Carnitine resembles choline in the induction of cholinesterase, acid phosphatase, and phospholipase C and in its action as an osmoprotectant in Pseudomonas aeruginosa.

The present study demonstrates that under conditions of iso or hyperosmolarity, P. aeruginosa utilized carnitine as the carbon, nitrogen or carbon and nitrogen sources. As occurred in the case of choline, the bacteria synthesized cholinesterase (ChE), acid phosphatase (Ac.Pase) and phospholipase C (PLC) under any of these conditions and in the presence of high or low Pi concentrations. Carnitine acted as an osmoprotectant when the cells were grown in the presence of preferred carbon and nitrogen sources and high NaCl concentrations. Under these conditions the three enzyme activities were not produced. The osmotically stressed bacteria grown under any of the above conditions accumulated betaine. Its presence indicated that carnitine may be metabolized by P. aeruginosa to produce betaine which could account for the induction of the three enzyme activities or its action as an osmoprotectant. The phosphatidylcholine encountered in the host cell membranes allows the bacteria to obtain free choline by the coordinated action of PLC and Ac.Pase. Since the consequence of this action may be cell disruption, the increase of free carnitine in the natural environment of the bacteria is also possible. These two compounds, choline and carnitine, acting in conjunction or separately, may increase the production of PLC and Ac.Pase activities by P. aeruginosa and thus enhance the degradative effect upon the host cells.

Pseudomonas aeruginosa acid phosphatase. Activation by divalent cations and inhibition by aluminium ion.

In Pseudomonas aeruginosa, the effect of different cations on the acid phosphatase activity was studied in order to acquire more information related to a previously proposed mechanism, involving the coordinated action of this enzyme with phospholipase C. Although the natural substrate of this enzyme is phosphorylcholine, in order to avoid the possible interaction of its positive charge and those of the different cations with the enzyme molecule, the artificial substrate p-nitrophenylphosphate was utilized. Kinetic studies of the activation of acid phosphatase (phosphorylcholine phosphatase) mediated by divalent cations Mg2+, Zn2+ and Cu2+ revealed that all these ions bind to the enzyme in a compulsory order (ordered bireactant system). The Km values obtained for p-NPP in the presence of Mg2+, Zn2+ and Cu2+ were 1.4 mM, 1.0 mM and 3.5 mM, respectively. The KA values for the same ions were 1.25 mM, 0.05 mM and 0.03 mM, respectively. The Vmax obtained in the presence of Cu2+ was about twofold higher than that obtained in the presence of Mg2+ or Zn2+. The inhibition observed with Al3+ seems to be a multi-site inhibition. The K'app and n values, from the Hill plot, were about 0.25 mM and 4.0 mM, respectively, which were independent of the metal ion utilized as activator. It is proposed that the acid phosphatase may exert its action under physiological conditions, depending on the availability of either one of these metal ions.

Pseudomonas aeruginosa cholinesterase and phosphorylcholine phosphatase: two enzymes contributing to corneal infection.

Choline, acetylcholine and betaine used as the sole carbon, nitrogen or carbon and nitrogen source increase cholinesterase activity in addition to phosphorylcholine phosphatase and phospholipase C activities in Pseudomonas aeruginosa. The cholinesterase activity catalyses the hydrolysis of acetylthiocholine (Km approx. 0.13 mM) and propionylthiocholine (Km approx. 0.26 mM), but not butyrylthiocholine, which is a pure competitive inhibitor (Ki 0.05 mM). Increasing choline concentrations in the assay mixture decreased the affinity of cholinesterase for acetylthiocholine, but in all cases prevented inhibition raised by high substrate concentrations. Considering the properties of these enzymes, and the fact that in the corneal epithelium there exists a high acetylcholine concentration and that Pseudomonas aeruginosa produces corneal infection, it is proposed that these enzymes acting coordinately might contribute to the breakdown of the corneal epithelial membrane.

Identification of the Pseudomonas aeruginosa acid phosphatase as a phosphorylcholine phosphatase activity.

Choline, betaine and N,N-dimethylglycine as the sole carbon and nitrogen source induced a periplasmic acid phosphatase activity in Pseudomonas aeruginosa. This enzyme produced the highest rates of hydrolysis in phosphorylcholine and phosphorylethanolamine among the various phosphoric esters tested. At saturating concentrations of Mg2+, the Km values were 0.2 and 0.7 mM for phosphorylcholine and phosphorylethanolamine respectively. At high concentrations both compounds were inhibitors of the enzyme activity. The Ksi values for phosphorylcholine and phosphorylethanolamine were 1.0 and 3.0 mM respectively. The higher catalytic efficiency was that of phosphorylcholine. Considering these results it is possible to suggest that the Pseudomonas aeruginosa acid phosphatase is a phosphorylcholine phosphatase. The existence of this activity which is induced jointly with phospholipase C by different choline metabolites, in a high phosphate medium, suggests that the attack of Pseudomonas aeruginosa on the cell host may also be produced under conditions of high phosphate concentrations, when the alkaline phosphatase is absent.

Choline derivatives increase two different acid phosphatases in Rhizobium meliloti and Pseudomonas aeruginosa.

In Pseudomonas aeruginosa and Rhizobium meliloti several choline derivatives, utilized as the sole carbon and nitrogen source, increase acid phosphatase activity. The enzyme activity of both bacteria could be released into the surrounding medium by EDTA-lysozyme treatment. The R. meliloti acid phosphatase activity of crude periplasmic extracts measured with p-nitrophenylphosphate was not inhibited by the presence of 5 mM choline, betaine, trimethylammonium or phosphorylcholine. The activity could not be detected using phosphorylethanolamine or phosphorylcholine as substrates. Among several phosphoesters tested only pyridoxal-5-phosphate was hydrolyzed at a considerable rate. In 7.5% polyacrylamide slab gel electrophoresis (non-denaturing conditions) of crude extracts obtained from bacteria grown in the presence of serine, glutamate, aspartate or dimethylglycine a phosphatase activity with identical mobility could be detected with alpha-naphthylphosphate or pyridoxal-5-phosphate were used as substrates. In conclusion, although the choline metabolites are capable of increasing acid phosphatase activities in R. meliloti and in P. aeruginosa, there are two different enzymes involved, apparently in different metabolisms.

Choline and betaine as inducer agents of Pseudomonas aeruginosa phospholipase C activity in high phosphate medium.

In Pseudomonas aeruginosa, choline or betaine employed as the sole carbon and nitrogen source in a high phosphate medium induced a phospholipase C and an acid phosphatase activity but not an alkaline phosphatase activity. The P. aeruginosa strain utilized in this work does not possess a constitutive phospholipase C, since under culture conditions identical to those utilized by other authors (J. Bacteriol. 93, 670-674 (1967) and J. Bacteriol. 150, 730-738 (1982), our phospholipase C proved to be an inorganic phosphate-repressible enzyme. These findings enable us to conclude that although the phosphate control for the synthesis of phospholipase C may exist, it is expressed only under certain favorable culture conditions.

Choline transport in Pseudomonas aeruginosa.

Choline used as the sole carbon or carbon and nitrogen source induces in Pseudomonas aeruginosa an active transport system. The induction of the choline uptake is repressed by succinate independently of the presence of ammonium ion in the culture medium. The repression mediated by succinate was insensitive to cyclic AMP. Substitution for dibutyryl-cyclic AMP was without effect. Choline metabolites that also support the growth of Pseudomonas aeruginosa were poor inducer agents of the choline transport. Kinetic evidence and the employment of choline metabolites as effectors indicated that the choline uptake system of this bacterium is formed by at least two components: one of high affinity (Km = 3 microM) and another of low affinity (Km = 400 microM). Contrary to what occurs in the synaptosome system, the high affinity form for the choline uptake was not dependent on Na+ ions and is not inhibited by hemicholinium-3. Since Pseudomonas aeruginosa can utilize choline as the sole carbon and nitrogen source, the induction of the choline transport with two components in this bacterium may be related to its own strategy to survive and grow in an adverse environment.

Pseudomonas aeruginosa acid phosphatase contains an anionic site with a trimethyl subsite. Kinetic evidences obtained with alkylammonium ions.

In this work the action of the following compounds upon Ps. aeruginosa acid phosphatase has been studied: 1) alkylammonium compounds; 2) aminoalcohols and aminoacids with different substituents (-H, -CH2OH and -CH3) attached to the nitrogen atom; 3) alcohols analogous to some compounds of the above series, but without the amino group. It was found that the enzyme inhibition was more effective with N-trimethylated compounds than with the triethylated ones. The degree of inhibition depended on the number of methyl groups bound to the nitrogen atom. Taking into account the choline and betaine series the hydroxyl derivatives showed more affinity for the enzyme than the carboxylated ones. In each series the Ki values increased with the decrease of methyl groups bound to the nitrogen atom. The presence of a positively charged nitrogen atom in the molecule of the effector was essential. These results enable us to confirm that in the molecule of Ps. aeruginosa acid phosphatase there exists an anionic site with one subsite with affinity for methyl groups.

Pseudomonas aeruginosa acid phosphatase and cholinesterase induced by choline and its metabolic derivatives may contain a similar anionic peripheral site.

Different compounds derived from choline, and obtained by demethylation or by oxidation of the primary alcohol group with subsequent N-demethylation, were tested as inducer agents of acid phosphatase and cholinesterase in Ps. aeruginosa. It was found that betaine and dimethylglycine were the most effective inducers of both enzyme activities. These metabolites including choline itself, were not inducers of acid phosphatase and cholinesterase in other Gram-negative bacteria such as: Escherichia coli, Salmonella typhimurium, Shigella flexneri, Enterobacter liquefacciens and Proteus mirabilis. The acid phosphatase activities found in these bacteria were not inhibited in vitro by choline, betaine and phosphorylcholine. From these results it may be concluded that the acid phosphatase activity from Ps. aeruginosa is different from the same activity observed in the other bacteria. In addition, it is also shown that Ps. aeruginosa acid phosphatase and cholinesterase were inhibited by a number of compounds containing a positively charged amino group, with methyl or ethyl groups bound to it. These results seem to confirm that Ps. aeruginosa acid phosphatase and cholinesterase may contain a similar anionic site.

Induction of acid phosphatase and cholinesterase activities in Ps. aeruginosa and their in-vitro control by choline, acetylcholine and betaine.

Choline, acetylcholine and betaine used as a sole carbon source, effectuate in Ps. aeruginosa an acid phosphatase activity in addition to a cholinesterase activity. Induction of both enzyme activities was repressed by succinate or glucose. Cyclic AMP failed to relieve the repression produced by these compounds. Substrates not related to choline and used as a sole source of carbon, were inefficient to produce induction of both enzymes. The in-vitro action of choline, acetylcholine and betaine on Ps. aeruginosa acid phosphatase and cholinesterase has also been studied. To perform these studies periplasmic extracts obtained by EDTA-lysozyme treatment of the cells grown on choline or betaine as sole source of carbon, were used. Acid phosphatase activity was competitively inhibited by betaine, whereas the inhibition produced by choline and acetylcholine showed competitive and noncompetitive components. Cholinesterase activity was noncompetitively inhibited by betaine. At low acetylthiocholine concentration choline was an inhibitor of cholinesterase, whereas at high substrate concentration choline raised the hydrolysis rate of acetylthiocholine. These findings allow the conclusion that acid phosphatase and cholinesterase are specifically induced by choline and its metabolites derivatives. Kinetic results led us to postulate that acid phosphatase and cholinesterase contain a similar allosteric site. This site would either be of an anionic nature or show affinity to a methyl group or display both characteristics.

Acetylcholinesterase from rat red cells and cholinesterase of Pseudomonas aeruginosa: different types of inhibition by atropine.

The inhibition by atropine of cholinesterase from Pseudomonas aeruginosa has been studied in parallel with the membrane bound acetylcholinesterase from rat red cells. Acetylcholinesterase of rat red cells, like other animal cholinesterases, was competitively inhibited while the cholinesterase from Pseudomonas aeruginosa was partially non competitively inhibited by atropine. These results clearly indicated a different behavior of cholinesterase from Pseudomonas aeruginosa in comparison with the enzyme of Pseudomonas fluorescens and other animal cholinesterases.