Vol. 124, No. 1

JOURNAL OF BACTERIOLOGY, Oct. 1975, p. 353-363 Copyright 0 1975 American Society for Microbiology

Printed in U.S.A.

Aminopeptidases of Bacillus subtilis EDWARD P. DESMOND, WILLIS L. STARNES, AND FRANCIS J. BEHAL* Department of Biochemistry, Texas Tech University School of Medicine, Lubbock, Texas 79409

Received for publication 13 May 1975

Three enzymes with L- and one enzyme with D-aminopeptidase (EC 3.4.11; a-aminoacyl peptide hydrolase) activity have been separated from each other and partially purified from Bacillus subtilis 168 W. T., distinguished with respect to their molecular weights and catalytic properties, and studied in relation to the physiology of this bacterium. One L-aminopeptidase, designated aminopeptidase I, has a molecular weight of 210,000 i 20,000, is produced early in growth, and hydrolyzes L-alanyl-,-naphthylamide most rapidly. Another, designated aminopeptidase II, molecular weight 67,000 i 10,000, is also produced early in growth and hydrolyzes L-lysyl-,@-naphthylamide most rapidly. A third, aminopeptidase III, molecular weight 228,000 i 20,000, is produced predominantly in early stationary phase and most efficiently utilizes L-a-aspartyl-,-naphthylamide as substrate. The synthesis of aminopeptidase III in early stationary phase suggests that selective catabolism of peptides occurs at this time, perhaps related to the cessation of growth or the onset of early sporulation-associated events. A D-aminopeptidase which hydrolyzes the carboxyl-blocked dipeptide D-alanyl-Dalanyl-B-naphthylamide (as well as D-alanyl-,B-naphthylamide and D-alanyl-Dalanyl-D-alanine) has also been identified, separated from aminopeptidase II, and purified 170-fold. D-Aminopeptidase, molecular weight 220,000 1 20,000, is localized predominantly in the cell wall and periplasm of the organism. This evidence and the variation of the activity during the growth cycle suggest an important function in cell wall or peptide antibiotic metabolism. The appearance of aminopeptidases, intracellularly and extracellularly, in cultures of many bacterial species has been noted (e.g., 4, 5, 18, 22). In the genus Bacillus, Aubert and Millet (2) reported an aminopeptidase in B. megaterium which increases fivefold in activity during sporulation. Hall et al. reported an extracellular aminopeptidase in culture filtrates of B. licheniformis which has a pH optimum of 8.5 to 9.0 and was activated by Co'+ ions (10). Two aminopeptidases have also been reported in B. stearothermophilus (20, 23). Matsumura et al. (17) and Minamiura et al. (19) reported two L-aminopeptidases in B. subtilis which could be separated by means of diethylaminoethyl (DEAE)-cellulose chromatography. At least one of these activities appeared chromatographically at the same elution volume with a n-aminopeptidase (APD). This APD was not separated from the L-aminopeptidase activity. Wagner et al. (26) described an extracellular L-aminopeptidase in cultures of B. subtilis which they suggest is secreted, under appropriate conditions, by intact cells into the medium. The pH optimum of this enzyme was 8.0, and Co'" was found to activate it.

Other enzymes which hydrolyze D-aminoacyl peptides have also been reported in Bacillus species. A D,D-carboxypeptidase and a D-transpeptidase have been extensively studied with respect to their role in cell wall synthesis and sensitivity to antibiotics. These activities have been reviewed recently, and it has been suggested that a single protein might be responsible for these two activities (7). Other transpeptidase or aminoacyl transferase activities specific for both D-aminoacyl and L-aminoacyl residues must be presumed to be present in these organisms, since cells in early stationary phase synthesize peptide antibiotics containing both configurations in a nonribosomal process (e.g., 13). The present work was undertaken to identify and study the biosynthesis and properties of some aminopeptidases of B. subtilis in order to provide a foundation for studies of the function(s) they have in the physiology of this organism.

353

MATERIALS AND METHODS Organisms. Bacillus subtilis 168 W.T., obtained from I. C. Felkner, Texas Tech University, is a strain

354

DESMOND, STARNES, AND BEHAL

J. BACTERIOL.

of B. subtilis 168 which has been converted to To prepare samples for ion exchange chromatogranutritional prototrophy by transformation with de- phy, cultures were grown in the same way, but larger oxyribonucleic acid of B. subtilis W 23. B. subtilis 3Y, (500-ml) samples were utilized and the clarified lysate a ribonucleic acid polymerase mutant, was obtained was dialyzed overnight against 0.01 M tris(hydroxyfrom Richard Losick, the Biological Laboratories, methyl)aminomethane-maleate buffer (pH 7.0, 0.005 Harvard University. B. subtilis 9V, a stage 0 sporula- M KCI) before placing it on the column. tion-negative strain, was obtained from P. Schaeffer, Heat-resistant spore titers. Culture samples were Institut de Microbiologie, Orsay, France. diluted, heated to 80 C for 10 min, and plated in Chemicals. Aminoacyl-,B-naphthylamides were ob- duplicate on nutrient agar plates (Difco) for colony tained from Mann Research Laboratories, New York, counts. N.Y., and from Fox Chemical Co., Los Angeles, Calif. Ion exchange chromatography. A column (0.9 by DEAE-cellulose was obtained from Sigma Chemical 22 cm) was packed with DEAE-cellulose under 1 atm Co., St. Louis, Mo. of nitrogen pressure. The column was equilibrated Aminopeptidase assays. For quantitative deter- with 0.01 M tris(hydroxymethyl)aminomethaneminations of aminopeptidase activity, a previously maleate buffer, pH 7.0, with 0.005 M KCI. Lysates of described colorimetric assay was used (3). The sub- B. subtilis which had been dialyzed overnight against strate was the appropriate aminoacyl-B-naphthyl- the same buffer were placed on the column. A linear amide (0.001 M), and the unit of activity was defined gradient of KCI (0.005 M to 0.4 M; flow rate, 25 ml/h) as that amount of enzyme which hydrolyzes 1 jmol of in the same buffer (500 ml, total volume) was used to substrate per min. elute proteins from the column. Since the L-aminopeptidases reported here all have Molecular weight estimation by gel filtration. A significant activity with L-alanyl-,-naphthylamide as column (1.5 by 96 cm) was packed with Sephadex substrate, it was used for all growth studies and for G-200 and equilibrated with 0.10 M borate buffer, pH assay of all DEAE-cellulose columns. Except when 8.0, which was also 0.50 M with respect to NaCl. To specified in the figure legends, D-alanyl-,-naphthyl- calibrate the column, 5 mg of each of the following amide was the substrate for the APD. All colorimetric was dissolved in 2.5 ml of the column buffer: blue assays for the L-aminopeptidases were performed in dextran, gamma globulin, bovine serum albumin, the presence of 0.001 M Co2+, which stimulates these lysozyme, and myoglobin. This mixture was pumped activities, at pH 8.0 in 0.01 or 0.02 M tris(hydroxy- through the column at a rate of 2.5 ml/h. methyl)aminomethane-maleate buffer. The APD acElution volumes (v.) were determined by measurtivity is assayed without Co2+ since Co2" has no ing the absorbance of the column fractions at 280 nm. effect on this enzyme. For molecular weight estimation of the aminopeptiFor qualitative deternmination of the hydrolysis of dases separated by ion exchange, pooled fractions short peptides, 0.01 to 0.10 ml of enzyme was incu- were concentrated by pressure dialysis with a Diaflo bated with 0.02 ml of 0.01 M Co'+, 0.1 ml of the PM10 ultrafiltration membrane. A 2.5-ml aliquot of peptide (3 to 4 mM), and sufficient buffer to make a the concentrated enzyme sample was then dialyzed 0.32-ml reaction mixture. The reaction was stopped against the column buffer and loaded onto the by addition of 0.10 ml of 1.0 M acetic acid. The column together with 2.5 mg of blue dextran and 5 mg mixture was analyzed with a Beckman model 121 HP of an internal protein standard (either gamma globuautomatic amino acid analyzer which had been stan- lin or bovine serum albumin). The elution volumes of dardized with the appropriate amino acids and pep- the protein standard and blue dextran were detertides. mined by measuring the absorbance of the fractions at Protein assays. Protein was routinely determined a wavelength of 280 nm; the aminopeptidase elution by measuring absorbancy at 280 nm. In experiments volume was measured by colorimetric enzymatic where specific activity was determined, or where assay. The approximate molecular weight was deternucleic acids were present, protein was measured by mined by reference to a plot made using standard the method of Lowry et al. (16). proteins, according to the method of Andrews (1). Determination of pH optimum. The effect of Growth of cells. A 12-h culture (100 ml) of the strain of B. subtilis being tested was inoculated into 3 hydrogen ion concentration on enzyme activity was liters of Hanson complex medium for sporulation of B. measured in a buffer which was 0.20 M with respect to subtilis (12). The cells were grown at 37 C with maleate, phosphate, and borate. This buffer system vigorous aeration. Growth was measured by estimat- has significant buffering capacity in the pH range ing the turbidity of the cultures at 650 nm (Bausch from 5.0 to 9.6. The colorimetric assay was used; no and Lomb Spectronic 20 colorimeter). Samples of 50 attempt was made to differentiate between irreversito 100 ml were removed at specified intervals, and ble inactivation and inactivation which is reversible harvested immediately by centrifugation at 4 C, by restoring the enzyme to optimal pH. 12,000 x g. Cellular localization of the APD. APD was localThe cells were washed three times in cold 0.01 M ized in the cell by a technique adapted from Weibull tris(hydroxymethyl)aminomethane-maleate buffer, and Bergstrom (27) and Nugent et al. (21). B. subtilis pH 8.0, and lysed by sonification for 2 min in a 168 W. T. was grown in 500 ml of nutrient broth for 12 Branson model S125 Sonifier. The lysates were clari- h and harvested by centrifugation (400 x g, 15 min). fied by centrifugation (4 C, 30,000 x g, 20 min), and The cells were then suspended in 100 ml of 0.02 M the specific activity was determined. phosphate buffer, pH 7.0, to which the following were

BACILLUS SUBTILIS AMINOPEPTIDASES

VOL. 124, 1975

added: 55 ml of 2 M sucrose, 2.5 ml of 1 M NaCl, and 1.67 ml of 4% lysozyme. The cells in isotonic medium with lysozyme were incubated for 30 min at 37 C, and then centrifuged at 10,000 x g for 1 h to sediment protoplasts, the presence of which was confirmed microscopically. The supernatant, containing digested cell wall components and material from the periplasmic space between cell membrane and cell wall, was saved and the sedimented protoplasts were lysed in a hypotonic solution, 0.02 M phosphate buffer, pH 7.0. This preparation was centrifuged at 2,000 x g for 10 min to sediment intact cells. The supernatant was then centrifuged at 20,000 x g for 20 min to separate membrane (sediment) and cytoplasmic (supernatant) fractions.

355 43

0-

3

z

-

0

n >-

2

to 0

I4

w 0

1-

0.

RESULTS

w

g

Fig. 1 and 2 show the variation of aminopeptidase activity, specific activity, and soluble D 0 0 protein with growth. Here both L-alanyl- (Fig. 03 1A and 1B) and D-alanyl-fl-naphthylamide ' 023 (Fig. 2A and 2B) have been used as substrate. In cr tL addition, the appearance of heat-resistant spores is indicated in Fig. 1A, although the time of appearance of heat resistance is the same in 5 24 10 15 20 both of the experiments shown in Fig. 1 and 2. TIME (HRS.) In general, whether the activity observed is FIG. 2. (A) The specific activity of APD and cell hydrolyzing either substrate, the pattern of (turbidity at 650 nm) as a function of culture variation is the same during log-phase growth. number time. (B) The total activity of APD and total protein U.

4

01i

_

t0

'.5

of cell lysates as a function of culture time. The development of heat-resistant spores here is coincident with the time shown in Fig. 1. OD,., Optical density at 650 nm.

/]~ 2t*There is a large increase in activity which peaks < at or near the onset of the maximum stationary , 1r< phase. D-Alanyl-f,-naphthylamide-hydrolyzing

,o 0

O

0o

con

activity routinely peaks slightly later than

.-6

B .' varies with total activity. At or near the onset of = stationary phase, both types of activities begin 0.2 0 to decrease. This decreasing phase of activity is

i4 D

relatively short, lasting -x

22 1

-4

L-

.,_______ 1.4 .alanyl-,8-naphthylamide-hydrolyzing activity. _ During this phase of growth, specific activity

a

a 0° F °

with the

12

TIME (HOURS)

0

1

to

2

h, in all

-alanyl-0-naphthylamide as substrate to be somewhat later in the time

again seem course

FIG. 1. (A) The variation of aminopeptidase specific activity with L-alanyl-fi-ni strate, heat-resistant spore titer andcelnumber(as turbidity at 650 nm) as functic anf The variation in total aminop eptidase activity per unit volume of the culture with L-alanyl-p-nraphthylamide as substrate and total so luble protein per unit volume of culture as a function of culture age. OD.5O, Optical density at 650 nm.

from

experiments (six with L-substrates and two with D-substrates), and it does not coincide with the pH minimum for the medium. The minima

of growth than the minima when L-ala-

nyl-0-naphthylamide is the substrate. Subsesubquent to the decrease in specific and total activity, both types of activity begin increasing. celtlureumage(Be) D-Alanyl-Il-naphthylamide-hydrolyzing activity continues to increase throughout the remainder of 24 h while L-alanyl-,B-naphthylamide-hydrolyzing activity passes through a maximum at about 8 h and subsequently decreases through-

356

J. BACTIOL.

DESMOND, STARNES, AND BEHAL

out the 24-h period. Specific activitty for both substrates increases throughout the 2!4-h period, and for the L-substrate this appears tAo be due to a more rapid decrease in the amountt of soluble protein. Experiments not rpported here wit;h a variety of L-aminoacyl-B-naphthylamides indicated that there was a somewhat differentt substrate specificity between the total amin opeptidase (naphthylamidase) produced in log-phase growth and that produced in the maximum stationary phase during the events jiast preceding and throughout sporulation. At l east a part of the difference in specificity may be attributed to the appearance of another enzymaltic activity during the later stage of growth (Fiig. 3). The growth conditions for this experimenkt are identical to those used in the experiment,-s described in Fig. 1 and 2, except that the solublle fractions obtained from cells harvested at 3, 4.1 5, 9, and 11 h were chromatographed on DEAE-ce,llulose columns. At 3 as well as at 4.5 h two Ipeaks with activity are seen. At 9 and 11 h a th ird peak is found. The aminopeptidase which e,lutes from the column first has been designated aminopeptidase I (AP I), and the two aminopeptidases which elute subsequently have been 4designated aminopeptidase II (AP II), and amin opeptidase III (AP Ill), respectively, in their or der of elu-

tion. Each of these aminopeptidases has significant activity toward L-alanyl-,B-naphthylamide (see Table 2), although AP II has a low activity with this substrate when compared with the rate observed with L-lysyl-#-naphthylamide. However, the absolute rate with L-alanyl-,B-naphthylamide is still quite high, and this substrate was used for these column studies. The identity of each peak with activity was confirmed by studying its substrate activity profile, since some differences in elution volumes do occur from experiment to experiment. The first rise in aminopeptidase activity, between 2 and 5 h (Fig. 1) is attributed to a marked increase in the activity of AP II. On the average, AP I decreased only slightly during the period, and the decrease shown here (Fig. 3) is greater than average. As previously noted, the second rise in Laminopeptidase specific activity (Fig. 1) between 7 and 12 h, may be largely attributed to the appearance of AP III during this stage of growth. This rise in activity briefly precedes the increase in heat-resistant spore formation. The data shown in Fig. 4 are the results from DEAE-cellulose chromatography of cell lysates of 12-h cultures of B. 8ubtilia 3Y, a sporulationnegative RNA polymerase mutant, and B. 8ubtili8 9Y, a stage 0 sporulation-negative mutant derived from strain 168 W.T. Different amounts of protein were used in these experiments than that used in Fig. 3. However, 3 hr separate studies not included here show that both of these spore-negative strains formed AP III to roughly the same degree as that found in strain 168 W.T. Experiments similar to those described in 4.5 hr Fig. 3 and 4 have also been conducted with N D-alanyl-,-naphthylamide-hydrolyzing activity. Only one peak of activity has been observed .____ upon DEAE-cellulose chromatography (Fig. 5), .E and the elution volume of this peak coincides with the elution volume of AP II. Enzyme with 9 hr similar properties is found in extracts from cells of the two sporulation-negative strains indicated in the previous paragraph, and again the levels found are similar to those found in the wild type (data not shown). 11 hr The enzyme responsible for the hydrolysis of D-alanyl-,8-naphthylamide can be separated from the L-aminopeptidase activity (Fig. 6). Here the fractions included in the bar in Fig. 5 400 100 300 200 ml have been chromatographed on Sephadex The residual activity against L-lysyl-,6G-200. FIG. 3. DEAE-cellulose chromatogr aphy of Laminopeptidase activity of B. subtilis 16i8 W.T. with naphthylamide, about 3.0% of the D-alanyl-,L-alanyl-,6-naphthylamide as substrate. The product naphthylamidase activity, does not remain in the more purified preparation (Table 1) used in measured was ,8-naphthylamine. 0

-

-

357

BACILLUS SUBTILIS AMINOPEPTIDASES

VOL. 124, 1975

the substrate specificity studies discussed here. The enzyme thus partially purified (Table 1) is not a homogeneous protein upon acrylamide gel electrophoresis, but remains stable and active over long periods of time. Attempts to further purify the enzyme have led to loss of activity, I

,z

33Y

020

2

A 0

I5

40

320

240

120

200

ELUTION

VOLUME

(ML)

FIG. 5. DEAE-cellulose chromatography of a partially purified preparation of APD showing that the activity co-elutes with AP II. Their respective substrates are shown. DNA, #-Naphthylamide. 0

; 0

8

SUBTILIS

SV

0

0310 05

o0

Z 0.2 ~ ~ 0

~

~

~

04

~

03

0~~~~~~~~~~~~

LE

I

-i I o ID

s 02 a

m0 10

20

30

40

50

00

o.l

FRACTION NO.

FIG. 4. DEAE-cellulose chromatography of Laminopeptidase activity of two B. subtilis mutants with L-alanyl-gl-naphthylamide as substrate. (A) LAminopeptidases of B. subtilis 3Y. (B) L-Aminopeptidases of B. subtilis 9V. AP III is present in both the sporulation-negative RNA polymerase mutant (3Y) and the stage 0 sporulation-negative mutant (9V). The optical density at 580 nm (OD5..) may be converted to micromoles per minute by multiplying by 0.02. Fraction volume was 10 ml. The experiments shown in Fig. 3 and 4 do not use comparable amounts of protein.

60

s0 ELUTION

100 VOLUME

(ML)

FIG. 6. G-200 gel filtration chromatography of a partially purified preparation of APD. The activity is separated from APII by this step. The elution volume for APD is characteristic of a protein with a molecular weight of 220,000-iX 20,000. The respective enzymatic substrates for this study are shown in the legend of the ordinate. #NA, ,8-Naphthylamide; OD,.., optical density at 280.

TABLE 1. Summary of purification of APD Units Step in (.omol/min) purification

1. Cell lysate 2. Bioglas with

Protein

(mg)

Sp act

(Mmol/min per mg)

%

Purification

Recovery

'2,360

6,160

0.38

1

454

555

0.82

2.2

100 19.2

14.5 40 170

20.1 9.9 7.1

(NH4) 2SO4

3. DEAE-cellulose 4. Sephadex G-200 5. DEAE-cellulose

475 233 168

86 15.3 2.6

5.5 15.2 64.7

358

DESMOND, STARNES, AND BEHAL

but homogeneous preparations with substantially diminished activity have been obtained. Considerable activity is lost in this preparation, with only about 9% of the original activity recovered. Aminopeptidases I and III were used for substrate specificity studies (Table 2) in the maximum stage of purity obtained from DEAEcellulose separation of AP I and AP III, followed by G-200 chromatography of the separated activities. AP II was used after it had been separated from APD by G-200 chromatography (Fig. 6) and after both AP II and APD had been separated from AP I and III by DEAE-cellulose chromatography. Neither AP I, II, or III has yet been purified to homogeneity, but acrylamide gels indicate, in the case of each activity, only one protein band which demonstrates the ability to hydrolyze L-alanyl-,B-naphthylamide. The rate at which these aminopeptidases hydrolyze nine aminoa6yl-ft-naphthylamides was measured to distinguish the individual substrate specificities of these enzymes (Table 2). The substrate most rapidly hydrolyzed by AP I is L-alanyl-f,-naphthylamide. Naphthylamides with acidic and basic amino acid residues are hydrolyzed at rates exceeding the rate of hydrolysis of L-phenylalanyl- and L-leucyl-,Bnaphthylamide. AP II hydrolyzes L-lysyl-fnaphthylamide preferentially at a rate exceeding 10 times the rate at which it hydrolyzes the other substrates tested. L-Arginyl- and L-histidyl-,B-naphthylamide were not assayed with this enzyme. L-Aspartyl- and L-alanyl-,6-naphthylamide are the best substrates for AP III, and the rate of hydrolysis of the other substrates

J. BACTERIOL.

tested does not exceed 15% of the rate at which L-aspartyl-,B-naphthylamide is hydrolyzed. LGlutamyl-,8-naphthylamide is hydrolyzed at only 10% the rate of L-aspartyl-,-naphthylamide even though it is structurally similar. D-Alanyl-o-naphthylamide is not a substrate for the L-aminopeptidases nor are the L-aminoacyl,@-naphthylamides substrates for APD. An amino-protected compound, N-acetyl-L-phenylalanyl-,8-naphthylamide, was not a substrate for any of the L-aminopeptidases, even at enzyme levels which provided significant rates of hydrolysis for L-phenylalanyl-,B-naphthylamide. Amino-protected substrates have not been tested with APD. Di- and tetra-L-alanine are good substrates for hydrolysis by AP I, AP II, and AP III, and none of the three enzymes hydrolyzes di- or tri-D-alanine, while APD effectively hydrolyzes the latter D-peptides but not the former ipeptides. D-Leucylglycine is only slowly hydrolyzed by APD. APD effectively hydrolyzes the carboxyl-protected dipeptide D-alanyl-D-alanyl-,8-naphthylamide (Fig. 7). In the case of L-aminopeptidase hydrolysis of tetraL-alanine, all of the expected products, alanine, di-L-alanine, and tri-L-alanine are observed. In the case of APD hydrolysis of tri-Dalanine, alanine and di-D-alanine were products. When D-alanyl-B-naphthylamide is the substrate for APD, the release of ,B-naphthylamine is initially linear with time up to about 1 h (Fig. 7). The initial substrate concentration was 1 mM. At about 1 h substrate is exhausted and very little product is released. However, when D-alanyl-D-alanyl-,8-naphthylamide is the sub-

TABLE 2. Relative rates of hydrolysis of

aminoacyl-,j-napthylamides by the aminopeptidases of Bacillus subtilis 168 W. T.

~~~~~~Enzymea

* L-Aminoacyl

residue

L-Alanyl L-Lysyl L-Aspartyl L-Glutamyl L-Phenylalanyl L-Leucyl L-Seryl L-Threonyl D-Alanyl

AP I

100 50.6

21.2 12.7 9.5 9.0 4.2 3.4