Abstract
Pseudoalteromonas atlantica AR06 is a marine bacterial strain that can utilize alginate as a sole source of carbon and energy. The extracellular protein fraction prepared from the AR06 cultivation media exhibited alginate lyase activity to depolymerize the alginate molecules having homopolymeric and heteropolymeric forms of mannuronate and guluronate so as to mainly convert into the dimer to tetramer. A DNA fragment encoding a portion of alginate lyase was amplified from AR06 genomic DNA by PCR using a set of degenerated primers, and then the whole alginate lyase gene, named alyA, and its flanking regions were obtained from a cosmid library of AR06 genomic DNA. The alyA mutant of AR06 showed (1) the loss of alginate depolymerization activity on alginate agar plate and (2) significant growth defects in alginate minimal medium; these defects were complemented by the introduction of the alyA gene. Furthermore, zymography and biochemical analyses revealed that three extracellular protein bands of AR06 had alginate lyase activities and that all three protein bands were derived from the nascent alyA gene product. These results clearly indicated that the alyA gene greatly contributes to the assimilation of alginate in AR06. The transcription of the alyA gene was induced by the presence of alginate in minimal medium, but its obvious induction was not observed in rich medium even in the presence of alginate.
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References
Bach E, Schollmeyer E (1992) An ultraviolet-spectrophotometric method with 2-cyanoacetamide for the determination of the enzymatic degradation of reducing polysaccharides. Anal Biochem 203:335–339
Boyen C, Bertheau Y, Barberyron T, Kloareg B (1990) Preparation of guluronate lyase from Pseudomonas alginovora for protoplast isolation in Laminaria. Enzyme Microb Technol 12:885–890
Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459–472
Dennis JJ, Zylstra GJ (1998) Plasposons: modular self-cloning minitransposon derivatives for rapid genetic analysis of gram-negative bacterial genomes. Appl Environ Microbiol 64:2710–2715
Doubet RS, Quatrano RS (1982) Isolation of marine bacteria capable of producing specific lyases for alginate degradation. Appl Environ Microbiol 44:754–756
Duilio A, Tutino ML, Marino G (2004) Recombinant protein production in Antarctic gram-negative bacteria. Methods Mol Biol 267:225–237
Figurski DH, Helinski DR (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci USA 76:1648–1652
Hashimoto W, Momma K, Mishima Y, Mikami B, Murata K (2001) Super-channel in bacteria: function and structure of a macromolecule import system mediated by a pit-dependent ABC transporter. Biosci Biotechnol Biochem 65:1949–1956
Haug A, Larsen B, Smidsrød O (1966) A study of the constitution. of alginic acid by partial acid hydrolysis. Acta Chem Scand 20:183–190
Heeb S, Itoh Y, Nishijyo T, Schnider U, Keel C, Wade J, Walsh U, O'Gara F, Haas D (2000) Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria. Mol Plant Microbe Interact 13:232–237
Iwamoto M, Kurachi M, Nakashima T, Kim D, Yamaguchi K, Oda T, Iwamoto Y, Muramatsu T (2005) Structure-activity relationship of alginate oligosaccharides in the induction of cytokine production from RAW264.7 cells. FEBS Lett 579:4423–4429
Iwamoto Y, Iriyama K, Osatomi K, Oda T, Muramatsu T (2002) Primary structure and chemical modification of some amino acid residues of bifunctional alginate lyase from a marine bacterium Pseudoalteromonas sp. strain no. 272. J Protein Chem 21:455–463
Iwamoto Y, Hidaka H, Oda T, Muramatsu T (2003) A study of tryptophan fluorescence quenching of bifunctional alginate lyase from a marine bacterium Pseudoalteromonas sp. strain No. 272 by acrylamide. Biosci Biotechnol Biochem 67:1990–1992
Kitamikado M, Tseng CH, Aoki K, Yamaguchi K, Araki T (1989) Isolation of bacteria capable of producing alginate-degrading enzyme from natural environment. Nippon Suisan Gakkaishi 55:709–713
Kovach ME, Phillips RW, Elzer PH, Roop RM 2nd, Peterson KM (1994) pBBR1MCS: a broad-host-range cloning vector. Biotechniques 16:800–802
Momcilovic D, Wittgren B, Wahlund KG, Karlsson J, Brinkmalm G (2003) Sample preparation effects in matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry of partially depolymerised carboxymethyl cellulose. Rapid Commun Mass Spectrom 17:1107–1115
Murata K, Inose T, Hisano T, Abe S, Yonemoto Y, Yamashita T, Takagi M, Sakaguchi K, Kimura A, Imanaka T (1993) Bacterial alginate lyase: enzymology, genetics and application. J Ferment Bioeng 76:427–437
Natsume M, Kamo Y, Hirayama M, Adachi T (1994) Isolation and characterization of alginate-derived oligosaccharides with root growth-promoting activities. Carbohydr Res 258:187–197
Onsøyen E (1996) Commercial applications of alginates. Carbohydrates in Europe 14:26–31
Ruvkun GB, Ausubel FM (1981) A general method for site-directed mutagenesis in prokaryotes. Nature 289:85–88
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Sawabe T, Takahashi H, Ezura Y, Gacesa P (2001) Cloning, sequence analysis and expression of Pseudoalteromonas elyakovii IAM 14594 gene (alyPEEC) encoding the extracellular alginate lyase. Carbohydr Res 335:11–21
Sutherland IW (1995) Polysaccharide lyases. FEMS Microbiol Rev 16:323–347
Uchida M, Nakayama A (1993) Isolation of Laminaria-frond decomposing bacteria from japanese costal waters. Nippon Suisan Gakkaishi 59:1865–1871
Uchida M, Maeda T, Shiba T (2002) Phylogenic analysis of three marine bacteria that have an ability to decompose Laminaria japonica. Fish Sci 68:703–705
Wong TY, Preston LA, Schiller NL (2000) Alginate lyase: review of major sources and enzyme characteristics, structure-function analysis, biological roles, and applications. Annu Rev Microbiol 54:289–340
Yoon HJ, Hashimoto W, Miyake O, Okamoto M, Mikami B, Murata K (2000) Overexpression in Escherichia coli, purification, and characterization of Sphingomonas sp. A1 alginate lyases. Protein Expr Purif 19:84–90
Zhang Z, Yu G, Zhao X, Liu H, Guan H, Lawson AM, Chai W (2006) Sequence analysis of alginate-derived oligosaccharides by negative-ion electrospray tandem mass spectrometry. J Am Soc Mass Spectrom 17:621–630
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This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology and the Ministry of Agriculture, Forestry, and Fisheries, Japan.
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Matsushima, R., Danno, H., Uchida, M. et al. Analysis of extracellular alginate lyase and its gene from a marine bacterial strain, Pseudoalteromonas atlantica AR06. Appl Microbiol Biotechnol 86, 567–576 (2010). https://doi.org/10.1007/s00253-009-2278-z
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DOI: https://doi.org/10.1007/s00253-009-2278-z