SummaryWe report the identification of an ATP-binding cassette (ABC) transporter and an associated large cellsurface protein that are required for biofilm formation by Pseudomonas fluorescens WCS365. The genes coding for these proteins are designated lap for l arge a dhesion p rotein. The LapA protein, with a predicted molecular weight of ~ 900 kDa, is found to be loosely associated with the cell surface and present in the culture supernatant. The LapB, LapC and LapE proteins are predicted to be the cytoplasmic membranelocalized ATPase, membrane fusion protein and outer membrane protein component, respectively, of an ABC transporter. Consistent with this prediction, LapE, like other members of this family, is localized to the outer membrane. We propose that the lapEBCencoded ABC transporter participates in the secretion of LapA, as strains with mutations in the lapEBC genes do not have detectable LapA associated with the cell surface or in the supernatant. The lap genes are conserved among environmental pseudomonads such as P. putida KT2440, P. fluorescens PfO1 and P. fluorescens WCS365, but are absent from pathogenic pseudomonads such as P. aeruginosa and P. syringae . The wild-type strain of P. fluorescens WCS365 and its lap mutant derivatives were assessed for their biofilm forming ability in static and flow systems. The lap mutant strains are impaired in an early step in biofilm formation and are unable to develop the mature biofilm structure seen for the wild-type bacterium. Time-lapse microscopy studies determined that the lap mutants are unable to progress from reversible (or transient) attachment to the irreversible attachment stage of biofilm development. The lap mutants were also found to be defective in attachment to quartz sand, an abiotic surface these organisms likely encounter in the environment.

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 ...

Bacterial life in the rhizosphere

A global analysis of Pseudomonas putida gene expression performed during the interaction with maize roots revealed how a bacterial population adjusts its genetic program to the specific conditions of this lifestyle.