Alkaline phosphatase of Pseudomonas aeruginosa: the mechanism of secretion and release of the enzyme from whole cells

1973 ◽  
Vol 19 (11) ◽  
pp. 1407-1415 ◽  
Author(s):  
J. M. Ingram ◽  
K. -J. Cheng ◽  
J. W. Costerton
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Cell Wall ◽  
Culture Filtrate ◽  
Outer Wall ◽  
Outer Cell ◽  
Periplasmic Space ◽  
Electron Microscopic ◽  
Whole Cells ◽  
Variable Quantity

The release of alkaline phosphatase from whole cells of Pseudomonas aeruginosa as a function of the MgCl2 concentration is proportional to the release of lipopolysaccharide from the cells. Cells grown under conditions where APase is almost completely secreted to the culture filtrate, i.e. growth at pH 7.6, also secrete lipopolysaccharide. Twenty percent sucrose releases a variable quantity of whole cell phosphatase. Localization of this portion of enzyme by biochemical and electron-microscopic techniques showed that it is located on the cell surface exterior to the outer tripartite layer. Phosphatase, which is not released by sucrose, but which is released by MgCl2, is located in the periplasmic space. Phosphatase is located in three areas; the culture filtrate, the outer cell wall surface, and the periplasmic space. The results suggest that A Pase is associated with, and bound to, a cell wall fraction which contains lipopolysaccharide and that the enzyme is "transported" through the outer wall in complex with this fraction. Liberation of the complex from the outer wall may be accomplished by the mechanical shearing forces developed during growth or during the sucrose suspension procedure.

In vitro studies of an alkaline phosphatase – cell wall complex from Pseudomonas aeruginosa

1975 ◽  
Vol 21 (1) ◽  
pp. 9-16 ◽  
Author(s):  
D. F. Day ◽  
J. M. Ingram
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Cell Wall ◽  
Complex Formation ◽  
Wall Material ◽  
Outer Cell ◽  
Whole Cells ◽  
Wall Components ◽  
Outer Cell Wall

Alkaline phosphatase (APase) of Pseudomonas aeruginosa exists primarily in the periplasmic region of the cell, i.e., between the cytoplasmic membrane and the outer tripartite layer. The enzyme is also found in the culture filtrate or associated with the outer layer of the cell wall. APase forms a complex with released outer cell wall material, and lipopolysaccharide (LPS) is associated with the complex. Since the enzyme was purified to homogeneity, it became desirable to determine whether complex formation with LPS, or the outer cell wall, affected any properties of the purified phosphatase. The ratio of activities of purified APase with p-nitrophenylphosphate and β-glycerolphosphate as substrates is about 4:1. The ratio of activities with enzyme complexed with LPS is about 1:1. The energy of activation of sucrose or magnesium released enzyme is 9500 cal/mol whereas the values for purified enzyme plus LPS, purified enzyme, purified enzyme plus phosphatidylethanolamine (PE), and purified enzyme plus LPS plus PE range from 3400 to 8700 cal/mol. These changes occur in the physiological temperature range, 27 to 39C, of this organism. Sucrose-released enzyme in the presence of substrate is inactivated at 47C whereas pure enzyme plus substrate is affected at 41C. The addition of LPS, PE, or a combination of both increases the temperature of inactivation from 45 to 51C. The results suggest that certain properties of the purified enzyme differ from those of the enzyme released from whole cells by either sucrose or magnesium resuspension. The addition of cell wall components such as LPS and PE to purified APase restores these properties. The evidence suggests that artificial complex formation changes the environment of the enzyme protein such that the environment now resembles that which exists within the whole cell wall.

Alkaline phosphatase localization and spheroplast formation of Pseudomonas aeruginosa

1970 ◽  
Vol 16 (12) ◽  
pp. 1319-1324 ◽  
Author(s):  
K. -J. Cheng ◽  
J. M. Ingram ◽  
J. W. Costerton
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Cell Wall ◽  
Electron Microscopic ◽  
Erroneous Conclusion ◽  
Lead Salts ◽  
Phosphatase System ◽  
Low Levels ◽  
Cell Wall Deposition ◽  
Periplasmic Enzyme

Pseudomonas aeruginosa cells grown in the presence of low levels of inorganic phosphate contain an inducible alkaline phosphatase system. The enzyme has been localized by electron microscopic techniques in the region between the cytoplasmic membrane and the tripartite layer of the cell wall, i.e. the periplasmic space. No deposits of lead salts are observed upon examination of either uninduced cells or cells in which the enzyme has been completely removed by 0.2 M magnesium washing. Samples of cells which were treated with glutaraldehyde before enzyme localization studies show cell wall deposition of lead salts which could give rise to the erroneous conclusion that the alkaline phosphatase was located in the tripartite layer. Cytochemical and biochemical studies are presented which show that discontinuities within the cell wall are insufficient to account for the release of this periplasmic enzyme and that dissociation by a divalent metal, increased pH, or both is required. As a consequence of this study it was possible to prepare true spheroplasts of P. aeruginosa.

Release of rhodanese from Pseudomonas aeruginosa by cold shock and its localization within the cell

1979 ◽  
Vol 25 (3) ◽  
pp. 340-351 ◽  
Author(s):  
R. W. Ryan ◽  
M. P. Gourlie ◽  
R. C. Tilton
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Outer Membrane ◽  
Electron Micrographs ◽  
Cold Shock ◽  
Cytoplasmic Membrane ◽  
Lactic Dehydrogenase ◽  
Periplasmic Space ◽  
Whole Cells ◽  
Periplasmic Enzyme

Whole cells of Pseudomonas aeruginosa possess rhodanese activity. The enzyme can be released by rapidly resuspending the cells in cold Tris–HCl buffer. Approximately 95% of the rhodanese activity is released by cold shock. Release of the enzyme can be inhibited either by preincubating the cells with Mg2+ or by incorporating Mg2+ into the shocking buffer. The effect of Mg2+ can be reversed by washing the cells twice with buffer prior to cold shock. While rhodanese can be released from P. aeruginosa by cold shock, lactic dehydrogenase, a cytoplasmic enzyme, remains within the cell. Diazo-7-amino-1,3-naphthalenedisulfonic acid, a compound which does not penetrate the cytoplasmic membrane, completely inactivated rhodanese and alkaline phosphatase, a periplasmic enzyme, whereas lactic dehydrogenase retained its full activity. These data suggest that rhodanese in P. aeruginosa, like alkaline phosphatase, is located distal to the cytoplasmic membrane in the periplasmic space. Electron micrographs also show that portions of the lipopolysaccharide outer membrane are shed from the cell during cold shock, while cells preincubated with Mg2+ did not release segments of their outer membrane.

Purification and characterization of Pseudomonas aeruginosa alkaline phosphatase

1973 ◽  
Vol 19 (10) ◽  
pp. 1225-1233 ◽  
Author(s):  
D. F. Day ◽  
J. M. Ingram
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Sodium Dodecyl ◽  
Current Theory ◽  
Outer Cell ◽  
Ph Optimum ◽  
Nitrophenyl Phosphate ◽  
A Subunit

Alkaline phosphatase and a subunit form of the enzyme have been isolated from Pseudomonas aeruginosa. The enzyme is pure as judged by molecular-sieve chromatography, sodium dodecyl gel electrophoresis, and ultracentrifugation. The enzyme possesses the following properties: (a) existence of three forms: monomer mol. wt. 39 000, dimer mol. wt. 68 000, and tetramer mol. wt. 139 000; (b) pH optimum 10.5; (c) Michaelis constant Km = 6.6 × 10−5 M p-nitrophenyl phosphate; and (d) energy of activation 5647 cal/mol. Amino acid analysis indicates a protein that is hydrophobic. Its physical behavior in solution supports this conclusion. These results explain the observed association of alkaline phosphatase and lipopolysaccharide and substantiate the current theory that the alkaline phosphatase of P. aeruginosa is bound to the outer cell wall in vivo.

The Outer Cell-Wall Membrane of Pseudomonas aeruginosa

1974 ◽  
Vol 130 (Supplement) ◽  
pp. S81-S93 ◽  
Author(s):  
J. C. Sadoff ◽  
M. S. Artenstein
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Wall ◽  
Outer Cell ◽  
Outer Cell Wall

The effect of complement and other cell wall reagents on tetracycline and streptomycin resistance in Pseudomonas aeruginosa

1974 ◽  
Vol 20 (8) ◽  
pp. 1101-1107 ◽  
Author(s):  
J. T. Tseng ◽  
L. E. Bryan
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Wall ◽  
Cell Membrane ◽  
Resistant Strain ◽  
Streptomycin Resistance ◽  
Outer Cell ◽  
Similar Magnitude ◽  
Peptidoglycan Layer ◽  
Outer Cell Wall

Lysozyme-free antiserum and complement treatment of strain 1885 of Pseudomonas aeruginosa was observed to destroy the penetration barrier of the outer cell wall to lysozyme but not to induce leakage of acid-soluble nucleotides through the cell membrane. The same treatment did not produce a significant increase in uptake of 3H-tetracycline or 3H-streptomycin by the resistant strain 1885 in spite of the destruction of the penetration barrier to lysozyme. A significant increase in both streptomycin and tetracycline uptake occurred in carbenicillin-treated strains but the increase was similar for both susceptible and resistant (to tetracycline and streptomycin) strains of P. aeruginosa. These data suggest (1) the outer cell wall is not a significant penetration barrier to these drugs; (2) the peptidoglycan layer does function as a penetration barrier of similar magnitude in resistant and susceptible cells; (3) the resistance of the strains is a property of the cell membrane or materials intimately associated with the cell membrane. The latter conclusion was further supported by the differential uptake of streptomycin in NaCl-lysozyme-induced spheroplasts of strains 1885 and 2379.

Spheroplasts of Methanospirillum hungatii formed upon treatment with dithiothreitol

1979 ◽  
Vol 25 (6) ◽  
pp. 730-738 ◽  
Author(s):  
G. D. Sprott ◽  
J. R. Colvin ◽  
R. C. McKellar
Keyword(s):  
Cell Wall ◽  
Attachment Site ◽  
Outer Wall ◽  
Methanogenic Bacteria ◽  
Methanosarcina Barkeri ◽  
Outer Cell ◽  
Alkaline Ph ◽  
Adhesive Material ◽  
Thin Sections ◽  
Outer Cell Wall

Strains of Methanospirillum hungatii, when treated with dithiothreitol at alkaline pH, formed spheroplasts which lysed in the absence of an osmotic stabilizer. Other methanogenic bacteria showed no response to this treatment, including Methanobacterium thermoautotrophicum. Methanobacterium strain M.o.H., Methanobacterium strain G2R, and Methanosarcina barkeri. The reaction with M. hungatii proceeded slowly at pH 8.0, but increased dramatically up to a pH of 10. Spheroplasts did not form in the presence of MgCl2 or if large amounts of FeS precipitate had been deposited on the cells during growth. Ultra-thin sections of M. hungatii GPI confirmed that individual cells, surrounded by a membrane and an inner wall, were contained within an outer wall or sheath. Adhesive material, which stained in the region of the cell spacer, was observed to bind together the inner and outer wall layers. After treatment with dithiothreitol, the spheroplasts, which retained the flexible inner wall and membrane, were released from the rigid outer wall. Examination of the isolated outer cell wall revealed that dithiothreitol (and dodecyl sodium sulfate) dissolved the adhesive material and damaged the cell spacer at the attachment site to the outer wall. It is proposed that dithiothreitol at alkaline pH loosens both the cell spacer and inner wall attachments to the outer wall, thereby allowing the osmotic pressure of the cytoplasm to eject the spheroplast. Resistance of the cells to lysis by Triton X-100 is lost upon spheroplast formation, indicating that protection to the cell is conferred by the outer cell wall.

Secretion and export of amylolytic activities in Schwanniomyces alluvius

1985 ◽  
Vol 63 (5) ◽  
pp. 366-371 ◽  
Author(s):  
C. V. Lusena ◽  
C. C. Champagne ◽  
G. B. Calleja
Keyword(s):  
Cell Wall ◽  
Sodium Dodecyl Sulfate ◽  
Cell Membrane ◽  
Molecular Species ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Periplasmic Space ◽  
Whole Cells ◽  
Sequence Of Events

We define secretion as the passage from the cytoplasm, across the cell membrane, to the periplasmic space. By contrast, export is the passage across the cell wall into the medium. Operationally we distinguish the two processes by comparing the molecular species in the medium, in whole cells, and in spheroplasts. Two techniques make the task possible: complete spheroplast preparation and detection of activities in bands obtained by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. The capability of Schwanniomyces alluvius to export α-amylase during stationary phase gradually increased with continual successive transfers from a slant culture to a liquid medium containing starch until a maximum was reached. Only cells which had developed full capability to export α-amylase were used in these studies. About 1 h after the end of the log phase of growth, α-amylase and glucoamylase start to be exported above constitutive levels and a concentration 10 times the constitutive level is reached 3 h later. Electrophoretic results show that at least three active molecular species of α-amylase appear in the cytoplasm at the end of log phase and that the smaller component (52 000 daltons) is secreted into the periplasm 0.5 h later and starts to be exported 1 h after that. The sequence of events suggests that the larger species are precursors of the 52 000 dalton molecules. Amylolytic activities in the cytoplasm and periplasm in late log phase are not detectable.

scholarly journals Interactions of Alkaline Phosphatase and the Cell Wall of Pseudomonas aeruginosa

1971 ◽  
Vol 107 (1) ◽  
pp. 325-336 ◽  
Author(s):  
K.-J. Cheng ◽  
J. M. Ingram ◽  
J. W. Costerton
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Cell Wall

Outer (cell wall) membrane proteins of Pseudomonas aeruginosa

1973 ◽  
Vol 19 (12) ◽  
pp. 1469-1471 ◽  
Author(s):  
J. D. Stinnett ◽  
R. G. Eagon
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Wall ◽  
Cell Envelope ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Outer Cell ◽  
Sucrose Density Gradient Centrifugation ◽  
Protein Components ◽  
Envelope Membranes ◽  
Outer Cell Wall

Cell envelope membranes were isolated from Pseudomonas aeruginosa. These membranes were resolved into cytoplasmic membrane rich and outer (cell wall) membrane rich fractions by discontinuous sucrose density gradient centrifugation. The resolution was based on the separation of enzyme activities and 2-keto-3-deoxyoctonate. Analysis by gel electrophoresis revealed that two of the three major cell envelope protein components were found in the fraction rich in outer (cell wall) membrane. These two protein components were previously shown to occur in a protein–lipopolysaccharide complex in this microorganism.

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