Sequence Analysis of Mutations Arising during Prolonged Starvation of Salmonella Typhimurium

We have examined the effects of prolonged histidine deprivation on the reversion of Salmonella typhimurium histidine auxotrophs containing either hisG46, a missense mutation (CTC----CCC), or hisG428, an ochre mutation (CAA----TAA). Both of these mutants can revert to His+ via intragenic and extragenic mechanisms. Whereas the hisG46 mutant site consists of G/C base pairs, extragenic suppression of hisG46 requires mutation at an A/T site. Conversely, the hisG428 site itself contains only A/T base pairs, and extragenic suppression of hisG428 occurs principally at G/C sites. Thus, by examining the mutational spectrum of hisG46 and hisG428 revertants that occurred in the presence and in the absence of histidine, it was possible to determine the effects of histidine starvation on mutations at G/C vs. A/T sites as well as on intragenic sites vs. extragenic suppressor sites. Using DNA-colony hybridization, we determined the DNA sequences of over 1300 hisG46 and hisG428 revertants. Histidine-independent revertants that arose during growth in liquid medium that contained histidine included both intragenic and extragenic suppressor mutations. The relative frequency of such extragenic suppressors was greatly reduced among the His+ revertants that were isolated after 5-10 days of histidine starvation on agar medium. Moreover, DNA sequence analysis revealed striking differences in the distribution of particular transversions at the hisG428 locus in revertants arising after prolonged histidine starvation as compared to those arising after growth in the presence of histidine.     

Isolation of glutamate-inserting ochre suppressor mutants of Salmonella typhimurium and Escherichia coli.

Glutamate-inserting ochre suppressors have been identified among late-arising, spontaneous revertants of a hisG428 mutant of Salmonella typhimurium and an argE3 mutant of Escherichia coli. The S. typhimurium suppressors mapped in the tRNA2(Glu) gene gltU at 82 min; those in E. coli were found to be in tRNA2(Glu) genes gltW at 56 min, gltU at 85 min, and gltT at 90 min.     

Methyl methanesulphonate (MMS) is clearly mutagenic in S. typhimurium strain TA1535; a comparison with strain TA100

No mutagenicity or an uncertain mutagenic response has been reported in the literature for methyl methanesulphonate (MMS) in S. typhimurium strain TA1535 when using the plate assay. In our studies we found a reproducible mutagenic activity of 62 revertants/mumole and plate for MMS in strain TA1535 when using the preincubation assay. A dose-dependent increase in revertants was, however, observed only at fairly high doses (exceeding 4 mumole). Two different slopes were observed in the dose-response curve when testing MMS with strain TA100. Slope A is dependent on the error-prone response, possible only in strain TA100 due to the pKm101 plasmid (R factor) but not possible in strain TA1535 due to its umuDC deficiency. Slope B observed at higher doses (as in strain TA1535) could be explained through a GC----AT transition initiated by the O6-methylation of guanine. Our findings demonstrate that MMS induces back mutation in S. typhimurium strains carrying the hisG46 missense mutation due to the formation of O6-methylguanine. In the case of strain TA100 the pKm101 plasmid-mediated error-prone mechanism is, however, the predominant process in MMS mutagenesis which leads to a higher mutagenic response at much lower doses than the GT----AT transition in strain TA1535.     

Plasmid-dosage effects on ultraviolet, visible light and methyl methanesulfonate mutagenesis in Salmonella typhimurium

Salmonella typhimurium LT2 strains bearing plasmids pKM101, R64 or pColIb-P9 demonstrated enhanced UV survival when compared with strains not bearing plasmids. A strain of S. typhimurium bearing both pKM101 and pColIb-P9 survived UV irradiation slightly better than either of the single-plasmid strains. Spontaneous reversion of the hisG46 and trpE8 missense alleles was enhanced in each single-plasmid strain, and for the dual-plasmid strain containing pKM101 and pColIb-P9 enhancement represented a near additivity of the response seen for the single-plasmid strains. Following exposure to UV or visible-light irradiation, reversion of hisG46 and trpE8 was also enhanced in each single-plasmid strain, but quantitatively greater in the dual-plasmid strain and was equal to or slightly greater than additive the responses of the single-plasmid strains. In contrast to visible-light irradiation, UV exposure resulted in two phenotypic Trp+-revertant classes. One Trp+ class, having normal colony size (2.0 mm) and similar in number to His+ revertants, was comprised of intragenic revertants of trpE8, while the predominant Trp+ class, having smaller colony size (0.8 mm), represented intergenic suppressor revertants, illuminating the differences in mutation and/or repair specificity for UV and visible-light exposure. Methyl methane-sulfonate (MMS)-induced reversion of hisG46 was similar in effect to that seen with UV or visible-light irradiation. Plasmids pKM101 or pColIb-P9 enhanced the frequency of hisG46 reversion, while a more than additive response was seen in a strain with both plasmids. Furthermore, MMS-induced reversion of hisG46 was also observed to be greatest in a strain bearing plasmid R64 (incompatibility group I alpha) and pKM101, when compared with single-plasmid strains bearing either R64 or pKM101.     

Reduction of the translation fidelity by kanamycin: effects on growth and mutant frequency in S. typhimurium TA102

Kanamycin reduces the translation fidelity in prokaryotes. By read-through of the ochre stop codon (hisG428) of S. typhimurium TA102 enough functional enzyme is produced to allow the his- cells to form a dense background growth on the minimal agar plates. The influence on the revertant colony numbers is similar to the effect of histidine supplementation.     

Effects of pH on the mutagenicity of sodium azide in Neurospora crassa and Salmonella typhimurium

Sodium azide at various pH values did not cause a significant increase in the frequency of forward mutation above the control frequency at the adenine-3 (ad-3) region in resting conidia and in conidia from growing cultures of heterokaryons 12 and 59 of Neurospora crassa. Conidia from ad-3 mutants were plated with sodium azide at various pH values, and no obvious increase in reverse mutation above the controls was observed. Data are presented showing that sodium azide at pH 3 is inactivating conidia by interacting with the cytoplasma rather than the nucleus, and this may be the primary reasons that no mutation at the ad-3 region was detected. The dependence of sodium azide mutagenicity on pH was investigated in histidine-requiring mutants of Salmonella typhimurium using a suspension test. There were no significant differences in the reversion frequencies among the pH values (3-8) tested. Thus, no pH dependence is associated with sodium azide mutagenicity, nor are growth and/or DNA replication required for mutagenicity by sodium azide, in S. typhimurium.     

Effect of the homokaryotic state of the uvs-2 allele in Neurospora crassa on formaldehyde-induced killing and ad-3 mutation

Formaldehyde was tested for its killing and mutagenic activities in the ad-3 forward-mutation test in Neurospora crassa. The test was conducted in 3 two-component heterokaryons (dikaryons) of N. crassa in order to determine the effect of the uvs-2 allele, which causes a defect in nucleotide excision repair, on formaldehyde-induced killing and the induction of ad-3 mutants. These dikaryons were homokaryotic for uvs-2+ (H-12), homokaryotic for usv-2 (H-59), and heterokaryotic for uvs-2 (H-71). Formaldehyde induced killing and ad-3 mutants in H-12, but the presence of uvs-2 in the homokaryotic state (H-59) resulted in a 9-fold increase in killing and a 40-fold increase in the induction of ad-3 mutants. This increased sensitivity to formaldehyde-induced killing and mutation conferred by uvs-2 in the homokaryotic state (H-59 vs. H-12) is similar to that noted by others in Escherichia coli. Salmonella typhimurium and Saccharomyces cerevisiae. The dikaryon heterokaryotic for uvs-2 (H-71) has the same sensitivity to formaldehyde-induced ad-3 mutation as H-12, indicating that uvs-2 is recessive to uvs-2+.     

The role of the excision and error-prone repair systems in mutagenesis by fluorinated quinolones in Salmonella typhimurium.

Patterns of reversion produced by ciprofloxacin, enoxacin and ofloxacin in Salmonella typhimurium strains carrying the hisG428 ochre mutation have been studied. These fluorinated quinolones produce a significant increase in reversion of this mutation, even when it is located on the chromosome. Nevertheless, reversion is higher when the hisG428 mutation is on the multicopy plasmid pAQ1 than when it is on the chromosome. Reversion of hisG428 induced by fluorinated quinolones is abolished both in a uvrB genetic background and in the absence of the plasmid pKM101. Therefore, mutagenesis produced by fluorinated quinolones in the Salmonella mutagenicity assay is significantly affected by both the excision repair and the error-prone repair systems. Furthermore, fluorinated quinolones are also detected as moderate mutagens with the base substitution hisG46 mutation when both repair systems are functional in the tester strain.     

Influence of uvrB and pKM101 on the spectrum of spontaneous, UV- and gamma-ray-induced base substitutions that revert hisG46 in Salmonella typhimurium

Oligonucleotide probes were used to identify base substitutions in 1089 revertants of hisG46 in Salmonella typhimurium that arose spontaneously or following irradiation with UV- or gamma-rays. The hisG46 allele, carrying a mutant CCC codon (Pro) in place of the wild-type codon CTC (Leu69) reverted via 6 distinguishable mutational events--C to T transitions at codon sites 1 or 2, C to A or C to G transversions at codon site 1, C to A at codon site 2, and an extragenic suppressor mutation. The distribution of hisG46 revertants differed among treatments and was influenced by the DNA-repair capacity of the bacteria. Plasmid pKM101 enhanced the frequencies of both spontaneous and induced mutations; transversion events were enhanced more efficiently by pKM101 than were transition events. Compared to Uvr+ bacteria, Uvr- bacteria had higher frequencies of spontaneous and induced mutations; transition mutations were enhanced more efficiently than were transversion mutations. The influence of DNA-repair activities on the mutational spectra provides some insights on the origins of spontaneous and UV-induced mutations.     

Genetic effects of 5-hydroxymethyl-2'-deoxyuridine, a product of ionizing radiation

Ionizing radiation causes formation of heterogeneous types of damage to DNA. Among those, 5-hydroxymethyl-2'-deoxyuridine (HMdU) was identified as a major thymidine derivative in gamma-irradiated HeLa cells [G.W. Teebor, K. Frenkel and M.S. Goldstein (1984) Proc. Natl. Acad. Sci. (U.S.A.), 81, 318-321]. We report here that HMdU is a strong inducer of lambda prophage in Escherichia coli WP2s(lambda) and is highy mutagenic in Salmonella typhimurium. HMdU causes his+ revertants in strains TA100, which reverts predominantly by base-pair substitution at G-C sites, and TA97, which reverts mainly by frameshift mutation at G-C sites. It does not cause reversion in TA98, another frameshift-sensitive strain, nor in strains TA1535 and TA1537. Of those tested, only the last two strains do not contain pkM101, a plasmid which enhances mutagenic effects of ionizing radiation. HMdU also causes reversion in strains TA102 and TA104, which detect oxidative damage and can revert by base-pair substitution at A-T base pairs at the hisG428 site. We show that HMdU can be incorporated into DNA of TA100 and that, in addition to causing point mutations, it causes suppressor mutations as well. The ability of HMdU to induce lambda prophage and its strong mutagenicity in Salmonella typhimurium provide evidence that the presence of HMdU in DNA is biologically significant and may play a major role in the genetic consequences of ionizing radiation and other types of oxidative damage.     

Deletions fusing the hisG and hisD genes in Salmonella typhimurium.

Frameshift mutation hisD497 occurs in the operator-proximal portion of the Salmonella typhimurium gene coding for the dimeric protein, L-histidinol dehydrogenase (HDH). Rare revertants of hisD497 are deletions fusing the hisD gene to the adjacent preceding structural gene, hisG (adenosine 5'-triphosphate-PR transferase). HDH purified from one revertant, hisGD4908, contains subunits of approximately normal molecular weight but with no clearly demonstrable unique amino-terminal sequence. We propose that a combined inactive G-D polypeptide is synthesized and then cleaved at a number of closely juxtaposed sites by endoproteolytic activity. At least some of the resulting fragments then participate in formation of active HDH dimers.     

Effects of amount and type of agar on the number of spontaneous revertants in the Ames test

It has been shown that the spontaneous revertants in the Ames test can depend on the amount and type of bottom agar on the plates. The most clear-cut effect was found with the his G46 strains, carrying the delta uvrB mutation, and with the new hisG428 strain, TA102. Evidence of mutagenic impurities in agar has been found.     

Preferential induction of AT-TA transversion, but not deletions, by chlorambucil at the hisG428 site of Salmonella typhimurium TA102.

The chemotherapeutic agent chlorambucil effectively induces deletion mutations in mouse germ cells. The possibility that this chemical also effectively induces deletion mutations in bacterial DNA was examined using Ames Salmonella tester strains. Chlorambucil was mutagenic only to strains TA102 (hisG428, rfa/pKM101) and YG2975 (hisG46, rfa/pKM101) when S9 mix was absent. Since strain TA102 can detect short deletions, the mutational changes of TA102 induced by this agent without S9 mix were directly determined by the DNA sequencing technique. It turned out that chlorambucil did not induce deletion mutations but preferentially induced AT-TA transversions at the hisG428 site of plasmid pAQ1 of strain TA102. These results caution that the positive results induced by chlorambucil in mutagenicity tests do not necessarily mean the occurrence of deletions.     

Analysis of spontaneous and psoralen-induced Salmonella typhimurium hisG46 revertants by oligodeoxyribonucleotide colony hybridization: use of psoralens to cross-link probes to target sequences

An improved DNA colony-hybridization method for the rapid characterization of Salmonella typhimurium hisG46 revertants is described. Oligodeoxyribonucleotides (15-mers) complementary to each of 6 possible transition or transversion mutations and an extragenic suppressor mutation, underlying the His+ phenotype, were prepared. Optimal sequence discrimination was achieved by hybridizing 15-mers at the apparent dissociation temperature (Td) for 2 h with chromosomal DNA of revertant colonies affixed to Whatman 541 filters. Subsequent exposure of filters to UVA radiation (320-400 nm) in the presence of 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) resulted in cross-linking of perfectly matched probes and target DNA sequences while sequences containing a single base-pair mismatch could be discriminated with a brief denaturing wash. No false negative results were obtained with the new procedure. An analysis of 204 spontaneous and 174 PUVA-induced TA100 revertants is presented.     

Mutational studies with diquat and paraquat in vitro.

Diquat and paraquat were assayed in the following tests. (1) Ames test in Salmonella typhimurium (strains TA1535, TA1537, TA1538, TA98 and TA100) with and without rat-liver microsomal fractions. (2) Resistance to 8-azaguanine in Salmonella typhimurium (strain hisG46, TA92 and TA1535. (3) Repair test in Salmonella typhimurium (strains TA1538 and TA1978). (4) Gene mutations in Aspergillus nidulans: 8-AG resistance and methionine suppression (meth A1 locus). (5) Lethal recessive damage in Aspergillus nidulans. (6) Unscheduled DNA synthesis (UDS) in human epithelial-like cells (EUE). Diquat and paraquat were positive in S. typhimurium (in the repair test and the 8-AG resistance system), in A. nidulans (for gene mutations and lethal recessive damage induction) and in EUE cells (UDS induction).     

Isolation of a mutant of Salmonella typhimurium strain TA1535 with decreased levels of glutathione (GSH-). Primary characterization and chemical mutagenesis studies

A mutant of Salmonella typhimurium strain TA1535 with decreased glutathione (GSH) levels was isolated after treatment with UV and selection for N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) resistance; this GSH- mutant also exhibited increased resistance to MNNG, the methyl analog of ENNG. Estimation of the cellular GSH content showed that the GSH- derivative contained about 20% of the GSH levels found in TA1535. In mutagenicity tests (hisG46 leads to His+), the GSH- strain required the presence of GSH or L-cysteine in the medium for an optimal phenotypic expression and/or growth of spontaneous and induced His+ revertants, and may, therefore, be allelic to cys mutants of Salmonella described earlier. The mutagenic activity of MNNG, ENNG and 1,2-dibromoethane (DBE), but not that of N-ethylnitrosourea (ENU), was strongly reduced in TA1535/GSH-; pretreatment of the strain with GSH restored the mutagenicity of the first 3 chemicals to levels normally found in TA1535. The results support the current view that MNNG, ENNG and DBE, but not ENU, can be activated via reaction with GSH to species of higher reactivity and mutagenicity. It is concluded that the present GSH- strain can be used to study more systematically the role of GSH in the bioactivation and -deactivation of xenobiotics to mutagenic factors.     

Mutation induction by the antischistosomal drug F30066 in various test systems.

The genetic activity of furapromidium (F30066), an antischistosomal drug, was studied in Salmonella typhimurium, Saccharomyces cerevisiae, Neurospora crassa and cultured Chinese hamster cells. The results show that F30066 induces gene mutations in S. typhimurium, N. crassa and Chinese hamster cells. This compound also causes gene conversions in S. cerevisiae.     

Mutagenic activity and specificity of N-nitrosomethylaniline and N-nitrosodiphenylamine in Salmonella

The carcinogenic nitrosamines, N-nitrosomethylaniline (NMA) and N-nitrosodiphenylamine (NDphA), which have been previously reported negative or very weakly mutagenic in the Salmonella/microsome assay, were found to be mutagenic in the hisG428 Salmonella strain, TA104. NMA was moderately potent and NDphA was about 10% as potent. Mutagenesis by both compounds was dependent on the uvrB mutation and enhanced in strains harboring the plasmid, pKM101. The mutational specificities of NMA and NDphA for base-pair substitutions were determined by assaying their activities in several mutants which are reverted by a limited number, or a single type of base-pair substitution mutation, and additionally by subclassification of revertants. NMA induced predominantly AT----CG transversions and NDphA induced AT----TA transversions. The specificity of NMA and NDphA for mutagenesis at AT base pairs and the lack of sensitivity of the previously employed hisG46 strains for these base changes may be the reason for the previous reports on the lack of mutagenic activity of these compounds. This specificity is quite unusual for nitrosamines and is consistent with the hypothesis that NMA and NDphA lead to DNA damage of different nature than that produced by other nitrosamines.     

Mutagenicity of fractions of cigarette smoke condensate in Neurospora crassa and Salmonella typhimurium

The mutagenicities of selected fractions of cigarette smoke condensate (CSC) were studied in Neurospora crassa for the presence of direct-acting mutagens. CSCs from the University of kentucky Reference Cigarette 1R1 were assayed in a forward-mutation test at the adenine-3 (ad-3) region in resting conidia of a 2-component heterokaryon. Direct-acting mutagenic activity was found in an enriched polycyclic aromatic hydrocarbons (EPAH) fraction and in a basic fraction (Swain 5). No direct-acting mutagenic activity was detected in an acidic fraction (Swain 8), although it was highly toxic to resting conidia. The EPAH fraction also was tested in the presence of S9 mix prepared from Aroclor-1254-induced rat liver. It was found to be mutagenic, but higher doses were required than in the absence of S9 mix. In addition, the mutagenicities of CSC and 10 fractions of CSC were investigated in Salmonella typhimurium TA1538 by the incorporation and preincubation methods. In general, preincubation did not enhance the mutagenicities of the fractions, and the two rankings of mutagenic potency of the condensates that were obtained by the two methods were not significantly different. This is the first report of the presence of potent direct-acting mutagenicity in the EPAH and Swain 5 fractions of CSC.     

Tests for the genotoxicity of m-AMSA, etoposide, teniposide and ellipticine in Neurospora crassa

The antitumor agents m-AMSA, etoposide, teniposide and ellipticine have been reported to be potent clastogens in mammalian cells but non- or weakly mutagenic in bacteria; these observations have been correlated to the interference of these chemicals with DNA topoisomerase II activity in the former, but not in the latter, organisms. The genotoxicity of these 4 agents was evaluated using ad-3 reverse- and forward-mutation tests in Neurospora crassa. These agents (up to 0.8 mumole/plate) did not cause reversion in conidia of the ad-3A frameshift strains N24 and 12-9-26 using the overlay plate test, as contrasted to the positive control frameshift mutagen ICR-170. Heterokaryon 12 (H-12) of N. crassa permits the recovery of all classes of forward mutation at the ad-3+ region, including multilocus deletions. Using resting conidia of H-12 in a suspension assay, ellipticine was moderately mutagenic but no increase in ad-3 mutants was noted with the other 3 agents at a dose of 100 micrograms/ml. In vegetative cultures of H-12 grown in the presence of these agents, all 4 agents were nonmutagenic at a dose of 100 micrograms/ml. The positive control mutagen ICR-170 was mutagenic in both resting conidia and growing cultures of H-12. A similarity between the topoisomerase II of N. crassa and DNA gyrase of bacteria is suggested.