Department of Physiology & Cell Biology, University of Nevada School of Medicine, Reno, NV, United States.
Microbiol Res. 2012 Sep 6;167(8):461-9. doi: 10.1016/j.micres.2012.03.004. Epub 2012 Apr 12.
β-Glucosidases are widespread in bacteria and involved in the metabolism of various carbohydrate substrates. Studying of β-glucoside utilization (bgl) operons on operon of Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34) will help us understanding how β-glucoside utilization (bgl) operon can cooperate with other systems in bacterium caused soft-rot disease. Pcc LY34 causes soft-rot disease in plants and expresses multiple enzymatic forms of β-glucosidases. To fully explore the β-glucoside utilization system in Pcc LY34, we have isolated a bgl operon from a genomic library for screening of β-glucosidase activities. Sequence analysis of a 3050bp cloned DNA fragment (accession number AY870655) shows two open reading frames (bglY and bglK) that are predicted to encode proteins of 474 and 278 amino acid residues, respectively. Pair wise similarity analysis suggests BglY is a beta-glucosidase (a member of glycosyl hydrolase family 1) and BglK is a transcriptional antiterminator protein. bglY promoter region contains an inverted repeat sequence similar to transcriptional terminator. Different from other four β-glucoside utilization operons of Pcc LY34 strain, BglY contains signal peptide sequences as extracellular β-glucosidase. Comparisons of five β-glucoside utilization operons of Pcc LY34 strain showed that bglYK operon does not have phosphotransferase system domain which are responsible for sugar transportation. BglY shares 33-44% identity with other four β-glucosidases of Pcc LY34 strain. Enzyme assay showed that purified BglY enzyme hydrolyzed salicin, arbutin, pNPG, and MUG, and exhibited maximal activity at pH 7.0 and 40°C. This activity was enhanced Mg(2+). Site-directed mutagenesis revealed E166 and E371 are critical of BglY's β-glucosidase activity.
β-葡萄糖苷酶广泛存在于细菌中，参与各种碳水化合物底物的代谢。研究果胶杆菌亚种胡萝卜软腐病菌（Pectobacterium carotovorum subsp. carotovorum LY34，Pcc LY34）的β-葡萄糖苷利用（bgl）操纵子有助于我们了解β-葡萄糖苷利用（bgl）操纵子如何与细菌中引起软腐病的其他系统合作。Pcc LY34 会引起植物软腐病，并表达多种β-葡萄糖苷酶的酶形式。为了充分探索 Pcc LY34 中的β-葡萄糖苷利用系统，我们从基因组文库中分离了一个 bgl 操纵子，用于筛选β-葡萄糖苷酶活性。对克隆的 3050bp DNA 片段（登录号 AY870655）进行序列分析表明，该片段包含两个开放阅读框（bglY 和 bglK），分别预测编码 474 和 278 个氨基酸残基的蛋白质。两两相似性分析表明，BglY 是一种β-葡萄糖苷酶（糖基水解酶家族 1 的成员），BglK 是一种转录终止子蛋白。bglY 启动子区域包含与转录终止子相似的反向重复序列。与 Pcc LY34 菌株的其他四个β-葡萄糖苷利用操纵子不同，BglY 包含信号肽序列作为细胞外β-葡萄糖苷酶。对 Pcc LY34 菌株的五个β-葡萄糖苷利用操纵子的比较表明，bglYK 操纵子没有负责糖运输的磷酸转移酶系统结构域。BglY 与 Pcc LY34 菌株的其他四种β-葡萄糖苷酶共享 33-44%的同一性。酶活性测定表明，纯化的 BglY 酶水解水杨苷、熊果苷、pNPG 和 MUG，在 pH7.0 和 40°C 时表现出最大活性。这种活性增强了 Mg2+。定点突变揭示 E166 和 E371 对 BglY 的β-葡萄糖苷酶活性至关重要。
