The mismatch repair system (mutS, mutL and uvrD genes) in Pseudomonas aeruginosa: molecular characterization of naturally occurring mutants

We have recently described the presence of a high proportion of Pseudomonas aeruginosa isolates (20%) with an increased mutation frequency (mutators) in the lungs of cystic fibrosis (CF) patients. In four out of 11 independent P. aeruginosa strains, the high mutation frequency was found to be complemented with the wild-type mutS gene from P. aeruginosa PAO1. Here, we report the cloning and sequencing of two additional P. aeruginosa mismatch repair genes and the characterization, by complementation of deficient strains, of these two putative P. aeruginosa mismatch repair genes (mutL and uvrD). We also describe the alterations in the mutS, mutL and uvrD genes responsible for the mutator phenotype of hypermutable P. aeruginosa strains isolated from CF patients. Seven out of the 11 mutator strains were found to be defective in the MMR system (four mutS, two mutL and one uvrD). In four cases (three mutS and one mutL), the genes contained frameshift mutations. The fourth mutS strain showed a 3.3 kb insertion after the 10th nucleotide of the mutS gene, and a 54 nucleotide deletion between two eight nucleotide direct repeats. This deletion, involving domain II of MutS, was found to be the main one responsible for mutS inactivation. The second mutL strain presented a K310M mutation, equivalent to K307 in Escherichia coli MutL, a residue known to be essential for its ATPase activity. Finally, the uvrD strain had three amino acid substitutions within the conserved ATP binding site of the deduced UvrD polypeptide, showing defective mismatch repair activity. Interestingly, cells carrying this mutant allele exhibited a fully active UvrABC-mediated excision repair. The results shown here indicate that the putative P. aeruginosa mutS, mutL and uvrD genes are mutator genes and that their alteration results in a mutator phenotype.     

Spontaneous mutators of salmonella typhimurium LT2 generated by insertion of transposable elements.

Spontaneous mutators of Salmonella typhimurium LT2 were generated by inserting the transposable element Tn5 or Tn10 into the bacterial chromosome. Two mutators mapped at the position of the mutH and mutL loci of S. typhimurium, and two other mutators mapped at positions corresponding to the mutS and uvrD loci of Escherichia coli. A fifth mutator, mutB, did not map at a position corresponding to any of the known mutators of S. typhimurium or E. coli. The mutH,L,S and uvrD alleles increased the frequency of both spontaneous base substitution and frameshift mutations, whereas the mutB allele increased the frequency only of spontaneous base substitution mutations. The increased frequency of base substitution mutations was recA+ independent in the mutH, mutL, and uvrD strains and partially recA+ independent in the mutS strain. The uvrD mutation decreased the resistance of the cells to killing by ultraviolet irradiation. The mutH,L,S and uvrD strains showed an increased sensitivity to mutagenesis by the alkylating agents methyl methane sulfonate and ethyl methane sulfonate, but not to mutagenesis by 4-nitroquinoline-1-oxide.     

Competition between the dam mutator and the isogenic wild-type of Escherichia coli

Previous studies have shown that the mutT, mutH, mutL and mutS mutators of Escherichia coli confer a marked selective advantage on their respective hosts in competition with otherwise isogenic wild-type strains. We have conducted competition experiments between dam- and dam+ strains of Escherichia coli and have found that dam mutator strains are negatively selected. Although dam- is the first mutator to have a lower fitness than wild-type under chemostat conditions our result does not contradict the hypothesis that increased mutation rates are of evolutionary advantage under environmental stress conditions. Only in the special case of dam- does the advantage of higher mutation rates not outweigh the disadvantage due to the dam- -caused heavy pleiotropic effects.     

Detection of mutator subpopulations in Salmonella typhimurium LT2 by reversion of his alleles

Defects in the methyl-directed mismatch repair lead to both the hypermutability phenotype and removal of a barrier to genetic exchange between species. Mutator bacteria carrying such defects occur frequently among bacterial pathogens, suggesting that subpopulations of mutators are contained within pathogen clones and give rise to the genetic variants that are acted upon by selective forces to allow survival or successful infection. We report here on the detection of the mutator subpopulation in Salmonella typhimurium and determination of its frequency in laboratory cultures. The analysis involved screening for mutators among revertants of S. typhimurium histidine auxotrophs selected for the His⁺ phenotype, since the frequency of mutators is expected to be increased in the selected mutant population they helped to spawn. The increases in spontaneous reversion of histidine mutations were first measured in isogenic strains carrying mismatch repair-defective mutH, mutL, mutS, or uvrD alleles, relative to their mismatch repair-proficient counterparts. Screening for the mutator phenotype in nearly 12,000 revertants of repair-proficient strains carrying his mutations highly stimulated for reversion in mutator backgrounds, the base substitution in hisG428 and frameshift in hisC3076, yielded five mutator strains (0.04%). the his⁺ reversion mutations contained within the newly-arisen mutator strains were characteristic of the predominant nucleotide changes expected in such mutators, as assessed by comparison with the spectra for reversion events in wild-type and mismatch correction-defective backgrounds. The results show that subpopulations of mutators, residing in normal populations at a finite frequency, can be culled from the culture by strong selection for a required phenotype. We calculate that the frequency of mutators in the unselected population of S. typhimurium is 1–4×10⁻⁶, an incidence of 10-fold lower than that expected based on studies of laboratory cultures of Escherichia coli.     

Evolution in Fast Forward: a Potential Role for Mutators in Accelerating Staphylococcus aureus Pathoadaptation

Pathogen evolution and subsequent phenotypic heterogeneity during chronic infection are proposed to enhance Staphylococcus aureus survival during human infection. We tested this theory by genetically and phenotypically characterizing strains with mutations constructed in the mismatch repair (MMR) and oxidized guanine (GO) system, termed mutators, which exhibit increased spontaneous-mutation frequencies. Analysis of these mutators revealed not only strain-dependent increases in the spontaneous-mutation frequency but also shifts in mutational type and hot spots consistent with loss of GO or MMR functions. Although the GO and MMR systems are relied upon in some bacterial species to prevent reactive oxygen species-induced DNA damage, no deficit in hydrogen peroxide sensitivity was found when either of these DNA repair pathways was lost in S. aureus. To gain insight into the contribution of increased mutation supply to S. aureus pathoadaptation, we measured the rate of α-hemolysin and staphyloxanthin inactivation during serial passage. Detection of increased rates of α-hemolysin and staphyloxanthin inactivation in GO and MMR mutants suggests that these strains are capable of modifying virulence phenotypes implicated in mediating infection. Accelerated derivation of altered virulence phenotypes, combined with the absence of increased ROS sensitivity, highlights the potential of mutators to drive pathoadaptation in the host and serve as catalysts for persistent infections.     

Phylogenetic evidence for horizontal transfer of mutS alleles among naturally occurring Escherichia coli strains

mutS mutators accelerate the bacterial mutation rate 100- to 1,000-fold and relax the barriers that normally restrict homeologous recombination. These mutators thus afford the opportunity for horizontal exchange of DNA between disparate strains. While much is known regarding the mutS phenotype, the evolutionary structure of the mutS(+) gene in Escherichia coli remains unclear. The physical proximity of mutS to an adjacent polymorphic region of the chromosome suggests that this gene itself may be subject to horizontal transfer and recombination events. To test this notion, a phylogenetic approach was employed that compared gene phylogeny to strain phylogeny, making it possible to identify E. coli strains in which mutS alleles have recombined. Comparison of mutS phylogeny against predicted E. coli "whole-chromosome" phylogenies (derived from multilocus enzyme electrophoresis and mdh sequences) revealed striking levels of phylogenetic discordance among mutS alleles and their respective strains. We interpret these incongruences as signatures of horizontal exchange among mutS alleles. Examination of additional sites surrounding mutS also revealed incongruous distributions compared to E. coli strain phylogeny. This suggests that other regional sequences are equally subject to horizontal transfer, supporting the hypothesis that the 61.5-min mutS-rpoS region is a recombinational hot spot within the E. coli chromosome. Furthermore, these data are consistent with a mechanism for stabilizing adaptive changes promoted by mutS mutators through rescue of defective mutS alleles with wild-type sequences.     

Comparative study of mutator-gene prv and several other mutator-genes of Escherichia coli K-12]

Mutations prv1, prv2 and mutR34, increasing frequencies of intragenic recombinations, are found not to complement and therefore to be alleles of one gene. Checking for the influence of mutator genes mutS3, mutT1 and uvrE502 on the intragenic recombination in conjugational crossings has shown that mutators mutS3 and uvrE502 increase the frequency of intragenic recombinations while mutT1 does not change it. None of the examined mutator genes influence the conjugational frequencies of recombination. A supplementary analysis for the mutability of the mutant prv1 has been carried out. The prv1 mutation can induce mutations of the frameshift type. Mutations uvrA6, recB21, recC22 and lexA produce no influence on the display of a mutator effect of the prv1 mutation.     

MutS deficiency and activity of the error-prone DNA polymerase IV are crucial for determining mucA as the main target for mucoid conversion in Pseudomonas aeruginosa

Pseudomonas aeruginosa colonizes the respiratory tract of cystic fibrosis (CF) patients, where mutators along with mucoid variants emerge leading to chronic infection. Mucoid conversion generally involves mutations inactivating the mucA gene. This study correlates the frequency and nature of mucA mutations with the activity of factors determining the mutation rate, such as MutS and polymerase IV (Pol IV). Results show that: (i) the emergence frequency of mucoid variants was higher in isolates arising from mutS populations compared with the wild-type strain; (ii) in both strains mucoid conversion occurred mainly by mucA mutations; (iii) however, the mutator strain harboured mostly mucA22 (a common allele in CF isolates), while the wild type showed a wider spectrum of mucA mutations with low incidence of mucA22; (iv) disruption of dinB in the wild-type and mutS strains decreased drastically the emergence frequency of mucoid variants; (v) furthermore, the incidence of mucA mutations diminished in the mutS dinB double mutant strain which consisted only in mucA22; (vi) finally, the mucoid isolates obtained from the dinB strain showed an unexpected absence of mucA mutations. Taken together results demonstrate the implication of both MutS and Pol IV in determining mucA as the main target for conversion to mucoidy.     

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.     

Mutator dynamics in fluctuating environments

Populations with high mutation rates (mutator clones) are being found in increasing numbers of species, and a clear link is being established between the presence of mutator clones and drug resistance. Mutator clones exist despite the fact that in a constant environment most mutations are deleterious, with the spontaneous mutation rate generally held at a low value. This implies that mutator clones have an important role in the adaptation of organisms to changing environments. Our study examines how mutator dynamics vary according to the frequency of environmental fluctuations. Although recent studies have considered a single environmental switch, here we investigate mutator dynamics in a regularly varying environment, seeking to mimic conditions present, for example, under certain drug or pesticide regimes. Our model provides four significant new insights. First, the results demonstrate that mutators are most prevalent under intermediate rates of environmental change. When the environment oscillates more rapidly, mutators are unable to provide sufficient adaptability to keep pace with the frequent changes in selection pressure and, instead, a population of 'environmental generalists' dominates. Second, our findings reveal that mutator dynamics may be complex, exhibiting limit cycles and chaos. Third, we demonstrate that when each beneficial mutation provides a greater gain in fitness, mutators achieve higher densities in more rapidly fluctuating environments. Fourth, we find that mutators of intermediate strength reach higher densities than very weak or strong mutators.     

Mutator Gene of Escherichia coli B

An azaserine-resistant derivative of Escherichia coli B/UV, AZA/R(1), was found to carry a mutator gene. This gene, designated mutS1, was mapped by means of conjugation and P1kc-mediated transduction. The mutS1 gene was cotransduced with argB at a frequency of 2.4%; the gene order in this region of the chromosome is thy argB mutS1. To determine whether a relationship commonly exists between azaserine resistance and the mutator property, 12 additional azaserine-resistant derivatives of B/UV were developed and tested for the mutator phenotype. None of the twelve was a mutator strain. The level of azaserine resistance was not increased over that of the recipient parent when mutS1 was transduced to an azaserine-susceptible strain. Reversion studies indicated that mutS1 induced adenosine-ribosylthymine to guanosine-cytidine and guanosine-cytidine to adenosine-ribosylthymine transitions. Because such mutational changes are suppressible with deoxynucleosides when induced by base analogues, an attempt was made to suppress the mutator activity of mutS1 by the addition of deoxyribonucleosides to the medium. No suppression was found. Recombinants were prepared containing mutS1 and the Treffers mutator gene of E. coli K-12. The effect of the mutator genes appears to be additive.     

Evolution and adaptation in Pseudomonas aeruginosa biofilms driven by mismatch repair system-deficient mutators

Pseudomonas aeruginosa is an important opportunistic pathogen causing chronic airway infections, especially in cystic fibrosis (CF) patients. The majority of the CF patients acquire P. aeruginosa during early childhood, and most of them develop chronic infections resulting in severe lung disease, which are rarely eradicated despite intensive antibiotic therapy. Current knowledge indicates that three major adaptive strategies, biofilm development, phenotypic diversification, and mutator phenotypes [driven by a defective mismatch repair system (MRS)], play important roles in P. aeruginosa chronic infections, but the relationship between these strategies is still poorly understood. We have used the flow-cell biofilm model system to investigate the impact of the mutS associated mutator phenotype on development, dynamics, diversification and adaptation of P. aeruginosa biofilms. Through competition experiments we demonstrate for the first time that P. aeruginosa MRS-deficient mutators had enhanced adaptability over wild-type strains when grown in structured biofilms but not as planktonic cells. This advantage was associated with enhanced micro-colony development and increased rates of phenotypic diversification, evidenced by biofilm architecture features and by a wider range and proportion of morphotypic colony variants, respectively. Additionally, morphotypic variants generated in mutator biofilms showed increased competitiveness, providing further evidence for mutator-driven adaptive evolution in the biofilm mode of growth. This work helps to understand the basis for the specific high proportion and role of mutators in chronic infections, where P. aeruginosa develops in biofilm communities.     

The role of Bacillus anthracis RecD2 helicase in DNA mismatch repair

DNA mismatch repair (MMR) systems can be classified as either MutH-dependent or MutH-independent. In bacteria, extensive studies have been conducted with the MutH-dependent MMR in Escherichia coli and its close relatives. The picture of MutH-independent MMR in other bacteria is less clear, as MMR components other than MutS and MutL have not been identified in the majority of bacteria. Bacillus anthracis is one of the MutH-less Gram(+) bacteria in the phylum of Firmicutes. We used papillation as a tool to search for B. anthracis new mutator strains and identified a spontaneous mutator that carries a minitransposon insertion in the BAS4289 locus. The mutational frequency and specificity exhibited in this mutant were comparable to that of MMR-deficient strains with knockouts of mutL or mutS. It retained a similar UV sensitivity profile as that of the wild type. BAS4289 encodes a putative DNA helicase RecD2 that shares 30% sequence identity with Deinococcus radiodurans RecD2, a well characterized superfamily 1B helicase whose homologs are widely present in Firmicutes complete genomes. We demonstrated that the N-terminal region of RecD2, a unique sequence extension used to distinguish RecD2 from RecD1, was important for B. anthracis RecD2, as mutations in the N-terminal conserved motifs affected its DNA repair function. This is the first report of a RecD2 helicase being associated with MMR. RecD2 and our recently described YycJ protein are likely to be two additional components in the B. anthracis MutH-independent MMR system.     

Interplay between Pleiotropy and Secondary Selection Determines Rise and Fall of Mutators in Stress Response

Mutators are clones whose mutation rate is about two to three orders of magnitude higher than the rate of wild-type clones and their roles in adaptive evolution of asexual populations have been controversial. Here we address this problem by using an ab initio microscopic model of living cells, which combines population genetics with a physically realistic presentation of protein stability and protein-protein interactions. The genome of model organisms encodes replication controlling genes (RCGs) and genes modeling the mismatch repair (MMR) complexes. The genotype-phenotype relationship posits that the replication rate of an organism is proportional to protein copy numbers of RCGs in their functional form and there is a production cost penalty for protein overexpression. The mutation rate depends linearly on the concentration of homodimers of MMR proteins. By simulating multiple runs of evolution of populations under various environmental stresses—stationary phase, starvation or temperature-jump—we find that adaptation most often occurs through transient fixation of a mutator phenotype, regardless of the nature of stress. By contrast, the fixation mechanism does depend on the nature of stress. In temperature jump stress, mutators take over the population due to loss of stability of MMR complexes. In contrast, in starvation and stationary phase stresses, a small number of mutators are supplied to the population via epigenetic stochastic noise in production of MMR proteins (a pleiotropic effect), and their net supply is higher due to reduced genetic drift in slowly growing populations under stressful environments. Subsequently, mutators in stationary phase or starvation hitchhike to fixation with a beneficial mutation in the RCGs, (second order selection) and finally a mutation stabilizing the MMR complex arrives, returning the population to a non-mutator phenotype. Our results provide microscopic insights into the rise and fall of mutators in adapting finite asexual populations.     

Experimental adaptation of Salmonella typhimurium to mice

Experimental evolution is a powerful approach to study the dynamics and mechanisms of bacterial niche specialization. By serial passage in mice, we evolved 18 independent lineages of Salmonella typhimurium LT2 and examined the rate and extent of adaptation to a mainly reticuloendothelial host environment. Bacterial mutation rates and population sizes were varied by using wild-type and DNA repair-defective mutator (mutS) strains with normal and high mutation rates, respectively, and by varying the number of bacteria intraperitoneally injected into mice. After <200 generations of adaptation all lineages showed an increased fitness as measured by a faster growth rate in mice (selection coefficients 0.11-0.58). Using a generally applicable mathematical model we calculated the adaptive mutation rate for the wild-type bacterium to be >10(-6)/cell/generation, suggesting that the majority of adaptive mutations are not simple point mutations. For the mutator lineages, adaptation to mice was associated with a loss of fitness in secondary environments as seen by a reduced metabolic capability. During adaptation there was no indication that a high mutation rate was counterselected. These data show that S. typhimurium can rapidly and extensively increase its fitness in mice but this niche specialization is, at least in mutators, associated with a cost.     

The evolution of mutator genes in bacterial populations: the roles of environmental change and timing

Recent studies have found high frequencies of bacteria with increased genomic rates of mutation in both clinical and laboratory populations. These observations may seem surprising in light of earlier experimental and theoretical studies. Mutator genes (genes that elevate the genomic mutation rate) are likely to induce deleterious mutations and thus suffer an indirect selective disadvantage; at the same time, bacteria carrying them can increase in frequency only by generating beneficial mutations at other loci. When clones carrying mutator genes are rare, however, these beneficial mutations are far more likely to arise in members of the much larger nonmutator population. How then can mutators become prevalent? To address this question, we develop a model of the population dynamics of bacteria confronted with ever-changing environments. Using analytical and simulation procedures, we explore the process by which initially rare mutator alleles can rise in frequency. We demonstrate that subsequent to a shift in environmental conditions, there will be relatively long periods of time during which the mutator subpopulation can produce a beneficial mutation before the ancestral subpopulations are eliminated. If the beneficial mutation arises early enough, the overall frequency of mutators will climb to a point higher than when the process began. The probability of producing a subsequent beneficial mutation will then also increase. In this manner, mutators can increase in frequency over successive selective sweeps. We discuss the implications and predictions of these theoretical results in relation to antibiotic resistance and the evolution of mutation rates.     

Determination of molecular phylogenetics of Vibrio parahaemolyticus strains by multilocus sequence typing

Vibrio parahaemolyticus is an important human pathogen whose transmission is associated with the consumption of contaminated seafood. There is a growing public health concern due to the emergence of a pandemic strain causing severe outbreaks worldwide. Many questions remain unanswered regarding the evolution and population structure of V. parahaemolyticus. In this work, we describe a multilocus sequence typing (MLST) scheme for V. parahaemolyticus based on the internal fragment sequences of seven housekeeping genes. This MLST scheme was applied to 100 V. parahaemolyticus strains isolated from geographically diverse clinical (n = 37) and environmental (n = 63) sources. The sequences obtained from this work were deposited and are available in a public database (http://pubmlst.org/vparahaemolyticus). Sixty-two unique sequence types were identified, and most (50) were represented by a single isolate, suggesting a high level of genetic diversity. Three major clonal complexes were identified by eBURST analysis. Separate clonal complexes were observed for V. parahaemolyticus isolates originating from the Pacific and Gulf coasts of the United States, while a third clonal complex consisted of strains belonging to the pandemic clonal complex with worldwide distribution. The data reported in this study indicate that V. parahaemolyticus is genetically diverse with a semiclonal population structure and an epidemic structure similar to that of Vibrio cholerae. Genetic diversity in V. parahaemolyticus appears to be driven primarily by frequent recombination rather than mutation, with recombination ratios estimated at 2.5:1 and 8.8:1 by allele and site, respectively. Application of this MLST scheme to more V. parahaemolyticus strains and by different laboratories will facilitate production of a global picture of the epidemiology and evolution of this pathogen.     

Infaunal burrows are enrichment zones for Vibrio parahaemolyticus

Vibrio parahaemolyticus, a species that includes strains known to be pathogenic in humans, and other Vibrionaceae are common, naturally occurring bacteria in coastal environments. Understanding the ecology and transport of these organisms within estuarine systems is fundamental to predicting outbreaks of pathogenic strains. Infaunal burrows serve as conduits for increased transport of tidal waters and V. parahaemolyticus cells by providing large open channels from the sediment to salt marsh tidal creeks. An extensive seasonal study was conducted at the North Inlet Estuary in Georgetown, SC, to quantify Vibrionaceae and specifically V. parahaemolyticus bacteria in tidal water, fiddler crab (Uca pugilator, Uca pugnax) burrow water, and interstitial pore water. Numbers of V. parahaemolyticus bacteria were significantly higher within burrow waters (4,875 CFU ml(-1)) than in creek water (193 CFU ml(-1)) and interstitial pore water (128 CFU ml(-1)), demonstrating that infaunal burrows are sites of V. parahaemolyticus enrichment. A strong seasonal trend of increased abundances of Vibrionaceae and V. parahaemolyticus organisms during the warmer months of May through September was observed. Multilocus sequence typing (MLST) analysis of isolates presumed to be V. parahaemolyticus from creek water, pore water, and burrow water identified substantial strain-level genetic variability among V. parahaemolyticus bacteria. Analysis of carbon substrate utilization capabilities of organisms presumed to be V. parahaemolyticus also indicated physiological diversity within this clade, which helps to explain the broad distribution of these strains within the estuary. These burrows are "hot spots" of Vibrionaceae and V. parahaemolyticus cell numbers and strain diversity and represent an important microhabitat.     

Resistance of Deinococcus radiodurans to Mutagenesis Is Facilitated by Pentose Phosphate Pathway in the mutS1 Mutant Background

MutS1 is a key protein involved in mismatch repair system for ensuring fidelity of replication and recombination in Deinococcus radiodurans. The zwf gene encodes glucose-6-phosphate dehydrogenase (G6PD) in the pentose phosphate (PP) pathway, which provides adequate metabolites as precursors of DNA repair. In this study, mutS1 and zwf were disrupted by homologous recombination. The zwf mutant (Deltazwf) and the zwf/mutS1 double mutant (Deltazwf/mutS1) were sensitive to ultraviolet (UV) light, H(2)O(2), and DNA cross-linking agent mitomycin C (MMC), whereas the mutS1 mutant (DeltamutS1) showed resistance to UV light, H(2)O(2) and MMC as the wild-type strain. Inactivation of mutS1 resulted in a 3.3-fold increase in frequency of spontaneous rifampicin-resistant mutagenesis and a 4.9-fold increment in integration efficiency of a donor point-mutation marker during bacterial transformation. Although inactivation of zwf had no obvious effect compared with the wild-type strain, dual disruption of zwf and mutS1 resulted in a 4.7-fold increase in mutation frequency and a 7.4-fold increase in integration efficiency. These results suggest that inactivation of the PP pathway decreases the resistance of D. radiodurans cells to DNA damaging agents and increases mutation frequency and integration efficiency in the mutS1 mutant background.     

A recA-dependent mutator of Escherichia coli K12: Method of isolation and initial characterization

A number of mutator strains of E. coli were isolated using histochemical techniques which allow the identification of a single mutator colony on agar plates with as many as 2000 colonies. Several mutators isolated in this way were found by P1-mediated transduction to map to the proA-proB region of the E. coli chromosome. The map position of these mutators is very close to that of the conditional mutator, mutD. However, in contrast to mutD, one of these newly isolated mutators was suppressed in thermosensitive recA strain at 43°C, but not at 30°C. This mutator mutation has been named mut-8. Besides being dependent upon recA, mut-8 is also dependent upon growth in enriched medium for the expression of its mutator activity. The mutator activity of mut-8 was found to be recessive to the wild-type allele.