title: A novel papillation assay for the identification of genes affecting mutation rate in Pseudomonas putida and other pseudomonads.
abstract: Formation of microcolonies (papillae) permits easy visual screening of mutational events occurring in single colonies of bacteria. In this study, we have established a novel papillation assay employable in a wide range of pseudomonads including Pseudomonas aeruginosa and Pseudomonas putida for monitoring mutation frequency in distinct colonies. With the aid of this assay, we conducted a genome-wide search for the factors affecting mutation frequency in P. putida. Screening ∼27,000 transposon mutants for increased mutation frequency allowed us to identify 34 repeatedly targeted genes. In addition to genes involved in DNA replication and repair, we identified genes participating in metabolism and transport of secondary metabolites, cell motility, and cell wall synthesis. The highest effect on mutant frequency was observed when truA (tRNA pseudouridine synthase), mpl (UDP-N-acetylmuramate-alanine ligase) or gacS (multi-sensor hybrid histidine kinase) were inactivated. Inactivation of truA elevated the mutant frequency only in growing cells, while the deficiency of gacS affected mainly stationary-phase mutagenesis. Thus, our results demonstrate the feasibility of the assay for isolating mutants with elevated mutagenesis in growing as well as stationary-phase bacteria.
authors.affiliation: Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.
authors.forename: Mari
authors.initials: M
authors.lastname: Tagel
authors.affiliation: Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.
authors.forename: Kairi
authors.initials: K
authors.lastname: Tavita
authors.affiliation: Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.
authors.forename: Rita
authors.initials: R
authors.lastname: Hõrak
authors.affiliation: Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.
authors.forename: Maia
authors.initials: M
authors.lastname: Kivisaar
authors.affiliation: Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia. Electronic address: heili.ilves@ut.ee.
authors.forename: Heili
authors.initials: H
authors.lastname: Ilves
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D015169:Colony Count, Microbial; D004249:DNA Damage; D004260:DNA Repair; D004269:DNA, Bacterial / Q000235:genetics*; D005798:Genes, Bacterial; D019751:Intramolecular Transferases / Q000235:genetics; D016296:Mutagenesis; D009154:Mutation; D059645:Mutation Rate; D010957:Plasmids; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics*; D013395:Sucrose / Q000737:chemistry; D014157:Transcription Factors / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D014157:Transcription Factors; D013395:Sucrose; C074165:lemA protein, bacterial; D019751:Intramolecular Transferases; C023199:tRNA-pseudouridine synthase I
keywords: Mutagenesis; Mutator; Papilla; Pseudomonads; gacS; truA