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

Abstract

Phenotypic variants of Pseudomonas fluorescens F113 showing a translucent and diffuse colony morphologyshow enhanced colonization of the alfalfa rhizosphere. We have previously shown that in the biocontrol agent P. fluorescens F113, phenotypic variation is mediated by the activity of two site-specific recombinases, Sss and XerD. By overexpressing the genes encoding either of the recombinases, we have now generated a large number of variants (mutants) after selection either by prolonged laboratory cultivation or by rhizosphere passage. All the isolated variants were more motile than the wild-type strain and appear to contain mutations in the gacA and/or gacS gene. By disrupting these genes and complementation analysis, we have observed that the Gac system regulates swimming motility by a repression pathway. Variants isolated after selection by prolonged cultivation formed a single population with a swimming motility that was equal to the motility of gac mutants, being 150% more motile than the wild type. The motility phenotype of these variants was complemented by the cloned gac genes. Variants isolated after rhizosphere selection belonged to two different populations: one identical to the population isolated after prolonged cultivation and the other comprising variants that besides a gac mutation harbored additional mutations conferring higher motility. Our results show that gac mutations are selected both in the stationary phase and during rhizosphere colonization. The enhanced motility phenotype is in turn selected during rhizosphere colonization. Several of these highly motile variants were more competitive than the wild-type strain, displacing it from the root tip within 2 weeks.Pseudomonas fluorescens F113 is a biocontrol agent isolated from the sugar beet rhizosphere (11) and capable of protecting this crop against the pathogenic fungus Pythium ultimum (17,31). In addition, derivatives of this strain with the ability to degrade polychlorinated biphenyls have been constructed by the integration of the Burkholderia sp. strain LB400 bph operon under the control of different regulatory elements (4, 38). P. fluorescens F113 is a good rhizosphere colonizer and can colonize the rhizospheres of different plants such as alfalfa (37), tomato (33), and pea (23).During alfalfa rhizosphere colonization, F113 undergoes phenotypic variation (27) characterized by the appearance of variants with a translucent and diffuse colony morphology. These variants were more prevalent in distal parts of the root (1, 27). Phenotypic variation in this strain appears to be mediated by the activity of two site-specific recombinases, Sss and XerD, since mutants with mutations in either of the genes encoding these recombinases show a severe reduction in the appearance of phenotypic variants after rhizosphere colonization and prolonged laboratory cultivation (22). Phenotypic variation seems to be an important trait for rhizosphere colonization, and mutants of different Pseudomonas strains affected in the sss (10, 22) or xerD (22) genes are ...

Importance of positioning for microbial evolution

“…The production of numerous extracellular secretions appears to correspond to the relative activity of the Gac system. Activity in the Gac pathway is variable in the pseudomonads (26,27) and Pf-01 is known to have a suppressed Gac system (32). This finding suggests that Pf-01 has a relatively low level of secretions to begin with and, under conditions that favor secretion, it may be more likely to lose rsmE function than a natural isolate that is already a strong secretor.…”

“…cated in metabolism, host-colonization, and pathogenesis in a wide range of organisms (25). Finding mutations in the Gac regulon is consistent with other data from Pseudomonas spp., which suggests that the Gac pathway is a common mutational target, which includes evidence that gac genes undergo reversible mutations (26) and gac mutants frequently evolve during rhizosphere colonization (27). RsmE is one of three Rsm proteins in the Pf0-1 genome.…”

Roles of Cyclic Di-GMP and the Gac System in Transcriptional Control of the Genes Coding for the Pseudomonas putida Adhesins LapA and LapF

“…However, under static conditions and shorter incubation times, similar to those used here, a gacA mutant was shown to be defective in biofilm maturation (45). In the plant root-colonizing strain P. fluorescens F113, mutations in gacS or gacA cause a hypermotile phenotype (46,47) as well as the loss of biocontrol activity (48). Although the production of certain secondary metabolites is regulated by this system, biocontrol activity also requires efficient colonization of the rhizosphere and of root surfaces.…”

Phase variation has a role in Burkholderia ambifaria niche adaptation

“…Interestingly, in P. fluorescens F113, phenotypic variation occurs during rhizosphere colonization (Martinez-Granero et al, 2006). For B. ambifaria HSJ1, an increase in the proportion of variants generated by the wild-type during the colonization of the pea rhizosphere was not observed.…”