Dataset Information

Characterisation of the MutS and MutL Proteins from the Pseudomonas avellanae Mismatch Repair (MMR) System.

ABSTRACT: The identification and analysis of the Pseudomonas avellanae mismatch repair system (MMR) were performed via sequencing and cloning the mutS and mutL genes and then analyzing the characteristics of the corresponding proteins studying their function and biological role in an E. coli heterologous system. In these studies, the P. avellanae MutS and MutL proteins were shown to localise at the nucleoid level, in a MutS-dependent manner as far as MutL is concerned, and were also able to complement the defect observed in both the mutS and mutL knockout strains of E. coli. In addition, their ability to form both homo and heterodimers between each other was shown by using the prokaryotic two-hybrid assay. Our results represent a first step to elucidate the MMR mechanism in plant pathogenic pseudomonads since the MMR genes were identified in P. syringae pathovars but there was no evidence on their action as effective repair products.

SUBMITTER: Grenga L

PROVIDER: S-EPMC3367300 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Characterisation of the MutS and MutL Proteins from the Pseudomonas avellanae Mismatch Repair (MMR) System.

Grenga Lucia L Gervasi Fabio F Paolozzi Luciano L Scortichini Marco M Ghelardini Patrizia P

The open microbiology journal 20120525

The identification and analysis of the Pseudomonas avellanae mismatch repair system (MMR) were performed via sequencing and cloning the mutS and mutL genes and then analyzing the characteristics of the corresponding proteins studying their function and biological role in an E. coli heterologous system. In these studies, the P. avellanae MutS and MutL proteins were shown to localise at the nucleoid level, in a MutS-dependent manner as far as MutL is concerned, and were also able to complement the ...[more]

PMID: 22670163

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Project description:BackgroundAcinetobacter baylyi ADP1 is an ideal bacterial strain for high-throughput genetic analysis as the bacterium is naturally transformable. Thus, ADP1 can be used to investigate DNA mismatch repair, a mechanism for repairing mismatched bases. We used the mutS deletion mutant (XH439) and mutL deletion mutant (XH440), and constructed a mutS mutL double deletion mutant (XH441) to investigate the role of the mismatch repair system in A. baylyi.ResultsWe determined the survival rates after UV irradiation and measured the mutation frequencies, rates and spectra of wild-type ADP1 and mutSL mutant via rifampin resistance assay (RifR assay) and experimental evolution. In addition, transformation efficiencies of genomic DNA in ADP1 and its three mutants were determined. Lastly, the relative growth rates of the wild type strain, three constructed deletion mutants, as well as the rifampin resistant mutants obtained from RifR assays, were measured. All three mutants had higher survival rates after UV irradiation than wild type, especially the double deletion mutant. Three mutants showed higher mutation frequencies than ADP1 and favored transition mutations in RifR assay. All three mutants showed increased mutation rates in the experimental evolution. However, only XH439 and XH441 had higher mutation rates than the wild type strain in RifR assay. XH441 showed higher transformation efficiency than XH438 when donor DNA harbored transition mutations. All three mutants showed higher growth rates than wild-type, and these four strains displayed higher growth rates than almost all their rpoB mutants. The growth rate results showed different amino acid mutations in rpoB resulted in different extents of reduction in the fitness of rifampin resistant mutants. However, the fitness cost brought by the same mutation did not vary with strain background.ConclusionsWe demonstrated that inactivation of both mutS and mutL increased the mutation rates and frequencies in A. baylyi, which would contribute to the evolution and acquirement of rifampicin resistance. The mutS deletion is also implicated in increased mutation rates and frequencies, suggesting that MutL may be activated even in the absence of mutS. The correlation between fitness cost and rifampin resistance mutations in A. baylyi is firstly established.

Dataset Information

Characterisation of the MutS and MutL Proteins from the Pseudomonas avellanae Mismatch Repair (MMR) System.

Publications

Characterisation of the MutS and MutL Proteins from the Pseudomonas avellanae Mismatch Repair (MMR) System.

Similar Datasets

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