Moreover, in this TEM analysis, the lipC mutant revealed no significant differences in piliation (Fig. 2). The cells used for a series of TEM experiments were taken from swarming plates because this motility form requires both cell appendages, respectively. The fact that both were present in the lipC mutant in combination with the residual, but the considerable level of all motility forms suggests that LipC does not affect the biosynthesis of type IV pilus and flagella, but is required for the proper function of these organelles. Rhamnolipids as self-produced biosurfactants have been
shown to be involved in the modification of the cell surface properties of P. aeruginosa and influence Cilomilast motility (Al-Tahhan et al., 2000; Caiazza et al., 2005). In the presence of hydrophobic compounds, rhamnolipids mediate the release of lipopolysaccharide molecules from the cell surface (Al-Tahhan et al., 2000). A reduction in cell surface hydrophobicity observed for the lipC mutant (data not shown) may therefore indicate an altered production level of rhamnolipids. Hence, we have analysed and quantified the rhamnolipids present
in culture supernatants ACP-196 obtained from the wild type and the lipC mutant of P. aeruginosa (Fig. 3). Compared with the wild type, the lipC mutant showed reduced levels of rhamnolipids, which were found to be statistically significant. Interestingly, the total yield of rhamnolipid increased over wild-type levels when LipC was overexpressed from the plasmid pBBLCH, indicating that the amount of LipC enzyme present within the cells directly influences rhamnolipid production. Pseudomonas aeruginosa biofilm formation depends on several cellular functions. Flagella and type IV pili have been described to be essential for initial adhesion, spreading of the cells on the substratum and maturation of the typical mushroom-like structures of P. aeruginosa Cyclooxygenase (COX) biofilms, respectively (Klausen et al., 2003). In addition, rhamnolipids play a role in the development and maintenance of these structures (Davey et al., 2003). Because the lipC mutant was also impaired in motility, we assumed that biofilm formation would also be affected. Analysis
by CLSM revealed major qualitative and quantitative differences in the three-dimensional composition of biofilms formed by P. aeruginosa wild type and the lipC mutant. Whereas the wild type formed well-structured biofilms after 4 days of growth, the biofilms of the lipC mutant were smooth, with most of the cells being evenly spread on the substratum (Fig. 4). In these biofilms, only a few isolated very large colony-like structures silhouetted against an otherwise flat, but dense layer of cells. These mound-like structures lacked the apical caps of typical mushroom-like structures and appeared with a considerable space between each other. The biomass of the mutant biofilms measured with the comstat analysis software was increased by a factor of two (Table 2).