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.     

HmbR, a hemoglobin-binding outer membrane protein of Neisseria meningitidis, undergoes phase variation

Neisseria meningitidis uses hemoglobin (Hb) as an iron source via two TonB-dependent outer membrane receptors, HmbR and HpuB. Analysis of 25 epidemiologically unrelated clinical isolates from serogroups A, B, C, and Y revealed that 64% strains possessed both Hb receptor genes. Examination of the hmbR expression pattern in strains in which the hpuB gene was genetically inactivated revealed two distinct Hb utilization phenotypes. Five strains retained the ability to grow as a confluent lawn, while seven grew only as single colonies around Hb discs. The single-colony phenotype observed for some hpuB mutants is suggestive of phase variation of hmbR. The length of the poly(G) tract starting at position +1164 of hmbR absolutely correlated with the two Hb utilization phenotypes. All five strains that grew as confluent lawns around Hb discs possessed either 9 or 12 consecutive G residues. All seven strains that grew as single colonies around Hb discs had poly(G) tracts of a length other than 9 or 12. These single-colony variants that arose around the Hb discs had poly(G) tracts with either 9 or 12 consecutive G residues restoring the hmbR reading frame. Inactivation of hmbR in these strains resulted in a loss of Hb utilization, demonstrating that the change in the hmbR gene was responsible for the phenotypic switch. The switching rates from hmbR phase off to phase on were approximately 5 x 10(-4) in four serogroup C strains, 2 x 10(-2) in the serogroup A isolate, and 7 x 10(-6) in the serogroup B isolate.     

Bromouracil mutagenesis and mismatch repair in mutator strains of Escherichia coli.

A screening procedure based on the formation of papillae on individual bacterial colonies was used to isolate mutants of Escherichia coli with high mutation rates in the presence of bromouracil. Most of the mutants obtained had high spontaneous mutation rates and mapped close to the previously known mutators mutT, mutS, mutR, uvrE and mutL. Except for mutants of mutT type, these mutators also showed high mutability by bromouracil. Transfection experiments were performed with heteroduplex lambda DNA to test for mismatch repair. The results suggest a reduced efficiency of repair of mismatched bases in mutators mutS, mutR, uvrE and mutL, whereas mutants mapping as mutT appear normal. The results support a connection between spontaneous and bromouracil-induced mutability and repair of mismatched bases in DNA.     

Identification and characterization of the mutL and mutS gene products of Salmonella typhimurium LT2.

The gene products of the mutL and mutS loci play essential roles in the dam-directed mismatch repair in both Salmonella typhimurium LT2 and Escherichia coli K-12. Mutations in these genes result in a spontaneous mutator phenotype. We have cloned the mutL and mutS genes from S. typhimurium by generating mutL- and mutS-specific probes from an S. typhimurium mutL::Tn10 and an mutS::Tn10 strain and using these to screen an S. typhimurium library. Both the mutL and mutS genes from S. typhimurium were able to complement E. coli mutL and mutS strains, respectively. By a combination of Tn1000 insertion mutagenesis and the maxicell technique, the products of the mutL and mutS genes were shown to have molecular weights of 70,000 and 98,000 respectively. A phi (mutL'-lacZ+) gene fusion was constructed; no change in the expression of the fusion could be detected by treatment with DNA-damaging agents. In crude extracts, the MutS protein binds single-stranded DNA, but not double-stranded DNA, with high affinity.     

The extreme mutator effect of Escherichia coli mutD5 results from saturation of mismatch repair by excessive DNA replication errors.

Escherichia coli mutator mutD5 is the most potent mutator known. The mutD5 mutation resides in the dnaQ gene encoding the proofreading exonuclease of DNA polymerase III holoenzyme. It has recently been shown that the extreme mutability of this strain results, in addition to a proofreading defect, from a defect in mutH, L, S-encoded postreplicational DNA mismatch repair. The following measurements of the mismatch-repair capacity of mutD5 cells demonstrate that this mismatch-repair defect is not structural, but transient. mutD5 cells in early log phase are as deficient in mismatch repair as mutL cells, but they become as proficient as wild-type cells in late log phase. Second, arrest of chromosomal replication in a mutD5-dnaA(Ts) strain at a nonpermissive temperature restores mismatch repair, even from the early log phase of growth. Third, transformation of mutD5 strains with multicopy plasmids expressing the mutH or mutL gene restores mismatch repair, even in rapidly growing cells. These observations suggest that the mismatch-repair deficiency of mutD strains results from a saturation of the mutHLS-mismatch-repair system by an excess of primary DNA replication errors due to the proofreading defect.     

Mutation spectrum in Escherichia coli DNA mismatch repair deficient (mutH) strain

The Dam-directed post-replicative mismatch repair system of Escherichia coli removes base pair mismatches from DNA. The products of the mutH, mutL and mutS genes, among others, are required for efficient mismatch repair. Absence of any of these gene products leads to persistence of mismatches in DNA with a resultant increase in spontaneous mutation rate. To determine the specificity of the mismatch repair system in vivo we have isolated and characterized 47 independent mutations from a mutH strain in the plasmid borne mnt repressor gene. The major class of mutations comprises AT to GC transitions that occur within six base pairs of the only two 5'-GATC-3' sequences in the mnt gene. In the wild type control strain, insertion of the IS1 element was the major spontaneous mutational event. A prediction of the Dam-directed mismatch repair model, that the mutation spectra of dam and mutH strains should be the same, was confirmed.     

Mutations in Haemophilus influenzae mismatch repair genes increase mutation rates of dinucleotide repeat tracts but not dinucleotide repeat-driven pilin phase variation rates

High-frequency, reversible switches in expression of surface antigens, referred to as phase variation (PV), are characteristic of Haemophilus influenzae. PV enables this bacterial species, an obligate commensal and pathogen of the human upper respiratory tract, to adapt to changes in the host environment. Phase-variable hemagglutinating pili are expressed by many H. influenzae isolates. PV involves alterations in the number of 5' TA repeats located between the -10 and -35 promoter elements of the overlapping, divergently orientated promoters of hifA and hifBCDE, whose products mediate biosynthesis and assembly of pili. Dinucleotide repeat tracts are destabilized by mismatch repair (MMR) mutations in Escherichia coli. The influence of mutations in MMR genes of H. influenzae strain Rd on dinucleotide repeat-mediated PV rates was investigated by using reporter constructs containing 20 5' AT repeats. Mutations in mutS, mutL, and mutH elevated rates approximately 30-fold, while rates in dam and uvrD mutants were increased 14- and 3-fold, respectively. PV rates of constructs containing 10 to 12 5' AT repeats were significantly elevated in mutS mutants of H. influenzae strains Rd and Eagan. An intact hif locus was found in 14 and 12% of representative nontypeable H. influenzae isolates associated with either otitis media or carriage, respectively. Nine or more tandem 5' TA repeats were present in the promoter region. Surprisingly, inactivation of mutS in two serotype b H. influenzae strains did not alter pilin PV rates. Thus, although functionally analogous to the E. coli MMR pathway and active on dinucleotide repeat tracts, defects in H. influenzae MMR do not affect 5' TA-mediated pilin PV.     

Spontaneous mutagenesis in Escherichia coli strains lacking 6-methyladenine residues in their DNA: an altered mutational spectrum in dam- mutants.

The mutational spectrum at the lacI locus in a dam-4 strain of Escherichia coli was examined. The observed 20-fold increase in spontaneous mutagenesis in a dam- strain was found to be due to base substitutions, primarily transitions, which had increased 140-fold. Using the trpE997 mutation it was found that the dam mutations also resulted in an increase in frameshift mutagenesis. The mutational spectrum of dam- strains was similar to that found with strains carrying the mutH, mutL, mutS and uvrE mutations thought to result in a defect in the repair of mismatched bases. These results are taken to be consistent with, and to support the hypothesis that, dam- strains are deficient in a post-replicative error-avoidance pathway which allows the directed elimination of mismatch lesions by a mechanism in which parental strands are recognized by their level of DNA methylation.     

Dominant negative mutator mutations in the mutS gene of Escherichia coli.

The MutS protein of Escherichia coli is part of the dam-directed MutHLS mismatch repair pathway which rectifies replication errors and which prevents recombination between related sequences. In order to more fully understand the role of MutS in these processes, dominant negative mutS mutations on a multicopy plasmid were isolated by screening transformed wild-type cells for a mutator phenotype, using a Lac+ papillation assay. Thirty-eight hydroxylamine- and 22 N-methyl-N'-nitro-N-nitrosoguanidine-induced dominant mutations were isolated. Nine of these mutations altered the P-loop motif of the ATP-binding site, resulting in four amino acid substitutions. With one exception, the remaining sequenced mutations all caused substitution of amino acids conserved during evolution. The dominant mutations in the P-loop consensus caused severely reduced repair of heteroduplex DNA in vivo in a mutS mutant host strain. In a wild-type strain, the level of repair was decreased by the dominant mutations to between 12 to 90% of the control value, which is consistent with interference of wild-type MutS function by the mutant proteins. Increasing the wild-type mutS gene dosage resulted in a reversal of the mutator phenotype in about 60% of the mutant strains, indicating that the mutant and wild-type proteins compete. In addition, 20 mutant isolates showed phenotypic reversal by increasing the gene copies of either mutL or mutH. There was a direct correlation between the levels of recombination and mutagenesis in the mutant strains, suggesting that these phenotypes are due to the same function of MutS.     

Natural transformation and phase variation modulation in Neisseria meningitidis

Neisseria meningitidis has evolved the ability to control the expression-state of numerous genes by phase variation. It has been proposed that the process aids this human pathogen in coping with the diversity of microenvironments and host immune systems. Therefore, increased frequencies of phase variation may augment the organism's adaptability and virulence. In this study, we found that DNA derived from various neisserial co-colonizers of the human nasopharynx increased N. meningitidis switching frequencies, indicating that heterologous neisserial DNA modulates phase variation in a transformation-dependent manner. In order to determine whether the effect of heterologous DNA was specific to the Hb receptor, HmbR, we constructed a Universal Rates of Switching cassette (UROS). With this cassette, we demonstrated that heterologous DNA positively affects phase variation throughout the meningococcal genome, as UROS phase variation frequencies were also increased in the presence of neisserial DNA. Overexpressing components of the neisserial mismatch repair system partially alleviated DNA-induced changes in phase variation frequencies, thus implicating mismatch repair titration as a cause of these transformation-dependent increases in switching. The DNA-dependent effect on phase variation was transient and may serve as a mechanism for meningococcal genetic variability that avoids the fitness costs encountered by global mutators.     

Cloning and characterization of mutL and mutS genes of Vibrio cholerae: nucleotide sequence of the mutL gene.

The mutL and mutS genes of Vibrio cholerae have been identified using interspecific complementation of Escherichia coli mutL and mutS mutants with plasmids containing the gene bank of V. cholerae. The recombinant plasmid pJT470, containing a 4.7 kb fragment of V. cholerae DNA codes for a protein of molecular weight 92,000. The product of this gene reduces the spontaneous mutation frequency of the E. coli mutS mutant. The plasmid, designated pJT250, containing a 2.5 kb DNA fragment of V. cholerae and coding for a protein of molecular weight 62,000, complements the mutL gene function of E. coli mutL mutants. These gene products are involved in the repair of mismatches in DNA. The complete nucleotide sequence of mutL gene of V. cholerae has been determined.     

Recombination is essential for viability of an Escherichia coli dam (DNA adenine methyltransferase) mutant

Double mutants of Escherichia coli dam (DNA adenine methyltransferase) strains with ruvA, ruvB, or ruvC could not be constructed, whereas dam derivatives with recD, recF, recJ, and recR were viable. The ruv gene products are required for Holliday junction translocation and resolution of recombination intermediates. A dam recG (Holliday junction translocation) mutant strain was isolated but at a very much lower frequency than expected. The inviability of a dam lexA (Ind(-)) host was abrogated by the simultaneous presence of plasmids encoding both recA and ruvAB. This result indicates that of more than 20 SOS genes, only recA and ruvAB need to be derepressed to allow for dam mutant survival. The presence of mutS or mutL mutations allowed the construction of dam lexA (Ind(-)) derivatives. The requirement for recA, recB, recC, ruvA, ruvB, ruvC, and possibly recG gene expression indicates that recombination is essential for viability of dam bacteria probably to repair DNA double-strand breaks. The effect of mutS and mutL mutations indicates that DNA mismatch repair is the ultimate source of most of these DNA breaks. The requirement for recombination also suggests an explanation for the sensitivity of dam cells to certain DNA-damaging agents.     

Discontinuous DNA replication in a lig-7 strain of Escherichia coli is not the result of mismatch repair, nucleotide-excision repair, or the base-excision repair of DNA uracil

After pulse-labeling with 3H-thymidine for 30 s at 42 degrees C, the newly-synthesized DNA from uvrB5 lig-7, uvrB5 lig-7 ung-1 (or ung152), uvrB5 lig-7 mutL218 (or mutS215), and uvrB5 lig-7 ung-1 mutL218 (or mutS215) cells sedimented very slowly in alkaline sucrose gradients. The bulk of these DNA molecules were smaller than 2,000 nucleotides long (i.e., about the size of Okazaki fragments), and none of the 3H-radioactivity was found to sediment as high-molecular-weight DNA. These results indicate that the apparent discontinuous DNA replication observed in lig-7 strains is not the result of mismatch repair, nucleotide-excision repair, or the base-excision repair of DNA uracil.     

Mutagenesis, by methylating and ethylating agents, in mutH, mutL, mutS, and uvrD mutants of Salmonella typhimurium LT2.

Salmonella typhimurium LT2 mutH, mutL, mutS, and uvrD mutants were especially sensitive to mutagenesis by both the recA+-dependent mutagen methyl methane sulfonate and the recA+-independent mutagen ethyl methane sulfonate, but not to mutagenesis by agents such as 4-nitroquinoline-1-oxide and UV irradiation. Similarly, these mutator strains were very sensitive to mutagenesis by the methylating agents N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea. The increased susceptibility to mutagenesis by small alkylating agents due to mutH, mutL, mutS, and uvrD mutations was not accompanied by an increased sensitivity to killing by these agents. Various models are discussed in an effort to explain why strains thought to be deficient in methyl-instructed mismatch repair are sensitive to mutagenesis by methylating and ethylating agents.     

Specificity of N-acetoxy-N-2-acetylaminofluorene-induced frameshift mutation spectrum in mismatch repair deficient Escherichia coli strains mutH, L, S and U

The mismatch repair system of Escherichia coli is known to contribute to the fidelity of the replicational process. This system involves the functions of mutH, mutL, mutS and mutU (uvrD) loci which recognize mispaired bases as a consequence of errors due to the polymerase itself. Chemical modifications of DNA have also been suspected to create mispaired bases which, if the mispaired bases are removed, will lead to mutations by frameshift. Using the pBR322 plasmid DNA modified by the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) we have investigated this possibility in a forward mutational assay (tetracycline sensitivity). This fluorene derivative has been shown to induce predominantly frameshift mutations. Our results show that: The sensitivity of the deficient strains mutH, mutL and mutS to the AAF adducts is similar to that of the corresponding wild-type strain. However, the mutU strain appears much more sensitive to those adducts although less than a uvrA, B or C-deficient strain. This suggests that the mutU gene product is involved in the repair of AAF adducts. For the four mut deficient strains, and as it was shown with the wild-type strain, AAF adducts induced mutations to tetracycline sensitivity are only observed when the SOS system of the host bacteria is induced by irradiation of the cells prior to transformation with the modified plasmid. The mutation frequencies depend upon the ultraviolet light doses and similar maxima were found for the four mut strains and the corresponding wild-type strain. In agreement with the results obtained with wild-type or uvrA strains we observe that AAF adducts induce mostly frameshift mutations in the mut strains. Two types of hot spots of mutagenesis were described in wild-type and uvrA strains occurring either at repetitive sequences or at sequences of the type 5' G-G-C-G-C-C 3' (NarI restriction enzyme recognition sequence). While the second type of mutational hot spot does exist in the mismatch repair-deficient strains, we observe that the repetitive sequences are no longer hot spots of mutations in these strains, suggesting that the mismatch repair protein complex is involved in the establishment of AAF-induced frameshift mutations at repetitive sequences.     

Genetic interactions of DNA repair pathways in the pathogen Neisseria meningitidis

The current increase in the incidence and severity of infectious diseases mandates improved understanding of the basic biology and DNA repair profiles of virulent microbes. In our studies of the major pathogen and model organism Neisseria meningitidis, we constructed a panel of mutants inactivating genes involved in base excision repair, mismatch repair, nucleotide excision repair (NER), translesion synthesis, and recombinational repair pathways. The highest spontaneous mutation frequency among the N. meningitidis single mutants was found in the MutY-deficient strain as opposed to mutS mutants in Escherichia coli, indicating a role for meningococcal MutY in antibiotic resistance development. Recombinational repair was recognized as a major pathway counteracting methyl methanesulfonate-induced alkylation damage in the N. meningitidis. In contrast to what has been shown in other species, meningococcal NER did not contribute significantly to repair of alkylation-induced DNA damage, and meningococcal recombinational repair may thus be one of the main pathways for removal of abasic (apurinic/apyrimidinic) sites and strand breaks in DNA. Conversely, NER was identified as the main meningococcal defense pathway against UV-induced DNA damage. N. meningitidis RecA single mutants exhibited only a moderate decrease in survival after UV exposure as opposed to E. coli recA strains, which are extremely UV sensitive, possibly reflecting the lack of a meningococcal SOS response. In conclusion, distinct differences between N. meningitidis and established DNA repair characteristics in E. coli and other species were identified.     

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.