Summary: We report the identification of an ATP‐binding cassette (ABC) transporter and an associated large cell‐surface protein that are required for biofilm formation by Pseudomonas fluorescens WCS365. The genes coding for these proteins are designated lap for large adhesion protein. 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 membrane‐localized 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 lapEBC‐encoded 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.

Background: Mutualistic interactions less well known than those between rhizobia and legumes are commonly found between plants and bacteria, frequently pseudomonads, which colonize roots and adjacent soil areas (the rhizosphere). Results: A global analysis of Pseudomonas putida genes expressed during their interaction with maize roots revealed how a bacterial population adjusts its genetic program to this lifestyle. Differentially expressed genes were identified by comparing rhizosphere-colonizing populations with three distinct controls covering a variety of nutrients, growth phases and life styles (planktonic and sessile). Ninety rhizosphere up-regulated (rup) genes, which were induced relative to all three controls, were identified, whereas there was no repressed gene in common between the experiments. Genes involved in amino acid uptake and metabolism of aromatic compounds were preferentially expressed in the rhizosphere, which reflects the availability of particular nutrients in root exudates. The induction of efflux pumps and enzymes for glutathione metabolism indicates that adaptation to adverse conditions and stress (oxidative) response are crucial for bacterial life in this environment. The finding of a GGDEF/EAL domain response regulator among the induced genes suggests a role for the turnover of the secondary messenger c-diGMP in root colonization. Several mutants in rup genes showed reduced fitness in competitive root colonization. Conclusion: Our results show the importance of two selective forces of different nature to colonize the rhizosphere: stress adaptation and availability of particular nutrients. We also identify new traits conferring bacterial survival in this niche and open a way to the characterization of specific signalling and regulatory processes governing the plant-Pseudomonas association.

L -Lysine catabolism in Pseudomonas putida KT2440 was generally thought to occur via the aminovalerate pathway. In this study we demonstrate the operation of the alternative aminoadipate pathway with the intermediates d -lysine, l -pipecolate, and aminoadipate. The simultaneous operation of both pathways for the use of l -lysine as the sole carbon and nitrogen source was confirmed genetically. Mutants with mutations in either pathway failed to use l -lysine as the sole carbon and nitrogen source, although they still used l -lysine as the nitrogen source, albeit at reduced growth rates. New genes were identified in both pathways, including the davB and davA genes that encode the enzymes involved in the oxidation of l -lysine to δ-aminovaleramide and the hydrolysis of the latter to δ-aminovalerate, respectively. The amaA , dkpA , and amaB genes, in contrast, encode proteins involved in the transformation of Δ ¹ -piperidine-2-carboxylate into aminoadipate. Based on l -[U- ¹³ C, U- ¹⁵ N]lysine experiments, we quantified the relative use of pathways in the wild type and its isogenic mutants. The fate of ¹³ C label of l -lysine indicates that in addition to the existing connection between the d - and l -lysine pathways at the early steps of the catabolism of l -lysine mediated by a lysine racemase, there is yet another interconnection at the lower end of the pathways in which aminoadipate is channeled to yield glutarate. This study establishes an unequivocal relationship between gene and pathway enzymes in the metabolism of l -lysine, which is of crucial importance for the successful colonization of the rhizosphere of plants by this microorganism.