Among 21 different polysaccharides tested, 5 greatly enhanced the spontaneous and cyclic AMP-induced formation of exolipase: glycogen, hyaluronate, laminarin, pectin B, and gum arabic. These polysaccharides have in common the tendency to form highly ordered networks because of the branching or helical arrangement, or both, of their molecules. None of the polysaccharides could be utilized by the cells as the sole carbon source. Strong lipid extraction of four different polysaccharides did not reduce their exolipase-enhancing efficacy. At a constant cell density the stimulation of exolipase formation by various concentrations of glycogen followed saturation kinetics, suggesting a limited number of "sites" for the glycogen to act. The active principle present in a solution of pectin was destroyed by degradation (,8-elimination) of the polymer. Hyaluronate lost its

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was excreted by Pseudomonas aeruginosa PAClR during the late logarithmic growth phase. Characterization of cell-free culture supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of significant amounts of lipopolysaccharide, part of which seemed to be tightly bound to lipase. After concentration of culture supernatants by ultrafiltration, lipase-lipopolysaccharide complexes were dissociated by treatment with EDTA-Tris buffer and subsequent sonication in the presence of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized lipase was purified by isoelectric focusing in an agarose gel containing the same detergent; the lipase activity appeared in a single peak corresponding to a distinct band in the silver-stained gel. The isoelectric point was 5.8. Analysis of purified lipase by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and scanning revealed an apparent molecular weight of 29,000 and a specific activity of 760 ,ikat/mg of protein. Estimations based on these data showed that a single P. aeruginosa cell excreted about 200 molecules of lipase, each having a molecular activity of 2.2 x 104 per s.Pseudomonas aeruginosa excretes several enzymes, three of which possess lipolytic activity: an extracellular phospholipase C (heat-labile hemolysin), a membrane-bound esterase, and an extracellular lipase. Phospholipase C (2) and esterase (26) have been purified to homogeneity and extensively characterized. Lipase (acylglycerol acylhydrolase, EC 3.1.1.3) has been only partially purified by ammonium sulfate precipitation from the growth medium of P. aeruginosa and subsequent column chromatographic steps (8,24).

The velamen radicum, a spongy, usually multiple epidermis of the roots, which at maturity consists of dead cells, is frequently described as an important adaptation of epiphytic orchids. Yet, quantitative evidence for the alleged functions, e.g., efficient water and nutrient uptake, nutrient retention, reduction of water loss, mechanical protection, or the avoidance of overheating, is rare or missing. We tested the notion originally put forward by Went in 1940 that the velamen allows plants to capture and immobilize the first solutions arriving in a rainfall, which are the most heavily charged with nutrients. In a series of experiments, we examined whether all necessary functional characteristics are given for this scenario to be realistic under ecological conditions. First, we show that the velamen of a large number of orchid species takes up solutions within seconds, while evaporation from the velamen takes several hours. Charged ions are retained in the velamen probably due to positive and negative charges in the cell walls, while uncharged compounds are lost to the external medium. Finally, we demonstrate that nutrient uptake follows biphasic kinetics with a highly efficient, active transport system at low external concentrations. Thus, our results lend strong support to Went's hypothesis: the velamen fulfills an important function in nutrient uptake in the epiphytic habitat. Most of the other functions outlined above still await similar experimental scrutiny.

Alginate-producing (mucoid) strains of Pseudomonas aeruginosa possess a 54-kDa outer membrane (OM) protein (AlgE) which is missing in nonmucoid bacteria. The coding region of the algE gene from mucoid P. aeruginosa CF3/M1 was subcloned in the expression vector pT7-7 and expressed in Escherichia coli. The level of expression of recombinant AlgE was seven times higher than that of the native protein in P. aeruginosa. Recombinant AlgE was found mainly in the OM. A putative precursor protein (56 kDa) of AlgE could be immunologically detected in the cytoplasmic membrane (CM). Surface exposition of AlgE in the OM of E. coli was indicated by labeling lysine residues with N-hydroxysuccinimide-biotin. Secondary-structure analysis suggested that AlgE is anchored in the OM by 18 membrane-spanning P-strands, probably forming a P-barrel.Recombinant AlgE was purified, and isoelectric focusing revealed a pI of 4.4. Recombinant AlgE was spontaneously incorporated into planar lipid bilayers, forming ion channels with a single-channel conductance of 0.76 nS in 1 M KCI and a mean lifetime of 0.7 ms. Single-channel current measurements in the presence of other salts as well as reversal potential measurements in salt gradients revealed that the AlgE channel was strongly anion selective. For chloride ions, a weak binding constant (Km = 0.75 M) was calculated, suggesting that AlgE might constitute an ion channel specific for another particular anion, e.g., polymannuronic acid, which is a precursor of alginate. Consistent with this idea, the open-state probability of the channel decreased when GDP-mannuronic acid was added. The AlgE channel was inactivated when membrane voltages higher than +85 mV were applied. The electrophysiological characteristics of AlgE, including its rectifying properties, are quite different from those of typical porins.Pseudomonas aeruginosa is an opportunistic human pathogen which infects particularly persons who are suffering from cystic fibrosis. The initial infection occurs mostly with nonmucoid bacteria, which convert to mucoid strains in an early stage of infection (13,26). Mucoid bacteria produce copious amounts of alginate, which seems to be the most important virulence factor (12). The initial steps of the alginate biosynthesis, leading to the putative precursor GDP-mannuronic acid, are well-known (21). However, there is a lack of information about the final steps of the biosynthesis, i.e., the polymerization and export of alginate. Recently, the genes involved in acetylation (algF) and degradation (algL) of alginate were cloned and mapped in the alginate biosynthesis gene cluster (34 min) (10, 30). In addition, the gene product of algG (34 min) was purified and identified as an epimerase located in the periplasm that introduces guluronic acid residues into the alginate (9). We identified a 54-kDa protein (AlgE) in the outer membrane (OM) of P. aeruginosa which appears in mucoid strains only (15,26). This protein was purified and the N-terminal amino acid sequence was determined (14). At the same time...

Tank epiphytes are adapted to low and intermittent nutrient supply by different mechanisms. They possess an effective mechanism to take up phosphate, minimizing dilution and loss of phosphorus captured in the tank. Available phosphorus is taken up from the tank solution almost quantitatively, and the surplus not needed for current metabolism is accumulated in reserves, i.e. plants show luxury consumption. Young, developing leaves are preferentially supplied with this nutrient element. Taken together, these features allow epiphytes the efficient use of scarce and variable nutrient supplies.