Emergence of phenotypic variants upon mismatch repair disruption in Pseudomonas aeruginosa

Abstract

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.

Hypermutation Is a Key Factor in Development of Multiple-Antimicrobial Resistance inPseudomonas aeruginosaStrains Causing Chronic Lung Infections

“…Certainly, the extremely high prevalence of hypermutable strains found in the present work suggests that hypermutation plays a role in the bacterial adaptation to the lung environment required for long-term persistence because hypermutation increases the rates of generation of any mutant phenotype (not only antibiotic resistance), including those involved in the development of resistance to the host immune system. In this sense, it has recently been found in vitro that mismatch repair disruption in P. aeruginosa determines the emergence at a high frequency of multiple morphological variants resembling what naturally occurs in chronic infections (39). Furthermore, the link between hypermutation and chronic infections seems not to be restricted to just P. aeruginosa, since recent works have found a significantly higher prevalence (14%) of hypermutable Staphylococcus aureus and Haemophilus influenzae strains in CF patients than in patients with other infections (1%) (32,37).…”

The Thymidine-Dependent Small-Colony-Variant Phenotype Is Associated with Hypermutability and Antibiotic Resistance in Clinical Staphylococcus aureus Isolates

“…In addition, the emergence of various morphotypic variants has been described for a P. aeruginosa strain with a disrupted mutS gene (42). While the component MutS is generally implicated in the detection of DNA mismatches and initiation of the MMR machinery, the function of MutL is to make a connection between the recognition of a mismatch and the excision of the mismatch from the strand within which it is contained (19).…”

Genetic adaptation of Pseudomonas aeruginosa during chronic lung infection of patients with cystic fibrosis: strong and weak mutators with heterogeneous genetic backgrounds emerge in mucA and/or lasR mutants

“…Sequence of mucA and lasR in consecutive CF P. aeruginosa isolates from different time points of the chronic lung infection Inactivation of the MMR system was shown in vitro to favour the emergence of mucA22 and lasR mutants (Smania et al, 2004;Lujan et al, 2007;Moyano et al, 2007), and an association between hypermutability and mucoidy has been reported in clinical CF isolates (Waine et al, 2008).…”

“…Inactivation of the MMR system was shown to favour the emergence in vitro of phenotypic variants considered typical markers of CF lung infection such as mucoidy due to mutations in mucA and loss of quorum sensing due to mutations in lasR (Smania et al, 2004). However, the time sequence in which the hypermutable, mucoid and quorum-sensing negative phenotypes emerge during chronic lung infection has not been investigated before in clinical CF P. aeruginosa isolates.…”