State Key Laboratory of Microbial Technology, College of Life Science, Shandong University, Jinan 250100, China.
Appl Microbiol Biotechnol. 2012 Oct;96(1):161-70. doi: 10.1007/s00253-012-4051-y. Epub 2012 Apr 29.
Cytophaga hutchinsonii is a Gram-negative gliding bacterium, which can rapidly degrade crystalline cellulose via a novel strategy without any recognizable processive cellulases. Its mechanism of cellulose binding and degradation is still a mystery. In this study, the mutagenesis of C. hutchinsonii with the mariner-based transposon HimarEm3 and gene complementation with the oriC-based plasmid carrying the antibiotic resistance gene cfxA or tetQ were reported for the first time to provide valuable tools for mutagenesis and genetic manipulation of the bacterium. Mutant A-4 with a transposon mutation in gene CHU_0134, which encodes a putative thiol-disulfide isomerase exhibits defects in cell motility and cellulose degradation. The cellulose binding ability of A-4 was only half of that of the wild-type strain, while the endo-cellulase activity of the cell-free supernatants and on the intact cell surface of A-4 decreased by 40%. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of proteins binding to cellulose in the outer membrane showed that most of them were significantly decreased or disappeared in A-4 including some Gld proteins and hypothetical proteins, indicating that these proteins might play an important role in cell motility and cellulose binding and degradation by the bacterium.
纤维弧菌是一种革兰氏阴性滑行细菌，它可以通过一种新颖的策略快速降解结晶纤维素，而不需要任何可识别的过程性纤维素酶。其纤维素结合和降解的机制仍然是一个谜。在这项研究中，首次报道了基于 mariner 的转座子 HimarEm3 对纤维弧菌的诱变和带有抗生素抗性基因 cfxA 或 tetQ 的 oriC 质粒的基因互补，为该细菌的诱变和遗传操作提供了有价值的工具。突变体 A-4 在基因 CHU_0134 中发生了转座子突变，该基因编码一种假定的巯基-二硫键异构酶，表现出细胞运动和纤维素降解缺陷。A-4 的纤维素结合能力仅为野生型菌株的一半，而细胞游离上清液和完整细胞表面的内切纤维素酶活性降低了 40%。外膜中与纤维素结合的蛋白质的十二烷基硫酸钠聚丙烯酰胺凝胶电泳表明，其中大多数在 A-4 中显著减少或消失，包括一些 Gld 蛋白和假设蛋白，表明这些蛋白可能在细菌的细胞运动和纤维素结合及降解中发挥重要作用。
