aeruginosa strain SG81 and its derivates were made using the fluorigenic lipase substrate ELF®-97-palmitate (Figure 1). An emulsion of the water insoluble ELF-97®-palmitate was prepared using sodium desoxycholate and gum arabic for emulsification and stabilisation of the substrate according to the well-established method for lipase activity determination with pNPP as a substrate [45]. Biofilms were grown on agar medium (PIA) supplemented with 0.1 M CaCl2 for stabilization of the biofilm matrix, since Ca2+ ions enhance the mechanical stability of P. aeruginosa biofilms by complexing the polyanion alginate LY3039478 in vivo [25, 28, 46]. This facilitates

the treatment of the biofilms necessary for activity staining and subsequent observation by confocal laser scanning microscopy (CLSM).

Figure 1 Visualization of lipase activity in biofilms of P. aeruginosa. Membrane filter biofilms (PIA + Ca2+, 24 h, 36°C) of the parent strain P. aeruginosa SG81, the lipA overexpression strain SG81lipA+, the lipA defect mutant SG81ΔlipA and their corresponding complementation strain SG81ΔlipA::lipA were stained using the lipase substrate ELF®-97-palmitate. Shown are CLSM micrographs (optical section in the vertical middle of the biofilms) at a 400-fold magnification. For cell staining SYTO 9 (green) were used. Lipase activity, red; cells, green; overlay, yellow. The bars indicate 20 μm. A heterogeneous distribution of lipase activity within the biofilms was observed (Figure 1). Cellular activity in most

of the cells indicated Blasticidin S solubility dmso by the yellow colour and extracellular red-coloured regions surrounding the cells could be distinguished. Significantly more extracellular lipase activity was detected in the LipA overproducing strain P. aeruginosa SG81lipA+, indicating that the Glutamate dehydrogenase visualized extracellular lipase activity was mainly based on the activity of LipA. No extracellular but weak cell-associated activity was observed in the lipase mutant P. aeruginosa SG81ΔlipA. This can be explained by the activity of other lipolytic enzymes such as the outer-membrane bound esterase EstA, which is able to degrade palmitate [14, 47]. The second extracellular lipase LipC of P. aeruginosa is unable to degrade palmitate ester substrates (personal communication). Furthermore, a deletion within the foldase gene lipH may also affect folding and activity of LipC [39]. The defect of extracellular lipolytic activity could be complemented by the expression of lipA in trans from the MK-2206 chemical structure plasmid pBBL7. Accordingly, the complementation strain P. aeruginosa SG81ΔlipA::lipA revealed a level of lipase activity staining of the biofilms similar to the parent strain P. aeruginosa SG81. The biochemical detection of lipase activity in cell-free material from biofilms and the in situ visualization of lipase activity in the intercellular space of biofilms using palmitate-based enzyme substrates indicate that extracellular lipase is expressed in biofilms of mucoid P.