Emergence of phenotypic variants upon mismatch repair disruption in Pseudomonas aeruginosa

Microbiology ◽  
2004 ◽  
Vol 150 (5) ◽  
pp. 1327-1338 ◽  
Author(s):  
Andrea M. Smania ◽  
Ignacio Segura ◽  
Roberto J. Pezza ◽  
Cecilia Becerra ◽  
Inés Albesa ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
High Frequency ◽  
Type Strain ◽  
Wild Type Strain ◽  
Point Mutations ◽  
Repair System ◽  
Wild Type ◽  
Antibiotic Resistant ◽  
Prolonged Incubation ◽  
Resistant Mutants

MutS is part of the bacterial mismatch repair system that corrects point mutations and small insertions/deletions that fail to be proof-read by DNA polymerase activity. In this work it is shown that the disruption of the P. aeruginosa mutS gene generates the emergence of diverse colony morphologies in contrast with its parental wild-type strain that displayed monomorphic colonies. Interestingly, two of the mutS morphotypes emerged at a high frequency and in a reproducible way and were selected for subsequent characterization. One of them displayed a nearly wild-type morphology while the other notably showed, compared with the wild-type strain, increased production of pyocyanin and pyoverdin, lower excretion of LasB protease and novel motility characteristics, mainly related to swarming. Furthermore, it was reproducibly observed that, after prolonged incubation in liquid culture, the pigmented variant consistently emerged from the mutS wild-type-like variant displaying a reproducible event. It is also shown that these P. aeruginosa mutS morphotypes not only displayed an increase in the frequency of antibiotic-resistant mutants, as described for clinical P. aeruginosa mutator isolates, but also generated mutants whose antibiotic-resistant levels were higher than those measured from spontaneous resistant mutants derived from wild-type cells. It was also found that both morphotypes showed a decreased cytotoxic capacity compared to the wild-type strain, leading to the emergence of invasive variants. By using mutated versions of a tetracycline resistance gene, the mutS mutant showed a 70-fold increase in the reversion frequency of a +1 frameshift mutation with respect to its parental wild-type strain, allowing the suggestion that the phenotypical diversity generated in the mutS population could be produced in part by frameshift mutations. Finally, since morphotypical diversification has also been described in clinical isolates, the possibility that this mutS diversification was related to the high frequency hypermutability observed in P. aeruginosa CF isolates is discussed.

scholarly journals Isolation of Rifampin-Resistant Mutants ofListeria monocytogenes and Their Characterization byrpoB Gene Sequencing, Temperature Sensitivity for Growth, and Interaction with an Epithelial Cell Line

1999 ◽  
Vol 37 (9) ◽  
pp. 2913-2919 ◽  
Author(s):  
Robert Morse ◽  
Karen O’Hanlon ◽  
Mumtaz Virji ◽  
Matthew D. Collins
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Point Mutations ◽  
Gene Sequencing ◽  
Epithelial Cell Line ◽  
Wild Type ◽  
Partial Sequencing ◽  
Resistant Mutants ◽  
Oflisteria Monocytogenes ◽  
Rna Polymerase Holoenzyme

The sequence of the rpoB gene from Listeria monocytogenes was determined. Rifampin-resistant (Rifr) mutants arising from L. monocytogenes cultures exposed to rifampin were isolated, and by partial sequencing of their rpoB genes, seven different point mutations affecting five different amino acids (473Asp→Asn or Gly, 479Gly→Asp, 483His→Tyr or Leu, 528Ile→Phe, and 530Ser→Tyr), which led to MICs of 0.5 to 100 μg/ml for the organisms, were determined. These mutants showed various deficiencies for growth at 42°C, with only one being comparable to the wild-type strain. The interaction of these Rifr mutants with human Caco-2 cells was examined by using an immunofluorescence technique. Three mutants failed to interact, while three showed a reduced interaction compared to that of the wild type. It is believed that these pleiotropic phenotypes have arisen as a result of mutations within the DNA-dependent RNA polymerase holoenzyme.

scholarly journals Mutations Conferring Aminoglycoside and Spectinomycin Resistance in Borrelia burgdorferi

2006 ◽  
Vol 50 (2) ◽  
pp. 445-452 ◽  
Author(s):  
Daniel Criswell ◽  
Virginia L. Tobiason ◽  
J. Stephen Lodmell ◽  
D. Scott Samuels
Keyword(s):  
16S Rrna ◽  
Borrelia Burgdorferi ◽  
Type Strain ◽  
Wild Type Strain ◽  
Small Subunit ◽  
Wild Type ◽  
Spectinomycin Resistance ◽  
E Coli ◽  
Resistant Mutants

ABSTRACT We have isolated and characterized in vitro mutants of the Lyme disease agent Borrelia burgdorferi that are resistant to spectinomycin, kanamycin, gentamicin, or streptomycin, antibiotics that target the small subunit of the ribosome. 16S rRNA mutations A1185G and C1186U, homologous to Escherichia coli nucleotides A1191 and C1192, conferred >2,200-fold and 1,300-fold resistance to spectinomycin, respectively. A 16S rRNA A1402G mutation, homologous to E. coli A1408, conferred >90-fold resistance to kanamycin and >240-fold resistance to gentamicin. Two mutations were identified in the gene for ribosomal protein S12, at a site homologous to E. coli residue Lys-87, in mutants selected in streptomycin. Substitutions at codon 88, K88R and K88E, conferred 7-fold resistance and 10-fold resistance, respectively, to streptomycin on B. burgdorferi. The 16S rRNA A1185G and C1186U mutations, associated with spectinomycin resistance, appeared in a population of B. burgdorferi parental strain B31 at a high frequency of 6 × 10−6. These spectinomycin-resistant mutants successfully competed with the wild-type strain during 100 generations of coculture in vitro. The aminoglycoside-resistant mutants appeared at a frequency of 3 × 10−9 to 1 ×10−7 in a population and were unable to compete with wild-type strain B31 after 100 generations. This is the first description of mutations in the B. burgdorferi ribosome that confer resistance to antibiotics. These results have implications for the evolution of antibiotic resistance, because the 16S rRNA mutations conferring spectinomycin resistance have no significant fitness cost in vitro, and for the development of new selectable markers.

scholarly journals A genetic analysis of resistance to nystatin inSaccharomyces cerevisiae

1967 ◽  
Vol 9 (2) ◽  
pp. 179-193 ◽  
Author(s):  
K. A. Ahmed ◽  
R. A. Woods
Keyword(s):  
Genetic Analysis ◽  
Resistance Genes ◽  
Type Strain ◽  
Wild Type Strain ◽  
Second Step ◽  
Wild Type ◽  
Recessive Resistance ◽  
Low Concentrations ◽  
Effects Of Temperature ◽  
Resistant Mutants

1. A number of stable nystatin-resistant mutants of the yeastSaccharomyces cerevisiaehave been isolated from platings of a sensitive wild-type strain on low concentrations of the antibiotic.2. These mutants were found to be resistant to 10, 15 or 60 units of drug/ml.3. Analysis of meiotic segregants from crosses of these mutants to wild-type indicate that resistance is determined by two types of genes; resistance genes and modifiers.4. Functional analysis of the mutants demonstrated the existence of three recessive resistance genes,nys-l,nys-2 andnys-3 and thatnys-1 andnys-2 were linked.5. Genetic analysis showed thatnys-1 was affected by two modifiers,Mnys-1 andMnys-2, but that onlyMnys-2 affectednys-2 andnys-3.6. The modifiersMnys-1 andMnys-2 are dominant.7. An investigation of the effects of temperature and medium on resistance demonstrated marked interactions between genotype and environment for both the resistance genes and the modifiers.8. Second-step mutants have been isolated by plating first-step mutants on higher concentrations of the drug. Some of these are resistant to 800 units/ml.9. Some possible mechanisms of nystatin resistance are discussed.

scholarly journals Role of β-Lactamases and Porins in Resistance to Ertapenem and Other β-Lactams in Klebsiella pneumoniae

2004 ◽  
Vol 48 (8) ◽  
pp. 3203-3206 ◽  
Author(s):  
George A. Jacoby ◽  
Debra M. Mills ◽  
Nancy Chow
Keyword(s):  
Klebsiella Pneumoniae ◽  
Outer Membrane ◽  
Type Strain ◽  
Wild Type Strain ◽  
Outer Membrane Proteins ◽  
Wild Type ◽  
Resistant Mutants ◽  
High Level ◽  
Level Resistance

ABSTRACT High-level resistance to ertapenem was produced by β-lactamases of groups 1, 2f, and 3 in a strain of Klebsiella pneumoniae deficient in Omp35 and Omp36. From a wild-type strain producing ACT-1 β-lactamase, ertapenem-resistant mutants for which the ertapenem MICs were up to 128 μg/ml and expression of outer membrane proteins was diminished could be selected.

scholarly journals Mismatch Repair Proteins Regulate Heteroduplex Formation during Mitotic Recombination in Yeast

1998 ◽  
Vol 18 (11) ◽  
pp. 6525-6537 ◽  
Author(s):  
Wenliang Chen ◽  
Sue Jinks-Robertson
Keyword(s):  
Gene Conversion ◽  
Mismatch Repair ◽  
Sister Chromatid ◽  
Type Strain ◽  
Wild Type Strain ◽  
Sequence Data ◽  
Mitotic Recombination ◽  
Assay System ◽  
Wild Type ◽  
Mmr Proteins

Mismatch repair (MMR) proteins actively inhibit recombination between diverged sequences in both prokaryotes and eukaryotes. Although the molecular basis of the antirecombination activity exerted by MMR proteins is unclear, it presumably involves the recognition of mismatches present in heteroduplex recombination intermediates. This recognition could be exerted during the initial stage of strand exchange, during the extension of heteroduplex DNA, or during the resolution of recombination intermediates. We previously used an assay system based on 350-bp inverted-repeat substrates to demonstrate that MMR proteins strongly inhibit mitotic recombination between diverged sequences inSaccharomyces cerevisiae. The assay system detects only those events that reverse the orientation of the region between the recombination substrates, which can occur as a result of either intrachromatid crossover or sister chromatid conversion. In the present study we sequenced the products of mitotic recombination between 94%-identical substrates in order to map gene conversion tracts in wild-type versus MMR-defective yeast strains. The sequence data indicate that (i) most recombination occurs via sister chromatid conversion and (ii) gene conversion tracts in an MMR-defective strain are significantly longer than those in an isogenic wild-type strain. The shortening of conversion tracts observed in a wild-type strain relative to an MMR-defective strain suggests that at least part of the antirecombination activity of MMR proteins derives from the blockage of heteroduplex extension in the presence of mismatches.

scholarly journals Frameshift intermediates in homopolymer runs are removed efficiently by yeast mismatch repair proteins.

1997 ◽  
Vol 17 (5) ◽  
pp. 2844-2850 ◽  
Author(s):  
C N Greene ◽  
S Jinks-Robertson
Keyword(s):  
Mismatch Repair ◽  
Wild Type Strain ◽  
Frameshift Mutation ◽  
Protein A ◽  
Repair System ◽  
Wild Type ◽  
Base Pairs ◽  
Replication Errors ◽  
Mismatch Repair System ◽  
Mismatch Repair Proteins

A change in the number of base pairs within a coding sequence can result in a frameshift mutation, which almost invariably eliminates the function of the encoded protein. A frameshift reversion assay with Saccharomyces cerevisiae that can be used to examine the types of insertions and deletions that are generated during DNA replication, as well as the editing functions that remove such replication errors, has been developed. Reversion spectra have been obtained in a wild-type strain and in strains defective for defined components of the postreplicative mismatch repair system (msh2, msh3, msh6, msh3 msh6, pms1, and mih1 mutants). Comparison of the spectra reveals that yeast mismatch repair proteins preferentially remove frameshift intermediates that arise in homopolymer tracts and indicates that some of the proteins have distinct substrate or context specificities.

scholarly journals Fitness Cost of Rifampin Resistance in Neisseria meningitidis:In VitroStudy of Mechanisms Associated withrpoBH553Y Mutation

2015 ◽  
Vol 59 (12) ◽  
pp. 7637-7649 ◽  
Author(s):  
Roberta Colicchio ◽  
Chiara Pagliuca ◽  
Gabiria Pastore ◽  
Annunziata Gaetana Cicatiello ◽  
Caterina Pagliarulo ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Capsular Polysaccharide ◽  
Culture Media ◽  
Point Mutations ◽  
Clinical Isolates ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Rifampin Resistance

ABSTRACTRifampin chemoprophylaxis againstNeisseria meningitidisinfections led to the onset of rifampin resistance in clinical isolates harboring point mutations in therpoBgene, coding for the RNA polymerase β chain. These resistant strains are rare in medical practice, suggesting their decreased fitness in the human host. In this study, we isolated rifampin-resistantrpoBmutants from hypervirulent serogroup C strain 93/4286 and analyzed their different properties, including the ability to grow/survive in different culture media and in differentiated THP-1 human monocytes and to compete with the wild-type strainin vitro. Our results demonstrate that differentrpoBmutations (H553Y, H553R, and S549F) may have different effects, ranging from low- to high-cost effects, on bacterial fitnessin vitro. Moreover, we found that the S549F mutation confers temperature sensitivity, possibly explaining why it is observed very rarely in clinical isolates. Comparative high-throughput RNA sequencing analysis of bacteria grown in chemically defined medium demonstrated that the low-cost H553Y substitution resulted in global transcriptional changes that functionally mimic the stringent response. Interestingly, many virulence-associated genes, including those coding for meningococcal type IV pili, porin A, adhesins/invasins, IgA protease, two-partner secretion system HrpA/HrpB, enzymes involved in resistance to oxidative injury, lipooligosaccharide sialylation, and capsular polysaccharide biosynthesis, were downregulated in the H553Y mutant compared to their level of expression in the wild-type strain. These data might account for the reduced capacity of this mutant to grow/survive in differentiated THP-1 cells and explain the rarity of H553Y mutants among clinical isolates.

scholarly journals The Role of the Mismatch Repair Machinery in Regulating Mitotic and Meiotic Recombination Between Diverged Sequences in Yeast

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1299-1313 ◽  
Author(s):  
Wenliang Chen ◽  
Sue Jinks-Robertson
Keyword(s):  
Mismatch Repair ◽  
Meiotic Recombination ◽  
Type Strain ◽  
Wild Type Strain ◽  
Sequence Divergence ◽  
Rate Data ◽  
Wild Type ◽  
Recombination Rates ◽  
Sequence Identity

Abstract Nonidentical recombination substrates recombine less efficiently than do identical substrates in yeast, and much of this inhibition can be attributed to action of the mismatch repair (MMR) machinery. In this study an intron-based inverted repeat assay system has been used to directly compare the rates of mitotic and meiotic recombination between pairs of 350-bp substrates varying from 82% to 100% in sequence identity. The recombination rate data indicate that sequence divergence impacts mitotic and meiotic recombination similarly, although subtle differences are evident. In addition to assessing recombination rates as a function of sequence divergence, the endpoints of mitotic and meiotic recombination events involving 94%-identical substrates were determined by DNA sequencing. The endpoint analysis indicates that the extent of meiotic heteroduplex DNA formed in a MMR-defective strain is 65% longer than that formed in a wild-type strain. These data are consistent with a model in which the MMR machinery interferes with the formation and/or extension of heteroduplex intermediates during recombination.

scholarly journals Pseudomonas aeruginosa Virulence Analyzed in a Dictyostelium discoideum Host System

2002 ◽  
Vol 184 (11) ◽  
pp. 3027-3033 ◽  
Author(s):  
Pierre Cosson ◽  
Laurence Zulianello ◽  
Olivier Join-Lambert ◽  
François Faurisson ◽  
Leigh Gebbie ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Type Strain ◽  
Wild Type Strain ◽  
Model System ◽  
Wild Type ◽  
Strain Pao1 ◽  
Antibiotic Resistant ◽  
Sensing System ◽  
Quorum Sensing System ◽  
Simple Model System

ABSTRACT Pseudomonas aeruginosa is an important opportunistic pathogen that produces a variety of cell-associated and secreted virulence factors. P. aeruginosa infections are difficult to treat effectively because of the rapid emergence of antibiotic-resistant strains. In this study, we analyzed whether the amoeba Dictyostelium discoideum can be used as a simple model system to analyze the virulence of P. aeruginosa strains. The virulent wild-type strain PAO1 was shown to inhibit growth of D. discoideum. Isogenic mutants deficient in the las quorum-sensing system were almost as inhibitory as the wild type, while rhl quorum-sensing mutants permitted growth of Dictyostelium cells. Therefore, in this model system, factors controlled by the rhl quorum-sensing system were found to play a central role. Among these, rhamnolipids secreted by the wild-type strain PAO1 could induce fast lysis of D. discoideum cells. By using this simple model system, we predicted that certain antibiotic-resistant mutants of P. aeruginosa should show reduced virulence. This result was confirmed in a rat model of acute pneumonia. Thus, D. discoideum could be used as a simple nonmammalian host system to assess pathogenicity of P. aeruginosa.

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