JOURNAL OF BACTERIOLOGY, Sept. 1973, p. 746-751Copyright © 1973 American Society for Microbiology

Vol. 115, No. 3Printed in U.S.A.

Preparation of Metabolically ActiveStaphylococcus aureus Protoplasts by Use of the

Aeromonas hydrophila Lytic EnzymeN. W. COLES AND R. GROSS

Commonwealth Serum Laboratories, Parkuille, Victoria, 3052, Australia

Received for publication 15 March 1973

Stable, metabolically active protoplasts of Staphylococcus aureus have beenprepared by the use of a staphylolytic enzyme produced by Aeromonashydrophila. Respiratory and glycolytic rates and synthesis of nucleic acids,protein, and lipid in these protoplasts, stabilized variously in 1.1 M sucrose, 0.37M sodium succinate, or 0.37 M sodium sulfate, have been shown to becomparable with the same parameters in intact cells under the same conditions.

Since the original studies on protoplasts ofBacillus megaterium by Weibull (16), proto-plasts of many organisms susceptible to theaction of lysozyme have been studied. Due tothe intractability of the cell wall of Staph-ylococcus aureus to the action of this enzyme,protoplasts of this organism were not readilyprepared until the availability of active staph-ylolytic enzymes. One of the earliest reportsdealing with the metabolic activity of proto-plasts of S. aureus prepared with enzymes ofknown specificity was that of Hash et al. (8)who used a fungal N-acetylhexosaminidase forremoval of the cell wall. These protoplasts werestabilized by 0.5 M sucrose, and the onlymetabolic activity investigated was the incorpo-ration of 14C-alanine into cold trichloroaceticacid-insoluble material, which proceeded atabout 60% of the rate of control cells.Schuhardt and Klesius (15) obtained osmoti-

cally fragile bodies from S. aureus using lyso-staphin for cell wall removal. The resultant pro-toplasts required 24% (4 M) NaCl or 64% (1.9M) sucrose for stabilization, and the only meta-bolic function studied (10) was respiration in theabsence and presence of glucose. Hirachi et al.(9) concentrated their efforts on studying theantigenic properties of' S. aureus protoplastmembranes. No extensive investigations of met-abolic capabilities of S. aureus protoplasts ap-pear to have been undertaken.The present report describes a method for the

preparation of metabolically active protoplastsfrom S. aureus using the Aeromonas hydrophilastaphylolytic enzyme (2). The protoplastsformed respire, glycolyze, are active in mac-

romolecular syntheses, and, as reported in apreceding communication (7), are very effectivein synthesizing inducible penicillinase.

MATERIALS AND METHODSBacterial strains. Two strains of' S. aureus induci-

ble for penicillinase were used and have been de-scribed previously (3, 4, 6) together with their methodof cultivation (6). The phage type 80/81 organismoriginally described has now been designated 2237.Cells were harvested in the early exponential phaseand washed three times with 0.02 M tris(hydroxy-methyl)aminomethane (Tris)-glycine buffer (pH 8.2at 37 C).

Cells for the production of' protopla.ts were resus-pended in 1.8 M sucose in 0.02 M Tris-glycine, pH8.2, to give a concentration of 1 mg dry weight per ml.Sufficient Aeromonas lytic enzyme (0.02 to 0.08 ml/mlof cell suspension) was added to produce protoplasts(considered complete when the absorbance was 0.04at 660 nm on dilution of the suspension with threevolumes of distilled water) within 30 min of gentleswirling at 37 C. Four volumes of 1.8 M sucrosecontaining 102 M MgSO4 were added as soon asremoval of the cell wall was complete as the presenceof magnesium in the medium during protoplast for-mation is known to inhibit severely the action of thelytic enzyme. This protoplast suspension was used inexperiments reported below except for measurementsof respiration and glycolysis when protoplasts werenot diluted with sucrose solution containing Mg2- butwere used as such after the addition of solid magne-sium sulfate to a final concentration of 7.5 x 10-3 M.In the preparation of protoplasts for some experi-ments, equiosmolar sodium succinate or sodium sul-f'ate was substituted for sucrose. When comparisons of'metabolic activity between cells and protoplasts weremade, cell suspensions were treated as above exceptf'or exposure to the lytic enzyme.

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Cell concentrations were determined at 660 nm on aBausch and Lomb Spectronic 20 colorimeter by relat-ing the absorbance to either dry weight or viable cellcounts estimated from calibration curves. Viable cellcounts were performed in duplicate by spreading0.1-ml samples of a suitable dilution of cells on thesurface of nutrient agar plates and incubating over-night at 37 C. The dry weight or numbers of proto-plasts quoted in experiments refer to the dry weight ornumbers of cells from which the protoplasts werederived. In this way direct comparisons of the meta-bolic activities of cells and protoplasts may be made,with no correction necessary for the amount of cellwall material removed.

Reconstituted "4C-protein hydrolysate containing13 radioactive L-amino acids and 3H-thymidine wereobtained from Schwarz BioResearch, Inc., Orange-burg, N.Y., sodium acetate-1- 14C from Interna-tional Chemical and Nuclear Corp., City of Industry,California, and uracil-2- 4C from the RadiochemicalCentre, Amersham, England.

A. hydrophila staphylolytic enzyme was preparedby the method of Coles, Gilbo, and Broad (2) with thefollowing modification. Excess triethylammoniumacetate was removed from the pooled fractions of theAG 50 W resin eluate by Diaflo membrane (UM 10)ultrafiltration in place of Sephadex G-10 chromatog-raphy. This produced a concentrated solution of lyticenzyme with low salt content.

Incubation mixtures for incorporation of radioac-tive precursors were the same as induction mixtures(7) with the omission of inducer, but with the follow-ing additions: for protein synthesis, 0.1 mg of aminoacid mixture (4) containing 0.1 MCi of "4C-proteinhydrolysate; for nucleic acid synthesis, 0.1 ml of 3 mM"C-uracil (0.1 MCi/smol) or 0.1 ml of 3 mM 3H-thymi-dine (16.7 ACi/umol); for lipid synthesis, 0.1 ml of 25mM '4C-sodium acetate (1 IACi/umol). Radioactivecompounds were added after temperature equilibra-tion at 37 C. At the end of the incubation periodsamples for the measurement of radioactivity incorpo-rated into nucleic acids and proteins were treated aspreviously described (3, 5) and counted in a Nuclear-Chicago gas-flow counter.

Lipid synthesis was determined by following theincorporation of "4C-acetate into membrane lipids. Atthe end of the incubation period, protoplasts werelysed with 1.5 vol of water and membranes weresedimented by centrifugation at 33,000 x g for 30 min.The pellet was then extracted by incubating withchloroform: methanol (1: 1) for 30 min at 37 C. Thechloroform-methanol solution was extracted twicewith 0.5 vol of 0.3% aqueous NaCl solution, and aportion of the organic phase was placed on a planchet,allowed to evaporate, and counted in a Nuclear-Chicago gas flow counter. Incubation mixtures con-taining intact cells were diluted with 1.5 vol of 0.1 Msodium acetate. The cells were sedimented, washedwith 0.02 M Tris-glycine buffer, and resuspended inthis buffer to an absorbance of 0.5 to 0.8 at 660 nm.They were treated with lytic enzyme at 37 C until theabsorbance had fallen below 0.04 and membranessedimented as for protoplasts. Subsequent lipid ex-traction was as described above.

Respiration and glycolysis were measured by con-ventional manometric techniques. For respirationmeasurement, 2 ml of cells or protoplasts (0.8 to 1mg/ml) and 0.5 ml of 0.08 M KH2PO4 (adjusted to pH7.4 with KOH) were placed in the main vessel; 0.5 mlof 0.3 M glucose was tipped from the side arm aftertemperature equilibration. The temperature was 37C, the atmosphere was air, and 0.2 ml of 20% KOHwas in center well. For glycolysis measurement, 2 mlof cells or protoplasts (0.8 to 1 mg/ml), 0.1 ml of 0.04M KH2PO4 (adjusted to pH 7.4 with KOH), 0.2 ml of0.255 M NaHCO,, and 0.2 ml of water were placed inthe main vessel. A 0.5-mi amount of 0.3 M glucose wastipped from the side arm after temperature equilibra-tion at 37 C. The aerobic glycolysis atmosphere was02:CO2 (95:5); anaerobic glycolysis atmosphereN2:C02 (95:5).

Electron microscopy. Protoplasts were prefixed in0.3% glutaraldehyde, centrifuged, fixed by themethod of Kellenberger, Ryter, and Sechaud (12),dehydrated in acetone series, embedded in Araldite,and sectioned with an LKB Ultramicrotome. Sectionswere stained with lead citrate (14) for 5 min andviewed in a Siemens Elmiskop I Microscope. Photo-graphs were taken using Agfa-Gevaert Scientia 23D56film.

RESULTSThe majority of the experiments reported

below were carried out using both strains oforganism, and, although the results are gener-ally reported for one strain only, the conclusionsdrawn are equally applicable to both strains.The conditions determined by Coles, Gilbo,

and Broad (2) to be optimal for the removal ofcell walls of S. aureus were assumed to beunsuitable for the formation of metabolicallyactive protoplasts because of the high tempera-ture (45 C) involved. A pH of 8.0 to 8.5 at 37 Cwas selected as being the optimum for the lyticenzyme and unlikely to be detrimental to theprotoplasts. The above conditions were found toproduce stable, metabolically active protoplastsas shown by the following criteria.Morphology. Many photomicrographs such

as those shown in Fig. 1 show the completeabsence of residual cell wall material adheringto the outside membrane of the great majorityof staphylococcal cells treated as above. Thisfact, together with their osmotic fragility and aninability to resynthesize visible cell wall afterincubation in nutrient media (Fig. ld) promptus to refer to these structures as protoplasts.Viable counts in many representative experi-ments showed the number of cells resistant tothe effect of lytic enzyme to be between 0.003and 0.01%. It can be noted from Fig. 1 that thelarge number of mesosomes known to exist inintact cells (unpublished results; Beaton [11published such data for the penicillinase con-

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FIG. 1. Electron micrographs of protoplasts of S. aureus stabilized in 1.1 M sucrose containing 0.017 MNaHCOs and 5 x 10-' M MgSO4. Samples were fixed immediately on preparation (a), after 90 min at 37 C (b),after incubation with 0.02 Mglucose for 90 min at 37 C (c), and after incubation with 0.02 M glucose and 0.27%tryptose for 90 min at 37 C (d). Same magnification throughout, bar represents 200 nm.

stitutive mutant 8325ai-p+) are apparentlylost, and small vesicles accumulate in the sur-rounding medium.The absorbance of a suspension of protoplasts

prepared as above in 1.8 M sucrose was main-tained constant at 37 C over a period of 1 h(longest time tested). This osmotic stability wasalso maintained when the concentration ofsucrose used was as low as 0.9 M. Electronmicrographs of protoplasts in various sucrosemedia taken at the beginning and end of thisperiod showed no morphological changes instructural appearance or size. It was found,however (7), that 1.1 M sucrose was the lowestconcentration to give consistently reproduciblelevels of induced penicillinase formation inprotoplasts. Two other compounds consideredas potential osmotic stabilizers, sodium succi-nate and sodium sulfate, were also tested at thisosmolarity (0.37 M) for their effect on themorphology of protoplasts, with similar results.No decrease in stability or changes in morphol-ogy of S. aureus protoplasts were noted on

incubation in 1.1 M sucrose containing 0.01 Mglucose at 37 C for 90 min. On the other hand,marked changes in size but not stability oc-curred when S. aureus protoplasts were in-cubated in 1.1 M sucrose containing peptone(0.27% Fisher Tryptose) and 0.02 M glucose. Asthe density of the cytoplasm and structuressuch as polysomes has not diminished and therehas been a great increase in the amount ofnuclear material (light areas in Figs. lb, c, andd), it is evident (Fig. 1) that the increase in thesize of the protoplasts supplied with nutrientshas been brought about by synthesis of proto-plasm rather than a mere swelling of the initialbody. Replacement of sucrose with either so-dium succinate or sodium sulfate as stabilizer inthe above nutrient medium resulted in similarrates of growth of the protoplasts.

Respiration and glycolysis. The respiratoryand glycolytic rates of whole cells and preparedprotoplasts were compared, and the resultsare presented in Tables 1 and 2. Respiratoryrates of protoplasts did not vary in the different

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TABLE 1. Respiration of cells and protoplasts in thepresence of various osmotic stabilizers

Q0, (pliters of 0, per hStabilizer Final concn per mg [dry wt ] of cells)Stabiizer (M)

Cells Protoplasts

Sucrose 1.1 65 77Succinate 0.37 95 80Sulfate 0.37 79 77

TABLE 2. Glycolysis of cells and protoplasts in thepresence of various osmotic stabilizers

02(Q( .,2,)a N2(Q('())aStabilizer Final concn(M) ClsProto- ClsProto-Clsplasts Clsplasts

Sucrose 1.1 51 48 96 83Succinate 0.37 40 29 79 66Sulfate 0.37 54 41 84 54

Qco, = gliters of CO, per h per mg [dry wt] ofcells.

stabilizing media, whereas respiratory activityof intact cells was markedly influenced by themedium. In general, aerobic and anaerobicglycolytic rates of protoplasts were somewhatless than those of whole cells.Protein synthesis. Figure 2 shows that proto-

plasts in either sucrose- or succinate-stabilizingmedium are capable of incorporating 'IC-aminoacids into hot trichloroacetic acid-insoluble ma-terial. In sucrose medium, comparable ratesbetween protoplasts and intact cells are ob-served for about 60 min when cells rapidlyincrease their metabolic activity, whereas, insuccinate-stabilizing medium, protoplasts andcells retain comparable incorporating ability.

Nucleic acid synthesis. Comparison of nu-cleic acid synthesis between protoplasts andcells was made using as a basis the incorpora-tion of "4C-uracil and 3H-thymidine into coldtrichloroacetic acid-insoluble material. Figures3 and 4 compare the incorporation, respectively,of thymidine and uracil by cells and proto-plasts. It can be seen that protoplasts activelyincorporate both isotopes at rates initially onlyslightly less than those of whole cells.

Lipid synthesis. "4C-acetate incorporationinto membrane lipids was always more exten-sive with protoplasts than with intact cells inboth sucrose- and succinate-stabilizing media(Table 3). Results expressed in Table 3 werederived from separate experiments and there-fore do not permit a direct comparison of therelative effectiveness of these two stabilizers inpromoting lipid synthesis.

DISCUSSIONThe data presented indicate that protoplasts

of S. aureus, prepared using the Aeromonasstaphylolytic enzyme under the conditions de-

121

z0

Ir-

0

0z

0 30 60 90TIME (min)

FIG. 2. Comparison of incorporation of "C-aminoacids by cells and protoplasts of S. aureus in twostabilizer media. Symbols: 0 0, cells in 1.1 Msucrose; A- -A, protoplasts in 1.1 M sucrose;* *, cells in 0.37 M sodium succinate; andA---- A, protoplasts in 0.37 M sodium succinate.Incorporation measured in counts per minute per 106cells or protoplasts.

z0I-

2000uz

0 50 100TIME (min)

FIG. 3. Comparison of incorporation of 3H-thymi-dine by cells and protoplasts of S. aureus in twostabilizer media. Symbols: 0 0, cells in 1.1 Msucrose; A- -A, protoplasts in 1.1 M sucrose or 0.37Msodium succinate; and 0-----0, cells in 0.37 Msodium succinate. Incorporation measured in countsper minute per 10' cells or protoplasts.

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i

I

I

I

z

.

cr6I~~~~~~~~~~

0

U

z

31

0 30 60 90TIME (min)

FIG. 4. Comparison of incorporation of 14C-uracil bycells and protoplasts of S. aureus in two stabilizermedia. Symbols: 0--- -0, cells in 1.1 M sucrose;A /A, protoplasts in 1.1 M sucrose; v- -0, cells in0.37M sodium succinate; and A A, protoplasts in0.37 M sodium succinate. Incorporation measured in

counts per minute per 106 cells or protoplasts.

TABLE 3. The incorporation of sodium 14C-acetateinto membrane lipids by intact cells and protoplasts

of S. aureusa

Expt 1 Expt 2

Stabilizer (1.1 M (0.37 Msucrose) succinate)

Cells 28,300 34,120Protoplasts 35,460 48,610

aMeasured in counts per minute per milligram(dry wt) of cells, corrected for zero time incorporation(280 counts per min per mg).

scribed and devoid of most of their mesosomalstructures, are metabolically active and thattheir anabolic and catabolic activities are com-parable to those of intact cells.The initial studies of' Weibull (16) showed

that protoplasts having similar respiratory val-ues to intact cells could be prepared from B.megaterium. Our own results and those of'Mitchell and Moyle (13) and Huber andSchuhardt (10) showed that protoplasts having

respiratory rates similar to those of' intact cellscould also be prepared from S. aureus. Howev-er, marked differences in absolute Q,2 valuesexist between our results and, for example,those of Huber and Schuhardt, who report Q,2values about 1.2% of those determined on ourpreparations. This is probably due to the inhibi-tory effect on the respiration of both cells andprotoplasts of the high concentration of NaCl (4M) necessary to stabilize protoplasts preparedwith lysostaphin. The concentrations of sucroseneeded to stabilize the protoplasts producedwith the Aeromonas lytic enzyme were morpho-logically 0.9 M and functionally (as measuredby induction of penicillinase [7]) 1.1 M, concen-trations similar to those reported by others (8,13).

It is interesting to note the higher respirationof cells in sodium succinate than in sodiumsulfate or sucrose, whereas protoplasts show nodifferences in the different stabilizing media. If'this result is an indication of a greater rate oftransport of succinate in cells than in proto-plasts, then the cell wall may contribute to thiseffect, possibly by confining some aspect of thetransport mechanism to the periplasm of thecell.Although the possibility of a slight swelling of'

our protoplasts in stabilizer medium supple-mented with glucose and peptone cannot beexcluded, the great increase in volume occur-ring under these conditions is compatible withnormal growth without cell division. The ob-served synthetic capabilities of the protoplastsare consistent with this electron microscopeevidence. In Fig. 1, the examples depicted werechosen by eye from lower magnification picturesto portray as far as possible sections thatrevealed the maximum diameter of' the proto-plasts. The great increase in size of protoplastsincubated with glucose and peptone is immedi-ately obvious. From the electron microscopeevidence alone, the extent of the increase can-not be stated with certainty because it is notproven that the sections shown are of maximumdiameter. An estimated increase of' four- tofivefold would probably not be in great error.

In all cases where comparisons of' metabolicactivities between cells and protoplasts havebeen made, the cells were subjected to the sameconditions as the protoplasts except for expo-sure to lytic enzyme. It should be pointed outhere that, in all stabilizers tested, the activitiesof' the cells tend to be less than those ot' the samenumber of cells in the absence of stabilizer. Inall parameters measured except the incorpora-tion of' acetate into membrane lipids, the activi-

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ties of protoplasts have been less than those ofthe corresponding number of intact cells. Theexception, where lipid synthesis by protoplastsexceeded that of whole cells, may be a reflectionof rapid membrane synthesis now uncoupledfrom restrictive cell wall synthesis.

ACKNOWLEDGMENT

The authors are indebted to R. C. Hamilton for theelectron micrographs.

LITERATURE CITED

1. Beaton, C. D. 1968. An electron microscope study of themesosomes of a penicillinase-producing staphylococ-cus. J. Gen. Microbiol. 50:37-42.

2. Coles, N. W., C. M. Gilbo, and A. J. Broad. 1969.Purification, properties and mechanism of action of a

staphylolytic enzyme produced by Aeromonas hydro-phila. Biochem. J. 111:7-15.

3. Coles, N. W., and R. Gross. 1965. The effect of mitomycinC on the induced synthesis of penicillinase in Staph-ylococcus aureus. Biochem. Biophys. Res. Commun.20:366-371.

4. Coles, N. W., and R. Gross. 1965. The induced formationof penicillinase in Staphylococcus aureus. Aust. J. Exp.Biol. Med. Sci. 43:725-736.

5. Coles, N. W., and R. Gross. 1967. Influence of organicanions on the liberation of penicillinase from Staph-ylococcus aureus. Biochem. J. 102:748-752.

6. Coles, N. W., and R. Gross. 1969. Exopenicillinasesynthesis in Staphvlococcus aureus. J. Bacteriol.98:659-661.

7. Coles, N. W., and R. Gross. 1973. Formation and secre-

tion of induced penicillinase in protoplasts of Staph-ylococcus aureus. Antimicrob. Ag. Chemother.3:000-000.

8. Hash, J. H., M. Wishnick, and P. A. Miller. 1964.Formation of 'protoplasts" of Staphylococcus aureuswith a fungal N-acetylhexosaminidase. J. Bacteriol.87:432-437.

9. Hirachi, Y., S. Kotani, H. Suginaka, and K. Kato. 1971.Preparation of cytoplasmic membranes of Staphylococ-cus aureus FDA 209P through protoplasts made withthe Lll enzyme and a preliminary analysis of mem-

brane antigens. Biken J. 14:11-28.10. Huber, T. W., and V. T. Schuhardt. 1970. Lysostaphin-

induced, osmotically fragile Staphylococcus aureus

cells. J. Bacteriol. 103:116-119.11. Kato, K., T. Matsubara, Y. Mori, and S. Kotani. 1960.

"Protoplast" formation in Staphvlococcus aureus usingthe lytic enzyme produced by a Flavobacterium. BikenJ. 3:201-203.

12. Kellenberger, E., A. Ryter, and J. Sechaud. 1958. Elec-tron microscope study of DNA-containing plasms. II.

Vegetative and mature phage DNA as compared withnormal bacterial nucleoids in different physiologicalstates. J. Biophys. Biochem. Cytol. 4:671-678.

13. Mitchell, P., and J. Moyle. 1957. Autolytic release andosmotic properties of 'protoplasts' from Staphylococcusaureus. J. Gen. Microbiol. 16:184-194.

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

an electron-opaque stain in electron microscopy. J. CellBiol. 17:208-212.

15. Schuhardt, V. T., and P. H. Kiesius. 1968. Osmoticfragility and viability of lysostaphin-induced staph-ylococcal spheroplasts. J. Bacteriol. 96:734-737.

16. Weibull, C. 1953. The isolation of protoplasts fromBacillus megaterium by controlled treatment withlysozyme. J. Bacteriol. 66:688-695.

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