DNA Replication and Copy-number Control of Bacillus plasmid

已经表征了从解淀粉芽孢杆菌菌株分离的8.2-kb隐蔽质粒pFTB 14的DNA复制所必需和足够的1.5-kb DNA序列的结构。1.5 kb的DNA序列包含一个开放的阅读框架，rep，延伸1017 bp，rep表达的启动子区，和一个可能的复制起点。通过缺失和插入突变的遗传分析表明，rep产物是反式的，是质粒自动复制所必需的。预测的rep蛋白是碱性蛋白，如在葡萄球菌中发现并用作芽孢杆菌载体的pT 181的RepC蛋白、pUB 101的RepB和pC 194的蛋白A。从蛋白分子上看，该蛋白与pC 194的A蛋白和pUB 101的RepB蛋白具有高度相似的氨基酸序列，而与pT 181的RepC和pC 194编码的启动复制的RepH蛋白不具有高度相似的氨基酸序列。rep蛋白的分子量估计为39，600 dal，与从DNA序列中预期的分子量相同。凝胶电泳分析表明，该蛋白在1.5kb的限制性DNA序列上具有结合活性，与pC 194和pUB 101的复制起点DNA序列相似，因此认为该蛋白是DNA结合蛋白。上述结果表明，rep蛋白通过与DNA复制起点结合来调节PFTB 14的DNA复制。

The structure of a 1.5-kb DNA sequence that is necessary and sufficient for the DNA replication of an 8.2-kb cryptic plasmid, pFTB14, isolated from a strain of Bacillus amyloliquefaciens has been characterized. The 1.5-kb DNA sequence contains an open reading frame, rep, stretching for 1017 bp, a promoter region for rep expression, and a possible replication origin. Genetic analysis by deletion and insertion mutations suggested that the rep product is trans-active and essential for plasmid automonous replication. The predicted rep protein is a basic protein, as are the RepC protein of pT181, RepB of pUB101 and protein A of pC194 which are found in staphylococci and used as Bacillus vectors. The redicted rep protein has highly similar amino acid sequences with protein A of pC194 and RepB of pUB101 through the protein molecule, but not with RepC of pT181 or protein RepH encoded by and initiating the replication of pC194.In order to obtain rep protein, it was overproduced in Escherichia coli by the promoter system of lambda phage, and purified partially through DEAE-Toyopearl chromatography. The molecular weight of the rep protein was estimated to be 39,600 dal as same as that expected from DNA sequences. By the result of gel-shift assay analysis the protein was considered as DNA binding protein because of the binding activity on the restricted DNA sequence of 1.5-kb region, which is similar to the DNA regions for the replication origin of pC194 and pUB101.The above results suggested the rep protein regulates DNA replication of PFTB14 through binding with the origin of DNA replication.