JOURNAL OF FERMENTATION AND BIOENGINEERJW Vol. 80, No. 3, 229-236. 1995

Structure of the Gene Encoding b-1,3-Glucanase B of Bacillus circulans WL- 12

TAKESHI OKADA, MAKOTO AISAKA, KAZUHIKO AIDA, NAOKI NIKAIDOU, HIROSATO TANAKA, AND TAKESHI WATANABE*

Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, 8050 Ikarashi-2, Niigata-shi, Niigata 950-21, Japan

Received 6 April 1995/Accepted 30 May 1995

,&l,S-Glucanases B (GlcB) and C (GlcC) are the major @-1,fglucanases of Bacillus circulans WL-12 detected in the culture supernatant grown in ,%l,fgiucan-free medium. The gene (glcB) encoding GicB was cloned into Escherichia coli and its nucleotide sequence was determined. The open reading frame of the glcB gene encodes a polypeptide of 412 amino acid residues. The N-terminal amino acid sequences of GlcB and GlcC purified from B. circukms culture supematant were determined to be identical to each other and to the deduced sequence downstream of Ala-29. The N-terminal amino acid sequence, isoelectric point and estimated size of the p- 1,fglucanase produced by E. coli cells carrying the cloned gZcB gene agreed well with those of GlcB of B. circukzns WL-12. The N-terminal to central region of GlcB exhibited high sequence similarity to ,9-1,3- glucanases of aikalophilic BaciZZus AG-430, GlcAl of B. circulans WL-12 and the 87 kDa p-1,3-glucanase H of B. circulans IAM1165. The C-terminal region of GlcB exhibited sequence similarity to the C-terminal regions of XlnA of Streptomyces lividans, ,8-1,3-giucanases of Oerskovia xanthineolytica and Arthrobacter sp. YCWD3, and yeast lytic protease I of Rarobacter faecitabidus. Biochemical and sequence data strongly sug gested that GlcC detected in the culture supematant of B. circulans WL-12 corresponded to the catalytic domain of GlcB generated by loss of a C-terminal region of GlcB.

wey words: j-1,3-glucanase, Bacillus circulans WL-12, glcB]

Six distinct ,8-1,3-glucanases (GlcAl, GlcA2, GlcA3, GlcA4, GlcB and GlcC) were detected in the culture supernatant of Bacillus circulans WL-12 when induced with pachyman, an insoluble p-1,3-glucan (1). Produc- tion of multiple ,9-1,3-glucanases has been reported for various microorganisms (2-7). However, the reason for the production of multiple ,8-l,f-glucanases, how they are produced and how they function in the degradation of j-1,3-glucan are not well understood. Among the six ,B-1,3-glucanases detected in B. circulans culture super- natant, GlcA2, GlcA3 and GlcA4 are derived from GlcAl, the product of the glcA gene (1, 8, 9). The other j-1,3_glucanases, GlcB and GlcC, are thought to be de- rived from another glucanase gene, glcB. Thus, the j-1,3- glucan-degrading enzyme system of this bacterium is assumed to consisted of products of the two distinct ,9- 1,3-glucanase genes, glcA and glcB. Identification of the glcB gene and elucidation of the properties and function of GlcB and GlcC are very important for understanding the p-1,3-glucan-degrading enzyme system of this bacteri- um (9). Considerable differences in the enzymatic proper- ties between GlcAl and GlcB are described elsewhere (10). Here, we describe the cloning and nucleotide se- quence of a newly identified B-1,3-glucanase gene (glcB) of B. circulans WL-12 encoding GlcB (and GlcC).

MATERIALS AND METHODS

Bacterial strains and media Escherichia coli JM109 cells carrying pUC19 or derivatives thereof were grown in L-broth medium containing 100 pg/mi of ampicillin. B. circulans WL-12 was grown in modified yeast-nitro- gen base (YNB, 11) medium containing 0.5% casein,

* Corresponding author.

0.2% pachyman and 0.5% yeast extract for enzyme production. For preparation of chromosomal DNA, the bacterium was grown in L-broth medium.

Construction of recombinant piasmid and manipuia- tion of DNA Chromosomal DNA was prepared from B. circulans WL-12 by the method described by Silhavy et al. (12). The chromosomal DNA was partially digest- ed with TaqI, and 2-10 kb DNA fragments were collect- ed by the method described by Maniatis et al. (13). The DNA fragments were ligated to dephosphorylated AccI- digested pUC19. E. coli JM109 cells were transformed with the ligated DNA and transformants carrying recom- binant plasmids were selected on L-broth agar plates con- taining isopropylthio-P-D-galactoside (IPTG, 40 pg/ml), 5-bromo-4-chloro-3-indolyl-B_Dgalactoside (X-gal, 40 pg/ ml) and ampicillin (100 pg/ml). The transformants were transferred onto L-broth agar plates containing 0.5% (w/v) pachyman, 0.02% (w/v) Congo red and lOOpg/ml of ampicillin. The plates were incubated at 33°C for 24 to 72 h, and the production of p-1,3-glucanase was detected by the formation of an orange-colored zone around the colony on the red background. Rapid prepa- ration of piasmid DNA was performed by the method described by Silhavy et al. (12). Large-scale preparation of plasmid DNA was carried out by the method de- scribed by Maniatis et al. (13).

Nucieotide sequencing The recombinant plasmid pMTOO2 carrying the glcB gene was digested with SalI, and two SalI fragments carrying a region complementary to the glcB gene were separately subcloned to obtain the piasmids pMTOl1 and pMTO12. Overlapping deletions were introduced into the inserted DNA fragment of each of these two piasmids and a piasmid with the fragment in reverse orientation with a deletion kit purchased from Takara Shuzo Co. Ltd. (Osaka). Regions of appropriate

229

230 OJCADA ET AL.

size of the deletion derivatives were sequenced by the dideoxy chain termination method of Sanger et al. (14) using Sequenase purchased from U.S. Biochemical (Cleaveland, USA). The nucleotide sequence of the junc- tion region of the two San fragments was confirmed by sequencing pMTOO2 using a synthetic sequencing primer complementary to the region near the SafI junction.

SDS-PAGE Sodium dodecyl sulfate-polyacryl- amide gel electrophoresis (SDS-PAGE) in 10% slabs was carried out as described by Ames (15), using the buffer system of Laemmli (16). After the electrophoresis was completed, renaturation of the enzymes and detec- tion of the P-1,3-glucanase activity in the gel were per- formed as described previously (1).

Preparation of the crude GlcB from E. coli E. coli JM109 cells carrying the plasmid pMTOO1 were grown in L-broth medium containing ampicillin at 33’C for 15 h. The cells were collected by centrifugation, and b-1,3- glucanase was extracted from the periplasmic space of E. coli by the osmotic shock procedure as described by Manoil and Beckwith (17). The extract was dialyzed against 0.02 M succinate buffer (pH 6.0) and lyophilized. The purification procedure for GlcB produced by E. coli was described elsewhere (10).

N-terminal amino acid sequence analysis The pro- teins to be subjected to analysis of the N-terminal amino acid sequence were separated from the other proteins by SDS-PAGE. The protein band was either electrophoreti- tally extracted from the polyacrylamide gel after brief staining with Coomassie brilliant blue R-250 as described previously (18), or electrophoretically blotted onto poly- vinylidene difluoride (PVDF) membrane by the method described by Matsudaira with some modifications (19). The N-terminal amino acid sequences of these proteins were determined with a gas-phase protein sequencer (Applied Biosystems, model 47OA).

Enzyme assay p-1,3Glucanase activity was mea- sured by 3,5dinitrosalicylic acid methods using laminarin as an assay substrate (20).

Chemicals Pachyman was prepared from the tree fungus Poria cocus Wolf and laminarin was prepared from Laminaria japonica as described previously (20).

RESULTS

Some properties of the &1,3-glucanases B and C When B. circulans WL-12 was grown in the presence of pachyman, a P-1,3-glucose poIymer, at least six distinct B-1,3-glucanase species were detected in the culture medi- um as described previously (1). When pachyman was sub- stituted with either glucose or chitin, the bacterium still produced a low level of /3-1,3-glucanase activity as shown in Fig. 1. The p-1,3-glucanase activity detected in the medium containing glucose was less than one-tenth that detected in the medium containing pachyman. The activity detected in the medium containing chitin was markedly higher than that detected in the medium con- taining glucose, and approximately one-fifth that detect- ed in the medium containing pachyman. The b-1,3- glucanase species produced in the medium containing either pachyman, chitin, or glucose were analyzed by SDS-PAGE as shown in Fig. 2. The results revealed that GlcB (and GlcC) is/are the major ,9-1,3glucanase species produced in the absence of P-1,3-glucan (uninduced con- dition). The protein bands corresponding to GlcB and GlcC were electrophoretically extracted from the poly-

J. FERMENT. BIOENG.,

0 24 49 72 96 120 Time lh)

P-1,3-Glucanase activities detected in the culture super- natant of B. circulans WL-12 during cultivation. B. circulans WL-12 was grown in the modified YNB medium containing casein and various supplements. Supplements: yeast extract and pachyman ( l ), yeast extract and chitin (A), yeast extract and glucose ( q ), glucose ( n ).

acrylamide gel and N-terminal amino acid sequences were determined by automated Edman degradation. The two b-1,3-glucanases exhibited apparently identical amino acid sequences, ATNWNLVWSDEFXXXSLNT- for GlcB and ATNWNLVWSXEFNGSSLNT- for GlcC. The results suggested that GlcC was derived from GlcB by loss of a C-terminal region of the protein. Although an amino acid region with some sequence similarity to the N-terminal region of GlcB (and GlcC) was found in the catalytic domain of Al, the N-terminal regions of GlcB and GlcC did not perfectly match any region of the amino acid sequence deduced from the glcA gene nucleo- tide sequence (8). Thus, the gene encoding GlcB (and

1 2 3

a b a b a b FIG. 2. SDS-PAGE analysis of the crude enzymes prepared from

culture supernatant of B. circulans WL-12. B. circulans WL-12 was grown in the medium containing yeast extract and either pachyman (lane 1, 40 pg protein was applied), chitin (lane 2, 40 ,ug protein), or glucose (lane 3, 15 ,~g protein). a, Protein staining with Coomassie brilliant blue R250; b, ,9-l ,3-glucanase activity detected on agar replica of the polyacrylamide gel.

VOL. 80, 1995 CLONING AND SEQUENCING OF glcB GENE 231

~MT002 .-....,

;'.,, ' ' !.:, I, II I

,:' ,: ..,_ ,/,' ,I' '.,

'.._

; ;' '... '.._

,.,I '.., '..,

.._ ,.1' '._ '.._

pMTOl1 pMT012

FIG. 3. Restriction map of the DNA fragments carrying the B- 1,3-glucanase gene on the recombinant plasmids pMTOO1, pMTOO2, pMTOl1 and pMTOl2. Bold lines indicate inserted DNA fragments derived from B. circulans WL-12. Thin lines represent the DNA of vector plasmids pUC19.

GlcC) must be different from the glcA gene. Cloning of the gene encoding GlcB Chromosomal

DNA prepared from B. circulans was partially digested with TaqI and DNA fragments 2 to 10 kb in size were collected. The fragments were ligated to AccI-digested and alkaline phosphatase treated pUC19, and were used to transform JM109 cells. Approximately 500 transfor- mants carrying recombinant plasmids were obtained in a single transformation experiment, and all of the transfor- mants were transferred onto L-broth agar plates contain-

(A)

(B) 66

FIG. 4. Clearing zones and orange-colored zones formed by E. coli transformants and purified j-1,3_glucanases. (A) E. coli JM109 carrying either pNTOO3 or pMTOO1 was inoculated on the L-broth agar plate containing either pachyman (left plate) or pachyman plus Congo red (right plate). The plates were incubated at 33’C for 2 d. (B) GlcAl and GlcB were spotted on the agar plate containing either pachyman (left plate) or pachyman plus Congo red (right plate). The plates were incubated at 33°C for 24 h.

a

Tube number

FIG. 5. Isoelectric focusing analysis of /S-1,3-glucanase produced by E. coli JM109 (pMTOO1). E. coli JM109 (pMTOO1) was grown at 30°C for 15 h and the periplasmic fraction was prepared as described in the text. l **, pH; 0, j-1,3-glucanase activity.

ing pachyman, Congo red, and ampicillin. After 24 h incubation, orange-colored zones on the red background were observed around two transformant colonies. The recombinant plasmids detected in these transformants were designated as pMTOO1 and pMT002. pMTOO1 car- ries a 9 kb inserted DNA fragment and pMTOO2 carries a 6 kb inserted DNA fragment derived from B. circulans chromosomal DNA. Restriction maps indicated that the inserted DNA fragments in the two plasmids overlapped each other (Fig. 3). The restriction maps of the overlap- ping region of the plasmids did not match that of the inserted DNA fragment of pNTOO3 which carried the g/CA gene (1). Thus, the p-1,3glucanase gene cloned in the present study is distinct from the glcA gene previously

kDa

32 +C

+B

-C

a b a b

FIG. 6. SDS-polyacrylamide gel electrophoresis of the cloned /% 1,3-glucanase produced by E. coii JM109 (pMTOO1). Lane 1, Crude enzyme (1OOpg protein) prepared from culture supernatant of B. circulans WL-12 grown in the medium containing chitin; lane 2, periplasmic protein (40 ,ug) prepared from E. cob JM109 (pMTOO1). a, Protein staining with Coomassie brilliant blue R-250; b, b-1,3- glucanase activity.

232 OKADAETAL. J. FERMENT. BIOENG

10 20 30 40 50 60 ATGTCCCCAATATATTAAAGGCAACAAGGTATGGACCGTTCATTCGCACAGGCCTGTTF&

70 80 90 100 110 120 AATACTTC~UATGCTGTCTTGTCCATGGTATATCCTGTTCG~TGGTAGAAATAATCGAGT --

130 140 150 160 170 180 TGCATGCGGAATACATTTAATGTAAACGTTGTTACGGATTGA GAAAAATGGGGTATCGTC

190 200 210 220 230 240 GTTCAGAATCAGTCCGTTAATATGGGGAGGGAAAA GATTGTTGTCGT

MLRKRKIV

250 260 270 280 290 300 AAGTATGCTGGTGCTGTTCAGTTTCCTAATCGcTTTACTGcCTGGcACGACGAcAAACGc SMLVLFSFLIALLPGTTTNA

310 320 330 340 350 360 TGCGACCAATTGGAATCTGGTGTGGAGTGAATTCAATC 4 ATNWNLVWSDEFNGSSLNTS

370 380 390 400 410 420 GAATTGGTCTGCGGAGATTGGTACCGGCAGcGGcGGAT GGGGAAATAATGAATTGCAGTA NW S AE IGTGSGGWGNNELQY

430 440 450 460 470 480 TTATACGAATCGCACCGAGAACCTGCAAGTTACAGGTGGcAATCTGG!l'CATcACTGcACG YTNRTE'NLQVTGGNLVITAR

490 500 510 520 530 540 CAAAGAAAATTACAATGGCAGCAGCTATACCTCCGCACGAAT~CCCAAGGCTTGAA KENYNGSSYTSARIXTQGLK

550 560 570 580 590 600 GGACTTTACTTAC GGGAAAGTTGUGCACGCATCAAGCTGCCGTCCGGTCAGGGGCTGTG DFTYGKVEARIKLPSGQGLW

610 620 630 640 650 660 GCCTGCTTTTTGGATGCTGGGAAGCAACATTAATTCCGTCGGCTGGCCTAAGAGCGGAGA PAFWMLGSNINSVGWPKSGE

670 680 690 700 710 720 GATCGACATTATGGAGCGGGTCAACAACAATGCTTTTGTAAACGGTACCGTGCACTGGGA IDIMERVNNNAFVNGTVHWD

730 740 750 760 770 780 TGCTAACGGGCATGCCGATTATGGAAAGATATCGGAGAATCTTGATTTTTCCCAATTTCA ANGHADYGKISENLDFSQFH

790 800 810 820 830 840 TGTCTACAGCATCt%ATGGGATTCCAAATATATAAGATGGTTTGTGGATGGcAAACAATT VYSIEWDSKY IRWFVDGRQF

850 860 870 880 890 900 CAATGAGTTTTACATCGGGAACAGGCAATACGGAGGAGTTCCAACGTCCGTTTTT NEFYIENGTGNTEEFQRPFF

910 920 930 940 950 960 CCTCCTGTTGAATATGGCTGTGGGTGGGAATTGGCCTGGCAGCCCTAATAATGCGACACC LLLNMAVGGNWPGSPNNATP

970 980 990 1000 1010 1020

TTTCCCATCACkAATGCTGGTGGATTATGTGCGGGTATATCAAGCAGCGAGTACACCGAA

FPSQMLVDYVRVYQAASTPN

VOL. 80, 1995 CLONING AND SEQUENCING OF glcB GENE 233

1030 1040 1050 1060 1070 1080

CATTGTCAGCGGGGGT CTCTATACGATCGGTTCCAAAGCAAGCGGTAAAGTGTTGGATAT

IVSGGLYT IGSKASGKVLDI

1090 1100 1110 1120 1130 1140

TGTGGATGTATCCACGGCAAGTGGAGCCAAAGTACAACAATGGACCAACTACAGCGCAAG

VDVSTASGAK V Q Q W TNYSAS

1150 1160 1170 1180 1190 1200

CAACCAAACGTTCAGAGTCGACAGCACAGGGGATGGTTTCTATAAACTCACGGCTGTTCA

NQTFRVDSTGDGFYKLTAVH

1210 1220 1230 1240 1250 1260

CAGCGGAAAAGTTCTGGATGTGCCTAACTCAACAGCATCACCAGGCGTGCAGCTCCAGCA

SGKVLDVPN STASPGVQLQQ

1270 1280 1290 1300 1310 1320

ATGGGATGATAACGGCTCTAACGCCCAAAGGTGGAGCATCCAGGATGCGGGT~CGGTTA

WDDNGSNAQRWS IQDAGNGY

1330 1340 1350 1360 1370 1380

CTATAAAATCATTTCCAAGGTGAATGGATTGGCAGTAGATGTTTCGAGCTCTTCGACGGC Y K I I SKVNGLAVDV SSSSTA

1390 1400 1410 1420 1430 1440

GGATGGGGCTGCAATCCAGCAATGGAATGATAACGGTACAGATGCACAAAGATGGTCGTT

DGAAIQQWN DNGTDAQRWSF

1450 1460 1470 1480 1490 1500

TAACAAATTGAATTAATTCAAAGACGATACTTTTTGGGGATTCCTTC

N K L N *

1510 1520 1530 1540 1550

TsAATATATTGTCTCAACZ#CTTGCAAGTGATGTTTACCCGGTGGAAAAT

FIG. 7. Nucleotide sequence of the glcB gene and deduced amino acid sequence of the gene product. Possible promoter sequences are un- derlined and a Shine-Daigarno sequence is doubly underlined. The vertical arrow indicates the cleavage site of the signal sequence. The horizontal arrows indicate an inverted repeat.

reported by our laboratory. In subcloning experiments, the newly identified B-1,3-glucanase gene was shown to be located within the two San fragments in the inserted DNA fragment carried by the plasmid pMTOO2 (Fig. 3).

bl,f-Giucanase produced in E. coli harboring pMToo1 The appearances of the pachyman clearing zone and orange-colored zone formed by the E. coli transformant harboring pMTOO1 and by E. coli harbor-

ing pNTOO3, the glcA clone, were compared (Fig. 4A). Orange-colored zones similar in appearance were formed around the colonies of the two clones grown on the plate containing pachyman and Congo red. On the other hand, the clearing zones of pachyman formed around the colonies of the two clones grown on the agar plate without Congo red were quite different. The clearing zone of pachyman formed around the colony of the E. coli transformant carrying pMTOO1 was very faint and hardly detectable, while a transparent clearing zone was formed around the colony of the glcA clone. In order to compare clearing zones formed around the colonies of the transformants with those formed by the j-1,3- glucanases produced by B. circulans WL-12, the same amounts (20 units) of GlcAl and GlcB preparations were spotted on the agar plate containing pachyman and incu- bated at 33°C for 24 h. As shown in Fig. 4B, the clear- ing zone formed by GlcB was very faint, while that formed by GlcAl was transparent, although orange-

colored zones similar in appearance were formed by the two glucanases on the plate containing pachyman and Congo red. The clearing zone and orange-colored zone formed around the colony of the transformant carrying pMTOO1 was very similar in appearance to these formed by GlcB.

A periplasmic protein fraction was extracted from E. coli JM109 cells carrying pMTOO1 and the ,8-1,3- glucanase in the fraction was analyzed. The isoelectric point of this /3-1,3-glucanase was determined to be 6.0 by isoelectric focusing analysis as shown in Fig. 5. This value is consistent with the isoelectric point of GlcB produced by B. circulans WL-12. When the crude en- zyme was analyzed by SDS-PAGE, two distinct b-1,3- glucanases with different sizes were detected on the agar replica of the polyacrylamide gel (Fig. 6). The estimated sizes of the P-1,3-glucanases were 40 kDa and 28 kDa. The sizes of the two ,&1,3glucanases produced by E. coli agreed well with those of GlcB and GlcC of B. cir- culans. The protein band corresponding to the 28 kDa /3-1,3-glucanase was not visible after staining with Coomassie brilliant blue R-250. The band representing the protein of the size corresponding to that of GlcB was electrophoretically blotted onto the PVDF mem- brane and the N-terminal amino acid sequence was ana- lyzed by automated Edman degradation. The obtained sequence, ATNWNLVWSDEFNGS-, was apparently iden-

234 OKADA ET AL. J. FERMENT. BIOENG.,

GlcB 29 AG-430 29 E GlcAl 420 IA NDSKSFPQDPNRYA BglH 422 IE NBKSFPQDPSRYA

_g_ . _ A 264 _Y____ S 264 FYKVTN

Fug Q 682

VTR G 685

FIG. 8. Alignment of N-terminal to central region of GlcB with the homologous regions of other ,&1,3glucanases. Identical amino acid residues to those of GlcB are indicated by a black background. BglH, 87 kDa j3-1,3-glucanase of B. circulans MM1 165; GlcAl, B. circulans WL-12 B-1,3-glucanase Al; AG-430, ,3-1,3glucanase of alkaliphilic Bacillus AG-430. Numbers represent positions in each protein sequence.

tical to the N-terminal amino acid sequences of GlcB and GlcC of B. circulans WL-12.

Nucleotide sequence of the cloned gene Two SalI fragments of pMTOO2 carrying a region complementary to the gfcB gene were subcloned into pUC19 to obtain pMTOl1 and pMT012. Overlapping deletions were in- troduced into the inserted DNA fragments in the two plasmids and nucleotide sequences were determined. One open reading frame of 1,239 bp encoding a polypeptide of 413 amino acid residues with a predicted A4, of 45,379 was found in the sequenced region as shown in Fig. 7. A presumed ribosomal binding site was found immediately preceding the TTG initiation codon which is a relatively common initiation codon in gram-positive bacteria (21). Downstream of the translation termination codon, a 10 bp inverted repeat which may serve as a transcription termination signal was observed. Three possible pro- moter sequences were predicted but the one that acts as the actual promoter in the transcription of this gene could not be deduced.

The N-terminal amino acid sequences of GlcB and GlcC of B. circulans and the cloned p-1,3-glucanase matched exactly the deduced sequence region down- stream of Ala-29. The N-terminal region, from Met-l to Ala-28, exhibited a typical signal sequence feature, i.e., a hydrophilic segment containing positively charged amino acid(s) followed by a hydrophobic amino acid se- quence. The calculated isoelectric point of mature GlcB

is 5.84. The calculated A4, of the mature GlcB (42,280) agrees well with the M, of GlcB estimated by SDS- PAGE analysis (40,000).

All of the results described above indicated that the ,!3- 1,3-glucanase gene cloned in the present study encodes GlcB (and GlcC).

Sequence similarities with other proteins Sequence comparisons with enzymes having similar activities and a database search using Swiss-Prot protein databank were performed. The highest sequence similarity was observed between GlcB and /3-1,3-glucanase of the alkaliphilic Bacillus AG-430 (22) followed by those between GlcAl of this bacterium and GlcB and the 87 kDa p-1,3- glucanase (BglH) of B. circulans IAM (23) and GlcB. The region in GlcAl which shows high sequence homology to the N-terminal to central region (234 amino acid residue region) of the mature GlcB corresponded to the catalytic domain of GlcAl (9). This observation is very interesting because GlcA and GlcB exhibit a striking difference in terms of the hydrolysis of j-1,3-1,Cglucan; that is, GlcAl does not hydrolyze it while GlcB does. The calculated size of the region showing the high sequence homology to the catalytic domain of GlcAl is 26,334 Da which is comparable to the estimated size of GlcC (28 kDa). Alignment of the N-terminal to central region of GlcB with the homologous region of b-1,3- glucanase from Bacillus AG-430, GlcAl and the 87 kDa /3-1,3-glucanase of B. circulans IAM is shown in

AVWIYTC AVWIYTC EV&MNC

FIG. 9. Sequence similarity of C-terminal region of GlcB with C-terminal regions of the other enzymes. Identical amino acid residues to those of GlcB are indicated by a black background. XYNA-STRLI, xylanase A of Streptomyces lividans; E13B-OEBXA, p-1,3-glucanase of 0. xunthineolyticu (23); GlcI, b-1,3glucanase I of Arthrobucter sp. YCWD3 (GenBank D23668); BFRPI, yeast lytic protease I of Rurobacter faecitabidus (24). Numbers represent positions in each protein sequence.

VOL. 80, 1995 CLONING AND SEQUENCING OF glcl3 GENE 235

Fig. 8. In addition to sequence homology to j-1,3- glucanases, the putative catalytic domain of GlcB shows weak sequence similarities to endo+1,3-1 ,Cglucanases of various bacteria and laminarinase of Clostridium ther- moserum (24) (data not shown). These sequence similari- ties were expected based on the sequence similarities of GlcAl and BglH to various endo-,4-1,3-1,Cglucanases which were already described (8, 23).

As shown in Fig. 9, the C-terminal region of GlcB exhibited the highest sequence similarity (30.3% amino acid residue match) to the C-terminal region of XlnA of Streptomyces lividans (25). C-terminal region sequence similarities were also observed between GlcB and laminaripentaose-producing j-1,3-glucanases of Oersko- via xanthineolytica (26) and Arthrobacter sp. YCWD3 (GenBank D23668), and yeast lytic protease I of Rarobacter faecitabidus (27). The C-terminal region of /3-1,3-glucanase of 0. xanthineolytica have been shown to have some sequence similarity to agglutinin and sug- gested to be involved in yeast cell wall lysis (26). Similar- ly, the C-terminal region of protease I of R. faecitabidus has been shown to have sequence similarity to the ricin B chain and is termed lectin-like domain (27).

Taken together with the biochemical data, the amino acid sequence results described above strongly suggest that the GlcC detected in the culture supernatant of B. circulans WL-12 is generated by loss of the C-terminal region of GlcB and corresponds to the catalytic domain of GlcB.

DISCUSSION

The glcA gene encoding the precursor of GlcAl was cloned into E. coli and the nucleotide sequence was de- termined as described in our previous reports (1, 8). In the experiment we obtained a glcA clone, B. circulans chromosomal DNA was digested with Hind111 and the glucanase-positive clones were detected by the formation of the clearing zone of pachyman around colonies. The strategy used in the experiment might not be suitable to clone the gene encoding GlcB (and GlcC), since we ob- tained only the glcA gene in repeated attempts using a similar strategy. The possible reasons for the failure in obtaining the glcB gene were assumed to be as follows: (i) Hind111 might inactivate the glcB gene by cleavage inside the gene, and (ii) an apparent clearing zone might not be formed around a colony of the transformant car- rying the glcB gene when grown on the plate containing pachyman. Accordingly, in order to clone the glcB gene, partial digestion with TaqI was employed to prepare chromosomal DNA fragments, and glucanase-positive clones were detected by color change of the medium con- taining pachyman and Congo red around transformant colonies, in the present study. Restriction maps of the recombinant plasmids carrying the glcB gene and the ap- pearance of the clearing zone formed around the colony of the transformant carrying the glcB gene demonstrated that these modifications in cloning strategy made it possi- ble to clone the glcB gene. The difference in appearance of the clearing zone of pachyman reflects differences in enzymatic properties between GlcAl and GlcB. In fact, the clearing zones formed by the purified GlcB prepared from B. circulans culture supernatant and the periplas- mic protein fraction of E. coli carrying the glcB gene were both faint in contrast to the transparent clearing zone formed by the purified GlcAl.

The N-terminal to central region (catalytic domain) of GlcB showed markedly high sequence similarity to the catalytic domain of GlcAl of this bacterium. In spite of the high sequence similarity, marked differences in the enzymatic properties were observed between the two glucanases. GlcB hydrolyzed mixed-linkage ,9-l ,3-l ,4-glu- can at a high rate while GlcAl did not hydrolyze it (10). The difference in the substrate specificity is not attributa- ble to the C-terminal region of this enzyme, since a pro- teolytic derivative corresponding to the catalytic domain of GlcB (and corresponding to GlcC) hydrolyzed mixed- linkage j-1,3-1,Cglucan at a high rate (manuscript in preparation). Therefore, amino acid residues in GlcB which are not conserved between the catalytic domains of GlcAl and GlcB are speculated to be essential to the ability to hydrolyze mixed-linkage ,&1,3-l ,4-glucan.

In addition to that to P-1,3-glucanases, GlcB showed weak sequence similarity to various j- 1,3- 1 ,Cglucanases . This result was expected based on the presence of weak sequence similarities of GlcAl of B. circulans WL-12 and 87 kDa j3-1,3-glucanase of B. circulans IAM to various j-1,3-1,Cglucanases (lichenases) which have been described (8, 23). On the other hand, Zverlov et al. reported that laminarinase from Clostridium thermose- rum, which has similar substrate specificity to that of GlcB, shows extensive sequence homology to j-1,3-1,4- glucanases from B. subtilis and B. amyloliquefaciens (24). This relationship indicates that the difference in the substrate specificity between the laminarinase and lichenase has been acquired by a small amino acid sequence alteration. GlcB and C. thermoserum lami- narinase have similar substrate specificities; however, the extent of sequence similarity between the two en- zymes is much lower than those between C. thermose- rum laminarinase and the b-1,3-1,4-glucanases, and be- tween GlcB and other j-1,3_glucanases. This observation may suggest that the two enzymes with similar substrate specificities evolved through different pathways.

Bueno and coworkers reported the cloning and nucleo- tide sequence of the gene encoding 1,3-1,4-B-D-glucanase of B. circulans WL-12 (28, 29). The estimated size of the mature form of the 1,3-l,C/%glucanase (40.5 kDa) is very close to the estimated size of GlcB (40 kDa). However, there are several marked differences in the properties between their glucanase and our GlcB. The enzyme reported by Bueno and coworkers was not produced until the stationary phase of growth, while GlcB was detected from the early stage of cultivation. GlcB hydrolyzed laminarin in addition to mixed-linkage 1,3-l ,C,&glucan (10) but their 1,3-1,4-,!%glucanase did not hydrolyze former (28). Nevertheless, we first as- sumed that GlcB may be closely related to their 1,3-1,4- p-glucanase because both hydrolyze mixed-linkage ,3-l ,3- 1,Cglucan. However, contrary to our expectation, no sig- nificant similarity was observed between the amino acid sequences of the two glucanases.

The C-terminal region of GlcB showed sequence similarity to the C-terminal regions of xylanase A of S. lividans (25), P-1,3-glucanases of 0. xanthineolytica (26) and Arthrobacter sp. YCWD3 (GenBank D23668), and yeast lytic protease I of R. faecitabidus (27). In spite of the high sequence similarity to the N-terminal region of GlcB, Bacillus AG-430 lacks the region corresponding to the C-terminal region of GlcB. Deletion of the C-termi- nal region of 0. xanthineolytica glucanase abolished the lytic activity of viable yeast cells (26). Similarly, deletion

236 OKADA ET AL. J . FERMENT. BIOENG.,

of the C-terminal region of protease I of R. faecitabidus abolished the mannose-binding and yeast-lytic activities (27). These observations strongly suggest that the C-ter- minal region of GlcB interacts with the yeast cell wall. This assumption is consistent with the fact that B. circu- lam WI_.-12 was originaly isolated as a fungal cell wall lytic bacterium and the hypothesis that glucanases play a major role in yeast cell wall lysis. Experiments focused on the role of the C-terminal region of GlcB are now underway.

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ACKNOWLEDGMENTS

We are grateful to Dr. Shoji Odani, Niigata University, for help in N-terminJ amino acid sequence analysis. This work was partly supported by a grant from Nakano Vinegar Co. Ltd.

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ration and purification of lytic enzymes. J. Bacterial., 89, 1570-1580 (1965). Silhavy, T. J., Berman, M. L., and Enquist, L. W.: Experi- ments with gene fusions. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1984). Maniatis, T., Fritsch, E. F., and Sambrook, J.: Molecular clon- ing: laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982). Sanger, F., Nicklen, S., and Coulson, A. R.: DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA, 74, 5463-5467 (1977). Ames, G. F. L.: Resolution of bacterial proteins by poly- acrylamide gel electrophoresis on slabs. J. Biol. Chem., 249, 634-644 (1974). Laemmli, U. K.: Cleavage of structural proteins during the as- sembly of the head of bacteriophage T4. Nature (London), 227, 680-685 (1970). Manoil, C. and Beekwith, J.: A genetic approach to analysing membrane protein topology. Science, 233, 1403-1408 (1986). Watanabe, T., Oyanagi, W., Suzuki, K., and Tanaka, H.: Chitinase system of Bacillus circulans WL-12 and importance of chitinase Al in chitin degradation. J. Bacterial., 172. 4017- 4022 (1990). Matsudaira, P.: Sequence from picomole quantities of proteins electroblotted onto polyvinylidene membrane. J. Biol. Chem., 262, 10035-10038 (1987). Tanaka, H., Ogasawara, N., Nakajima, T., and Tamari, K.: Cell walls of Pyricularia oryzae. I. Selective enzymolysis of Pyricularia oryzae walls by wall lytic enzymes of Bacillus circu- lans WL-12. J. Gen. Appl. Microbial., 16, 39-60 (1970). Hager, P. W. and Rabinowitz, J. C.: Translational specificity in Bacillus subtilis, p. 1-32. In Dubnau, D.A. (ed.), The molecular biology of Bacilli, vol. 2. Academic Press Inc., New York (1985). -_ No@, Y.: Extremely thermostable p-1,3-glucanases produced bv alkaliohilic Bacillus SD. AG-430, p. 262-269. In Horikoshi, i. and &ant, W. D. (ed.), Superb&s. Springer-Verlag, Berlin (1991). Yamamoto, M., Aono, R., and Horikoshi, K.: Structure of the 87-kDa p- 1,3-glucanase gene of Bacillus circulans IAMl165 and properties of the enzyme accumulated in the periplasm of Escherichia coli carrying the gene. Biosci. Biotech. Biochem., 57, 1518-1525 (1993). Zverlov, V. V., Laptev, D. A., Tishkov, V. I., and Velikodvor- skaya, G. A.: Nucleotide sequence of the Clostridium ther- mocellum laminarinase gene. Biochem. Biophys. Res. Com- mun., 12, 811-816 (1990). Shareck, F., Roy, C., Yaguchi, M., Morsoli, R., and Kluepfel, D.: Sequences of three genes specifying xylanases in Strep- tomyces lividans. Gene, 107, 75-83 (1991). Shen, S.-H., Chr&ien, P., Bastien, L., and Slllaty, S. N.: Pri- mary sequence of the glucanase gene from Oerskovia xan- thineolytica. J. Biol. Chem., 266, 1058-1063 (1991). Shiioi, H., Iimura, Y., Obata, T., and Tadenuma, M.: Molecular structure of Rarobacter faecitabidus protease I. J. Biol. Chem., 267, 25189-25195 (1992). Bueno, A., Vazquez de Aldana, C. R., Correa, J., Vlla, T. G., and del Rel, F.: Synthesis and secretion of a Bacillus circulans WL-12 1,3-1,4-P-D-glucanase in Escherichia coli. J. Bacterial., 172, 2160-2167 (1990). Bueno, A., Vazquez de Aldana, C. R., Correa, J., and del Rey, F.: Nucleotide sequence of a 1,3-1,4-b-o-glucanase-encod- ing gene in Bacillus circulans WL-12. Nucl. Acids Res., 18, 4248 (1990).

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

REFERENCES

Watanabe, T., Yahata, N., Nakamura, Y., Muramato, Y., Suzuki. K.. Kamlmiva. S.. and Tanaka, H.: Expression in Escherichia’coli of thk bacihus circulars WL-12 structural gene for b-1,3-glucanase A. Agric. Biol. Chem., 53, 1759-1767 (1989). Doi, K., Doi, A., Ozaki, T., and Fukui, T.: Further studies on the heterogeneity of the lytic activity for isolated yeast cell wall of the components of an Arthrobacter glucanase system: properties of the two components of a ,5’-(1-3)-glucanase. Agric. Biol. Chem., 40, 1355-1362 (1976). Marl, H., Yamamoto, S., and Nagasaki, S.: Multiple forms of the lytic glucanase of Flavobacterium dormitator var. glucanolyticae and the properties of the main component en- zyme. Agric. Biol. Chem., 41, 611-613 (1977). Obata, T., Fujioka, K, Hara, S., and Namba, Y.: The synergis- tic effects among I-1,3-glucanases from Oerskovia sp. CK on lysis of viable yeast cells. Agric. Biol. Chem., 41, 671-677 (1977). Phaff, H. J.: Enzymatic yeast cell wall degradation. Adv. Chem. Ser., 160, 244-282 (1977). Vrsanska, M., Biely, P., and Kratky, Z.: Enzymes of the yeast lytic system produced by Arthrobacter GJM-1 bacterium and their role in the lysis of yeast cell walls. Z. Allg. Mikrobiol., 17, 465-480 (1977). Aono, R., Hammura, M., Yamamoto, M., and Asano, T.: Isolation of extracellular 28- and 42-kilodalton ,9-1,3-glucanases and comparison of three ,9-1,3-glucanases produced by Bacillus circulans IAM1165. Appl. Environ. Microbial., 61, 122-129 (1995). Yahata, N., Watanabe, T., Nakamura, Y., Yamamoto, Y., Kamimiya, S., and Tanaka, I-I.: Structure of the gene encoding B-1,3-glucanase Al of Bacillus circulans WL-12. Gene, 86, 113-117 (1990). Watanabe, T., Kasahara, N., Aida, K., and Tanaka, H.: Three N-terminal domains of ,3-1,3-glucanase Al are involved in bind- ing to insoluble p-1,3-glucan. J. Bacterial., 174, 186-190 (1992). Aida, K., Okada, T., Kasakara, N., Nikaidou, N., Tanaka, H., and Watanabe, T.: Comparative studies of j3-1,3-glucanase Al and B of Bacillus circulans WL-12: purification and en- zymatic properties. J. Ferment. Bioeng., (1995). in press Tanaka, H. and Phalf, H. J.: Enzymatic hydrolysis of yeast cell walls. I. Isolation of wall-decomposing organism and sepa-

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.