Search

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.

Both reaction-product localization and ferritin-coupled antibody studies have shown that alkaline phosphatase is evenly distributed throughout the peri-plasmic space of Escherichia coli and a marine pseudomonad. This space is not locally enlarged except in cases where plasmolysis has occurred.

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.

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.

Ingram, Jordan M. (Michigan State University, East Lansing), and W. A. Wood. Enzymatic basis for d-arabitol production by Saccharomyces rouxii. J. Bacteriol. 89:1186-1194. 1965.-The enzymatic steps in d-arabitol synthesis by Saccharomyces rouxii were studied. The fermentation of d-glucose-6-C(14) gave rise to d-arabitol labeled at C-5; d-ribose of ribonucleic acid had the same isotope pattern. Crude extracts were able to reduce d-ribulose with reduced nicotinamide adenine dinucleotide phosphate (NADPH(2)) and d-xylulose with reduced nicotinamide adenine dinucleotide (NADH(2)). These extracts also oxidized d-arabitol with nicotinamide adenine dinucleotide phosphate and xylitol with nicotinamide adenine dinucleotide. No reduction of d-ribulose-5-phosphate or d-xylulose-5-phosphate was observed. An enzyme which reduced d-xylulose with NADH(2) was purified 33-fold and characterized as a xylitol (--> d-xylulose) dehydrogenase. Similarly, an enzyme reducing d-ribulose with NADPH(2) was purified 12-fold and characterized as a d-arabitol (--> d-ribulose) dehydrogenase. Alkaline and acid phosphatases were purified 50- and 40-fold, respectively, and their specificities were determined. Only the acid phosphatase had detectable activity on d-ribulose-5-phosphate. The data support the postulate that d-arabitol arises by dephosphorylation of d-ribulose-5-phosphate and reduction of d-ribulose by a NADPH(2)-linked d-arabitol (--> d-ribulose) dehydrogenase.