Fleming A B, Tangney M, Jørgensen P L, Diderichsen B, Priest F G
Department of Biological Sciences, Heriot Watt University, Edinburgh, United Kingdom.
Appl Environ Microbiol. 1995 Nov;61(11):3775-80. doi: 10.1128/aem.61.11.3775-3780.1995.
A deletion of the spoIIAC gene of Bacillus licheniformis was prepared in vitro by using the splicing-by-overlap-extension technique. This gene was introduced into B. licheniformis on a temperature-sensitive plasmid, and following integration and excision from the chromosome, a precisely located deletion on the chromosomal gene was prepared. The mutated bacterium was totally asporogenous and formed abortively disporic cells characterized by asymmetric septa at the poles of the cells. Qualitative plate tests revealed that the bacterium synthesized normal levels of DNase, polygalacturonate lyase, protease, RNase, and xylanase, but the hydrolysis zones due to beta-1,3-glucanase and carboxymethyl cellulase activity were smaller in the mutant than in the parent strain. The synthesis of alkaline protease was the same in batch cultures of the mutant and the parent during prolonged incubation for 72 h, but the alpha-amylase yields were reduced by about 30% by the mutation.
通过重叠延伸剪接技术在体外构建了地衣芽孢杆菌spoIIAC基因的缺失突变体。该基因通过温度敏感型质粒导入地衣芽孢杆菌，经染色体整合和切除后，在染色体基因上获得了精确的定位缺失。突变菌株完全不产芽孢，形成了异常双芽孢细胞，其特征是细胞两极有不对称隔膜。定性平板试验表明，该菌株能合成正常水平的脱氧核糖核酸酶、聚半乳糖醛酸裂解酶、蛋白酶、核糖核酸酶和木聚糖酶，但与亲本菌株相比，突变体中β-1,3-葡聚糖酶和羧甲基纤维素酶活性产生的水解圈较小。在延长培养72小时的分批培养中，突变体和亲本的碱性蛋白酶合成情况相同，但突变使α-淀粉酶产量降低了约30%。
