Journal of Environmental Biotechnology
Vol. 7, No. 1, 27–32, 2007

Original paper (regular paper)

Analyses of ChiA and ChiB Production by Bacillus cereus CH:
Induction, Gene Expression, and Localization of Two Chitinases
YUZURU SATO* and YOSHIO ARAKI
Laboratory of Environmental Molecular Biology, Graduate School of Environmental Science, Hokkaido University,
Sapporo-shi, Hokkaido 060-0810, Japan
Present address: Company Promotion Department, Chiba Industry Advancement Center,
Kisarazu-shi, Chiba 292-0818, Japan* TEL/FAX: +81–(0)438–20–5740* E-mail: y-satou@kazusabio.net(Received; 10 October, 2006/Accepted; 2 November, 2006)
Bacillus cereus CH produces two chitinases (ChiA and ChiB). In the present work diﬀerent chitin derivatives (chitooligosaccharides) and colloidal chitin were examined for the induction of the genes expression and productivity of enzymes.
Enzyme activity in the culture supernatant increased as the length of GlcNAc increased up to six. Gene expression levels ofboth chiA and chiB were elevated in the presence of the inducers. However, little amount of ChiA was produced extracellularly while ChiB was secreted normally. It was demonstrated that the ChiB is a dominant extracellular chitinase induced bychitooligosaccharides and colloidal chitin.
Key words: chitinase induction, chitin oligomers, secretion, ELISA, RT-PCR

1.

Introduction

Chitin, a homopolymer of β-1,4-linked N-acetyl-D-glucosamine (GlcNAc), is one of the most abundant carbohydrate
polymers in nature. In a portion of fungi and most ofnematodes, insects, and crustaceans, this carbohydrate
generally occurs in forms of complexes with several uniqueproteins and other materials. Its structural characteristic isbelieved to provide rigid and Ca2+-complexed entities with agreat advantage for the construction of very robust exoskeletons. In terrestrial biosphere the recycling of chitin seems
to be carried out predominantly through co-operative actionof chitinolytic and other non-chitinolytic microorganisms.
Taking account of a large amount of annual chitin production (about 1011 tonnes/year), a variety of chitinolytic enzyme
systems may play an important role in recycling of chitin
produced in both aquatic and terrestrial biospheres7). In thechitin degradation process, at least two distinct enzymes are
required for complete degradation of chitin: one is chitinase(EC 3.2.1.14) to depolymerize the polysaccharide structure
into dimer or oligomers of GlcNAc and the other is exohydrolytic chitinase or chitobiose-specific N-acetylglucosaminidase (chitobiase, EC 3.2.1.14) to hydrolyze a variety ofchitin oligomers into monosaccharide GlcNAc. Bacterial
species belonging to genera Alteromonas18,19), Bacillus1,11),
Nocardiopsis20), Pseudomonas2,3), Serratia 4,17), and Vibrio13)
have been reported to produce and secrete multiple chitinolytic enzymes. Probably, multiple chitinolytic enzymes provide an eﬀective chitin degradation system through theirsynergistic action13). In order to develop chitin degradation
systems, we believe that basic understanding of production
mechanisms of chitin producers is important10,13).
Previous studies on Bacillus cereus CH11,12) showed thatcolloidal chitin can serve as an inducer in the production ofchitinases A and B (ChiA and ChiB) and the respective
genes, chiA and chiB, have been cloned and sequenced.
However, detailed mechanisms for their induction, production, and secretion remain to be elucidated. In the present
study, several chitin oligomers were tested for inducers ofchitin production. We also prepared specific rabbit antisera
against recombinant ChiA and ChiB and used for the
analysis of ChiA and ChiB production. Moreover, RT-PCR
was performed for the analysis of chiA and chiB transcription levels during induction.

2.
2.1.

Materials and Methods

Cultivation, induction, and enzyme activity

Bacillus cereus CH was precultivated to exponential
phase (6 h) in Luria-Bartini medium (pH 7.2). For chitinaseinduction, aliquots (1 ml) of the preculture were transferred
to each second fresh medium (25 ml, pH 7.2) containing
0.2% triptone, 0.1% yeast extract, 0.2% NaCl, 0.025%
KH2PO4, 0.025% K2HPO4, 0.01% calcium acetate, and
0.01% (w/v) magnesium acetate. After 12 h cultivation, avariety of inducer at a concentration of 0.1% (w/v) was
added to the second medium. At indicated time periods, a
portion (1 ml) of each induction culture was withdrawn andSATO and ARAKI

28

centrifuged to separate the culture supernatant and cellpellet. Enzyme activity recovered in each of the supernatants
was measured using 1.5 mM p-nitrophenyl-β-chitobioside{(GlcNAc) 2-pNP} as a substrate. Detailed reaction conditions were the same as those described previously11). Protein
concentration was determined using DC protein assay kit(Bio-Rad, CA, U.S.A.) and bovine serum albumin as standard.
2.2.

Preparation of rabbit antisera against recombinant
ChiA and ChiB

According to the procedure described in our previous
report, recombinant ChiA and ChiB (rChiA and rChiB)
were produced by Escherichia coli harboring pCA-S or
pCB-XE and were purified12). Both recombinant proteins
were used as the immunogens and reference proteins. After
dialysis against phosphate-buﬀer saline (PBS, pH 7.2) andemulsifying with complete Freund adjuvant, each of thepurified rChiA and rChiB (0.1 mg) was subcutaneously injected to each group (three) of young female New Zealandwhite rabbits (7 to 11 weeks, Hokudo, Sapporo, Japan).
Each of the same immunogens (0.05 mg) was injected
8 times in intervals of 2 to 3 weeks. At seven days after the
final immunization, bloods were collected from the respective groups of immunized rabbits, and anti-sera were prepared in usual procedure and stored at −20°C.
2.3. Western blot analysis and ELISA method
Each sample to be analyzed was denatured by heating(95°C, 3 min) in 0.1% (w/v) SDS and then subjected to12.5% SDS-PAGE according to the method of Laemmli9).
After electrotransfer of proteins to PVDF membrane andtreatment with blocking solution (KPL, MD, U.S.A.), themembrane was subjected to primary immunoreaction (25°C,
1 h) with adequately diluted anti-rChiA (1 : 500) or antirChiB (1 : 8000) serum. After thoroughly washing with
0.05% Tween 20-containing PBS (TPBS), the secondary
immunoreaction was carried out with peroxidase-labeledgoat anti-rabbit IgG (H+L) (1 : 3000, American Qualex,
CA, U.S.A.). Color development by coupled horseradish
peroxidase was measured after incubation (30 min, rt) in
TPBS containing 0.06% 4-chloro-1-naphtol, 20% ethanol,and 0.1% H2O26). For determination of the respective antiserum titers, an adequate volume of the recombinant
ChiA or ChiB solution was added to 96 well plates (Nalgen
Nunk, NY, U.S.A.). After standing overnight at 4°C, theunbound protein was removed. The antigen-coated plates
were thoroughly washed with 0.05% Tween 20-containing
PBS (TPBS), masked with blocking solution (KPL), andthen washed with TPBS. To the antigen-coated wells 50 µlof the respective diluted anti-serum solutions was added andincubated at room temperature for 1 h. After thoroughly
washing, 50 µl each of diluted secondary antibody and peroxidase-labeled goat anti-rabbit IgG (H+L) (American
Qualex) was added. Incubation and washing were carriedout as those described in the above. The coupled enzyme

activity on formed ternary immunocomplex was measured
using TMB peroxidase ELA substrate kit (Bio-Rad) in according to the procedure recommended by the supplier. To
determine ranges suitable for quantifing amounts of the
ChiA and ChiB, ELISA similar to the above was carriedout. For measurement of the ChiA and ChiB amounts insupernatants and fractions, each supernatant from cellcollected at the various induction periods and fractions was
thoroughly dialyzed against PBS, and a portion of each supernatant protein (anti-rChiA: 64 ng/well and anti-rChiB:
32 ng/well) was subjected to ELISA.
2.4.

Preparation of cytosolic, solubilized membrane,
and extracellular fractions

To study cellular localization of the ChiA and ChiB proteins, three cell fractions including cytosolic, membrane,
and extracellular (culture sup.) were separated as follows. B.
cereus CH cell was cultivated under similar induction conditions in the presence or absence of 15 mM sodium azide.
The harvested cell (5 g, wet weight) was suspended in 10 ml(same volume of culture medium) of 20 mM Tris-HCl
buﬀer (pH 7.2) and sonicated for 1.5 min (15 s intervals forsonication and cooling). After removal of cell debris andwall (13,000 g, 10 min), supernatant was subjected to ultracentrifugation (100,000 g, 30 min). The resulting supernatantand pellet were used as cytosolic and membrane fractions,
respectively. The later fraction was further suspended in
1 ml of 20 mM Tris-HCl buﬀer (pH 7.2) containing 0.6%
Triton X-100, 1 mM DTT, 5 mM MgCl2, and 1 mM phenylmethysulfonyl fluoride. The suspension was stirred withmagnetic stirrer at 4°C for 30 min and subjected to ultracentrifugation. The obtained supernatant was used as solubilized membrane fraction after dialysis. Chitin oligomers(n=2–6) were purchased from Seikagaku Corp. Tokyo,
Japan; other chemicals, Nacalai Tesque, Kyoto, Japan. All
experiments in this study were carried out for three times.
2.5.

RNA extraction and RT-PCR

B. cereus CH was cultivated for 12 h in the second medium
and then each inducer was added at a concentration of
0.025%. At indicated periods, aliquots (0.5–0.7 ml) of therespective culture were well mixed with the same volume ofbacterial RNA reagent (Qiagen, CA, U.S.A.). The resulting
cell mixture was centrifuged. After removal of the supernatant, wet weight of cell pellet was prepared in the same
weight. Extraction and purification of total RNA werecarried out according to the procedure of Gilman andChamberlin5). The pellet was dissolved in a mixture of lysing
matrix B (Q-BIO gene, CA, U.S.A.) and a solution containing 0.6% CTAB and 1 mM DTT. Purification of total RNA
was carried out by phenol-chloroform extraction, ethanol
precipitation, and sequential digestion with DNase I (Takara
Bio Inc, Otsu, Japan). Each of the resulting RNAs was
resuspended in 0.1 ml DEPC-treated water and stored at–80°C until it was used for RT-PCR. To determine theexpression levels of chiA and chiB mRNAs, RT-PCR wasAnalysis of ChiA and ChiB Production

carried out using the same concentration of prepared total
RNA. On the basis of nucleotide sequences previously
reported for chiA and chiB genes12), most suitable primer
pairs were constructed as follows: forward and reverse primers for chiA were 5'-GTGGACAAAATGGAGTCGTTT-3'(corresponding to 520–540 sequence of chiA gene) and
5'-TAGGCGGTGAACCTCGACGGC-3' (1057–1077),
respectively; those for chiB, 5'-AAAGCAGGTGCTGAAGATGG-3' (786–805 sequence of chiB gene) and 5'-ACCTGAACATGCCGTTA-GAG-3' (1408–1427), respectively.
The first strands of cDNAs against the respective total
RNAs (1 µg each) were prepared by using reverse transcriptase (Invitrogen Corp., CA, U.S.A.) and random hexamers (50 µg) as primers. The reaction mixtures for RTPCR contained 1 mM dNTPs, 5 mM MgCl2, and 20 mM
DTT. After preincubation (rt, 5 min), reverse transcriptase(20 U) was added to the mixture. Annealing (25°C, 10 min)and extension (42°C, 50 min) were carried out and the reaction was terminated by heating (70°C, 15 min). A primer
pair of 16S rRNA sequences (341F and 926R)21) were used
as an external standard. Reaction mixture for PCR contained
50 ng template DNA or cDNA, 12.5 µM primer pair,
200 µM dNTPs, and 125 mU Taq Ex polymerase (Takara)in a total volume of 12.5 µM. The extension was initiatedby hot start method. The PCR reaction was done in following program: 94°C for 1 min, at 54°C for 30 s, and at 74°C
for 2 min. The RT-PCR product was electrophoresed on
1.2% agarose (Takara) gel for 1 h at 100 V with 1×TAE
running buﬀer. The gel was stained with ethidium bromide,
and visualized by UV light and documented.

3.
3.1.

Results and Discussion

Extracellular chitinolytic activities induced byvarious chitin oligomers

As shown in our previous paper11), colloidal chitin served
as a good inducer for production of extracellular chitinolytic
enzymes. However, colloidal chitin is a water-insoluble large29

molecular weight compound and hard to interact directlywith a cell surface receptor and transfer extracellular signal
to promote transcription of chi genes. Previously, Yu et al.
suggested a possibility that in Vibrio furnissii the inductionof chitinase may arise from any physical contact with solid
particles such as chitin-containing substance22,23). We tested
this possibility using a chitin derivative, fully de-N-acetylated
chitin, chitosan 10B. The level of extracellular chitinolytic
activity induced by chitosan 10B was very low (less than
1.5 mU/ml) as well as in the absence of any inducer. This
result suggests that physical contact of solid form polysaccharides (chitosan 10B) is not enough to induce chitinase
production of the strain. Since it is known that chitin oligomers could serve as inducers of other chitinases, monomer
to hexamer of GlcNAc was tested for B. cereus CH15). As
shown in Fig. 1, enzyme activity increased as the chain
length of chitin oligomer increased. In all of these cases, theextracellular chitinolytic activity reached a maximal levelwithin short induction periods (less than 12 h) without lag
period. In contrast, there was a significant lag period in thecase for induction by colloidal chitin. This may be due tothat reaction products (chitin oligomers) of chitinase fromcolloidal chitin function as autonomous inducers.
3.2.

Quality of anti-ChiA and anti-ChiB serum

B. cereus CH was known to produce two diﬀerent chitinase,
ChiA and ChiB. In order to analyze the productivity ofeach enzyme, we adopted ELISA method by using specificanti-rChiA and anti-rChiB sera prepared from rabbit. In
order to verify this method, ELISA was carried out in combination of antigen (rChiA or rChiB)-coated plates (250 ng/
well) and serially diluted anti-sera (1 : 250 to 1 : 512,000).
Up to the dilution folds 1 : 8000 and 1 : 128,000 for antirChiA and anti-rChiB sera, respectively, well linearity was
obtained in the range 0–64 ng/well in rChiA and 0–32 ng/
well in rChiB (Fig. 2). Specificity of anti-rChiA and antirChiB sera was ascertained by Western blot analysis (Fig. 3).
It was clearly shown that anti-rChiA and anti-rChiB sera

Fig. 1. Chitinolytic activity in the culture supernatants induced by various inducers.
The used inducer was presented in the next to the above figure, and 0.1% (w/v) inducer was applied to culture medium. All induction
conditions were also the same.

30

SATO and ARAKI

specifically bound to rChiA and rChiB, respectively (Fig.
3a, b, c). When the culture supernatant at 72 h was used,
only anti-rChiB serum showed bands (Fig. 3d, right). Thisshould be because the amount of ChiA in the supernatant
was too low to be detected by anti-rChiA serum (Fig. 3d,left). Upper and lower bands would be intact ChiB and aproteolytic product derived from ChiB, respectively (Fig. 3d,right). Above results demonstrate that anti-rChiA and antirChiB sera were sensitive and specific enough for detection
and quantification of ChiA and ChiB, respectively, in theculture or cell lysate.
The linearity of ELISA method and specificity of eachantiserum enabled us to determine extracellular and intracellular amounts of ChiA and ChiB under various induction
conditions. We used inducers at 0.025% in further experiments because the extracellular enzyme activity induced was
almost the same at 0.1%. As shown in Fig. 4, only a small
amount of extracellular ChiA (less than 0.65 µg/ml) wasFig. 2. Quantitativeness of ELISA method.
Each well of immunoassay plate was coated with various
amounts (0–512 ng/well) of rChiA or rChiB in a series of twofold dilutions, and then, immunostained by sequential additionof anti-ChiA (panel A) or ani-ChiB (panel B) serum followedby crossreaction with peroxidase-coupled anti-rabbit serumand developing reagent mixture. After incubation, absorbanceat 450 nm was measured.

Fig. 3. Immuno-specificity of anti-rChiA and anti-rChiB sera.
Lane a, 5 µg protein marker; lane b, 5 µg rChiA; lane c, 5 µg
rChiB; lane d, 30 µg of culture supernatant after induction for
72 h were subjected to 12.5% SDS-PAGE respectively, andthen proteins were electrotransferred to PVDF membrane. The
membrane was treated with TPBS containing anti-rChiA sera
(left panel, 1 : 500) or anti-rChiB sera (right panel, 1 : 8000).

Fig. 4. Amounts of ChiA, ChiB, and chitinolytic activity in the culture supernatant.
Inducers used was 0.025% each of GlcNAc, A; (GlcNAc)4, B;(GlcNAc)6, C; colloidal chitin, D; chitosan 10B, E. At indicatedtime periods, an adequate volume was withdrawn and chitinolytic activity (white circle) and amounts of extracellular ChiA
(black bar) and ChiB (white bar) in the culture supernatantwere measured.

Analysis of ChiA and ChiB Production

produced under the conditions except for GlcNAc monomer(Fig. 4A). We do not know the reason why the productionlevel of ChiA is higher when GlcNAc monomer was used as
an inducer. On the contrary, a large increment of extracellular ChiB (more than 15 µg/ml) was observed under the
every condition, and amount of extracellular ChiB correlateswith chitinolytic activity. These results clearly demonstrated
that ChiB is a dominant extracellular chitinase produced by
B. cereus CH. Furthermore, hexamer of GlcNAc induced
production of extracellular ChiB in a short time as compared with that of colloidal chitin. Therefore, we judgedthat hexamer of GlcNAc was the best inducer among tested.
3.3.

Expression levels of chiA and chiB mRNAs

We assumed that the lower production of ChiA than
ChiB would attribute to less amount of chiA mRNA thanthat of chiB. In order to compare the amount of mRNA ofchiA and chiB, we adopted RT-PCR method by using specificprimer pairs to chiA and chiB genes. Expected sizes of RTPCR products were 585 bp (16S rRNA), 557 bp (chiA), and641 bp (chiB), respectively, and 16S rRNA product was used
as an external standard to standardize the amount of total
mRNA used. As shown in Fig. 5A, amounts of chiA andchiB mRNAs were similarly elevated within 12 h after addition of monomer, tetramer, or hexamer of GlcNAc or
colloidal chitin. A contradiction between high level of chiAtranscription and low level of ChiA production may attributeto its low translation or secretion eﬃciency. It is worth tonote that even in the absence of inducer, mRNA of chiAand chiB were detectable. This result supports an idea that

Fig. 5. RT-PCR analyses for expression levels of chiA and chiB.
In this experiment a lowered concentration (0.025%) of monomer, tetramer, or hexamer of GlcNAc or colloidal chitin wereused. At indicated induction periods, an adequate volume waswithdrawn from the respective cultures and centrifuged. Total
RNA was extracted from the resulting cell pellet and subjected
to RT-PCR. Panel A, expression levels of chiA and chiB mRNAs.
Panel B, an example of external standard of 16S rRNA.

31

this constitutively transcribed and translated ChiA and ChiBdegraded colloidal chitin, and the product chitin oligomers
functioned as autonomous inducer (Fig. 1).
3.4.

Eﬀects of sodium azide on the production of ChiA
and ChiB

In order to get further insight into the low-level production of extracellular ChiA irrespective of high-level of transcription, cytosolic, membrane, and extracellular fraction
were prepared and analyzed for localization of ChiA
(Fig. 6A). It was found that production level of ChiA wassignificantly lower than ChiB and most of ChiA was produced in the cytosolic and membrane fractions. While, morethan half of ChiB was produced extracellularly. These
results show that both translation and secretion eﬃciency of
ChiA are lower than ChiB. Total amount of enzyme remained
low level over the time, about 0.8 µg/ml for ChiA and2.0 µg/ml for ChiB, respectively, in the absence of inducer.
In a separate experiment, cytosolic chitinase was found to
be enzymatically inactive (data not shown). This is probably

Fig. 6. Localizations of ChiA and ChiB and eﬀect of sodium azide.
Amounts of ChiA and ChiB were measured by ELISA method.
Cytosolic (zebra bar), solubilized membrane (white bar), andextracellular fractions (black bar) were prepared from cells cultivated in the presence of 0.025% colloidal chitin, panel A. In
panel B, 15 mM sodium azide was added to the culture at 0 h.

SATO and ARAKI32

due to an improper folding of the enzyme with N-terimnal
signal peptide. Homology search to accessible genomic
DNA sequences, several nucleotide sequences involving in
the regulation of Sec secretion pathway were found in ourcloning chiB gene12). Furthermore, a previous study for
Vibrio cholerae showed that mRNAs of chitinase and secA
were similarly induced13). It is known that sodium azide
inhibits the activity of SecA-related ATPases8,14,24). We wereinterested in testing the eﬀect of sodium azide, at 15 mM,
on the secretion of ChiA and ChiB by B. cereus CH. Cellgrew normally in the presence of sodium azide at thisconcentration. It was found that secretion of ChiB wascompletely impaired and the amount of ChiB in the cytosolic
fraction slightly increased in the presence of 15 mM sodium
azide in the culture (Fig. 6B). Eﬀect of sodium azide on thesecretion of ChiA was not clear because secretion level oforiginal culture without sodium azide was very low (Fig. 6A).
In contrast to ChiB, the amounts of ChiA produced in thecytosolic and membrane fractions increased by two folds.
In this report, we demonstrated that ChiB is a dominantchitinase produced by B. cereus CH, is excreted from the cellby way of general Sec-pathway. Gradual increase in the production of chitinase upon addition of colloidal chitin was
suggested to be attributed to the gradual accumulation ofchitin oligomers produced by constitutive and inducible chitinase activities. Analyses of translation and secretion mechanism of ChiA remain to be elucidated on the molecular level.

Acknowledgments
The authors deeply thank Dr. Yoshiyuki Deguchi (Center
for Advanced Science and Technology of Hokkaido University) for generous advice about the preparation of antisera.
The authors also acknowledge the critical reading of manuscript by Prof. Masaaki Morikawa (Laboratory of Environmental Molecular Biology, Graduate School of Environmental Science, Hokkaido University).

References
1) Alam, M.D.M., T. Mizutani, M. Isono, N. Nikaidou, and T.
Watanabe. 1996. Three chitinase genes (chiA, chiC, and chiD)comprise the chitinase system of Bacillus circulans WL-12. J.
Ferment. Bioeng. 82: 28–36.
2) Folders, J., J. Algra, M.S. Roelofs, L.C. van Loon, J. Tommassen,
and W. Bitter. 2001. Characterization of Pseudomonas
aeruginosa chitinase, a gradually secreted protein. J. Bacteriol.
183: 7044–7052.
3) Folders, J., J. Tommassen, L.C. van Loon, and W. Bitter.
2000. Identification of a chitin-binding protein secreted by
Pseudomonas aeruginosa. J. Bacteriol. 182: 1257–1263.
4) Gal, S.W., J.Y. Choi, C.Y. Kim, Y.H. Cheong, Y.J. Choi, J.D.
Bahk, S.Y. Lee, and M.J. Cho. 1997. Isolation and characterization of the 54-kDa and 22-kDa chitinase genes of Serratia
marcescens KCTC2172. FEMS Microbiol. Lett. 151: 197–204.
5) Gilman, M.Z., and M.J. Chamberlin. 1983. Developmental
and genetic regulation of Bacillus subtilis genes transcribedby σ28-RNA polymerase. Cell 35: 285–293.
6) Hawkes, R., E. Niday, and J. Gordon. 1982. A dot-immunobinding assay for monoclonal and other antibodies. Anal. Biochem. 119: 142–147.

7) Jerde, C.W., and R. Lasker. 1966. Molting of euphausiid
shrimps: shipboard observations. Oceanogr. 11: 120–124.
8) Jongbloed, J.D.H., H. Antelmann, M. Hecker, R. Nijland, S.
Bron, U. Airaksinen, F. Pries, W.J. Quax, J.M. van Dijl, andP.G. Braun. 2002. Selective contribution of the twin-argininetranslocation pathway to protein secretion in Bacillus subtilis.
J. Biol. Chem. 277: 44068–44078.
9) Laemmli, U.K. 1970. Cleavage of structural proteins during
the assembly of the head of bacteriophage T4. Nature 227:
680–685.
10) Li, X., and S. Roseman. 2004. The chitinolytic cascade inVibrios is regulated by chitin oligosaccharides and a two-component chitin catabolic senser/kinase. Proc. Natl. Acad. Sci.
USA 101: 627–631.
11) Mabuchi, N., I. Hashizume, and Y. Araki. 2000. Characterization of chitinases excreted by Bacillus cereus CH. Can. J.
Microbiol. 46: 370–375.
12) Mabuchi, N., and Y. Araki. 2001. Cloning and sequencing of
two genes encoding chitinases A and B from Bacillus cereus
CH. Can. J. Microbiol. 47: 895–902.
13) Meibom, K.L., X.B. Li, A.T. Nielsen, C. Wu, S. Roseman, andG.K. Schoolnik. 2004. The Vibrio cholerae chitin utilization
program. Proc. Natl. Acad. Sci. USA 101: 2524–2529.
14) Oliver, D.B., R.J. Cabelli, K.M. Dolan, and G.P. Jarosik. 1990.
Azide-resistant mutants of Escherichia coli alter the SecA
protein, an azide-sensitive component of the protein export
machinery. Proc. Natl. Acad. Sci. USA 87: 8227–8231.
15) Roby, D., A. Gadelle, and A. Toppan. 1987. Chitin oligosaccharides as elicitors of chitinase activity in melon plants. Biochem. Biophys. Res. Commun. 143: 885–92.
16) Sheridan, G.E.C., C.I. Masters, J.A. Shallcross, and B.M.
Mackey. 1998. Detection of mRNA by reverse transcriptionPCR as an indicator of viability in Escherichia coli cells. Appl.
Environ. Microbiol. 64: 1313–1318.
17) Suzuki, K., N. Sugawara, M. Suzuki, T. Uchiyama, F. Katouno,
N. Nikaidou, and T. Watanabe. 2002. Chitinases A, B, and C1of Serratia marcescens 2170 produced by recombinant
Escherichia coli: Enzymatic properties and synergism on chitin
degradation. Biosci. Biotechnol. Biochem. 66: 1075-1083.
18) Tsujibo, H., H. Orikoshi, N. Baba, M. Miyahara, K. Miyamoto,
M. Yasuda, and Y. Inamori. 2002. Identification and characterization of the gene cluster involved in chitin degradation in amarine bacterium, Alteromonas sp. strain O-7. Appl. Environ.
Microbiol. 68: 263–270.
19) Tsujibo, H., N. Kondo, K. Tanaka, K. Miyamoto, N. Baba,
and Y. Inamori. 1999. Molecular analysis of the gene encoding
a novel transglycosylative enzyme from Alteromonas sp. strain
O-7 and its physiological role in the chitinolytic system. J.
Bacteriol. 181: 5461–5466.
20) Tsujibo, H., T. Kubota, M. Yamamoto, K. Miyamoto, and Y.
Inamori. 2003. Characterization of chitinase genes from analkaliphilic actinomycete, Nocardiopsis prasina OPC-131.
Appl. Environ. Microbiol. 69: 894–900.
21) Watanabe, K., Y. Kodama, and S. Harayama. 2001. Designand evaluation of PCR primers to amplify bacterial 16S
ribosomal DNA fragments used for community fingerprinting.
J. Microbiol. Methods 44: 253–262.
22) Yu, C., A.M. Lee, B.L. Bassler, and S. Roseman. 1991. Chitin
utilization by marine bacteria: A physiological function forbacterial adhesion to immobilized carbohydrates. J. Biol.
Chem. 266: 24260–24267.
23) Yu, C., A.M. Lee, and S. Roseman. 1987. The sugar-specificadhesion/deadhesion apparatus of the marine bacterium
Vibrio furnissii is a sensorium that continuously monitors
nutrient levels in the environment. Biochem. Biophys. Res.
Commun. 149: 86–92.
24) Zito, C.R., and D. Oliver. 2003. Two-stage binding of SecA to
the bacterial translocon regulates ribosome-translocon interaction. J. Biol. Chem. 278: 40640–40646.