- Email: [email protected]
Characterization of the Bacillus subtilis CwbA protein which stimulates cell wall lytic amidases
FEMS Microbiology Letters 95 (1992) 1if- 114 ,~, 1992 Federation of European Microbit,logicalStvcicties0378-10t~7/92/$05.110 Published by Elsevier
FEMSLE 114082
Characterization of the Bacillus subtilis CwbA protein which stimulates cell wall lytic amidases Akio Kuroda and Junichi Sckiguchi Department t~t"Al~pli,'d IJioh~,t,%Faculty c~t"T~ttih" Sch'ncc and Tet'hnoh,gy. Sh/,I,~hu Umt cr,sity, Nagano, Japan Recek'ed II May 1002 Accepted 12 May 1092 Key words: Autolysin; N-Acetylmuramoyl-i.-alanine amidasc: Modifier protein; Bacillus subtilis; Bacillus lichenifonnis
1. S U M M A R Y T h e Bacillus subtilis cell wall binding protein, CwbA, stimulated the cell wall lytic activities of the B. subtilis and B. lichenilbrmis autolysins (CwlA and CwlM, respectively) in addition to that of the major B. subtilis autolysin (CwlB). Even though the substrate for the enzyme reaction was changed from B. subtili,~ cell wall conraining a teichoic acid to Micrococcus haeus cell wall containing a teichuronic acid, the stimulatory effect of CwbA on CwlA activity was observed.
2. I N T R O D U C T I O N In Bacillus subtilis the major autolysin is an N-acetyhmtramoyl-L-alanine amidase ('amidase') which is responsible tot cell wall turnover [ I - 3 ] and cell lysis at the stationary phase [4]. Herbold and Glaser reported the purification of a major Correspondence to: J. Sekiguchi. Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, 3-15-I Tokida, Ueda-shi, Nagan~, 386, Japan.
B. s:tblilis ATCC 61)51 amidase to homogeneity, as well as the purtfication of a second protein (modifier pre,,ein), which in combination with the amidase stimulates enzyme activity [5,6]. Recently, we reportc6 the cloning and sequencing of a major amidase (AI r 46000) gene (cwIB) from B. subtili,~ 168S [4]. Moreover, a cwbA gcne which encodes a cell wall binding protein of B. subtilis 168S was located in the same operon [4,7]. Since CwbA stimulated the enzyme activity of the major autolysin, CwlB, about 3-fold, and the M r (75000) and amino acid composition of CwbA were similar to those of the modifier protein, wc could regard CwbA as a modifier protein from strain 168S [7]. On the other hand, B. subtilis 168S produces another type of amidase (CwlA; M r 23000) [8], which reflects the difference in enzyme specificity from CwIB [4,8,9]. B. licheniforints also produces an amidase (CwIM; M r 291H1()) which has the ability to digest B. licheniforints, 13. subtilis and Micrococcus haeus cell walls [ I0]. We report here the effect of CwbA on the amidasc activities of CwlA and CwlM on 13. subtilts a n d / o r Micrococcus hlteus cell walls.
3. M A T E R I A L S A N D M E T H O D S
3.1. l:,'nzyme preparation Purification o f CwlA from Escherichia colt was carried out as follows. E. colt J M I 0 9 cells harboring p B A 4 7 E S R containing the cwlA g e n e [8] w e r e cultured with shaking in LB m e d i u m [8] (100 ml) s u p p l e m e n t e d with ampicillin (50 # g / m l ) at 37°C until the early exponential phase, lsopropyl-fl-Dthiogalactopyranoside (final c o n c e n t r a t i o n 0.3 m M ) was t h e n a d d e d to the culture, followed by
f u r t h e r incubation for 2 h. A f t e r centrifugation at 6000 × g for 10 min, the cells w e r e s u s p e n d e d in 5 ml o f 0.1 M NaCI in buffer A (50 m M T r i s . HCI ( p H 8.0), 10 m M MgCI 2, I m M E D T A ) [4] and t h e n d i s r u p t e d by ultrasonication [8]. A f t e r centrifugation at l ( 1 0 0 0 x g for Ill min, the supernatant was applied to a column (2.2 by Ill cm) o f polyacrylamide gel containing B. subtilis cell wall (2(1{) mg o f cell wall was a d d e d to 20 ml o f a 12% ( w / v ) acrylamide solution and t h e n solidified, followed by h o m o g e n i z a t i o n with an A c e h o m o -
A 1oo I
=o 200
~o 50 I00
I
10 Time
!
20
!
30
t
0
10
I,
20
!
30
(min) Time (min) Fig. 1. Effect of CwbA on the cell wall lytie activity toward CwlA. The reaction mixture comprised B. subtilis cell wall (I.5 rag) partially purified with SDS and I /.tg of CwlA (e, o), 4.7 p.g of (\vbA ( II, El ), or I ~tg of CwlA and 4.7/.tg of CwbA ( A. zx). A. Cell wall turbidity at As.~t~was monitored at 37°C. B. after tl-, Ill-, 20- and 30-min incubation, 50t}-p.Isamples were removed, heated at gS°C for 10 min and then labelled with l-fluoro-2,4-dinitrobenzene to detect wall cleavage products as dinitrophenyl (DNP) amino acids [11]. Open and closed symbols indicate DNP-diaminopimelic acid (Dap) and DNP-t.-alanine (Ala). respectively. In this experiment, D-alaninc binding to a teichoic acid had been abolished with a lea (w/v) K., B407 solution (f~0°C, I h) [14].
g~'nizer (Nissei)) equilibrated with 0.1 M NaCI in buffer A. The column was washed with 3 volumes of 0.1 M NaCI in buffer A and then eluted with a linear gradient of NaCI in buffer A, from 0.1 M to 2 M NaCI. CwlA was eluted at a concentration of 0.5 M NaCI, and then dialysed against 1 M KCI in buffer A. CwbA was purified as described previously [7] except for the longer incubation of B. subtilis A D I harboring pHWABg containing cwbA [7], increased from 10 h to 14 h, and the higher concentration of B. subtilis cell wall, increased from 2.5 m g / m l to 10 m g / m l , in the gel for column chromatography. These changes enabled us to eliminate the second purification step which was previously performed, i.e., hydroxyapatite gel chromatography [7]. CwIM was purified as described previously [10]. CwbA and CwIM gave single bands on SDS-PAGE (data not shown). Specific activities of CwlA and CwIM used in this study for B. subtilis cell wall were 64(X) and 30 units per mg protein, respectively. Lysozyme of chicken egg white and N-acctylmuramidase SG of Streptomyccs globisponls 1829
A looq~
were purchased from Sigma and Seikagaku Kogyo Co., Tokyo, respectively.
Cell wall preparation B. subtilis vegetative cells and Micrococcus hlteus (lysodeikticus) cells (Sigma) were disrupted
3.Z
with a beadbeater (Biospec) and then partially purified with a SDS solution (4f4 w / v ) as described previously [8].
3.3. A,~say of cell waU lyric actirity Lytic activity was measured as follows. Thc reaction mixture (5 ml) comprised B. subtilis or M. haeus cell wall (to a final absorbancc at 540 nm (A540) of {).3) in buffer A containing 0.16 M KCI for CwiA or 0.1 M KCI for CwIM, lysozymc and muramidase SG, and was incubated with shaking at 37°C. Cell wall turbidity was mot.toted at 540 nm. The wall cleavage products w~-re identified as described previously [11]. One unit of enzyme activity was defined as the amount of enzyme necessary to decrease the A54() by 0.{X)I in 1 min.
B
C
1oo
ioo
~0
5o
,q r. -
~o
~ _ . , t _ .
....... 30
Time {min)
~. . . . . . . 60 o
i 30
Tir~e (min)
u 60
o
I 30 Time (mtn)
Fig. 2. Eflect of CwbA on the cell-wall lyric activities of CwlM, lysozyme and muramidase SG. The hydrolysis of B. subtilis cell wall by CwIM {42 p,g) (A), lysozyme (30 # g ) (B) or muramidase S G (20 ttg) (C), with (4.7 #,g) (o) or without CwbA {©), was monitored at
540 nm.
3.4. Protein determhtation Protein was determined using BCA protein assay reagent (Pierce), with btwine serum albumin as the standard.
4. RESULTS A N D DISCUSSION One-step purification of B. subtilis cell wall hydrolase CwlA was achieved by cell wall immobilized acrylamide gel chromatography. It gave a single protein band on S D S - P A G E and its M~ (23000) was similar to that reported previously [11]. Figure IA shows that the addition of CwbA to CwlA stimulates the cell wall hydrolysis, as judged when the rate of the reduction in cell wall turbidity was measured at 5411 nnl. The stimulation was not observed on the addition of CwlA to the substrate mixture containing B. subtilis cell wall which had been preincubated with CwbA at 37°C for 30 min, followed by heat inactivation at 95°C for 10 rain (data not shown). This result suggests that the stimulation is not due to irreversible degradation of the cell wall. Examination of the cell wall cleavage products obtained in the absence and presence of CwbA indicated that the effect of CwbA is not a change in the enzyme velocity (Fig. IB). Herbold and Glaser reported a similar result for the modifier protein and the major autolysin from B. subtilis A T C C 6051, and proposed a mechanism by which the modifier protein changes the pattern of cleavage of the major autolysin from a random one to a sequential one [6]. Figure 2 shows the effect of CwbA on the activities of B. licheniformis amidase (CwiM), egg white lysozyme and Streptomyces globi.~l~orus Nacetylmuramidase SG (muramidase SG). CwbA also stimulated the amidase activity of CwlM, but did not stimulate the activities of lysozyme and muramidase SG, which cleave glycan strands in cell walls. These results indicate that the effects of CwbA seem to be specific to amidases, but not always specific to the major autolysin (CwlB) ([7]; Fig. 2). CwlA exhibits no amino acid sequence homology with CwlB, CwlM o r CwbA, and the N-terminal amino acid sequence of CwlM is only homologous to the C-terminal of CwlB [4,8,10].
1.o,
o
~
CwbA ( Itg ) Fig. 3. Effect of the ('wbA concentration on the {'wlA activity. The r~tes of hydrolysis of B. subtilis cell wall (e) and M. bin,us
cell wall (o) by CwlA (I ~tLg)were measured in ,454"with the indicated amounts of CwbA. Relative activity was defined as the ratio of the time necessaD' to reach 81|¢~.;of the initial As~, without CwbA to lhat with the indicated amount of CwbA. For example, the mol~lrralio of 5 .u.g of CwbA to 1 .ug of CwlA is 1.53.
Although direct binding of the modifier protein to the major autolysin was reported by Herbold and Glaser [5], CwbA may not bind to CwlA a n d / o r CwlM and thus the binding may be unnecessary for stimulation of the amidase activity. CwlB has a narrow substrate specificity and does not lyse M. haeus cell wall ([Ill]; unpublished results). But CwlA lyses various types of cell walls, e.g., M. haeus cell wall, B. sphaericus vegetative cell wall and B. megaterium KM spore cortex peptidoglycan [9]. Figure 3 shows the stimulation by CwbA of the hydrolysis of Micrococcus cell wall with CwlA. Previous reports suggested that a teichoic acid is essential for stimulation by the modifier protein of the major autolysin activity [5,6], but M. haeus does not contain a teichoic acid but a tcichuronic acid [12,13]. Therefore teichuronic acid may play a similar role to tei-
113 choic acid, or teichoic acid and teichuronic acid may be unnecessary for the effect of CwbA on the CwlA activity. We report here the stimulatory effect of CwbA on at least three amidases (all of the tested amidases). The N-terminal amino acid sequence of CwbA is highly homologous to the sequence of the N-terminal non-catalytic and cell wall-binding domain of CwlB [4,7]. The C-terminal sequence of CwbA is highly homologous to about one half of the whole amino acid sequence of the S o o l I D protein [7]. Since the S o o l l D protein is required for the removal of septal peptidoglycan at sporulation stage II [15], the effect of CwbA on amidases will be very important for investigating the properties of SpollD. it is very likely that the N-terminal domains of CwbA and CwiB play roles in the specific binding with a teichoic acid [4-7]. CwbA and ( ~ ' I B bound to B. subtilis cell wall in buffer A containing 1 M LiCI, but did not bind to M. luteus cell wall, even in buffer A containing 0.5 M LiCI {data not shown). O n the o t h e r hand, CwlA did not bind to either cell wall in buffer A containing 0.5 M LiCI (data not shown). Since neither CwbA nor CwlA binds tightly to Microctu'cus cell wall (data not shown), there seems to be no relation between tight binding and stimulation by CwbA.
REFERENCES [ I ] Mauck, J.. Chan. l,. and Gla,~r, L. (1971) J. Biol. Chem. 246, 1820- I,~27. [2] Rogers. ll.J.. Perkins, II.R. and Ward, J.B. (19~I} Microbial ('ell Walls and Membranes. Chapman and llall, London. [3] Ward, J.B. and William.'~m, R. 119841 In: Microbial con Wall Synthesis and Autolysis. (Nombela, C.. Ed.), pp. 159-166, Elsevier. Amsterdam. [41 Kuroda, A. and Sekiguchi, 1. 119911 J. Bactcriol. 173, 73114- 7312. [51 llcrl~fld, D R . and Gla,~r. L 110751 J. Biol. Chem. 2511, 1676-16S2. [6] tlerl'~fld. D.R. and Glaser, L (19751 J. Biol. ('hem. 2511, 7231-7238. [7] Kuroda, A., Rashid, M.il. and Sekiguehi, J. (It1921 J. Gen. Microbiol. 13,vl, in pr¢~s. [8] Kuroda, A. and Sekiguchi. J. 119901 J. Gen. Mierobiol.
136, 2211¢)-2216. [9] Foster, SJ. (1~911J. Gen. Mierobiol. 137, 1987-1¢~8. Jill] Kumda. A.. Sugimoto, Y.. Funahashi, T. and Sekiguchi, J. (19~21 Mol. Gen. Genet.. in press. [I I1 Kuroda, A., Imazeki. M and Sekiguchi. J. (1991) FEMS Microbiol. Len. 81, 9-14. [12] ttase, S. and Matsushima. Y. (11~77) J. Biochem. ~1, 1181-1186. [131 Nasir-ud-Din, Lhermine. M.. Lamblin, G. and Jeanloz. R.W. {19851J. Biol. ('hem. 2~1, '-~81-9987. [141 Matsuda, T., Kotani. S. and Kato, K. (1968) Riken J. I I , 111-126.
[15] llling, N. and Errington, J. (1991) J. Bacteriol. 173. 31593169.
FEMSLE 114082
Characterization of the Bacillus subtilis CwbA protein which stimulates cell wall lytic amidases Akio Kuroda and Junichi Sckiguchi Department t~t"Al~pli,'d IJioh~,t,%Faculty c~t"T~ttih" Sch'ncc and Tet'hnoh,gy. Sh/,I,~hu Umt cr,sity, Nagano, Japan Recek'ed II May 1002 Accepted 12 May 1092 Key words: Autolysin; N-Acetylmuramoyl-i.-alanine amidasc: Modifier protein; Bacillus subtilis; Bacillus lichenifonnis
1. S U M M A R Y T h e Bacillus subtilis cell wall binding protein, CwbA, stimulated the cell wall lytic activities of the B. subtilis and B. lichenilbrmis autolysins (CwlA and CwlM, respectively) in addition to that of the major B. subtilis autolysin (CwlB). Even though the substrate for the enzyme reaction was changed from B. subtili,~ cell wall conraining a teichoic acid to Micrococcus haeus cell wall containing a teichuronic acid, the stimulatory effect of CwbA on CwlA activity was observed.
2. I N T R O D U C T I O N In Bacillus subtilis the major autolysin is an N-acetyhmtramoyl-L-alanine amidase ('amidase') which is responsible tot cell wall turnover [ I - 3 ] and cell lysis at the stationary phase [4]. Herbold and Glaser reported the purification of a major Correspondence to: J. Sekiguchi. Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, 3-15-I Tokida, Ueda-shi, Nagan~, 386, Japan.
B. s:tblilis ATCC 61)51 amidase to homogeneity, as well as the purtfication of a second protein (modifier pre,,ein), which in combination with the amidase stimulates enzyme activity [5,6]. Recently, we reportc6 the cloning and sequencing of a major amidase (AI r 46000) gene (cwIB) from B. subtili,~ 168S [4]. Moreover, a cwbA gcne which encodes a cell wall binding protein of B. subtilis 168S was located in the same operon [4,7]. Since CwbA stimulated the enzyme activity of the major autolysin, CwlB, about 3-fold, and the M r (75000) and amino acid composition of CwbA were similar to those of the modifier protein, wc could regard CwbA as a modifier protein from strain 168S [7]. On the other hand, B. subtilis 168S produces another type of amidase (CwlA; M r 23000) [8], which reflects the difference in enzyme specificity from CwIB [4,8,9]. B. licheniforints also produces an amidase (CwIM; M r 291H1()) which has the ability to digest B. licheniforints, 13. subtilis and Micrococcus haeus cell walls [ I0]. We report here the effect of CwbA on the amidasc activities of CwlA and CwlM on 13. subtilts a n d / o r Micrococcus hlteus cell walls.
3. M A T E R I A L S A N D M E T H O D S
3.1. l:,'nzyme preparation Purification o f CwlA from Escherichia colt was carried out as follows. E. colt J M I 0 9 cells harboring p B A 4 7 E S R containing the cwlA g e n e [8] w e r e cultured with shaking in LB m e d i u m [8] (100 ml) s u p p l e m e n t e d with ampicillin (50 # g / m l ) at 37°C until the early exponential phase, lsopropyl-fl-Dthiogalactopyranoside (final c o n c e n t r a t i o n 0.3 m M ) was t h e n a d d e d to the culture, followed by
f u r t h e r incubation for 2 h. A f t e r centrifugation at 6000 × g for 10 min, the cells w e r e s u s p e n d e d in 5 ml o f 0.1 M NaCI in buffer A (50 m M T r i s . HCI ( p H 8.0), 10 m M MgCI 2, I m M E D T A ) [4] and t h e n d i s r u p t e d by ultrasonication [8]. A f t e r centrifugation at l ( 1 0 0 0 x g for Ill min, the supernatant was applied to a column (2.2 by Ill cm) o f polyacrylamide gel containing B. subtilis cell wall (2(1{) mg o f cell wall was a d d e d to 20 ml o f a 12% ( w / v ) acrylamide solution and t h e n solidified, followed by h o m o g e n i z a t i o n with an A c e h o m o -
A 1oo I
=o 200
~o 50 I00
I
10 Time
!
20
!
30
t
0
10
I,
20
!
30
(min) Time (min) Fig. 1. Effect of CwbA on the cell wall lytie activity toward CwlA. The reaction mixture comprised B. subtilis cell wall (I.5 rag) partially purified with SDS and I /.tg of CwlA (e, o), 4.7 p.g of (\vbA ( II, El ), or I ~tg of CwlA and 4.7/.tg of CwbA ( A. zx). A. Cell wall turbidity at As.~t~was monitored at 37°C. B. after tl-, Ill-, 20- and 30-min incubation, 50t}-p.Isamples were removed, heated at gS°C for 10 min and then labelled with l-fluoro-2,4-dinitrobenzene to detect wall cleavage products as dinitrophenyl (DNP) amino acids [11]. Open and closed symbols indicate DNP-diaminopimelic acid (Dap) and DNP-t.-alanine (Ala). respectively. In this experiment, D-alaninc binding to a teichoic acid had been abolished with a lea (w/v) K., B407 solution (f~0°C, I h) [14].
g~'nizer (Nissei)) equilibrated with 0.1 M NaCI in buffer A. The column was washed with 3 volumes of 0.1 M NaCI in buffer A and then eluted with a linear gradient of NaCI in buffer A, from 0.1 M to 2 M NaCI. CwlA was eluted at a concentration of 0.5 M NaCI, and then dialysed against 1 M KCI in buffer A. CwbA was purified as described previously [7] except for the longer incubation of B. subtilis A D I harboring pHWABg containing cwbA [7], increased from 10 h to 14 h, and the higher concentration of B. subtilis cell wall, increased from 2.5 m g / m l to 10 m g / m l , in the gel for column chromatography. These changes enabled us to eliminate the second purification step which was previously performed, i.e., hydroxyapatite gel chromatography [7]. CwIM was purified as described previously [10]. CwbA and CwIM gave single bands on SDS-PAGE (data not shown). Specific activities of CwlA and CwIM used in this study for B. subtilis cell wall were 64(X) and 30 units per mg protein, respectively. Lysozyme of chicken egg white and N-acctylmuramidase SG of Streptomyccs globisponls 1829
A looq~
were purchased from Sigma and Seikagaku Kogyo Co., Tokyo, respectively.
Cell wall preparation B. subtilis vegetative cells and Micrococcus hlteus (lysodeikticus) cells (Sigma) were disrupted
3.Z
with a beadbeater (Biospec) and then partially purified with a SDS solution (4f4 w / v ) as described previously [8].
3.3. A,~say of cell waU lyric actirity Lytic activity was measured as follows. Thc reaction mixture (5 ml) comprised B. subtilis or M. haeus cell wall (to a final absorbancc at 540 nm (A540) of {).3) in buffer A containing 0.16 M KCI for CwiA or 0.1 M KCI for CwIM, lysozymc and muramidase SG, and was incubated with shaking at 37°C. Cell wall turbidity was mot.toted at 540 nm. The wall cleavage products w~-re identified as described previously [11]. One unit of enzyme activity was defined as the amount of enzyme necessary to decrease the A54() by 0.{X)I in 1 min.
B
C
1oo
ioo
~0
5o
,q r. -
~o
~ _ . , t _ .
....... 30
Time {min)
~. . . . . . . 60 o
i 30
Tir~e (min)
u 60
o
I 30 Time (mtn)
Fig. 2. Eflect of CwbA on the cell-wall lyric activities of CwlM, lysozyme and muramidase SG. The hydrolysis of B. subtilis cell wall by CwIM {42 p,g) (A), lysozyme (30 # g ) (B) or muramidase S G (20 ttg) (C), with (4.7 #,g) (o) or without CwbA {©), was monitored at
540 nm.
3.4. Protein determhtation Protein was determined using BCA protein assay reagent (Pierce), with btwine serum albumin as the standard.
4. RESULTS A N D DISCUSSION One-step purification of B. subtilis cell wall hydrolase CwlA was achieved by cell wall immobilized acrylamide gel chromatography. It gave a single protein band on S D S - P A G E and its M~ (23000) was similar to that reported previously [11]. Figure IA shows that the addition of CwbA to CwlA stimulates the cell wall hydrolysis, as judged when the rate of the reduction in cell wall turbidity was measured at 5411 nnl. The stimulation was not observed on the addition of CwlA to the substrate mixture containing B. subtilis cell wall which had been preincubated with CwbA at 37°C for 30 min, followed by heat inactivation at 95°C for 10 rain (data not shown). This result suggests that the stimulation is not due to irreversible degradation of the cell wall. Examination of the cell wall cleavage products obtained in the absence and presence of CwbA indicated that the effect of CwbA is not a change in the enzyme velocity (Fig. IB). Herbold and Glaser reported a similar result for the modifier protein and the major autolysin from B. subtilis A T C C 6051, and proposed a mechanism by which the modifier protein changes the pattern of cleavage of the major autolysin from a random one to a sequential one [6]. Figure 2 shows the effect of CwbA on the activities of B. licheniformis amidase (CwiM), egg white lysozyme and Streptomyces globi.~l~orus Nacetylmuramidase SG (muramidase SG). CwbA also stimulated the amidase activity of CwlM, but did not stimulate the activities of lysozyme and muramidase SG, which cleave glycan strands in cell walls. These results indicate that the effects of CwbA seem to be specific to amidases, but not always specific to the major autolysin (CwlB) ([7]; Fig. 2). CwlA exhibits no amino acid sequence homology with CwlB, CwlM o r CwbA, and the N-terminal amino acid sequence of CwlM is only homologous to the C-terminal of CwlB [4,8,10].
1.o,
o
~
CwbA ( Itg ) Fig. 3. Effect of the ('wbA concentration on the {'wlA activity. The r~tes of hydrolysis of B. subtilis cell wall (e) and M. bin,us
cell wall (o) by CwlA (I ~tLg)were measured in ,454"with the indicated amounts of CwbA. Relative activity was defined as the ratio of the time necessaD' to reach 81|¢~.;of the initial As~, without CwbA to lhat with the indicated amount of CwbA. For example, the mol~lrralio of 5 .u.g of CwbA to 1 .ug of CwlA is 1.53.
Although direct binding of the modifier protein to the major autolysin was reported by Herbold and Glaser [5], CwbA may not bind to CwlA a n d / o r CwlM and thus the binding may be unnecessary for stimulation of the amidase activity. CwlB has a narrow substrate specificity and does not lyse M. haeus cell wall ([Ill]; unpublished results). But CwlA lyses various types of cell walls, e.g., M. haeus cell wall, B. sphaericus vegetative cell wall and B. megaterium KM spore cortex peptidoglycan [9]. Figure 3 shows the stimulation by CwbA of the hydrolysis of Micrococcus cell wall with CwlA. Previous reports suggested that a teichoic acid is essential for stimulation by the modifier protein of the major autolysin activity [5,6], but M. haeus does not contain a teichoic acid but a tcichuronic acid [12,13]. Therefore teichuronic acid may play a similar role to tei-
113 choic acid, or teichoic acid and teichuronic acid may be unnecessary for the effect of CwbA on the CwlA activity. We report here the stimulatory effect of CwbA on at least three amidases (all of the tested amidases). The N-terminal amino acid sequence of CwbA is highly homologous to the sequence of the N-terminal non-catalytic and cell wall-binding domain of CwlB [4,7]. The C-terminal sequence of CwbA is highly homologous to about one half of the whole amino acid sequence of the S o o l I D protein [7]. Since the S o o l l D protein is required for the removal of septal peptidoglycan at sporulation stage II [15], the effect of CwbA on amidases will be very important for investigating the properties of SpollD. it is very likely that the N-terminal domains of CwbA and CwiB play roles in the specific binding with a teichoic acid [4-7]. CwbA and ( ~ ' I B bound to B. subtilis cell wall in buffer A containing 1 M LiCI, but did not bind to M. luteus cell wall, even in buffer A containing 0.5 M LiCI {data not shown). O n the o t h e r hand, CwlA did not bind to either cell wall in buffer A containing 0.5 M LiCI (data not shown). Since neither CwbA nor CwlA binds tightly to Microctu'cus cell wall (data not shown), there seems to be no relation between tight binding and stimulation by CwbA.
REFERENCES [ I ] Mauck, J.. Chan. l,. and Gla,~r, L. (1971) J. Biol. Chem. 246, 1820- I,~27. [2] Rogers. ll.J.. Perkins, II.R. and Ward, J.B. (19~I} Microbial ('ell Walls and Membranes. Chapman and llall, London. [3] Ward, J.B. and William.'~m, R. 119841 In: Microbial con Wall Synthesis and Autolysis. (Nombela, C.. Ed.), pp. 159-166, Elsevier. Amsterdam. [41 Kuroda, A. and Sekiguchi, 1. 119911 J. Bactcriol. 173, 73114- 7312. [51 llcrl~fld, D R . and Gla,~r. L 110751 J. Biol. Chem. 2511, 1676-16S2. [6] tlerl'~fld. D.R. and Glaser, L (19751 J. Biol. ('hem. 2511, 7231-7238. [7] Kuroda, A., Rashid, M.il. and Sekiguehi, J. (It1921 J. Gen. Microbiol. 13,vl, in pr¢~s. [8] Kuroda, A. and Sekiguchi. J. 119901 J. Gen. Mierobiol.
136, 2211¢)-2216. [9] Foster, SJ. (1~911J. Gen. Mierobiol. 137, 1987-1¢~8. Jill] Kumda. A.. Sugimoto, Y.. Funahashi, T. and Sekiguchi, J. (19~21 Mol. Gen. Genet.. in press. [I I1 Kuroda, A., Imazeki. M and Sekiguchi. J. (1991) FEMS Microbiol. Len. 81, 9-14. [12] ttase, S. and Matsushima. Y. (11~77) J. Biochem. ~1, 1181-1186. [131 Nasir-ud-Din, Lhermine. M.. Lamblin, G. and Jeanloz. R.W. {19851J. Biol. ('hem. 2~1, '-~81-9987. [141 Matsuda, T., Kotani. S. and Kato, K. (1968) Riken J. I I , 111-126.
[15] llling, N. and Errington, J. (1991) J. Bacteriol. 173. 31593169.