Two site-specific recombinases are implicated in phenotypic variation and competitive rhizosphere colonization in Pseudomonas fluorescens

Abstract

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.

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).…”

Evolutionary history predicts the stability of cooperation in microbial communities

“…We use an isogenic signal-blind mutant of one of the used genotypes (Pseudomonas fluorescens CHA0DgacS 30 ) as model defector of defined genetic background. This mutant is typical for spontaneous mutations deactivating secondary metabolism in pseudomonads 28 and lacks extracellular proteases required for growing on albumin 3,30 . It grows better than the wild type when cooperation is not required 4 , but preliminary experiments showed that it is incapable of growing on QSM medium alone.…”

“…The phenotype of the isolated defectors is typical for mutations in the gacS/gacA QS cascade 27 , a rapidly mutating system whose deactivation results in signal blindness and accounts for virtually all protease-deficient mutants in Pseudomonas spp. 9,28 .…”

“…Bacteria were serialdiluted and enumerated on milk agar plates (tryptic soy broth 3 g l À 1 , agar 15 g l À 1 and skim milk 150 ml l À 1 ). In Pseudomonads, protease-negative phenotypes emerge as a consequence of deactivation of the gac-rsm QS cascade 9,28 . To confirm that 'defectors' were not simply protease-deficient mutants, we looked for colonies lacking, in addition to proteolytic activity, tryptophane dioxygenase activity, another trait regulated by the gac-rsm pathway 27 .…”

“…Bacteria were grown in QSM 3 , a minimal medium containing 1% bovine serum albumin as the sole carbon source, at 25°C with agitation for 48 h. This time lapse corresponds to about 25 generations and is known to allow the evolution and spread of new phenotypes 28 . Bacteria were serialdiluted and enumerated on milk agar plates (tryptic soy broth 3 g l À 1 , agar 15 g l À 1 and skim milk 150 ml l À 1 ).…”