Gene, 136(1993)247-251 0 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/93/%06.00 247 GENE 07490 Cloning and sequence of an alkaline serine protease-encoding gene from the marine bacterium Alteromonas sp. strain O-7 (Proteinase; class-II subtilase; subtilisin-type serine protease; nucleotide sequence; amino acid sequence; secretion; chaperone; Gram negative) Hiroshi Tsujibo”, Katsushiro Miyamotoa, Kazumi Tanakaa, Minoru Kawaia, Kenji Tainaka”, Chiaki Imadab, Yoshiro Okamib and Yoshihiko Inamoria zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJ “Osaka University of Pharmaceutical Sciences, lo-65 Kawai 2-chome, Matsubara city, Osaka 580, Japan. Tel. (81-723) 32- 1015; and blnstitute of Microbial Chemistry, Kamiosaki, Shinagawa, Tokyo 141, Japan. Received by A.J. Podhajska: 7 April 1993; Revised/Accepted: 1 July/Z July 1993; Received at publishers: 5 August 1993 SUMMARY The gene (uprll) encoding alkaline serine protease (AprII; subtilase) from Alteromonas sp. strain O-7 was cloned in plasmid pUC19 and transformed into Escherichia coli JM109. The nucleotide (nt) sequence of aprll has been determined. A single open reading frame (ORF) encoded a protein consisting of 621 amino acids (aa) with a M, of 63958. The results of aa sequence analysis indicated that AprII is produced as a large precursor consisting of four domains: the signal sequence, the N-terminal pro-region (AprII-N), the mature AprII (AprII-M) and the C-terminal pro-region (AprII-C). The aa sequence of AprII-M shows high sequence homology with those of class-II subtilases. Two conserved sequences were found in AprII-N which might play a critical role in the maintenance of chaperone-like activity. Repeated aa sequences were observed in AprII-C (AprII-Cl and AprII-C2). The aa sequences of AprII-Cl and AprIIC2 show high sequence homology with those of the C-terminal pro-region of the other known proteases. INTRODUCTION Alteromonas sp. strain O-7 is a Gram-, flagellated, motile, aerobic, rod-shaped bacterium of marine origin Correspondence to: Dr. H. Tsujibo, Osaka University of Pharmaceutical Sciences, lo-65 Kawai, Matsubara city, Osaka 580, Japan. Tel. (81-723) 32-1015; Fax (81-723) 32-9929; e-mail: C62017g@centerOSAKA-Uacjp Abbrevations: aa, amino acid(s); Ap, ampicillin; AprII, Alteromonas pro- tease II; aprll, gene encoding AprII; AprII-C, C-terminal pro-region of AprII; AprII-M, mature AprII; AprII-N, N-terminal pro-region of AprII; bp, base pair(s); kb, kilobase or 1000 bp; nt, nucleotide(s); ORF, open reading frame; PAGE, PA-gel electrophoresis; PCMB, p- chloromercuribenzoic acid; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene difluoride; SD, Shine-Dalgarno (sequence); SDS, sodium dodecyl sulfate; subtilase, subtilisin-type serine protease (Siezen et al., 1991); 7?:, Thermus: K, Vibrio; X., Xanthomonas. excreting alkaline serine proteases into the growth medium (Tsujibo et al., 1991). Microbial proteases are divided into four groups on the basis of catalytic mecha- nism. Siezen et al. (1991) further classified the subtilisin- like family (subtilases) into class-I and class-II by the homology of primary structure. Extensive studies, related to thermostability (Takagi et al., 1990), catalytic mecha- nism (Carter and Wells, 1988) and substrate specificity (Carter and Wells, 1987) were performed on class-I subti- lases. Furthermore, functional analysis on domain- structure (Silen and Agard, 1989; Ohta et al., 1991), secre- tory mechanism (Yanagida et al., 1986; Pohlner et al., 1987) and induction mechanism of microbial proteases (Samal et al., 1990) were studied. However, there is still much left to be studied hereafter on these subjects. To elucidate these mechanisms, it is essential that the aa sequence of the precursor protein be determined. The aim