JOURNAL OF BACTERIOLOGY, Apr. 1978, p. 108e-114 0021-9193/78/0134-0108$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 134, No. 1
Printed in U.S.A.
Alkaline Phosphatase Possessing Alkaline Phosphodiesterase Activity and Other Phosphodiesterases in Bacillus subtilis KUNIO YAMANE* AND BUNJI MARUO Institute ofApplied Microbiology, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
Received for publication 21 July 1977
In Bacillus subtilis Marburg strain, single-point mutations in the phoP locus brought about simultaneous losses of the major activities of alkaline phosphatase (APase) and alkaline phosphodiesterase (APDase). Revertants recovered the two activities. APases with APDase activity were purified from the membrane fraction of B. subtilis 6160-BC6 and from the culture fluid of an APase-secreting B. subtilis mutant strain, RAN 1. In addition to these major APases with APDase activity, at least two kinds of phosphodiesterase (PDase) without phosphatase activity were found in the cytoplasmic supernatants of RAN 1 and an APase-less B. subtilis mutant strain, SP25. Another minor APase with a molecular weight of about 80,000, which had almost no PDase activity, was isolated from the membrane fraction of strain 6160-BC6. Enzyme distribution in subcellular fractions from various strains cultured in high- and low-phosphate media was analyzed. The PDases did not cross-react with rabbit antiserum against the RAN 1 APase with APDase activity. The main component of the PDases had a molecular weight of about 80,000 and was most active at pH 8.0. These results suggest that APase with APDase activity is different from PDases detected in cytoplasmic supernatants and that phoP is the structural gene for the phosphate-repressible APase with APDase activity. In Bacillus subtilis strain Marburg, the formation of alkaline phosphatase (APase) and that of phosphodiesterase (PDase) are closely related. The two activities are lowered in the presence of Pi and are simultaneously increased upon exhaustion of Pi in the culture medium (R. Takata, M. Tanifuji, Y. Hirota, and A. Tsugita, Jpn. J. Genet. 38:208, 1963). A single-point mutation event in the locus phoP brings about the simultaneous loss of APase and PDase activities (3). Furthermore, an APase-constitutive strain, B. subtilis 6160-BC6, is also constitutive for PDase (9). However, whether phoP is the structural gene for the APase or not has not been determined as yet (3,12), because APases and PDases were separately purified from cultures of Marburg strain, B. subtilis SB15 (7, 8), and B. subtilis 168 M (3), and they were reported to be structurally and immunologically unrelated to each other (3, 7, 8). The APase of B. subtilis is mainly located in and tightly attached to the cell membrane and is not easily brought into solution at low ionic strength (3, 7, 9). We have isolated a mutant, B. subtilis RAN 1, that produces an extracellular soluble APase (10). The PDase of the mutant was also an extracellular soluble enzyme. An APase (APase-Rl) purified as a homogeneous material from the culture fluid of RAN 1 con-
tained PDase activity at the same time. We also purified an insoluble APase (APase-BC6) with a PDase activity from the membrane preparation of strain 6160-BC6. These PDase activities, with pH optima of 9.5, were tentatively named alkaline PDase (APDase) activities. APase and APDase activities of APase-Rl, as well as those of APase-BC6, behaved similarly against inhibitors, ethylenediaminetetraacetate, metals, and other parameters. We suggested from these results that the two enzyme activities in these preparations were contained by the same enzyme proteins or by the same active sites of the enzyme proteins (9). In this paper, we describe physiological, immunological, and genetic evidence that APase and APDase activities are found in the same protein. We also show that APases with APDase activity are unrelated to other PDases localized in the cytoplasmic supernatant of the Marburg strain.
MATERILS AND METHODS Organisms and media. B. subtilis 6160 (purB6 trpB3 metB5) is a derivative of B. subtilis Marburg 168. An APase-constitutive strain, 6160-BC6, and a mutant, RAN 1, producing extracellular soluble APase and derived from 6160-BC6 were described in our previous papers (9, 10). APase-less B. subtilis mutants
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VOiL. 134, 1978
Vll, V12, and SP25 (phoP argA Strr) were obtained from Y. Ikeda (Institute of Applied Microbiology, The University of Tokyo, Tokyo, Japan). Mutational sites in the APase-less mutants are very closely linked to each other and are distributed within the putative structural gene (phoP) for APase. The mutational site(s) of 6160-BC6 for the constitutive production of APase is not linked to phoP (4). The composition of high-phosphate (HP) medium, containing 2.2 mM KH2PO4, was described in our previous papers (9, 10). Low-phosphate (LP) medium had the same composition as HP medium except that it contained 0.2 mM KH2PO4. Assay of APase, APDase, and PDase activities. APase activity against p-nitrophenyl phosphate (NPP) and APDase activity against bis(p-nitrophenyl)phosphate (bisNPP) were assayed at 400C in 0.1 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer, pH 9.5 (designated NPPase-9.5 and bisNPPase-9.5 activities, respectively) (9). PDase activity at pH 7.5 (designated bisNPPase-7.5 activity) was assayed under the same conditions as those of the APase assay, except that 0.1 M Tris-hydrochloride, pH 7.5, containing bisNPP (0.2 mg/ml) was used and the reaction was terminated by adding a mixture of 13% K2HPO4 and 2% KOH. p-Nitrophenol liberated was measured by the absorbance at 420 nm. One unit of enzyme activity is defined as that amount of enzyme preparation that liberated 1 nmol of p-nitrophenol per min. NPPase-9.5 and bisNPPase-9.5 activities in washed cells were assayed after the cells were treated with toluene. Assay of APase and APDase on agar plates. Filter-paper disks soaked with NPP or bisNPP (2.0 mg/ml of 0.2 M Tris-hydrochloride, pH 9.5) were pressed down on colonies growing on agar plates. Colonies that gave yellow color instantly were judged as positive for enzyme activity. Isolation of enzyme preparations. Isolation of membrane-bound insoluble APase (APase-BC6) from the membrane preparation of 6160-BC6 and of the extracellular soluble APase (APase-Rl) from the culture fluid of RAN 1 has been described (9). A minor APase with a molecular weight of 80,000 (APase 80,000) with very low APDase activity was extracted from a membrane preparation of 6160-BC6 with a mixture of 1 M Tris-hydrochloride, pH 7.5, and 1 M MgCl2 containing 2 mM calcium acetate-0.1 mM cobalt chloride (Ca-Co), and it was partially purified by hydroxylapatite-Celite 545 (1:1, wt/wt) column chromatography and by gel filtration on a Sephadex G-200 column equilibrated with a mixture of 0.1 M Trishydrochloride (pH 7.5), 0.2 M MgSO4 7H20, and 1.2 M KCI containing Ca-Co. Fractionation of the ceils. The media incubated at 30°C for 24 h were separated into extracellular fluid and cells by centrifugation at 1,000 x g for 20 min. The ultracentrifugal supernatant was the supernatant of the extracellular fluid obtained by centrifugation at 60,000 x g for 60 min. The cells were washed and suspended in the original volume of 10 mM Tris-hydrochloride, pH 7.5, containing Ca-Co (washed cells). Protoplast lysate in 10 mM Tris-hydrochloride, pH 7.5, containing Ca-Co was prepared after protoplasts were formed by treatment with lysozyme (200 ug/ml)
APase AND PDases IN B. SUBTILIS
109
in 0.1 M Tris-hydrochloride, pH 7.5, containing 0.4 M sucrose and Ca-Co, and the protoplasts were precipitated by centrifugation at 10,000 x g for 20 min. The supernatant and pellet of the protoplast lysate obtained by centrifugation at 60,000 x g for 60 min were termed the cytoplasmic supernatant and membrane, respectively. Procedure of transformation. Transforming DNA was prepared by the phenol (pH 9.0) method of Saito and Miura (6). Transfornation experiments were carried out according to the method of Yoshikawa (11). Chemicals. Lysozyme (egg white) was obtained from Seikagaku Kogyo Co. Ltd. (Tokyo, Japan), NPP disodium salt was from Wako Pure Chemicals Co. (Osaka, Japan), bisNPP sodium salt was from Daiichi Fine Chemicals Co. (Tokyo, Japan), 2'-deoxythymidine-3'-NPP and 2'-deoxythymidine-5'-NPP were from Boehringer Mannheim GmbH, Biochemica (Mannheim, West Germany), and Sephadex G-200 was from Pharmacia Fine Chemicals Inc. (Uppsala, Sweden). All other chemicals were of reagent grade.
RESULTS
Production and localization of APase and PDase activities. The activities to hydrolyze NPP at pH 9.5 (NPPase-9.5) and to hydrolyze bisNPP at pH 9.5 (bisNPPase-9.5) of the parental 6160 and 6160-BC6 and of various mutants either starved for phosphate or with high concentrations of phosphate were compared. The cells were cultured at 30°C in LP or HP medium for 24 h. APase activity reached a maximum at this time. Table 1 shows the results. Differential centrifugation of cultured media revealed that more than 90% of the N,PPase-9.5 activities of 6160 cultured in LP medium and of 6160-BC6 cultured in both LP and HP media were present in the membrane fractions. Most of NPPase-9.5 activity in the extracellular fractions from these strains was sedimented by centrifugation at 60,000 x g for 60 min. BisNPPase-9.5 activity was found to be similarly localized. On the other hand, a mutant strain, RAN 1, secreted NPPase9.5 and bisNPPase-9.5 activities into the ultracentrifugal supernatant. In the APase-less mutant Vll, almost no NPPase-9.5 activity was detected, but a considerable amount of bisNPPase-9.5 activity was observed in all subcellular fractions, among which it was highest in the cytoplasmic supernatant. Essentially the same results were obtained with two other APase-less mutants, V12 and SP25 (data not shown). Comparison of bisNPPase-9.5 activities in the cytoplasmic supematants of the four strains, in which no or quite a small amount of NPPase-9.5 was present, showed that a similar amount of bisNPPase-9.5 was present in each strain cultured in each medium. Higher activity was found in the cells
110
YAMANE AND MARUO
J. BACTERIOL.
TABLE 1. Fractional localization of NPPase-9.5 and bisNPPase-9.5 activities in parental strains and mutantsa NPPase-9.5 activity (U/ml) in: Extracellular fluid
Strain
Culture medium
UltraUnfractionated
cen-
trifugal supema-
BisNPPase-9.5 activity (U/ml) in:
Extracellular fluid
Washed cells
Unfractionated
tan
CytoCell lysate
laspr
super-
ntnt 13.7 0.4 1.0 0.3 50.0 0.7 24.8 0.7 2.9 0.2 0.8 0.2 0.1 0.0 0.7 0.0
1.8 0.5 15.5 LP HP 0.5 0.4 1.8 0.6 45.7 LP 4.2 6160-BC6 HP 3.5 0.7 29.5 LP 19.9 19.2 3.1 RAN 1 HP 20.3 19.4 2.2 LP 0.0 0.0 0.1 Vi 0.1 0.1 1.3 HP a Cells were cultured in LP or HP medium at in the text.
6160
Membrane
300C
Unfractionated
Ultracentrifugal sutan
Washed cells
UnCytofrac- Cell ly- ml. Memtionsate spr brane super-
tantntn
4.2 28.1 22.0 2.9 20.2 0.8 3.0 3.1 9.7 9.0 7.9 1.6 48.0 7.8 5.3 68.7 66.0 1.0 57.1 26.7 6.9 2.8 50.6 47.9 8.8 37.8 2.8 28.8 28.7 16.0 14.7 1.6 10.0 0.6 35.9 34.8 12.9 9.3 8.5 1.6 0.0 1.2 5.0 4.4 3.1 0.8 _b 1.0 3.1 7.3 6.5 6.9 1.4 for 24 h. Subcellular fractions were obtained as described 13.0
5.6
b-, Not determined.
cultured in HP medium than in those cultured in LP medium. These results raised the possibility that at A. least two kinds of PDase are present in B. subs X tilis; one of them behaves the same as NPPase. 1.0 9.5, which would be APase with APDase activity, relative to synthesis and location, and the no Xother is localized mainly in the cytoplasmic suthe production of which is regulated E.pernatant, c 0. from that of APase. independently OJ(B)| Il Relation of enzymes that show APase . . and PDase activities. To understand the molecular relationship between APase and PDase w activities found in various fractions of B. subtilis, z the molecular sizes of the enzymes responsible C.S . . m : : these activities were examined. A culture ^(Cfor 54 * s o ° o. \ ~~~~fluid ofRAN 1 and the cytoplasmic supematants of RAN 1 and SP25 were prepared. RAN 1 was . C cultured in HP medium containing 0.3 M NaCl, 2.0 ( C ) z l l and SP25 was cultured in HP medium at 30°C 4l 57 for 24 h. The three preparations were subjected to gel filtration on a Sephadex G-200 column (1.2 by 85 and cm). bisNPPase-9.5 Gel filtrationactivities, behavior and of 1.0 NPPase-9.5 the activity of the preparations to hydrolyze bisNPP at pH 7.5 (bisNPPase-7.5), are shown in . :~ ~ .JL.4 i~ 1. Main peaks of bisNPPase-7.5 activities in 0.0 Fig. 1.36 1.13 1.59 1.82 2.05 Vel Vo hydrochloride, pH 7.5, containing 0.3 M KCI and CaCo. Fractions of 3.2 ml each were collected. NPPaseFIG. 1. Gel filtration behavior of NPPase-9.5, 9.5 (0), bisNPPase-9.5 (0), and bisNPPase-7.5 (A) bisNPPase-9.5, and bisNPPase-7.5 activities in the activities were determined in each tube. Arrows incytoplasmic supernatants ofSP25 (A) and RAN 1 (B) dicate the eluted positions of the molecular weight and in the extracellular fraction of RAN 1 (C) on a markers ([a] rabbit muscle aldolase, 158,00(); [bl Sephadex G-200 column. The three preparations were bovine serum albumin, 67,000; [cl ovalbumin, 45,000; successively chromatographed over a column (1.2 by [dl chymotrypsinogen A, 25,000; and [el cytochrome 85 cm) equilibrated and eluted with 10 mM Tris- c, 12,50o).
2.0 (A)
a
i.c B
b J,
c
t,
d
I
e
APase AND PDases IN B. SUBTILIS
VOL. 134, 1978
the cytoplasmic supernatants of SP25 and RAN 1 were equally eluted at fractions around Ve/Vo = 1.59. Their molecular weight was about 80,000. The presence of at least two peaks in the elution curves of SP25 and RAN 1 suggested the presence of more than one enzyme with bisNPPase7.5 activity in the preparations. A small amount of bisNPPase-9.5 activity detected in parallel with bisNPPase-7.5 activity was most likely due to PDases with optima of pH 8.0, since PDases had approximately 50 to 60% of the activity at pH 9.5 (Fig. 2). NPPase-9.5 activity was not detected in any fraction (Fig. 1A and B). Fractions around the bisNPPase-7.5 activity peaks (Ve/Vo = 1.54 to 1.64) were pooled and used in the next experiments. They were tentatively named PDase-SP25 and PDase-Rl. On the other hand, NPPase-9.5 and bisNPPase-9.5 activities of the culture fluid of RAN 1 were eluted at the same time from the column with a single peak (Fig. 10). The elution position of this peak was equivalent to a molecular weight of approximately 45,000 (Ve/Vo = 1.8). As described in the accompanying paper (9), we purified an extraceilular soluble enzyme (APase-Rl) with APase and APDase activities from the culture fluid of RAN 1, and an insoluble enzyme (APase-BC6) with APase and APDase activities from a membrane fraction of B. sub-
111
TABLE 2. Substrate specificity ofAPase and PDase preparationsa pH of Enzyme
Substrate specificity
reac-
NPP bisNPP T3'NPP T5'NPP ture 30 20 130 9.5 100 APase-BC6 11 4.8 15 100 9.5 APase-80,000 21 30 140 9.5 100 APase-Rl 9.4 5.4 0 100 7.5 PDase-Rl 6.8 3.8 0 100 7.5 PDase-SP25 a Reactions were conducted at 40°C for 10 and 20 min and were terminated by adding a mixture of 13% K2HPO4 and 2% KOH. Then, liberatedp-nitrophenol was measured at 420 nm. Each substrate was tested at an initial concentration of 0.5 mM. Rates of p-nitrophenol liberated were expressed relative to NPP in APase preparations (relative ratio = 100) and to bisNPP in PDase preparations (relative ratio = 100). Abbreviations: T3'NPP, 2'-deoxythymidine-3'-NPP; T5'NPP, 2'-
prepn
mix-
deoxythymidine-5'-NPP.
tilis 6160-BC6 as homogeneous materials. The pH optima for NPP-hydrolyzing activity of APase-Rl and APase-BC6 were both at 9.5, and they agreed with those for bisNPP-hydrolyzing activity (9). In contrast, the pH optima for bisNPP-hydrolyzing activity of PDase-SP25 and PDase-Rl were at 8.0, and no NPP-hydrolyzing activity was detected at any pH (Fig. 2). Substrate specificity of the enzyme preparations was analyzed by usingp-nitrophenyl derivatives. Each substrate was tested at an initial 0.75 1( a ) PDase-SP25 concentration of 0.5 mM. Table 2 shows the relative activities of the enzymes on the four iXA. substrates. APase-Rl and APase-BC6 hydrolyzed the four substrates. The hydrolyzing activ0.50. ities of the two enzymes against each of the four substrates were almost equal. PDase-SP25 and PDase-Rl hydrolyzed bisNPP, 2'-deoxythymi2 0.255 . dine-3'-NPP, and 2'-deoxythymidine-5'-NPP, but not NPP. Table 2 also includes the substrate specificity of another APase (APase-80,000) that x 0.0 f~ * had been isolated from the membrane fraction of 6160-BC6. This preparation showed at least three protein bands in sodium dodecyl sulfate < 0.5CD. discontinuous gel electrophoresis, and the moA6 lecular weight of the major band was approximately 46,000. This phosphatase had very low 0.25 bisNPP-hydrolyzing activity. APase-80,000 was also insoluble in solutions of low ionic strength. The relationship between APase-BC6 and APase-80,000 is not yet clear. OD 1 To test the possible structural relation of APase-Rl, and RAN 1 bisNPPaseAPase-BC6, FIG. 2. PDase activity pH optima for PDase-SP25 antiserum against APase-Rl was rabbit a 7.5, and PDase-Rl. Symbols: solid line, phosphatase activity; dashes, PDase activity; A, 0.1 M acetic acid- prepared. The method of preparation of this sodium acetate buffer containing Ca-Co (pH 3.2 to serum will be published elsewhere. The ultra6.5); 0, 0.1 M Tris-hydrochloride buffer containing centrifugal and cytoplasmic supernatants were Ca-Co (pH 7.0 to 9.5); *, 0.1 M glycine-sodium hy- prepared from RAN 1 cultures with LP and HP droxide buffer containing Ca-Co (pH 8.0-11.0). media, as described in Materials and Methods. E
c
.4
3
5
7
pH
9
112
J. BACTrERIOL.
YAMANE AND MARUO
The NPPase-9.5 and bisNPPase-9.5 activities in vertants of V12 were cultured in LP medium at the ultracentrifugal supernatants of RAN 1 and 30°C for 24 h. Their productivity of the major in APase-BC6 were equally well neutralized by membraneous APase and APDase was meathe serum, whereas bisNPPase-7.5 activities in sured. Representatives are shown in Table 5. the cytoplasmic supernatants were not (Table They produced the two enzyme activities in 3). Also, PDase-SP25 and PDase-Rl prepara- ratios similar to that of 6160. These results tions did not cross-react with the serum (data showed that the loss in the major membraneous not shown). These results suggested that APase- APase and APDase activities was induced by a Rl and APase-BC6, which possessed both single-point mutational event. NPPase-9.5 and bisNPPase-9.5 activities, are DISCUSSION structurally unrelated to PDases localized in the cytoplasmic supernatants. Extracellular soluble APase-Rl and memGenetic evidence for the presence of ma- brane-bound insoluble APase-BC6, which carjor membraneous APase and APDase ac- ried both APase and APDase activities, were tivities in a single enzyme molecule. To test essentially identical in optimum pH, substrate whether locus phoP is the structural gene for specificity, and immunological property, althe major APase and APDase, reversion of the though the enzyme preparations were absolutely two activities in APase-less mutants with differ- different in their solubility in solutions of low ent lesions within phoP was studied by transfor- ionic strength. These major APase and APDase mation experiments. The phenotypical APase- activities would be measured, resp9ctively, as and APDase-producing characters (APase+ and APDase+, respectively) of RAN 1 and 6160-BC6 TABLE 4. Cotransformation of APase+ and were transferred into V12 and SP25 (APasein APase-less mutants SP25 and V12 by APDase+ APDase- argA Strr) by DNA-mediated transDNAs from RAN 1 and 6160-BC6' formation (Table 4). Arg+ transformants were No. DNA donor DNA re- No. of selected. Three hundred transformants in each cipient . + APase+ cross were transferred on LP medium-agar APase+/ (APaset strain trargn on LP APDase plates, and then APase+ and APDase+ trans(APaseAPDase+ fomantsmediumformants were scored among them. Every airA)W) APDase- tested rmant pae argA) APase+ transformant on the plates was also 71 300 V12 APDase+. Spontaneous APase+ revertants were RAN 1 71/71 79 300 SP25 79/79 isolated from V12 on LP medium-agar plates RAN 1 101 300 101/101 with a reversion frequency of approximately 6160-BC6 V12 300 87 87/87 10'. The 16 APase+ revertants isolated were 6160-BC6 SP25 a also APDase+ on the plates. Ninety APase+ 1 High concentrations (about ,ug of DNA per ml) transformants of V12 and SP25 and the 16 re- were used for each transformation experiment. TABLE 3. Neutralization of NPPase-9.5, bisNPPase-9.5, and bisNPPase-7.5 in the ultracentrifugal and cytoplasmic supernatants of RAN ) and in APase-BC6 by rabbit antiserum against APase-Rla Enzyme activity in: Medium and treatment
Ultracentrifugal supernatant
Cytoplasmic supernatant
APase-BC6b
NPPase-9.5
NPPase-9.5 BisNPPase7.5
NPPase-9.5 BisNPPase-9.5
BisNPPase-9.5
LP medium None 100 __d 100 NDc 100 + Antiserum (1:20) 3.1 9.8 92 + Antiserum (1:100) 1.9 6.9 101 HP medium None 100 100 ND 100 100 100 + Antiserum (1:20) 1.0 3.7 99 9.3 8.2 + Antiserum (1:100) 0.8 3.0 100 2.8 1.6 a The preparations were mixed with the antiserum against APase-Rl diluted to 1:20 or to 1:100 with 10 mM Tris-hydrochloride, pH 7.5, containing 0.9% NaCI and Ca-Co, and immunological reaction was allowed to proceed for 1 h at room temperature. After the mixtures were centrifuged at 1,000 x g for 15 min, the enzymatic activities in the supernatants were assayed. b APase-BC6 was dissolved in a buffer of 1 M Tris-hydrochloride-2 M MgCl2-Ca-Co, pH 7.0. c ND, Not detected. d _, Not determined.
APaSe AND PDases IN B. SUBTILIS
VOL. 134, 1978
TABLE 5. Production of APase and APDase by, and ratio of the two enzyme activities
(APDase/APase) in, representative APase+ transformants and revertants of APase-less mutant V12a Enzyme activity (U/ml)
Strain APase
Transformants 1 2 3 4 V12 revertants 1 2 3 4
33 54 41 62
APDase
46 63 55 93
APDase/ APase
1.4 1.2 1.3 1.5
67 91 1.4 74 98 1.3 95 1.4 69 53 69 1.3 104 1.2 85 5 40 47 1.2 6160 0.1 3 V12 _b 0.1 3 SP25 a for 24 LP medium at 300C in cultured were Cells h. Transformants 1, 2,3, and 4 were the representative transformants of the crosses RAN 1 x SP25, RAN 1 x V12, 6160-BC6 x SP25, and 6160-BC6 x V12, re-
spectively. bh, Not calculated.
NPPase-9.5 and bisNPPase-9.5 activities in the subcellular fractions of the culture media (Tables 1 and 3). On the other hand, the bisNPPase9.5 activity found in the cytoplasmic supernatants should be due to other PDases, because the optimum pH, substrate specificity, and immunological property of their main activity were different from those of APase-Rl and APaseBC6. The PDase activity was most likely due to PDases with an optimum pH of 8.0. These results suggest that the major PDase activity, which was produced in Pi-deficient medium and was lost in APase-less mutants, was included in the phosphate-repressible APase but was different from enzymes with PDase activity localized in the cytoplasmic supernatant. No structural relationship between the two groups of enzymes could be detected by the immunological study. APase-constitutive mutants were also constitutive for APDase, and when the mutation(s) in B. subtilis 6160-BC6 responsible for the constitutive production of APase was transferred into other strains by the DNA-mediated transformation, constitutive production of the two enzyme activities was cotransferred (unpublished data). The simultaneous loss of APase and APDase characters would be caused by a singlepoint mutation in locus phoP, because APase+ strains isolated by transformation of V12 and SP25 and by spontaneous reversion of V12 were
113
also APDase+. Furthermore, the ratio of APase activity to APDase activity found in the APase+ strains was almost constant. These results suggest that phoP is the structural gene for APase with APDase activity. The notion that the locusphoP is the common regulator gene for the production of two different enzyme molecules, APase and APDase, is not likely, since APase-Rl and APase-BC6, which contained APase and APDase activities, were purified as homogeneous materials and since it was indicated that the two enzyme activities would be present in the same protein (9). The possibility, however, that the structural gene for APase with APDase activity is localized in another site of the locus phoP and that phoP regulates the production of an enzyme molecule that possesses both APase and APDase activities still remains to be tested. Formation of the major membraneous APase with APDase activity in B. subtilis would be regulated by at least two kinds of regulator genes, as suggested by Miki et al. (4). One of the putative regulator genes is phoRI, which is closely linked to phoP, and the other is phoRII, which would not be linked to phoP or phoRI. These genes in B. subtilis would correspond to phoR, phoS, and others for APase synthesis in Escherichia coli (1, 5). The APase formation system in B. subtilis seems to resemble essentially that of E. coli. The APase of B. subtilis Marburg strain was previously purified by Takeda and Tsugita (7), Glenn and Mandelstam (2), and Le Hegarat and Anagnostopoulos (3). The purified preparations were reported to have mainly NPP-hydrolyzing activity and little or no bisNPP-hydrolyzing activity. These authors extracted the enzyme from membrane preparations with a 1 M Mg2+ solution. We noticed that solubilization of the major APase from a membrane preparation of 6160BC6 was difficult. We also partially purified an APase (APase-80,000) that had properties similar to the preparations obtained by Takeda and Tsugita (7) and Le Hegarat and Anagnostopoulos (3), but the amount of that APase was quite small. It is possible that the poor bisNPP-hydrolyzing activity of APase-80,000 was induced by the dimerization of APase-BC6 (unpublished data). PDase(s) in the cytoplasmic supernatant of B. subtilis Marburg strain was purified by Taniguchi and Tsugita (8) and by Le Hegarat and Anagostopoulos (3). The preparation of Le Hegarat and Anagnostopoulos (3), which had a molecular weight of 150,000, seems to be different from the main PDase(s) in the cytoplasmic supernatants of SP25 and RAN 1. The molecular
114
YAMANE AND MARUO
weights of PDase-Rl and PDase-SP25 were around 80,000 (Fig. 1). This difference could be due to cultural conditions; SP25 and RAN 1 were cultured in HP medium. These minor PDases did not hydrolyze NPP and had no structural relation to APase-Rl or APase-BC6. At least two kinds of PDase were demonstrated in the cytoplasmic supernatants of SP25 and RAN 1 by gel filtration on a Sephadex G-200 column (Fig. 1). Taniguchi and Tsugita (8) reported the presence of three kinds of PDase from diethylaminoethyl-cellulose column chromatography and showed that one of them had ribonuclease activity. Synthesis of these PDases in the cytoplasmic supernatant seemed to be regulated independently by the concentration of Pi in the culture medium. The amount of PDase activity in the preparations was higher when cultured in HP medium than when cultured in LP medium (Table 1). PDases might participate in the intracellular metabolism of phosphates when the cells are allowed to utilize sufficient amounts of Pi from the outside medium. ACKNOWLEDGMENTS This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science and Culture, Japan. We thank I. Shibuya for helpful discussion and Y. Ikeda for a gift of APase-less mutants of B. subtiis. LITERATURE CITED 1. Echols, H., A. Garen, S. Garen, and A. Torrian. 1961. Genetic control of repression of alkaline phosphatase in E. coli. J. Mol. Biol. 3:425-438.
J. BACTERIOL. 2. Glenn, A. R., and J. Mandelstam. 1971. Sporulation in Bacillus subtilis 168: comparison of alkaline phosphatase from sporulating and vegetative cells. Biochem. J. 123:129-138. 3. Le Hegarat, J.-C., and C. Anagnostopoulos, 1973. Purification, subunit structure and properties of two repressible phosphohydrolases of Bacillus subtilis. Eur. J. Biochem. 39:525-539. 4. Miki, T., Z. Minami, and Y. Ikeda 1965. The genetics of alkaline phosphatase formation in Bacillus subtilis. Genetics 52:1093-1100. 5. Morris, H., M. J. Schlesinger, M. Bracha, and E. Yagil. 1974. Pleiotropic effects of mutations in-olved in the regulation of Escherichia coli K-12 alkaline phosphatase. J. Bacteriol. 119:583-592. 6. Saito, H., and K. Miura. 1963. Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta 72:619-629. 7. Takeda, K., and A. Tsugita. 1967. Phosphoesterases of Bacillus subtilis. II. Crystallization and properties of alkaline phosphatase. J. Biochem. (Tokyo) 61:231-241. 8. Taniguchi, K., and A. Tsugita. 1966. Phosphoesterases of Bacillus subtilis. I. Purification and properties of phosphodiesterases. J. Biochem. (Tokyo) 60:372-380. 9. Yamane, K., and B. Maruo. 1978. Purification and characterization of extracellular soluble and membranebound insoluble alkaline phosphatases possessing phosphodiesterase activities in Bacillus subtilis. J. Bacteriol. 134:100-107. 10. Yamane, K., T. Miki, H. Saito, Y. Ikeda, and B. Maruo. 1976. Isolation of a mutant secreting extracellular soluble alkaline phosphatase in BaciUus subtilis. Agric. Biol. Chem. 40:2181-2185. 11. Yoshikawa, H. 1970. Temperature-sensitive mutants of Bacillus subtilis. I. Multiforked replication and sequential transfer of DNA by a temperature-sensitive mutant. Proc. Natl. Acad. Sci. U.S.A. 65:206-213. 12. Young, F. E., and G. A. Wilson. 1975. Chromosomal map of Bacillus subtilis, p. 596-614. In P. Gerhardt, R. N. Costilow, and H. L. Sadoff (ed.), Spores VI. American Society for Microbiology, Washington, D.C.
Vol. 134, No. 1
Printed in U.S.A.
Alkaline Phosphatase Possessing Alkaline Phosphodiesterase Activity and Other Phosphodiesterases in Bacillus subtilis KUNIO YAMANE* AND BUNJI MARUO Institute ofApplied Microbiology, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
Received for publication 21 July 1977
In Bacillus subtilis Marburg strain, single-point mutations in the phoP locus brought about simultaneous losses of the major activities of alkaline phosphatase (APase) and alkaline phosphodiesterase (APDase). Revertants recovered the two activities. APases with APDase activity were purified from the membrane fraction of B. subtilis 6160-BC6 and from the culture fluid of an APase-secreting B. subtilis mutant strain, RAN 1. In addition to these major APases with APDase activity, at least two kinds of phosphodiesterase (PDase) without phosphatase activity were found in the cytoplasmic supernatants of RAN 1 and an APase-less B. subtilis mutant strain, SP25. Another minor APase with a molecular weight of about 80,000, which had almost no PDase activity, was isolated from the membrane fraction of strain 6160-BC6. Enzyme distribution in subcellular fractions from various strains cultured in high- and low-phosphate media was analyzed. The PDases did not cross-react with rabbit antiserum against the RAN 1 APase with APDase activity. The main component of the PDases had a molecular weight of about 80,000 and was most active at pH 8.0. These results suggest that APase with APDase activity is different from PDases detected in cytoplasmic supernatants and that phoP is the structural gene for the phosphate-repressible APase with APDase activity. In Bacillus subtilis strain Marburg, the formation of alkaline phosphatase (APase) and that of phosphodiesterase (PDase) are closely related. The two activities are lowered in the presence of Pi and are simultaneously increased upon exhaustion of Pi in the culture medium (R. Takata, M. Tanifuji, Y. Hirota, and A. Tsugita, Jpn. J. Genet. 38:208, 1963). A single-point mutation event in the locus phoP brings about the simultaneous loss of APase and PDase activities (3). Furthermore, an APase-constitutive strain, B. subtilis 6160-BC6, is also constitutive for PDase (9). However, whether phoP is the structural gene for the APase or not has not been determined as yet (3,12), because APases and PDases were separately purified from cultures of Marburg strain, B. subtilis SB15 (7, 8), and B. subtilis 168 M (3), and they were reported to be structurally and immunologically unrelated to each other (3, 7, 8). The APase of B. subtilis is mainly located in and tightly attached to the cell membrane and is not easily brought into solution at low ionic strength (3, 7, 9). We have isolated a mutant, B. subtilis RAN 1, that produces an extracellular soluble APase (10). The PDase of the mutant was also an extracellular soluble enzyme. An APase (APase-Rl) purified as a homogeneous material from the culture fluid of RAN 1 con-
tained PDase activity at the same time. We also purified an insoluble APase (APase-BC6) with a PDase activity from the membrane preparation of strain 6160-BC6. These PDase activities, with pH optima of 9.5, were tentatively named alkaline PDase (APDase) activities. APase and APDase activities of APase-Rl, as well as those of APase-BC6, behaved similarly against inhibitors, ethylenediaminetetraacetate, metals, and other parameters. We suggested from these results that the two enzyme activities in these preparations were contained by the same enzyme proteins or by the same active sites of the enzyme proteins (9). In this paper, we describe physiological, immunological, and genetic evidence that APase and APDase activities are found in the same protein. We also show that APases with APDase activity are unrelated to other PDases localized in the cytoplasmic supernatant of the Marburg strain.
MATERILS AND METHODS Organisms and media. B. subtilis 6160 (purB6 trpB3 metB5) is a derivative of B. subtilis Marburg 168. An APase-constitutive strain, 6160-BC6, and a mutant, RAN 1, producing extracellular soluble APase and derived from 6160-BC6 were described in our previous papers (9, 10). APase-less B. subtilis mutants
108
VOiL. 134, 1978
Vll, V12, and SP25 (phoP argA Strr) were obtained from Y. Ikeda (Institute of Applied Microbiology, The University of Tokyo, Tokyo, Japan). Mutational sites in the APase-less mutants are very closely linked to each other and are distributed within the putative structural gene (phoP) for APase. The mutational site(s) of 6160-BC6 for the constitutive production of APase is not linked to phoP (4). The composition of high-phosphate (HP) medium, containing 2.2 mM KH2PO4, was described in our previous papers (9, 10). Low-phosphate (LP) medium had the same composition as HP medium except that it contained 0.2 mM KH2PO4. Assay of APase, APDase, and PDase activities. APase activity against p-nitrophenyl phosphate (NPP) and APDase activity against bis(p-nitrophenyl)phosphate (bisNPP) were assayed at 400C in 0.1 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer, pH 9.5 (designated NPPase-9.5 and bisNPPase-9.5 activities, respectively) (9). PDase activity at pH 7.5 (designated bisNPPase-7.5 activity) was assayed under the same conditions as those of the APase assay, except that 0.1 M Tris-hydrochloride, pH 7.5, containing bisNPP (0.2 mg/ml) was used and the reaction was terminated by adding a mixture of 13% K2HPO4 and 2% KOH. p-Nitrophenol liberated was measured by the absorbance at 420 nm. One unit of enzyme activity is defined as that amount of enzyme preparation that liberated 1 nmol of p-nitrophenol per min. NPPase-9.5 and bisNPPase-9.5 activities in washed cells were assayed after the cells were treated with toluene. Assay of APase and APDase on agar plates. Filter-paper disks soaked with NPP or bisNPP (2.0 mg/ml of 0.2 M Tris-hydrochloride, pH 9.5) were pressed down on colonies growing on agar plates. Colonies that gave yellow color instantly were judged as positive for enzyme activity. Isolation of enzyme preparations. Isolation of membrane-bound insoluble APase (APase-BC6) from the membrane preparation of 6160-BC6 and of the extracellular soluble APase (APase-Rl) from the culture fluid of RAN 1 has been described (9). A minor APase with a molecular weight of 80,000 (APase 80,000) with very low APDase activity was extracted from a membrane preparation of 6160-BC6 with a mixture of 1 M Tris-hydrochloride, pH 7.5, and 1 M MgCl2 containing 2 mM calcium acetate-0.1 mM cobalt chloride (Ca-Co), and it was partially purified by hydroxylapatite-Celite 545 (1:1, wt/wt) column chromatography and by gel filtration on a Sephadex G-200 column equilibrated with a mixture of 0.1 M Trishydrochloride (pH 7.5), 0.2 M MgSO4 7H20, and 1.2 M KCI containing Ca-Co. Fractionation of the ceils. The media incubated at 30°C for 24 h were separated into extracellular fluid and cells by centrifugation at 1,000 x g for 20 min. The ultracentrifugal supernatant was the supernatant of the extracellular fluid obtained by centrifugation at 60,000 x g for 60 min. The cells were washed and suspended in the original volume of 10 mM Tris-hydrochloride, pH 7.5, containing Ca-Co (washed cells). Protoplast lysate in 10 mM Tris-hydrochloride, pH 7.5, containing Ca-Co was prepared after protoplasts were formed by treatment with lysozyme (200 ug/ml)
APase AND PDases IN B. SUBTILIS
109
in 0.1 M Tris-hydrochloride, pH 7.5, containing 0.4 M sucrose and Ca-Co, and the protoplasts were precipitated by centrifugation at 10,000 x g for 20 min. The supernatant and pellet of the protoplast lysate obtained by centrifugation at 60,000 x g for 60 min were termed the cytoplasmic supernatant and membrane, respectively. Procedure of transformation. Transforming DNA was prepared by the phenol (pH 9.0) method of Saito and Miura (6). Transfornation experiments were carried out according to the method of Yoshikawa (11). Chemicals. Lysozyme (egg white) was obtained from Seikagaku Kogyo Co. Ltd. (Tokyo, Japan), NPP disodium salt was from Wako Pure Chemicals Co. (Osaka, Japan), bisNPP sodium salt was from Daiichi Fine Chemicals Co. (Tokyo, Japan), 2'-deoxythymidine-3'-NPP and 2'-deoxythymidine-5'-NPP were from Boehringer Mannheim GmbH, Biochemica (Mannheim, West Germany), and Sephadex G-200 was from Pharmacia Fine Chemicals Inc. (Uppsala, Sweden). All other chemicals were of reagent grade.
RESULTS
Production and localization of APase and PDase activities. The activities to hydrolyze NPP at pH 9.5 (NPPase-9.5) and to hydrolyze bisNPP at pH 9.5 (bisNPPase-9.5) of the parental 6160 and 6160-BC6 and of various mutants either starved for phosphate or with high concentrations of phosphate were compared. The cells were cultured at 30°C in LP or HP medium for 24 h. APase activity reached a maximum at this time. Table 1 shows the results. Differential centrifugation of cultured media revealed that more than 90% of the N,PPase-9.5 activities of 6160 cultured in LP medium and of 6160-BC6 cultured in both LP and HP media were present in the membrane fractions. Most of NPPase-9.5 activity in the extracellular fractions from these strains was sedimented by centrifugation at 60,000 x g for 60 min. BisNPPase-9.5 activity was found to be similarly localized. On the other hand, a mutant strain, RAN 1, secreted NPPase9.5 and bisNPPase-9.5 activities into the ultracentrifugal supernatant. In the APase-less mutant Vll, almost no NPPase-9.5 activity was detected, but a considerable amount of bisNPPase-9.5 activity was observed in all subcellular fractions, among which it was highest in the cytoplasmic supernatant. Essentially the same results were obtained with two other APase-less mutants, V12 and SP25 (data not shown). Comparison of bisNPPase-9.5 activities in the cytoplasmic supematants of the four strains, in which no or quite a small amount of NPPase-9.5 was present, showed that a similar amount of bisNPPase-9.5 was present in each strain cultured in each medium. Higher activity was found in the cells
110
YAMANE AND MARUO
J. BACTERIOL.
TABLE 1. Fractional localization of NPPase-9.5 and bisNPPase-9.5 activities in parental strains and mutantsa NPPase-9.5 activity (U/ml) in: Extracellular fluid
Strain
Culture medium
UltraUnfractionated
cen-
trifugal supema-
BisNPPase-9.5 activity (U/ml) in:
Extracellular fluid
Washed cells
Unfractionated
tan
CytoCell lysate
laspr
super-
ntnt 13.7 0.4 1.0 0.3 50.0 0.7 24.8 0.7 2.9 0.2 0.8 0.2 0.1 0.0 0.7 0.0
1.8 0.5 15.5 LP HP 0.5 0.4 1.8 0.6 45.7 LP 4.2 6160-BC6 HP 3.5 0.7 29.5 LP 19.9 19.2 3.1 RAN 1 HP 20.3 19.4 2.2 LP 0.0 0.0 0.1 Vi 0.1 0.1 1.3 HP a Cells were cultured in LP or HP medium at in the text.
6160
Membrane
300C
Unfractionated
Ultracentrifugal sutan
Washed cells
UnCytofrac- Cell ly- ml. Memtionsate spr brane super-
tantntn
4.2 28.1 22.0 2.9 20.2 0.8 3.0 3.1 9.7 9.0 7.9 1.6 48.0 7.8 5.3 68.7 66.0 1.0 57.1 26.7 6.9 2.8 50.6 47.9 8.8 37.8 2.8 28.8 28.7 16.0 14.7 1.6 10.0 0.6 35.9 34.8 12.9 9.3 8.5 1.6 0.0 1.2 5.0 4.4 3.1 0.8 _b 1.0 3.1 7.3 6.5 6.9 1.4 for 24 h. Subcellular fractions were obtained as described 13.0
5.6
b-, Not determined.
cultured in HP medium than in those cultured in LP medium. These results raised the possibility that at A. least two kinds of PDase are present in B. subs X tilis; one of them behaves the same as NPPase. 1.0 9.5, which would be APase with APDase activity, relative to synthesis and location, and the no Xother is localized mainly in the cytoplasmic suthe production of which is regulated E.pernatant, c 0. from that of APase. independently OJ(B)| Il Relation of enzymes that show APase . . and PDase activities. To understand the molecular relationship between APase and PDase w activities found in various fractions of B. subtilis, z the molecular sizes of the enzymes responsible C.S . . m : : these activities were examined. A culture ^(Cfor 54 * s o ° o. \ ~~~~fluid ofRAN 1 and the cytoplasmic supematants of RAN 1 and SP25 were prepared. RAN 1 was . C cultured in HP medium containing 0.3 M NaCl, 2.0 ( C ) z l l and SP25 was cultured in HP medium at 30°C 4l 57 for 24 h. The three preparations were subjected to gel filtration on a Sephadex G-200 column (1.2 by 85 and cm). bisNPPase-9.5 Gel filtrationactivities, behavior and of 1.0 NPPase-9.5 the activity of the preparations to hydrolyze bisNPP at pH 7.5 (bisNPPase-7.5), are shown in . :~ ~ .JL.4 i~ 1. Main peaks of bisNPPase-7.5 activities in 0.0 Fig. 1.36 1.13 1.59 1.82 2.05 Vel Vo hydrochloride, pH 7.5, containing 0.3 M KCI and CaCo. Fractions of 3.2 ml each were collected. NPPaseFIG. 1. Gel filtration behavior of NPPase-9.5, 9.5 (0), bisNPPase-9.5 (0), and bisNPPase-7.5 (A) bisNPPase-9.5, and bisNPPase-7.5 activities in the activities were determined in each tube. Arrows incytoplasmic supernatants ofSP25 (A) and RAN 1 (B) dicate the eluted positions of the molecular weight and in the extracellular fraction of RAN 1 (C) on a markers ([a] rabbit muscle aldolase, 158,00(); [bl Sephadex G-200 column. The three preparations were bovine serum albumin, 67,000; [cl ovalbumin, 45,000; successively chromatographed over a column (1.2 by [dl chymotrypsinogen A, 25,000; and [el cytochrome 85 cm) equilibrated and eluted with 10 mM Tris- c, 12,50o).
2.0 (A)
a
i.c B
b J,
c
t,
d
I
e
APase AND PDases IN B. SUBTILIS
VOL. 134, 1978
the cytoplasmic supernatants of SP25 and RAN 1 were equally eluted at fractions around Ve/Vo = 1.59. Their molecular weight was about 80,000. The presence of at least two peaks in the elution curves of SP25 and RAN 1 suggested the presence of more than one enzyme with bisNPPase7.5 activity in the preparations. A small amount of bisNPPase-9.5 activity detected in parallel with bisNPPase-7.5 activity was most likely due to PDases with optima of pH 8.0, since PDases had approximately 50 to 60% of the activity at pH 9.5 (Fig. 2). NPPase-9.5 activity was not detected in any fraction (Fig. 1A and B). Fractions around the bisNPPase-7.5 activity peaks (Ve/Vo = 1.54 to 1.64) were pooled and used in the next experiments. They were tentatively named PDase-SP25 and PDase-Rl. On the other hand, NPPase-9.5 and bisNPPase-9.5 activities of the culture fluid of RAN 1 were eluted at the same time from the column with a single peak (Fig. 10). The elution position of this peak was equivalent to a molecular weight of approximately 45,000 (Ve/Vo = 1.8). As described in the accompanying paper (9), we purified an extraceilular soluble enzyme (APase-Rl) with APase and APDase activities from the culture fluid of RAN 1, and an insoluble enzyme (APase-BC6) with APase and APDase activities from a membrane fraction of B. sub-
111
TABLE 2. Substrate specificity ofAPase and PDase preparationsa pH of Enzyme
Substrate specificity
reac-
NPP bisNPP T3'NPP T5'NPP ture 30 20 130 9.5 100 APase-BC6 11 4.8 15 100 9.5 APase-80,000 21 30 140 9.5 100 APase-Rl 9.4 5.4 0 100 7.5 PDase-Rl 6.8 3.8 0 100 7.5 PDase-SP25 a Reactions were conducted at 40°C for 10 and 20 min and were terminated by adding a mixture of 13% K2HPO4 and 2% KOH. Then, liberatedp-nitrophenol was measured at 420 nm. Each substrate was tested at an initial concentration of 0.5 mM. Rates of p-nitrophenol liberated were expressed relative to NPP in APase preparations (relative ratio = 100) and to bisNPP in PDase preparations (relative ratio = 100). Abbreviations: T3'NPP, 2'-deoxythymidine-3'-NPP; T5'NPP, 2'-
prepn
mix-
deoxythymidine-5'-NPP.
tilis 6160-BC6 as homogeneous materials. The pH optima for NPP-hydrolyzing activity of APase-Rl and APase-BC6 were both at 9.5, and they agreed with those for bisNPP-hydrolyzing activity (9). In contrast, the pH optima for bisNPP-hydrolyzing activity of PDase-SP25 and PDase-Rl were at 8.0, and no NPP-hydrolyzing activity was detected at any pH (Fig. 2). Substrate specificity of the enzyme preparations was analyzed by usingp-nitrophenyl derivatives. Each substrate was tested at an initial 0.75 1( a ) PDase-SP25 concentration of 0.5 mM. Table 2 shows the relative activities of the enzymes on the four iXA. substrates. APase-Rl and APase-BC6 hydrolyzed the four substrates. The hydrolyzing activ0.50. ities of the two enzymes against each of the four substrates were almost equal. PDase-SP25 and PDase-Rl hydrolyzed bisNPP, 2'-deoxythymi2 0.255 . dine-3'-NPP, and 2'-deoxythymidine-5'-NPP, but not NPP. Table 2 also includes the substrate specificity of another APase (APase-80,000) that x 0.0 f~ * had been isolated from the membrane fraction of 6160-BC6. This preparation showed at least three protein bands in sodium dodecyl sulfate < 0.5CD. discontinuous gel electrophoresis, and the moA6 lecular weight of the major band was approximately 46,000. This phosphatase had very low 0.25 bisNPP-hydrolyzing activity. APase-80,000 was also insoluble in solutions of low ionic strength. The relationship between APase-BC6 and APase-80,000 is not yet clear. OD 1 To test the possible structural relation of APase-Rl, and RAN 1 bisNPPaseAPase-BC6, FIG. 2. PDase activity pH optima for PDase-SP25 antiserum against APase-Rl was rabbit a 7.5, and PDase-Rl. Symbols: solid line, phosphatase activity; dashes, PDase activity; A, 0.1 M acetic acid- prepared. The method of preparation of this sodium acetate buffer containing Ca-Co (pH 3.2 to serum will be published elsewhere. The ultra6.5); 0, 0.1 M Tris-hydrochloride buffer containing centrifugal and cytoplasmic supernatants were Ca-Co (pH 7.0 to 9.5); *, 0.1 M glycine-sodium hy- prepared from RAN 1 cultures with LP and HP droxide buffer containing Ca-Co (pH 8.0-11.0). media, as described in Materials and Methods. E
c
.4
3
5
7
pH
9
112
J. BACTrERIOL.
YAMANE AND MARUO
The NPPase-9.5 and bisNPPase-9.5 activities in vertants of V12 were cultured in LP medium at the ultracentrifugal supernatants of RAN 1 and 30°C for 24 h. Their productivity of the major in APase-BC6 were equally well neutralized by membraneous APase and APDase was meathe serum, whereas bisNPPase-7.5 activities in sured. Representatives are shown in Table 5. the cytoplasmic supernatants were not (Table They produced the two enzyme activities in 3). Also, PDase-SP25 and PDase-Rl prepara- ratios similar to that of 6160. These results tions did not cross-react with the serum (data showed that the loss in the major membraneous not shown). These results suggested that APase- APase and APDase activities was induced by a Rl and APase-BC6, which possessed both single-point mutational event. NPPase-9.5 and bisNPPase-9.5 activities, are DISCUSSION structurally unrelated to PDases localized in the cytoplasmic supernatants. Extracellular soluble APase-Rl and memGenetic evidence for the presence of ma- brane-bound insoluble APase-BC6, which carjor membraneous APase and APDase ac- ried both APase and APDase activities, were tivities in a single enzyme molecule. To test essentially identical in optimum pH, substrate whether locus phoP is the structural gene for specificity, and immunological property, althe major APase and APDase, reversion of the though the enzyme preparations were absolutely two activities in APase-less mutants with differ- different in their solubility in solutions of low ent lesions within phoP was studied by transfor- ionic strength. These major APase and APDase mation experiments. The phenotypical APase- activities would be measured, resp9ctively, as and APDase-producing characters (APase+ and APDase+, respectively) of RAN 1 and 6160-BC6 TABLE 4. Cotransformation of APase+ and were transferred into V12 and SP25 (APasein APase-less mutants SP25 and V12 by APDase+ APDase- argA Strr) by DNA-mediated transDNAs from RAN 1 and 6160-BC6' formation (Table 4). Arg+ transformants were No. DNA donor DNA re- No. of selected. Three hundred transformants in each cipient . + APase+ cross were transferred on LP medium-agar APase+/ (APaset strain trargn on LP APDase plates, and then APase+ and APDase+ trans(APaseAPDase+ fomantsmediumformants were scored among them. Every airA)W) APDase- tested rmant pae argA) APase+ transformant on the plates was also 71 300 V12 APDase+. Spontaneous APase+ revertants were RAN 1 71/71 79 300 SP25 79/79 isolated from V12 on LP medium-agar plates RAN 1 101 300 101/101 with a reversion frequency of approximately 6160-BC6 V12 300 87 87/87 10'. The 16 APase+ revertants isolated were 6160-BC6 SP25 a also APDase+ on the plates. Ninety APase+ 1 High concentrations (about ,ug of DNA per ml) transformants of V12 and SP25 and the 16 re- were used for each transformation experiment. TABLE 3. Neutralization of NPPase-9.5, bisNPPase-9.5, and bisNPPase-7.5 in the ultracentrifugal and cytoplasmic supernatants of RAN ) and in APase-BC6 by rabbit antiserum against APase-Rla Enzyme activity in: Medium and treatment
Ultracentrifugal supernatant
Cytoplasmic supernatant
APase-BC6b
NPPase-9.5
NPPase-9.5 BisNPPase7.5
NPPase-9.5 BisNPPase-9.5
BisNPPase-9.5
LP medium None 100 __d 100 NDc 100 + Antiserum (1:20) 3.1 9.8 92 + Antiserum (1:100) 1.9 6.9 101 HP medium None 100 100 ND 100 100 100 + Antiserum (1:20) 1.0 3.7 99 9.3 8.2 + Antiserum (1:100) 0.8 3.0 100 2.8 1.6 a The preparations were mixed with the antiserum against APase-Rl diluted to 1:20 or to 1:100 with 10 mM Tris-hydrochloride, pH 7.5, containing 0.9% NaCI and Ca-Co, and immunological reaction was allowed to proceed for 1 h at room temperature. After the mixtures were centrifuged at 1,000 x g for 15 min, the enzymatic activities in the supernatants were assayed. b APase-BC6 was dissolved in a buffer of 1 M Tris-hydrochloride-2 M MgCl2-Ca-Co, pH 7.0. c ND, Not detected. d _, Not determined.
APaSe AND PDases IN B. SUBTILIS
VOL. 134, 1978
TABLE 5. Production of APase and APDase by, and ratio of the two enzyme activities
(APDase/APase) in, representative APase+ transformants and revertants of APase-less mutant V12a Enzyme activity (U/ml)
Strain APase
Transformants 1 2 3 4 V12 revertants 1 2 3 4
33 54 41 62
APDase
46 63 55 93
APDase/ APase
1.4 1.2 1.3 1.5
67 91 1.4 74 98 1.3 95 1.4 69 53 69 1.3 104 1.2 85 5 40 47 1.2 6160 0.1 3 V12 _b 0.1 3 SP25 a for 24 LP medium at 300C in cultured were Cells h. Transformants 1, 2,3, and 4 were the representative transformants of the crosses RAN 1 x SP25, RAN 1 x V12, 6160-BC6 x SP25, and 6160-BC6 x V12, re-
spectively. bh, Not calculated.
NPPase-9.5 and bisNPPase-9.5 activities in the subcellular fractions of the culture media (Tables 1 and 3). On the other hand, the bisNPPase9.5 activity found in the cytoplasmic supernatants should be due to other PDases, because the optimum pH, substrate specificity, and immunological property of their main activity were different from those of APase-Rl and APaseBC6. The PDase activity was most likely due to PDases with an optimum pH of 8.0. These results suggest that the major PDase activity, which was produced in Pi-deficient medium and was lost in APase-less mutants, was included in the phosphate-repressible APase but was different from enzymes with PDase activity localized in the cytoplasmic supernatant. No structural relationship between the two groups of enzymes could be detected by the immunological study. APase-constitutive mutants were also constitutive for APDase, and when the mutation(s) in B. subtilis 6160-BC6 responsible for the constitutive production of APase was transferred into other strains by the DNA-mediated transformation, constitutive production of the two enzyme activities was cotransferred (unpublished data). The simultaneous loss of APase and APDase characters would be caused by a singlepoint mutation in locus phoP, because APase+ strains isolated by transformation of V12 and SP25 and by spontaneous reversion of V12 were
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also APDase+. Furthermore, the ratio of APase activity to APDase activity found in the APase+ strains was almost constant. These results suggest that phoP is the structural gene for APase with APDase activity. The notion that the locusphoP is the common regulator gene for the production of two different enzyme molecules, APase and APDase, is not likely, since APase-Rl and APase-BC6, which contained APase and APDase activities, were purified as homogeneous materials and since it was indicated that the two enzyme activities would be present in the same protein (9). The possibility, however, that the structural gene for APase with APDase activity is localized in another site of the locus phoP and that phoP regulates the production of an enzyme molecule that possesses both APase and APDase activities still remains to be tested. Formation of the major membraneous APase with APDase activity in B. subtilis would be regulated by at least two kinds of regulator genes, as suggested by Miki et al. (4). One of the putative regulator genes is phoRI, which is closely linked to phoP, and the other is phoRII, which would not be linked to phoP or phoRI. These genes in B. subtilis would correspond to phoR, phoS, and others for APase synthesis in Escherichia coli (1, 5). The APase formation system in B. subtilis seems to resemble essentially that of E. coli. The APase of B. subtilis Marburg strain was previously purified by Takeda and Tsugita (7), Glenn and Mandelstam (2), and Le Hegarat and Anagnostopoulos (3). The purified preparations were reported to have mainly NPP-hydrolyzing activity and little or no bisNPP-hydrolyzing activity. These authors extracted the enzyme from membrane preparations with a 1 M Mg2+ solution. We noticed that solubilization of the major APase from a membrane preparation of 6160BC6 was difficult. We also partially purified an APase (APase-80,000) that had properties similar to the preparations obtained by Takeda and Tsugita (7) and Le Hegarat and Anagnostopoulos (3), but the amount of that APase was quite small. It is possible that the poor bisNPP-hydrolyzing activity of APase-80,000 was induced by the dimerization of APase-BC6 (unpublished data). PDase(s) in the cytoplasmic supernatant of B. subtilis Marburg strain was purified by Taniguchi and Tsugita (8) and by Le Hegarat and Anagostopoulos (3). The preparation of Le Hegarat and Anagnostopoulos (3), which had a molecular weight of 150,000, seems to be different from the main PDase(s) in the cytoplasmic supernatants of SP25 and RAN 1. The molecular
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weights of PDase-Rl and PDase-SP25 were around 80,000 (Fig. 1). This difference could be due to cultural conditions; SP25 and RAN 1 were cultured in HP medium. These minor PDases did not hydrolyze NPP and had no structural relation to APase-Rl or APase-BC6. At least two kinds of PDase were demonstrated in the cytoplasmic supernatants of SP25 and RAN 1 by gel filtration on a Sephadex G-200 column (Fig. 1). Taniguchi and Tsugita (8) reported the presence of three kinds of PDase from diethylaminoethyl-cellulose column chromatography and showed that one of them had ribonuclease activity. Synthesis of these PDases in the cytoplasmic supernatant seemed to be regulated independently by the concentration of Pi in the culture medium. The amount of PDase activity in the preparations was higher when cultured in HP medium than when cultured in LP medium (Table 1). PDases might participate in the intracellular metabolism of phosphates when the cells are allowed to utilize sufficient amounts of Pi from the outside medium. ACKNOWLEDGMENTS This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science and Culture, Japan. We thank I. Shibuya for helpful discussion and Y. Ikeda for a gift of APase-less mutants of B. subtiis. LITERATURE CITED 1. Echols, H., A. Garen, S. Garen, and A. Torrian. 1961. Genetic control of repression of alkaline phosphatase in E. coli. J. Mol. Biol. 3:425-438.
J. BACTERIOL. 2. Glenn, A. R., and J. Mandelstam. 1971. Sporulation in Bacillus subtilis 168: comparison of alkaline phosphatase from sporulating and vegetative cells. Biochem. J. 123:129-138. 3. Le Hegarat, J.-C., and C. Anagnostopoulos, 1973. Purification, subunit structure and properties of two repressible phosphohydrolases of Bacillus subtilis. Eur. J. Biochem. 39:525-539. 4. Miki, T., Z. Minami, and Y. Ikeda 1965. The genetics of alkaline phosphatase formation in Bacillus subtilis. Genetics 52:1093-1100. 5. Morris, H., M. J. Schlesinger, M. Bracha, and E. Yagil. 1974. Pleiotropic effects of mutations in-olved in the regulation of Escherichia coli K-12 alkaline phosphatase. J. Bacteriol. 119:583-592. 6. Saito, H., and K. Miura. 1963. Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta 72:619-629. 7. Takeda, K., and A. Tsugita. 1967. Phosphoesterases of Bacillus subtilis. II. Crystallization and properties of alkaline phosphatase. J. Biochem. (Tokyo) 61:231-241. 8. Taniguchi, K., and A. Tsugita. 1966. Phosphoesterases of Bacillus subtilis. I. Purification and properties of phosphodiesterases. J. Biochem. (Tokyo) 60:372-380. 9. Yamane, K., and B. Maruo. 1978. Purification and characterization of extracellular soluble and membranebound insoluble alkaline phosphatases possessing phosphodiesterase activities in Bacillus subtilis. J. Bacteriol. 134:100-107. 10. Yamane, K., T. Miki, H. Saito, Y. Ikeda, and B. Maruo. 1976. Isolation of a mutant secreting extracellular soluble alkaline phosphatase in BaciUus subtilis. Agric. Biol. Chem. 40:2181-2185. 11. Yoshikawa, H. 1970. Temperature-sensitive mutants of Bacillus subtilis. I. Multiforked replication and sequential transfer of DNA by a temperature-sensitive mutant. Proc. Natl. Acad. Sci. U.S.A. 65:206-213. 12. Young, F. E., and G. A. Wilson. 1975. Chromosomal map of Bacillus subtilis, p. 596-614. In P. Gerhardt, R. N. Costilow, and H. L. Sadoff (ed.), Spores VI. American Society for Microbiology, Washington, D.C.
