Generation of Enhanced Competitive Root-Tip-Colonizing Pseudomonas Bacteria through Accelerated Evolution

Abstract

A recently published procedure to enrich for efficient competitive root tip colonizers (I. Kuiper, G. V. Bloemberg, and B. J. J. Lugtenberg, Mol. Plant-Microbe Interact. 14:1197-1205) after bacterization of seeds was applied to isolate efficient competitive root tip colonizers for both the dicotyledenous plant tomato and the monocotyledenous plant grass from a random Tn5luxAB mutant bank of the good root colonizer Pseudomonas fluorescens WCS365. Unexpectedly, the best-colonizing mutant, strain PCL1286, showed a strongly enhanced competitive root-tip-colonizing phenotype. Sequence analyses of the Tn5luxAB flanking regions showed that the transposon had inserted in a mutY homolog. This gene is involved in the repair of A ⅐ G mismatches caused by spontaneous oxidation of guanine. We hypothesized that, since the mutant is defective in repairing its mismatches, its cells harbor an increased number of mutations and therefore can adapt faster to the environment of the root system. To test this hypothesis, we constructed another mutY mutant and analyzed its competitive root tip colonization behavior prior to and after enrichment. As a control, a nonmutated wild type was subjected to the enrichment procedure. The results of these analyses showed (i) that the enrichment procedure did not alter the colonization ability of the wild type, (ii) that the new mutY mutant was strongly impaired in its colonization ability, but (iii) that after three enrichment cycles it colonized significantly better than its wild type. Therefore it is concluded that both the mutY mutation and the selection procedure are required to obtain an enhanced root-tip-colonizing mutant.Competitive root tip colonization by Pseudomonas strains can play an important role in the efficient control of soilborne crop diseases caused by fungi (17,30,36,40). Inadequate colonization is often the limiting factor in biocontrol (3,30,39). Pseudomonas fluorescens WCS365 is an excellent colonizer of various plant root systems. To study competitive root tip colonization, we decided to characterize colonization traits and genes. The results of multiple studies have recently been reviewed by Lugtenberg et al. (18). Among the colonization mutants a mutant was found which is impaired in competitive root tip colonization and mutated in a gene with homology to the sss gene from Pseudomonas aeruginosa (7). This gene encodes a protein belonging to the integrase family of sitespecific recombinases involved in DNA rearrangements. The role of the sss homologue in colonization is proposed to be through genetic rearrangements causing different phenotypes. This colonization mutant is assumed to be locked in a phase that is not suitable for competitive colonization in the rhizosphere. Introduction of the sss gene into the poor colonizer P. fluorescens WCS307 and into the good colonizer P. fluorescens F113 improved competitive root-tip-colonizing abilities of these strains (6). These results show that it is possible to improve colonization through genetic engineering (6).Efficient colo...

Oxidative DNA Damage Defense Systems in Avoidance of Stationary-Phase Mutagenesis in Pseudomonas putida

“…Additionally, a mutYdefective derivative of Pseudomonas fluorescens showed a strongly enhanced competitive root-tip-colonizing phenotype through accelerated evolution (20). The fact that the absence of MutY increases the mutation frequency of Pseudomonas species has also been demonstrated in our work with P. putida (67).…”

“…The absence of MutY or MutT causes a strong mutator phenotype in E. coli, whereas the lack of MutM has a milder effect on mutation frequency (17,45,59,81). Mutators isolated from natural populations of Pseudomonas species have been found to be defective in either enzymes of DNA mismatch repair or MutY, but no mutators were identified with inactivation of the mutT or mutM gene (20,60,61). This raised the question of how essential MutT and MutM are in the avoidance of mutations in pseudomonads.…”

Rhizosphere Selection of Highly Motile Phenotypic Variants of Pseudomonas fluorescens with Enhanced Competitive Colonization Ability

“…Since efficient rhizosphere colonization is a requirement for biotechnological applications in biocontrol and rhizoremediation (8,20), engineering of competitiveness can be rendered in more effective strains. It has been previously shown that after three enrichment cycles in the rhizosphere, a P. fluorescens strain harboring a mutation in the mutY gene showed enhanced competitive colonization (12). Since the MutY protein is implicated in DNA repair and a mutant is therefore prone to accumulate mutations that can be selected by the rhizosphere environment, a similar mechanism can be inferred for the overexpression of the recombinases and selection of hypermotile strains after a single rhizosphere passage.…”

Antimutator Role of DNA Glycosylase MutY in Pathogenic Neisseria Species

“…Such a link is further emphasized by the fact that Pseudomonas sp. mutY mutants have been found to exhibit a root-tip hypercolonizer phenotype (10) and that Oliver et al (35), in the search for Pseudomonas aeruginosa mutators in cystic fibrosis patients, found four mutY mutants. Moreover, DNA glycosylase deficiencies have been associated with human cancer (42) and ageing (26).…”