Cloning of the Microcystis aeruginosa M228 Lectin (MAL) Gene Mitsuru Jimbo,* ,1 Masato Yamaguchi,† Koji Muramoto,† and Hisao Kamiya* *Department of Marine Biochemistry, School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-0101, Japan; and †Department of Biological Resource Sciences, Graduate School of Agriculture, Tohoku University, Sendai 981-8555, Japan Received April 30, 2000 We have cloned and characterized the gene encod- ing Microcystis aeruginosa (strain M228) lectin (MAL). The gene contains 1551 nucleotides and an open read- ing frame for a protein of 517 amino acids with a pre- dicted molecular weight of 55,159 Da. The carboxy- terminal region of MAL has three tandemly repeated homologous domains composed of 61 amino acids. These regions show similarity to the corresponding regions of the -amylase of Clostridium beijerinckii (23% identity). The mal gene lies adjacent to an ORF that dislay homology to cytochrome P-450 and polyketide synthase. Southern hybridization showed that the genomic DNA of the strain M228 contained, in addition to MAL gene (mal), at least two other mal like gene. © 2000 Academic Press Key Words: cyanobacterium; Microcystis aeruginosa; lectin; gene; polyketide synthase. Lectin is a carbohydrate-binding protein found in a wide range of organisms, including animals, plants, and bacteria. A few prokaryotic lectins (1–3) are known to play an essential role in the initiation of infection, while other lectins are involved in fruit- body formation (4). The prokaryotic freshwater cyanobacteria (blue- green algae), Microcystis, often forms dense water- blooms in eutrophic lakes and ponds. These blooms produce toxic heptapeptide microcystins that cause the death of livestock and other animals in many countries (5, 6). It has been reported that hemagglutinating ac- tivities are present in the extracts of both natural blooms and laboratory cultures of Microcystis (7, 8). When we examined the hemagglutinating activities of laboratory cyanobacteria, few of them had hemagglu- tinating activity. We have recently reported that puri- fied lectin from Microcystis aeruginosa strain M228 (9) is inhibited by N-acetyl-D-galactosamine and lactose, and that an amino-terminal sequence of 18 amino acid residues shows no similarity to other known lectins. The physicochemical properties and the entire amino acid sequence of lectins have remained largely un- known. Here, we describe the cloning and sequencing of the gene mal and its adjacent regions from M. aeruginosa strain M228. MATERIALS AND METHODS Cyanobacterial and bacterial strains and plasmids. Laboratory- cultured strains used in this study were M. aeruginosa (M204, M228, NIES-98, NIES-298), M. viridis (NIES-102, TAC44), and M. wesen- berugii (NIES-106, NIES-108). All these strains were obtained from the following institutions: M; Tokyo Metropolitan Research Labora- tory, TAC; Tsukuba Algal Collection in the Department of Botany, National Science Museum, NIES; Global Environmental Forum. NIES-98 and 102 are axenic, while M204, M228, NIES-106, 108, and 298 are unialgal. Escherichia coli strain DH5 was used for cloning and DNA manipulations. The E. coli vector pGEM-T easy (Promega) was used for cloning and sequencing. The restriction endonucleases and other enzymes were purchased from TOYOBO (Osaka, Japan) and Takara Shuzo (Osaka, Japan). Media and growth conditions. E. coli was grown at 37°C in Ter- rific broth or Luria broth agar, with 100 g/ml ampicillin. Microcystis was grown in MA medium (10) at 25°C and 45 E/m 2 per s with continuous illumination. Enzymatic digestion and separation of peptides. MAL was re- duced with 10 mM dithiothreitol in 0.5 M Tris–HCl (pH 8.6) contain- ing 10 mM EDTA and 6 M guanidine hydrochloride at 37°C for 1 h and reacted with 20 mM monoiodoacetic acid for 30 min at room temperature. Excess reagent was removed by gel filtration on a HiTrap desalting column (5 ml, Amersham Pharmacia Biotech.) pre-equilibrated with 0.1 M NH 4 HCO 3 (pH 8.0). The lectin was dissolved with 1 ml of 0.1 M NH 4 HCO 3 (pH 8.0) and digested with Achromobacter protease I(S/E = 50:1) at 37°C for 16 h. Each digest was separated by reversed-phase HPLC on a TSKgel ODS 120-T column (5 m, inner diameter 4.6  250 mm) (Tosoh, Tokyo, Japan) using a gradient of acetonitrile in 0.1% trifluoroacetic acid. The purified peptide fragments were analyzed on a Shimazu gas-phase sequencer PSQ-1. DNA sequencing and analysis. Cloned DNAs were sequenced by the dideoxy termination method (11) using the BigDye terminater DNA sequencing kit and an ABI PRISM 377 DNA sequencer (PE 1 To whom correspondence should be addressed. Fax: +81-192-44- 3932. E-mail: mjinbo@kitasato-u.ac.jp. Biochemical and Biophysical Research Communications 273, 499 –504 (2000) doi:10.1006/bbrc.2000.2961, available online at http://www.idealibrary.com on 499 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.