The Bacillus subtilis 168 alkaline phosphatase III gene: impact of a phoAIII mutation on total alkaline phosphatase synthesis.
The Bacillus subtilis 168 alkaline phosphatase III gene: impact of a phoAIII mutation on total alkaline phosphatase synthesis.
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枯草芽孢杆菌 168 碱性磷酸酶 III 基因：phoAIII 突变对碱性磷酸酶总合成的影响。
DOI：
10.1128/jb.172.7.3730-3737.1990
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发表时间：
1990
影响因子：
3.2
通讯作者：
Hulett,FM
中科院分区：
文献类型：
--
作者：
Bookstein,C;Edwards,CW;Kapp,NV;Hulett,FM
枯草芽孢杆菌 168 的第一个碱性磷酸酶 (APase) 结构基因突变体是使用通过与合成简并寡核苷酸杂交鉴定的克隆构建的。探针的设计基于已测序的成熟 APase III 蛋白的前 29 个氨基酸，该蛋白是从磷酸盐饥饿的营养细胞的分泌部分中分离出来的。克隆的 DNA 测序揭示了 APase III 蛋白的前 80 个氨基酸，包括成熟蛋白起始点之前的 32 个氨基酸的典型原核信号序列。紧随信号序列之后的预测开放阅读框编码的 29 个氨基酸与测序成熟蛋白的前 29 个氨基酸相同。该区域与在大肠杆菌和哺乳动物 APase 中非常保守的 β 片层 A 链具有 80% 的同一性。 phoAIII结构突变体是通过插入诱变用编码区内部的片段构建的。在磷酸盐饥饿和孢子形成条件下分析了该突变对枯草芽孢杆菌 168 中 APase 产生的影响。 APase III 的突变使总营养 APase 比活性降低了大约 40%，孢子形成 APase 比活性降低了大约 45%。从处于 III 期的孢子形成细胞中分离出 APase 蛋白，并通过氨基末端的蛋白测序以及通过其在 phoAIII 突变体中的缺失将其鉴定为 APase III。 APase III 基因已被定位到枯草芽孢杆菌染色体上大约 50 度。
The first alkaline phosphatase (APase) structural gene mutant of Bacillus subtilis 168 was constructed by using a clone identified by hybridization to a synthetic degenerative oligonucleotide. The design of the probe was based on the first 29 amino acids of the sequenced mature APase III protein, which had been isolated from the secreted fraction of vegetative, phosphate-starved cells. DNA sequencing of the clone revealed the first 80 amino acids of the APase III protein, including a typical procaryotic signal sequence of 32 amino acids preceding the start of the mature protein. The 29 amino acids encoded by the predicted open reading frame immediately following the signal sequence are identical to the first 29 amino acids of the sequenced mature protein. This region shows 80% identity to strand A of the beta sheet that is very well conserved in Escherichia coli and mammalian APases. A phoAIII structural mutant was constructed by insertional mutagenesis with a fragment internal to the coding region. The effects of this mutation on APase production in B. subtilis 168 were analyzed under both phosphate starvation and sporulation conditions. The mutation in APase III reduced the total vegetative APase specific activity by approximately 40% and sporulation APase specific activity by approximately 45%. An APase protein was isolated from sporulating cells at stage III and was identified as APase III by protein sequencing of the amino terminus and by its absence in the phoAIII mutant. The APase III gene has been mapped to approximately 50 degrees on the B. subtilis chromosome.