The ability of plant-associated micro-organisms to colonize and compete in the rhizosphere is specially relevant for the biotechnological application of micro-organisms as inoculants. Pseudomonads are one of the best root colonizers and they are widely used in plant-pathogen biocontrol and in soil bioremediation. This study analyses the motility mechanism of the well-known biocontrol strain Pseudomonas fluorescens F113. A 6?5 kb region involved in the flagellar filament synthesis, containing the fliC, flaG, fliD, fliS, fliT and fleQ genes and part of the fleS gene, was sequenced and mutants in this region were made. Several non-motile mutants affected in the fliC, fliS and fleQ genes, and a fliT mutant with reduced motility properties, were obtained. These mutants were completely displaced from the root tip when competing with the wild-type F113 strain, indicating that the wild-type motility properties are necessary for competitive root colonization. A mutant affected in the flaG gene had longer flagella, but the same motility and colonization properties as the wild-type. However, in rich medium or in the absence of iron limitation, it showed a higher motility, suggesting the possibility of improving competitive root colonization by manipulating the motility processes. INTRODUCTIONThe study of rhizosphere colonization by micro-organisms is crucial for the efficient application of bacteria as inoculants, both in agricultural and in environmental biotechnology processes. Pseudomonas spp. can colonize the roots of a wide range of plants (Simons et al., 1996;Naseby & Lynch, 1998;Villacieros et al., 2003), being one of the best root colonizers, and are used as a model in root-colonization studies (Bloemberg et al., 2000; Chin-a-Woeng et al., 2000). The rhizosphere is a complex environment that supports a large and metabolically active microbial population, several orders of magnitude higher than the non-rhizospheric soil. Many bacterial genes and traits have been shown to be involved in plant-root colonization (Lugtenberg & Dekkers, 1999;Rainey, 1999;Lugtenberg et al., 2001). However, not only colonization but also the pseudomonads' ability to compete with the indigenous microbial population are essential to improve their biotechnological applications in the rhizosphere environment.The soil-borne fluorescent pseudomonads are used as biocontrol inoculants because of their ability to produce some antifungal metabolites (Dowling & O'Gara, 1994;Walsh et al., 2001). Other applications of pseudomonads include soil biofertilization and rhizoremediation (Ramos et al., 1991; Brazil et al., 1995; Höflich et al., 1995;Yee et al., 1998).The strain Pseudomonas fluorescens F113 was isolated from the sugarbeet rhizosphere and it is used as a biocontrol agent against the fungal pathogen Pythium ultimum, which causes damping-off disease in sugarbeet seedlings. The biocontrol abilities of this strain are due mainly to the production of the antifungal metabolite DAPG (2,4-diacetylphloroglucinol) (Shanahan et al., 1992). P. fluorescens ...
The biocontrol agent Pseudomonas fluorescens F113 undergoes phenotypic variation during rhizosphere colonization, and this variation has been related to the activity of a site-specific recombinase encoded by the sss gene. Here, it is shown that a second recombinase encoded by the xerD gene is also implicated in phenotypic variation. A putative xerD gene from this strain was cloned, and sequence analysis confirmed that it encoded a site-specific recombinase of the l integrase family. Mutants affected in the sss or xerD genes produced a very low quantity of phenotypic variants compared to the wild-type strain, both under prolonged cultivation in the laboratory and after rhizosphere colonization, and they were severely impaired in competitive root colonization. Overexpression of the genes encoding either recombinase resulted in a substantial increment in the production of phenotypic variants under both culture and rhizosphere colonization conditions, implying that both site-specific recombinases are involved in phenotypic variation. Overexpression of the sss gene suppressed the phenotype of a xerD mutant, but overexpression of the xerD gene had no effect on the phenotype of an sss mutant. Genetic analysis of the phenotypic variants obtained after overexpression of the genes encoding both the recombinases showed that they carried mutations in the gacA/S genes, which are necessary to produce a variety of secondary metabolites. These results indicate that the Gac system is affected by the activity of the site-specific recombinases. Transcriptional fusions of the sss and xerD genes with a promoterless lacZ gene showed that both genes have a similar expression pattern, with maximal expression during stationary phase. Although the expression of both genes was independent of diffusible compounds present in root exudates, it was induced by the plant, since bacteria attached to the root showed enhanced expression.
Pseudomonas fluorescens F113 is motile by means of type b flagella. Analysis of the region encoding the synthesis of the flagellar filament has shown a transcriptional organization different from that of type a flagella. Additionally to the promoters driving fliC, fliD, and fleQ expression, we have found promoters upstream of the flaG gene and the fliST operon. These promoters were functional in vivo. Both promoters have been mapped and appear to be dependent on the vegetative sigma factor and independent of FleQ, the master regulator of flagellum synthesis.Pseudomonads are ubiquitous bacteria that can adapt to different lifestyles, with species that are either saprophytic or pathogenic for plants and animals. Motility is an important trait for pseudomonads, and it has been shown to be involved in rhizosphere colonization (7,13,14,18), biofilm formation (9,20,27), and pathogenesis in plant (10, 22) and animal (4) models. Flagellar biosynthesis in pseudomonads is regulated in a hierarchical way, with the transcriptional activator fleQ on top of the regulatory cascade (2, 8). Besides, the alternative sigma factors encoded by fliA and rpoN are required for flagellum assembly (25,26). The regulation of flagellar biosynthesis has been analyzed using DNA microarrays and Pseudomonas aeruginosa PAK (12), which contains type a flagella (24). Other pseudomonads, like Pseudomonas fluorescens F113 or P. aeruginosa PAO1, contain type b flagella and are characterized by different syntenies in the region encoding the formation of the flagellar filament (7,12,24). In type b strains, this region contains fliC, encoding type b flagellin; flaG, encoding a protein of unknown function implicated in filament length (7); fliD, encoding the flagellar-cap protein (3); fliS, encoding a FliC chaperone required for flagellin export and assembly (7); fliT, encoding a protein of unknown function required for full motility and rhizosphere colonization in P. fluorescens F113 (7); fleQ (2, 8), encoding the master regulatory protein; and fleSR, encoding a two-component system required for flagellar biosynthesis (23).Analysis of the transcriptional organization of a region implicated in the synthesis of the flagellar filament in P. fluorescens F113. In order to establish the transcriptional organization of the fliC-fleQ region of Pseudomonas fluorescens F113, the upstream regions of the fliC, flaG, fliS, fliT, and fleQ genes were amplified and cloned into the reporter vector pMP220 to form transcriptional fusions with a promoterless lacZ gene. These constructs were introduced into strain F113 by triparental mating, and -galactosidase activity was tested (19). Substantial activity was shown for the regions upstream of all genes (125 to 350 Miller units), except the region upstream of the fliT gene (Ͻ5 Miller units) (see Fig. S1 in the supplemental material). Previous studies of other pseudomonads have shown the existence of promoters upstream of the fliC, fliD, and fleQ genes. To our knowledge, no promoters have been found upstream of the flaG or...
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