Analysis of extracellular alginate lyase and its gene from a marine bacterial strain, Pseudoalteromonas atlantica AR06.
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.
National Research Institute of Fisheries Science, Fisheries Research Agency, 2-12-4 Fukuura, Yokohama, 236-8648, Japan.
Ryoji
R
Matsushima
Hiroko
H
Danno
Motoharu
M
Uchida
Kenji
K
Ishihara
Toshiyuki
T
Suzuki
Masaki
M
Kaneniwa
Yoshiyuki
Y
Ohtsubo
Yuji
Y
Nagata
Masataka
M
Tsuda
D000464:Alginates / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D015723:Gene Library; D020723:Glucuronic Acid / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011133:Polysaccharide-Lyases / Q000235:genetics* / Q000378:metabolism*; D031161:Pseudoalteromonas / Q000201:enzymology*; D017422:Sequence Analysis, DNA
D000464:Alginates; D004269:DNA, Bacterial; D006603:Hexuronic Acids; C007896:guluronic acid; D020723:Glucuronic Acid; C008324:mannuronic acid; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase