Generation of Enhanced Competitive Root-Tip-Colonizing Pseudomonas Bacteria through Accelerated Evolution
| Autor: | Guido V. Bloemberg, Sandra de Weert, Irene Kuiper, Linda C. Dekkers, Ben J. J. Lugtenberg |
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| Jazyk: | angličtina |
| Rok vydání: | 2004 |
| Předmět: |
Genetics
Transposable element Rhizosphere Pseudomonas Mutant Meristem Wild type Pseudomonas fluorescens Biology biology.organism_classification Microbiology digestive system diseases DNA Glycosylases Plant Microbiology Drug Resistance Bacterial Mutation Colonization Rifampin Pest Control Biological Molecular Biology Pseudomonadaceae |
| Popis: | 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 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 colonizers can be obtained after inoculation of seedlings with a mixture of different bacteria followed by growth of the seedling in a gnotobiotic sand system (32). The root tip contains the most efficient colonizing bacteria (32). Repeated use of this method yields enhanced root-tip-colonizing wild-type bacteria (14). In the present study we applied this procedure to a random collection of Tn5luxAB-marked mutants of the efficient colonizer P. fluorescens WCS365 in an attempt to isolate mutants that are efficient in competitive root tip colonization of both the dicotyledenous plant tomato and the monocotyledenous plant grass. Surprisingly, we found a mutant that proved to be a superior competitive colonizer compared to its parental strain. Its isolation and its genetic and functional analyses are described in this paper. |
| Databáze: | OpenAIRE |
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