Comparison of Salmonella typhimurium TA102 with Escherichia coli WP2 tester strains

In 1982, Levin et al. published a paper describing a new Salmonella typhimurium strain, TA102, for detecting mutagenic agents that react preferentially with AT base pairs. This strain has an AT base pair at the critical mutation site within the hisG gene, which is located on a multicopy plasmid, pAQ1; the chromosomal copy of the hisG gene has been deleted. It also has an intact excision repair system, thus facilitating the detection of cross-linking agents, and carries the mutator plasmid, pKM101. Although TA102 has been shown to be reverted by certain mutagenic agents that are not detected in the usual battery of strains (TA1535, TA1537, TA1538, TA98 and TA100), there has been a general reluctance within the field to include TA102 as one of the standard screening strains. This may in part result from the difficulties which have been experienced in many laboratories in maintaining the strain, and in obtaining reproducible spontaneous and induced revertant counts. At Glaxo we routinely include certain Escherichia coli strains in our microbial test battery, and were aware that some of the genetic features offered by TA102 were already being covered by these strains. For example, E.coli WP2 (pKM101) has an AT base pair at the critical mutation site within the trpE gene, is excision proficient (and thus will detect cross-linking agents) and carries the pKM101 plasmid to enhance error-prone repair. From the published literature it was apparent that a number of the 'TA102 specific' mutagens could be detected in E.coli e.g. neocarzinostatin, UV and 8-MOP plus UV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative mutagenic effects of structurally similar flavonoids quercetin and taxifolin on tester strains Salmonella typhimurium TA102 and Escherichia coli WP‐2 uvrA

Quercetin (QT) and Taxifolin (TF) are structurally similar plant polyphenols. Both have been reported to have therapeutic potential as anti‐cancer drugs and antioxidants. Mutagenic effects of QT and TF were evaluated using Salmonella typhimurium TA102 and Escherichia coli WP‐2 uvrA tester strains. Either in the presence or absence of S9 mix, QT was mutagenic to TA102 and WP2 uvrA. However, the mutagenicity of QT was significantly enhanced in the presence of S9 mix. Likewise, in the presence of Iron (Fe2+) and NADPH generating system (NGS) and absence of S9 mix, QT induced significantly high mutations in both TA102 and WP‐2 uvrA. Mutagenicity of QT decreased in both strains in the presence of Iron (Fe2+) or NGS alone. TF was not mutagenic in the presence or absence of S9 mix in both TA102 and WP‐2 uvrA 2, regardless of the presence of iron or NGS. Incorporation of antioxidants (ascorbate, superoxide dismutase (SOD), catalase (CAT)) and/or iron chelators (desferroxamine (DF) and ethylenediamine‐tetraacetate (EDTA)) in the test systems markedly decreased QT‐induced mutations in both tester strains. These results suggest that QT but not TF, could induce mutations in the presence or absence of rat liver S9 or Iron (Fe2+) and NGS in both tester strains by redox cycling and Fenton reactions to produce oxygen free radicals. Our results indicate that a minor structural variation between the two plant polyphenols could elicit a marked difference in their genotoxicities. These results provide a basis for further study into the potential use of QT in combination with iron supplements. Environ. Mol. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.     

Propyldazine is mutagenic in Salmonella typhimurium and Escherichia coli: distinct specificity for strains TA1537 and TA97

The antihypertensive drug propyldazine (Atensil) was demonstrated to be mutagenic with auxotrophic mutants of Salmonella typhimurium and Escherichia coli. Addition of liver S9 mix (postmitochondrial supernatant fraction supplemented with an NADPH-generating system) had little, if any, effect on the mutagenicity. The mutagenicity showed an unusual pattern of strain specificity. Increased frequencies of reversion were observed with all strains whose auxotrophy was caused by frame-shift mutations: the number of revertant colonies per plate from S. typhimurium TA98, TA1538, TA97, and TA1537 was increased up to 5-, 9-, 43-, and 160-fold, respectively, above background. Among the strains that became auxotrophic by substitution mutations, S typhimurium TA102, E. coli WP2, and E coli WP2 uvrA yielded positive results (twofold above background). S. typhimurium TA1535 and TA100 were not reverted by propyldazine. It should be noted that propyldazine, due to its low toxicity and good solubility, could be tested up to very high doses. Hence, although quite impressive mutagenic effects occurred, the mutagenic potency was moderate even in the most responsive strains, TA1537 and TA97 (about 0.3 and 1.0 revertants per nmole, respectively). With the limitation that the strain specificities were different, the mutagenic potency of propyldazine was in the same order of magnitude as that of hydralazine and dihydralazine, two related antihypertensive drugs which were already known to be mutagenic. In our hands, both compounds were mutagenic in S typhimurium TA1535, TA100, TA1537, and TA98. These results differ from data in the literature in that we found clear but weak effects even with strains for which others have reported negative results.     

Comparisons of mutation induction by six monocyclic aromatic amines in Salmonella typhimurium tester strains TA97, TA1537, and TA1538

The mutagenicity of six monocyclic aromatic amines (2,4-diaminoanisole, 2,4-diaminoethoxybenzene, 2,4-diaminopropoxybenzene, m-phenylenediamine, 2,4-diaminotoluene, and nitro-p-phenylenediamine) was investigated in Salmonella typhimurium strains TA97, TA1537, and TA1538 in the presence of two different amounts of Aroclor 1254-induced S9 preparations. Strain TA1538 was found to be the most responsive of the three strains with this group of compounds. Regarding the other strains, TA1537 responded to three of the compounds better than strain TA97, if one calculates responsiveness as the fold-increase in numbers of revertants per plate. However, if one calculates the number of revertants per nanomole or compares the number of induced revertants per plate, TA97 was more responsive than TA1537 for all six compounds. Comparisons of mutagenesis from tests involving strain TA97 are complicated by the large variations in spontaneous mutation frequencies in this strain. The amount of S9 per plate is another important variable in tests of monocyclic aromatic amines; in general, more revertants are detected when the S9 mix contains 10% S9 than when it contains 4% S9. Nevertheless, in all our tests of 2,4-diaminoanisole, 2,4-diaminoethoxybenzene, and 2,4-diaminopropoxybenzene, the same relationship between chemical structure and mutagenic activity was observed. In all three strains, the mutagenic responses become less when the alkoxy substituent on the molecule becomes larger.     

Cd2+ tolerance in Escherichia coli and Salmonella typhimurium

Relative tolerance of Escherichia coli strains to Cd2+ is induced by prior growth in medium containing low levels of either Cd2+ or Hg2+, and maximal induction appears to be dependent upon recA+ function. Biosynthesis of glutathione is not required for induction or for expression of induced resistance. Salmonella typhimurium strains are relatively resistant to Cd2+, and this resistance is essentially constitutive.     

Mutagenicity and toxicity of the DNA alkylation carcinogens 1, 2-dimethylhydrazine and azoxymethane in Escherichia coli and Salmonella typhimurium

DNA alkylating agents such as 1, 2-dimethylhydrazine (SDMH)and azoxymethane (AOM) are potent carcinogens and are widelyused to induce colon tumors in experimental animals. However,standard bacterial mutagenesis assays have failed to detectthe mutagenic effects of these chemicals. Using derivativesof a set of Escherichia coli test strains developed by Cupplesand Miller (Proc. NatL Acad. Set USA, 86, 5345, 1989), we havedemonstrated that under two conditions, SDMH and AOM inducedpoint mutations by several-fold in a dose-dependent manner:(i) of six possible base substitutions, they only induced GC     

Mutagenicity of 2-alkylpropenals in Salmonella typhimurium strain TA 100: structural influences.

alpha,beta-Unsaturated aldehydes are a class of mutagenic and carcinogenic compounds that form promutagenic 1,N(2)-propanodeoxyguanosine adducts. They are important industrial and environmental compounds, are formed endogenously, and are found in food. We recently published structure-mutagenicity relationships for 3-alkyl substituted alpha,beta-unsaturated aldehydes (beta-alkylacroleins) and here we present structural influences on the mutagenicity of the 2-alkyl substituted alpha,beta-unsaturated aldehydes (alpha-alkylacroleins), 2-methylacrolein, 2-ethylacrolein, 2-propylacrolein, and 2-butylacrolein, in Salmonella typhimurium TA 100. All four alkylacroleins are mutagenic without S9-mix; however, the results are strongly influenced by bacterial toxicity of the alkylacroleins. In general, toxicity increases with increasing length of the alkyl substituent. The increasing toxicity with increasing alkyl groups can be explained by increasing lipophilicity that allows the compounds to better penetrate into the bacterial cell. Other structural effects, such as steric hindrance of the deoxyguanosine binding (DNA-adduct formation) and the positive inductive effect of the alkyl groups, have only a slight effect on mutagenesis. Addition of S9-mix leads to an increase in the absolute revertant peak values but a decrease in mutagenic activities, as expressed by revertants per micromol. This effect is also observed with heat-inactivated S9-mix and does not depend on metabolic activation. The effect of S9-mix can be explained by partial detoxication of the substances by nucleophilic components of the S9-mix such as glutathione.     

Studies of error-prone DNA repair in Escherichia coli K-12 and Ames Salmonella typhimurium strains using a model alkylating agent

4-Acetoxy-3-acetoxymethyl acetophenone (AAMAP) is muta-genicin Ames Salmonella typhimurium tester strains TA100 and TA98,which carry plasmid pKM101, but not in the isogenic plasmid-lessstrains TA1535 and TA1538. Similarly, no AAMAP-induced reversionof the his-4 allele is detectable in Escherichia coli K-12 umuCstrains in the absence of the plasmid, even when the strainsare treated with ethylene-diaminetetraacetate to increase permeability,or when the uvrB allele is introduced to increase error-proneDNA repair. AAMAP is, however, mutagenic in umuC⁺ strains orin umuC strains in which plasmid pKM101 has been introduced,suggesting that the plasmid-encoded MucAB or the chromosomallydetermined UmuDC proteins are required for mutagenesis. Mutationfrequencies are higher in E. coli umuC (pKM101) strains, whichresemble Ames tester strains of S.typhimurium, than in E.coliumuC⁺ or even umuC⁺ (pKM101) strains. Therefore, providing thatthe recommended pKM101-containing tester strains are used, theapparent absence of Umu-like protein activity in S. typhimuriummay actually increase the sensitivity of the Ames test for thedetection of mutagens that require error-prone DNA repair foractivity. ^* Parts of this paper were communicated to the Fourth InternationalConference on Environmental Mutagens, Stockholm, 1985. ²To whom correspondence should be addressed      

Comparison of the sensitivities of Salmonella typhimurium strains TA102 and TA2638A to 16 mutagens

The qualitative and quantitative sensitivity of the genetically related, histidine-auxtrophic Salmonella typhimurium strains TA102 and TA2638a to 16 compounds was examined. The compounds were mainly cross-linking and oxidising mutagens, the effects of which were known to be detected by strain TA102 preferentially or by a combination of Escherichia coli WP2 (pkM101) and uvrA/pkM101. The morphology and number of spontaneous revertants was also compared. Fourteen of the 16 compounds caused reversion in both strains. Bleomycin and streptomycin induced reversion in strain TA102 but not TA2638a. The greater sensitivity of TA102 to these compounds may be associated with the extrachromosomal location of the target genes. The overall quantitative sensitivity of the two strains was similar for the other compounds. The number of compounds that caused reversions at lower doses or produced greater proportional increases were the same in TA102 as in TA2638a. The spontaneous number of revertants, without and with metabolic activation, respectively, was 98 and 130 for TA2638a and 322 and 465 for TA102. Strain TA2638a formed larger, more uniform colonies than TA102. The present results together with those of previous studies indicate a high degree of concordance between the sensitivity of strains TA102 and TA2638 for the detection of mutagens. The uniform colony size and lower spontaneous reversion frequency seen with strain TA2638a compared with TA102 would make it more reliable and convenient for routine testing. It is concluded that strain TA2638a should be considered as an alternative to TA102 and included, as well as the two E.coli strains, in the set of bacterial strains used in the standard test battery for mutagenicity testing.     

Collaborative study of interlaboratory variability in Salmonella typhimurium TA102 and TA2638 and Escherichia coli WP2/pKM101 and WP2 uvrA/pKM101

A collaborative study of interlaboratory variability in bacterialmutagenicity induced by mitomycin C (MMC) and bleomycin (BLM)was performed using the four strains Salmonella typhimuriumTA102 and TA2638 and Escherichia coli WP2/pKM101 and WP2 uvrA/pKM101.Thirteen laboratories participated in this study. The four strainsand two chemicals were sent from a central source to each laboratory.Each strain was cultured in nutrient broth containing antibioticsand laboratories were requested to ensure that the spontaneouscounts for marker check fell within a specified range in thepreparation of the original stock culture. Concerning the responseto the chemicals, most interlaboratory variability was withina 4-fold range for tests with TA102, TA2638 and WP2/pKM101.From the results of the statistical analysis using the linearregression test, positive results in TA102, TA2638 and WP2/pKM101and negative results in WP2 uvrA/pKM101 were obtained by MMCtesting in all of the laboratories. On the other hand, withBLM testing, considerable interlaboratory variability was observedbetween the strains, largely because of the variation in resultsof the repeat experiments. Overall mean spontaneous revertantcounts in all laboratories were within acceptable ranges forall four bacterial strains. In conclusion, it is judged thatamong Japanese laboratories, there is excellent agreement inthe tested response of these bacterial strains to a strong mutagensuch as MMC, as well as uniformity in the spontaneous reversionrates. However, for chemicals such as BLM that induce a weakresponse, it may be necessary to repeat experiments severaltimes to obtain clear results. In this study, TA102 was shownto be a useful strain for routine mutagenicity testing. Thisnecessitated control over culture maintenance by the additionof tetracycline and strict selection in the preparation of theoriginal stock cultures.     

Mutagenicity of carbon tetrachloride and chloroform in Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2uvrA/pKM101 and WP2/pKM101, using a gas exposure method

The volatile solvents carbon tetrachloride and chloroform are carcinogens that are often reported as nonmutagenic in bacterial mutagenicity assays. In this study, we evaluated the mutagenicity of these compounds in Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2uvrA/pKM101 and WP2/pKM101, with and without S9 mix, using a gas exposure method. Tests were also conducted with a glutathione-supplemented S9 mix. Carbon tetrachloride was mutagenic in TA98 without S9 mix, and in WP2/pKM101 and WP2uvrA/pKM101 with and without S9 mix; carbon tetrachloride was not mutagenic in TA100, TA1535 or TA1537. Chloroform was mutagenic in WP2/pKM101, but only in the presence of glutathione-supplemented S9 mix. Chloroform was not mutagenic in TA98, TA100, TA1535, TA1537, or WP2uvrA/pKM101 with or without S9 mix, and was not mutagenic in TA98, TA100, TA1535, TA1537, or WP2uvrA/pKM101 in the presence of glutathione-supplemented S9 mix. The data indicate that carbon tetrachloride and chloroform are bacterial mutagens when adequate exposure conditions are employed and suggest that a genotoxic mode of action could contribute to the carcinogenicity of these compounds.     

Isolation of streptomycin-dependent strains from salmonella typhimurium TA98 and TA100 and their use in mutagenicity tests

Streptomycin-dependent (SMd) mutant strains of Salmonella typhimurium TA98 and TA100 were isolated. A highly sensitive detection system was obtained by using these SMd derivatives for the SMd leads to SMind mutation. The system was available for testing the mutagenicities of samples containing histidine, such as food.     

HscA is involved in the dynamics of FtsZ-ring formation in Escherichia coli K12

BACKGROUND: FtsZ, a homologue of eukaryotic tubulin, localizes throughout the cytoplasm in non-dividing Escherichia coli. However, it assembles in cytokinetic rings at the early stages of septation. Factors controlling the dynamics of FtsZ ring formation are unknown, and the molecular mechanism governing these dynamics is yet to be determined. RESULTS: At 42 degrees C, JE10715 mutant bacteria formed multinucleated filaments with a highly reduced number of FtsZ-rings at potential division sites. The JE10715 phenotype resulted from a mis-sense mutation in the hscA gene which encodes a heat shock Hsp70 family protein, with a single alanine-to-valine substitution at position 192 within the ATPase domain. Both JE10715 and the hscA knockout strain of JE10715 were completely complemented by a plasmid-born, wild-type hscA gene, but not by a mutant-type hscA715 gene. An hscA conditional knockout of the wild-type strain under non-permissive conditions exhibited longer rod cells with an abnormal localization of FtsZ. The over-expression of dnaK partially complemented the JE10715 mutation. In vitro, the ATPase activity of the mutant protein HscA715 was reduced to 63% of wild-type HscA activity. HscA co-sedimented with FtsZ-polymers in the presence of GTP. CONCLUSION: HscA is involved in FtsZ-ring formation, through a chaperon-like interaction with FtsZ. Defects in hscA, however, can partially be compensated for by redundant genes, including the wild-type dnaK.     

Genotoxicity of quinoxaline 1,4-dioxide derivatives in Escherichia coli and Salmonella typhimurium

The mutagenicities of 5 quinoxaline 1,4-dioxide (QdO) derivatives were tested by 2 bacterial assays using forward mutation with Escherichia coli WP2uvrA/pKM101 and reverse mutation with Salmonella typhimurium TA100 and TA98. Potent mutagenic activities of all QdOs tested were observed in both mutation assays. Mutagenicities of these compounds were varied by addition of S9 mix. Their SOS-inducing activities were examined with a ‘Rec-lac test’ that has been newly developed by us for detecting genotoxins. A high level of SOS-inducing activity was observed in all samples tested. These results suggest that the mutagenicity of QdOs results from the error-prone repair involved in SOS responses.     

Different systems for iron supply of Salmonella typhimurium and Escherichia coli wild strains as tool for typing.

Hitherto the siderophore-pattern analysis included bioassays to detect enterobactin,aerobactin and other siderophores of Enterobacteriaceae. In addition, 2,3-dihydroxybenzoic acid, the precursor of enterobactin, and new hydroxamate siderophores could be examined by means of two new Salmonella mutants as indicator strains. In this manner the siderophore pattern analysis extends its discriminating ability. Among 167 Salmonella typhimurium strains tested, we detected 5 siderophore patterns. 6 siderophore patterns could be detected among a total of 204 E. coli strains. Using siderophore pattern analysis for clinical-epidemiological and ecological purposes appropriate technical methods can be recommended.     

Factors influencing the copy number of F-like plasmids in Escherichia coli and Salmonella typhimurium

The copy numbers of Flac, four F-like plasmids and pLT2 were estimated in two strains of Salmonella typhimurium and (for all except pLT2) one strain of Escherichia coli. For organisms grown in casamino acids minimal medium, the plasmids spanned a 7–8 fold range of copy number with ColB-K98 having the highest copy number in each strain and R124 the lowest. The copy number of ColB-K98 was substantially greater than 1 in each of the strains tested. There was no clear relation between the plasmid size and copy number, although the plasmids studied spanned only a narrow size range. The copy number of individual plasmids was slightly reduced or not affected at all by the presence of a second plasmid in the same strain. Derivatives harbouring each of the plasmids were grown in three different media to ascertain how plasmid copy number responds to changes in growth rate. For each plasmid, the copy number increased with decreasing growth rate. Extracts from each of the three strains harbouring ColB-K98 contained two distinct plasmid species. One appeared to be about twice as large as the other and both were absent from Col^? segregants.     

Selection of agar for use in Salmonella typhimurium and Escherichia coli mutation assays

The spontaneous and induced revertant frequency of four Salmonella typhimurium strains (TA1535, TA1537, TA98, and TA100) and Escherichia coli [WP2 uvrA (pKM101)] was evaluated using Vogel Bonner minimal plates prepared with ten different agars. In addition to the Difco Bacto agar originally recommended by Ames, Difco Noble, granulated and Bitek agars; BD grade A, BBL granulated and purified agars; Oxoid purified and No. 1 agars; and GIBCO select agar were tested. Several of these agars have been reported as acceptable alternatives for these Salmonella strains, but comparable studies with E. coli have not been done. The bacteria were treated with DMSO or an appropriate positive control in the presence or absence of an Aroclor 1254-induced rat liver activation system. With the exception of Noble agar in the presence of S9, there was little difference among the responses of the Salmonella strains on any of the agars. However, with E. coli the responses include either a reduction or an increase in spontaneous revertants numbers as well as a reduction in absolute and relative induced revertant frequency. Difco Bacto agar appears to be the most consistent agar for use with these strains. As an alternative, only BBL purified agar resulted in consistent results for all of these strains under all testing conditions. These results emphasize the need to evaluate the components of the standard mutation assay when incorporating additional bacterial strains. Suboptimal responses related to the agar or other components could compromise the detection of weak mutagens. Environ. Mol. Mutagen. 32:192–196, 1998 © 1998 Wiley-Liss, Inc.     

Induction of SOS genes in Escherichia coli and mutagenesis in Salmonella typhimurium by fluoroquinolones

The induction of several SOS genes of Escherichia coli by fluoroquinolones has been studied. Three different SOS gene fusions (recA::lacZ, umuC::lacZ and sulA::lacZ) have been introduced into the E.coli MC1061 strain to study the induction of these SOS genes in the same genetic background. Data on the basal level of expression of these fusions, as well as their induction by mitomycin C and N-methyl-N'-nitro-N-nitrosoguanidine are presented. Using these strains, we have found that, like nalidixic acid, ofloxacin, enoxacin and ciprofloxacin are strong inducers of the SOS genes tested, umuC gene expression being the highest. Furthermore, fluoroquinolones produced a significant increase in the reversion of the base substitution hisG428 mutation in the TA102 Salmonella tester strain, while no effect was found in strains TA98, TA100, TA1537 and TA1535. These data indicate that the error-prone repair pathway can participate in mutagenesis induced by fluoroquinolones and also that the damage produced by these chemicals may be similar to that produced by nalidixic acid.