Isolation and Characterization of the Genes Encoding Two ...

Biosci. Biotechnol. Biochem., 66 (2), 416–421, 2002

Note

Isolation and Characterization of the Genes Encoding Two Metalloproteases (MprI and MprII) from a Marine Bacterium, Alteromonas sp. Strain O-7 Katsushiro MIYAMOTO, Hiroshi TSUJIBO,† Eiji NUKUI, Hiroyuki ITOH, Yoshihiko KAIDZU, and Yoshihiko INAMORI Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan Received June 27, 2001; Accepted September 21, 2001

An extracellular alkaline metalloprotease (MprI) from Alteromonas sp. strain O-7 was puriêd and characterized. The molecular mass of the puriêd enzyme was estimated to be 56 kDa by SDS-PAGE. The optimum pH and temperature were pH 10.0 and 609C, respectively. The gene (mprI ) encoding MprI was cloned and its nucleotide sequence was analyzed. The deduced amino acid sequence of MprI showed signiˆcant similarity to metalloproteases classiêd into the thermolysin family. Furthermore, sequence analysis showed that another metalloprotease (MprII)-encoding gene was located downstream from mprI. The deduced amino acid sequence of MprII showed high similarity to metalloproteases of the aminopeptidase family. Similar repeated C-terminal extensions were found in both MprI and MprII. Key words:

metalloprotease; thermolysin; aminopeptidase

Microbial proteases play an important role in the use of protein or peptides as a nutrient source for bacterial growth, and in the pathogenesis and virulence of disease development. Thus, there are many reports on microbial proteases not only in scientiˆc êlds such as the relationship between structure and function but also for practical purposes such as potential cleaning agents and food additives. Recently, many enzymes of the subtilisin-like superfamily (subtilases) have been cloned and sequenced.1) Siezen and Leunissen classiêd the subtilases into families A (subtilisin family), B (thermitase family), C (proteinase K family), D (lantibiotic peptidase family), E (Kexin family), and F (pyrolysin family) based on amino acid sequence similarity.2) On the other hand, many metalloproteases, which are thought to play an important role in the pathogenesis, have been cloned and sequenced from pathogenic bacteria, such as Vibrio vulniˆcus 3) and Aeromonas hydrophila.4) The metalloproteases with the HEXXH zinc-binding motif can be further classiêd into thermolysin, serralysin, and neurotoxin families based on the loca†

tion of the third zinc ligands.5) Alteromonas sp. strain O-7 was isolated from Sagami Bay in Japan as an e‹cient producer of chitinolytic enzymes.6) The strain also secretes various enzymes into the milieu, such as proteases, lipases, amylases, and DNases. Among these enzymes, we have been studying the chitinolytic7–9) and proteolytic enzymes of the strain to clarify the roles of individual enzymes involved in chitin or protein degradation and the relationship between structure and function. The strain secretes several proteases into the culture medium. Of these proteases, we have already cloned and sequenced the genes encoding serine proteases (AprI and AprII) and the relationship between structure and function of these enzymes has been investigated.10,11) The mature AprI and AprII belong to families B and C, respectively. These proteases were produced as large precursors consisting of four domains: the signal sequence, the N-terminal pro-region, the mature AprI and AprII, and a conserved C-terminal extension. The repeated sequences in the C-terminal extension had high sequence similarities with the C-terminal regions of proteases from Vibrio (V.) vulniˆcus,3) V. cholerae,12) and Xanthomonas campestris.13) Furthermore, we found that the AprII gene was regulated by Fur protein, which plays an essential role in the iron acquisition system.14) In this paper we describe the puriˆcation and characterization of an extracellular alkaline metalloprotease (MprI) and the cloning the gene encoding the enzyme. In addition, we report the cloning of the metalloprotease (MprII)-encoding gene located immediately downstream from the mprI gene. Alteromonas sp. strain O-7 was cultured at 279 C in Bacto Marine Broth 2216 (Difco). Samples of the culture were taken at 2-h intervals and protease activity of the supernatant was measured as in our previous paper.15) The strain secreted proteases into the culture medium coincidentally with the release of a yellow pigment (Fig. 1(A)). The supernatant after 6 h of cultivation was incubated for 10 min with 10 mM

To whom correspondence should be addressed. TEL & FAX: +81-726-90-1057; E-mail: tsujibo＠gly.oups.ac.jp

Metalloproteases from Alteromonas sp. Strain O-7

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Fig. 1.

(A) Production Curve of Proteases from Alteromonas sp. Strain O-7; (B) Zymogram of Proteases; (C) SDS-PAGE of MprI. (A) Symbols: , bacterial growth; , protease activity of the supernatant. (B) Lanes 1, no addition; 2, supernatant treated with PMSF; 3, supernatant treated with OPA. (C) Lanes: 1, marker proteins; 2, MprI.

phenylmethylsulfonyl ‰uoride (PMSF, a serine protease inhibitor) or 10 mM o-phenanthroline (OPA, a metalloprotease inhibitor), and then the sample was separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) containing 0.01z sodium caseinate (Fig. 1(B)).16) The results indicate that at least three serine proteases and three metalloproteases are produced by Alteromonas sp. strain O-7. To purify the metalloprotease, the strain was cultured until the optical density at 600 nm reached 1.5. The culture supernatant (2 l) was collected by centrifugation at 10,000×g at 49C and was used as a crude enzyme. All puriˆcation steps were done at 49C. The crude enzyme was dialyzed overnight against 50 mM Tris-HCl buŠer, pH 8.0. The dialyzed enzyme solution was put on a DEAEToyopearl 650M column (3.0×15 cm; Tosoh, Tokyo, Japan) equilibrated with the same buŠer. The column was washed ˆrst with buŠer and then with a linear gradient of NaCl (0 to 1.0 M). The protease activities were eluted at 0.2 to 0.4 M NaCl and separated into four fractions (fractions I, II, III, and IV). The major active fraction (fraction II) was put on a DEAE-Toyopearl 650M column (1.9×55 cm) and eluted with a linear gradient of NaCl (0 to 0.5 M). Ammonium sulfate (ˆnal concentration: 2.0 M) was added to the active fraction and put on a phenylToyopearl 650M column (1.0×20 cm; Tosoh, Tokyo, Japan) equilibrated with 50 mM Tris-HCl buŠer, pH 8.0 containing 2.0 M ammonium sulfate. The column was washed ˆrst with buŠer and then with a linear gradient of ammonium sulfate (2.0 to 0 M). One active fraction was eluted at a concentration of about 0.4 M ammonium sulfate. The puriêd protease showed a single band on SDS-PAGE (Fig. 1(C)). The molecular mass of the protease was estimated to be 56 kDa by SDS-PAGE. When protease activity of fraction II was taken as 100z, the

ˆnal puriˆcation resulted in a yield of 17.6z of the activity and a 2.47-fold increase in speciˆc activity. The N-terminal amino acid sequence of the enzyme was ANATGPGGNQ. The optimum pH level and temperature for the puriêd protease activity were pH 10.0 and 609C, respectively, when casein was used as a substrate. Most of the alkaline metalloproteases have been shown to be active from pH 7 to 9.17,18) Recently, novel metalloproteases from Vibrio sp. NUF-BPP1 and psychrophilic Pseudomonas sp. TAC II-18, which have optimum pHs in the highly alkaline region (pH 9–10), were puriêd and characterized.19,20) MprI also has a quite similar optimum pH to that of these metalloproteases, however, details of the relationship between the structure and function of these novel enzymes remain to be examined. MprI was stable for 30 min at pH 8.0 up to 609 C. EDTA inhibited the protease activity completely at the concentration of 1 mM, but pchloromercuribenzoic acid and PMSF did not have the inhibitory eŠects at the same concentration. These results suggest that the puriêd protease (MprI) is an alkaline metalloprotease with a high optimum pH. To isolate the MprI-encoding gene from a genomic library of Alteromonas sp. strain O-7, transformants were screened for protease activity on LB agar plates containing 1.0z Bacto-skim milk and 100 mg W ml ampicillin. A genomic library of the strain was constructed in Escherichia ( E.) coli JM109 from Sau3AI partially digested chromosomal DNA ligated into the Bam HI site of pUC19. Among 2,500 transformants, seven clones formed clear halos around the colonies. The nucleotide sequence showed that two clones carrying aprI gene11) formed clear halos at day 1 and that two clones carrying aprII gene10) formed clear halos at day 2. On the other hand, three clones containing the same 5.3-kb Sau3AI insert (pSau53) formed clear

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Fig. 2.

(A) Restriction map of pPX54; (B) domain structures of MprI and MprII. (A) Arrows indicate the ORF and the direction of transcription. The hybridization probe (the XhoI-Sau3AI fragment) is represented , signal peptide; , N-terminal proregion; , metalby the shadow box. (B) The arrows indicate the repeat amino acid sequence. , C-terminal extension. loprotease region;

halos at day 3. To identify the coding region for the enzyme, various subclones were prepared and expression of the protease in E. coli was detected by the formation of clear halos around the colonies. These results indicated that the coding region for the protease gene was on the 3.1-kb Pst I-XhoI fragment (pPX31). The restriction map of pSau53 and pPX31 are shown in Fig. 2. The fragment contained a single complete open reading frame (ORF) of 2,184 bp coding for 727 amino acids with a molecular mass of 78,367 Da (Fig. 3). A putative Shine-Dalgarno sequence, AGGAG, was found 9 bp upstream from the start codon (GTG) of the ORF. A putative signal sequence of 23 amino acids was present with a predicted cleavage site after Ala-23. The amino terminal amino acid sequence of MprI perfectly matched the sequence starting from Ala-204 of the deduced amino acid sequence encoded by the mprI gene. The mature protein consequently has a length of 524 amino acids with a calculated molecular mass of 56,051 Da. This value agrees with that of the puriêd MprI (Fig. 1(C)). MprI (residues 344–348) had the highly conserved HEXXH zinc-binding motif, indicating that the enzyme is a metalloprotease. The BLAST search program found that MprI was signiˆcantly similar to metalloproteases of the thermolysin family (peptidaseäM4), such as a pro-aminopeptidase converting metalloprotease from Aeromonas ( A.) punctata (69z identity),21) VVP (vibriolysin) from V. vulniˆcus (64z identity),3) and LasB (pseudolysin) from Pseudomonas aeruginosa (60z identity).22) In the thermolysin family, another conserved amino acid sequence (the third zinc ligand-motif, GXXNEXXSD) is generally found together with the HEXXH-motif.5) The motif (GGLNEAFSD,

residues 364–372) was also found in prepro-MprI. The two repeated sequences, which showed sequence similarity with that commonly found in AprI and AprII, were in the C-terminal region of MprI. These results indicate that MprI is produced as a precursor consisting of four domains: the signal sequence, Nterminal pro-region, metalloprotease region, and Cterminal extension. The sequence analysis of the region downstream of the mprI gene revealed that there is a partial ORF, which shows similarity with metalloproteases of the aminopeptidase family (peptidaseäM28, Fig. 3).23) Thus, we cloned the downstream region of the ORF by inverse PCR. Southern hybridization showed that a 2.3-kb XhoI fragment hybridized to the probe (data not shown). The fragments in the range of 2.2- to 2.5-kb were cut from the gel and puriêd with a GenElute gel puriˆcation kit (Sigma). These were self-ligated and then PCR was done using the inverse primers, which were synthesized on the basis of nucleotide sequence of the XhoI-Sau3AI fragment from pSau53. The ampliêd DNA fragments were digested with KpnI and XhoI and cloned into pUC19 (pKX17). Plasmid pPX54, which contained the fulllength genes encoding MprI and the ORF, was constructed by combining a 3.7-kb Pst I-KpnI fragment from pSau53 and a 1.7-kb KpnI-XhoI fragment from pKX17 (Fig. 2). The ORF encoded a protein consisting of 609 amino acids with a molecular mass of 65,682 Da (Fig. 3). The BLAST search program revealed that the enzyme, designated MprII, was highly similar in sequence to M28 metalloproteases, such as aminopeptidase from A. punctata (50z identity),24) leucyl aminopeptidase from V. proteolyticus (46z identity),25) and aminopeptidase from A. proteolytica


Fig. 3.

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Nucleotide Sequence of mprI and mprII. The inverted repeat sequence is indicated by convergent arrows. The putative SD sequences are double-underlined. The deduced amino acid sequences of MprI and MprII are shown below the nucleotide sequence. The putative signal peptidase cleavage sites are shown with vertical arrows. N-terminal amino acid sequence of MprI is underlined. The HEXXH zinc-binding motif is bold letters. The third zinc ligand-motif is shaded. The C-terminal repeat sequences of MprI and MprII are solid and dashed boxes, respectively. The nucleotide sequence data will appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases with the accession number AB063611.

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(46z identity).26) Furthermore, MprII, like MprI, had a similar two-repeated C-terminal extension. These C-terminal polypeptides are found in various types of proteases produced by Gram-negative bacteria, such as Aeromonas,21) Alteromonas,10,11) Helicobacter,27) Schewanella,28) Vibrio,3) and Xanthomonas.13) Further research is planned to examine the enzymatic characterization of MprII and to clarify the function of C-terminal extension of AprI, AprII, MprI, and MprII from Alteromonas sp. strain O-7.

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