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Applied and Environmental Microbiology, April 2005, p. 1811-1815, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.1811-1815.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Roles of Four Chitinases (ChiA, ChiB, ChiC, and ChiD) in the Chitin Degradation System of Marine Bacterium Alteromonas sp. Strain O-7

Hideyuki Orikoshi, Shigenari Nakayama, Katsushiro Miyamoto, Chiaki Hanato, Masahide Yasuda, Yoshihiko Inamori, and Hiroshi Tsujibo^*

Department of Microbiology, Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka, Japan

Received 15 July 2004/ Accepted 22 October 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Alteromonas sp. strain O-7 secretes four chitinases (ChiA, ChiB, ChiC, and ChiD) in the presence of chitin. To elucidate why the strain produces multiple chitinases, we studied the expression levels of the four genes and proteins, their enzymatic properties, and their synergistic effects on chitin degradation. Among the four chitinases, ChiA was produced in the largest quantities, followed by ChiD, and the production of ChiB and ChiC changed at lower levels than those of ChiA and ChiD. The expression of the chiA, chiB, chiC, and chiD genes was investigated at the transcriptional level. The RNA transcript of chiA was most strongly induced in the presence of chitin, the expression of chiD followed, and the RNA transcripts of chiB and chiC changed at low levels. The hydrolyzing activities of the four chitinases against various substrates were examined. ChiA was the most active enzyme against powdered chitin, whereas ChiC was the most active against soluble chitin among the four chitinases. ChiD had activities closer to those of ChiA than to those of ChiB and ChiC. ChiB showed no distinctive feature against the chitinous substrates tested. When powdered chitin was treated with the proper combination of four chitinases, an approximately 2.0-fold increase in the hydrolytic activity was observed. These results, together with the results described above, indicate that ChiA plays a central role in chitin degradation for this strain.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chitin, an insoluble homopolymer of ß-(1,4)-linked N-acetylglucosamine (GlcNAc), is one of the most abundant organic compounds in nature. For the complete hydrolysis of chitin to GlcNAc, the concerted action of chitinase (EC 3.2.1.14) and ß-N-acetylglucosaminidase (EC 3.2.1.30) is considered to be essential (6). Chitinases are enzymes that randomly cleave glycosidic linkages of GlcNAc to produce soluble oligosaccharides, mainly chitobiose, which are further hydrolyzed to GlcNAc by ß-N-acetylglucosaminidases. Among these enzymes, chitinases play a central role in the degradation process by bacteria, although the process is exceedingly complex (9). Many chitinase genes have been cloned from bacteria from both terrestrial and marine environments, and their biochemical properties, catalytic mechanisms, and tertiary structures have been clarified. Our final goal is to clarify the chitinolytic system of Alteromonas sp. strain O-7 at the molecular level.

Alteromonas sp. strain O-7 is a gram-negative, flagellated, motile, aerobic, rod-shaped bacterium of marine origin and is an efficient producer of chitinolytic enzymes (14). We have already shown that the chitinolytic system of the strain consists of four chitinases (ChiA, ChiB, ChiC, and ChiD), three ß-N-acetylglucosaminidases (GlcNAcase A, GlcNAcase B, and GlcNAcase C), a transglycosylative enzyme (Hex99), a chitin-binding protein (Cbp1), and a chitin-binding protease (AprIV) (11, 12, 15-21). ChiA, ChiB, ChiC, and ChiD have sequence similarities to family 18 bacterial chitinases (4, 7, 8). These chitinases have a catalytic domain, the chitin-binding domain type 3, and a fibronectin type III-like domain as a common structural unit. However, the polycystic kidney disease (PKD) domain (3, 5, 10) is found only in the N-terminal region of ChiA. Chitinolytic bacteria produce multiple chitinases, but there is comparatively little information available about the properties and roles of the individual chitinases in a chitinolytic system. Furthermore, no studies have been performed at the gene and protein expression levels in the presence of chitin. The synergy between multiple chitinases is assumed to be necessary for effective chitin degradation, but how the expression of these chitinases is coordinated in the presence of chitin is not known. For the present study, we examined the enzymatic properties, expression levels, and synergistic effects of ChiA, ChiB, ChiC, and ChiD to elucidate why Alteromonas sp. strain O-7 produces multiple chitinases in the presence of chitin.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and culture conditions.
Alteromonas sp. strain O-7 was grown at 27°C in Bacto marine broth 2216 (Difco) and used as the source of chromosomal DNA. Escherichia coli JM109, BL21(DE3), and TOP10 were grown at 37°C in Luria-Bertani medium containing 100 µg of ampicillin per ml for the selection of transformants and for the production of recombinant proteins. For agar medium, Luria-Bertani medium was solidified with 1.5% (wt/vol) agar (Nacalai Tesque, Kyoto, Japan).

Construction of expression plasmids.
The recombinant ChiA, ChiB, and ChiD proteins were constructed as described previously (12, 21). The expression plasmid pET-ChiC, coding for ChiC, was constructed as follows. Two oligonucleotide primers, P-1 and P-2 (Table 1), which were modified to contain BamHI and HindIII restriction sites to facilitate cloning in frame into pET-20b(+) (Novagen), were synthesized. The chiC gene was amplified from chromosomal DNA digested with HindIII by a PCR with these primers. The PCR was performed for 30 cycles consisting of 94°C for 15 s, 50°C for 30 s, and 68°C for 2 min. The amplified DNA was digested with BamHI and HindIII, and the resulting fragment (1.4 kb) was inserted into the corresponding sites of pET-20b(+). The nucleotide sequences of the junction between the vector and the insert and of the whole amplified DNA were confirmed by use of a DYEnamic ET Terminator cycle sequencing premix kit (Amersham Bioscience) and a DNA sequencer (ABI Prism 310 genetic analyzer; Applied Biosystems).

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TABLE 1. Sequences of primers used for this study

Purification of recombinant proteins.
For the production of recombinant ChiA, ChiB, ChiC, and ChiD, E. coli BL21(DE3) cells harboring pET-ChiA, pET-ChiB, pET-ChiC, and pET-ChiD were grown, and recombinant proteins were induced by 1.0 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) at mid-exponential growth phase and further incubated for 4 h at 37°C. Cells were harvested by centrifugation, washed, and resuspended in phosphate-buffered saline. The cells were disrupted by sonication, and the lysate was centrifuged at 10,000 x g for 20 min. Each recombinant protein accumulated in the cells as an inclusion body. Each pellet was then solubilized in 6 M guanidine hydrochloride, and the recombinant protein was purified by use of a HisTrap column (Amersham Bioscience). The proteins were slowly dialyzed against 50 mM Tris-HCl buffer (pH 7.5) by several buffer changes. The supernatants were further chromatographed with a linear gradient of NaCl with a Resource Q (Amersham Bioscience) fast protein liquid chromatography column. Active fractions were combined and used as purified enzymes.

Protein determination.
Protein concentrations were determined by the Bradford method, with bovine serum albumin used as a standard (1).

Chitinase assay.
Chitinase activities were measured as described previously (12, 14), with powdered chitin, colloidal chitin, glycol chitin (Seikagaku Co., Tokyo, Japan), or p-nitrophenyl-N,N'-diacetylchitobiose [pNP-(GlcNAc)₂] (Seikagaku Co.) as the substrate. One unit of chitinase activity was defined as the amount of enzyme releasing 1 µmol of reducing sugar or 1 µmol of p-nitrophenol per min.

SDS-PAGE and Western blotting analysis.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done as described previously (14). Proteins in the gel were transferred to a Sequi-Blot polyvinylidene difluoride membrane (Bio-Rad). The membrane was incubated for 1 h at room temperature with an anti-ChiA, -B, -C, or -D polyclonal rabbit antiserum diluted to 1:1,000 in phosphate-buffered saline containing 2.0% skim milk (Difco). Bound antibodies were detected with an ECL anti-rabbit secondary antibody (Amersham Bioscience) conjugated to peroxidase, which allowed colorimetric detection by the use of 3-amino-9-ethylcarbazole. The detected bands were quantified by the use of Image GAUGE (Fuji Film) and Excel (Microsoft).

Real-time quantitative PCR analyses of chiA, -B, -C, and -D transcripts.
Alteromonas sp. strain O-7 was cultured in Bacto marine broth 2216 containing 1.0% powdered chitin at 27°C. The total RNA was extracted from 1.0 ml of a cell suspension of Alteromonas sp. strain O-7 by use of an SV total RNA isolation system (Promega) according to the manufacturer's instructions. The total RNA (5.0 µg) and primers P-4, P-6, P-8, and P-10 (Table 1) were used to reverse transcribe the chiA, chiB, chiC, and chiD transcripts, respectively. The reaction was carried out at 55°C for 60 min with Moloney murine leukemia virus reverse transcriptase (RNase H^–; Promega) and was terminated by heating at 70°C for 15 min. The amounts of reverse transcripts for the chiA, chiB, chiC, and chiD genes were measured by real-time quantitative PCR. Primers P-3 and P-4 were used for chiA, P-5 and P-6 were used for chiB, P-7 and P-8 were used for chiC, and P-9 and P-10 were used for chiD (Table 1). PCR amplification was monitored by use of a QuantiTect SYBR green PCR kit (QIAGEN) and a LightCycler instrument (Roche Diagnostics). Serial 10-fold dilutions of a known amount of plasmid carrying the target chitinase gene, which corresponded to 1.0 x 10³ to 1.0 x 10⁷ copies of each gene, were used as standard samples. The cycle threshold number (C_T; the intersection of the log-linear line by a baseline x axis distinguished from the signal background) of each sample was measured by the fit points method. The C_T values of standard samples were plotted against the logarithms of their copy numbers to generate the standard line. The copy number of each test sample was determined by relating its C_T value to the standard line. The data were calculated automatically by LightCycler software (version 3.53; Roche Diagnostics).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression and purification of recombinant chitinases.
To elucidate the roles of four chitinases on chitin degradation and to prepare a polyclonal antiserum for each chitinase, we produced recombinant ChiA, ChiB, ChiC, and ChiD in E. coli. These recombinant chitinases were expressed in E. coli as inclusion bodies. These enzymes were then refolded and purified to homogeneity by a combination of affinity and ion-exchange column chromatography. The apparent molecular masses of the recombinant ChiA, ChiB, ChiC, and ChiD proteins were estimated to be 90, 91, 53, and 114 kDa, respectively, by SDS-PAGE analysis. These were in close agreement with their theoretical molecular masses, although a six-His tag was added to the C-terminal amino acid residue of each chitinase. Their enzymatic properties (optimum temperature and pH and thermal stability) were almost the same as those of the purified native enzymes from Alteromonas sp. strain O-7 (15, 18, 21). These results indicate that the recombinant enzymes were completely refolded from inclusion bodies.

Expression level of each chitinase.
In order to investigate the expression levels of four chitinases in the culture supernatant, we performed Western blot analysis with a polyclonal rabbit antibody against each chitinase. The expression levels of ChiA, ChiB, ChiC, and ChiD were measured from working curves obtained by a Western blot analysis of each chitinase. The threshold concentrations that were able to detect ChiA, ChiB, ChiC, and ChiD were 0.196, 0.036, 0.041, and 0.044 µg/ml, respectively. The production of chitinases by Alteromonas sp. strain O-7 was examined in Bacto marine broth 2216 containing chitin as an inducer. Among the four chitinases, ChiA was produced at the highest level, followed by ChiD, and the production of ChiB and ChiC gradually increased at low expression levels (Fig. 1). When (GlcNAc)₂ was used as an inducer substrate, the experimental result showed a similar tendency to that for chitin (data not shown). On the other hand, when GlcNAc was used as an inducer, the expression level of each chitinase was about half that of chitin (data not shown). The mechanism of this phenomenon is not yet known.

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FIG. 1. Biosynthesis of ChiA, ChiB, ChiC, and ChiD. Alteromonas sp. strain O-7 was cultured at 27°C in Bacto marine broth 2216 containing 1% powdered chitin. Symbols: , ChiA; , ChiB; , ChiC; , ChiD.

To investigate the expression of the chiA, chiB, chiC, and chiD genes at the transcriptional level, we performed a real-time quantitative PCR analysis. The strain was cultured in Bacto marine broth 2216 containing chitin, and the amounts of mRNA were measured at 1-h intervals. The RNA transcript of chiA was most strongly induced in the presence of chitin, the expression of chiD followed this, and the RNA transcripts of chiB and chiC changed at low levels until 7 h (Fig. 2). These results indicate that the order of protein expression levels is the same as that of the mRNA transcript levels. Although there was a clear difference in the amounts of RNA transcript of each chitinase gene, the four chitinase genes were expressed simultaneously after 5 h.

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FIG. 2. Real-time quantitative PCR analysis of chiA, chiB, chiC, and chiD transcripts. Alteromonas sp. strain O-7 was cultured at 27°C in Bacto marine broth 2216 containing 1% powdered chitin. Symbols: , chiA transcript; , chiB transcript; , chiC transcript; , chiD transcript.

Hydrolyzing activities against various substrates.
To clarify the substrate specificities of ChiA, ChiB, ChiC, and ChiD, we compared the hydrolyzing activities of these enzymes against various substrates (Fig. 3). When glycol chitin was used as a substrate, ChiC showed the highest activity among the four chitinases, whereas ChiA had the lowest activity. When colloidal chitin was used as a substrate, ChiD showed the highest activity. ChiA, ChiB, and ChiC had 14, 6, and 33% of the ChiD activity, respectively, when the hydrolytic activity of ChiD was set to 100%. When powdered chitin was used as a substrate, ChiA showed the highest activity among the four chitinases, and ChiD had about half the activity of ChiA. On the other hand, the hydrolyzing activities of ChiB and ChiC showed 7 and 14% of the ChiA activity, respectively. These results indicate that ChiA plays a central role in the hydrolysis of powdered chitin and that ChiC is utilized for soluble chitins, such as glycol chitin. ChiD had activities closer to those of ChiA than to those of ChiB and ChiC.

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FIG. 3. Substrate specificities of ChiA, ChiB, ChiC, and ChiD. The reactions were performed at 27°C for 30 min in 50 mM citrate buffer, pH 6.0, with 0.12% glycol chitin (A), colloidal chitin (B), or powdered chitin (C) as a substrate. The data shown are the means ± standard deviations of three independent experiments.

Synergistic effects on chitin degradation.
To examine whether the four chitinases act synergistically on chitin degradation, we measured the synergistic effects against powdered chitin. First, the additive effects obtained by combining two different chitinases were examined. When the molar ratios of ChiA to ChiD, ChiA to ChiC, ChiB to ChiC, ChiB to ChiD, and ChiC to ChiD were 1:2, 3:2, 3:2, 1:2, and 1:3, respectively, the highest synergistic effects were observed for each combination (data not shown). Based on the above experimental results, we assumed that the molar ratio to obtain the highest synergistic effect of the four chitinases was three ChiA molecules to three ChiB molecules to two ChiC molecules to six ChiD molecules. To examine the synergistic effect, we used each chitinase in proportion to this molar ratio by keeping the total concentration of the four chitinases constant (14.0 pmol/ml). As shown in Fig. 4, a clear synergistic effect was observed. When powdered chitin was treated with the combination of ChiA, ChiB, ChiC, and ChiD, an approximately 2.0-fold increase in the hydrolytic activity was observed in comparison with the sum of all the chitinase activities.

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FIG. 4. Synergistic effect of ChiA, ChiB, ChiC, and ChiD. Chitinase activities were assayed in 200-µl reaction mixtures containing 0.1% powdered chitin and each enzyme in 50 mM citrate buffer, pH 6.0, at 27°C. ChiA, ChiB, ChiC, and ChiD were used at a molar ratio of 3:3:2:6. The total concentration of the chitinases was constant (14.0 pmol/ml). , difference between the activity obtained by the mixture of four chitinases and the sum of the chitinase activities. The data shown are the means ± standard deviations of three independent experiments.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The chitinolytic system of Alteromonas sp. strain O-7 includes four chitinases (12, 15, 18, 21), three ß-N-acetylglucosaminidases (16, 17, 20), a transglycosylative enzyme (19), a chitin-binding protein (21), and a chitin-binding protease (11). The present study was conducted to elucidate why the strain produces multiple chitinases in the presence of chitin. The recombinant ChiA, ChiC, and ChiD proteins shared several enzymatic properties, such as their optimum pH, optimum temperature, and stability, when pNP-(GlcNAc)₂ was used as a substrate (12). However, ChiB showed different enzymatic properties from the other three chitinases. The optimum temperature of ChiB was 30°C, and ChiB showed remarkable thermal lability compared to ChiA, ChiC, and ChiD. These results indicate that the production of ChiB may be advantageous for the strain, allowing it to easily acquire nutrients from chitin and to survive in cold environments.

When the expression levels of the chitinases in culture supernatants were compared, ChiA was produced in the largest quantities among the four chitinases, followed by ChiD. On the other hand, the production of ChiB and ChiC changed at lower levels than those of ChiA and ChiD until 40 h of cultivation. Furthermore, the order of protein expression levels was the same as that of mRNA transcript levels. These results indicate that ChiA plays a central role in chitin degradation in Alteromonas sp. strain O-7. The appearance of each chitinase in the culture medium was observed immediately after the expression of mRNA. The production of chitinases increased until 40 h. On the other hand, the expression level of mRNA increased until 7 h and declined sharply below the limit of detection afterwards. Although we could not give a reasonable explanation for this phenomenon, one possibility is that the translation rate of chitinase genes exceeded the secretion capacity. The strain also secretes four proteases in the presence of chitin. Therefore, the four chitinases are proteolytically modified to produce more chitinases after secretion into the culture medium. For example, Chi65 which lacks the C-terminal chitin-binding domain, is a proteolytic derivative of ChiA. ChiB, ChiC, and ChiD are also proteolytically cleaved to produce the catalytic domain and the chitin-binding domain. Therefore, chitin seems to be degraded efficiently through a quite complicated process by four chitinases and their truncated forms produced by the strain. However, we do not know why Altermonas sp. strain O-7 produces various truncated forms of ChiA, ChiB, ChiC, and ChiD for chitin degradation. The expression of the chitinase genes occurred at about the same time in the presence of chitin, indicating that the four chitinase genes are regulated by a common induction mechanism in the presence of chitin. An alignment of the nucleotide sequences of chiA, chiB, chiC, and chiD near the promoter regions revealed the presence of a conserved 8-bp sequence, 5'-ACAACATG-3', in these genes (unpublished data). Therefore, these chitinase genes might be coordinately controlled by the same regulatory protein(s). The differences in the amounts of RNA transcript might be due to the differences in the promoter activities of the chitinase genes.

To examine the roles of the four chitinases in chitin degradation, we examined their hydrolyzing activities against various chitinous substrates. ChiA and ChiD were adaptable for the hydrolysis of insoluble chitin, and ChiC was utilized for the hydrolysis of soluble chitin. ChiB showed no distinctive features against the chitinous substrates tested. Suzuki et al. described the hydrolyzing activities of ChiA, ChiB, and ChiC1 from Serratia marcescens 2170 against various chitinous substrates (13). ChiA was the enzyme that was adopted to hydrolyze insoluble chitin, and ChiC1 was used for the hydrolysis of soluble chitin. ChiB had activities closer to those of ChiC1 than to those of ChiA against insoluble chitin and closer to those of ChiA than to those of ChiC1 against soluble chitin. Judging from these experimental data from Alteromonas sp. strain O-7 and S. marcescens 2170, it seems likely that chitinolytic bacteria produce multiple chitinases which have different substrate specificities against various chitinous substrates. Next, the synergistic effects of ChiA, ChiB, ChiC, and ChiD on the hydrolysis of powdered chitin were examined. When ChiA, ChiB, ChiC, and ChiD were combined at a molar ratio of 3:3:2:6, clear synergistic effects on chitin degradation were observed. Suzuki et al. described synergism between ChiA and ChiB as well as ChiA and ChiC1 from S. marcescens 2170 for the hydrolysis of powdered chitin (13). Brurberg et al. also reported synergistic effects on the hydrolysis of colloidal chitin between ChiA and ChiB from S. marcescens BJL200 (2). These results indicate that chitinolytic bacteria such as Alteromonas sp. strain O-7 and S. marcescens produce multiple chitinases for the efficient degradation of chitin in the natural environment.

ChiA was produced in the largest quantities by Alteromonas sp. strain O-7 and showed the highest activity against powdered chitin among the four chitinases. This chitinase, as previously described, comprises the PKD domain, a catalytic domain, two fibronectin type III-like domains, and the C-terminal chitin-binding domain. The PKD domain was only found in the N-terminal region of ChiA among the four chitinases.

Therefore, characteristic features of the enzymatic properties of ChiA which are distinct from those of the other three chitinases may be attributable to the PKD domain of ChiA. In the future, it will be interesting to determine the crystal structure of Alteromonas ChiA to understand the mechanism for the efficient hydrolysis of insoluble chitin.

 
This study was supported in part by a grant-in-aid for high technology research and a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

 
* Corresponding author. Mailing address: Department of Microbiology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan. Phone: (81-726) 90-1057. Fax: (81-726) 90-1057. E-mail: tsujibo{at}gly.oups.ac.jp.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, April 2005, p. 1811-1815, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.1811-1815.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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• Lennon, J. T. (2007). Diversity and Metabolism of Marine Bacteria Cultivated on Dissolved DNA. Appl. Environ. Microbiol. 73: 2799-2805 [Abstract] [Full Text]  
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