INTERNATIONAL JOURNAL OF SYSTEMATICBACTERIOLOGY, July 1988, p. 273-278 Vol. 38, No. 3 0020-7713I881030273-06$02.0010 Copyright 0 1988, International Union of Microbiological Societies

Comparison of of in Two Members of the Mycoplasmataceae Family

BENJAMIN G. COCKS, RIMA YOUIL, AND LLOYD R. FINCH* Russell Grimwade School of , University of Melbourne, Parkville, Victoria 3052, Australia

The presence or absence of some enzymes of nucleotide metabolism has been suggested by other workers as being of possible value as criteria for some genera within the Mollicutes. We assayed subcellular fractions of Mycoplasma mycoides subsp. mycoides strain Y and Ureaplasma urealyticum 960T (T = type strain) for these activities. U. urealyticum 960T had characteristics similar to those noted previously (Williams and Pollack, Int. J. Syst. Bacteriol. 35227-230,1985) for some other species in the Mycoplasmataceae in that it (i) does not show a triphosphate nucleotidohydrolase, (ii) does possess deaminase, (iii) does not show a specific deoxyuridine monophosphate phosphatase, and (iv) does possess deoxycytidine monophosphate deaminase. M. mycoides differs from these other members of the Mycoplasmataceae in that it (i) does possess a deoxyuridine triphosphate nucleotidohydrolase and (ii) does not show deoxycytidine or deaminase activity, but it is similar in that it does not show a specific deoxyuridine monophosphate phosphatase and does possess deoxycytidine monophosphate deaminase. Extracts of U. urealyticum were fractionated and assayed for activities of a range of enzymes of nucleotide metabolism. Our results indicate a capacity for salvage pathways of nucleotide synthesis with similarities to those already proposed for M. mycoides and Acholeplasma ladlawii B-PG9.

The class Mollicutes consists of three orders, Mycopla- supernatant from sonicated cell extracts, whereas Williams smatales, Acholeplasmatales, and Anaeroplasmatales. The and Pollack (18) tested cytoplasmic and membrane fractions sterol-requiring Mycoplasmatales contains two families, isolated after osmotic lysis of the cells. Mycoplasmataceae (with genera Mycoplasma and Urea- We sought to clarify the questions of possible differences plasma) and Spiroplasmatacea (with the genus Spiro- for M. mycoides by testing activities in both sorts of prepa- plasma). The sterol-nonrequiring Acholeplasmatales con- ration and took the opportunity to extend the comparative sists of a single family and genus, while the survey to Ureaplasma urealyticum 960T (T = type strain), a Anaeroplasmatales have a single family for two distinct species from the other genus in the Mycoplasmataceae. genera (Anaeroplasrna and Asteroleplasma). Williams and Pollack (18) have examined six Mycoplasma species, one MATERIALS AND METHODS Spiroplasma species, and five Acholeplasma species for Organisms and culture media. M. mycoides subsp. my- activities involved in deoxynucleotide coides goat strain Y was obtained from A. W. Rodwell, metabolism. These authors concluded that it may be possi- Commonwealth Scientific and Industrial Research Organisa- ble, after more species are studied, to distinguish some tion, Parkville, Australia. Cultures of M. mycoides were genera within the Mollicutes by the presence or absence of grown in PPLO broth (15) as described previously (8). U. the enzymatic activities of (i) deoxyuridine triphosphate urealyticum serovar VIII strain 960T (= ATCC 27618=), nucleotidohydrolase (dUTPase) (EC 3.6.1.23), which is de- provided by G. Masover, was grown in modified PPLO broth fined as triphosphate (ATP) insensitive and spe- as described previously (2). cifically deoxyuridine triphosphate (dUTP) hydrolyzing, (ii) Preparation of cell extracts and membrane fractions. Ex- deoxycytidine (dC) deaminase (EC 3.5.4.14), (iii) deoxyuri- tracts of M. mycoides from 50-ml cultures were prepared by dine monophosphate (dUMP) phosphatase (dUMPase) (EC sonication as previously described (8), except that 0.2 mM 3.1.3 3, and (iv) deoxycytidine monophosphate (dCMP) pepstatin A was added to inhibit protease digestion of deaminase (EC 3.5.4.12). All of the Mycoplasma species cellular enzymes. The pellet from the high-speed centrifuga- which they tested had dC and dCMP deaminase activities, tion following sonication of M. mycoides was suspended in but no detectable dUTPase or specific dUMPase activities. 10 mM tris(hydroxymethy1)aminomethane (Tris) hydrochlo- In the studies of Williams and Pollack (17, 18), dUTPase ride (pH 8.1)-1 mM dithiothreitol (DTT) and was subjected and also deoxycytidine triphosphate (dCTP) nucleotidohy- to an additional 60 s of sonication. For preparation of U. drolase (dCTPase) were defined as being ATP-insensitive urealyticum extracts by sonication the cells from 8 liters of activities and dUMPase was defined as an adenosine mono- medium were washed with 0.25 M NaCl and then suspended phosphate-insensitive activity. Neale et al. (10) have re- in 1 ml of 1 mM Tris (pH 8.l)-l mM DTT. The subsequent ported dUTPase, dCTPase, and dUMPase activities from steps were as previously described (l),except that no bovine Mycoplasma mycoides subsp. mycoides strain Y, but made serum albumin was added. Cytoplasmic and membrane no test for ATP or sensitivity so fractions of both organisms were obtained after osmotic lysis that no conclusion is warranted as to whether M. mycoides as previously described by Razin (14). However, cells from may be intrinsically different from the Mycoplasma species 1 liter of a U. urealyticum culture were lysed in a volume of studied by Williams and Pollack (18). Another difference in 5 ml. the studies was that Neale et al. (10) used a high-speed Chemicals and radiochemicals. Horse serum for growth media was obtained from the Commonwealth Serum Labo- ratories, Parkville, Australia, and yeast extract and PPLO * Corresponding author broth were obtained from Difco Laboratories, Detroit, Mich.

273 274 COCKS ET AL. INT. J. SYST.BACTERIOL.

Thioglycollic acid was obtained from Ajax Chemicals Ltd., tained 0.1 mM [2-14C], 1 mM ATP, 2 mM DTT, Melbourne, Australia, and amino acids, antibiotics, and and 20 mM NaF. buffers were obtained from Sigma Chemical Co., St. Louis, (ii) Isolation of radiolabeled products on polyethyleneimine- Mo. The scintillation solute 2,5-diphenyloxazole, nucleo- cellulose thin layers. The dCMP deaminase (EC 3.5.4.12) tides, cofactors, intermediates, and enzymes were all ob- assay was performed as previously described (lo), except tained from Sigma. that 0.1 mM dCTP and 2 mM DTT were included for 2 min [2-14C]dC (30 Ci/mol) was obtained from New England of preincubation at 37°C before the reaction was begun by Nuclear Corp., Boston, Mass. [U-14C]dCMP (464 Ci/mol), adding . [U-14C] (502 Cilmol), [U-14C]dCTP The (deaxy)CMP kinase (EC 2.7.4.14) assay was per- (452 Ci/mol), [U-14C]deoxythymidine monophosphate formed as previously described (10). (dTMP) (528 Ci/mol), [U-14C]adenosine (500 Cilmol), de- The dTMP kinase (EC 2.7.4.9) assay was performed as ~xy[U-~~C]adenosine(512 Cilmol), [2-14C] (57 Cil previously described (lo), except that 0.1 mM [U-14C]dTMP mol), [2-14C](60 Ci/mol), [U-14C] (235 Ci/mol), was used as the substrate. [U-14C]cy tidine (510 Ci/mol) , [U- 14C]c y t idine dipho sphat e The uridine phosphorylase (EC 2.4.2.3) assay was per- (436 Ci/mol), [2-14C]thymidine (57 Cilmol), [U-14]deoxygua- formed as previously described (l),except that 0.1 mM nosine monophosphate (dGMP) (517 Cilmol), [U-14C]deoxy- [2-14C]uridine was used as the substrate. adenosine monophosphate (dAMP) (518 Ci/mol), [U-14C] The adenosine phosphorylase (EC 2.4.2.1) assay mixture glucose (275 Cilmol), [S3H]dCTP (21 Ci/mmol), [5-3H]dUTP contained 5 mM sodium orthophosphate and 0.1 mM [U- (20 Ci/mmol), [5-3H]dUMP (9.6 Ci/mmol), and [Y-~~P]ATP 14C]adenosine. Chromatography was performed as previ- (3,000 Cilmmol) were all obtained from Amersham Corp., ously described (8), and both [14C]adenine (Rf, 0.5) and Arlington Heights, Ill. [14C]-l-phosphate (Rf,0 to 0.1) were counted. Fractionation of cell-free extract. Partial purification of The adenine PRTase (EC 2.4.2.7) assay mixture contained enzyme activities in the U.urealyticum 960T cell-free extract 0.1 mM [U-14C]adenine and 0.4 mM 5-phosphoribosyl-l- was achieved by using a fast protein liquid chromatography diphosphate. Samples were spotted onto the origins, and system (Pharmacia Pty. Ltd., West Melbourne, Australia) as then the chromatograms were developed twice in distilled described previously (2); however, fractions were collected water. This separated the adenosine monophosphate prod- directly into tubes containing 50 pg of cytochrome c. All of uct (Rf, 0 to 0.1) from adenine (Rf,0.9 to 1). these fractions were assayed for the enzymes listed below, The uridine kinase (EC 2.7.1.48) assay mixture contained and proteins in fractions and in the cell-free extract were 0.1 mM [2-14C]uridine, 2 mM ATP, 1 mM DTT, and 20 mM estimated (5). For two-step partial purification of dUTPase NaF. Chromatography was performed as described previ- from sonicated cell-free extracts of M. mycoides by fast ously (7). protein liquid chromatography we used a Superase 12 col- The dCTPase (EC 3.6.1.12) and dUTPase (EC 3.6.1.23) umn (elution buffer, 50 mM NaCI-10 mM Tris hydrochlo- assays were performed as previously described (10); how- ride, pH 8.5) and then a Mono Q column and the elution ever, ATP was included in some assay mixtures, and various conditions described previously (2). concentrations of substrate were used, as indicated below. All assays were performed at 37°C in 40 mM Tris (pH 7.8)- The monophosphate was not eluted prior 5 mM MgC1, supplemented with additional components as to counting. indicated below. Unless specified otherwise, the concentra- The nucleoside diphosphate kinase (EC 2.7.4.6) assay tion of labeled substrate in all nucleotide enzyme assays was mixture contained 0.1 mM [U-14C]cytidine diphosphate, 1 0.1 M, and the specific activities were 5 to 20 Ci/mol for 14C- mM ATP, and 20 mM NaF. Chromatography was performed and 32P-labeled substrates and 100 Ci/mol for 3H-labeled as described above for the dCTPase assay. substrates. Each assay mixture contained up to 25 pg of cell The (EC 2.4.2.4) assay was protein, except for the phosphoribosyltrans- performed like the uridine phosphorylase assay (see above), ferase (PRTase), dGMP kinase, and dCMP deaminase assay except that 0.1 mM [U-14C]thymidine and 5 mM sodium mistures, which all contained 150 pg of cell protein. The orthophosphate were used. The chromatogram was sec- assay volumes were 12 to 20 pl, and reactions were begun by tioned to count the deoxyribose-1-phosphate (Rf,0 to 0.1) adding enzyme and were sampled at various times by adding product, which was separated from thymidine and 4 p1 of reaction mixture to the separation matrix for isolating (both of which had Rfvalues of 0.9 to 1.0). the product from the substrate. All assays, except the The dGMP kinase (EC 2.7.4.8) assay mixture contained (deoxy) assay, involved separations in 0.1 mM [U-14C]dGMP, 1 mM ATP, and 20 mM NaF. which we used polyethyleneimine-cellulose thin layers (10, Chromatography was performed as described above for 13) or ethylenediaminetetraacetate-treatedWhatman DE81 dTMP kinase, and the diphosphate product diethylaminoethyl-cellulose paper (1). (Rf,0.4 to 0.5) was counted. The Rfof dGMP was 0.7 to 0.8. Details of specific assays. (i) Isolation of radiolabeled prod- In the assays for deoxynucleotide phosphohydrolases (EC uct on diethylaminoethyl-cellulosepaper. The dC kinase (EC 3.1.3.5) the dUMPase assay was performed as previously 2.7.1.74) assay was performed as described previously (10). described (lo), except that the concentrations of [5-3H] The uracil PRTase (EC 2.4.2.9) assay mixture contained dUMP were 0.05 and 1.25 mM in the assays for enzymes 0.1 mM [2-14C]uracil, 1 mM triphosphate, and 0.4 from U.urealyticum and M. mycoides, respectively, and the mM 5-phosphoribosyl-1-diphosphate. [5-3H]uridine product was not eluted before counting. The The cytidine kinase (EC 2.7.1.48) assay mixture contained dAMP phosphatase, dCMP phosphatase, dGMP phospha- 0.1 mM [U-14C]cytidine, 2 mM ATP, 1 mM DTT, and 20 mM tase, and dTMP phosphatase assays were performed like the NaF. dUMPase assay, except that in the U. urealyticum 960T The hypoxanthine PRTase (EC 2.4.2.8) assay mixture assays the respective U-14C-labeled deoxynucleotide mono- contained 0.1 mM [8-14C]hypoxanthine, 0.4 mM 5-phospho- phosphates were used at concentrations of 0.1 mM. ribosyl-1-diphosphate, and 20 mM NaF. The adenosine triphosphatase (EC 3.6.1.3) assay mixture The thymidine kinase (EC 2.7.1.21) assay mixture con- contained 0.1 mM [Y-~~PIATP.The chromatography condi- VOL. 38, 1988 NUCLEOTIDE METABOLISM IN MYCOPLASMATACEAE 275

TABLE 1. Activities for the hydrolysis of dUTP and dCTP in different fractions from extracts of M. mycoides subsp. mycoides

Sp act (pnoUmin per g of total cell protein) Nucleotide(s) added Product assayed Sonication Sonication Osmotic lysis Osmotic lysis pellet supernatant membrane cytoplasm 0.25 mM [3H]dUTP dUMP 0.1 8.0 4.9 16.8 dUDP Nil Nil Nil 5.9 0.25 mM [3H]dUTP + 1.25 mM ATP dUMP 0.01 8.0 5.2 12.9 dUDP Nil Nil 2.5 Nil 0.25 mM [3H]dCTP dCMP 0.03 1.9 0.03 2.9 dCDP 0.4 Nil 2.6 0.02 0.25 mM [3H]dCTP + 1.25 mM ATP dCMP Nil 0.7 Nil 0.8 dCDP 0.3 1.4 2.5 Nil tions were the same as those described above for dCTPase, substrates were in the membrane fraction rather than the and the labeled orthophosphate product was counted (the Rf cytoplasm, with inhibition by ATP. This is similar to the values of ATP and orthophosphate were 0.1 to 0.2 and 0.8 to findings of Williams and Pollack (18) for the distribution of a 0.9, respectively). nonspecific activity for dUTP and dCTP hydrolysis in the (iii) Isolation of radiolabeled product by ion-exchange col- Mycoplasrnataceae species which they tested. With sonica- umns. The cytidine deaminase (EC 3.5.4.5) and dC deami- tion the activities were found in the supernatant, probably nase (EC 3.5.4.14) reaction mixtures contained [U-14C] through comminution of membrane fragments by the soni- cytidine or [2-14C]dC at a concentration of 0.1 mM. Samples cation. (5 1.1) of incubation mixture were taken at various times and For further investigation of the possible differences in placed into 1 ml of a settled 1:4 mixture of Dowex 50 resin these activities between the two Mycoplasmataceae species, and 10 mM HCl. This mixture was contained in a 1-ml sonication supernatants were subjected to fractionation by (Oxford) pipettor tip plugged with nylon wool and capped at fast protein liquid chromatography to achieve partial purifi- the pointed end. After the cap was removed, the liquid cation from possible interfering actions of other enzymes in containing the (de~xy)[’~C]uridineproduct was drained into the crude extracts. Extracts from M. mycoides were sub- vials for counting; this was followed by the addition of 8 ml jected to a two-step fractionation, first for size separation on of scintillation fluid (5 g of 2,5-diphenyloxazole per liter of a Superose 12 column and then on a Mono Q ion-exchange toluene and Teric-10 [2: 1, vol/vol]). Unchanged substrate column. The dUTPase eluted from the Superose column 12 remained bound to the resin. in the volume range of 13.8 to 14.4 ml. dCTPase activity was also present but was displaced toward the higher volumes in RESULTS the range. Both substrates gave the monophosphates as the Table 1 shows comparisons of the total activities for sole nucleotide products. The dCTPase activity showed hydrolysis of dUTP and dCTP in extracts of M. rnycoides inhibition by 1.25 mM ATP, but the dUTPase activity did subsp. rnycoides prepared as sonication supernatants and not. The fractions containing dUTPase activity were then pellets by the procedure of Neale et al. (10) and in membrane pooled for fractionation on Mono Q. Activity on dUTP and cytoplasmic fractions prepared by the osmotic lysis eluted in three fractions with NaCl concentrations of 0.20 to procedure of Razin (14). With all preparations dUMP was 0.22 M. Only the middle fraction showed activity on dCTP at the main product of dUTP hydrolysis, and there was no less than 5% of the activity on dUTP. Neither activity was strong inhibition of its formation by 1.25 mM ATP. Although inhibited by ATP. The dUTPase pooled from the three some activity was found in all preparations, the greatest total fractions showed essentially no inhibition (<2%) by 1 mM activity and highest specific activity were found in the dCTP and a K, value of approximately 5 +M for dUTP (Fig. cytoplasm from osmotic lysis. This fraction also showed 1). deoxyuridine diphosphate (dUDP) formation in the absence Extracts from U. urealyticurn were fractionated directly of ATP but not when ATP was present. With the lack of on a Mono Q column and were assayed for activity on dUTP dUMP kinase in extracts of M. rnycoides (lo), such produc- and dCTP and for a range of other enzymic activities (Fig. 2 tion of dUDP probably reflects activity of nucleoside diphos- and Table 2). The activities on dUTP and dCTP eluted phate kinase associated with residual nucleoside diphos- together, and both were subject to inhibition by ATP, phates in the extract or of nonspecific phosphatases. With although adenosine triphosphatase was not associated with dCTP as the substrate, dCMP was the major product with the fractions active on dUTP and dCTP (Fig. 2b). The the osmotic lysis cytoplasm and the sonication supernatant, monophosphates were the only nucleotide products from the but the membrane fraction and the sonication pellet pro- hydrolysis, and, as indicated by the rates observed at duced deoxycytidine diphosphate (dCDP). This dCDP pro- concentrations of 10 and 100 FM (Table 2), the K,, for dUTP duction was not inhibited by ATP, but the yield of dCMP was approximately 500 to 600 FM. The inhibition by ATP did with the cytoplasm and sonication supernatant was de- not appear to be competitive since the inhibition of 50% by creased. This decrease may have been due to inhibition of 200 pM ATP observed with 100 pM dUTP (Fig. 2b) was the the dCTPase activity, but loss of product may also have same as the inhibition observed with 10 pM dUTP (data not occurred because of the strong phosphorylating effects of the shown). dCMP kinase and nucleoside diphosphate kinase known to The data in Fig. 2b and Table 2 show activity for dC be in the preparations (10). deaminase coincident with cytidine deaminase activity in the Similar experiments with preparations from U. urealyti- fractionation, which is consistent with both activities being cum showed that the major hydrolytic activities for both properties of the one enzyme, as usually reported (16). In 276 COCKS ET AL. INT.J. SYST.BACTERIOL.

The data in Table 2 also show that U. urealyticum pos- V-’ sesses many of the other enzymes of nucleotide metabolism 8 previously reported in other members of the Mollicutes. Adenosine phosphorylase was previously identified in U. urealyticum extracts by Davis et al. (3), and Cocks et al. (1) 6 observed uridine phosphorylase and very high levels of adenylate kinase activity. Adenine and hypoxanthine PRT- ase activities were detected (Table 2), although they were not observed previously when they were sought in ureaplas- mas (12). Figure 2a shows that cytidine kinase and dC kinase chromatographed separately, in keeping with these enzymes 2 being different proteins, as observed in their isolation from other sources (4, 11). No uridine kinase activity was ob- served. The kinase activities for dCMP and cytidine mono- phosphate were coincident in the fractionation (the fraction with maximum activity is shown in Table 2), which is 0 1 2 3 consistent with previous results obtained with other orga- [d UTPI-’ pM-’ nisms (10, ll), suggesting that these are activities of one enzyme. Maximum activity in a single peak for adenylate FIG. 1. Double reciprocal plot for dUTPase from M. mycoides. kinase also occurred in the same fraction from the Mono Q 71 is the reaction rate, expresed as millimoles per minute per gram. column (Table 2). However, this activity was separated from cytidine monophosphate kinase activity after fractionation a.greement with previous reports (7, lo), we were not able to of cell-free extracts of U. urealyticum 960T by gel filtration. detect either of these activities in extracts or fractionated Nucleoside diphosphate kinase activity also co-eluted from extracts of M. mycoides. No dCMP deaminase was detected the Mono Q column with cytidine monophosphate kinase in the fractionated extracts from U. urecllyticum, but it was and adenylate kinase activities (Table 2), but we consider it found in the crude sonication supernatant (Table 2). unlikely that this activity is a function of either of those

1’ I I I I I I

0

i, U

FRACTION NUMBER FIG. 2. Fractionation of some enzymes of nucleotide metabolism from U.urealyticum. An extract of U.urealyticum was applied to a Mono Q anion exchange column which was then developed with 30 ml of a linear gradient of 0 to 1.0 M NaCl buffered with 5 mM Tris (pH K5). Collection of fraction 2 commenced with the start of the gradient. Fractions were assayed for enzymic activities as described in the text. (a) Symbols: A, dC kinase; A, cytidine kinase; 0, cell protein. (b) Symbols: 0, adenosine triphosphatase; 0,cytidine deaminase; 0, dC cleaminase; 0,dUTPase; m, dUTPase + 200 pM ATP; A, dCTPase; A,dCTPase + 200 pM ATP. (c) Symbols: 0,dAMP phosphatase; m, clGMP phosphatase; A,dCMP phosphatase; A, dTMP phosphatase; 0, dUMPase. The dotted line indicates NaCl concentration. VOL. 38, 1988 NUCLEOTIDE METABOLISM IN MYCOPLASMATACEAE 277

TABLE 2. Enzyme activities in chromatographic fractions tested for M. mycoides and U. urealyticurn (Table 3). The from extracts of U. urealyticum data showed that most activity on was in the Sp act (p,moVmin membrane fraction of U.urealyticum and illustrated the very Enzyme activity" Fraction no.b per g of cell high level of activity in extracts from this species compared protein)" with extracts from M. mycoides. With M. mycoides the strongest activity was clearly against dUMP and was equally Cytidine kinase 10 0.24 dC kinase 17 0.47 distributed between membrane and cytoplasmic fractions. Thymidine kinase 13 0.34 Studies on the activities in both fractions for both organisms dTMP kinase 10 0.2 showed competition between the various nucleotides and Cytidine monophosphate kinase 15 2.0 gave no evidence for a specific dUMPase activity. Fraction- dCMP kinase 15 0.23 ation of sonication supernatants from M. mycoides on a Adenylate kinase 15 71 Superose 12 column showed phosphatase activity against Nucleoside diphosphate kinase 15 1.1 dUMP, dTMP, dGMP, and dAMP, co-eluting over the range Uracil PRTase 11 2.6 from 12 to 13.5 ml. The activity against dAMP was only Adenine PRTase 10 1.5 about one-quarter of that against the other three nucleotides, Hypoxanthine PRTase -d 0.36 Uridine phosphorylase 10 0.74 and no activity was observed against dCMP. Adenosine phosphorylase 21 5.3 Thymidine phosphorylase 1 8.1 DISCUSSION Cytidine deaminase 16 0.8 Our results indicate that U.urealyticum 960T, a member of dC deaminase 16 1.8 the second genus of the Mycoplasmataceae, has character- dCMP deaminase - 2.9 dCTPase 25 2.3 istics similar to those observed for other species in this dUTPase (100 pM dUTP) 25 1.9 family tested by Williams and Pollack (18) in that it (i) does dUTPase (10 pM dUTP) 25 0.22 not show a dUTPase, (ii) does possess dC deaminase, (iii) dAMP phosphatase 28 2.6 does not show a specific dUMPase, and (iv) does possess dGMP phosphatase 28 3.1 dCMP deaminase. dCMP phosphatase 28 2.7 Overall, the enzymic activities detected in U.urealyticum dTMP phosphatase 20, 24, 28, 30 4.9 960T, including PRTases for uracil, adenine, and hypoxan- dUMPase 24 0.9 thine, indicate a capacity for salvage pathways of nucleotide Adenosine triphosphatase 12, 13 0.7 biosynthesis similar to those proposed for M. mycoides

a Enzyme activities were assayed as described in Materials and Methods. subsp. mycoides strain Y (6-10) and for A. laidlawii B-PG9 Assays were performed on fractions isolated by column chromatography (16, 17). Consistent with such pathways, experiments ana- as described in Materials and Methods. The number(s) of the fraction(s) lyzing the distribution of label into ribonucleic acid and showing the highest activity is indicated. The value shown is the sum of the activities in all fractions expressed in deoxyribonucleic acid of U. urealyticum (A. Mitchell and F. relation to the total cell protein applied to the column. Brake, unpublished data) have confirmed that [2-14C]uracil -, Activity was measured in the crude sonication supernatant but was not added to the growth medium is a precursor of uridine, detected in any individual fractions. cytidine, and deoxycytidine nucleotides and that [8-14C]- adenine is a precursor of adenosine and nucleotides. enzyme proteins. The fractions showing dTMP kinase activ- M. mycoides subsp. mycoides strain Y is similar to the ity were without activity on dUMP as a substrate, indicating other members of the Mycoplasmataceae examined by similar properties for this enzyme in U. urealyticum, M. Williams and Pollack (18) and to U. urealyticum 960T in that mycoides (ll), and A. laidlawii B-PG9 (18). it does not show a specific dUMPase and does possess Very strong phosphatase activity on deoxynucleoside dCMP deaminase. However, it differs from these other monophosphates and o-nitrophenylphosphate was observed members of the Mycoplasmataceae in that it does possess an in Ureapfasma extracts. Figure 2c shows that the activity ATP-insensitive dUTPase and does not show a dC or cyti- against dAMP, dGMP, dCMP, and dTMP had a wide distri- dine deaminase activity. Both of these observations may be bution in the fractions eluted from Mono Q, as did the considered as examples to raise some concern about the activity against o-nitrophenylphosphate (data not shown); reliability of enzymatic activities of nucleotide metabolism however, activity against dUMP was distributed as a single as possible taxonomic criteria. Initial assays in our labora- broad peak. The dUMPase in fractions from the Mono Q tory did not detect dUTPase in M. mycoides extracts; column was completely inhibited by 4 mM adenosine mono- however, ultimately (10) this enzyme was demonstrated. phosphate. The distribution of nucleotide phosphatase activ- Repeated assays (7, 10; this study) have still not detected ities between cytoplasmic and membrane preparations was cytidine deaminase (or dC deaminase) in M. mycoides ex-

TABLE 3. Distribution of deoxynucleoside monophosphate phosphohydrolase activities between membrane and cytoplasmic fractions in osmotic lysis extracts of M. mycoides subsp. mycoides and U. urealyticurn Rate of product formation (pmoymin per g of total cell protein) Organism Fraction dTMP dGMP dCMP dAMP dUMPase phosphatase phosphat ase phosphatase phosphatase M. mycoides Membrane 6.0 3.9 2.0 0.5 0.1 Cytoplasm 6.1 3.2 1.3 Nil 1.1 U. urealyticum Membrane 154 221 250 117 154 Cytoplasm 6.9 2.6 Nil Nil Nil 278 COCKS ET AL. INT.J. SYST.BACTERIOL. tracts. However, labeling studies (6,9), studies on the use of Bacteriol. 130:1047-1054. rnethyl cytidine derivatives as sources of thymine (G. A. M. 7. Mitchell, A., and L. R. Finch. 1979. Enzymes of pyrimidine Neale, Ph.D. thesis, University of Melbourne, Parkville, metabolism in Mycoplasma mycoides subsp. mycoides. J. Bac- Australia, 1984), and unreported data on the effects of teriol. 137: 1073-1080. tetrahydrouridine, a cytidine deaminase inhibitor, all suggest 8. Mitchell, A., I. L. Sin, and L. R. Finch. 1978. Enzymes of that the activity exists in vivo. Many factors may affect the metabolism in Mycoplasma mycoides subsp. mycoides. J. Bac- detectability of an enzyme so that a negative result to an teriol. 134:706-712. enzyme assay is inconclusive. Enzymes of nucleotide me- 9. Neale, G. A. M., A. Mitchell, and L. R. Finch. 1983. Pathways of pyrimidine biosynthesis in Mycoplasma tabolism in unfractionated extracts can be particularly sus- mycoides subsp. mycoides. J. Bacteriol. 154:17-22. ceptible to lack of detection because of interference from 10. Neale, G. A. M., A. Mitchell, and L. R. Finch. 1983. Enzymes of strong competing reactions to remove substrates or products pyrimidine deoxyribonucleotide metabolism in Mycoplasrna or both. These inferfering reactions can often be extremely mycoides subsp. mycoides. J. Bacteriol. 156:lOOl-1005. rapid compared with the reactions under study. 11. Neuhard, J. 1983. Utilization of preformed pyrimidine bases and , p. 95-148. In A. Munch-Petersen (ed.), Metabo- ACKNOWLEDGMENTS lism of nucleotides, nucleosides and in microorga- This work was carried out with support from the Australian nisms. Academic Press, Inc. (London), Ltd., London. Research Grants Scheme and during the tenure of a Commonwealth 12. O’Brien, S. J., J. M. Simonson, S. Razin, and M. F. Barile. 1983. Postgraduate Research Award to B.G.C. On the distribution and characteristics of isozyme expression in Mycoplasma, Acholeplasma and Ureaplasma species. Yale J. LITERATURE CITED Biol. Med. 56:701-708. 1. Cocks, B. G., F. A. Brake, A. Mitchell, and L. R. Finch. 1985. 13. Randerath, K., and E. Randerath. 1964. Thin layer separation Enzymes of intermediary carbohydrate metabolism in methods for derivatives. Methods Enzymol. 12: Ureaplasma urealyticum and Mycoplasma mycoides subsp. 323-347. mycoides. J. Gen. Microbiol. 131:2129-2135. 14. Razin, S. 1983. Cell lysis and isolation of membranes, p. 225- 2. Cocks, B. G., and L. R. Finch. 1987. Characterization of a 233. In S. Razin and J. G. Tully (ed.), Methods in mycoplas- restriction endonuclease from Ureaplasma urealyticum 960 and mology, vol. 1. Academic Press, Inc., New York. differences in the deoxyribonucleic acid modification of human 15. Rodwell, A. W., J. E. Peterson, and E. S. Rodwell. 1975. Striated ureaplasmas. Int. J. Syst. Bacteriol. 37:451453. fibers of the rho form of Mycoplasma: in vitro reassembly, 3. Davis, J. W., P. Nelson, and R. Ranglin. 1984. Enzyme activities composition, and structure. J. Bacteriol. 122:1216-1229. contributing to hypoxanthine production in Ureaplasma. Isr. J. 16. Wentworth, D. F., and R. Wolfenden. 1978. Cytidine deami- Med. Sci. 20:946-949. nases from Escherichia coli and human liver. Methods En- 4. Ives, D. H., and S.-M. Wang. 1978. from z ymol. 51:401-407. calf thymus. Methods Enzymol. 51:337-345. 17. Williams, M. V., and J. D. Pollack. 1985. Pyrimidine deoxyri- 5. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. bonucleotide metabolism in Acholeplasma laidlawii B-PG9. J. 1951. Protein measurements with the Folin phenol reagent. J. Bacteriol. 161:1029-1033. Biol. Chem. 193:265-275. 18. Williams, M. V., and J. D. Pollack. 1985. Pyrimidine deoxyri- 6. Mitchell, A., and L. R. Finch. 1977. Pathways of nucleotide bonucleotide metabolism in members of the class Mollicutes. biosynthesis in Mycoplasma mycoides subsp. mycoides. J. Int. J. Syst. Bacteriol. 35227-230.