interactions of alkaline phosphatase andthe cell wall of … · alkaline phosphatase, reduced...

JOURNAL OF BACTERIOLOGY, JUly 1971, p. 325-336Copyright ( 1971 American Society for Microbiology

Vol. 107, No. IPrinted in U.S.A.

Interactions of Alkaline Phosphatase and the CellWall of Pseudomonas aeruginosa

K.-J. CHENG,' J. M. INGRAM, AND J. W. COSTERTON2

Department of Microbiology, Macdonald College of McGill Universitv, Quebec, Canada

Received for publication 9 April 1971

Spheroplasts prepared by lysozyme treatment of cells of Pseudomonas aerugi-nosa, suspended in 20% sucrose or 0.2 M MgCl2, were examined in detail. Prepara-tion of spheroplasts in the presence of 0.2 M Mg2+ released periplasmic alkalinephosphatase, whereas preparation in the presence of 20% sucrose did not, even

though untreated cells released phosphatase when suspended in sucrose in the ab-sence of lysozyme. Biochemical characterizations of the sucrose-lysozyme prepara-

tions indicated that lysozyme mediated a reassociation of the released phosphatasewith the spheroplasts. In addition, the enzyme released from whole cells suspendedin 20% sucrose (which represents 20 to 40% of the cell-bound phosphatase) reasso-

ciates with the cells in the presence of lysozyme. Electron microscopic examina-

tions of various preparations revealed that phosphatase released in sucrose reasso-

ciated with the external cell wall layers in the presence of lysozyme, that sucrose-

lysozyme prepared spheroplasts did not dissociate phosphatase which remained inthe periplasm of sucrose-washed cells, and that phosphatase was never observed tobe associated with the cytoplasmic membrane. A model to account for the binding ofP. aeruginosa alkaline phosphatase to the internal portion of the tripartite layer ofthe cell wall rather than to the cytoplasmic membrane or peptidoglycan layer ispresented.

It has been found (3, 4) that alkaline phospha-tase (orthophosphoric monoester phosphohy-drolase, EC3.1.3.1.) of Pseudomonas aerugi-nosa is inducible by phosphate starvation and isreadily removed from cells after washes in 0.2 MMgCI2, pH 8.4. The enzyme (3) has been local-ized by electron microscope techniques in theregion between the cytoplasmic membrane andthe tripartite layer of the cell wall, i.e., the peri-plasmic space (9). It is well established that pern-plasmic enzymes from Escherichia coli can beremoved by either the ethylenediaminetetraaceticacid (EDTA) osmotic shock procedure of Neuand Heppel (10) or by the procedure of Malamyand Horecker (8) involving the formation ofspheroplasts in the presence of lysozyme andEDTA. Because EDTA lyses P. aeruginosa cells(7), we developed alternative methods for theformation of spheroplasts from P. aeruginosasuch as the Mg2+-lysozyme method or sucrose-lysozyme method (3). During the formation ofspheroplasts by the Mg2+-lysozyme method, thealkaline phosphatase is largely released into the

National Research Council of Canada Postdoctoral Fel-low. 1969 1971.

2 Present address: Department of Biology, University ofCalgary, Calgary, Alberta, Canada.

325

supernatant fluid, whereas almost no alkalinephosphatase is released during the sucrose-lyso-zyme spheroplast procedure although suspensionof lysozyme-untreated cells in 20% sucrose waspreviously shown to remove 20 to 40% of thecell-bound alkaline phosphatase.

This communication presents evidence whichdemonstrates that, in the absence of high concen-trations of Mg2", the lysozyme present in thespheroplast-forming solution mediates a reasso-ciation of alkaline phosphatase with the cell wallof P. aeruginosa. In addition, it was possible toestablish the specific location of alkaline phos-phatase within the periplasmic space of P. aeru-ginosa cells. Electron micrographs of sucrose-lysozyme treated cells and sucrose-lysozyme pre-pared spheroplasts showed that the alkalinephosphatase is closely associated with the innerregion of the tripartite layer and is never ob-served to be associated with the cytoplasmicmembrane.

MATERIALS AND METHODS

Organism and culture conditions. P. aeruginosaATCC 9027 was cultivated at 37 C on a rotary shaker ina glucose-ammonium salt-proteose peptone medium as

previously described (4). Under these conditions theculture actively synthesizes alkaline phosphatase be-

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CHENG, INGRAM, AND COSTERTON

tween the 5th and 14th hr. The proteose peptone servesas the only source of organic or inorganic phosphate.

Chemicals. p-Nitrophenylphosphate, tris(hydroxy-methyl)aminomethane (Tris) and lysozyme (murami-dase) were purchased from the Sigma Chemical Co.,St. Louis, Mo. Sodium-,8-glycerophosphate was ob-tained from Fisher Scientific Co., Pittsburgh, Pa.

Release of alkaline phosphatase from cells. Standardquantities of cells obtained from 14-hr cultures grown

to 1.2 to 1.4 optical density units (OD, 660 nm; Gilfordmodel 300-N spectrophotometer) were washed with 0.2M MgC12 in 0.01 M Tris (pH 8.4) or 20% sucrose in 0.01M Tris (pH 8.4) as described previously (4). A calibra-tion curve relating OD to dry weight was constructedand it was determined that I OD corresponded to 18.5mg (dry weight). Cell-free extracts were prepared byultrasonic disruption of cells resuspended in 0.01 M Tris,pH 8.4, as previously described (4).

Enzyme assays. Alkaline phosphatase, reduced nico-tinamide adenine dinucleotide (NADH) oxidase, andglucose-6-phosphate (G-6-P) dehydrogenase were as-

sayed as described previously (4). The effect of the var-

ious washing components upon alkaline phosphataseactivity, as compared to release, were found to be neg-ligible as determined previously (4). One unit of en-

zyme activity corresponds to the conversion of I Mmoleof substrate to product per minute.

Preparation of spheroplasts. Sucrose-lysozyme sphe-roplasts were prepared by centrifuging the cells from20 ml of a 14-hr culture (13,000 x g) and then resus-

pending in 20 ml of 20% sucrose (with 0.01 M Tris, pH8.4); the resulting suspension was 0.5 mg/ml in lyso-zyme. The cell suspension was incubated at 25 C in a

water bath shaker for 30 min; cells were centrifugedand resuspended in 0.01 M MgCI2 and 0.01 M Trisbuffer (pH 8.4) for the formation of spheroplasts. Su-crose-0.01 M Mg2+-lysozyme spheroplasts were pre-pared in the same way except that MgCl2, 0.01 M finalconcentration, was added to the sucrose. Magnesium-lysozyme spheroplasts were prepared in a similar man-

ner, except that the 20% sucrose was replaced by 0.2 M

MgCl2 (pH 8.4) and 0.01 M Tris. During the processesused to convert P. aeruginosa cells to spheroplasts, allsupernatant fluids were collected and assayed (as inTable 1) for alkaline phosphatase, NADH oxidase, andG-6-P dehydrogenase.

Wbole cell assay system for alkaline phosphataselocalization studies. P. aeruginosa cells were suspendedfor 2 min in 0.2 M MgCI2 and 0.01 M Tris buffer (pH8.4), or 20% sucrose in 0.01 M Tris buffer (pH 8.4) con-

taining 0.5 mg of lysozyme per ml. The suspensionswere centrifuged at 13,000 x g and incubated for 30min in a modified Gomori (5) mixture of the followingcomposition: sodium-,B-glycerophosphate, 0.5%; so-

dium-barbitol, 0.5%; Ca(No3)2, 0.02 M; MgCl2, 0.01 M;and Tris buffer, 0.01 M (pH 8.4). Control treatmentswith cells obtained from inorganic phosphate dere-pressed and repressed cultures (4), sucrose-lysozymespheroplasts, magnesium-lysozyme spheroplasts, su-

crose-0.01 M MgCI2-lysozyme spheroplasts, and su-

crose-lysozyme spheroplasts which had been washedtwice with 0.2 M MgCI2 were also incubated with theabove mentioned modified Gomori mixture to localizethe alkaline phosphatase.

Electron microscopy. The methods for fixation andembedding were those described previously (3). Thinsections were cut with a Sorvall Porter-Blum modelMT-2 ultramicrotome, stained with lead citrate (13),and examined with an AEI-EM-(801) electron micro-scope by using 60-kv electron acceleration voltage.

RESULTS

Release of enzymes during Mg2+lysozyme or

sucrose-lysozyme spheroplast formation. Theresults presented in Table I relating to the re-

lease of alkaline phosphatase from cells of P.aeruginosa as mediated by 0.2 M MgCI2 or 20%sucrose washing are in agreement with previouslyreported findings (4). It is further observed thatwhen P. aeruginosa cells are treated with lyso-zyme in the presence of 0.2 M Mg2+, almost allof the alkaline phosphatase is released to thesupernatant fluid as compared to the cell-freeextract (Table 1), whereas NADH oxidase andG-6-P dehydrogenase remain entirely within thespheroplasts.When lysozyme-treated cells of P. aeruginosa

are maintained in a medium of high osmoticpressure, i.e., 0.2 M Mg2+ which was 0.01 M inTris (pH 8.4), only some of the cells becomespherical. When suspensions of these cells are

centrifuged and resuspended in 0.01 Mg2` and0.01 M Tris (pH 8.4), 100% of the cells are

changed to spheres (Fig. 1 C) as compared tountreated cells (Fig. 1 A). Assay of the superna-tant fluid from the 0.01 M Mg2` suspension ofspheroplasts showed essentially no further releaseof alkaline phosphatase and no significant releaseof the internal enzymes, G-6-P dehydrogenase,or NADH oxidase (Table 1). Spheroplasts pre-

pared by this method are stable overfight, and,furthermore, actinomycin D was unable to blockthe uptake of '4C-uracil by these preparations(Cheng, Costerton, and Ingram, unpublisheddata). This can be taken as evidence of the intactstate of the cytoplasmic membrane of these sphe-roplasts.When cells of P. aeruginosa were suspended

into 20% sucrose and 0.5 mg of lysozyme per ml,no alkaline phosphatase, NADH oxidase, or G-6-P dehydrogenase was released into the super-natant fluid (Table 1). When the cells obtainedby the above treatment were suspended into 0.01M MgCI2 and 0.01 M Tris (pH 8.4), more than99% of the cells were converted to spheres (Fig.I B). The supernatant fluid obtained after cen-

trifugation of this suspension contained no alka-line phosphatase or NADH oxidase, but therewas a considerable amount of G-6-P dehydro-genase (Table 1). This indicated that sucrose-ly-sozyme spheroplasts were more susceptible toosmotic shock than Mg2+-lysozyme spheroplasts.

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ALKALINE PHOSPHATASE OF P. AERUGINOSA

TABLE 1. Enzyme release from cells and spheroplasts of Pseudomonas aeruginosa

Magnesiuma Sucrose

Treatment Alkaline NADH G-6-P Alkaline NADH G-6-P

phosphatase oxidase dehydro- phosphatase oxidase dehydro-genase genase

0.2 M MgCl2 or 20% sucrose washes of cells5 ...... 156.0' 0 0 60.0 0 0Supernatant fluid obtained from cells treated with

lysozyme in either 0.2M MgCl2 or 20% sucrose . . 154.8 0 0 0 0 0Supernatant fluid obtained from lysozyme-treated

cells after resuspension into 0.01 M MgCl2 (sphe-roplast formation) ......... ............... 1.3 0 0 0 0 18.3

0.2 M MgCl2 wash of spheroplast pellets ........ 0 0 0 165.0 0 0Cell-free extract ............. ............... 155.8 15;6 144.5 155.8 15.6 144.5

a Magnesium and sucrose refer to the wash in which each sample of cells was initially suspended. NADH, re-duced nicotinamide adenine dinucleotide; G-6-P, glucose-6-phosphate.

h Twenty milliliters of 14-hr cells [480 mg (dry weight) of a culture grown to 1.3 optical density units] were cen-trifuged and resuspended in an equal volume of wash solution as a control to determine the release of periplasmic,cytoplasmic membrane, and cytoplasmic marker enzymes. A similar volume of cells was resuspended in each ofthe two wash solutions to which lysozyme (0.5 mg/ml) was added, and the suspension was incubated at 25 C for30 min in a water bath shaker. The suspension was centrifuged, the supernatant fluids were assayed for each en-zyme, and the cell pellets were resuspended in 0.01 M MgCI2 and 0.01 M tris(hydroxymethyl)aminomethane, pH8.4, for the formation of spheroplasts. The suspension was centrifuged and the supernatants were assayed for eachenzyme. Spheroplast pellets were washed with 0.2 M MgCI2, a treatment which was previously shown (4) to effec-tively remove all remaining cell-bound alkaline phosphatase, and the supernatant fluids obtained after this treat-ment were assayed for each enzyme.c Units of enzyme per 25 g (dry weight).

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BFIG. 1. Phase contrast micrographs of (A) whole cells; (B) spheroplasts prepared in sucrose; (C) spheroplasts

prepared in 0.2 M Mg2+. x2,000.

It was also noted that when 20% sucrose-lyso-zyme or 0.2 M Mg2+-lysozyme cells were sus-

pended in distilled water, the Mg2+-lysozymecells changed to spheres and were stable formore than I hr, but sucrose-lysozyme cells werelysed within 10 min. This result demonstrates theimportance of Mg2+ in the maintenance of

spheroplast integrity. The uptake of '4C-uracilby sucrose-lysozyme spheroplasts was notblocked by actinomycin D (Cheng, Costerton,and Ingram, unpublished data) and the amountof G-6-P dehydrogenase released did not increasewith time. It is obvious, therefore, that the G-6-Pdehydrogenase released into the medium initially

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is due to the bursting of some cells during os-motic shock. The phase microscopic count ofthese spheroplasts also confirmed this conclu-sion. The sucrose-lysozyme spheroplasts whichare not lysed by osmotic shock are stable in lowconcentrations of MgCI2, i.e., 0.01 M, for morethan I hr.From these results, it is apparent that, even

though some sucrose-lysozyme spheroplastsburst, the alkaline phosphatase is still associatedwith the cells. Since we have shown that suspen-sion of whole cells in 20% sucrose releases ap-proximately one-third of the cell-bound alkalinephosphatase if lysozyme is not present (Table 1),it is clear that lysozyme either prevents the re-lease of alkaline phosphatase or mediates itsreassociation with the cell.

Reassociation of alkaline phosphatase with cellsof P. aeruginosa. To delineate between the possi-bilities (i) that lysozyme prevents the dissociationof cell-bound alkaline phosphatase from 20%sucrose-treated cells or (ii) that lysozyme medi-ates a reassociation of released alkaline phospha-tase with cells, a method was required wherebythe reassociation of dissociated alkaline phospha-tase could be observed in the presence of cellsand lysozyme. Based upon our evidence, a rea-sonable approach was to attempt to recombinethe phosphatase released by the 20% sucrosewashing procedure (reference 3, and experiment2, Table 2) with washed cells in the presence andabsence of 0.5 mg of lysozyme per ml.The results in Table 2 show that the alkaline

phosphatase released by washing cells in 20%sucrose is converted almost instantly, upon theaddition of lysozyme, to a state which sedimentsafter a 10-min centrifugation at 13,000 x g (ex-periment 6, Table 2). Since the reassociation isalmost instantaneous, there is little likelihood ofdamage to the cells due to the lysozyme, and theonly logical site for alkaline phosphatase reasso-ciation is the cell wall. The reaction also showsan absolute requirement for washed cells;without washed cells the addition of lysozyme toa 20% sucrose supernatant solution containingalkaline phosphatase did not affect its removalfrom solution upon centrifugation for 10 min at13,000 x g (experiment 4, Table 2).Once the reassociation of alkaline phosphatase

with the sucrose-washed cells occurred, the com-plex was stable to suspension in 0.01 M Mg2"solution, but a 0.2 M Mg2+ wash completelyremoved the bound enzyme (experiment 8, Table2). The enzyme removed by this 0.2 M Mg2"wash includes the component which was not re-leased upon initial exposure to 20% sucrose andthe component which was reassociated upon theaddition of lysozyme (Table 2). Table 2 also

shows that added lysozyme cannot mediate thereassociation of the enzyme with cells washedand suspended in the presence of 0.2 M MgCl2(experiments 5 and 7, Table 2). This clearly indi-cates that the lysozyme-mediated reassociationof alkaline phosphatase with cells of P. aerugi-nosa requires low ionic concentration.

Ultrastructural localization of alkaline phos-phatase. Previously (3) it was shown that alkalinephosphatase of untreated cells is completely lo-calized between the cytoplasmic membrane andthe tripartite layer of the cell wall, and the samedistribution is seen in the untreated cells used inthese experiments (Fig. 2). Similar studies per-formed with inorganic phosphate-repressed cells,or with cells which were devoid of alkaline phos-phatase activity after washes with 0.2 M MgCI2showed a complete absence of lead phosphatedeposits within the periplasmic space, indicatingthat such deposits are indeed the result of alka-line phosphatase activity (3). Since enzyme anal-ysis showed that washing in 20% sucrose releasedapproximately 30% of the alkaline phosphatasepresent in the cell (Table 2), whereas all of theenzyme remains bound to the cells after suspen-sion in 20% sucrose containing 0.5 mg of lyso-zyme per ml, it became desirable to localize thealkaline phosphatase in these treated cells. Cellstreated by the modified Gomori reaction and ini-tially suspended in sucrose and lysozyme for 2min showed that some of the enzyme is still lo-cated between the cytoplasmic membrane andthe tripartite layer of the cell wall, i.e., the peri-plasmic space (reference 9 and Fig. 3, arrows),whereas a substantial proportion is also asso-ciated with the outer surface of the cells.When sucrose-lysozyme treated cells are resus-

pended into 0.01 M Mg2" and Tris, 0.01 M (pH8.4), they assume a spherical shape, and the dis-tribution of alkaline phosphatase in their cellenvelopes remains the same as that observed insucrose-lysozyme treated cells (Fig. 4). An innerdense deposit of lead phosphate is observed in-side the tripartite layer of the cell wall where thislayer is clearly resolved (Fig. 4, arrows), and amore loosely packed mass of crystals is asso-ciated with the outer surface of the cells. Theamorphous aggregates seen between the sphero-plasts are probably cellular debris, and, althoughthe most striking reassociation of the alkalinephosphatase is to the surface of the cell wall,there is probably some association of alkalinephosphatase with debris.When the cytoplasmic membrane is separated

from the cell wall (Fig. 4, "S"), both the innerand outer deposits of lead phosphate are asso-ciated with the cell wall. In sucrose-plasmolyzedcells (Fig. 5) and in fragments of the cell enve-

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lope (Fig. 5, inset), both layers of electron-densematerial are associated with the cell wall and no

alkaline phosphatase activity is associated withthe cytoplasmic membrane.When spheroplasts are formed by incubating

cells in 20% sucrose, 0.5 mg of lysozyme per ml,and 0.01 M Mg2+ with subsequent resuspensioninto 0.01 M MgCl2, an enzyme localization pat-tern identical with that of sucrose spheroplasts isobtained (Fig. 6). The inclusion of 0.01 M Mg2+in the sucrose-lysozyme treatment solutions ap-pears to enhance the separation of the cyto-plasmic membrane from the cell wall in a largenumber of cells and, as in the case of the su-

crose-lysozyme spheroplasts, the enzyme activityis exclusively associated with the cell wall. Thedeposition of electron-dense material in the cellenvelope of this preparation shows that lowMg2+ concentration neither increases the releaseof alkaline phosphatase nor interferes with thelysozyme-mediated reassociation to the outersurface of the cell. Examinations of electron mi-crographs of magnesium-lysozyme spheroplastsshowed no evidence of electron-dense depositsindicating the complete absence of alkaline phos-phatase, a result consistent with the biochemicaldata.Enzyme assays showed that, when either su-

crose-lysozyme spheroplasts or sucrose-0.01 M

Mg2+-lysozyme spheroplasts are resuspendedinto 0.2 M Mg2+, the alkaline phosphatase wascompletely released (Table I), and magnesiumwashed spheroplasts after treatment by Gomorireaction show a complete absence of lead phos-phate deposits in the cell envelope (Fig. 7).Taken with our previous finding (3) that theGomori rea6tion occurs only in cells induced tosynthesize alkaline phosphatase, the absence ofreaction in cells treated to remove the enzymeestablishes that the original deposition of theelectron-dense material described here is due tothe production of inorganic phosphate from 3-

glycerophosphate by this enzyme.

DISCUSSIONIn a previous study (3), we reported the pro-

duction of spheroplasts of P. aeruginosa afterwashes of the cells in either 0.2 M MgCI2 or 20%sucrose, 0.5 mg of lysozyme per ml, and resus-pension of the cells into 0.01 M MgC92. Thepresent investigations were undertaken in an ef-fort to describe more fully some of the character-istics of spheroplasts produced by these twomethods. From the results presented here, appar-ently alkaline phosphatase, a periplasmic enzymeof P. aeruginosa, is released by 0.2 M MgCl2prior to the production of spheroplasts. Sphero-plasts prepared in this manner do not release

TABLE 2. Conditions for release and reassociation ofalkaline phosphatase with cells ofPseudomonas

aeruginosa

Alkaline Alkalinephospha- phospha-Expt tase inTreatment tase re-no. super- mainingnatant in cellsfluid

1 0.2M Mg2+ wash of cellsa 150.Ob 1.32 20% Sucrose wash of cells 48.7 101.83 Supernatant fluid from 150.0 -

experiment I + lyso-zyme (0.5 mg/ml)

4 Supernatant fluid from 48.7 -

experiment 2 + lyso-zyme (0.5 mg/ml)

5 Supernatant fluid from 150.0 1.3experiment 3 + 0.2 MMg2+-washed cells

6 Supernatant fluid from 0 147.4experiment 4 + 20%sucrose-washed cells

7 0.2 M Mg2+ wash of cells 1.3 0from experiment 5

8 0.2 M Mg2+ wash of cells 147.0 1.3from experiment 6

9 Cell-free extract 145.0 -

a Twenty milliliters of a 14-hr culture [480 mg (dryweight) of a culture grown to 1.3 optical density units]was centrifuged and resuspended in 20 ml of thewashing solutions as indicated for 10 min. Suspensionsfrom each experiment were centrifuged and the super-natant fluids were assayed for alkaline phosphatase ac-tivity. The cell pellets obtained were resuspended into0.01 M Tris, pH 8.4, and subjected to sonic disintegra-tion to determine the alkaline phosphatase remainingin the cells. Experiments I and 2 were performed induplicate to obtain the appropriately treated cells usedin experiments 4 and 6. Similarly, experiments 5 and 6were performed in duplicate to obtain the cell pelletsused in experiments 7 and 8. A dash (-) indicates thatthe test was not performed.

b Units of enzyme per 25 g (dry weight) of cell cul-ture.

cytoplasmic membrane or cytoplasmic markerenzymes such as NADH oxidase or G-6-P dehy-drogenase, a result consistent with that foundafter whole cell washing with 0.2 M MgCl2 (4). Inaddition, phase microscopic examinations andinternal enzyme release suggest that cells treatedby the above procedures and diluted into 0.01 MMgCI2 yield spheroplasts which are stable for atleast 24 hr.

Spheroplasts of P. aeruginosa prepared bysuspending cells in 20% sucrose and 0.5 mg oflysozyme per ml appear to be similar to thoseprepared after suspension in 0.2 M MgCl2. Nei-ther internal nor membrane marker enzymes arereleased during the spheroplast formation step.

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FIG. 2. Electron micrograph of a thin section of cells grown at pH 6.8 and subjected to the Gomori reactionfor the localization of alkaline phosphatase. Note the heavy deposition oflead phosphate inside the tripartite layerof the cell wall (arrows). x 139,000. Bar represents 0.1 inm.

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FIG. 3. Electron micrograph of a sucrose-lysozyme treated cell which was subjected to the Gomori reaction.Note the dense deposits of lead phosphate lying inside the tripartite layer of the cell wall (arrows) and large,loosely organized deposits at the cell surface. x 112,000. Bar represents 0.1 fI m.

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FIG. 4. Electron micrograph of sucrose-lysozyme spheroplasts which were subjected to the Gomori reaction.Note the inner dense layer of lead phosphate which clearly lies inside the tripartite layer of the cell wall (arrows)and the loosely organized deposits on the surface of the cells and on the aggregates of cellular debris. In the area (S)where the cytoplasmic membrane is separated from the cell wall, the enzyme activity is clearly associated with thewall alone. x84,000. Bar represents 0.1 uim.

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FIG. 5. Electron micrograph of a sucrose-lysozyme treated cell which was subjected to the Gomori reaction.The cytoplasmic membrane is clearly resolved (arrow) in this plasmolyzed cell and the enzyme activity is asso-ciated only with the cell wall. Inset shows that both the inner dense deposit of lead phosphate and the outer,loosely organized deposit have remained associated with a fragment of the cell envelope even though the cyto-plasmic membrane has been essentially lost. x 102,600. Bar represents 0.1 uim.

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FIG. 6. Electron micrograph of sucrose-0.01 M Mg"+ lysozyme spheroplasts which were subjected to the Go-mori reaction. Note that both inner dense and outer, loosely organized deposits of lead phosphate are formed andthat both of these deposits are clearly associated with the cell wall when this structure is separated from the cyto-plasmic membrane. x 75,000. Bar represents 0.1 pm.

FIG. 7. Electron micrograph of a sucrose-lysozyme spheroplast which was washed in 0.2 M Mg2+ and subjectedto the Gomori reaction. Note the absence of enzyme activity and the distortion of the cell wall. x 111,500. Barrepresents 0.1 gm.

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Based upon the small, significant release of G-6-P dehydrogenase from the sucrose-lysozymepreparations, however, it appears that thesespheroplasts are more fragile than those obtainedby suspension in 0.2 M MgCI2.From the results obtained in this study, appar-

ently lysozyme mediates a reassociation of alka-line phosphatase with the cell wall of P. aerugi-nosa. The electron micrographs suggest that thereassociated alkaline phosphatase is confined tothe outer cell wall layers, whereas enzyme whichis not disssociated by 20% sucrose remains in theperiplasmic space. Since the reassociated alkalinephosphatase appears as nonuniform, looselybound fragments attached to the outer cell walllayers and since periplasm-localized enzymewithin the spheroplasts appears uniform, it isconcluded that neither lysozyme nor spheroplastformation releases enzyme from the periplasmicspace.The involvement of lysozyme as a direct or

mediating binding agent has been described inother systems. Lysozyme is known to bind ribo-nucleic acid (12), deoxyribonucleic acid (2), thecell surface of E. coli (15), membranes and ribo-somes of E. coli (11), and liposomes (14). It hasalso been suggested that this enzyme binds to thecell surface of P. aeruginosa (6). From these dis-cussions, it appears that the reassociation of re-leased alkaline phosphatase with 20% sucrose-washed cells of P. aeruginosa is mediated by ly-sozyme and that the binding may be to the lipo-polysaccharide or lipoprotein components of thecell wall. This latter conclusion is strengthenedby the fact that even cell debris reassociates withthe phosphatase in the presence of lysozyme. Thepossibility of cross reactivity between E. coli al-kaline phosphatase and P. aeruginosa cell wall,as mediated by lysozyme, is currently under in-vestigation.

Although previous studies with P. aeruginosa(3) and E. coli (10) have shown that alkalinephosphatase is a periplasmic enzyme that is lo-cated between the cytoplasmic membrane andthe outer tripartite layer, it is not clear at presentwhether the enzyme is bound to the cytoplasmicmembrane, or the peptidoglycan layer, and pro-jects outwardly to the periplasm or whether it isbound to the tripartite layer and inwardly pro-jects into the periplasm. The fact that sucrose-lysozyme spheroplasts of P. aeruginosa still con-tain uniformly located periplasmic alkalinephosphatase as already mentioned suggests that,in this organism, at least, the peptidoglycan isnot responsible for phosphatase binding. Evenmore important is the fact that in cells treatedfor a short time with sucrose and lysozyme (Fig.

. OUTER LAYERTRIPARTITE LAYER

_~~U~" * _ _~-w,' - 4- ALKALINE PHOSPHATASE- PERIPLASMIC SPACE

4- PEPTIDOGLYCAN_}ijJ }(- CYTOPLASMAC MEMBRANE

-CYTOPLASM

FIG. 8. Diagrammatic representation of alkalinephosphatase location within the periplasmic space of P.aeruginosa. The peptidoglycan layer is illustrated nextto the cytoplasmic membrane for convenience only,since its true location has not been established in thisorganism.

5), sucrose-lysozyme spheroplasts (Fig. 4 "S"),sucrose-0.01 M Mg2+-lysozyme spheroplastswhich show enhanced separations of the cyto-plasmic membrane from the tripartite layer (Fig.6), or sucrose-plasmolyzed cells (Fig. 5, inset),unreleased phosphatase is not associated with thecytoplasmic membrane but rather with the in-ternal region of the tripartite layer. Although notentirely ruled out by the discussions presentedhere, the remote possibility exists that lysozymeitself reassociates periplasmic alkaline phospha-tase with the internal region of the tripartite layerof the cell wall. However, based upon considera-tions such as the uniform distribution of undisso-ciated periplasmic alkaline phosphatase in su-crose-lysozyme spheroplasts, the complete ab-sence of alkaline phosphatase from the cyto-plasmic membrane, and the translocation ofalkaline phosphatase from its periplasmic loca-tion to an external cell wall location (3) after glu-taraldehyde fixation of untreated cells, it is con-cluded that the possibility of a lysozyme-medi-ated binding and translocation of alkaline phos-phatase to the inner tripartite layer is unlikely. Itis proposed, therefore, that in untreated cells,periplasmic alkaline phosphatase is not boundby the cytoplasmic membrane or the peptido-glycan layer but rather by the internal region ofthe tripartite layer. The proposed model for thespecific location of alkaline phosphatase withinthe periplasmic space of P. aeruginosa cells isillustrated in Fig. 8.

Studies on the forces responsible for thebinding to this layer as well as the actual compo-nent of the layer involved in binding phosphataseare currently under investigation.

ACKNOWLEDGMENTS

The financial assistance of the National Research Councilof Canada is gratefully acknowledged.We would also like to thank A. C. Blackwood for his con-

tinued interest in this project.

335VOL. 107, 1971


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LITERATURE CITED

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2. Catten, O., and D. Bourgoin. 1968. Interactions betweenDNA and hen's egg white lysozyme. Effects of composi-tion and structural changes of DNA, ionic strength andtemperature. Biochim. Biophys. Acta 161:56 57.

3. Cheng, K.-J., J. M. Ingram, and J. W. Costerton. 1970.Alkaline phosphatase localization and spheroplast for-mation of Pseudomonas aeruginosa. Can. J. Microbiol.12:1319-1324.

4. Cheng, K.-J., J. M. Ingram, and J. W. Costerton. 1970.Release of alkaline phosphatase from cells of Pseudo-monas aeruginosa by manipulation of cation concentra-tion and ofpH. J. Bacteriol. 104:748-753.

5. Done, J., C. 0. Shorey, J. P. Lake., and J. K. Pollak.1965. The cytochemical localization of alkaline phospha-tase in Escherichia coli at the electron microscope level.Biochem. J. 96:27C 28C.

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EDTA on Pseudomonas aeruginosa. J. Appl. Bacteriol.28:153-164.

8. Malamy, H. M., and B. L. Horecker. 1964. Release ofalkaline phosphatase from cells of E. coli upon lysozyme

spheroplast formation. Biochemistry 3:1889 -1893.9. Mitchell, P. 1961. In T. W. Goodwin and 0. Lindberg

(ed.), Biological structure and function, vol. 2. AcademicPress Inc., New York.

10. Neu, H. C., and L. A. Heppel. 1965. Tbe release of en-

zymes from Escherichia coli by osmotic shock andduring the formation of spheroplasts. J. Biol. Chem.240:3685 -3692.

11. Patterson, D., M. Weinstein, R. Nixon, and D. Gillespie.1970. Interaction of ribosomes and the cell envelope ofEscherichia coli mediated by lysozyme. J. Bacteriol. 101:584-591.

12. Preiss, J. W. 1968. The complexing of lysozyme with polyC and other homopolymers. Biophys. J. 8:1201-1210.

13. Reynolds, E. S. 1963. The use of lead citrate at high pH as

electron-opaque stain in electron microscopy. J. CellBiol. 17:208 242.

14. Sessa, G.. and G. Weismann. 1970. Incorporation of lyso-

zyme into liposomes. J. Biol. Chem. 245:3295-3301.15. Wilson, L. A., and J. K. Spitznagel. 1968. Molecular and

structural damage to Escherichia coli produced by anti-body, complement and lysozyme systems. J. Bacteriol.%:1339-1348.

336 J. BACTERIOL.


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