Genetic analysis of functions involved in adhesion of Pseudomonas putida to seeds - PubMed (original) (raw)

Genetic analysis of functions involved in adhesion of Pseudomonas putida to seeds

M Espinosa-Urgel et al. J Bacteriol. 2000 May.

Abstract

Many agricultural uses of bacteria require the establishment of efficient bacterial populations in the rhizosphere, for which colonization of plant seeds often constitutes a critical first step. Pseudomonas putida KT2440 is a strain that colonizes the rhizosphere of a number of agronomically important plants at high population densities. To identify the functions involved in initial seed colonization by P. putida KT2440, we subjected this strain to transposon mutagenesis and screened for mutants defective in attachment to corn seeds. Eight different mutants were isolated and characterized. While all of them showed reduced attachment to seeds, only two had strong defects in their adhesion to abiotic surfaces (glass and different plastics). Sequences of the loci affected in all eight mutants were obtained. None of the isolated genes had previously been described in P. putida, although four of them showed clear similarities with genes of known functions in other organisms. They corresponded to putative surface and membrane proteins, including a calcium-binding protein, a hemolysin, a peptide transporter, and a potential multidrug efflux pump. One other showed limited similarities with surface proteins, while the remaining three presented no obvious similarities with known genes, indicating that this study has disclosed novel functions.

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Figures

FIG. 1

FIG. 1

Summary of the screening strategy used to isolate mus derivatives of P. putida KT2440. Details are presented in the text.

FIG. 2

FIG. 2

Adhesion of P. putida KT2440 (wild type [wt]) and mus mutants to corn seeds. (A) Quantitation of attachment of P. putida KT2440 and eight mus mutants to corn seeds. After 1 h of incubation with each bacterial suspension, seeds were washed and disrupted, and the number of attached cells was estimated as CFU after plating serial dilutions. Results are presented as percentage of attached cells with respect to the number of cells inoculated (average of at least three independent experiments). (B) Adhesion to corn seeds (left) of KT2440 and mus mutants harboring plasmid pDLDLUX, visualized by overnight exposure of film (right). (C) Adhesion of mus mutants (white bars) coinoculated with KT2442 (grey bar). Relative percentages of cells of each strain attached to the seeds (average of three independent assays) are shown.

FIG. 3

FIG. 3

Biofilm formation on different abiotic surfaces, quantified by staining attached cells with crystal violet and measuring _A_600 after solubilization of the stain with ethanol. Results are the average of four independent assays. wt, wild type.

FIG. 4

FIG. 4

Surface proteins of KT2440 and mus strains. Surface proteins were analyzed by SDS-PAGE on a 12% polyacrylamide gel and staining with Coomassie brilliant blue. Bands showing less intensity in the mutants than in the parental strain (wild type [wt]) are marked with black arrows; bands with more intensity in the mutants than in KT2440 are indicated with white arrows. Positions of molecular weight markers are indicated in kilodaltons.

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