Phenotypic Selection and Phase Variation Occur during Alfalfa Root Colonization by Pseudomonas fluorescens F113

Abstract

During colonization of the alfalfa rhizosphere, Pseudomonas fluorescens F113 undergoes phenotypic variation, resulting in the appearance of colonies with different morphology. Among phenotypic variants, three isolates, C, F, and S were selected, with the C variant showing colony morphology identical to that of the inoculated wild-type strain and F and S having a translucent and diffuse morphology. Phenotypic variants F and S were shown to preferentially colonize distal parts of the roots and showed alterations in motility, swimming faster than the C variant and swarming under conditions that did not allow swarming of the C variant. The motility behavior correlated with overproduction of the fliC-encoded protein flagellin but not with hyperflagellation. Flagella of the F and S variants were several times longer than those of the C variant, and overproduction of flagellin was regulated at the transcriptional level. Variant F showed alterations in traits that have been shown to be important for rhizosphere colonization, such as siderophore, cyanide, and exoprotease production, and these phenotypes were complemented by a cloned gacA. Sequence analysis of the gacA alelle in variant F suggested selection of the phenotype in the rhizosphere. Variant F was also affected in other phenotypes, such as lipopolysaccharide structure and flocculation in unshaken liquid medium, which were not complemented by the gacA or gacS gene. Mutation of the F113 sss gene, encoding a site-specific recombinase, showed that most of the phenotypic variation was due to the activity of this recombinase, indicating that phase variation occurs during rhizosphere colonization.Bacterial phase variation consists of the diversification of a population into subpopulations which present genotypic and phenotypic differences. It has been described for a variety of species, mostly within gram-negative bacteria (reviewed in reference 26). Phase variation is typical of bacteria that occupy heterogeneous ecological niches and has been related to adaptation to different environmental situations and to sudden changes in the ecosystem (49). Under these conditions, phase variation would generate a mixed population able to colonize different parts of the ecosystem, and in the case of rapid environmental changes, part of the initial population would survive (17).Phase variation predominantly affects surface components of cells, such as membrane antigens, flagella, and fimbriae, causing morphological alterations in colonies that allow easy detection (26). Typical examples are fimbrial variation in Escherichia coli, flagellar variation in Salmonella spp., and variation in surface antigens in several species of the genus Neisseria. From a genetic point of view, phase variation often results from genomic rearrangements that can be caused by several molecular mechanisms. The best-studied mechanisms are the action of a site-specific recombinase that can produce inversions or deletions of specific sites of the genome (1), mutations in homopolymeric tracts that produce fr...

Analysis of Pseudomonas fluorescens F113 genes implicated in flagellar filament synthesis and their role in competitive root colonization

“…1 shows the genetic organization of a 6?5 kb DNA region from the P. fluorescens F113 genome that was isolated from a cosmid that contained the fliC gene, from an F113 gene bank (Sanchez-Contreras et al, 2002). This region also contains another five ORFs and a partial ORF.…”

“…This mutant was as poor a competitor as aflagellate mutants, showing that not only flagella and chemotactic motility (de Weert et al, 2002), but also a wild-type level of motility are necessary for competitive rhizosphere colonization. Furthermore, although no differences in motility or colonization were observed for a flaG mutant under the standard conditions used, the fact that this mutant showed higher motility under certain conditions and the preferred location of hypermotile variants in distal parts of the root (Sanchez-Contreras et al, 2002) suggest the possibility of improving competitive root colonization by manipulating the motility processes. …”

Predators promote defence of rhizosphere bacterial populations by selective feeding on non-toxic cheaters

“…The resulting mutants are impaired in cooperative group traits, such as extracellular enzymes and toxin production (Lapouge et al, 2008), but under laboratory conditions show an improved growth rate compared to their wild-type ancestors due to the cessation of secondary metabolism (Bull et al, 2001). Despite being weak competitors when inoculated alone (Natsch et al, 1994), gacS/gacA mutants multiply rapidly within soil pseudomonad populations (Chancey et al, 2002;Sanchez-Contreras et al, 2002;Martinez-Granero et al, 2005). Consequently, we hypothesized that these mutants gain advantage by exploiting the exoproducts of the wild-type population, with their competitiveness being the highest at low frequency in a dense wild-type population (Velicer et al, 2000).…”

Cell Density-Dependent Gene Contributes to Efficient Seed Colonization by Pseudomonas putida KT2440

“…They include the two-component system colR/colS and a site-specific recombinase (13,14). Site-specific recombinases cause DNA rearrangements, which might correlate with the phenotypic variability observed in P. fluorescens populations isolated from roots (40). Other regulatory processes may be mediated by cell-cell communication via quorum-sensing signaling.…”