g of Difco Bacto agar per liter; top agar media were made by adding 6 g of agar per liter. The media used for mutagenicity studies were as de- scribed by Maron and Ames (1983) except that the Vogel-Bonner base agar contained 0.5% glu- cose rather than 2% glucose. VBB(HL) is Vogel-Bonner medium supplemented with 2.5/~M biotin and 2.5 mM histidinol dihydrochloride. VBBH is Vogel-Bonner medium supplemented with 2.5/~M biotin and 0.5 mM histidine hydro- chloride. Ara-VBBH is VBBH medium containing 0.2% L-arabinose in place of glucose. VBBH(Amp) is VBBH medium containing 25/~g of ampicillin per ml. The "Gas-Pak" anaerobic system was obtained from BBL, Cockeysville, MD.

Oxoid Nutrient Broth No. 2 (CM67) was ob- tained from K.C. Biological, Lenexa, KS. 9- Aminoacridine, 2-aminofluorene, 1-aminopyrene, benzo[a]pyrene, bis(2-chloroethyl)amine, 1,2-di- bromoethane (ethylene dibromide), 1,2-epoxybu- tane, ethyl methanesulfonate, 2-fluorenylaceta- mide (2-AAF), hydrazine hydrate, 2-hydroxyethyl- hydrazine, N-methyl-N '-nitro-N-nitrosoguanidine (MNNG), 3-methylcholanthrene, 4-nitro-o-phen-

ylenediamine, phenylhydrazine, styrene oxide, and 2,2',4,4'-tetrahydroxybenzophenone were ob- tained from Aldrich Chemical Co., Milwaukee, WI. Ethidium bromide, nitrofurantoin [N-(5- nitro-2- furfurylidene)-l-aminohydantoin], N-ni- troso-N-methylurea, 1-nitrosopiperidine, and sodi- um azide were obtained from Sigma Chemical Co., St. Louis, MO. Sodium nitrite and 2,3,5-tri- phenyl tetrazolium chloride were obtained from Fisher Scientific, Fair Lawn, N J; 4-nitroquinoline- N-oxide was obtained from K & K Laboratories, Plainview, NY; and ICR-191 was obtained from Polysciences, Inc., Warrington, PA. Aflatoxin B~ was a gift from Dr. R.M. Eppley, U.S. Food and Drug Administration.

Bacterial and bacteriophage strains Escherichia coli K-12 strain 514NR (lac dele-

tion, trp-, naiR) was obtained from Dr. Karin Ihler, Texas A&M University, College Station, TX. S. typhimurium strains hisD3052, TA1530, TA1532, TA1535, TA1537, TA1538 and TA1964 (Ames et al., 1975) were obtained from the labora- tory of Dr. Bruce Ames, University of California, Berkeley. The generalized transducing bacterio- phage PlcmC1-100 (Rosner, 1972) was obtained from Dr. Stuart Austin, Frederick Cancer Re- search Center, Frederick, MD. This phage carries a gene (cm) for resistance to chloramphenicol as well as a temperature-sensitive repressor (C1. 100) which permits lysogens to be induced simply by raising the temperature to 40 ° C. Bacteriophage C21, which lyses gal deletion mutants that are rfa + but not rfa- (Wilkinson et al., 1972), was obtained from Dr. Peter Gemski, Walter Reed Army Institute of Research, Washington, DC.

Phage stock preparation Stocks of bacteriophage C21 were prepared by

adding phage at a multiplicity of infection (m.o.i.) of about 1 to log phase S. typhimurium strain TA1964 at a titer of about 106 cells per ml in Oxoid Nutrient Broth No. 2 containing 4 mM CaC12. The cells were shaken overnight at 37°C and centrifuged. The supernatant phage suspen- sion, containing 4-6 × 107 PFU/ml, was stored at 2-4 ° C. The phage titer was determined by plaqu- ing on strain TA1964 in LB top agar on LB base agar plates.

Stocks of the generalized transducing phage PlcmC1. 100 were prepared by adding phage PlcmC1. 100 at an m.o.i, of about 0.5-1 to mid log-phase cells of E. coli K-12 strain 514NR or C600 in LBC medium. The culture was then grown overnight at 37 °C and lysed with chloroform, and the bacteria were removed by centrifugation. The supernatant containing the phage was stored at 2-40C.

Isolation of galactose deletion mutant A deletion was introduced through the uvrB-

chi-bio-gal region of the genome of S. typhi- murium strain hisD3052 by first isolating sponta- neous chlorate-resistant mutants that could not utilize galactose as the sole source of carbon and energy. Cells that are chl- are resistant to the usual toxic effects of chlorate under anaerobic conditions because such mutants lack the chlorate reductase which reduces chlorate to a toxic prod- uct, probably chlorite. Strain hisD3052 was plated on agar containing Difco antibiotic medium No. 2 at 25.5 g per liter and supplemented with potas- sium chlorate at 2 g/l, 2,3,5-triphenyl-2H-tetra- zolium chloride at 50 mg/l, and galactose at 5 g/1. Plates were then incubated at 37°C in a "Gas- Pak" anaerobic system for 1 day and incubated aerobically at room temperature for another day to permit the colonies to enlarge. The resulting red (gai-) colonies were picked, purified on LB agar, and tested for resistance to chlorate under anaerobic conditions (chl-) and for their inability to utilize galactose as the sole source of carbon and energy (gal-). Those strains that were gal- and chl- were then tested for UV sensitivity (uvrB-) and biotin auxotrophy (bio-). Since any resulting chlorate-resistant, galactose-nonutilizing, UV-sensitive, biotin-requiring strain had simulta- neously lost the functions of several closely linked genes, it was judged to contain a deletion through the uvrB-chl-bio-gal region. One of these dele- tion-containing strains was retained and named TP164.

Isolation of rfa mutants To isolate an rfa (deep rough) mutant from

strain TP164, the cells were grown in Oxoid Nutri- ent Broth No. 2 overnight. Then 0.1 ml was plated in LB top agar on LB base agar medium with 0.1

457

ml of stock C21 phage. After overnight incuba- tion, resistant colonies were picked and tested for sensitivity to phage PlcmC1. 100 (Ornellas and Stocker, 1974). One of these Pl-sensitive rfa strains isolated was designated S. typhimurium strain TP138 and corresponds to the Ames tester strain TA1538 (see Fig. 1 and Table 1).

Preparation of transducing lysates To transfer the hisG46 and hisC3076 mutations

into strain TP138, transducing lysates of PlcmCl • 100 were prepared from lysogens of the donor strains. S. typhimurium strains TA1530 and TA1532 were first lysogenized by infecting mid-log phase cells with approx. 2 × 109 pfu/ml of phage PlcmCl. 100 in LBC medium. The culture was shaken for 1 h at 310C, diluted, and plated on LBC agar containing chloramphenicol at 20 #g/ml. After 2 days of incubation, chlor- amphenicol-resistant colonies, which were lyso- genic for the phage, were picked, purified and saved.

Lysogens of strains TA1530 and TA1532 were induced by growing the cells in LBC medium at 31°C to log phase and then raising the temper- ature to 40 o C. After 3 h, a few drops of chloro- form were added to each culture. The cultures were centrifuged at 8000 rpm for 15 min and sterilized by filtration. The resulting lysates were titered on E. coli strain 514NR in LBC top agar on LBC base agar plates.

Transduction To transduce the hisG46 and hisC3076 alleles

into strain TP138, log phase LB cultures of the recipient TP138 ceils were infected with PlcmC1. 100 transducing lysates grown on TA1530 or TA1532. The multiplicity of infection was ap- proximately 0.1. Absorption and infection were allowed to proceed at 30 °C for 30 min; then 0.05 ml of 0.2 M sodium citrate was added to prevent further infection. This mixture was then plated on VBB(HL) and incubated for 2 days at 37 ° C. The original recipient strain, TP138, carrying the hisD3052 mutation, cannot grow on VBB(HL) agar medium because it contains a mutation in the hisD gene. This gene codes for histidinol dehy- drogenase, the last enzyme in the pathway of histidine biosynthesis, which converts the meta-

458

bolic intermediate histidinol into histidine. Strain TP138, which carries the hisD3052 mutation, is therefore unable to convert the histidinol in the medium to histidine.

Strains carrying either the hisG46 or hisC3076 mutation lack the function of the hisG and hisC genes, respectively. These genes code for enzymes that are earlier in the pathway than the hisD gene product (histidinol dehydrogenase). Therefore, strains that are hisG -,hisD +, or hisC -,hisD +, will grow on the VBB(HL) medium because they can synthesize the hisD gene product, histidinol dehy- drogenase, and thus convert the histidinol in the medium to histidine. Any hisG-,hisD + cells that would result from the transduction in which TA1530 was the donor would therefore be able to grow on the selective VBB(HL) medium, as would the hisC-,hisD ÷ cells arising from the transduc- tion in which TA1532 was the donor. Trans- ductant colonies were picked, purified, and checked for histidine auxotrophy (his-), sensitiv- ity to crystal violet (rfa-), and UV sensitivity (uvrB deletion). A hisG-,hisD ÷ transductant was designated as strain TP15 and a hisC-,hisD + transductant as strain TP17.

To eliminate the possibility that genetic material other than that in the his region was transferred from the donors to the recipient cells, the trans- duction was repeated. For the repeat, the trans- ducing phage PlcmC1. 100 was grown on each of the transductants, TP15 and TP17, which are iso- genic to the recipient strain TP138. The resulting lysates were used to transduce strain TP138; selec- tion for histidinol utilizers on VBB(HL) agar medium and checking for histidine auxotrophy (his-), sensitivity to crystal violet (rfa-), and UV sensitivity (uvrB deletion) were again performed. The resulting hisG46, chl(uvrB-bio-gal deletion), rfa strain was named TP135, and the hisC3076, chl(uvrB-bio-gal deletion), rfa strain was named TP137. Isogenic strains TP135 and TP137 corre- spond to Ames tester strains TA1535 and TA1537, respectively.

Transfer of plasmid pKMI01 The plasmid pKM101 was transferred to strains

TP135 and TP138 from S. typhimurium trp dele- tion strain SB3507/pKM101 (Cebula, 1986). Log phase cultures of the donor and recipient strains

were prepared at about 2 × 10 s cells/ml in Oxoid Nutrient Broth No. 2 supplemented with t-tryp- tophan at 20 gg/ml. The donor and recipient strains were mixed by adding 1 ml of each culture to 8 ml of the same medium. The cell mixtures then were incubated for 2 h at 37 °C with gentle shaking, diluted 1/100, and plated on VBBH (Amp) agar plates. Isolated colonies were picked, purified, and checked to confirm that they were rfa- (sensitive to crystal violet and sodium de- oxycholate by the methods of Ames et al., 1975), and contained the plasmid pKM101 (resistant to ampicillin by the method of Zeiger et al., 1981). An ampicillin-resistant derivative of TP135 was saved and designated as strain TP200. An ampicil- lin-resistant derivative of TP138 was designated TP198. These strains correspond to the Ames tes- ter strains TA100 and TA98, respectively.

Mutagenicity testing The plate-incorporation assay described by

Ames et al. (1975) was used to determine the responsiveness of the newly constructed strains to a variety of mutagens. The in vitro metabolic activation system used contained 10% Aroclor 1254-induced liver $9 from 7-8-week-old male Sprague-Dawley rats. The data shown for each chemical in Table 2 represent the means of dupli- cate or triplicate platings from a single experi- ment; the data in all of the figures represent the means of triplicate platings.

Results and discussion

Phenotype of isogenie strains The 5 strains, TP135, TP137, TP138, TP198

and TP200, all exhibit sensitivity to crystal violet, which indicates the presence of an rfa (deep rough) mutation (Ames et al., 1975 ). Strains TP135, TP137 and TP138 are sensitive to ampicillin, while strains TP198 and TP200 are ampiciUin-resistant, indicat- ing the presence of the plasmid pKM101.

We evaluated the ability of the Ames tester strains and the isogenic tester strains to utilize arabinose by streaking them on Ara-VBBH medium. In agreement with the report of Busch et al. (1986), we found that TA1535 is ara + while TA1537 and TA1538 are both ara-. As expected, all three of the isogenic tester strains, TP138,

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TABLE 2

RESPONSES OF ISOGENIC STRAINS TO MUTAGENS REPORTED TO BE SPECIFIC FOR PARTICULAR AMES TESTER STRAINS

Chemical Dose $9 Ames Revertants/plate

(/~g/plate) Spec. a TP135 TP137 TP138

Sodium azide 2.5 Spontaneous controls

Ethyl methanesulfonate 5000 Spontaneous controls

Streptozotocin 1 Bis(2-chloroethyl)amine 50 2- Hydroxyethylhydrazine 1100 1,2-Dibromoethane 4344 Hydrazine hydrate 450 N-Nitroso-N-methylurea 140 Styrene oxide 282 Spontaneous controls

N-Methyl-N '-nitro- N-nitrosoguanidine 3

Spontaneous controls

1,2-Epoxybutane 28,877 /~-Propiolactone 360 Spontaneous controls

Phenylhydrazine 433 Spontaneous controls

2,2',4,4 '-Tetrahydroxy- benzophenone 600

Spontaneous controls

ICR191 5 Spontaneous controls

4-Nitro-o-phenylenediamine 10 Spontaneous controls

Benzol a ]pyrene 5 2-Aminofluorene 10 Spontaneous controls

4-Aminobiphenyl 50 1 -Aminopyrene 50 3-Methylcholanthrene 50 Spontaneous controls

m

TA1535 662 9 20 22 5 21

TA1535 423 10 26 21 7 18

TA1535 1546 18 10 TA1535 646 7 14 TA1535 433 7 18 TA1535 256 6 14 TA1535 95 4 7 TA1535 1128 13 16 TA1535 282 31 31

22 5 18

TA1535 753 11 21 12 6 24

TA1535 650 6 31 TA1535 1977 10 17

36 16 18

TA1537 31 198 23 36 16 18

+ TA1537 22 50 27 + 16 13 23

- FS b 23 957 330 - - 2 2 5 21

- - FS 22 48 981 21 7 18

+ FS 32 148 222 + FS 38 77 1408 + FS 16 13 23

+ FS 30 51 392 + FS 19 884 1349 + FS 13 47 146 + 15 9 20

a "Ames Spec.": the Ames tester strain for which the chemical is specific. The source of this specificity information is: hydrazine hydrate and phenylhydrazine (Parodi et al., 1981); 4-nitro-o-phenylenediarnine (Dunkel et al., 1985); 2,2',4,4'-tetrahydroxybenzo- phenone (Prival et al., 1982); all other compounds (MeCarm et al., 1975a).

b "FS": the chemical has been reported to be mutagenic in frameshift strains TA1537 and TA1538 but not mutagenic in base-pair substitution strain TA1535.

T P 1 3 5 a n d T P 1 3 7 , w e r e f o u n d to b e ara-; t h e y

w e r e al l i s o l a t e d f r o m t he ara-9 m u t a n t s t r a i n

hisD3052 ( H a r t m a n , 1987) .

W e c o n s i d e r e d t h e p o s s i b i l i t y t h a t t h e i s o g e n i c

s t r a i n s T P 1 3 5 a n d T P 1 3 7 m i g h t h a v e b e c o m e lyso-

g e n i c fo r t h e P l c m C 1 . 100 b a c t e r i o p h a g e u s e d i n

460

their construction. However, these strains were found to be non-lysogenic for this phage. This was demonstrated by showing that cells were sensitive to chloramphenicol, did not make infective centers when seeded on a lawn of E. coli C600 cells, and grew well when streaked on LBC agar medium and incubated at 41°C. Lysogens of TP135 and TP137 were prepared to serve as a control; these lysogens were resistant to chloramphenicol, did make infective centers when seeded on a lawn of E. coli C600, and grew poorly when streaked on LBC agar medium and incubated at 41°C as a result of the temperature-inducible phage within the cells.

Sensitivity to mutagens We evaluated the responsiveness of the isogenic

strains to a series of mutagens. To test whether the range of sensitivity of the isogenic strains is the same as that of the original Ames strains, we first selected mutagens that have been reported to give a specific response in one or two of the three Ames strains TA1535, TA1537 and TA1538. Strains TA1535 and TP135 contain the base pair substitution mutation hisG46; strains TA1537 and TP137 contain the frameshift mutation hisC3076; strains TA1538 and TP138 contain the frameshift mutation hisD3052.

Two chemicals that are known to be mutagenic in base-pair substitution strain TA1535 but not in frameshift strains TA1537 or TA1538 are sodium nitrite (McCann et al., 1975a) and 1-nitrosopi- peridine (McCann et al., 1975a). As shown in Figs. 2 and 3, these chemicals induced a muta- genie response in strain TP135 similar to the re- sponse induced in TA1535, but they were not mutagenic to strains TP137 or TP138.

The mutagen 9-aminoacridine is effective when tested in strain TA1537, but is not mutagenic in strain TA1535 or TA1538 (McCann et al., 1975a). As shown in Fig. 4, the effect in the isogenic strains was similar: TP137 responded but TP135 and TP138 did not give any mutagenic response. Ethidium bromide is mutagenic to Ames tester strain TA1538, but not to strains TA1535 or TA1537 (McCann et al., 1975a; Fig. 5). Isogenic tester strain TP138, which corresponds to strain TA1538, was sensitive to this chemical, but strains TP135 and TP137 were not (Fig. 5). Similarly,

600

Sodium Nitrite 5oo

~- 400

e¢-

loo

0 3,000 6,000 9,000

/~g per Plate Fig. 2. Mutagenicity of sodium nitrite on Ames base-pair substitution tester strain TA]535 (o ) and in isogenic strains TP135 (e), TP137 (&) and TP138 (11). Data shown are results

obtained without $9.

2-fluorenylacetamide is mutagenic to strains TA1538 and TP138 in the dose range of 1.25-5 /~g/plate; it is not mutagenic in this dose range to the other Ames tester strains, TA1537 and TA1538, or to the other isogenic tester strains, TP137 and TP138 (Fig. 6).

Aflatoxin B 1 and 4-nitroquinoline-N-oxide are more mutagenic to strain TA1538 than to strains

1501 1-Nitrosopiperidine ~

~.1

o 1250 2500 3750 5000

/z g per Plate

Fig. 3, Mutagenicity of 1-nitrosopiperidine in Ames tester strains TA1535 (o), TA1537 (zx) and TA1538 (E3) and in isogenic strains TP135 (O), TP137 ( - ) and TP138 (11). Data

shown are results obtained with $9.

TA1535 and TA1537 in the dose range used (Figs. 7, 8). The responses of the isogenic strains TP135, TP137 and TP138 were similar to those of the corresponding Ames strains.

A number of additional chemicals were tested for mutagenicity in the 3 isogenic strains, TP135, TP137 and TP138 (Table 2). These chemicals have been reported in the literature to be specific for one or two of the nonplasmid-bearing Ames tester strains TA1535, TA1537 or TA1538. Each chem- ical was tested at a single dose that has been reported in the literature to be on the rising por- tion of its dose-response curve. As shown in Table 2, the specificities of the responses in the 3 isogenic strains appear to be the same as those reported in the Ames tester strains.

We used two chemicals to demonstrate the increased responsiveness of the plasmid-bearing strains TP198 and TP200, as compared to their corresponding plasmid-free strains, TP138 and TP135. As shown in Fig. 9, aflatoxin B 1 at doses of 0.025-0.10 /~g/plate was mutagenic to hisD3052, plasmid-containing strains TA98 and TP198 but not to the corresponding hisD3052,

1600

9 -Aminoacr id ine 14oo

12oo

e~

¢ r

200

o , , B •

0 25 50 75 100 /xg per Plate

Fig. 4. Mutagenicity of 9-aminoacridine in Ames frameshift tester strain TA1537 (/,) and in isogenic swains TP135 (e), TP137 (A) and TP138 (B). Data shown are results obtained

without $9.

461

1000

800

a -6oo

O.

¢-

oo

O)

o>400 r t -

200

Ethid ium Bromide

0 1 ~ 0 1.25 2.5

#g per Plate

Fig. 5. Mutagenicity of ethidium bromide in Ames tester strains TA1535 (o), TA1537 (zx) and TA1538 (ra) and in isogenic strains TP135 (O), TP137 (A) and TP138 (11). Data

shown are results obtained with $9.

plasmid-free strains, TA1538 and TP138. Simi- laxly, the hisG46, plasmid-containing strains TA100 and TP200 were sensitive to aflatoxin B 1 (Fig. 10) and nitrofurantoin (Fig. 11), whereas the hisG46, plasmid-free strains TA1535 and TP135 were not responsive to these rnutagens. These re- suits axe consistent with the reports of McCann et al. (1975a, b) on the pKM101-dependence of aflatoxin B1 mutagenicity at low doses and on the unique responsiveness of TA100 to nitrofuran mutagenesis.

In summary, we have isolated strains of S. typhimurium that contain the relevant mutat ions of the standard Ames tester strains but that are isogenic with respect to the remainder of their genomes. To do this we transferred the hisG46 and hisC3076 mutations by transduction to the hisD3052 mutant strain TP138. This transduction maintains the isogenicity of the strains. The possi- bility that heterologous D N A sequences might

462

160

140

120

~_ 100

40

20 i

0 l t 1 t I 0 1.25 2.5 3,75 5

/~g per Plate

Fig. 6. Mutagenicity of N-(2-fluorenyi)acetamide in Ames tes- ter strains TA1535 (o), TA1537 (zx) and TA1538 (El) and in isogenie strains TP135 (O), TP137 (A) and TP138 (B). Data

shown are results obtained with $9.

500

4-Nitroquinoline-N-oxide

40O

0-300

2 ® e¢-

100

i I 00 0.5 1.0

#g per Plate Fig. 8, Mutagenicity of 4-rtitroquinoline-N-oxide in Ames tes- ter strains TA1535 (o), TA1537 (zx) and TA1538 (n) and in isogenic strains TP135 (O), TP137 (&) and TP138 (B). Data

shown are results obtained without $9.

200

Aflatoxin B 1

150

== 100

>= t 'n"

50

0 ~ "r -r 0.1 0.2

vg per Plate

Fig. 7. Mutagenicity of aflatoxin B l in Ames tester strains TA1535 (o), TA1537 (tx) and TA1538 (O) and in isogenic strains TP135 (e), TP137 (A) and TP138 (ram). Data shown are

results obtained with $9.

h a v e b e e n i n t r o d u c e d b y m u l t i p l e i n f e c t i o n d u r i n g

the t r a n s d u c t i o n was m i n i m i z e d b y us ing a l o w

m u l t i p l i c i t y o f i n f e c t i o n a n d b y ser ia l ly t r ans fe r -

r ing the hisG46 and hisC3076 al le les two t imes

1200

Aflatoxin B 1

" 600

e r

200

O r ~P T , l l

0 0.025 0.050 0.075 0.10

/~g per Plate

Fig. 9. Mutagenicity of aflatoxin B] in Ames frameshift tester strain TA1538 ([3) and its pKM101-containing derivative TA98 (z~) and in the corresponding isogenic strains TP138 (==) and

TP198 (A). Data shown are results obtained with $9.

463

1800

1400

1200

~- 1000

400

200

0 0.025 0.050 0.075

#g per Plate

Fig. 10. Mutagenicity of aflatoxin B 1 in Ames base-pair sub- stitution tester strain TA1535 (o) and its pKM101-eontaining derivative TA100 (zx) and in the corresponding isogenic strains TP135 (I) and TP200 (a). Data shown are results obtained

with $9.

into recipient strain TP138. The degree of re- latedness of the isogenic tester strains can be compared to that of the Ames tester strains by examining Fig. 1. This figure shows that the Ames tester strains are separated by many steps, some involving treatment with mutagens; the isogenic strains were derived directly from one another by transduction or by plasmid transfer.

The specific responsiveness of the isogenic "TP" strains to a number of mutagens is similar to that which has been observed with the Ames "TA" strains. The limited number of chemicals tested enables us to conclude only that this similarity is qualitative; any general conclusions concerning the quantitative similarities or differences in sensi- tivity between the isogenic strains and the Ames strains will have to await additional studies. The results presented here demonstrate that the specificity of a wide range of mutagens for the various Ames tester strains is the result of the differences in the nature of the his mutation, and not the result of any of the other unknown genetic differences that exist among the various non-iso- genie Ames tester strains.

800

600

_R a .

~400 ¢ -

r r

200

Nitrofurantoin

- ~ @ q~ 0.1 0.2 0.3

#g per Plate

Fig. 11. Mutagenicity of nitrofurantoin in Ames base-pair substitution tester strain TA1535 (O) and its pKM101-contain- ing derivative TA100 (zx) and in the corresponding isoganic strains TP135 (I) and TP200 (A). Data shown are results

obtained without $9.

Acknowledgement

We thank Thomas Cebula, Rosalie Elespuru, and Kristien Mortelmans for their valuable com- ments on this manuscript.

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