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Mutagenesis, Vol. 17, No. 4, 313-316, July 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press

Characterization of Trp+reversions in Escherichia coli strain WP2uvrA

Toshihiro Ohta1, Shin-ichi Tokishita, Rumi Tsunoi, Satoru Ohmae and Hideo Yamagata

School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and Discussion
 References
 
The Escherichia coli strain WP2uvrA is widely used in general mutagenicity screening tests because of its high sensitivity to many kinds of mutagens and it serves as a supplement to the standard Salmonella typhimurium tester strains. In contrast to Salmonella His+ revertants, E.coli Trp+ revertants have not been characterized at the molecular level. In this study we found that in the trpE65 allele of WP2uvrA the triplet that codes for the fourth amino acid from the N-terminus of anthranilate synthetase was an ochre stop codon (TAA) instead of a glutamine codon (CAA). In spontaneous Trp+ revertants the ochre codon had been changed to glutamine (CAA), lysine (AAA), glutamic acid (GAA), leucine (TTA), serine (TCA) or tyrosine (TAC, TAT). Since tryptophan prototrophy could also be restored by ochre suppressor mutations at the anticodon sites in the genes for tRNAGlu (glnU), tRNALys (lysT) and tRNATyr (tyrT, tyrU), the Trp+ reversion system with E.coli WP2uvrA detected five types of base substitutions, A·T->T·A, A·T->C·G, A·T->G·C, G·C->A·T and G·C->T·A. About 30–50% of Trp+ revertants induced by N-ethyl-N'-nitro-N-nitrosoguanidine, captan and angelicin plus UVA irradiation were attributable to reversion at the trpE65 ochre locus; the others were attributable to suppressor mutations. In contrast, almost all revertants induced by N-methyl-N'-nitro-N-nitrosoguanidine, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone and furylfuramide were caused by suppressor mutations. Thus, the high mutagen sensitivity of WP2uvrA is due to several target sites consisting of A·T base pairs (trpE65, lysT) and G·C base pairs (glnU, tyrT, tyrU).


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and Discussion
 References
 
In bacterial mutagenicity screening tests for chemicals Escherichia coli tester strain WP2uvrA or WP2uvrA/pKM101 is recommended as a supplement to the standard Salmonella typhimurium tester strains. Some mutagens induce base substitutions in E.coli tester strains but not in S.typhimurium tester strains TA1535, TA100 and TA102 (Matsushima et al., 1981Go; Gatehouse et al., 1994Go), which were designed to detect base substitutions. Therefore, it is important to elucidate the difference between Trp+ reversion and His+ reversion events. Salmonella typhimurium TA100 and TA1535 utilize the hisG46 missense mutation, which substitutes a proline triplet (CCC) for a leucine triplet (CTC) at position 434 of the histidine operon (Barnes et al., 1982Go). Cells containing hisG46 can revert to histidine prototrophy via a mutation that changes the CCC codon to a codon for leucine (CTC, the wild-type), serine (TCC), alanine (GCC), histidine (CAC) or threonine (ACC) (Miller and Barnes, 1986Go). Salmonella typhimurium TA102 carries the hisG428 nonsense mutation, which encodes an ochre stop codon (TAA) in place of a glutamine codon (CAA) at position 847 of the histidine operon (Levin et al., 1982Go). Strain TA102 can revert to histidine prototrophy by base substitutions at either the ochre site in the hisG428 or extragenic ochre suppressor sites (anticodon change of tRNA genes). Reversions at the ochre site include five types of mutations, glutamine (CAA), leucine (TTA), tyrosine (TAT, TAC) and lysine (AAA), among the seven possible sense mutations induced by a single base substitution. So far no base substitution to glutamic acid (GAA) or serine (TCA) has been detected (Levin and Ames, 1986Go). Suppressor mutations include supB, supC, supG and supM mutations of the tRNA genes glnU, tyrT, lysT and tyrU, respectively.

In contrast to His+ reversions, little is known about Trp+ reversions other than that the trpE65 allele contains an ochre nonsense mutation (Osborn and Person, 1967Go). The presence of that mutation was deduced based on the observation that a mutant T4 phage containing an ochre mutation in an essential gene could grow in many WP2 Trp+ revertant strains, but the mutation was not characterized. In the present study we have identified the ochre stop codon in the trpE65 gene of WP2uvrA and, by DNA sequencing, analyzed the base substitutions in Trp+ revertants.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and Discussion
 References
 
Bacterial strains, media and chemicals
Escherichia coli B/r WP2 [trpE65 (ochre)] and its nucleotide excision repair-deficient derivative WP2uvrA [trpE65] (Green and Muriel, 1976Go) were used. WP2008 [trpE65, argE3] was constructed by introducing argE3 (ochre) from IC3830 [argE3, btuB::Tn10] into WP2uvrA by P1 phage-mediated transduction (Ohta et al., 1998Go). Strain IC3830 was provided by Dr M.Blanco (FVIB, Valencia, Spain). The MG agar medium consisted of Vogel–Bonner E medium (0.2% citric acid monohydrate, 1% K2HPO4, 0.35% NaNH4HPO4·4H2O and 0.02% MgSO4·7H2O) supplemented with 0.5% glucose and 1.5% agar. TA top agar contained 10 µg/ml tryptophan, 250 µg/ml arginine, 0.5% NaCl and 0.6% agar. Nutrient broth (Oxoid No. 2) was used for preculture of the bacterial strains. 3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), furylfuramide (AF-2) and captan were obtained from Wako Pure Chemical (Tokyo, Japan). N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and angelicin were purchased from Sigma-Aldrich Japan K.K. (Tokyo, Japan). Mutagens were dissolved in dimethylsulfoxide (Wako Pure Chemical).

Cloning and sequencing the trpE65 gene
Chromosomal DNA was prepared from WP2 and WP2uvrA. The entire trpE65 region from each strain was amplified by PCR with the LA PCR Kit (Takara Biomedicals, Tokyo, Japan) and cloned into HindIII- and BamHI-digested pUC118 vector. Oligonucleotides 5'-CCCAAGCTTGTATTCACCATGCG-TAAAG-3' and 5'-CGCGGATCCAGCAGAATGTCAGCCATCA-3' were used, respectively, as specific 5'- and 3'-primers for PCR. The nucleotide sequence was determined with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems Japan, Tokyo) using an ABI-377 DNA sequencer (Applied Biosystems Japan). The sequencing was performed on two independent clones for confirmation.

Isolation of trpE+ revertants
An overnight culture (0.1 ml) of WP2008 (Trp- and Arg-) was added with or without 0.1 ml of mutagen solution to 0.5 ml of sodium phosphate buffer (100 mM, pH 7.4). After preincubation at 37°C for 20 min the treated cells were poured onto MG agar plates with 2 ml of molten (45°C) TA top agar. Angelicin mutagenesis included UVA irradiation (320–400 nm, 0.26 mW/cm2, black light FL15BL-B; National Co., Osaka, Japan) for 10 min of the suspension of bacterial cells and angelicin in a 24-well multiplate (Ohta et al., 2001Go). The irradiated cells in the presence of angelicin were plated on MG agar plates with TA top agar. Trp+ revertants were counted after incubation for 2 days at 37°C. The average number of revertants in control plates was 17. The numbers of revertants per plate were 171 at 2 µg/plate captan, 217 at 0.5 µg/plate ENNG, 90 at 4 µg/plate angelicin, 213 at 0.5 µg/plate MNNG, 224 at 0.02 µg/plate AF-2 and 202 at 0.2 µg/plate MX. This means that 10, 8, 19, 8, 8 and 8% of Trp+ revertants, respectively, are expected to be spontaneous in origin. Trp+ revertant colonies were isolated from these plates (50 colonies each) and checked for Arg auxotrophy. Cells showing both the Trp+ and Arg+ phenotypes were considered to contain tRNA ochre suppressor mutations. Trp+ revertants showing the Arg- phenotype were considered to contain mutations within the trpE gene and subjected to sequence analysis. We prepared chromosomal DNA from the revertants, then amplified a 0.6 kb DNA fragment encoding the 5' region of trpE by PCR. The 5'-primer was the same as described above and the 3'-primer was 5'-CGCGGATCCTGACAGTTGCGGTAAATCTTC-3'. The nucleotide sequences of the amplified fragments were directly determined by the dideoxy chain termination method.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and Discussion
References
 
DNA sequencing revealed three base substitutions in the trpE65 gene derived from WP2 and WP2uvrA compared with the wild-type trpE gene of E.coli strain K-12 (Yanofsky et al., 1981Go) (Figure 1Go). A substitution of G for C at position 240 did not change the corresponding amino acid leucine, while that of A for T at position 29 caused a change from leucine to glutamine. A substitution of T for C at position 10 resulted in an ochre stop codon. The remainder of trpE65 was identical to the wild-type and the trpE65 gene was the same in WP2 and WP2uvrA. The amino acid change Leu->Gln would not seriously affect the activity of anthranilate synthetase, because all the Trp+ revertants of WP2uvrA analyzed in this study had Gln10. We concluded that the Trp auxotrophy exhibited by WP2 and WP2uvrA is due to substitution in trpE65 of an ochre stop codon (TAA) for a glutamine codon (CAA), altering the encoded peptide at the fourth amino acid from the N-terminus.



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  		Fig. 1. . Nucleotide sequence of the wild-type trpE gene from E.coli K-12 and the mutant trpE65 gene from E.coli B/r WP2uvrA. The first 300 nt are shown. The predicted amino acid sequence is shown below the nucleotide sequence. Codons differing in the two strains are underlined. The Gln4 codon (CAA) and Leu10 codon (CTG) were changed to an ochre stop codon (TAA) and Gln codon (CAG), respectively, in the trpE65 gene. A G->C substitution at position 240 did not change the corresponding amino acid leucine. The nucleotide sequences of the trpE65 gene from WP2 and WP2uvrA were completely identical.

 
Single base substitutions in the TAA triplet can result in codons for glutamine (CAA), glutamic acid (GAA), lysine (AAA), leucine (TTA), tyrosine (TAT, TAC) and serine (TCA). To investigate which amino acid substitutions restored the Trp+ phenotype, we isolated spontaneous WP2008 Trp+ revertants and performed sequence analyses. Among the 66 spontaneous Trp+ revertants collected from four control plates, we excluded from analysis 38 colonies that showed the Arg+ as well as Trp+ phenotype, assuming them to be sup mutants. Besides reversion to the wild-type glutamine triplet (CAA) by an A·T->G·C transition, we found all possible amino acid changes (Figure 2Go). Mutations to leucine (TTA), tyrosine (TAT) and lysine (AAA) triplets were formed by A·T->T·A transversions, and mutations to tyrosine (TAC), serine (TCA) and glutamic acid (GAA) triplets were formed by A·T->C·G transversions. In E.coli at least four kinds of suppressor mutations, supC in the tyrT gene, supM in the tyrU gene, supG (supL) in the lysT gene and supB in the glnU gene, suppress ochre mutations by inserting the respective amino acids (Miller, 1992Go). In strains WP2 and WP2uvrA these suppressor mutations would also restore the Trp+ phenotype because tyrosine and lysine can replace glutamine at position 4. Since the anticodon changes of supC and supM are GUA->UUA and those of supB and supG are UUG->UUA and UUU->UUA, respectively, the mutations are formed by a G·C->T·A transversion, a G·C->A·T transition and an A·T->T·A transversion. Mutational target sites in the Trp+ reversion test with strains WP2 and WP2uvrA consist of A·T (trpE65, lysT) and G·C (glnU, tyrT, tyrU) base pairs and, therefore, these strains can detect all types of base substitutions except G·C->C·G transversions. The present results also indicate that there is no fundamental difference in detectable base substitution events between the trpE65 ochre locus in E.coli WP2uvrA and the hisG428 ochre allele in S.typhimurium TA102 (Koch et al., 1996Go), as summarized in Table IGo.



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  		Fig. 2. . Base substitutions found in 66 spontaneous Trp+ revertants expressed as a percentage. Seven unique single base substitutions at the ochre locus were recovered. Glu, Lys, Leu, Tyr and Ser could replace Gln4.

 

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  		Table I. . Reversion characteristics in E.coli WP2uvrA and S.typhimurium TA102
 
Treatment with captan resulted in equal recovery of reversions due to mutations in the trpE65 gene and those due to mutations in suppressor genes (Figure 3Go). Captan induced predominantly A·T->C·G transversions, rather than A·T->T·A transversions or A·T->G·C transitions, which is consistent with the mutation spectrum in the Lac+ reversion system (Lu et al., 1995Go; Ohta et al., 2000Go). About one-third of the ENNG- and angelicin-induced revertants of WP2008 had mutations in the trpE65 gene (Figure 3Go), but in contrast to the captan mutation spectrum, A·T->T·A transversions were predominant and we found no angelicin-induced A·T->C·G transversions. This contrasts with the S.typhimurium mutation spectrum (Koch et al., 1996Go), in which angelicin efficiently induced A·T->C·G transversions as well as A·T->T·A transversions in hisG428 ochre strain TA104. In our previous study with the E.coli Lac+ reversion system angelicin did induce A·T->C·G transversions (unpublished observation). Since angelicin increased Trp+ mutants a modest 5.3-fold and we analyzed only 16 of the revertants, it is difficult to conclude whether there was a quantitative difference in angelicin-induced mutations in E.coli trpE65 and S.typhimurium hisG428. In contrast, almost all (94–98%) Trp+ revertants recovered from cells treated with MNNG, AF-2 and XM were suppressor mutations (Figure 4Go). The small number of A·T->C·G and A·T->T·A transversions we observed were likely to have been spontaneous in origin. The specific induction of base substitutions at G·C base pairs by these mutagens was also consistent with our previous studies with the Lac+ reversion system (Ohta et al., 1998Go, 2000Go).



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  		Fig. 3. . Distribution of base substitutions at the ochre locus in trpE65 and at suppressor mutations in tRNA genes derived from revertants induced by captan (left), ENNG (center) and angelicin (plus UVA irradiation) (right). The y-axis indicates the number of Trp+ revertants. TAC, TCA and GAA are formed by A·T->C·G transversions and TAT, TTA and AAA are formed by A·T->T·A transversions. A wild-type CAA reversion is formed by an A·T->G·C transition.

 


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  		Fig. 4. . Distribution of base substitutions at the ochre locus in trpE65 and those at suppressor mutations in tRNA genes derived from revertants induced by MNNG (left), AF-2 (center) and MX (right). The y-axis indicates the number of Trp+ revertants.

 
As far as detectable base substitutions are concerned, the Trp+ and His+ reversion systems do not differ. Both can detect all types of base substitutions except G·C->C·G transversions. Therefore, differences in mutagenicity of a compound to E.coli WP2uvrA and WP2uvrA/pKM101versus S.typhimurium TA102 may be due to differences in cell permeability, cytotoxic responses, metabolism or nucleotide excision repair capabilities.


   Acknowledgments
 
We thank Saori Fujisawa for technical assistance and Dr Miriam Bloom for critical reading of the manuscript.


   Notes
 
1 To whom correspondence should be addressed. Tel: +81 426 76 7093; Fax: +81 426 76 7081; Email: ohta{at}ls.toyaku.ac.jp Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results and Discussion
 References
 

    Barnes,W., Tuley,E. and Eisenstadt,E. (1982) Base-sequence analysis of His+ revertants of the hisG46 missense mutation in Salmonella typhimurium. Environ. Mutagen., 4, 297.

    Gatehouse,D., Haworth,S., Cebula,T., Gocke,E., Kier,L., Matsushima,T., Melcion,C., Nohmi,T., Ohta,T., Venitt,S. and Zeiger,E. (1994) Recommendations for the performance of bacterial mutation assays. Mutat. Res., 312, 217–233.[Web of Science][Medline]

    Green,M.H.L. and Muriel,W.J. (1976) Mutagen testing using Trp+ reversion in Escherichia coli. Mutat. Res., 38, 3–32.[Web of Science][Medline]

    Koch,W.H., Henrikson,E.N. and Cebula,T.A. (1996) Molecular analysis of Salmonella hisG428 ochre revertants for rapid characterization of mutational specificity. Mutagenesis, 11, 341–348.[Abstract/Free Full Text]

    Levin,D.E. and Ames,B.N. (1986) Classifying mutagens as to their specificity in causing the six possible transitions and transversions: a simple analysis using the Salmonella mutagenicity assay. Environ. Mutagen., 8, 9–28.[Web of Science][Medline]

    Levin,D.E., Hollstein,M., Christman,M.F., Schwiers,E.A. and Ames,B.N. (1982) A new Salmonella tester strain (TA102) with A:T base pairs at the site of mutation detects oxidative mutagens. Proc. Natl Acad. Sci. USA, 79, 7445–7449.[Abstract/Free Full Text]

    Lu,C., Pfeil,R.M. and Rice,C.P. (1995) Determination of mutational spectrum of the pesticide, captan, with an improved set of Escherichia coli lacZ mutants. Mutat. Res., 343, 219–227.[Web of Science][Medline]

    Matsushima,T., Takamoto,Y., Shirai,A., Sawamura,M. and Sugimura,T. (1981) Reverse mutation test on 42 coded compounds with the E.coli WP2 system. In de Serres,F.J. and Ashby,J. (eds) Evaluation of Short-term Tests for Carcinogens. Elsevier North-Holland, Amsterdam, The Netherlands, pp. 351–360.

    Miller,J.H. (1992) A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Miller,J.K. and Barnes,W.M. (1986) Colony probing as an alternative to standard sequencing as a means of direct analysis of chromosomal DNA to determine the spectrum of single-base changes in regions of known sequence. Proc. Natl Acad. Sci. USA, 83, 1026–1030.[Abstract/Free Full Text]

    Ohta,T., Watanabe-Akanuma,M., Tokishita,S., Shiga,Y. and Yamagata,H. (1998) Development of new tester strains derived from E.coliWP2uvrA for the determination of mutational specificity. Mutat. Res., 413, 219–225.[Web of Science][Medline]

    Ohta,T., Watanabe-Akanuma,M. and Yamagata,H. (2000) A comparison of mutation spectra detected by the E.coli Lac+reversion assay and the S. typhimurium His+reversion assay. Mutagenesis, 15, 317–323.[Abstract/Free Full Text]

    Ohta,T., Ohmae,S., Yamaya,K., Kanemichi,Y., Tokishita,S. and Yamagata,H. (2001) Characterization of the mutational specificity of DNA cross-linking mutagens by the Lac+ reversion assay with Escherichia coli. Teratog. Carcinog. Mutagen., 21, 275–282.[Web of Science][Medline]

    Osborn,M. and Person,S. (1967) Characterization of revertants of E.coli WU36-10 and WP2 using amber mutants and an ochre mutant of bacteriophage T4. Mutat. Res., 4, 504–507.[Web of Science][Medline]

    Yanofsky,C., Platt,T., Crawford,I.P., Nichols,B.P., Christie,G.E., Horowitz,H., VanCleemput,M. and Wu,A.M. (1981) The complete nucleotide sequence of the tryptophan operon of Escherichia coli. Nucleic Acids Res., 9, 6647–6668.[Abstract/Free Full Text]

Received on December 7, 2001; accepted on February 19, 2002.


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