Characterization of phenotypic changes in Pseudomonas putida in response to surface-associated growth

The formation of complex bacterial communities known as biofilms begins wit h the interaction of planktonic cells with a surface. A switch between plan ktonic and sessile growth is believed to result in a phenotypic change in b acteria. In this study, a global analysis of physiological changes of the p lant saprophyte Pseudomonas putida following 6 h of attachment to a silicon e surface was carried out by analysis of protein profiles and by mRNA expre ssion patterns. Two-dimensional (2-D) gel electrophoresis revealed 15 prote ins that were up-regulated following bacterial adhesion and 30 proteins tha t were down-regulated. N-terminal sequence analyses of 11 of the down-regul ated proteins identified a protein with homology to the ABC transporter, Po tF; an outer membrane lipoprotein, NIpD; and five proteins that were homolo gous to proteins involved in amino acid metabolism. cDNA subtractive hybrid ization revealed 40 genes that were differentially expressed following init ial attachment of P. putida. Twenty-eight of these genes had known homologs . As with the 2-D gel analysis, NIpD and genes involved in amino acid metab olism were identified by subtractive hybridization and found to be down-reg ulated following surface-associated growth. The gene for PotB was up-regula ted, suggesting differential expression of ABC transporters following attac hment to this surface. Other genes that showed differential regulation were structural components of flagella and type IV pili, as well as genes invol ved in polysaccharide biosynthesis. Inummoblot analysis of PHA and FliC con firmed the presence of flagella in planktonic cultures but not in 12- or 24 -h biofilms. In contrast, PHA was observed in 12-h biofilms but not in plan ktonic culture. Recent evidence suggests that quorum sensing by bacterial h omoserine lactones (HSLs) may play a regulatory role in biofilm development . To determine if similar protein profiles occurred during quorum sensing a nd during early biofilm formation, HSLs extracted from P. putida and pure C -12-HSL were added to 6-h planktonic cultures of P. putida, and cell extrac ts were analyzed by 2-D gel profiles. Differential expression of 16 protein s was observed following addition of HSLs. One protein, PotF, was found to be down-regulated by both surface-associated growth and by HSL addition. Th e other 15 proteins did not correspond to proteins differentially expressed by surface-associated growth. The results presented here demonstrate that P. putida undergoes a global change in gene expression following initial at tachment to a surface. Quorum sensing may play a role in the initial attach ment process, but other sensory processes must also be involved in these ph enotypic changes.