mutM, a second mutator locus in Escherichia coli that generates G.C----T.A transversions.

We used strains carrying specific lacZ alleles to identify a new mutator locus in Escherichia coli which generates only G.C----T.A transversions among base substitutions. The locus, mutM, mapped near the cysE locus, which is at 81 min on the genetic map.     

Yersinia pestis lacZ expresses a beta-galactosidase with low enzymatic activity

Although very little, if any, beta-galactosidase activity is detected in Yersinia pestis by a standard Miller assay, we found that Y. pestis KIM6+ cells formed blue colonies on plates containing 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal). Searches of the Y. pestis genome databases revealed the presence of noncontiguous sequences highly homologous to Escherichia coli lacZ, lacY, and lacI. Yersinia pestis lacZ is predicted to encode a 1060 amino-acid protein with 62% identity and 72% similarity to beta-galactosidase from E. coli. A deletion in the Y. pestis lacZ gene caused the formation of white colonies on X-gal-containing plates and beta-galactosidase activity was at background levels in the KIM6+lacZ mutant, while the complemented strain expressed about 190 Miller units. The Y. pestis lacZ promoter was not regulated by isopropylthiogalactoside or glucose. Finally, uptake of lactose by Y. pestis may be impaired.     

Analysis of spontaneous base substitutions generated in mutator strains of Bacillus subtilis

In the current studies, we investigated base substitutions in the Bacillus subtilis mutT, mutM, and mutY DNA error-prevention system. In the wild type strain, spontaneous mutations were mainly transitions, either G:C --> A:T or A:T --> G:C. Although both transitions and transversions were observed in mutY and mutM mutants, mutM/mutY double mutants contain strictly G:C --> T:A transversions. In the mutT strain, A:T --> C:G transversion was not observed, and over-expression of the B. subtilis mutT gene had no effect on the mutation rate in the Escherichia coli mutT strain. Using 8-oxo-dGTP-induced mutagenesis, transitions especially A:T --> G:C were predominant in the wild type and mutY strains. In contrary, transversion was high on mutY and double mutant (mutM mutY). Finally, the opuBC and yitG genes were identified from the B. subtilis chromosome as mutator genes that prevented the transition base substitutions.     

Interactions among the Escherichia coli mutT,mutM, and mutY damage prevention pathways

We have investigated in detail the interactions between the Escherichia coli mutT, mutM, and mutY error-prevention systems. Jointly, these systems protect the cell against the effects of the oxidative stress product, 8-oxoguanine (8-oxoG), a base analog with ambiguous base-pairing properties, pairing with either A or C during DNA synthesis. mutT mutator strains display a specific increase in A.T-->C.G transversions, while mutM and mutY mutator strains show specific G.C-->T.A increases. To study in more detail the in vivo processing of the various mutational intermediates leading to A.T-->C.G and G.C-->T.A transversions, we analyzed defined A.T-->C.G and G.C-->T.A events in strains containing all possible combinations of these mutator alleles. We report three major findings. First, we do not find evidence that the mutT allele significantly increases G.C-->T.A transversions in either mut(+), mutM, mutY or mutMmutY backgrounds. We interpret this result to indicate that incorporation of 8-oxodGTP opposite template C may not be frequent relative to incorporation opposite template A. Second, we show that mutT-induced A.T-->C.G transversions are significantly reduced in strains carrying mutY and mutMmutY deficiencies suggesting that 8-oxoG, when present in DNA, preferentially mispairs with dATP. Third, the mutY and mutMmutY deficiencies also decrease A.T-->C.G transversions in the mutT(+) background, suggesting that, even in the presence of functional MutT protein, A.T-->C.G transversions may still result from 8-oxodGTP misincorporation.     

Escherichia coli mutT mutator effect during in vitro DNA synthesis. Enhanced A.G replicational errors

The mechanism of the Escherichia coli mutT mutator effect was investigated using single-stranded phage as a mutational target. In vivo experiments showed that two M13mp2 lacZ alpha nonsense mutants reverted at a higher rate on a mutT1 host than on the wild-type host. The specificity of this mutator effect was identical to that observed for E. coli genes: A.T----C.G transversions. The mutT effect was subsequently demonstrated in vitro during DNA replication of M13mp2 DNA in cell-free extracts of E. coli. Replication (the single-stranded----replicative form conversion) in mutT1 extracts proceeded with a higher error rate than in wild-type extracts, and DNA sequence analysis of the in vitro revertants revealed the specific induction of A.T----C.G transversions. On the basis of the template specificity of the mutT effect in vitro, we conclude that the mutT effect involves the aberrant processing of A.G rather than T.C mispairs.     

RecA-independent mutagenesis in Escherichia coli may be subject to glucose repression

The frameshift mutagen 9-aminoacridine (9AA) causes DNA damage via a recA+-independent mechanism in Escherichia coli. In this study we have exposed E. coli cells carrying the lacZ19124 frameshift marker to 9AA in defined minimal media, washed them, and plated to score for Lac+ revertants. Our results show that 9AA-induced reversion to Lac+ occurs in the absence of any exogenous carbon source and when cells are plated on media which do not allow much, if any, cell replication prior to expression of the revertant phenotype. When glycerol (1% w/v) was added to the liquid treatment medium, the number of Lac+ E. coli revertants was similar to that obtained when no carbon source was present. By contrast the addition of glucose (1% w/v) during the mutagenesis treatment caused a significant decrease in the number of revertants. Further experiments indicate that the repressing effects of glucose may be due to a reduction in cAMP concentration, since 9AA mutagenesis was abolished in a cya strain in which no adenylate cyclase is produced. These results are consistent with (but do not prove) the notion that at least one part of the process leading to 9AA mutagenesis is subject to catabolite repression.     

Mutagenic specificity of imidazole ring-opened 7-methylpurines in M13mp18 phage DNA

The most abundant lesion formed in DNA upon modification with methylating agents 7-methylguanine, under alkaline conditions is converted into 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7MeGua). We have previously shown that treatment of dimethylsulfate methylated DNA with NaOH creates mutagenic base derivatives leading to a 60-fold increase in the frequency of A-->G transitions and a 2-3-fold increase of G-->T and G-->C transversions. We have analyzed which lesions lead to these mutations. We compared mutagenic spectra in the lacZ gene of M13mp18 phage DNA modified with dimethylsulfate and NaOH after selective elimination of damaged bases from molecules used for transfection into SOS-induced E. coli. Partial elimination of Fapy-7MeGua from phage DNA performed by its digestion with formamidopyrimidine-DNA glycosylase resulted in a 2-3-fold decrease of G-->T and G-->C transversions. Selective depurination of methylated bases (9 h, 37 degrees C, pH 7.0) resulting in almost complete loss of 7MeAde as demonstrated by HPLC analysis of [3H]MNU alkylated phage DNA used as a probe, caused a dramatic, 9-fold decrease of A-->G transitions. Alkali-catalysed rearrangement of 7MeAde was followed by HPLC analysis of [3H]MNU alkylated poly(A) and poly(dA). After incubation of these oligonucleotides in NaOH, 7MeAde disappeared from both chromatograms, but only in polyA, 2 new peaks migrating with retention time different from that of 1MeAde, 3MeAde or 7MeAde were detected, suggesting formation of two rotameric forms of Fapy-7MeAde as observed for Fapy-7MeGua. Thus the miscoding lesion, giving rise to A-->G transitions derived from 7MeAde was Fapy-7MeAde. Fapy-7MeGua was at least an order of magnitude less mutagenic, but in SOS-induced cells it gave rise to G-->T and G-->C transversions.     

Escherichia coli MutY protein has a guanine-DNA glycosylase that acts on 7,8-dihydro-8-oxoguanine:guanine mispair to prevent spontaneous G:C-->C:G transversions.

Low rates of spontaneous G:C-->C:G transversions would be achieved not only by the correction of base mismatches during DNA replication but also by the prevention and removal of oxidative base damage in DNA. Escherichia coli must have several pathways to repair such mismatches and DNA modifications. In this study, we attempted to identify mutator loci leading to G:C-->C:G transversions in E.coli. The strain CC103 carrying a specific mutation in lacZ was mutagenized by random miniTn 10 insertion mutagenesis. In this strain, only the G:C-->C:G change can revert the glutamic acid at codon 461, which is essential for sufficient beta-galactosidase activity to allow growth on lactose. Mutator strains were detected as colonies with significantly increased rates of papillae formation on glucose minimal plates containing P-Gal and X-Gal. We screened approximately 40 000 colonies and selected several mutator strains. The strain GC39 showed the highest mutation rate to Lac+. The gene responsible for the mutator phenotypes, mut39 , was mapped at around 67 min on the E.coli chromosome. The sequencing of the miniTn 10 -flanking DNA region revealed that the mut39 was identical to the mutY gene of E.coli. The plasmid carrying the mutY + gene reduced spontaneous G:C-->T:A and G:C-->C:G mutations in both mutY and mut39 strains. Purified MutY protein bound to the oligonucleotides containing 7,8-dihydro-8-oxo-guanine (8-oxoG):G and 8-oxoG:A. Furthermore, we found that the MutY protein had a DNA glycosylase activity which removes unmodified guanine from the 8-oxoG:G mispair. These results demonstrate that the MutY protein prevents the generation of G:C-->C:G transversions by removing guanine from the 8-oxoG:G mispair in E.coli.     

DNA alkylation repair limits spontaneous base substitution mutations in Escherichia coli.

The Escherichia coli Ada and Ogt DNA methyltransferases (MTases) are known to transfer simple alkyl groups from O6-alkylguanine and O4-alkylthymine, directly restoring these alkylated DNA lesions to guanine and thymine. In addition to being exquisitely sensitive to the mutagenic effects of methylating agents, E. coli ada ogt null mutants display a higher spontaneous mutation rate than the wild type. Here, we determined which base substitution mutations are elevated in the MTase-deficient cells by monitoring the reversion of six mutated lacZ alleles that revert via each of the six possible base substitution mutations. During exponential growth, the spontaneous rate of G:C to A:T transitions and G:C to C:G transversions was elevated about fourfold in ada ogt double mutant versus wild-type E. coli. Furthermore, compared with the wild type, stationary populations of the MTase-deficient E. coli (under lactose selection) displayed increased G:C to A:T and A:T to G:C transitions (10- and 3-fold, respectively) and increased G:C to C:G, A:T to C:G, and A:T to T:A transversions (10-, 2.5-, and 1.7-fold, respectively). ada and ogt single mutants did not suffer elevated spontaneous mutation rates for any base substitution event, and the cloned ada and ogt genes each restored wild-type spontaneous mutation rates to the ada ogt MTase-deficient strains. We infer that both the Ada MTase and the Ogt MTase can repair the endogenously produced DNA lesions responsible for each of the five base substitution events that are elevated in MTase-deficient cells. Simple methylating and ethylating agents induced G:C to A:T and A:T to G:C transitions in these strains but did not significantly induce G:C to C:G, A:T to C:G, and A:T to T:A transversions. We deduce that S-adenosylmethionine (known to e a weak methylating agent) is not the only metabolite responsible for endogenous DNA alkylation and that at least some of the endogenous metabolites that cause O-alkyl DNA damage in E. coli are not simple methylating or ethylating agents.     

Insertion of an E. coli lacZ gene in Acetobacter xylinus for the production of cellulose in whey

A mini-Tn10:lacZ:kan was inserted into a wild-type strain of Acetobacter xylinus by random transposon mutagenesis, generating a lactose-utilising and cellulose-producing mutant strain designated ITz3. Antibiotic selection plate assays and Southern hybridisation revealed that the lacZ gene was inserted once into the chromosome of strain ITz3 and was stably maintained in non-selective medium after more than 60 generations. The modified strain had, on the average, a 28-fold increase in cellulose production and a 160-fold increase in beta-galactosidase activity when grown in lactose medium. beta-Galactosidase activity is present in either lactose or sucrose medium indicating that the gene is constitutively expressed. Cellulose and beta-galactosidase production by the modified strain was also evaluated in pure and enriched whey substrates. Utilisation of lactose in whey substrate by ITz3 reached 17 g l(-1) after 4 days incubation.     

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.     

Studies on the replication of the ring opened formamidopyrimidine, Fapy.dG in Escherichia coli

Fapy.dG is produced in DNA as a result of oxidative stress from a precursor that also forms OxodG. Bypass of Fapy.dG in a shuttle vector in COS-7 cells produces G --> T transversions slightly more frequently than does OxodG (Kalam, M. A., et al. (2006) Nucleic Acids Res. 34, 2305). The effect of Fapy.dG on replication in Escherichia coli was studied by transfecting M13mp7(L2) bacteriophage DNA containing the lesion within the lacZ gene in 4 local sequence contexts. For comparison, experiments were carried out side-by-side on OxodG. The efficiency of lesion bypass was determined relative to that of a genome containing native nucleotides. Fapy.dG was bypassed less efficiently than OxodG. Bypass efficiency of Fapy.dG and OxodG increased modestly in SOS-induced cells. Mutation frequencies at the site of the lesions in the originally transfected genomes were determined using the REAP assay (Delaney, J. C., Essigmann, J. M. (2006) Methods Enzymol. 408, 1). G --> T transversions were the only mutations observed above background when either Fapy.dG or OxodG was bypassed. OxodG mutation frequencies ranged from 3.1% to 9.8%, whereas the G --> T transversion frequencies observed upon Fapy.dG bypass were T transversions.     

A Search for a General Phenomenon of Adaptive Mutability

The most prominent systems for the study of adaptive mutability depend on the specialized activities of genetic elements like bacteriophage Mu and the F plasmid. Searching for general adaptive mutability, we have investigated the behavior of Salmonella typhimurium strains with chromosomal lacZ mutations. We have studied 30 revertible nonsense, missense, frameshift, and insertion alleles. One-third of the mutants produced >=10 late revertant colonies (appearing three to seven days after plating on selective medium). For the prolific mutants, the number of late revertants showed rank correlation with the residual β-galactosidase activity; for the same mutants, revertant number showed no correlation with the nonselective reversion rate (from fluctuation tests). Leaky mutants, which grew slowly on selective medium, produced late revertants whereas tight nongrowing mutants generally did not produce late revertants. However, the number of late revertants was not proportional to residual growth. Using total residual growth and the nonselective reversion rate, the expected number of late revertants was calculated. For several leaky mutants, the observed revertant number exceeded the expected number. We suggest that excess late revertants from these mutants arise from general adaptive mutability available to any chromosomal gene.     

Mutation spectra of chemical mutagens determined by Lac+ reversion assay with Escherichia coli WP3101P-WP3106P tester strains

We previously reported the development of mutation-specific Escherichia coli B tester strains WP3101 to WP3106 from strain WP2uvrA. In this study we constructed their pKM101-containing derivatives WP3101P to WP3106P, and further isolated their rfa derivatives WP4101-WP4106 and WP4101P-WP4106P. The six kinds of F' plasmids (lacI-, lacZ-, proAB+), each of which carries a different lacZ allele, contained in the above strains were originally derived from E. coli K-12 strains CC101-CC106. All the tester strains show Lac- and Trp- phenotype. Assays for transitions and transversions are based upon Lac+ reversion of a specific mutation located within the lacZ gene on an F' plasmid. The trpE65(ochre) allele in the same strains enables them to be used for Trp+ reversion assays as well. In the present paper, we evaluated the sensitivity, specificity, and usefulness of the newly developed tester strains. Strains WP3101P-WP3106P were highly sensitive to determine mutational profile of heterocyclic amines with S9 mix-mediated metabolic activation and most of the oxidative mutagens and free radical generators tested. Every type of base-pair substitutions induced by 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) or 5-diazouracil were detected in strains WP3101P-WP3106P, while A:T-->C:G and G:C-->A:T mutations induced by MeIQ, and A:T-->C:G, G:C-->A:T, and G:C-->C:G by 5-diazouracil were not detected in pKM101-free tester strains. In pKM101-carrying strains, cumene hydroperoxide induced all types of base substitutions, while formaldehyde preferentially induced G:C-->T:A transversions. Phenazine methosulfate induced predominantly G:C-->A:T transitions and G:C-->T:A transversions, while H2O2 induced predominantly G:C-->T:A and A:T-->T:A transversions. Introduction of the rfa mutation considerably enhanced sensitivity to bulky mutagens such as polycyclic aromatic compounds. All six possible base substitutions induced by 9, 10-dimethyl-1,2-benzanthracene (DMBA) were detected in tester strains WP4101P-WP4106P. In conclusion, our tester strains WP3101P-WP3106P and WP4101P-WP4106P permitted rapid and simple detection of specific mutations induced by variety of mutagens.     

Miscoding and misincorporation of 8-oxo-guanine during leading and lagging strand synthesis in Escherichia coli

We examined whether strand identity with respect to DNA replication influences strand bias for 8-oxo-7,8-dihydroguanine (8-oxoG) mutagenesis. The specificity of 8-oxoG mutagenesis was determined in a mutM mutY or a mutT strain carrying the supF gene on one of two vectors that differed only in the orientation of supF with respect to a unique origin of replication. Most of the supF mutations in the mutM mutY strain were base substitutions (67%), predominantly G:C-->T:A transversions (> 64%), while the majority in the mutT strain were base substitutions (> 92%), predominantly A:T-->C:G transversions (> 91%). The distributions of frequently mutated sites of G:C-->T:A and A:T-->C:G transversions in the supF gene in the mutM mutY and mutT strains, respectively, did not differ markedly between the two vectors. These results suggest that gene orientation is not an important determinant of the strand bias of 8-oxoG mutagenesis.     

Effect of endogenous carotenoids on "adaptive" mutation in Escherichia coli FC40

The appearance over many days of Lac(+) frameshift mutations in Escherichia coli strain FC40 incubated on lactose selection plates is a classic example of apparent "adaptive" mutation in an episomal gene. We show that endogenously overproduced carotenoids reduce adaptive mutation under selective conditions by a factor of around two. Carotenoids are known to scavenge singlet oxygen suggesting that the accumulation of oxidative base damage may be an integral part of the adaptive mutation phenomenon. If so, the lesion cannot be 7,8-dihydro-8-oxoguanine since adaptive mutation in FC40 is unaffected by mutM and mutY mutations. If active oxygen species such as singlet oxygen are involved in adaptive mutation then they should also induce frameshift mutations in FC40 under non-selective conditions. We show that such mutations can be induced under non-selective conditions by protoporphyrin photosensitisation and that this photodynamic induction is reduced by a factor of just over two when endogenous carotenoids are present. We argue that the involvement of oxidative damage would in no way be inconsistent with current understanding of the mechanism of adaptive mutation and the role of DNA polymerases.     

Population Dynamics of a Lac(-) Strain of Escherichia Coli during Selection for Lactose Utilization

During selection for lactose utilization, Lac(+) revertants of FC40, a Lac(-) strain of Escherichia coli, appear at a high rate. Yet, no Lac(+) revertants appear in the absence of lactose, or in its presence if the cells have another, unfulfilled requirement for growth. This study investigates more fully the population dynamics of FC40 when incubated in the absence of a carbon source or when undergoing selection for lactose utilization. In the absence of a carbon source, the viable cell numbers do not change over 6 days. When incubated in liquid lactose medium, Lac(-) cells do not undergo any measurable increase in numbers or in turbidity for at least 2 days. When FC40 is plated on lactose minimum medium in the presence of scavenger cells, the upper limit to the amount of growth of Lac(-) cells during 5 days is one doubling, and there is no evidence for turnover (i.e., a balance between growth and death). The presence of a minority population that could form microcolonies was not detected. The implications of these results, plus the fact that the appearance of Lac(+) revertants during lactose selection is nearly constant with time, are discussed in reference to several models that have been postulated to account for adaptive mutations.     

Mutagenesis and repair in Bacillus anthracis: the effect of mutators

We have generated mutator strains of Bacillus anthracis Sterne by using directed gene knockouts to investigate the effect of deleting genes involved in mismatch repair, oxidative repair, and maintaining triphosphate pools. The single-knockout strains are deleted for mutS, mutY, mutM, or ndk. We also made double-knockout strains that are mutS ndk or mutY mutM. We have measured the levels of mutations in the rpoB gene that lead to the Rif(r) phenotype and have examined the mutational specificity. In addition, we examined the mutational specificity of two mutagens, 5-azacytidine and N-methyl-N'-nitro-N-nitroso-guanidine. The mutY and mutM single knockouts are weak mutators by themselves, but the combination of mutY mutM results in very high mutation rates, all due to G:C --> T:A transversions. The situation parallels that seen in Escherichia coli. Also, mutS knockouts are strong mutators and even stronger in the presence of a deletion of ndk. The number of sites in rpoB that can result in the Rif(r) phenotype by single-base substitution is more limited than in certain other bacteria, such as E. coli and Deinococcus radiodurans, although the average mutation rate per mutational site is roughly comparable. Hotspots at sites with virtually identical surrounding sequences are organism specific.