The pel genes of the Pseudomonas aeruginosa PAK strain are involved at early and late stages of biofilm formation Perrine Vasseur, 1 Isabelle Vallet-Gely, 1 3 Chantal Soscia, 1 Ste ´ phane Genin 2 and Alain Filloux 1 Correspondence Alain Filloux filloux@ibsm.cnrs-mrs.fr 1 Laboratoire d’Inge ´ nierie des Syste ` mes Macromole ´ culaires, CNRS-IBSM-UPR9027, 31 Chemin Joseph Aiguier, 13402 Marseille cedex 20, France 2 Laboratoire de Biologie Mole ´ culaire des Relations Plantes-Micro-organismes, INRA-CNRS, Castanet-Tolosan, France Received 16 June 2004 Revised 9 November 2004 Accepted 9 November 2004 Pseudomonas aeruginosa is a Gram-negative bacterium associated with nosocomial infections and cystic fibrosis. Chronic bacterial infections are increasingly associated with the biofilm lifestyle in which microcolonies are embedded in an extracellular matrix. Screening procedures for identifying biofilm-deficient strains have allowed the characterization of several key determinants involved in this process. Biofilm-deficient P. aeruginosa PAK strains affected in a seven-gene cluster called pel were characterized. The pel genes encode proteins with similarity to components involved in polysaccharide biogenesis, of which PelF is a putative glycosyltransferase. PelG was also identified as a putative component of the polysaccharide transporter (PST) family. The pel genes were previously identified in the P. aeruginosa PA14 strain as required for the production of a glucose-rich matrix material involved in the formation of a thick pellicle and resistant biofilm. However, in PA14, the pel mutants have no clear phenotype in the initiation phase of attachment. It was shown that pel mutations in the PAK strain had little influence on biofilm initiation but, as in PA14, appeared to generate the least robust and mature biofilms. Strikingly, by constructing pel mutants in a non-piliated P. aeruginosa PAK strain, an unexpected effect of the pel mutation in the early phase of biofilm formation was discovered, since it was observed that these mutants were severely defective in the attachment process on solid surfaces. The pel gene cluster is conserved in other Gram-negative bacteria, and mutation in a Ralstonia solanacearum pelG homologue, ragG, led to an adherence defect. INTRODUCTION Bacteria predominantly exist in sessile communities, rather than as free-living cells, and develop as biofilms on any surfaces. The establishment of the biofilm architecture follows a sequence of events, going from initial attach- ment of a single cell to formation of a mushroom-shaped mature biofilm (Stoodley et al., 2002). Such structural development of a biofilm could be dependent on environ- mental conditions. For example, depending on the carbon source, Pseudomonas aeruginosa forms a flat and very dynamic biofilm (citrate), or a heterogeneous biofilm containing mushroom-shaped multicellular structures separated by water-filled channels (glucose) (Klausen et al., 2003). A mature biofilm is engulfed in a matrix containing extracellular polymeric substances. The main components of this matrix are exopolysaccharides; in addition, nucleic acids and proteins are also found. The role of exopoly- saccharides in biofilm formation has not been fully elucidated. On the one hand, as with Vibrio cholerae (Watnick & Kolter, 1999), mutations that abolish exo- polysaccharide production are linked to a severe defect in the initial stages of attachment. On the other hand, as with Escherichia coli (Danese et al., 2000), it has been shown that colanic acid is required for biofilm architecture rather than for initial binding to surfaces. Consequently, these observations indicate that components involved in exopolysaccharide biogenesis may play a role at various stages of biofilm formation. In addition to exopolysacchar- ides, bacterial surface lipopolysaccharides (LPS) (Makin & Beveridge, 1996) or lipooligosaccharides (LOS) (Swords et al., 2004) are also involved in biofilm formation. Moreover, several determinants of adhesion and biofilm formation, such as pili, flagella and surface adhesins, appear to be glycosylated in several micro-organisms (Power & 3Present address: Children’s Hospital and Harvard Medical School, Boston, MA 02115, USA. Abbreviations: KDO, 2-keto-3-deoxyoctonate; PST, polysaccharide transporters. 0002-7410 G 2005 SGM Printed in Great Britain 985 Microbiology (2005), 151, 985–997 DOI 10.1099/mic.0.27410-0