Tris(hydroxymethyl)aminomethane buffer modification of Escherichia coli outer membrane permeability.

The effect of tris(hydroxymethyl)aminomethane (Tris) buffer on outer membrane permeability was examined in a smooth strain (D280) and in a heptose-deficient lipopolysaccharide strain (F515) of Escherichia coli O8. Tris buffer (pH 8.00) was found to increase outer membrane permeability on the basis of an increased Vo of whole-cell alkaline phosphatase activity and on the basis of sensitivity to lysozyme and altered localization pattern of alkaline phosphatase. The Tris buffer-mediated increase in outer membrane permeability was found to be dependent upon the extent of exposure to and concentration of the Tris buffer. The Tris buffer effects were demonstrated not to be due to allosteric activation of cell-associated alkaline phosphatase and were specific for Tris buffer. Exposure of cells to Tris resulted in the release of a limited amount of cell envelope component. Investigators utilizing Tris buffer are cautioned that Tris is not physiologically inert and that it may interact with the system under investigation.     

Effect of saline on the releasability of alkaline phosphatase from cells of Serratia marcescens.

The effect of 0.9% sodium chloride solution on the release of alkaline phosphatases from cells of four strains of Serratia marcescens was studied. Saline had a greater action in the releasability of the enzyme on cells of the polymyxin B sensitive strains than those of the polymyxin B resistant strains. SDS-polyacrylamide gel electrophoresis of the released materials showed the presence of proteins and lipopolysaccharide components of the outer membrane as well as enzyme activity in all four strains. Cells from strains harvested under higher temperatures contained more releasable activity in the salin wash fraction than those harvested under refrigerated condition. Active components with molecular weights of 190,000 and 110,000 daltons were either absent or present to a lesser degree in the extracts released by the polymyxin B treatment of the washed cells. However, active components not released by saline were found in the polymyxin B extracts. Contrary to other reports, results of this study clearly showed the ubiquitous nature of alkaline phosphatase in S. marcescens. It appears that their releasability is related to the polymyxin B susceptibility as well as the instability of the outer membrane of the cell envelope.     

Glutaraldehyde reactions with alkaline phosphatase of Pseudomonas aeruginosa

Glutaraldehyde, the biological fixative of choice in the cytochemical localization of the phosphatases, was investigated for its effects on Pseudomonas aeruginosa alkaline phosphatase. Comparative studies on the inactivation of alkaline phosphatase by glutaraldehyde showed significant differences when the purified protein was compared with whole, cell-bound enzyme. The effects of the reagent on the kinetics of the purified enzyme were studied and some conclusions drawn as to the mode of inactivation. The reaction of glutaraldehyde with the cell envelope of P. aeruginosa was also investigated, and it was found not to modify the extraction of lipopolysaccharides from the outer membrane. This study emphasizes the care that must be taken to interpret data, cytochemical or otherwise, obtained when glutaraldehyde is used as a fixative or cross-linking reagent.     

Biochemical Localization of Alkaline Phosphatase in the Cell Wall of a Marine Pseudomonad

The various layers of the cell envelope of marine pseudomonad B-16 (ATCC 19855) have been separated from the cells and assayed directly for alkaline phosphatase activity under conditions established previously to be optimum for maintenance of the activity of the enzyme. Under conditions known to lead to the release of the contents of the periplasmic space from the cells, over 90% of the alkaline phosphatase was released into the medium. Neither the loosely bound outer layer nor the outer double-track layer (cell wall membrane) showed significant activity. A small amount of the alkaline phosphatase activity of the cells remained associated with the mureinoplasts when the outer layers of the cell wall were removed. Upon treatment of the mureinoplasts with lysozyme, some alkaline phosphatase was released into the medium and some remained with the protoplasts formed. Cells washed and suspended in 0.5 M NaCl were lysed by treatment with 2% toluene, and 95% of the alkaline phosphatase in the cells was released into the medium. Cells washed and suspended in complete salts solution (0.3 M NaCl, 0.05 M MgSO(4), and 0.01 M KCl) or 0.05 M MgSO(4) appeared intact after treatment with toluene but lost 50 and 10%, respectively, of their alkaline phosphatase. The results suggest that the presence of Mg(2+) in the cell wall is necessary to prevent disruption of the cells by toluene and may also be required to prevent the release of alkaline phosphatase by toluene when disruption of the cells by toluene does not take place.     

Citrate-tris(hydroxymethyl)aminomethane-mediated release of outer membrane sections from the cell envelope of a deep-rough (heptose-deficient lipopolysaccharide) strain of Escherichia coli O8.

A heptose-deficient lipopolysaccharide strain of Escherichia coli O8, strain F515, was found to release portions of its outer membrane when cells were exposed to 10 mM citrate buffer (pH 2.75) for 30 min and subsequently exposed to 100 mM tris(hydroxymethyl)aminomethane buffer (pH 8.00). The outer membrane component release was found to be composed of protein, lipopolysaccharide, phospholipid (cardiolipin, phosphatidylethanolamine, and phosphatidylglycerol), and alkaline phosphatase. The outer membrane component was released from the cell envelope in the absence of cell lysis, as no glucose-6-phosphate dehydrogenase activity or succinic dehydrogenase activity was detected. Morphologically, the outer membrane component appeared to consist of laminar fragments and vesicles which had an associated alkaline phosphatase activity.     

Presence and activity of alkaline phosphatase in two human osteosarcoma cell lines

The presence and activity of alkaline phosphatase in SAOS-2 and TE-85 human osteosarcoma cells grown in culture were examined at the ultrastructural level. A monoclonal antibody raised against purified human bone osteosarcoma alkaline phosphatase was used to localize the enzyme in cultures of the osteosarcoma cells. Similar cultures were analyzed for alkaline phosphatase activity using an enzyme cytochemical method with cerium as the capture agent. Alkaline phosphatase was immunolocalized at the light microscopic level in an osteogenic sarcoma and ultrastructurally on the SAOS-2 cell membrane and the enclosing membrane of extracellular vesicular structures close to the cells. In contrast, the TE-85 cells were characterized by the absence of all but a few traces of immunolabeling at the cell surface. Enzyme cytochemical studies revealed strong alkaline phosphatase activity on the outer surface of the SAOS-2 cell membrane. Much lower enzyme activity was observed in the TE-85 cells. The results support biochemical data from previous studies and confirm that SAOS-2 cells have a significantly greater concentration of alkaline phosphatase at the plasma membrane.     

Heat-induced blebbing and vesiculation of the outer membrane of Escherichia coli.

Thermal damage to the outer membrane of Escherichia coli W3110 was studied. When E. coli cells were heated at 55 degrees C in 50 mM Tris-hydrochloride buffer at pH 8.0, surface blebs were formed on the cell envelope, mainly at the septa of dividing cells. Membrane lipids were released from the cells during the heating period, and part of the released lipids formed vesicle-like structures from the membrane. This vesicle fraction had a lipopolysaccharide to phospholipid ratio similar to that of the outer membrane of intact cells, whereas it had a lower content of protein than the isolated outer membrane. After heating bacterial cells at 55 degrees C for 30 min, the resulting leakage from the cells of a periplasmic enzyme, alkaline phosphatase, amounted to 52% of the total activity, whereas no release of a cytoplasmic enzyme, glucose-6-phosphate dehydrogenase, was detected. The results obtained suggest that surface blebs formed by heat treatment almost completely consist of the outer membrane and that the blebs may be gradually released from the cell surface into the heating menstruum to partially form vesicles.     

Alkaline phosphatase fusions: sensors of subcellular location.

Alkaline phosphatase fusions allow genes to be identified solely on the basis of their protein products being exported from the cytoplasm. Thus, the use of such fusions helps render biological processes which involve cell envelope and secreted proteins accessible to a sophisticated genetic analysis. Furthermore, alkaline phosphatase fusions can be used to locate export signals. Specifying such signals is an important component of studies on the structure of individual cell envelope proteins. The basis of the alkaline phosphatase fusion approach is the finding that the activity of the enzyme responds differently to different environments. Thus, the activity of the fusion protein gives evidence as to its location. This general approach of using sensor proteins which vary in their function, depending on their environment, could be extended to the study of other sorts of problems. It may be that certain enzymes will provide an assay for localization to a particular subcellular compartment, if the environment of the compartment differs from that of others. For instance, the lysosome is more acidic than other intracellular organelles. A gene fusion system employing a reporter enzyme that could show activity only at the pH of the lysosome could allow the detection of signals determining lysosomal localization. Analogous types of enzymes may be used as probes for other subcellular compartments.     

Alkaline phosphatase activity of rumen bacteria.

Of the 54 strains of rumen bacteria examined for alkaline phosphatase (APase) production, 9 of 33 gram-negative strains and none of 21 gram-positive strains produced the enzyme. The APase of the cells of the three strains of Bacteroides ruminicola that produced significant amounts of the enzyme was located in the periplasmic area of the cell envelope, whereas the enzyme was located in the strains of Selenomonas ruminantium and Succinivibrio dextrinosolvens was associated with the outer membrane. The localization of APase production in the cells of natural populations of rumen bacteria from hay-fed sheep was accomplished by reaction product deposition, and both the proportion of APase-producing bacteria and the location of the enzyme in the cell envelope of the producing cells could be determined. We suggest that this procedure is useful in detecting shifts in the bacterial population and the release of cell-bound APase that accompany feedlot bloat and other sequelae of dietary manipulation in ruminants.     

Alkaline phosphatase activity of rumen bacteria

Of the 54 strains of rumen bacteria examined for alkaline phosphatase (APase) production, 9 of 33 gram-negative strains and none of 21 gram-positive strains produced the enzyme. The APase of the cells of the three strains of Bacteroides ruminicola that produced significant amounts of the enzyme was located in the periplasmic area of the cell envelope, whereas the enzyme was located in the strains of Selenomonas ruminantium and Succinivibrio dextrinosolvens was associated with the outer membrane. The localization of APase production in the cells of natural populations of rumen bacteria from hay-fed sheep was accomplished by reaction product deposition, and both the proportion of APase-producing bacteria and the location of the enzyme in the cell envelope of the producing cells could be determined. We suggest that this procedure is useful in detecting shifts in the bacterial population and the release of cell-bound APase that accompany feedlot bloat and other sequelae of dietary manipulation in ruminants.     

Relationship of a Wall-Associated Enzyme with Specific Layers of the Cell Wall of a Gram-Negative Bacterium

In untreated cells of the marine pseudomonad studied here, alkaline phosphatase was found to be located in the periplasmic space, at the cell surface, and in the medium into which it had been shed during growth. Washing in 0.5 M NaCl, which removed the loosely bound outer layer, caused a shift of periplasmic enzyme to the outer aspect of the double-track layer and released some of the cell surface-associated enzyme. When the double-track layer of the cell wall was partially deranged, large amounts of this cell wall-associated enzyme were released, and, when the double-track was removed from the cells to produce mureinoplasts, alkaline phosphatase was released into the menstruum. There was no significant association of the enzyme with the peptidoglycan layer of the cell wall, which is the outermost structure of the mureinoplast, and no association of the enzyme with the cytoplasmic membrane of these modified cells. This study has shown that alkaline phosphatase is specifically associated with the outer layers of the cell walls of cells of this organism and is retained within the cell wall by virtue of this association.     

Changes in the composition of anionic membrane phospholipids influence the protein secretion and biogenesis of cell envelope in Escherichia coli

The secretion of alkaline phosphatase (PhoA) and peculiarities of biogenesis of the cell envelope were studied in Escherichia coli strains HD30/pHD 102 and HDL11 with controlled synthesis of anionic phospholipids, phosphatidylglycerol, and cardiolipin. Inactivation of the pgsA gene encoding the synthesis of anionic phospholipids or changes in the regulation of its expression by an environmental factor caused changes in the metabolism and composition of membrane phospholipids, which resulted in a decrease in the secretion of alkaline phosphatase through the cytoplasmic membrane and an increase in PhoA secretion from the periplasm into the culture medium. A conforming increase was observed in exopolysaccharide secretion, as well as a decrease in the contents of lipopolysaccharide and lipopolyprotein of the outer membrane that determine the membrane barrier properties. The results obtained testify that anionic phospholipids play a significant role in protein secretion and are probably involved in the interrelation between the protein secretion and biogenesis of cell envelope components.     

Cell surface-localized alkaline phosphatase of Escherichia coli as visualized by reaction product deposition and ferritin-labeled antibodies.

When cells of a wild-type Eschericia coli O8 strain bearing a complete lipopolysaccharide were incubated for alkaline phosphatase reaction product and examined by electron microscopy, the depostion of lead salts was to be observed primarily within the periplasmic space. A similar treatment of cells derived from this strain, which bears a highly abbreviated lipopolysaccharide, showed a mixed cell surface and periplasmic localization of reaction product, suggesting a surface association of a portion of the enzyme. To further explore this possibility, ferritin-antibody conjugates against the active enzyme and its irreversibly dissociated subunits were prepared and allowed to react with cells of both strains. The results obtained from these experiments revealed the presence of both the active enzyme and inactive subunits of the enzyme at the cell surface of the mutant strain. The evidence obtained offers further proof of the validity of the reaction product deposition technique and indicates that alkaline phosphatase may be associated with some component of the outer membrane in this organism. The observation of enzyme subunits at the cell surface further suggests that an association of these subunits with structural components of the cell envelope may provide a locus at which they may dimerize to form active enzyme.     

Production of two phosphatases by Lysobacter enzymogenes and purification and characterization of the extracellular enzyme.

Lysobacter enzymogenes produces an extracellular phosphatase (EC. 3.1.3.1) during the stationary phase of growth. The cells also produce a cell-associated alkaline phosphatase. This enzyme is found in the particulate fraction of cell extracts and may be membrane bound. The production of both phosphatases, especially the extracellular enzyme, is reduced by inorganic phosphate. The extracellular phosphatase was purified to a specific activity of 270 U/mg primarily by chromatography on carboxymethyl cellulose and gel filtration. The enzyme is stable under normal storage conditions but is rapidly inactivated above 70 degrees. It consists of one polypeptide with an approximate molecular weight of 25,000. The pH optimum is 7.5, and the Km for p-nitrophenylphosphate is 2.2 X 10(-4) M. The enzyme degrades a number of other phosphomonoesters but at a reduced rate compared with the rate obtained with p-nitrophenylphosphate. Phosphate and arsenate inhibit the enzyme, but EDTA and other chelating agents have no effect. The lack of a metal ion requirement for activity, the lower molecular weight, the soluble nature of the enzyme, and the lower pH optimum clearly distinguish the extracellular phosphatase from the cell-associated phosphatase and from other bacterial phosphatases.     

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

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-napthalenedisulfonic 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.     

Lactoperoxidase-125I localization of salt-extractable alkaline phosphatase on the cytoplasmic membrane of Bacillus licheniformis.

Previous histochemical and biochemical localizations of alkaline phosphatase in Bacillus licheniformis MC14 have shown that the membrane-associated form of the enzyme is located on the inner surface of the cytoplasmic membrane, and soluble forms are located in the periplasmic space and in the growth medium. The distribution of salt-extractable alkaline phosphatase on the surfaces of the cytoplasmic membrane of B. licheniformis MC14 was determined by using lactoperoxidase-125I labeling techniques. Cells harvested during rapid alkaline phosphatase production were converted to protoplasts or lysed protoplasts and labeled. Analysis of the data obtained indicated that 30% of the salt-extractable, membrane-associated alkaline phosphatase was located on the outer surface of the cytoplasmic membrane, whereas 70% of the membrane-associated enzyme was localized on the inner surface. Controls for protoplast integrity (release of tritiated thymidine or examination of cytoplasmic proteins for label content) indicated excellent protoplast stability. Controls indicated that chemical labeling was not a factor in the apparent distribution of alkaline phosphatase on the membrane. These results support the previously reported histochemical localization of alkaline phosphatase on the membrane inner surface. The presence of alkaline phosphatase on the membrane outer surface is reasonable, considering the soluble forms of the enzyme found in the periplasmic region and in the culture medium.     

Genetic and biochemical characterization of periplasmic-leaky mutants of Escherichia coli K-12.

Periplasmic-leaky mutants of Escherichia coli K-12 were isolated after nitrosoguanidine-induced mutagenesis. They released periplasmic enzymes into the extracellular medium. Excretion of alkaline phosphatase, which started immediately in the early exponential phase of growth, could reach up to 90% of the total enzyme production in the stationary phase. Leaky mutants were sensitive to ethylenediaminetetraacetic acid, cholic acid, and the antibiotics rifampin, chloramphenicol, mitomycin C, and ampicillin. Furthermore, they were resistant to colicin E1 and partially resistant to phage TuLa. Their genetic characterization showed that the lky mutations mapped between the suc and gal markers, near or in the tolPAB locus. A biochemical analysis of cell envelope components showed that periplasmic-leaky mutants contained reduced amounts of major outer membrane protein OmpF and increased amounts of a 16,000-dalton outer membrane protein.     

Comparison of the phosphatases of Lysobacter enzymogenes with those of related bacteria

Lysobacter enzymogenes ATCC 29487 (UASM 495) produces an outer-membrane-associated phosphatase and an excreted phosphatase. The cell-associated enzyme was compared to phosphatases of nine other Gram-negative gliding bacteria and to that of Escherichia coli. The other three species of the genus Lysobacter also produce a particulate, cell-associated phosphatase. Antiserum prepared against the phosphatase from the outer membrane of L. enzymogenes effectively precipitated the phosphatases of two other L. enzymogenes strains and the enzymes of L. antibioticus, L. brunescens and L. gummosus. Some inhibition of the enzyme by the antiserum also was observed. No significant reaction could be detected between the antiserum and the cell-associated phosphatases of species of Cytophaga johnsonae, 'C. compacta', Myxococcus xanthus, E. coli and the excreted phosphatase of L. enzymogenes. The results indicate that the four species of the genus Lysobacter are closely related despite their physiological differences and that the outer-membrane-associated phosphatases of these organisms have different structural characteristics than the phosphatases of the other Gram-negative bacteria that were used. Furthermore, differences in the amino acid compositions of the cell-associated and the excreted phosphatase of L. enzymogenes confirm the immunological results and are in agreement with the physical and chemical differences noted between the two enzymes.     

Intracellular localization of alkaline phosphatase in freshly isolated foetal rat hepatocytes

Summary The cytochemical localization of alkaline phosphatase activity in foetal rat hepatocytes was examined in relation to the pattern of cell to cell attachment during cell isolation and culture. In foetal hepatocytesin vivo, alkaline phosphatase was exclusively localized on the bile canalicular membrane. In freshly isolated foetal hepatocytes, however, the activity was present in the endoplasmic reticulum, nuclear envelope, Golgi apparatus, tubulo-vesicular organelles, and over the entire plasma membrane. In monolayer cells cultured for one or two days, the activity was localized on the reconstituted bile canalicular membrane, plasma membrane sites adjacent to neighbouring cells and on the bottom surface of the monolayer, but was detected in none of the intracellular organelles. Biochemical alkaline phosphatase activity did not change during isolation of the cells. These results suggest that, in foetal hepatocytes, loss of cell—cell contact may induce a temporal disturbance, or dedifferentiation, in their membrane system.     

Localization of Acid and Alkaline Phosphatases in Staphylococcus aureus

The localization of acid and alkaline phosphatases in Staphylococcus aureus was studied by fractionation of cells after treatment with the L-11 enzyme and by electron microscopic histochemistry. The two enzyme activities were located in distinctly different positions at the surface of the cells. Acid phosphatase appeared to be localized around the cell membrane of the bacteria, because the enzyme was recovered exclusively in the membrane fraction and because deposition of lead phosphate was detected by electron microscopic histochemistry on the inner surface of the cell membrane of intact bacteria and spheroplasts. The highest specific activity of alkaline phosphatase was also associated with the membrane fraction. However, on electron microscopic histochemistry of intact cells, the deposition of lead phosphate was only seen on the outer surface of the cell wall.