Journal of Environmental Biotechnology
Vol. 8, No. 2, 119–121, 2008

Original paper (short paper)

Identification of Inducers for Chitinase B (ChiB) Production
in Bacillus cereus CH and Estimation of Its Induction Mechanism
YUZURU SATO1,2* and YOSHIO ARAKI1
Laboratory of Environmental Molecular Biology, Graduate School of Environmental Science, Hokkaido University,Sapporo, Hokkaido 060-0810, Japan2
Department of Material Science, Interdisciplinary Graduate School of Science and Engineering, Shimane University,
Matsue, Shimane 690-8504, Japan1* E-mail: msato310@spice.ocn.ne.jp(Received; 14 February, 2008/Accepted; 5 November, 2008)
Chtinolytic Bacillus cereus CH produces chitinase A (ChiA) and chitinase B (ChiB) and colloidal chitin and N-acetylglucosamine (GlcNAc) oligomers induce their production. Deacetylated chitin products (chitosan 7B, 9B, and 10B) andglucosamine oligomers showed much decrease activity to induce ChiB production campared with that of colloidal chitin andGlcNAc oligomers, indicating that acetylation of chitin and chitin oligosaccharides are essential to their induction activity.
Histidine protein kinase inhibitors (closantel and 3,3’,4’,5-tetrachlorosalicylanilide) inhibited ChiB production even in thepresence of an inducer (GlcNAc hexamer). This result suggests that ChiB production in B. cereus CH is regulated by twocomponent regulatory system.
Key words: chitinase B, chitooligosaccharides, two-component regulatory system, Bacillus cereus

Chitin is the main component of exoskeleton (cuticle) ofcrabs. We consume large amounts of crabs and shrimps forfood and as a result, enormous amounts of their shell
wastes are produced every year. Although chitin is utilizedin environmental technology and food medical industries,
most of shell wastes are discarded by incineration and burying. Thus, reutilization of shell wastes is an important issue
in ecological concern. Cuticles are biopolymers composedof rigid complex of N-acetylglucosamine polymers, proteins,and Ca2+ and highly insoluble in water and organic solvents.
Chitinolytic and non-chitinolytic microorganisms cooperatively degrade cuticles. To understand chitin degradation
system, we isolated chitinolytic bacterium Bacillus cereus
CH and investigated its chitinolytic enzyme system3,4,7). B.
cereus CH produces two chitinases, chitinase A (ChiA) andchitinase B (ChiB) and ChiB is responsible for the major
extracellular chitinase activity. ChiA and ChiB production isinducible and colloidal chitin promotes transcription of thechiA and chiB genes. In this study, we report that the extent
of acetylation of chitooligosaccharides affects induction ofchiB transcription, and that there is the possibility that a
two-component regulatory system is involved in the controlof chiB transcription.
B. cereus CH was cultivated in medium containing 0.2%
triptone, 0.1% yeast extract, 0.2% NaCl, 0.025% KH2PO4,0.025% K2HPO4, 0.01% calcium acetate, and 0.01% magnesium acetate (pH 7.2) at 33°C. At 12 h after the initiation of cultivation, 0.025% of test compounds were added

to induce ChiB production. Extracellular and cytoplasmic
ChiB was quantified by enzyme-linked immunosorbent assays(ELISA) with anti-ChiB serum solution as described in theprevious report7). Quantification of chiB mRNA was performed by reverse transcription PCR (RT-PCR) as describedpreviously7).
In the previous study, we demonstrated that ChiA andChiB production in B. cereus CH is induced by colloidal
chitin and N-acetylglucosamine oligomers, but not bydeacetylated chitin (chitosan 10B)7). This result suggeststhat the extent of acetylation of chitin affects the ability ofchitin to induce chitinase genes in B. cereus CH. To investigate an effect of the extent of acetylation of chitin, we examined chitin products with different deacetylation ratio, i.e.
chitosan 7B, 9B, and 10B for their ability to induce extracellular ChiB production. The deacetylation ration of chitosan
7B, 9B, and 10B is 70, 90, and 100%, respectively. As
shown in Fig. 1, colloidal chitin showed the best induction
activity and the induction activity decreased with increasingof the deacetylation ratio of chitin. To confirm an effect ofacetylation of chitin on induction of ChiB production, we
examined the activity of oligomers (dimer to hexamer,[(GlcN)2, (GlcN)4, (GlcN)5, and (GlcN)6]) of glucosamine(GlcN) to induce ChiB production and compared it withthat of N-acetylglucosamine (GlcNAc) oligomers ((GlcNAc)2to (GlcNAc)6). As reported in the previous study, GlcNAc
oligomers showed remarkable induction activity which increased with residue numbers of the oligomers (Fig. 2).

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SATO and ARAKIFig. 1. Induction of extracellular ChiB production in B. cereus CH
by colloidal chitin and chitosan products.
B. cereus CH was cultivated at 33°C and at 12 h after theinitiation of cultivation, 0.025% (w/v) colloidal chitin (closedcircles), chitosan 7B (closed squares), chitosan 9B (open circles),and chitosan 10B (closed triangles) were added to medium.
Culture broth was withdrawn at each time point and subjected
to quantification of extracellular ChiB by ELISA. Open triangles
represent extracellular ChiB production in the negative control(no addition of inducer).

Although the induction activity of GlcN oligomers also increased with residue numbers, they showed much decreased
activity compared with that of GlcNAc oligomers. These
results clearly demonstrate that the extent of acetylation ofchitin and chitooligosaccharides affects their activity to induce
ChiB production. It is interesting to mention that (GlcNAc)6
induced higher ChiB production than colloidal chitin withshort induction time (~12 h) (Fig. 2) although colloidal
chitin induced higher ChiB production than (GlcNAc)6 withlonger induction time (48-72 h)7). It suggests that acetylated
chitin oligosaccharides (degradation products of chitin),rather than chitin itself, are inducers.
It was reported that two-component regulatory system isinvolved in induction of chitinase production by chitin oligosaccharides in Streptmyces thermoviolaceus, Vibrio spp.,
and Vibrio cholerae2,6,9). We investigated whether ChiB production is regulated by two-component regulatory systemusing inhibitors for autophosphorylation of histidine protein
kinases of two-component regulatory systems. Closanteland 3,3’,4’,5-tetrachlorosalicylanilide (TCSA) were shownto act as general inhibitors of autophosphorylation of histidine protein kinases1,5,8). Both 15 mM closantel and TCSA
inhibited induction of extracellular ChiB production by(GlcNAc)6 (Fig. 3). Closantel and TCSA rarely affected
growth of B. cereus CH (data not shown). In our previous
study, we demonstrated that sodium azide inhibits secretionof ChiB protein but not ChiB production7). ChiB protein
was accumulated inside of cells in the presence of inducer(colloidal chitin) and sodium azide. Therefore, there is thepossibility that closantel and TCSA also inhibit secretion ofChiB but do not affect ChiB production. To rule out thispossibility, cytoplasmic ChiB was measured for cells simultaneously exposed to (GlcNAc)6 (inducer) and inhibitors(closantel, TCSA, and sodium azide) by ELISA. Cells exposedFig. 2. Induction of extracellular ChiB production in B. cereus CHby GlcNAc and GlcN oligomers.
Cultivation and induction of B. cereus CH, and quantificationof extracellular ChiB protein were performed as described inFig. 1. (A) 0.025% (w/v) (GlcNAc)2 (open squares), (GlcNAc)4(closed triangles), (GlcNAc)5 (open circles), (GlcNAc)6 (closedcircles), and colloidal chitin (closed squares) were added tomedium as inducers. (B) 0.025% (w/v) (GlcN)2 (open squares),(GlcN)4 (closed triangles), (GlcN)5 (open circles), (GlcN)6(closed circles), and colloidal chitin (closed squares) were added to medium as inducers.

to (GlcNAc)6 and sodium azide accumulated significant
amounts of cytoplasmic ChiB, whereas virtually no cytoplasmic ChiB was detected with cells exposed to (GlcNAc)6and histidine protein kinase inhibitors (Fig. 3). To further
confirm that closantel inhibits ChiB production, RT-PCR
was performed to measure chiB mRNA. Significant
amounts of PCR product derived from chiB mRNA was detected when cells were incubated with (GlcNAc)6, no PCR
product was detected with cells exposed to (GlcNAc)6 andclosantel. Thus, it was clearly demonstrated that closantel
and TCSA inhibit ChiB production in B. cereus CH. Theseresults suggest that ChiB production in B. cereus CH is regulated by two-component regulatory system. Further molecularbiological research is needed to elucidate the induction
mechanism of chitinase production in B. cereus CH.
Acknowledgments
This work was partly supported by the 21st century COE
program of prediction and voidance of an abrupt change inthe bio-geosphere system at the Graduate School of Environmental Earth Science, Hokkaido University. The authors
deeply thank members of the COE program for employment as a research assistant in the school and education bythe project.

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ReferencesFig. 3. Inhibition of extracellular ChiB production (A) and cytoplasmic ChiB production (B) by closantel, TCSA and sodiumazide.
B. cereus CH was cultivated at 33°C and at 12 h after theinitiation of cultivation, 15 mM closantel (open circles), TCSA
(closed squares), and sodium azide (closed triangles) together
with 0.025% (w/v) (GlcNAc)6 were added to medium. Culture
broth was withdrawn at each time point and subjected toquantification of extracellular and cytoplasmic ChiB by ELISA.
Closed circles represent ChiB concentrations in positive control(no addition of inhibitors).

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