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A Metalloprotease (MprIII) Involved in the Chitinolytic System of a Marine Bacterium, Alteromonas sp. Strain O-7

Department of Microbiology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan

Received 23 May 2002/ Accepted 22 August 2002

	ABSTRACT

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Alteromonas sp. strain O-7 secretes several proteins in addition to chitinolytic enzymes in response to chitin induction. In this paper, we report that one of these proteins, designated MprIII, is a metalloprotease involved in the chitin degradation system of the strain. The gene encoding MprIII was cloned in Escherichia coli. The open reading frame of mprIII encoded a protein of 1,225 amino acids with a calculated molecular mass of 137,016 Da. Analysis of the deduced amino acid sequence of MprIII revealed that the enzyme consisted of four domains: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The C-terminal extension (PkdDf) was characterized by four polycystic kidney disease domains and two domains of unknown function. Western and real-time quantitative PCR analyses demonstrated that mprIII was induced in the presence of insoluble polysaccharides, such as chitin and cellulose. Native MprIII was purified to homogeneity from the culture supernatant of Alteromonas sp. strain O-7 and characterized. The molecular mass of mature MprIII was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 115 kDa. The optimum pH and temperature of MprIII were 7.5 and 50°C, respectively, when gelatin was used as a substrate. Pretreatment of native chitin with MprIII significantly promoted chitinase activity. Furthermore, the combination of MprIII and a novel chitin-binding protease (AprIV) remarkably promoted the chitin hydrolysis efficiency of chitinase.

	INTRODUCTION

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Chitin, an insoluble linear ß-1,4-linked polymer of N-acetylglucosamine (GlcNAc), is the second most abundant organic compound found in nature. This polysaccharide is a major component of the cell walls of fungi and the cuticles of insects and crustaceans (11) and is used by many microorganisms as a source of carbon and nitrogen. Since carbon and nitrogen are generally limited in the marine environment, chitin is a particularly important nutrient source for marine organisms (8).

If the insoluble form of chitin could not be returned to the ecosystem in a biologically usable form, the marine environment would be completely depleted of a carbon and nitrogen source in a relatively short time (38). Chitinolytic marine bacteria, such as zooplankton, play a crucial role in the recycling of chitinous materials (38). To degrade chitin, chitinolytic bacteria produce two classes of enzymes: chitinases (EC 3.2.1.14) and ß-N-acetylglucosaminidases (GlcNAcases; EC 3.2.1.30). In crustacean cuticles, chitin is tightly associated with proteins, inorganic salts, such as calcium carbonate, and lipids, including pigments (25). Thus, proteases and lipases together with chitinases could be necessary for efficient degradation of crustacean cuticles in the natural environment.

We have been studying the chitinolytic system of the marine bacterium Alteromonas sp. strain O-7 to clarify the roles of individual enzymes in chitin degradation, the relationship between structure and function, and the regulation of gene expression. We have already cloned and sequenced the genes encoding three chitinases (ChiA, ChiB, and ChiC), three GlcNAcases (GlcNAcase A, GlcNAcase B, and GlcNAcase C), chitinase-like enzyme (ChiD), and chitin-binding protein (Cbp1), and the role of each protein in chitin degradation has been clarified (29-31, 33, 35, 36). Recently, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that several proteins besides chitinolytic enzymes were induced in the presence of chitin. It has been reported that one of the proteins, a novel chitin-binding protease (AprIV), participates in chitin degradation by Alteromonas sp. strain O-7 (16). AprIV is a modular enzyme consisting of three domains: the family A subtilase region, the polycystic kidney disease domain (PkdD), and chitin-binding domain type 3 (ChtBD3). Pretreatment of native chitin with AprIV significantly promoted chitinase activity. In this study, we report the cloning, sequence, and analysis of the gene encoding a metalloprotease (MprIII) induced by chitin. Furthermore, the purification and characterization of the enzyme from the culture supernatant of Alteromonas sp. strain O-7 are described.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and growth conditions.
Alteromonas sp. strain O-7 was cultured at 27°C in Bacto Marine Broth 2216 (Difco) supplemented with 50 mM HEPES, pH 7.0. Escherichia coli JM109 and BL21(DE3) were grown at 37°C on Luria-Bertani medium supplemented with appropriate antibiotics for the selection of transformants and for the production of recombinant protein, respectively. Plasmids pUC18 and pUC19 were used as cloning vectors. Plasmid pProEX HTa (Invitrogen) was used as the expression vector.

Nucleotide sequence determination.
Nucleotide sequencing was carried out by the dideoxy chain termination method using the DYEnamic ET terminator cycle sequencing premix kit (Amersham Bioscience) on a DNA sequencer (ABI Prism 310 genetic analyzer; Applied Biosystems). Sequence data were analyzed using the GENETYX-WIN program (Software Development Co., Ltd.).

N-terminal amino acid sequence of MprIII.
SDS-PAGE was done according to the method of Laemmli (13). Alteromonas sp. strain O-7 was cultured at 27°C in Bacto Marine Broth 2216 equilibrated with 50 mM HEPES, pH 7.0, containing 1.0% powdered chitin from crab shells (Nacalai Tesque, Kyoto, Japan) until the optical density at 600 nm reached 1.5. The culture supernatant was collected by centrifugation (24,650 x g for 5 min at 4°C), and 0.1 volumes of 20% trichloroacetic acid were added to the supernatant. After centrifugation, the pellet was dissolved in SDS-PAGE sample buffer. Proteins were separated by SDS-PAGE and transferred to a Sequi-Blot polyvinylidene difluoride membrane (Bio-Rad Laboratories) with a Trans-Blot SD semidry electrophoretic transfer cell (Bio-Rad Laboratories). The chitin-induced protein band corresponding to MprIII was directly sequenced using an ABI Procise 491 HT protein sequencer (Applied Biosystems) that was connected to an online phenylthiohydantoin derivative analyzer.

Cloning of the mprIII gene.
Alteromonas sp. strain O-7 total DNA was digested with various restriction enzymes and electrophoresed on a 1.0% agarose gel. The fragments in the range of 0.3 to 1.0 kb were excised from the gel and purified with a GenElute gel purification kit (Sigma). These were self-ligated and used as template DNAs. The degenerate inverse primers mprN, 5'-CC(A/T)(C/G)(A/T)(A/G)CC(A/G)CCTTCATA(A/T)AC(A/T)AC-3', and mprC, 5'-GG(C/T)AA(C/T)GATAAAGT(A/T)(A/T)(C/G)(A/T)CG(C/T)CC-3', were synthesized based on the N-terminal amino acid sequence of MprIII. PCR amplification was performed with KOD-Plus DNA polymerase (Toyobo, Osaka, Japan) for 30 cycles consisting of 94°C for 15 s, 53°C for 30 s, and 68°C for 1 min. The 0.4-kb product was amplified by using the HindIII-digested template and phosphorylated by T4 polynucleotide kinase (Toyobo). The phosphorylated fragment was digested with HindIII. The resulting 0.3-kb fragment (pXH03) was cloned into the SmaI and HindIII sites of pUC19. However, analyses of the entire nucleotide sequences of the inserted DNAs indicated that the 5' upstream and 3' downstream regions of the mprIII gene were missing. To clone the full length of the mprIII gene, Southern hybridization was performed using the 0.3-kb fragment (pXH03) as a probe. The probe was labeled with alkaline phosphatase according to the manufacturer's instructions (AlkPhos DIRECT; Amersham Bioscience). The probe hybridized with the 4.6-kb chromosomal fragment digested with EcoRV. The DNA fragments corresponding to 4.6 kb were excised from the gel and purified with a GenElute gel purification kit. These were ligated into the dephosphorylated SmaI site of pUC19, and the recombinant plasmids were introduced into competent E. coli JM109. The library was screened by colony hybridization with the labeled probes as previously described (16).

Real-time quantitative PCR analysis of the mprIII transcript.
PCR amplification of the reverse transcriptase product was monitored by using a QuantiTect SYBR Green PCR kit (Qiagen) in a LightCycler (Roche Diagnostics). For each sample, a long linear line was fitted automatically by selecting two points above the threshold band to determine the fractional cycle number of the crossing point. The data were calculated automatically with LightCycler software (version 3.53; Roche Diagnostics).

Construction of the expression plasmid.
The expression plasmid, pProPkdDf, encoding the full length of the C-terminal extension (residues Ala539 to Asn1225) of MprIII was constructed as follows. Two oligonucleotide primers (EV2505E, 5'-TACTGGTGCTGAATTCGTAATGAGACAAAC-3', and EV4618S, 5'-TCTACAAGGTAGTGTCGACTTTCTCCGCTC-3') which were modified to contain EcoRI and SalI recognition sites to facilitate in-frame cloning into the His-tagged protein expression vector pProEX HTa (Invitrogen) were synthesized. PCR was performed with the plasmid pMP46 as a template for 30 cycles consisting of 94°C for 15 s, 57°C for 30 s, and 68°C for 2 min. The amplified DNA was digested by EcoRI and SalI, and the resulting fragment (2,113 bp) was inserted into the corresponding sites of pProEX HTa. The nucleotide sequence of the PCR fragment was confirmed by DNA sequencing.

Purification and binding study of PkdDf.
E. coli BL21(DE3) cells harboring pProPkdDf were induced at the mid-exponential growth phase with 0.1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) and further incubated for 1.5 h at 37°C. Cells were harvested by centrifugation and washed and resuspended with phosphate-buffered saline. The cells were disrupted by sonication, and the lysate was centrifuged at 10,000 x g for 10 min. The His-tagged protein accumulated in the cells as inclusion bodies. The pellet was then solubilized in 6 M guanidine hydrochloride and purified with HisTrap (Amersham Bioscience). The purified protein (PkdDf) was dialyzed against 50 mM Tris-HCl buffer, pH 8.0. The N-terminal amino acid sequence of PkdDf was confirmed by protein sequencing. The purified PkdDf (5 µg) was mixed with 5 mg of -chitin, ß-chitin, chitosan, or cellulose. The binding assay mixtures were incubated on ice for 30 min, and then the pellets were directly dissolved with SDS-PAGE sample buffer. After SDS-PAGE, the amount of protein was measured as a band with Image Gauge version 3.45 (Fujifilm, Tokyo, Japan).

Western blotting and immunodetection.
As described above for the determination of the N-terminal amino acid sequence, proteins from the culture supernatant were separated by SDS-PAGE and transferred to a Sequi-Blot polyvinylidene difluoride membrane. The membrane was incubated for 1 h at room temperature with anti-PkdDf polyclonal mouse antiserum at a 1:2,000 dilution in phosphate-buffered saline containing 2.0% skim milk (Difco). The anti-PkdDf polyclonal mouse antiserum was prepared by the following procedure. Five mice were immunized intramuscularly with 250 µg of the purified PkdDf. Subsequent immunizations were performed after 14 days. Mice were bled at day 28, and antiserum reactivity was evaluated by both enzyme-linked immunosorbent assay and immunoblot analysis. Bound antibody was detected as previously described (34). The amount of MprIII production was measured with Image Gauge version 3.45.

Enzyme assay.
Gelatinolytic activity was assayed according to the method of Sasagawa et al. (24) with slight modifications. The reaction mixture consisted of 20 µl of enzyme solution and 30 µl of 0.2% gelatin in 50 mM boric acid-borate buffer, pH 7.5. After incubation at 50°C for 2 h, the reaction was terminated by the addition of 60 µl of 0.1 N HCl. The free -amino groups produced were measured by the ninhydrin method (19). One unit of gelatinolytic enzyme was defined as the amount of enzyme that liberated 1 µmol of glycine in 1 min under the conditions described above. Caseinolytic activity was examined as described previously (27). The hydrolysis of keratin azure (Sigma) and elastin-orcein (Elastin Products Co., Inc.) was assayed by the methods of Bressollier et al. (3) and Grimwood et al. (9), respectively. Characterization of MprIII was determined with gelatin as a substrate. The enzyme activity was measured at pH values from 5 to 9 under the standard assay conditions. The buffers used were 50 mM acetate buffer (pH values 5 and 6) and 50 mM boric acid-borate buffer (pH values 7 to 9).

Purification of MprIII.
Alteromonas sp. strain O-7 was cultured in Bacto Marine Broth 2216 containing 1.0% powdered chitin from crab shells (Nacalai Tesque) until the optical density at 600 nm reached 1.5. The supernatant was dialyzed against 50 mM Tris-HCl buffer, pH 8.0, and applied to a DEAE-Toyopearl column (3.0 by 15 cm; Tosoh, Tokyo, Japan). The protease activities were eluted at 0.2 to 0.5 M NaCl and separated into three active fractions (fractions I, II, and III). Ammonium sulfate was added to fraction I until the final concentration reached 1.0 M. The fraction was applied to a phenyl-Toyopearl 650 M column (1.0 by 20 cm; Tosoh) equilibrated with 50 mM Tris-HCl buffer (pH 8.0) containing 1.0 M ammonium sulfate, and the protein was eluted with a decreasing ammonium sulfate gradient. The active fraction corresponding to native MprIII was eluted at a concentration of about 0.55 M ammonium sulfate and dialyzed against 50 mM Tris-HCl buffer, pH 8.0. The dialyzed fraction was put on a DEAE-Toyopearl column (1.9 by 55 cm) and eluted with a linear gradient of NaCl (0 to 0.5 M). MprIII protein was eluted at a concentration of about 0.2 M NaCl. All purification steps were done at 4°C. The protein concentration was determined by the method of Bradford with bovine serum albumin as a standard (2).

Effect of MprIII on chitin degradation.
The effect of MprIII on chitinase activity was assessed as described previously (16). The crude native chitin was suspended with 400 µl of 50 mM Tris-HCl buffer (pH 8.0) containing 1 pmol of purified AprIV, MprIII, or both AprIV and MprIII and incubated at 27°C for 3 h. The reaction mixtures were then centrifuged, and the peptides removed from the chitin were assayed by the ninhydrin method (19). The chitin was washed twice with 1 ml of 50 mM Tris-HCl buffer (pH 8.0) and resuspended with 500 µl of the buffer containing 0.5 µg of purified chitinase (Chi85) from the strain. The reaction mixtures were incubated at 50°C for 6 h. Samples were taken at 1-h intervals. The chitinase activity was measured as described previously (29).

Nucleotide sequence accession number.
The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession no. AB084466.

	RESULTS

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Cloning and nucleotide sequence of mprIII.
Alteromonas sp. strain O-7 was cultured in the presence of chitin, and extracellular proteins were separated by SDS-PAGE. The N-terminal amino acid sequence (VVYEGGSGPSGNDKVSRPDY) of a chitin-induced protein corresponding to 115 kDa was determined. The BLAST search program revealed that the protein, designated MprIII, is homologous to enzymes belonging to the thermolysin family of microbial metalloproteases. Thus, to isolate the gene encoding MprIII, degenerate inverse primers (mprN and mprC) were synthesized based on the N-terminal amino acid sequence. The inverse PCR was performed using template DNAs produced by circularization of HindIII fragments from the genomic DNA as described in Materials and Methods. The amplified DNA was about 0.4 kb, and the nucleotide sequences of the HindIII digest, pXH03, were analyzed (Fig. 1). The PCR product encoded a portion near the N terminal of MprIII and lacked the 5' upstream and 3' downstream regions of the mprIII gene. To obtain the full length of the mprIII gene, the EcoRV library was screened by colony hybridization using a 0.3-kb DNA fragment from pHX03 as a probe. Among 1,000 transformants, one positive clone (pMP46) which hybridized to the probe was isolated by colony hybridization. The clone was analyzed by restriction endonuclease digestion and found to contain a 4.6-kb insert with the full-length mprIII gene (Fig. 1A).

View larger version (13K): [in this window] [in a new window]   FIG. 1. (A) Restriction map of pMP46. The arrow indicates the ORF and the direction of transcription. (B) Domain structure of MprIII and PkdDf. igwidth>, signal peptide; igwidth>, N-terminal proregion; , protease region; , PkdD; igwidth>, domain of unknown function.

Structural features of MprIII.
BLAST search analysis of the deduced amino acid sequence of prepro-MprIII revealed that the gene encoded a modular enzyme consisting of four domains: the signal sequence (20 amino acids), the N-terminal proregion (192 amino acids), the protease region (326 amino acids), and the C-terminal extension (687 amino acids) (Fig. 1B). MprIII includes the highly conserved HEXXH zinc-binding motif (residues 371 to 375), indicating that the enzyme is a zinc metalloprotease belonging to the zincins superfamily (17). The protease region (residues Val213 to Gln538) of MprIII showed sequence homology to metalloproteases belonging to the zincins superfamily (peptidase_M4), such as MprI from Alteromonas sp. strain O-7 (41.2% identity) (15), a proaminopeptidase-converting metalloprotease from Aeromonas punctata (40.6% identity) (20), elastase from Aeromonas hydrophila (37.8% identity) (5), VVP (vibriolysin) from Vibrio vulnificus (37.2% identity) (6), and thermolysin from Bacillus thermoproteolyticus (17.7% identity) (26). The zinc ligand motif of MprIII is aligned with those of the other metalloproteases, as shown in Fig. 2. As mentioned previously, the HEXXH zinc-binding motif was conserved in MprIII; however, the glycine residue in the third zinc ligand motif (GXXNEXXSD) of the thermolysin family was replaced by an arginine residue. The C-terminal extension consisted of four PkdDs and two domains of unknown function (Fig. 1B). PkdD, which was first identified in human polycystin-1 (7), has also been found in bacterial collagenases (14), proteases (16, 21), cellulases (1), and chitinases (22). This domain has a ß-sandwich fold, and the sequence WDFGDG is highly conserved in the domain (4). The four regions of MprIII from residues Asp570 to Asp650, Asn791 to Thr875, Arg877 to Glu950, and Ser962 to Met1047 had sequences similar to that of PkdD, with the conserved sequence at residues Trp601 to Gly606, Trp824 to Gly829, Trp904 to Gly909, and Trp993 to Gly998, respectively. The region from residues Tyr651 to Glu790 showed 23.4% identity to that from Tyr1059 to Asn1225. However, the domains had no similarity to the other domains in the National Center for Biotechnology Information conserved domain database (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml).

View larger version (20K): [in this window] [in a new window]   FIG. 2. Alignment of the active-site region of MprIII with those of other metalloproteases (MprI from Alteromonas sp. strain O-7; proaminopeptidase-converting metalloprotease from Aeromonas punctata [PA protease]; elastase from Aeromonas hydrophila [AhpB]; vibriolysin [VVP] from Vibrio vulnificus; and thermolysin from Bacillus thermoproteolyticus). Identical residues are indicated by bold letters. The HEXXH motif and GXXNEXXSD sequence are indicated by the box and shaded box, respectively.

Gene expression.
To confirm whether the expression of mprIII was induced by chitin or its related compounds, real-time quantitative PCR analysis was performed. Alteromonas sp. strain O-7 was cultured in Bacto Marine Broth containing GlcNAc, N-acetylchitobiose, powdered chitin, or skim milk. The RNA transcript of mprIII was induced by 3.4-fold in the presence of 1.0% powdered chitin. However, GlcNAc, N-acetylchitobiose, and skim milk did not influence the expression of mprIII (Fig. 3). These results indicate that chitin induces MprIII production in Alteromonas sp. strain O-7.

View larger version (13K): [in this window] [in a new window]   FIG. 3. Real-time quantitative PCR analysis of the mprIII transcript. C, control (no addition); G1, 1.0% GlcNAc; G2, 1.0% N-acetylchitobiose; PC, 1.0% powdered chitin; SM, 1.0% skim milk.

View larger version (39K): [in this window] [in a new window]   FIG. 4. (A) SDS-PAGE of the culture supernatants. (B) Western blot analysis of MprIII. Lanes: M, prestained molecular weight marker; 1, control (no addition); 2, 1.0% glucose; 3, 1.0% GlcNAc; 4, 1.0% N-acetylchitobiose; 5, 1.0% powdered chitin.

View larger version (14K): [in this window] [in a new window]   FIG. 5. Characterization of MprIII from Alteromonas sp. strain O-7. (A) SDS-PAGE of MprIII. (B) Effect of pH on MprIII activity. The amount of enzyme was 0.32 mU. (C) Effect of temperature on MprIII activity. The reaction was carried out at the various temperatures for 2 h with 50 mM boric acid-borate buffer, pH 7.5. The amount of enzyme was 0.29 mU.

View larger version (16K): [in this window] [in a new window]   FIG. 6. Participation of MprIII in chitin degradation. (A) The protease activities of AprIV and MprIII were measured with the native chitin of the substrates. Control experiments were carried out without enzyme. (B) The effects of AprIV and MprIII on chitinase activity were examined using the native chitin. •—•, Chi85; —, Chi85 plus AprIV; —, Chi85 plus MprIII; —, Chi85 plus AprIV and MprIII. Aliquots were taken at the indicated times and centrifuged, and chitinase activity was measured. The value after incubation for 6 h using AprIV, MprIII, and Chi85 was taken as 100%. Data are from three independent experiments; standard deviations are indicated by vertical lines.

	DISCUSSION

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The mprIII gene encoded a modular enzyme consisting of four domains. The amino acid sequence of the protease region of MprIII showed similarity to that of zinc-containing metalloproteases belonging to the thermolysin family. Holden et al. have shown that two histidine residues in the consensus sequence HEXXH and the glutamic acid residue in the GXXNEXXSD sequence participate in binding to a zinc(II) ion based on the three-dimensional structure of thermolysin from Bacillus thermoproteolytics (10). In the thermolysin family, glutamic acid, which functions as the third zinc ligand, is usually located 25 residues from the HEXXH motif towards the C terminal (17). However, glutamic acid 399, which seems to be the third zinc ligand of MprIII, was located 29 residues from the HEXXH motif towards the C terminal (Fig. 2). In the angiotensin-converting enzyme family, glutamic acid, which is the third zinc ligand, is located 29 residues from the HEXXH motif towards the C terminal. However, the amino acid sequence surrounding the third zinc ligand of MprIII (RAINEAFSD) showed significant similarity to that of the enzymes in the thermolysin family but not to that of the angiotensin-converting enzyme family. Thus, the effect of the insertion of four amino acid residues in this region on the substrate specificity of MprIII was examined by using MprI as a contrast (Table 2). It has been reported that MprI, which is a metalloprotease produced by Alteromonas sp. strain O-7, is one of the typical enzymes in the thermolysin family (15). MprIII showed a high activity towards gelatin among the substrates tested, whereas MprI showed a high activity towards casein (Table 2). These results may suggest that the insertion sequence of MprIII plays an important role in substrate specificity. To confirm a role for the insertion sequence, we are currently preparing a series of deletion mutants of MprIII.

The results of Western and real-time quantitative PCR analyses demonstrated that the synthesis of MprIII was induced in the presence of chitin. Cellulose, which is structurally similar to chitin, showed weak induction activity of MprIII. However, Western blotting demonstrated that the production of MprIII was slightly repressed by the addition of GlcNAc or N-acetylchitobiose, although GlcNAc and N-acetylchitobiose did not influence the expression of mprIII at the transcriptional level. AprIV, a chitin-binding protease involved in chitin degradation in the strain, was induced in the presence of GlcNAc, N-acetylchitobiose, or chitin (16). When chitin was used as an inducer, AprIV tightly bound chitin molecules in the milieu until chitin was hydrolyzed into soluble oligomers. On the other hand, when GlcNAc or N-acetylchitobiose was used as an inducer, AprIV diffused into the culture medium. Thus, the apparent decrease in the production of MprIII seems to be due to proteolysis by AprIV.

In chitinase-producing microorganisms, chitin is a common inducer of chitinase production. However, since chitin is insoluble and impermeable to microorganisms, a soluble degradation product(s) such as GlcNAc, N-acetylchitobiose, or a higher oligomer is considered to act as a direct inducer of chitinase. However, MprIII was induced by chitin, not by GlcNAc, N-acetylchitobiose, and higher oligomers. These findings suggest the following possibilities: (i) a membrane-localized chitin-binding protein might interact with insoluble chitin, and the signal might then be transmitted to a regulatory protein that activates the expression of the target gene; (ii) the expression of mprIII might be switched on depending on fluctuations and stimuli due to chitin present in the immediate environment of the cell. It has been suggested that marine chitinolytic bacteria, such as Vibrio furnissii (12), V. harveyi (18), and V. alginolyticus (23), adhering to chitin surfaces have evolved specific systems to recognize and associate with this substrate. It has been reported that these chitinolytic bacteria could specifically attach to chitin particles through chitin-binding proteins associated with cell membranes. We are currently examining whether Alteromonas sp. strain O-7 has a chitin-binding protein on the cell membrane that is able to recognize chitin particles.

As previously reported, AprIV was expressed selectively by Alteromonas sp. strain O-7 in the presence of GlcNAc, N-acetylchitobiose, or chitin (16). Chitinases produced from the strain were also induced by GlcNAc, N-acetylchitobiose, or chitin. These results suggest that chitinases and AprIV involved in the chitin degradation system of Alteromonas sp. strain O-7 might be coordinately controlled by the same regulatory system. The alignment of the nucleotide sequences of aprIV, 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). However, the conserved sequence was not found in the upstream region of the mprIII gene. These results suggest that the expression of mprIII might be controlled differently from that of aprIV.

Pretreatment of native chitin with MprIII, as with AprIV, stimulated the chitinase activity of Chi85. Furthermore, the combination of MprIII and AprIV remarkably promoted the chitin hydrolysis efficiency of Chi85 compared with the use of AprIV or MprIII alone. MprIII and AprIV differed from each other in substrate specificities; that is, MprIII showed a high activity towards fibrous proteins, such as gelatin and elastin, and AprIV showed a high activity towards casein (Table 2). These results suggest that MprIII and AprIV hydrolyze the different constituent proteins in the surface cuticles to facilitate the interaction between chitinase and chitin molecules.

	FOOTNOTES

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

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Ahsan, M. M., T. Kimura, S. Karita, K. Sakka, and K. Ohmiya. 1996. Cloning, DNA sequencing, and expression of the gene encoding Clostridium thermocellum cellulase CelJ, the largest catalytic component of the cellulosome. J. Bacteriol. 178:5732-5740.[Abstract/Free Full Text]
2. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.[CrossRef][Medline]
3. Bressollier, P., F. Letourneau, M. Urdaci, and B. Verneuil. 1999. Purification and characterization of a keratinolytic serine proteinase from Streptomyces albidoflavus. Appl. Environ. Microbiol. 65:2570-2576.[Abstract/Free Full Text]
4. Bycroft, M., A. Bateman, J. Clarke, S. J. Hamill, R. Sandford, R. L. Thomas, and C. Chothia. 1999. The structure of a PKD domain from polycystin-1: implications for polycystic kidney disease. EMBO J. 18:297-305.[CrossRef][Medline]
5. Cascon, A., J. Yugueros, A. Temprano, M. Sanchez, C. Hernanz, J. M. Luengo, and G. Naharro. 2000. A major secreted elastase is essential for pathogenicity of Aeromonas hydrophila. Infect. Immun. 68:3233-3241.[Abstract/Free Full Text]
6. Chuang, Y. C., T. M. Chang, and M. C. Chang. 1997. Cloning and characterization of the gene (empV) encoding extracellular metalloprotease from Vibrio vulnificus. Gene 189:163-168.[CrossRef][Medline]
7. Gluecksmann-Kuis, M. A., O. Tayber, E. A. Woolf, L. Bougueleret, N. Deng, G. D. Alperin, F. Iris, F. Hawkins, C. Munro, N. Lakey, G. Duyk, M. C. Schneider, L. Geng, F. Zhang, Z. Zhao, S. Torosian, S. T. Reeders, P. Bork, M. Pohlschmidt, C. Loehning, B. Kraus, U. Nowicka, A. L. S. Leung, and A.-M. Frischauf. 1995. Polycystic kidney disease: the complete structure of the PKD1 gene and its protein. Cell 81:289-298.[CrossRef][Medline]
8. Gooday, G. W. 1990. The ecology of chitin degradation, p. 387-430. In K. C. Marshall (ed.), Advances in microbial ecology, vol. 11. Plenum Press, New York, N.Y.
9. Grimwood, B. G., T. H. Plummer, Jr., and A. L. Tarentino. 1994. Purification and characterization of a neutral zinc endopeptidase secreted by Flavobacterium meningosepticum. Arch. Biochem. Biophys. 311:127-132.[CrossRef][Medline]
10. Holden, H. M., D. E. Tronrud, A. F. Monzingo, L. H. Weaver, and B. W. Matthews. 1987. Slow- and fast-binding inhibitors of thermolysin display different modes of binding: crystallographic analysis of extended phosphonamidate transition-state analogues. Biochemistry 26:8542-8553.[CrossRef][Medline]
11. Jannatipur, M., R. W. Soto-Gil, L. C. Childers, and J. W. Zynskind. 1987. Translocation of Vibrio harveyi N,N'-diacetylchitobiase to the outer membrane of Escherichia coli. J. Bacteriol. 169:3785-3791.[Abstract/Free Full Text]
12. Keyhani, N. O., and S. Roseman. 1996. The chitin catabolic cascade in the marine bacterium Vibrio furnissii. Molecular cloning, isolation, and characterization of periplasmic chitodextrinase. J. Biol. Chem. 271:33414-33424.[Abstract/Free Full Text]
13. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685.[CrossRef][Medline]
14. Matsushita, O., C.-M. Jung, S. Katayama, J. Minami, Y. Takahashi, and A. Okabe. 1999. Gene duplication and multiplicity of collagenases in Clostridium histolyticum. J. Bacteriol. 181:923-933.[Abstract/Free Full Text]
15. Miyamoto, K., H. Tsujibo, E. Nukui, H. Itoh, Y. Kaidzu, and Y. Inamori. 2002. Isolation and characterization of the genes encoding two metalloproteases (MprI and MprII) from a marine bacterium, Alteromonas sp. strain O-7. Biosci. Biotechnol. Biochem. 66:416-421.[CrossRef][Medline]
16. Miyamoto, K., E. Nukui, H. Itoh, T. Sato, T. Kobayashi, C. Imada, E. Watanabe, Y. Inamori, and H. Tsujibo. 2002. Molecular analysis of the gene encoding a novel chitin-binding protease from Alteromonas sp. strain O-7 and its role in the chitinolytic system. J. Bacteriol. 184:1865-1872.[Abstract/Free Full Text]
17. Miyoshi, S., and S. Shinoda. 2000. Microbial metalloproteases and pathogenesis. Microb. Infect. 2:91-98.[CrossRef][Medline]
18. Montgomery, M. T., and D. L. Kirchman. 1993. Role of chitin-binding proteins in the specific attachment of the marine bacterium Vibrio harveyi to chitin. Appl. Environ. Microbiol. 59:373-379.[Abstract/Free Full Text]
19. Moore, S., and W. H. Stein. 1948. Photometric ninhydrin method for use in the chromatography of amino acids. J. Biol. Chem. 176:367-388.[Free Full Text]
20. Nirasawa, S., Y. Nakajima, Z. Zhang, M. Yoshida, and K. Hayashi. 1999. Molecular cloning and expression in Escherichia coli of the extracellular endoprotease of Aeromonas caviae T-64, a pro-aminopeptidase processing enzyme. Biochim. Biophys. Acta 1433:335-342.[CrossRef][Medline]
21. Oda, K., M. Ito, K. Uchida, Y. Shibano, K. Fukuhara, and S. Takahashi. 1996. Cloning and expression of an isovaleryl pepstatin-insensitive carboxyl proteinase gene from Xanthomonas sp. T-22. J. Biochem. 120:564-572.
22. Perrakis, A., I. Tews, Z. Dauter, A. B. Oppenheim, I. Chet, K. S. Wilson, and C. E. Vorgias. 1994. Crystal structure of a bacterial chitinase at 2.3 Å resolution. Structure 2:1169-1180.[Medline]
23. Pruzzo, C., A. Crippa, S. Bertone, L. Pane, and A. Carli. 1996. Attachment of Vibrio alginolyticus to chitin mediated by chitin-binding domain. Microbiology 145:925-934.[Abstract]
24. Sasagawa, Y., Y. Kamio, Y. Matsubara, Y. Matsubara, K. Suzuki, H. Kojima, and K. Izaki. 1993. Purification and properties of collagenase from Cytophaga sp. L43-1 strain. Biosci. Biotechnol. Biochem. 57:1894-1898.
25. Shimahara, K., Y. Takiguchi, K. Ohkouchi, K. Kitamura, and O. Okada. 1984. Chemical composition and some properties of crustacean chitin prepared by use of proteolytic activity of Pseudomonas maltophilia LC102, p. 239-255. In J. P. Zikakis (ed.), Chitin, chitosan and related enzymes. Academic Press, Orlando, Fla.
26. Titani, K., M. A. Hermodson, L. H. Ericsson, K. A. Walsh, and H. Neurath. 1972. Amino-acid sequence of thermolysin. Nat. New Biol. 238:35-37.
27. Tsujibo, H., K. Miyamoto, T. Hasegawa, and Y. Inamori. 1990. Purification and characterization of two types of alkaline serine proteases produced by an alkaliphilic actinomycete. J. Appl. Bacteriol. 69:520-529.[Medline]
28. Tsujibo, H., K. Miyamoto, K. Tanaka, M. Kawai, K. Tainaka, C. Imada, Y. Okami, and Y. Inamori. 1993. Cloning and sequence of an alkaline serine protease-encoding gene from the marine bacterium Alteromonas sp. strain O-7. Gene 136:247-251.[CrossRef][Medline]
29. Tsujibo, H., H. Orikoshi, H. Tanno, K. Fujimoto, K. Miyamoto, C. Imada, Y. Okami, and Y. Inamori. 1993. Cloning, sequence, and expression of a chitinase gene from a marine bacterium, Alteromonas sp. strain O-7. J. Bacteriol. 175:176-181.[Abstract/Free Full Text]
30. Tsujibo, H., K. Fujimoto, H. Tanno, K. Miyamoto, C. Imada, Y. Okami, and Y. Inamori. 1994. Gene sequence, purification and characterization of N-acetyl-ß-glucosaminidase from a marine bacterium, Alteromonas sp. strain O-7. Gene 146:111-115.[CrossRef][Medline]
31. Tsujibo, H., K. Fujimoto, H. Tanno, K. Miyamoto, Y. Kimura, C. Imada, Y. Okami, and Y. Inamori. 1995. Molecular cloning of the gene which encodes ß-N-acetylglucosaminidase from a marine bacterium, Alteromonas sp. strain O-7. Appl. Environ. Microbiol. 61:804-806.[Abstract]
32. Tsujibo, H., K. Miyamoto, K. Tanaka, Y. Kaidzu, C. Imada, Y. Okami, and Y. Inamori. 1996. Cloning and sequence analysis of a protease-encoding gene from the marine bacterium Alteromonas sp. strain O-7. Biosci. Biotechnol. Biochem. 60:1284-1288.[Medline]
33. Tsujibo, H., H. Orikoshi, K. Shiotani, M. Hayashi, J. Umeda, K. Miyamoto, C. Imada, Y. Okami, and Y. Inamori. 1998. Characterization of chitinase C from a marine bacterium, Alteromonas sp. strain O-7, and its corresponding gene and domain structure. Appl. Environ. Microbiol. 64:472-478.[Abstract/Free Full Text]
34. Tsujibo, H., K. Miyamoto, T. Okamoto, H. Orikoshi, and Y. Inamori. 2000. A serine protease-encoding gene (aprII) of Alteromonas sp. strain O-7 is regulated by the iron uptake regulator (Fur) protein. Appl. Environ. Microbiol. 66:3778-3783.[Abstract/Free Full Text]
35. Tsujibo, H., J. Miyamoto, N. Kondo, K. Miyamoto, N. Baba, and Y. Inamori. 2000. Molecular cloning of the gene encoding an outer-membrane-associated ß-N-acetylglucosaminidase involved in chitin degradation system of Alteromonas sp. strain O-7. Biosci. Biotechnol. Biochem. 64:2512-2516.[CrossRef][Medline]
36. 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 a marine bacterium, Alteromonas sp. strain O-7. Appl. Environ. Microbiol. 68:263-270.[Abstract/Free Full Text]
37. von Heijne, G. 1983. Patterns of amino acids near signal sequence cleavage sites. Eur. J. Biochem. 133:17-21.[Medline]
38. Yu, C., A. M. Lee, B. L. Bassler, and S. Roseman. 1991. Chitin utilization by marine bacteria. A physiological function for bacterial adhesion to immobilized carbohydrates. J. Biol. Chem. 25:24260-24267.

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