JOURNAL oF BACTERIOLOGY, Apr. 1977, p. 297-302Copyright C 1977 American Society for Microbiology

Vol. 130, No. 1Printed in U.S.A.

Localization of Enzymes in Ureaplasma urealyticum(T-Strain Mycoplasma)

G. K. MASOVER,1* S. RAZIN,2 AND L. HAYFLICKDepartment of Medical Microbiology, Stanford University School of Medicine, Stanford, California 94305

Received for publication 31 August 1976

Ureaplasma urealyticum cells were lysed by osmotic shock or by digitonin.The membrane fraction contained four to ten times as much protein as thecytoplasmic fraction. These values are in large excess of those reported forclassical mycoplasmas, suggesting that the Ureaplasma membrane fraction washeavily contaminated with proteins derived from the growth medium. The U.urealyticum urease activity was localized in the cytoplasmic fraction, whereasthe adenosine triphosphatase activity was localized in the membrane fraction.Significant urease activity could be detected also in nonviable cells. Urea, atconcentrations above 0.25 M, was mycoplasmastatic to Acholeplasma laidlawii,Mycoplasma hominis, and U. urealyticum, so that the Ureaplasma urease didnot afford preferential protection against urea toxicity. The intracellular locali-zation of the urease would be expected to release ammonia from urea in thecytoplasm. The ammonia will take up protons to become ammonium ions. It canbe hypothesized that the intracellular NH4+ plays a role in proton elimination oracid-base balance, which might be coupled to an energy producing ion gradientand/or transport mechanisms.

Ureaplasmas (formerly T-strain mycoplas-mas) are now included as a genus in the familyMycoplasmataceae of the class Mollicutes (30)and are largely distinguished from Myco-plasma, the other genus in that family, by theirability to hydrolyze urea. Although the enzy-matic activities of phosphatases (1) and pro-teinases (32, 33) have been identified in urea-plasmas, knowledge oftheir metabolism is verymeager compared with the bacteria and, in-deed, even when compared with other Molli-cutes (30). Energy metabolism is still unknownfor ureaplasmas, and the enzymatic activitiesof adenosine triphosphatase and reduced nico-tinamide dinucleotide (NADH) oxidase, whichhave been measured and localized in other my-coplasmas (19), have not yet been reported forureaplasmas. This lack of information may beat least partly attributable to the low yield ofureaplasmas, in terms of both viable units andcell mass (30).The problems of understanding the metabo-

lism ofureaplasmas and obtaining larger yieldsare intertwined; a solution to either problemwill facilitate solving the other. Until that isaccomplished, however, we must use the avail-

1 Present address: Department of Surgery, Division ofUrology, Stanford University School of Medicine, Stanford,CA 94305.

2 Permanent address: Biomembrane Research Labora-tory, Department of Clinical Microbiology, the HebrewUniversity-Hadassah Medical School, Jerusalem, Israel.

able methodology on the available materialand/or develop micromethods to obtain the in-formation we seek about the biology of theureaplasmas. In this research, conventionalmethods were employed to demonstrate the lo-calization of urease, adenosine triphosphatase,and NADH oxidase for comparison with othermycoplasmas in which the localization of theseenzymes has phylogenetic and taxonomic appli-cation. In addition, we report the results ofexperiments to determine if Ureaplasmaurease activity enables that genus to survivehigh urea concentration in the growth milieuas compared with a Mycoplasma and an Acho-leplasma species.

MATERIALS AND METHODSOrganisms and growth conditions. The strains

used in this study were Ureaplasma urealyticum[strain 960-(cx8)I, Acholeplasma laidlawii (PG 8,ATCC 23226) and Mycoplasma hominis (PG 21,ATCC 23114). Two types of media were employed: amodified Hayflick medium (14) that contained 5 or10% (vol/vol) unheated whole horse serum (Microbi-ological Associates, Inc., Bethesda, Md.) and a mod-ified Edward medium (24) that contained Difco yeastextract (0.7% wt/vol) instead of fresh yeast extractand 5% (vol/vol) horse serum. For most experimentsthe media were filtered through a membrane filter(0.22-uAm pore diameter; Millipore Corp., Bedford,Mass.). For Ureaplasma growth, the medium wassupplemented with 0.01 M urea and 0.01 M putres-

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298 MASOVER, RAZIN, AND HAYFLICK

cine, unless otherwise specified, and the pH of themedium was adjusted to 6.5. The same medium, atpH 6.5, supplemented with 20 mM L-arginine wasused to grow M. hominis. To promote A. laidlawiigrowth, the medium was supplemented with 0.5%(wt/vol) glucose, and its pH was adjusted to 7.8. Forenzyme localization studies, U. urealyticum wasgrown in 10-liter batches of medium dispensed infour Erlenmeyer flasks (4-liter). The cultures whichhad been statically incubated at 37°C were subjectedto continuous bubbling of CO2 through the medium(17).Assessment of growth. Growth of the ureaplas-

mas was assessed by colony counts of diluted cul-tures on agar plates, and the results were expressedas colony-forming units per milliliter or as colorchange units per milliliter as determined by thetube dilution method which has been previouslydescribed (16).

Preparation of cell fractions. After a 24-h incuba-tion, the U. urealyticum cultures were usually har-vested at the late logarithmic phase of growth bycentrifugation at 15,000 x g for 60 min at 4°C with aSorvall RC2B centrifuge and a G3 rotor. The com-bined pellets from a 10-liter culture were suspendedin 200 ml of 0.25 M NaCl and centrifuged at 35,000 xg for 15 min at 4°C in the Sorvall SS34 rotor. Thewashed cells in the resulting pellets were lysedeither by osmotic shock or by digitonin according tothe methods outlined by Razin and Rottem (24). Thepellet containing the washed cells for osmotic lysiswas suspended in 2.5 to 6.0 ml of p-buffer (24) whichhad been diluted 1:20 with deionized water (dilute /3-buffer) and incubated at 37°C for 15 min. The smallvolume of diluent was used to prevent excessivedilution of cytoplasmic enzymes. Lysis by digitoninwas performed by resuspending the pellet in 3 ml of0.25 M NaCl containing 40 ,ug of digitonin per mland incubating it at 37°C for 15 min. The cytoplas-mic fraction was separated from the membrane frac-tion by centrifugation of the lysed cell suspensionsat 38,000 x g for 60 min at 4°C. The resulting mem-brane-containing pellets were washed once withdeionized water and then with 0.05 M NaCl in 0.01M phosphate buffer (pH 7.5). The washed membranefraction was resuspended in about 3 ml of dilute ,3-buffer, kept in ice, and tested with the cytoplasmicfraction for enzymatic activities, all on the sameday.

Radioisotopes and reagents. [14C]Urea with aspecific activity of 54.4 mCi/mmol was purchasedfrom Amersham/Searle (Arlington Heights, Ill.). N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid(HEPES) and digitonin were obtained from Calbi-ochem (La Jolla, Calif.). Disodium(ethylenedini-trilo)-tetraacetate (EDTA, disodium salt which che-lates 250 to 300 mg of CaCO3 per g) was a BakerAnalyzed reagent (J. T. Baker Chemical Co.,Philipsburg, N.J.).

Analytical procedures. Protein was determinedby the method ofLowry et al. (11). Urease activity incell fractions was assayed by the disappearance of[14C]urea (0.01 M; 0.1 uCi/ml). The test tubes wereincubated at 37°C, 0.5-ml samples were transferredat 15-min intervals to glass scintillation vials con-

J. BACTERIOL.

taining 0.5 ml of 3.6 N H2SO4, and radioactivity wasdetermined as described by Masover et al. (17). Re-sults were expressed as micromoles of urea hydro-lyzed per microgram of protein per unit of time.Urease activity in growing cultures was assayed asdescribed previously (18). Results were expressed asthe percentage of [14C]urea hydrolyzed in 1 h. Aden-osine triphosphatase activity was measured by therelease of inorganic phosphate from adenosine 5'-triphosphate (20). Results were expressed as micro-moles of inorganic phosphate released per milligramof protein in 30 min. NADH oxidase activity wasmeasured spectrophotometrically by following thedecrease in absorbance at 340 nm on addition ofNADH to the reaction mixture (20).

RESULTSCell yields and contamination with medium

components. To minimize the contamination ofthe sedimented cell pellets with proteins orother components from the serum-containinggrowth medium, the concentration of serumwas reduced from 10 to 5%, the fresh yeastextract which had been used in the Hayflickmedium was clarified by centrifugation, andthe complete medium was filtered through a0.22-,um membrane filter prior to its inocula-tion. These modifications resulted in a markeddecrease in the size and protein content of thepellet which had been obtained on centrifuga-tion of the U. urealyticum cultures, though thenumber of sedimented viable organisms wasabout the same as that which had been ob-tained on harvest of cultures grown in the origi-nal unfiltered Hayflick medium. The growth ofU. urealyticum in the modified Edward me-dium was about the same as that in Hayflickmedium, but the Edward medium had the ad-vantage of higher clarity and ease of filtration.The amount of protein in the washed pellets

obtained either from the filtered Hayflick me-dium or Edward medium was about 1 mg perliter of culture containing about 5 x 106 50%color change units per ml. Digitonin or osmoticlysis of the Ureaplasma cells included in thepellets resulted in the release of only about 10%of the total protein of the pellet as a solublefraction which is presumed to be cytoplasmic.Hence, the "membrane" fraction, sedimentableby centrifugation, contained about 10 timesmore protein than the cytoplasmic fraction. Ad-ditional washings of the membrane fractionwith water and dilute phosphate buffer resultedin a loss of protein from this fraction, reducingthe ratio of membrane-pellet protein to cyto-plasmic-soluble protein from 10 to 4.

Localization of urease. Figure 1 shows thatthe U. urealyticum urease is almost entirelylocalized in the cytoplasmic fraction, which wasobtained either by osmotic lysis of the organism

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ENZYME LOCALIZATION IN UREAPLASMA

12-

.CYTOPLASM

0. 10 DIGITONIN

8 0

W 6 */ ..' \ CYTOPLASMen *Z / / O OSMOTIC LYSIS

0

4

CC 2D MEMBRANE MEMBRANYE

DIGITONIN LYSIS\OSMOTIC.LYSIS

0 15 30 60 105

INCUBATION TIME AT 37 C (minutes)

FIG. 1. Localization of urease activity in fractionsof U. urealyticum cells lysed by digitonin or by os-motic shock.

or by their lysis with digitonin. The effects ofpH and culture age on the urease activity weretested in U. urealyticum cultures incubated ina CO2 or a N2 atmosphere. Figure 2 shows thatthe steep rise in the pH of the culture on itstransfer from CO2 to N2 caused a decrease inthe urease activity of the organisms. This is inaccordance with the finding that urease activ-ity in cell-free extracts of U. urealyticum de-clines steeply at pH values higher than 8.0(Swanberg and Masover, unpublished data).Figure 2 also shows the decrease in urease ac-tivity on aging of the culture. Nevertheless,significant urease activity was detected in theculture incubated with CO2 when the cells wereno longer viable. This finding can also be seenin Table 1. Urease activity was even detected 70h after the loss of viability. The filtration ofportions, which had been taken from culturesat different ages, through 0.22-,um membranefilters showed that the urease activity was re-tained on the filter, suggesting the associationof the enzyme with the cells, even after theirdeath.To test whether urease is a constitutive en-

zyme, U. urealyticum was transferred through12 consecutive 10-fold dilutions in a virtually"urea-free" Hayflick medium containing di-alyzed calf serum and putrescine as previouslydescribed (13). The urease activity in the cyto-plasmic fraction of the organisms grown with-out urea was comparable to that of organismsgrown with urea. From this experiment, it ap-

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3 7.0

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0 20 40 60 80 100

INCUBATION TIME AT 37 C (hours)

FIG. 2. Effect of (a) viability and (b) pH on ureaseactivity ofa U. urealyticum culture grown with 0.01M urea and 0.01 Mputrescine. At the time indicatedby the arrow, the culture grown under 100% C02 wasdivided into two equal portions; one was transferredto 100% N2 and the other remained under 100% Co2-At the time intervals indicated in the figure, sampleswere taken and tested for urease activity by the addi-tion of [P4C]urea and determination of the residualradioactivity in the medium after 1 h. CCU50, 50%color change units.

TABLE 1. Relationship between urease activity andU. urealyticum viability in a broth culture saturated

with C02a

Urease ac-Age of tivity (%

Flask culture CCU5o/mlb urea hy-(h) drolyzed in

1 h)A 24 6.3 x 106 55

27 1.6 x 107 5430 2.0 x 107 5247 3.2 x 105 37

B 71 0 2777 0 3095 0 13143 0 10

a Replicate cultures were incubated at 37°C in a100% CO2 atmosphere. At the indicated times,['4C]urea to a final concentration of 0.01 M and 0.01,uCi/ml was added to the flasks. Urease activity wastested by determination of residual radioactivity inthe flasks at various time intervals during the firsthour after the addition of urea (17).

b CCU50, 50% color change units.

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ot* * * ~~CO20, , C ,2

VOL. 130, 1977 299

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300 MASOVER, RAZIN, AND HAYFLICK

pears that the urease is a constitutive enzymein U. urealyticum.One role for the Ureaplasma urease activity

could be the detoxification of urea, which isable to penetrate into cells freely (3). Since thenatural habitat of U. urealyticum is the uro-genital tract (12, 30), the ureaplasmas might beexposed to a potentially toxic range of ureaconcentrations in their host. It was, therefore,of interest to investigate whether urease activ-ity has a survival advantage for the ureaplas-mas over other mycoplasmas which do not pos-sess this enzyme. The effects of increasing con-centrations of urea on the growth of U. urealy-ticum, M. hominis, and A. laidlawii weretherefore tested. Urea concentrations above0.25 M inhibited the growth of all three myco-plasmas tested, as determined by the absence ofa color change of the pH indicator included inliquid growth medium or by the inhibition ofcolony formation on solid media. However, theinhibition of a color change was not a result ofcell death, because colony counts showed thatthe number of viable organisms inoculated intoliquid media containing 1 M urea remainedvirtually constant during a 24-h incubation at370C.

Localization of adenosine triphosphataseactivity. Figure 3 shows that the adenosinetriphosphatase activity of U. urealyticum re-sembles that ofA. laidlawii as it is also local-ized in the membrane. The low activity in thecytoplasmic fractions of both organisms couldrepresent activity associated with minute mem-

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2

1

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INCUBATION TIME AT 37 C (minutes)

FIG. 3. Adenosine triphosphatase activity inmembrane and cytoplasmic fractions of U. urealyti-cum and A. laidlawii obtained by osmotic lysis. Theactivity at zero time (just after the addition of thesubstrate) of any of the fractions tested was taken as

i.00, so that the enzymatic activities with progressiveincubation time are related to this value.

J. BACTERIOL.

brane fragments that are not sedimentable bythe centrifugal force which had been applied toseparate the membrane from the cytoplasmicfraction. The specific activity of the Urea-plasma adenosine triphosphatase was abouthalf that of A. laidlawii in the experimentshown in Fig. 3 and somewhat lower in otherexperiments. Several attempts to assay the lo-calization of the U. urealyticum NADH oxidaseactivity failed to produce conclusive results, ap-parently due to the small amount of materialavailable for study.

DISCUSSIONThe propensity of ureaplasmas harvested

after growth in vitro to be associated with non-cellular materials from the growth medium hasbeen previously observed (16, 25, 27). This hasalso been observed for some classical mycoplas-mas and is known to influence the antigeniccharacter (2, 26, 28) and the ability of the myco-plasmas to react in various diagnostic tests (4).Biochemical measurements of ureaplasmas aremade more difficult because large amounts ofexogenous materials associated with the smallUreaplasma cell mass interferes with the quan-titative determination of cell yields and withcalculation of specific enzyme activities of cellsand cell fractions.We have previously reported (18) a value for

the protein content of ureaplasmas harvestedfrom Hayflick medium, which contained 10%whole unheated horse serum and a 10%o freshyeast extract preparation (9), and then washedin saline before lysis by digitonin (24). In thatcase, the ratio of protein in the washed mem-brane fraction to protein in the soluble fractionwas about 25. This ratio is much higher thanthe 0.5 to 1.0 ratios reported for similar frac-tions obtained from classical mycoplasmas (23)and apparently reflects noncellular protein co-sedimenting with or bound to the membranes ofthe lysed Ureaplasma cells; the precise natureofcontaminating protein is not known. Steps toclarify the media, such as centrifugation of thefresh yeast extract preparations included in theHayflick medium, reduction of the serum con-tent from 10 to 5%, and filtration (0.22-,ummembrane filter [millipore]) of the whole me-dium prior to use, reduced the ratio of proteinin the membrane fraction to that in the solublefraction to approximately 10. Several washingsofthe membrane fraction with water and dilutebuffers removed about half of the protein in themembrane fraction. However, extensive mem-brane washing may lead to their fragmenta-tion, so that the protein removed from themembrane fraction might include membrane

UREAPLASMAMEMBRANES o-A

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ENZYME LOCALIZATION IN UREAPLASMA

protein along with contaminating protein. It isclear, at least for ureaplasmas grown in vitro,that cell-associated exogenous materials mustbe accounted for in biological and biochemicaltests. In light of this, some recently reportedanalyses of Ureaplasma membrane fractions(34, 35) are questionable.The experiments shown here confirm our pre-

vious finding (18) that the Ureaplasma ureaseactivity is cytoplasmic. In addition, we wereable to show that the cytoplasmic localization ofurease can be demonstrated by using eitherdigitonin or osmotic lysis of the ureaplasmas,thus ruling out the possibility that digitonin issolubilizing a membrane-bound urease. Sinceurease is an extremely efficient enzyme (31), itis easy to test and, by virtue of its cytoplasmiclocalization, is useful for estimation of the de-gree of cytoplasmic contamination of mem-brane preparations. For example, the data pre-sented in Fig. 1 not only show that the urease isalmost entirely cytoplasmic but that the cyto-plasmic fraction of osmotically lysed cells is lessactive than the same fraction from cells of thesame batch which had been lysed by digitonintreatment. Since the data in Fig. 1 are ex-pressed as activity per microgram ofprotein perunit of time, the membrane fraction, whichcontains 10 times as much protein as the cyto-plasmic fraction, appears to have practically noactivity; that is not the case. Some urease activ-ity remained in the washed membrane fractionsuch that if the same data were expressed as aratio of the total activity (cytoplasm/mem-brane) in digitonin-lysed cells, the ratio wouldbe about 14.5, whereas that ratio in the osmoti-cally lysed cells is 4.5. Thus, there was contami-nation of the osmotically lysed membrane frac-tion with cytoplasmic urease. We believe thatthis is the result ofincomplete osmotic lysis dueto the small volume (ca. 6.0 ml) of hypotonicbuffer used to lyse the cells and is also, perhaps,due to the extraneous proteinaceous materialswhich may have interfered with osmotic lysis.That the urease is, and remains, cell associ-

ated was shown by the fact that it was possibleto remove the activity from the media by filtra-tion at all stages of the culture. These findingsare consistent with those of Furness, who sug-gested that Ureaplasma urease is not extracel-lular (7). The intracellular localization ofurease in ureaplasmas might have physiologi-cal significance regarding the role of urea inthese organisms. Urea is uncharged at physio-logical pH and is freely permeable across thecell membrane (3). When hydrolyzed within thecell, it yields CO2, which is completely recover-able (5), and ammonia (NH3), which will accepta proton and become an ammonium ion (NH4+)

at physiological pH. Although the precisemechanism for transport of the NH4+ from in-side the cell to the outside is not known, it isclear that NH4+ accumulates in the culture me-dium. We have previously suggested that thismight provide a mechanism for acid-balance orproton elimination (14) and, under appropriateconditions, perhaps an ion gradient coupled toan energy producing or a transport system (8).The observation that higher concentrations

of urea are toxic to ureaplasmas was first madeby Shepard and Lunceford (29), who observedan inhibition of the growth of the organisms by2.0% (wt/vol) urea. Our findings agree withtheirs by showing that concentrations higherthan 0.25 M urea (1.5% wt/vol) caused the inhi-bition of Ureaplasma growth. However, we alsofound that the effect of urea is mycoplasma-static and not mycoplasmacidal. In addition, wefound that the ureaplasmas were not more re-sistant to the growth-inhibiting effect of ureacompared to M. hominis and A. laidlawiiwhich lack urease. Thus, the urease activity ofureaplasmas does not make them any moretolerant to urea than any other Mollicuteswhich do not have urease activity.Our data show that the adenosine triphos-

phatase activity in U. urealyticum is localizedin the membrane as it is in A. laidlawii and inall other mycoplasmas tested so far (22). Thelower specific activity of the Ureaplasma aden-osine triphosphatase activity as compared withthat of A. laidlawii undoubtedly reflects con-tamination ofthe Ureaplasma membrane prep-aration with exogenous protein as discussedabove.

Several attempts to deternine NADH oxi-dase activity in Ureaplasma cell fractionsyielded equivocal results, the major problembeing the small amounts of cellular materialavailable for study. To overcome this problem,larger cell yields must be obtained or moresensitive methods must be employed for meas-urement of the NADH oxidase activity. If thisenzyme activity is found in the cytoplasm ofureaplasmas, it could relate them to the Myco-plasmas species, whereas localization of theNADH oxidase activity in Ureaplasma mem-branes would relate them to Acholeplasma spe-cies (20).

ACKNOWLEDGMENTSWe gratefully acknowledge the technical assistance of

Marina Palant and Stephen L. Swanberg.This work was suported by Public Health Service grant

AI 11805 from the National Institute of Allergy and Infec-tious Diseases.

LITERATURE CITED1. Black, F. T. 1973. Phosphatase activity in T-mycoplas-

mas. Int. J. Syst. Bacteriol. 23:65-66.

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8. Harold, F. M. 1972. Conservation and transformation ofenergy by bacterial membranes. Bacteriol. Rev.36:172-230.

9. Hayflick, L. 1965. Cell cultures and mycoplasmas. Tex.Rep. Biol. Med. 23(suppl. 1):285-303.

10. Lin, J. S., and E. H. Kass. 1970. Immune inactivation ofT-strain mycoplasmas. J. Infect. Dis. 122:93-95.

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13. Masover, G. K., J. R. Benson, and L. Hayflick. 1974.Growth of T-strain mycoplasmas in medium withoutadded urea: effect of trace amounts of urea and of aurease inhibitor. J. Bacteriol. 117:765-774.

14. Masover, G. K., and L. Hayflick. 1973. The growth ofT-strain mycoplasmas in media without added urea.Ann. N.Y. Acad. Sci. 225:118-130.

15. Masover, G. K., and L. Hayflick. 1974. Dialysis cultureof a T-strain mycoplasma. J. Bacteriol. 118:46-52.

16. Masover, G. K., R. P. Mischak, and L. Hayflick. 1975.Some effects of growth medium composition on theantigenicity of a T-strain mycoplasma. Infect. Im-mun. 11:530-539.

17. Masover, G. K., S. Razin, and L. Hayflick. 1977. Ef-fects of carbon dioxide, urea, and ammonia on growthofUreaplasma urealyticum (T-strain mycoplasma). J.Bacteriol. 130:292-296.

18. Masover, G. K., J. E. Sawyer, and L. Hayflick. 1976.Urea hydrolyzing activity of a T-strain mycoplasma:Ureaplasma urealyticum. J. Bacteriol. 125:581-587.

19. Pollack, J. D. 1975. Localization of reduced nicotina-mide adenine dinucleotide oxidase activity inAchole-plasma and Mycoplasma species. Int. J. Syst. Bacte-riol. 25:108-113.

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22. Razin, S. 1975. The mycoplasma membrane, p. 257-312.In D. A. Cadenhead, J. F. Danielli, and M. D. Rosen-berg (ed.), Progress in surface and membrane sci-ence, vol. 9. Academic Press Inc., New York.

23. Razin, S., M. Argaman, and J. Avigan. 1963. Chemicalcomposition of mycoplasma cells and membranes. J.Gen. Microbiol. 33:477-487.

24. Razin, S., and S. Rottem. 1976. Technics for the manip-ulation of mycoplasma membranes, p. 3-26. in A. H.Maddy (ed.), Biochemical analysis of membranes.Chapman and Hall Ltd., London.

25. Razin, S., J. Valdesuso, R. H. Purcell, and R. M.Chanock. 1970. Electrophoretic analysis of cell pro-teins of T-strain mycoplasmas isolated from man. J.Bacteriol. 103:702-706.

26. Riggs, S., and J. T. Sharp. 1970. Serologic reactions ofmycoplasmas, p. 82-103. In J. T. Sharp (ed.), The roleof mycoplasmas and L-forms of bacteria in disease.Charles C Thomas, Springfield, Ill.

27. Romano, N., P. F. Smith, and W. R. Mayberry. 1972.Lipids of a T-strain mycoplasma. J. Bacteriol.109:565-569.

28. Sethi, K. K., and H. Brandis. 1972. Killing ofmycoplas-mas by the antibodies to foreign antigens acquired bythe organisms from the growth medium. Med. Micro-biol. Immunol. 157:113-119.

29. Shepard, M. C., and C. D. Lunceford. 1967. Occurrenceof urease in T-strain of mycoplasma. J. Bacteriol.93:1513-1520.

30. Shepard, M. C., C. D. Lunceford, D. K. Ford, R. H.Purcell, D. Taylor-Robinson, S. Razin, and F. T.Black. 1974. Ureaplasma urealyticum gen. nov., sp.nov.: proposed nomenclature for the human T (T-strain) mycoplasmas. Int. J. Syst. Bacteriol. 24:160-171.

31. Sumner, J. B. 1951. Urease, p. 873-892. In J. B. Sumnerand K. Myrbach (ed.), The enzymes, vol. 1, part 2.Academic Press Inc., New York.

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33. Watanabe, T., K. Mishima, and T. Horikawa. 1973.Proteolytic activities of human mycoplasmas. Jpn. J.Microbiol. 17:151-153.

34. Whitescarver, J., F. Castillo and G. Furness. 1975. Thepreparation of membranes of some human T-myco-plasmas and the analysis of their carbohydrate con-tent. Proc. Soc. Exp. Biol. Med. 150:20-22.

35. Whitescarver, J., M. Trocoli, T. Campana, R. Marks,and G. Furness. 1976. A study of the amino acids andproteins of some human T-mycoplasma membranes.Proc. Soc. Exp. Biol. Med. 151:68-71.

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