Mapping of export signals of Pseudomonas aeruginosa pilin with alkaline phosphatase fusions.

Pili of Pseudomonas aeruginosa are assembled from monomers of the structural subunit, pilin, after secretion of this protein across the bacterial membrane. These subunits are initally synthesized as precursors (prepilin) with a six-amino-acid leader peptide that is cleaved off during or after membrane traversal, followed by methylation of the amino-terminal phenylalanine residue. This report demonstrates that additional sequences from the N terminus of the mature protein are necessary for membrane translocation. Gene fusions were made between amino-terminal coding sequences of the cloned pilin gene (pilA) and the structural gene for Escherichia coli alkaline phosphatase (phoA) devoid of a signal sequence. Fusions between at least 45 amino acid residues of the mature pilin and alkaline phosphatase resulted in translocation of the fusion proteins across the cytoplasmic membranes of both P. aeruginosa and E. coli strains carrying recombinant plasmids, as measured by alkaline phosphatase activity and Western blotting. Fusion proteins constructed with the first 10 amino acids of prepilin (including the 6-amino-acid leader peptide) were not secreted, although they were detected in the cytoplasm. Therefore, unlike that of the majority of secreted proteins that are synthesized with transient signal sequences, the membrane traversal of pilin across the bacterial membrane requires the transient six-amino-acid leader peptide as well as sequences contained in the N-terminal region of the mature pilin protein.     

Interactions of alkaline phosphatase and the cell wall of Pseudomonas aeruginosa

Spheroplasts prepared by lysozyme treatment of cells of Pseudomonas aeruginosa, suspended in 20% sucrose or 0.2 m MgCl(2), were examined in detail. Preparation of spheroplasts in the presence of 0.2 m Mg(2+) released periplasmic alkaline phosphatase, whereas preparation in the presence of 20% sucrose did not, even though untreated cells released phosphatase when suspended in sucrose in the absence of lysozyme. Biochemical characterizations of the sucrose-lysozyme preparations indicated that lysozyme mediated a reassociation of the released phosphatase with the spheroplasts. In addition, the enzyme released from whole cells suspended in 20% sucrose (which represents 20 to 40% of the cell-bound phosphatase) reassociates with the cells in the presence of lysozyme. Electron microscopic examinations of various preparations revealed that phosphatase released in sucrose reassociated with the external cell wall layers in the presence of lysozyme, that sucrose-lysozyme prepared spheroplasts did not dissociate phosphatase which remained in the periplasm of sucrose-washed cells, and that phosphatase was never observed to be associated with the cytoplasmic membrane. A model to account for the binding of P. aeruginosa alkaline phosphatase to the internal portion of the tripartite layer of the cell wall rather than to the cytoplasmic membrane or peptidoglycan layer is presented.     

Cell division in Pseudomonas aeruginosa: participation of alkaline phosphatase.

Pseudomonas aeruginosa grows at an apparent reduced rate at 46 C as compared with the rate at 37 C, when growth is measured as an increase in absorbance. Cells at 46 C are long, plasmolyzed, nonmotile filaments. The filaments contain phase-dark material that may be chromosomal in nature. When the 46 C culture is shifted to 37 C, the filaments fragment at polar ends after flagella form, and the final number of cells is equal to the number of chromosomal "packets" observed within the filament. The outer envelope of the filament appears to be structurally complete as determined by biochemical, thin section, and freeze-etch examination. When filaments are treated with lysozyme, they form large spheroplasts, suggesting that the outer wall and the cytoplasmic membrane are continuous within the filament. Filaments produce little or no periplasm-located alkaline phosphatase (APase), but activity appears immediately after a shift to 37 C. Cells grown at 37 C and shifted to 46 C remain as single, nonmotile, rods or doublets, and the APase formed at 37 C remains stable at 46 C. The addition of APase or inorganic phosphate is partially or completely effective as an inducer of filament fragmentation at 46 C. The results suggest that periplasm-located APase is an important enzyme in the final stages of cell division when P. aeruginosa is cultured on inorganic phosphate-limiting media.     

Mapping of a gene controlling the production of phospholipase C and alkaline phosphatase in Pseudomonas aeruginosa

Summary Using plasmid R68.45 mediated conjugation and transduction with bacteriophage F116L, a genetic locus controlling at least two orthophosphate repressible proteins in Pseudomonas aeruginosa has been mapped at about 22–23 min on the PAO chromosome.     

Release of alkaline phosphatase from cells of Pseudomonas aeruginosa by manipulation of cation concentration and of pH

Pseudomonas aeruginosa ATCC 9027 contains an inducible alkaline phosphatase. The enzyme is readily removed from 14-hr cells by washes in 0.2 m MgCl(2), pH 8.4. Similar washes in tris(hydroxymethyl)aminomethane buffer, 20% sucrose, monovalent ions, or water partially release enzyme from the cells. The release of alkaline phosphatase is correlated with an increased release of protein and retention of internal enzymes. The effect of 0.2 m MgCl(2) washing upon the cells is minimal since both viability and growth rates remain unchanged as compared to water washing. Although cells are plasmolyzed in both 0.2 m MgCl(2) and 20% sucrose, it is evident that plasmolysis alone is unable to account for total enzyme release and that a divalent metal, i.e. Mg(2+), augments the release pattern. Growing cells in the presence of increasing concentrations of MgCl(2) or at increased pH values results in an almost total secretion of the enzyme to the culture filtrate. The findings suggest that P. aeruginosa alkaline phosphatase is linked to the exocytoplasmic region through divalent metal ion, presumably Mg(2+), bridges.     

Pyocyanin reactions with Pseudomonas aeruginosa cells and their fragments

Pyocyanin added to suspensions of Pseudomonas aeruginosa P changes the structural organization of the cells depending on their physiological characteristics, in particular, the capability to liberate pyocyanin into the cultural broth. Exogenous pyocyanin does not interact with the cells of the parent strain producing the pigment. However, the structure of the isolated cell walls deformed after the fractionation becomes similar, upon contact with pyocyanin, to the structure of the freshly isolated cell walls. The fraction of the cytoplasmic membranes of the parent strain also slightly changes its spatial organization after addition of pyocyanin. The cells of the mutant which do not produce pyocyanin display the ability for structural interaction with exogenous pyocyanin. In contrast, the fraction of their cell walls does not react to addition of the pigment. The cytoplasmic membranes of the mutant interact with pyocyanin in the same manner as the whole cells. These changes caused by pyocyanin always involve certain parts of the molecules. As a result, the following features are observed in their spectra: the absorption at 1475 cm-1 becomes more intensive; the "knee" at the band Amide II is more pronounced in the range of 1500 cm-1; the absorption at 1520 cm-1 decreases as well as the intensity of the band Amide II as a whole. The interaction of pyocyanin with biological objects stems, apparently, from the presence of certain proteins in them since peroxidase was found to respond to pyocyanin in a manner similar to that described for the cells and their fractions.     

Pseudomonas aeruginosa alkaline proteinase might share a biological function with plasmin.

Pseudomonas aeruginosa alkaline proteinase, which is a zinc-dependent bacterial endopeptidase, preferentially hydrolyzed Boc-Val-Leu-Lys-methylcoumarylamide (MCA) which was originally designed as a specific substrate of plasmin, a plasma serine proteinase. The hydrolytic capacity was resistant to tosyl-lysine chloromethylketone at a concentration as high as 1 mM, but was blocked by a treatment with metal chelator such as o-phenanthroline at the concentration of 5 mM. Kinetic parameters of the amidolytic reaction were Km = 21 microM, kcat = 0.067 s-1 and kcat/Km = 3190 M-1 s-1. A synthetic peptide inhibitor which bore a possible ligand for zinc atom at the carboxy terminal was designed. This inhibitor, Ac-Val-Leu-Lys-4-mercaptoanilide, blocked the amidolytic activity of the pseudomonal alkaline proteinase in a competitive manner with the dissociation constant (Ki) value of 24 microM. The results imply that P. aeruginosa alkaline proteinase must be an unusual zinc-dependent 'C (COOH)-type' endopeptidase, which hydrolyzes the peptide bond of certain amino acid residues at the carboxyl group side by specific recognition, like serine- and cysteine-proteinases. In comparison, P. aeruginosa elastase which is a typical 'N (NH2)-type' metalloproteinase did not hydrolyze all of the commercially available peptide-MCA substrates tested at the present study. P. aeruginosa alkaline proteinase also hydrolyzed natural substrates of plasmin, such as fibrin and fibrinogen, with similar specific activities to plasmin. The susceptible subunits of fibrinogen were the A-alpha and B-beta ones, in this order. P. aeruginosa alkaline proteinase also exhibited an anti-coagulant activity in human plasma attributed to the direct fibrinogenolytic function. Such potential anti-coagulant capacity of the P. aeruginosa alkaline proteinase might explain, at least partly, the most characteristic pathologic feature of the P. aeruginosa septicemia, hemorrhagic lesions with lacking thrombi (Fetzer, A.E. et al. (1967) Am. Rev. Respirat. Dis. 96, 1121-1130).     

Phosphate regulation in Pseudomonas aeruginosa: cloning of the alkaline phosphatase gene and identification of phoB- and phoR-like genes

Summary In Pseudomonas aeruginosa, phosphate limitation results in the synthesis of several protein species. We report the cloning of the P. aeruginosa alkaline phosphatase structural gene, phoA, and we show that this gene is regulated normally in Escherichia coli. We have also identified and cloned two P. aeruginosa genes which can complement phoB and phoR mutations in E. coli. This suggests that a pho regulon system similar to that in E. coli may exist in P. aeruginosa, using at least two similar regulatory factors.     

Pseudomonas aeruginosa acid phosphatase contains an anionic site with a trimethyl subsite

Summary 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, –CH₃OH 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.     

Calcium-induced folding and stabilization of the Pseudomonas aeruginosa alkaline protease

Pseudomonas aeruginosa is an opportunistic pathogen that contributes to the mortality of immunocompromised individuals and patients with cystic fibrosis. Pseudomonas infection presents clinical challenges due to its ability to form biofilms and modulate host-pathogen interactions through the secretion of virulence factors. The calcium-regulated alkaline protease (AP), a member of the repeats in toxin (RTX) family of proteins, is implicated in multiple modes of infection. A series of full-length and truncation mutants were purified for structural and functional studies to evaluate the role of Ca(2+) in AP folding and activation. We find that Ca(2+) binding induces RTX folding, which serves to chaperone the folding of the protease domain. Subsequent association of the RTX domain with an N-terminal α-helix stabilizes AP. These results provide a basis for the Ca(2+)-mediated regulation of AP and suggest mechanisms by which Ca(2+) regulates the RTX family of virulence factors.     

Reverse hydrolysis reaction of a recombinant alkaline ceramidase of Pseudomonas aeruginosa

Recently, we purified an alkaline ceramidase (CDase) of Pseudomonas aeruginosa and found that the enzyme catalyzed a reversible reaction in which the N-acyl linkage of ceramide was hydrolyzed or synthesized [J. Biol. Chem. 273 (1998) 14368-14373]. Here, we report the characterization of the reverse hydrolysis reaction of the CDase using a recombinant enzyme. The reverse hydrolysis reaction of the CDase was clearly distinguishable from the reaction of an acyl-coenzyme A (CoA) dependent N-acyltransferase, because the CDase catalyzed the condensation of a free fatty acid to sphingosine (Sph) without cofactors but did not catalyze the transfer of a fatty acid from acyl-CoA to Sph. The reverse hydrolysis reaction proceeded most efficiently in the presence of 0.05% Triton X-100 at neutral pH, while the hydrolysis reaction tended to be favored with an increase in the concentration of the detergent at alkaline pH. The specificity of the reverse reaction for fatty acids is quite broad; saturated and unsaturated fatty acids were efficiently condensed to Sph. In contrast, the stereo-specificity of the reverse reaction for the sphingoid bases is very strict; the D-erythro form of Sph, not the L-erythro or D/L-threo one, was only acceptable for the reverse reaction. Chemical modification of the enzyme protein affected or did not affect both the hydrolysis and reverse reactions to the same extent, suggesting that the two reactions are catalyzed at the same catalytic domain.     

Some specific reactions of the purine-oxidizing system of Pseudomonas aeruginosa

• 1.1. Whole resting cells of Pseudomonas aeruginosa oxidise 2-aminopurine as well as the 2-methylamino- and 2-dimethylamino derivative at position 8, in contrast to previous observations with mammalian xanthine oxidase.
• 2.2. 6-Mercaptopurine is attacked first at C-2, then at C-8. The resulting 6-thiouric acid is degraded further.
• 3.3. 6-Mercaptopurine does not inhibit growing cells of P. aeruginosa, but increases production of the bacterial xanthine oxidase.
• 4.4. The 3-methyl derivatives of thioxanthines are oxidised at position 8, while 3-methylhypoxanthine is attacked first at C-2. The resulting complex, containing 3-methylxanthine, dissociates before further oxidation to 3-methyluric acid can take place, in contrast to xanthine.