National Research Institute of Fisheries Science, Fisheries Research Agency, 2-12-4 Fukuura, Yokohama, 236-8648, Japan.
Appl Microbiol Biotechnol. 2010 Mar;86(2):567-76. doi: 10.1007/s00253-009-2278-z. Epub 2009 Oct 21.
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.
海洋细菌大西洋假交替单胞菌 AR06 可以利用褐藻胶作为唯一的碳源和能源。从 AR06 培养介质中制备的胞外蛋白部分表现出褐藻胶裂解酶活性，可使具有均聚和共聚形式的甘露糖醛酸和古洛糖醛酸的褐藻胶分子解聚，主要转化为二聚体到四聚体。通过使用一组简并引物从 AR06 基因组 DNA 中通过 PCR 扩增了编码部分褐藻胶裂解酶的 DNA 片段，然后从 AR06 基因组 DNA 的 cosmid 文库中获得了全长的褐藻胶裂解酶基因，命名为 alyA 及其侧翼区域。AR06 的 alyA 突变体表现出：（1）在褐藻胶琼脂平板上丧失褐藻胶解聚活性，（2）在褐藻胶最小培养基中生长缺陷显著；这些缺陷可以通过引入 alyA 基因得到补偿。此外，同工酶和生化分析表明，AR06 的三种胞外蛋白带具有褐藻胶裂解酶活性，并且这三种蛋白带均源自新生的 alyA 基因产物。这些结果清楚地表明，alyA 基因对 AR06 中褐藻胶的同化有很大的贡献。alyA 基因的转录在最小培养基中存在褐藻胶时被诱导，但在富培养基中即使存在褐藻胶也没有明显的诱导。
