Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene

The mismatch repair (MMR) system is one of the key molecular devices that prokaryotic cells have for ensuring fidelity of DNA replication. While the canonical MMR of E. coli involves 3 proteins (encoded by mutS, mutL and mutH), the soil bacterium Pseudomonads putida has only 2 bona fide homologues (mutS and mutL) and the sensitivity of this abridged system to different types of mismatches is unknown. In this background, sensitivity to MMR of this bacterium was inspected through single stranded (ss) DNA recombineering of the pyrF gene (the prokaryotic equivalent to yeast's URA3) with mutagenic oligos representative of every possible mispairing under either wild-type conditions, permanent deletion of mutS or transient loss of mutL activity (brought about by the thermoinducible dominant negative allele mutL_E36K). Analysis of single nucleotide mutations borne by clones resistant to fluoroorotic acid (5FOA, the target of wild type PyrF) pinpointed prohibited and tolerated single-nucleotide replacements and exposed a clear grading of mismatch recognition. The resulting data unequivocally established the hierarchy A:G < C:C < G:A < C:A, A:A, G:G, T:T, T:G, A:C, C:T < G:T, T:C as the one prevalent in Pseudomonas putida. This information is vital for enabling recombineering strategies aimed at single-nucleotide changes in this biotechnologically important species.     

Interaction between the mismatch repair and nucleotide excision repair pathways in the prevention of 5-azacytidine-induced CG-to-GC mutations in Escherichia coli

5-Azacytidine induces CG-to-GC transversion mutations in Escherichia coli. The results presented in this paper provide evidence that repair of the drug-induced lesions that produce these mutations involves components of both the mismatch repair and nucleotide excision repair systems. Strains deficient in mutL, mutS, uvrA, uvrB or uvrC all showed an increase in mutation in response to 5-azacytidine. Using a bacterial two-hybrid assay, we showed that UvrB interacts with MutL and MutS in a drug-dependent manner, while UvrC interacts with MutL independent of drug. We suggest that 5-azacytidine-induced mismatches recruit MutS and MutL, but are poorly processed by mismatch repair. Instead, the stalled MutS–MutL complex recruits the Uvr proteins to complete repair.     

Transposon Tn951 (TnLac) is defective and related to Tn3

Summary Tn951 is flanked by two perfect inverted repeats of 41 bp which include the 38 bp sequence of the IR of Tn3. Tn951 also contains the last 100 bp of the tnpA gene but with at least two mutations. However, beyond nucleotide 137 the sequences diverge and hybridization experiments show that Tn951 lacks at least the first two thirds of the tnpA gene.In agreement with these observations Tn951 does not transpose by itself at a detectable frequency but can be complemented by the tnpA gene of Tn801 or Tn3. Tn501, Tn1721 and gamma delta do not complement Tn951 transposition.Transposition of Tn951 duplicates 5 bp of target DNA sequence.     

MutS and MutL Are Dispensable for Maintenance of the Genomic Mutation Rate in the Halophilic Archaeon Halobacterium salinarum NRC-1

Background

The genome of the halophilic archaeon Halobacterium salinarum NRC-1 encodes for homologs of MutS and MutL, which are key proteins of a DNA mismatch repair pathway conserved in Bacteria and Eukarya. Mismatch repair is essential for retaining the fidelity of genetic information and defects in this pathway result in the deleterious accumulation of mutations and in hereditary diseases in humans.

Methodology/Principal Findings

We calculated the spontaneous genomic mutation rate of H. salinarum NRC-1 using fluctuation tests targeting genes of the uracil monophosphate biosynthesis pathway. We found that H. salinarum NRC-1 has a low incidence of mutation suggesting the presence of active mechanisms to control spontaneous mutations during replication. The spectrum of mutational changes found in H. salinarum NRC-1, and in other archaea, appears to be unique to this domain of life and might be a consequence of their adaption to extreme environmental conditions. In-frame targeted gene deletions of H. salinarum NRC-1 mismatch repair genes and phenotypic characterization of the mutants demonstrated that the mutS and mutL genes are not required for maintenance of the observed mutation rate.

Conclusions/Significance

We established that H. salinarum NRC-1 mutS and mutL genes are redundant to an alternative system that limits spontaneous mutation in this organism. This finding leads to the puzzling question of what mechanism is responsible for maintenance of the low genomic mutation rates observed in the Archaea, which for the most part do not have MutS and MutL homologs.     

Development of a Tn10 Vehicle for Insertion Mutagenesis in Rhizobium

A transposon Tn10 vehicle was developed using a self transmissible (Tra⁺) plasmid pRK2013 having narrow host range ori of replication (ColEl). The construct pSA10-3 carrying Tn10 was useful in efficiently transferring transposon Tn10 from E. coli into various rhizobia. The ColEl replicon conferred suicidal property to vector in Rhizobium background where it falls to replicate stably. Thus this plasmid can be employed to cause independent insertion mutations in rhizobia by Tn10 transposition. The frequency of tetracycline resistant colonies of Rhizobium (Tn10 mutants) was approximately 10⁵ folds higher than the spontaneous Tet^R mutants. Reversion frequency of these mutants was less than 10^?8 indicating adequate stability of Tn10 mutations.     

Role of hypermutability in the evolution of the genus Oenococcus

Oenococcus oeni is an alcohol-tolerant, acidophilic lactic acid bacterium primarily responsible for malolactic fermentation in wine. A recent comparative genomic analysis of O. oeni PSU-1 with other sequenced lactic acid bacteria indicates that PSU-1 lacks the mismatch repair (MMR) genes mutS and mutL. Consistent with the lack of MMR, mutation rates for O. oeni PSU-1 and a second oenococcal species, O. kitaharae, were higher than those observed for neighboring taxa, Pediococcus pentosaceus and Leuconostoc mesenteroides. Sequence analysis of the rpoB mutations in rifampin-resistant strains from both oenococcal species revealed a high percentage of transition mutations, a result indicative of the lack of MMR. An analysis of common alleles in the two sequenced O. oeni strains, PSU-1 and BAA-1163, also revealed a significantly higher level of transition substitutions than were observed in other Lactobacillales species. These results suggest that the genus Oenococcus is hypermutable due to the loss of mutS and mutL, which occurred with the divergence away from the neighboring Leuconostoc branch. The hypermutable status of the genus Oenococcus explains the observed high level of allelic polymorphism among known O. oeni isolates and likely contributed to the unique adaptation of this genus to acidic and alcoholic environments.     

Bile-induced DNA damage in Salmonella enterica

In the absence of DNA adenine methylase, growth of Salmonella enterica serovar Typhimurium is inhibited by bile. Mutations in any of the mutH, mutL, and mutS genes suppress bile sensitivity in a Dam⁻ background, indicating that an active MutHLS system renders Dam⁻ mutants bile sensitive. However, inactivation of the MutHLS system does not cause bile sensitivity. An analogy with Escherichia coli, in which the MutHLS system sensitizes Dam⁻ mutants to DNA-injuring agents, suggested that bile might cause DNA damage. In support of this hypothesis, we show that bile induces the SOS response in S. enterica and increases the frequency of point mutations and chromosomal rearrangements. Mutations in mutH, mutL, or mutS cause partial relief of virulence attenuation in a Dam⁻ background (50- to 100-fold by the oral route and 10-fold intraperitoneally), suggesting that an active MutHLS system reduces the ability of Salmonella Dam⁻ mutants to cope with DNA-damaging agents (bile and others) encountered during the infection process. The DNA-damaging ability of bile under laboratory conditions raises the possibility that the phenomenon may be relevant in vivo, since high bile concentrations are found in the gallbladder, the niche for chronic Salmonella infections.     

Tn903 induces inverted duplications in the chromosome of bacteriophage lambda

Specialized transducing strains of bacteriophage lambda have been isolated that carry the transposable kanamycin resistance element, Tn903. Tn903 carries an inverted duplication of 1130 base-pairs flanking the kanamycin resistance gene. Often, when λ::Tn903 particles are infected into bacterial cells, the lambda chromosome is rearranged into a defective lambda plasmid which replicates with the bacterial cell. The formation of the defective plasmids (called Tn903λdv) is most likely induced by the Tn903 insertion itself. This follows from the fact that the novel DNA sequence found in these plasmids, with respect to the ancestral λTn903 chromosome, is always adjacent to the Tn903 element. Physical chromosomal mapping of these plasmids shows that they contain large inverted duplications of lambda sequences situated about the Tn903 insertion. The formation of the Tn903λdv plasmids from the ancestral λTn903 is not dependent on the recombination functions provided through the phage red gene or the host recA gene.     

Mucoidy,Quorum Sensing,Mismatch Repair and Antibiotic Resistance in Pseudomonas aeruginosa from Cystic Fibrosis Chronic Airways Infections

Survival of Pseudomonas aeruginosa in cystic fibrosis (CF) chronic infections is based on a genetic adaptation process consisting of mutations in specific genes, which can produce advantageous phenotypic switches and ensure its persistence in the lung. Among these, mutations inactivating the regulators MucA (alginate biosynthesis), LasR (quorum sensing) and MexZ (multidrug-efflux pump MexXY) are the most frequently observed, with those inactivating the DNA mismatch repair system (MRS) being also highly prevalent in P. aeruginosa CF isolates, leading to hypermutator phenotypes that could contribute to this adaptive mutagenesis by virtue of an increased mutation rate. Here, we characterized the mutations found in the mucA, lasR, mexZ and MRS genes in P. aeruginosa isolates obtained from Argentinean CF patients, and analyzed the potential association of mucA, lasR and mexZ mutagenesis with MRS-deficiency and antibiotic resistance. Thus, 38 isolates from 26 chronically infected CF patients were characterized for their phenotypic traits, PFGE genotypic patterns, mutations in the mucA, lasR, mexZ, mutS and mutL gene coding sequences and antibiotic resistance profiles. The most frequently mutated gene was mexZ (79%), followed by mucA (63%) and lasR (39%) as well as a high prevalence (42%) of hypermutators being observed due to loss-of-function mutations in mutL (60%) followed by mutS (40%). Interestingly, mutational spectra were particular to each gene, suggesting that several mechanisms are responsible for mutations during chronic infection. However, no link could be established between hypermutability and mutagenesis in mucA, lasR and mexZ, indicating that MRS-deficiency was not involved in the acquisition of these mutations. Finally, although inactivation of mucA, lasR and mexZ has been previously shown to confer resistance/tolerance to antibiotics, only mutations in MRS genes could be related to an antibiotic resistance increase. These results help to unravel the mutational dynamics that lead to the adaptation of P. aeruginosa to the CF lung.     

DNA base sequence changes induced by bromouracil mutagenesis of lambda phage

The base sequence changes induced by bromouracil mutagenesis in the cI gene of phage lambda have been determined by direct sequence analysis. Phage DNA mutagenized during prophage replication or during phage lytic growth showed predominantly A · T → G · C transitions. The frequency of this mutation was strongly sequence-dependent: 5′ A-C-G-C 3′ > A-C(A.C or T) > A(A.G or T). The difference in mutability of bases in the gene is not the result of specificity in mutL-dependent mismatch repair, since phage grown in mutL host cells showed the same distribution of bromouracil mutations. The observations made in phage mutagenized with bromouracil in the prophage state should be representative of bromouracil mutagenesis in the Escherichia coli chromosome.     

Inversions and deletions of the Salmonella chromosome generated by the translocatable tetracycline resistance element Tn10

Spontaneous tetraoyoline-sensitive derivatives of a Tn10 insertion in the hisG gene of Salmonella typhimurium were isolated and subjected to genetic analysis. All 123 of the drug-sensitive derivatives characterized have undergone stable alterations in the Tn10 element itself; over half of the derivatives have also undergone major alterations of neighboring regions of the Salmonella chromosome. These chromosomal rearrangements are of two types: inversions and deletions. Any single inversion or deletion affects a contiguous stretch of chromosomal material extending from the site of the original Tn10 element either leftward or rightward.The genetic properties of deletion and inversion derivatives suggest that these chromosomal alterations are promoted by the Tn10 element itself. The role of translocatable elements in promoting chromosomal deletions is well documented; the ability of an element to promote inversions of chromosomal material has not previously been reported. Possible analogies between the 1400-base-pair inverted repetition at the end of Tn10 and the small insertion sequence IS1 predict particular structures for Tn10-promoted deletions. A structural explanation or model for Tn10-promoted inversions is presented. The observation that Tn10 promotes the formation of inversions suggests that such elements could play a previously unanticipated role in promoting chromosomal inversions during evolution of prokaryotic organisms. Generally applicable genetic methods for the identification and characterization of chromosomal inversions are described.     

Phage-associated mutator phenotype in group A streptococcus

Defects in DNA mismatch repair (MMR) occur frequently in natural populations of pathogenic and commensal bacteria, resulting in a mutator phenotype. We identified a unique genetic element in Streptococcus pyogenes strain SF370 that controls MMR via a dynamic process of prophage excision and reintegration in response to growth. In S. pyogenes, mutS and mutL are organized on a polycistronic mRNA under control of a common promoter. Prophage SF370.4 is integrated between the two genes, blocking expression of the downstream gene (mutL) and resulting in a mutator phenotype. However, in rapidly growing cells the prophage excises and replicates as an episome, allowing mutL to be expressed. Excision of prophage SF370.4 and expression of MutL mRNA occur simultaneously during early logarithmic growth when cell densities are low; this brief window of MutL gene expression ends as the cell density increases. However, detectable amounts of MutL protein remain in the cell until the onset of stationary phase. Thus, MMR in S. pyogenes SF370 is functional in exponentially growing cells but defective when resources are limiting. The presence of a prophage integrated into the 5′ end of mutL correlates with a mutator phenotype (10⁻⁷ to 10⁻⁸ mutation/generation, an approximately a 100-fold increase in the rate of spontaneous mutation compared with prophage-free strains [10⁻⁹ to 10⁻¹⁰ mutation/generation]). Such genetic elements may be common in S. pyogenes since 6 of 13 completed genomes have related prophages, and a survey of 100 strains found that about 20% of them are positive for phages occupying the SF370.4 attP site. The dynamic control of a major DNA repair system by a bacteriophage is a novel method for achieving the mutator phenotype and may allow the organism to respond rapidly to a changing environment while minimizing the risks associated with long-term hypermutability.     

The global bacterial regulator H-NS promotes transpososome formation and transposition in the Tn5 system

The histone-like nucleoid structuring protein (H-NS) is an important regulator of stress response and virulence genes in gram-negative bacteria. In addition to binding regulatory regions of genes in a structure-specific manner, H-NS also binds in a structure-specific manner to sites in the Tn10 transpososome, allowing it to act as a positive regulator of Tn10 transposition. This is the only example to date of H-NS regulating a transposition system by interacting directly with the transposition machinery. In general, transposition complexes tend to include segments of deformed DNA and given the capacity of H-NS to bind such structures, and the results from the Tn10 system, we asked if H-NS might regulate another transposition system (Tn5) by directly binding the transposition machinery. We show in the current work that H-NS does bind Tn5 transposition complexes and use hydroxyl radical footprinting to characterize the H-NS interaction with the Tn5 transpososome. We also show that H-NS can promote Tn5 transpososome formation in vitro, which correlates with the Tn5 system showing a dependence on H-NS for transposition in vivo. Taken together the results suggest that H-NS might play an important role in the regulation of many different bacterial transposition systems and thereby contribute directly to lateral gene transfer.     

RecBC and RecF recombination pathways and the induced precise excision of Tn10 in Escherichia coli

Mitomycin C (MMC) treatment or mutations in uvrD enhance the frequency of Tn10 precise excision. We have shown previously that several repair-recombination genes, such as recA, ruv and recF are involved in the induced excision process. In this study, we find that other genes belonging to the RecBC and RecF sexual recombination pathways also participate in this process since mutations in recB, sbcB or recO diminish, though to different degrees, the frequency of Tn10 precise excision induced by MMC treatment or by uvrD mutants. Pairwise combinations of some of these mutations were also tested for Tn10 induced precise excision; most of these double mutants showed additive effects in reducing the frequency of the excision process. The results of these studies suggest that recombinational-repair genes, particularly recF, sbcB and recO have different roles in the induced excision of Tn10 than in recombinational mating.     

Site-specific Tn7 transposition into the human genome

The bacterial transposon, Tn7, inserts into a single site in the Escherichia coli chromosome termed attTn7 via the sequence-specific DNA binding of the target selector protein, TnsD. The target DNA sequence required for Tn7 transposition is located within the C-terminus of the glucosamine synthetase (glmS) gene, which is an essential, highly conserved gene found ubiquitously from bacteria to humans. Here, we show that Tn7 can transpose in vitro adjacent to two potential targets in the human genome: the gfpt-1 and gfpt-2 sequences, the human analogs of glmS. The frequency of transposition adjacent to the human gfpt-1 target is comparable with the E.coli glmS target; the human gfpt-2 target shows reduced transposition. The binding of TnsD to these sequences mirrors the transposition activity. In contrast to the human gfpt sequences, Tn7 does not transpose adjacent to the gfa-1 sequence, the glmS analog in Saccharomyces cerevisiae. We also report that a nucleosome core particle assembled on the human gfpt-1 sequence reduces Tn7 transposition by likely impairing the accessibility of target DNA to the Tns proteins. We discuss the implications of these findings for the potential use of Tn7 as a site-specific DNA delivery agent for gene therapy.     

Analysis of tetracycline resistance encoded by transposon Tn10: deletion mapping of tetracycline-sensitive point mutations and identification of two structural genes

Deletions in the tet genes derived from Tn10 were formed from different tet::Tn5 insertion mutations by removing DNA sequences located between a HindIII site in Tn5 and a HindIII site adjacent to the tet genes. Tetracycline-sensitive point mutations were mapped in recombination tests with the deletions and were thus aligned with the genetic and physical map of the tet region. Plasmids carrying point mutations were tested for complementation with derivatives of pDU938, a plasmid carrying cloned tet genes derived from Tn10 which had been inactivated by Tn5 insertions. Complementation occurred between promoter-proximal tet point mutations and distal tet::Tn5 insertions, suggesting the existence of two structural genes, tetA and tetB. These results, together with the analysis of polypeptides in minicells harboring pDU938tet::Tn5 mutants, suggested that tetA and tetB are expressed coordinately in an operon. The tetB gene encodes the previously characterized 36,000-dalton cytoplasmic membrane TET protein, but the product of tetA was not identified. Point mutations in either tetA or tetB led to the defective expression of the resistance mechanism involving tetracycline efflux. It is suggested that the tetA and tetB products interact cooperatively in the membrane to express resistance.     

Evidence that YycJ is a novel 5′–3′ double-stranded DNA exonuclease acting in Bacillus anthracis mismatch repair

The most important system for correcting replication errors that survive the built in editing system of DNA polymerase is the mismatch repair (MMR) system. We have identified a novel mutator strain yycJ in Bacillus anthracis. Mutations in the yycJ gene result in a spontaneous mutator phenotype with a mutational frequency and specificity comparable to that of MMR-deficient strains such as those with mutations in mutL or mutS. YycJ was annotated as a metallo-β-lactamase (MβL) super family member with unknown activity. In this study we carried out a biochemical characterization of YycJ and demonstrated that a recombinant YycJ protein possesses a 5′–3′ exonuclease activity at the 5′ termini and at nicks of double-stranded DNA. This activity requires a divalent metal cofactor Mn²⁺ and is stimulated by 5′-phosphate ends of duplex DNA. The mutagenesis of conserved amino acid residues revealed that in addition to the five MβL family conserved motifs, YycJ appears to have its specific motifs that can be used to distinguish YycJ from other closely related MβL family members. A phylogenetic survey showed that putative YycJ homologs are present in several bacterial phyla as well as in members of the Methanomicrobiales and Thermoplasmales from Archaea. We propose that YycJ represents a new group of MβL fold exonucleases, which is likely to act in the recognition of MMR entry point and subsequent removal of the mismatched base in certain MutH-less bacterial species.