Overexpression of the mexC–mexD–oprJ efflux operon in nfxB-type multidrug-resistant strains of Pseudomonas aeruginosa

OprJ, overproduced in nfxB multidrug-resistant strains of Pseudomonas aeruginosa, and OprK, overproduced in the multidrug-resistant strain K385, were demonstrated to be immunologically cross-reactive using an OprJ-specific monoclonal antibody. Treatment of the purified proteins with trypsin or chymotrypsin yielded virtually indistinguishable digestion patterns, and the N-terminal sequence of two trypsin fragments was identical for both proteins, indicating that OprJ and OprK share identity. The N-terminal amino acid sequences were used to facilitate cloning of the oprJ gene on a 5kbp KpnI fragment and a 10kbp BamHI fragment. Nucleotide sequencing of portions of these fragments revealed that oprJ was the terminal gene in a putative three-gene operon, The predicted mexC–mexD–oprJ gene products exhibit homology to the MexA–MexB–OprM components of the multidrug-resistance efflux pump of P. aeruginosa (43–46% identity). Consistent with an implied role for mexC–mexD–oprJ in drug efflux, the mexC–mexD–oprJ-hyperexpressing strain K385 showed reduced accumulation of a variety of antibiotics as compared with its parent strain, and this drug ‘exclusion’ was abrogated by energy inhibitors. The mexC and oprJ products are putative lipoproteins of a molecular mass of 40707 and 51742Da, respectively, while mexD was predicted to encode a protein of 111936Da. Sequencing upstream of mexC revealed the presence of the nfxB gene transcribed divergently from the efflux genes. Overproduction of OprJ and the attendant multiple-antibiotic resistance of strain K385 was shown to result from a point mutation in nfxB, resulting in a H87→R change in the predicted NfxB polypeptide. OprJ overproduction and multidrug resistance in K385 was reversed by the cloned nfxB gene, suggesting that nfxB encodes a repressor of mexC–mexD–oprJ expression. Consistent with this, the cloned nfxB gene repressed synthesis of a mexC–lacZ fusion in Escherichia coli. nfxB also repressed expression of a nfxB–lacZ fusion, indicating that NfxB negatively regulates its own expression. These data indicate that the multidrug resistance of nfxB strains is due to overexpression of an efflux operon, mexC–mexD–oprJ, encoding components of a second efflux pump in P. aeruginosa.     

Simple Sequence Repeats and Mucoid Conversion: Biased mucA Mutagenesis in Mismatch Repair-Deficient Pseudomonas aeruginosa

In Pseudomonas aeruginosa, conversion to the mucoid phenotype marks the onset of an irreversible state of the infection in Cystic Fibrosis (CF) patients. The main pathway for mucoid conversion is mutagenesis of the mucA gene, frequently due to −1 bp deletions in a simple sequence repeat (SSR) of 5 Gs (G⁵-SSR₄₂₆). We have recently observed that this mucA mutation is particularly accentuated in Mismatch Repair System (MRS)-deficient cells grown in vitro. Interestingly, previous reports have shown a high prevalence of hypermutable MRS-deficient strains occurring naturally in CF chronic lung infections. Here, we used mucA as a forward mutation model to systematically evaluate the role of G⁵-SSR₄₂₆ in conversion to mucoidy in a MRS-deficient background, with this being the first analysis combining SSR-dependent localized hypermutability and the acquisition of a particular virulence/persistence trait in P. aeruginosa. In this study, mucA alleles were engineered with different contents of G:C SSRs, and tested for their effect on the mucoid conversion frequency and mucA mutational spectra in a mutS-deficient strain of P. aeruginosa. Importantly, deletion of G⁵-SSR₄₂₆ severely reduced the emergence frequency of mucoid variants, with no preferential site of mutagenesis within mucA. Moreover, although mutagenesis in mucA was not totally removed, this was no longer the main pathway for mucoid conversion, suggesting that G⁵-SSR₄₂₆ biased mutations towards mucA. Mutagenesis in mucA was restored by the addition of a new SSR (C⁶-SSR₄₃₁), and even synergistically increased when G⁵-SSR₄₂₆ and C⁶-SSR₄₃₁ were present simultaneously, with the mucA mutations being restricted to −1 bp deletions within any of both G:C SSRs. These results confirm a critical role for G⁵-SSR₄₂₆ enhancing the mutagenic process of mucA in MRS-deficient cells, and shed light on another mechanism, the SSR- localized hypermutability, contributing to mucoid conversion in P. aeruginosa.     

Genetic studies of the lac repressor. X. Analysis of missense mutations in the lacI gene.

Approximately 2000 non-suppressible mutations in the lacI gene of Escherichia coli have been extensively analyzed. The majority consists of missense mutations resulting in amino acid substitutions in the lac repressor. We characterized each mutation with respect to the resulting altered phenotype, and also mapped them against a large set of deletions. The correlation of the genetic and physical map reported previously has been used to localize the part of the protein affected by each mutation with a high degree of precision (within several amino acids). In particular, we examined the distribution of mutational sites along the gene leading to the i^?, i^s, i^r and i^ts phenotypes. Certain regions of the protein, such as the amino-terminal end, are very sensitive to amino acid exchanges with regard to the i^? phenotype, whereas other regions are relatively insensitive to substitutions. Of particular interest is the C-terminal half of the gene-protein map, where many I^s, and I^ts mutational sites cluster in very small regions separated by distinct and nearly regularly spaced intervals. The possible significance of these results with respect to repressor structure and function, and to protein structure in general, is discussed. In the following paper we consider the results reported here together with the data from suppressed nonsense mutations, which are described in the preceding paper.     

Role of the Outer Membrane Protein OprD2 in Carbapenem-Resistance Mechanisms of Pseudomonas aeruginosa

We investigated the relationship between the outer membrane protein OprD₂ and carbapenem-resistance in 141 clinical isolates of Pseudomonas aeruginosa collected between January and December 2013 from the First Affiliated Hospital of Anhui Medical University in China. Agar dilution methods were employed to determine the minimum inhibitory concentration of meropenem (MEM) and imipenem (IMP) for P. aeruginosa. The gene encoding OprD₂ was amplified from141 P. aeruginosa isolates and analyzed by PCR and DNA sequencing. Differences between the effects of IMP^R and IMP^S groups on the resistance of the P. aeruginosa were observed by SDS-poly acrylamide gel electrophoresis (SDS-PAGE). Three resistance types were classified in the 141 carbapenem-resistant P. aeruginosa (CRPA) isolates tested, namely IMP^RMEM^R (66.7%), IMP^RMEM^S (32.6%), and IMP^RMEM^S (0.7%). DNA sequencing revealed significant diverse gene mutations in the OprD₂-encoding gene in these strains. Thirty-four strains had large fragment deletions in the OprD₂gene, in 6 strains the gene contained fragment inserts, and in 96 resistant strains, the gene featured small fragment deletions or multi-site mutations. Only 4 metallo-β-lactamase strains and 1 imipenem-sensitive (meropenem-resistant) strain showed a normal OprD₂ gene. Using SDS-PAGE to detect the outer membrane protein in 16 CRPA isolates, it was found that 10 IMP^RMEM^R strains and 5 IMP^RMEM^S strains had lost the OprD₂ protein, while the IMP^SMEM^R strain contained a normal 46-kDa protein. In conclusion, mutation or loss of the OprD₂-encoding gene caused the loss of OprD₂, which further led to carbapenem-resistance of P. aeruginosa. Our findings provide insights into the mechanism of carbapenem resistance in P. aeruginosa.     

Mutational Spectrum Drives the Rise of Mutator Bacteria

Understanding how mutator strains emerge in bacterial populations is relevant both to evolutionary theory and to reduce the threat they pose in clinical settings. The rise of mutator alleles is understood as a result of their hitchhiking with linked beneficial mutations, although the factors that govern this process remain unclear. A prominent but underappreciated fact is that each mutator allele increases only a specific spectrum of mutational changes. This spectrum has been speculated to alter the distribution of fitness effects of beneficial mutations, potentially affecting hitchhiking. To study this possibility, we analyzed the fitness distribution of beneficial mutations generated from different mutator and wild-type Escherichia coli strains. Using antibiotic resistance as a model system, we show that mutational spectra can alter these distributions substantially, ultimately determining the competitive ability of each strain across environments. Computer simulation showed that the effect of mutational spectrum on hitchhiking dynamics follows a non-linear function, implying that even slight spectrum-dependent fitness differences are sufficient to alter mutator success frequency by several orders of magnitude. These results indicate an unanticipated central role for the mutational spectrum in the evolution of bacterial mutation rates. At a practical level, this study indicates that knowledge of the molecular details of resistance determinants is crucial for minimizing mutator evolution during antibiotic therapy.     

Mutational spectrum analysis of RNase H(35) deficient Saccharomyces cerevisiae using fluorescence-based directed termination PCR

Mutational spectrum analysis has become an informative genetic tool to understand those protein functions involved in mutation avoidance pathways since specific types of mutations are often associated with particular protein defects involved in DNA replication and repair. In this study, we describe a novel, fluorescence-based procedure for direct determination of deletions and insertions with 100% accuracy. We performed two complementary directed termination PCR with near infrared dye-labeled primers, followed by visualization of termination fragments using an automated Li-cor DNA sequencer. This method is used for rapid analysis of mutational spectra generated in nuclease-defective strains of Saccharomyces cerevisiae to elucidate the role of RNase H(35) in RNA primer removal during DNA replication and in mutation avoidance. Strains deficient in RNH35 displayed a distinct spontaneous mutation spectrum of deletions characterized by a unique 4 bp deletion in a lys2-Bgl allele. This was in sharp contrast to strains deficient in rad27 that displayed duplication mutations. Further analysis of mutations in a rnh35/rad27 double mutant revealed a mixed spectrum. These results indicate that RNase H(35) may participate in a redundant pathway in Okazaki fragment processing and that mutational spectra caused by protein deficiencies may be more intermediate-specific than pathway-specific.     

The Fixation Probability of Rare Mutators in Finite Asexual Populations

A mutator is an allele that increases the mutation rate throughout the genome by disrupting some aspect of DNA replication or repair. Mutators that increase the mutation rate by the order of 100-fold have been observed to spontaneously emerge and achieve high frequencies in natural populations and in long-term laboratory evolution experiments with Escherichia coli. In principle, the fixation of mutator alleles is limited by (i) competition with mutations in wild-type backgrounds, (ii) additional deleterious mutational load, and (iii) random genetic drift. Using a multiple-locus model and employing both simulation and analytic methods, we investigate the effects of these three factors on the fixation probability P_fix of an initially rare mutator as a function of population size N, beneficial and deleterious mutation rates, and the strength of mutations s. Our diffusion-based approximation for P_fix successfully captures effects ii and iii when selection is fast compared to mutation (). This enables us to predict the conditions under which mutators will be evolutionarily favored. Surprisingly, our simulations show that effect i is typically small for strong-effect mutators. Our results agree semiquantitatively with existing laboratory evolution experiments and suggest future experimental directions.     

Molecular characterization of Rifr mutations in Pseudomonas aeruginosa and Pseudomonas putida

The rpoB gene encoding for β subunit of RNA polymerase is a target of mutations leading to rifampicin resistant (Rif^r) phenotype of bacteria. Here we have characterized rpoB/Rif^r system in Pseudomonas aeruginosa and Pseudomonas putida as a test system for studying mutational processes. We found that in addition to the appearance of large colonies which were clearly visible on Rif selective plates already after 24 h of plating, small colonies grew up on these plates for 48 h. The time-dependent appearance of the mutant colonies onto selective plates was caused by different levels of Rif resistance of the mutants. The Rif^r clusters of the rpoB gene were sequenced and analyzed for 360 mutants of P. aeruginosa and for 167 mutants of P. putida. The spectrum of Rif^r mutations characterized for P. aeruginosa grown at 37 °C and that characterized for P. putida grown at 30 °C were dissimilar but the differences almost disappeared when the mutants of both strain were isolated at the same temperature, at 30 °C. The strong Rif^r phenotype of P. aeruginosa and P. putida was accompanied only with substitutions of these residues which belong to the putative Rif-binding pocket. Approximately 70% of P. aeruginosa mutants, which were isolated at 37 °C and expressed weak Rif^r phenotype, contained base substitutions in the N-terminal cluster of the rpoB gene. The differences in the spectra of mutations at 30 °C and 37 °C can be explained by temperature-sensitive growth of several mutants in the presence of rifampicin. Thus, our results imply that both the temperature for the growth of bacteria and the time for isolation of Rif^r mutants from selective plates are critical when the rpoB/Rif^r test system is employed for comparative studies of mutagenic processes in Pseudomonas species which are conventionally cultivated at different temperatures.     

Rates and Genomic Consequences of Spontaneous Mutational Events in Drosophila melanogaster

Because spontaneous mutation is the source of all genetic diversity, measuring mutation rates can reveal how natural selection drives patterns of variation within and between species. We sequenced eight genomes produced by a mutation-accumulation experiment in Drosophila melanogaster. Our analysis reveals that point mutation and small indel rates vary significantly between the two different genetic backgrounds examined. We also find evidence that ∼2% of mutational events affect multiple closely spaced nucleotides. Unlike previous similar experiments, we were able to estimate genome-wide rates of large deletions and tandem duplications. These results suggest that, at least in inbred lines like those examined here, mutational pressures may result in net growth rather than contraction of the Drosophila genome. By comparing our mutation rate estimates to polymorphism data, we are able to estimate the fraction of new mutations that are eliminated by purifying selection. These results suggest that ∼99% of duplications and deletions are deleterious—making them 10 times more likely to be removed by selection than nonsynonymous mutations. Our results illuminate not only the rates of new small- and large-scale mutations, but also the selective forces that they encounter once they arise.     

Spontaneous Unstable UNC-22 IV Mutations in C. ELEGANS Var. Bergerac

This paper describes a mutator system in the nematode Caenorhabditis elegans var. Bergerac for the gene unc-22. Of nine C. elegans and two C. briggsae strains tested only the Bergerac BO strain yielded mutant animals at a high frequency and the unc-22 IV gene is a preferred mutational target. The forward spontaneous mutation frequency at the unc-22 locus in Bergerac BO is about 1 x 10^-4 , and most of these spontaneous unc-22 mutations revert at frequencies between 2 x 10^-3 and 2 x 10 ^-4. Both the forward mutation frequency and the reversion frequency are sensitive to genetic background. Spontaneous unc-22 mutations derived in a Bergerac background and placed in a primarily Bristol background revert at frequencies of <10^-6. When reintroduced into a Bergerac/Bristol hybrid background the mutations once again become unstable.

The mutator activity could not be localized to a discrete site in the Bergerac genome. Nor did mutator activity require the Bergerac unc-22 gene as a target since the Bristol unc-22 homolog placed in a Bergerac background also showed high mutation frequency. Intragenic mapping of two spontaneous unc-22 alleles, st136 and st137, place both mutations in the central region of the known unc-22 map. However, these mutations probably recombine with one another, suggesting that the unstable mutations can occur in more than one site in unc-22. Examination of the phenotypic effect of these mutations on muscle structure indicates that they are less severe in their effect than a known amber allele. We suggest that this mutator system is polygenic and dispersed over the nematode genome and could represent activity of the transposable element Tc1.

     

Polynucleotide Phosphorylase Plays an Important Role in the Generation of Spontaneous Mutations in Escherichia coli

Polynucleotide phosphorylase (PNP) plays a central role in RNA degradation, generating a pool of ribonucleoside diphosphates (rNDPs) that can be converted to deoxyribonucleoside diphosphates (dNDPs) by ribonucleotide reductase. We report here that spontaneous mutations resulting from replication errors, which are normally repaired by the mismatch repair (MMR) system, are sharply reduced in a PNP-deficient Escherichia coli strain. This is true for base substitution mutations that occur in the rpoB gene leading to Rif^r and the gyrB gene leading to Nal^r and for base substitution and frameshift mutations that occur in the lacZ gene. These results suggest that the increase in the rNDP pools generated by polynucleotide phosphorylase (PNP) degradation of RNA is responsible for the spontaneous mutations observed in an MMR-deficient background. The PNP-derived pool also appears responsible for the observed mutations in the mutT mutator background and those that occur after treatment with 5-bromodeoxyuridine, as these mutations are also drastically reduced in a PNP-deficient strain. However, mutation frequencies are not reduced in a mutY mutator background or after treatment with 2-aminopurine. These results highlight the central role in mutagenesis played by the rNDP pools (and the subsequent dNTP pools) derived from RNA degradation.     

Premature Termination of MexR Leads to Overexpression of MexAB-OprM Efflux Pump in Pseudomonas aeruginosa in a Tertiary Referral Hospital in India

ObjectivesThe present study was undertaken to investigate the mutations that are present in mexR gene of multidrug resistant (MDR) isolates of Pseudomonas aeruginosa collected from a tertiary referral hospital of north east India.Methods76 MDR clinical isolates of P. aeruginosa were obtained from the patients who were admitted to or attended the clinics of Silchar medical college and hospital. They were screened phenotypically for the presence of efflux pump activity by an inhibitor based method. Acquired resistance mechanisms were detected by multiplex PCR. Real time PCR was performed to study the expression of mexA gene of MexAB-OprM efflux pump in isolates with increase efflux pump activity. mexR gene of the isolates with overexpressed MexAB-OprM efflux pump was amplified, sequenced and analysed.ResultsOut of 76 MDR isolates, 24 were found to exhibit efflux pump activity phenotypically against ciprofloxacin and meropenem. Acquired resistance mechanisms were absent in 11 of them and among those isolates, 8 of them overexpressed MexAB-OprM. All the 8 isolates possessed mutation in mexR gene. 11 transversions, 4 transitions, 2 deletion mutations and 2 insertion mutations were found in all the isolates. However, the most significant observation was the formation of a termination codon at 35^th position which resulted in the termination of the polypeptide and leads to overexpression of the MexAB-OprM efflux pump.ConclusionsThis study highlighted emergence of a novel mutation which is probably associated with multi drug resistance. Therefore, further investigations and actions are needed to prevent or at least hold back the expansion and emergence of newer mutations in nosocomial pathogens which may compromise future treatment options.     

Genetic adaptation of Pseudomonas aeruginosa to the airways of cystic fibrosis patients is catalyzed by hypermutation

In previous work (E. E. Smith, D. G. Buckley, Z. Wu, C. Saenphimmachack, L. R. Hoffman, D. A. D'Argenio, S. I. Miller, B. W. Ramsey, D. P. Speert, S. M. Moskowitz, J. L. Burns, R. Kaul, and M. V. Olson, Proc. Natl. Acad. Sci. USA 103:8487-8492, 2006) it was shown that Pseudomonas aeruginosa undergoes intense genetic adaptation during chronic respiratory infection (CRI) in cystic fibrosis (CF) patients. We used the same collection of isolates to explore the role of hypermutation in this process, since one of the hallmarks of CRI is the high prevalence of DNA mismatch repair (MMR) system-deficient mutator strains. The presence of mutations in 34 genes (many of them positively linked to adaptation in CF patients) in the study collection of 90 P. aeruginosa isolates obtained longitudinally from 29 CF patients was not homogeneous; on the contrary, mutations were significantly concentrated in the mutator lineages, which represented 17% of the isolates (87% MMR deficient). While sequential nonmutator lineages acquired a median of only 0.25 mutation per year of infection, mutator lineages accumulated more than 3 mutations per year. On the whole-genome scale, data for the first fully sequenced late CF isolate, which was also shown to be an MMR-deficient mutator, also support these findings. Moreover, for the first time the predicted amplification of mutator populations due to hitchhiking with adaptive mutations in the course of natural human infections is clearly documented. Interestingly, increased accumulation of mutations in mutator lineages was not a consequence of overrepresentation of mutations in genes involved in antimicrobial resistance, the only adaptive trait linked so far to hypermutation in CF patients, demonstrating that hypermutation also plays a major role in P. aeruginosa genome evolution and adaptation during CRI.     

Detection and characterisation of large SERPINC1 deletions in type I inherited antithrombin deficiency

Methods routinely used for investigating the molecular basis of antithrombin (AT) deficiency do not detect large SERPINC1 rearrangements. Between 2000 and 2008, 86 probands suspected of having AT-inherited type I deficiency were screened for SERPINC1 mutations in our laboratory. Mutations causally linked to the deficiency were identified by sequencing analysis in 63 probands. We present here results of multiplex ligation-dependent probe amplification (MLPA) analysis performed in 22 of the 23 remaining probands, in whom sequencing had revealed no mutation. Large deletions, present at the heterozygous state, were detected in 10 patients: whole gene deletions in 5 and partial deletions removing either exon 6 (n = 2), exons 1–2 (n = 1) or exons 5–7 (n = 2) in 5 others. Exon 6 partial deletions are a 2,769-bp deletion and a 1,892-bp deletion associated with a 10-bp insertion, both having 5′ and/or 3′ breakpoints located within Alu repeat elements. In addition, we identified the 5′ breakpoint of a previously reported deletion of exons 1–2 within an extragenic Alu repeat. Distinct mutational mechanisms explaining these Alu sequence-related deletions are proposed. Overall, in this series, large deletions detected by MLPA explain almost half of otherwise unexplained type I AT-inherited deficiency cases.     

Dynamics of Adaptation During Three Years of Evolution Under Long-Term Stationary Phase

Many bacterial species that cannot sporulate, such as the model bacterium Escherichia coli, can nevertheless survive for years, following exhaustion of external resources, in a state termed long-term stationary phase (LTSP). Here we describe the dynamics of E. coli adaptation during the first three years spent under LTSP. We show that during this time, E. coli continuously adapts genetically through the accumulation of mutations. For nonmutator clones, the majority of mutations accumulated appear to be adaptive under LTSP, reflected in an extremely convergent pattern of mutation accumulation. Despite the rapid and convergent manner in which populations adapt under LTSP, they continue to harbor extensive genetic variation. The dynamics of evolution of mutation rates under LTSP are particularly interesting. The emergence of mutators affects overall mutation accumulation rates as well as the mutational spectra and the ultimate spectrum of adaptive alleles acquired under LTSP. With time, mutators can evolve even higher mutation rates through the acquisition of additional mutation rate–enhancing mutations. Different mutator and nonmutator clones within a single population and time point can display extreme variation in their mutation rates, resulting in differences in both the dynamics of adaptation and their associated deleterious burdens. Despite these differences, clones that vary greatly in their mutation rates tend to coexist within their populations for many years, under LTSP.     

Resistance to glyphosate in the cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> as result of pre-selective mutations

Adaptation of Microcystis aeruginosa (Cyanobacteria) to resist the herbicide glyphosate was analysed by using an experimental model. Growth of wild-type, glyphosate-sensitive (G^s) cells was inhibited when they were cultured with 120 ppm glyphosate, but after further incubation for several weeks, occasionally the growth of rare cells resistant (G^r) to the herbicide was found. A fluctuation analysis was carried out to distinguish between resistant cells arising from rare spontaneous mutations and resistant cells arising from other mechanisms of adaptation. Resistant cells arose by rare spontaneous mutations prior to the addition of glyphosate, with a rate ranging from 3.1 × 10⁻⁷ to 3.6 × 10⁻⁷ mutants per cell per generation in two strains of M. aeruginosa; the frequency of the G^r allele ranged from 6.14 × 10⁻⁴ to 6.54 × 10⁻⁴. The G^r mutants are slightly elliptical in outline, whereas the G^s cells are spherical. Since G^r mutants have a diminished growth rate, they may be maintained in uncontaminated waters as the result of a balance between new resistants arising from spontaneous mutation and resistants eliminated by natural selection. Thus, rare spontaneous pre-selective mutations may allow the survival of M. aeruginosa in glyphosate-polluted waters via G^r clone selection.     

A trade-off between oxidative stress resistance and DNA repair plays a role in the evolution of elevated mutation rates in bacteria

The dominant paradigm for the evolution of mutator alleles in bacterial populations is that they spread by indirect selection for linked beneficial mutations when bacteria are poorly adapted. In this paper, we challenge the ubiquity of this paradigm by demonstrating that a clinically important stressor, hydrogen peroxide, generates direct selection for an elevated mutation rate in the pathogenic bacterium Pseudomonas aeruginosa as a consequence of a trade-off between the fidelity of DNA repair and hydrogen peroxide resistance. We demonstrate that the biochemical mechanism underlying this trade-off in the case of mutS is the elevated secretion of catalase by the mutator strain. Our results provide, to our knowledge, the first experimental evidence that direct selection can favour mutator alleles in bacterial populations, and pave the way for future studies to understand how mutation and DNA repair are linked to stress responses and how this affects the evolution of bacterial mutation rates.     

Mutational clusters generated by non-processive polymerases: A case study using DNA polymerase β in vitro

Available DNA mutational spectra reveal that the number of mutants with multiple mutations (“multiples”) is usually greater than expected from a random distribution of mutations among mutants. These overloads imply the occurrence of non-random clusters of mutations, probably generated during episodes of low-fidelity DNA synthesis. Excess multiples have been reported not only for viruses, bacteria, and eukaryotic cells but also for the DNA polymerases of phages T4 and RB69 in vitro. In the simplest case of a purified polymerase, non-random clusters may be generated by a subfraction of phenotypic variants able to introduce more errors per cycle of DNA synthesis than the normal enzyme. According to this hypothesis, excess multiples are not expected with non-processive polymerases even if they harbor rare mutator variants. DNA polymerase β (Pol β) is a mammalian DNA-repair polymerase with very low processivity. Although several Pol β mutational spectra have been described, there is conflicting evidence on whether or not excess multiples occur, with spectra based on the HSV-tk system tending to show excess multiples. Excess multiples generated by Pol β or any of its mutants might imply that the excesses of multiples observed in numerous other systems, especially those with processive polymerases, could be artifactual. Here, the distributions of mutations generated by native and recombinant rat Pol β and by the Pol β^Y265C mutator were analyzed in the M13mp2 lacZα system. Our results present no evidence for a significant excess of multiples over the expected numbers with any of the Pol β enzymes tested in this system. The reported excess of Pol β-generated multiples in the HSV-tk system may reflect a reduced efficiency of detection of base substitutions that cause weak phenotypes, which in turn may artifactually increase the frequency of multiples.