Bezzate S, Steinmetz M, Aymerich S
Institut National de la Recherche Agronomique, Thiverval-Grignon, France.
J Bacteriol. 1994 Apr;176(8):2177-83. doi: 10.1128/jb.176.8.2177-2183.1994.
The Bacillus polymyxa CF43 lelA gene, expressing both sucrose and fructan hydrolase activities, was isolated from a genomic library of B. polymyxa screened in Bacillus subtilis. The gene was detected as expressing sucrose hydrolase activity; B. subtilis transformants did not secrete the lelA gene product (LelA) into the extracellular medium. A 1.7-kb DNA fragment sufficient for lelA expression in Escherichia coli was sequenced. It contains a 548-codon open reading frame. The deduced amino acid sequence shows 54% identity with mature B. subtilis levanase and is similar to other fructanases and sucrases (beta-D-fructosyltransferases). Multiple-sequence alignment of 14 of these proteins revealed several previously unreported features. LelA appears to be a 512-amino-acid polypeptide containing no canonical signal peptide. The hydrolytic activities of LelA on sucrose, levan, and inulin were compared with those of B. subtilis levanase and sucrase, confirming that LelA is indeed a fructanase. The lelA gene in the chromosome of B. polymyxa was disrupted with a chloramphenicol resistance gene (cat) by "inter-gramic" conjugation: the lelA::cat insertion on a mobilizable plasmid was transferred from an E. coli transformant to B. polymyxa CF43, and B. polymyxa transconjugants containing the lelA::cat construct replacing the wild-type lelA gene in their chromosomes were selected directly. The growth of the mutant strain on levan, inulin, and sucrose was not affected.
从在枯草芽孢杆菌中筛选的多粘芽孢杆菌基因组文库中分离出了表达蔗糖和果聚糖水解酶活性的多粘芽孢杆菌CF43 lelA基因。该基因被检测为表达蔗糖水解酶活性；枯草芽孢杆菌转化体未将lelA基因产物（LelA）分泌到细胞外培养基中。对在大肠杆菌中足以实现lelA表达的1.7 kb DNA片段进行了测序。它包含一个548个密码子的开放阅读框。推导的氨基酸序列与成熟的枯草芽孢杆菌果聚糖酶显示出54%的同一性，并且与其他果聚糖酶和蔗糖酶（β-D-果糖基转移酶）相似。对其中14种蛋白质的多序列比对揭示了几个以前未报道的特征。LelA似乎是一个512个氨基酸的多肽，不包含典型的信号肽。将LelA对蔗糖、果聚糖和菊粉的水解活性与枯草芽孢杆菌果聚糖酶和蔗糖酶的水解活性进行了比较，证实LelA确实是一种果聚糖酶。通过“革兰氏间”接合用氯霉素抗性基因（cat）破坏了多粘芽孢杆菌染色体中的lelA基因：将可移动质粒上的lelA::cat插入片段从大肠杆菌转化体转移到多粘芽孢杆菌CF43，并直接选择在其染色体中含有取代野生型lelA基因的lelA::cat构建体的多粘芽孢杆菌接合子。突变菌株在果聚糖、菊粉和蔗糖上的生长不受影响。
