SummaryWe have investigated the role of LapF, one of the two largest proteins encoded in the genome of Pseudomonas putida KT2440, in bacterial colonization of solid surfaces. LapF is 6310 amino acids long, and is localized on the cell surface. The C-terminal region of the protein is essential for its secretion, which presumably requires the ABC transporter encoded by an operon (lapHIJ) adjacent to the lapF gene. Although the initial attachment stages are not different between the wild type and a lapF mutant, microcolony formation and subsequent development of a mature biofilm is impaired in the mutant. This is consistent with the expression pattern of lapF; activation of its promoter takes place at late stages of growth and is regulated by the alternative sigma factor RpoS. A lapF mutant is also affected in individual and competitive plant root colonization. In these assays, mixed microcolonies formed by cells of both the wild-type and the mutant strains could be observed but microcolonies of the mutant alone were not found. These data and the localization of the protein at discrete spots in areas of contact between cells in biofilms suggest that LapF determines the establishment of cell-cell interactions during sessile growth.

Motility is a key trait for rhizosphere colonization by Pseudomonas fluorescens. Mutants with reduced motility are poor competitors, and hypermotile, more competitive phenotypic variants are selected in the rhizosphere. Flagellar motility is a feature associated to planktonic, free-living single cells, and although it is necessary for the initial steps of biofilm formation, bacteria in biofilm lack flagella. To test the correlation between biofilm formation and rhizosphere colonization, we have used P. fluorescens F113 hypermotile derivatives and mutants affected in regulatory genes which in other bacteria modulate biofilm development, namely gacS (G), sadB (S) and wspR (W). Mutants affected in these three genes and a hypermotile variant (V35) isolated from the rhizosphere were impaired in biofilm formation on abiotic surfaces, but colonized the alfalfa root apex as efficiently as the wild-type strain, indicating that biofilm formation on abiotic surfaces and rhizosphere colonization follow different regulatory pathways in P. fluorescens. Furthermore, a triple mutant gacSsadBwspR (GSW) and V35 were more competitive than the wild-type strain for root-tip colonization, suggesting that motility is more relevant in this environment than the ability to form biofilms on abiotic surfaces. Microscopy showed the same root colonization pattern for P. fluorescens F113 and all the derivatives: extensive microcolonies, apparently held to the rhizoplane by a mucigel that seems to be plant produced. Therefore, the ability to form biofilms on abiotic surfaces does not necessarily correlates with efficient rhizosphere colonization or competitive colonization.

A number of genetic determinants required for bacterial colonization of solid surfaces and biofilm formation have been identified in different micro-organisms. There are fewer accounts of mutations that favour the transition to a sessile mode of life. Here we report the isolation of random transposon Pseudomonas putida KT2440 mutants showing increased biofilm formation, and the detailed characterization of one of them. This mutant exhibits a complex phenotype, including altered colony morphology, increased production of extracellular polymeric substances and enhanced swarming motility, along with the formation of denser and more complex biofilms than the parental strain. Sequence analysis revealed that the pleiotropic phenotype exhibited by the mutant resulted from the accumulation of two mutations: a transposon insertion, which disrupted a predicted outer membrane lipoprotein, and a point mutation in lapG, a gene involved in the turnover of the large adhesin LapA. The contribution of each alteration to the phenotype and the possibility that prolonged sessile growth results in the selection of hyperadherent mutants are discussed. INTRODUCTIONThe development of a multicellular community associated with a surface and surrounded by an exopolymeric matrix, referred to as a biofilm, is common to a variety of bacteria under different environmental conditions. Biofilm formation has received increasing attention due to its importance in medicine, since biofilm populations are considered relevant to chronic infection, and are more resistant to the action of antibiotics and biocidals than planktonic populations (Anderson & O'Toole, 2008;Høiby et al., 2010). Biofilm development is also relevant in industrial settings and in the design of bioreactors (Nicolella et al., 2000;Singh et al., 2006). Genetic determinants that play a role in biofilm formation have been unravelled in different bacterial species, and environmental and cellular signals that influence this process have also been described. These include iron availability, quorum sensing, and the intracellular secondary messenger cyclic di-GMP (Banin et al., 2006;Patriquin et al., 2008;Ueda & Wood, 2009;Coenye, 2010). Changes in the levels of cyclic di-GMP correlate with phenotypic changes associated with virulence, motility, colony morphology, production of exopolysaccharides, and the transition between planktonic and sessile lifestyles (Hengge, 2009; Römling & Simm, 2009, and references therein). Changes in the expression of different functions are also associated with one or the other lifestyle; in Pseudomonas aeruginosa and Pseudomonas putida, for example, differential expression of flagellar components has been observed when comparing planktonic and biofilm populations, and even during the various stages of biofilm development (Sauer & Camper, 2001;Sauer et al., 2002;Toutain et al., 2007). In the former organism, swarming motility and surface attachment are inversely regulated through a pathway that involves BifA, a cyclic-di-GMP phosphodiesterase, and the membra...