Genetic Analysis of Functions Involved in Adhesion of Pseudomonas putida to Seeds

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.Soil bacteria belonging to the species Pseudomonas fluorescens and Pseudomonas putida show metabolic versatility and a variety of characteristics that makes them attractive for environmental and agricultural uses (26). They can colonize the surface of plant roots and the surrounding soil regions (rhizosphere) in a mutualistic association in which the bacteria obtain nutrients from root exudates. In turn, some bacterial strains can promote plant growth and have biocontrol potential against certain pathogens (39). Also, some P. putida strains have the ability to degrade toxic organic compounds, which are frequently present as contaminants in the environment (26). P. putida KT2440, a derivative of the soil isolate mt-2 which has been widely studied in relation to biodegradation processes (17,21,28), can also colonize the rhizosphere of agronomically relevant plants at high population densities, making it a suitable candidate for its use in rhizoremediation (20).Agricultural uses of microorganisms often involve coating seeds with bacterial suspensions. Adhesion to the seed appears as a key element, since it determines the subsequent colonization of the root system. Establishment of the bacterial population in the root and colonization of the rhizosphere are essential for biocontrol efficiency (8). These latter events, root colonization and survival in the rhizosphere, as well as responses to root exudates, are being extensively studied at the molecular level (5,25,30,32,33), but very little is known regarding the elements that are important for bacterial colonization of seeds. Results obtained by DeFlaun and coworkers have shown that some P. fluorescens mutants defective in attachment to ...

Selection of hyperadherent mutants in Pseudomonas putida biofilms

“…Transposon mutagenesis with mini-Tn5 was done by triparental conjugation using the protocol previously described (Espinosa-Urgel et al, 2000). Kanamycin-resistant clones were selected in minimal M9 medium with citrate as carbon source.…”

“…Our own observations of adhesion of the plantbeneficial bacterium P. putida KT2440 to plant seeds indicate that only a small percentage of the population initiates attachment to the surface (Espinosa-Urgel et al, 2000;Yousef-Coronado et al, 2008). This leaves open the possibility that in the planktonic population a subpopulation specialized in surface colonization arises, and therefore Abbreviations: CLSM, confocal laser-scanning microscopy; EPS, extracellular polymeric substances.…”

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

“…Serial dilutions were plated to estimate the initial number of bacteria inoculated per seed. After 1 h, seeds were washed and attached bacteria were recovered by vortexing in the presence of glass beads (17). Dilutions were plated in selective medium to quantify the number of cells that had attached to the seeds.…”

GacS-Dependent Regulation of Polyhydroxyalkanoate Synthesis in Pseudomonas putida CA-3

“…1B). The mini-Tn5 insertion site was mapped in each isolate using two consecutive rounds of arbitrary PCR as previously described (25), and the resulting amplicons were sequenced. Comparative analyses of the sequence data against the GenBank nonredundant nucleotide database were performed using the NCBI BLASTn algorithm (http://blast.ncbi.nlm.nih.gov/).…”

“…Arbitrarily primed PCR was employed to map the gene disruption sites by utilizing previously published oligonucleotide primers and appropriate thermal cycling parameters (25). Products were visualized on 1% agarose gels, purified using a Qiagen QIAquick gel extraction kit, and sequenced using the mini-Tn5 internal primer, TNINT (see Table S1 in the supplemental material).…”