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Cloning and Sequencing of the Genes Encoding Vacuolar-type Na^+-ATPase in Enterococcus hirae

海拉肠球菌液泡型Na+-ATP酶基因的克隆与测序

基本信息

批准号：
03808020
负责人：
KAKINUMA Yoshimi
金额：
$ 0.38万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03808020/
关键词：
Enterococcus hirae Vacuolar-type ATPase Na^+-ATPase Gene structure NA^+ D2- D2ATPase Na^+ーATPase

项目摘要

1. A 1000-bp fragment of Enterococcus hirae genomic DNA was amplified by polymerase chain reaction, using the oligonucleotide primers designed from amino acid sequences of both amino-terminal and a tryptic fragment of the Na^+-ATPase A subunit in this organism. DNA sequencing of this product revealed that the amino acid sequence of this subunit is highly homologous to the corresponding sequences of large (a) subunits of vacuolar(archaebacterial) type H^+-ATPases.2. The eubacterium Enterococcus hirae ATCC 9790 possesses a H^+-translocating ATPase, and the deduced amino acid sequences of the genes coding for this enzyme have indicated that it is a typical F_0F_1-type ATPase. We cloned the ntpA and ntpB genes coding for the A and B subunits, respectively, of Na^+-ATPase from the same bacterium, and the full amino acid sequences of the two subunits were deduced from the nucleotide sequence. The A (593 amino acid residues) and B (458 amino acid residues) subunits were highly homologous (48-60 % identical) to the A (large or a) and the B (small or b) subunits, respectively, of various vacuolar-type H^+-ATPases which have been found in eukaryotic endomembrane systems and archaebacterial cell membranes. The A and B subunits of Na^+-ATPase showed about 23-28 % identities with the b and a subunits of E. hirae F_1-ATPase and of Escherichia coli F_1-ATPase, respectively. These results indicate that E. hirae Na^+-ATPase belongs to the vacuolar-type ATPase. This is the first demonstration that both genes for V- and F-type ATPases are functionally expressed in one bacterial cell.

1.根据肠球菌Na^+-ATP酶A亚基氨基端和胰蛋白酶片段的氨基酸序列设计寡核苷酸引物，通过聚合酶链反应扩增出肠球菌基因组DNA的1000 bp片段。DNA测序结果表明，该亚基的氨基酸序列与液泡型（古细菌）H^+-ATP酶的大（a）亚基的相应序列高度同源.肠球菌ATCC 9790具有H^+转运ATP酶，其编码基因的氨基酸序列表明该酶为典型的F_0F_1型ATP酶。我们克隆了同一细菌Na^+-ATP酶A和B亚基的ntpA和ntpp B基因，并根据核苷酸序列推导出这两个亚基的氨基酸全序列。A（593个氨基酸残基）和B（458个氨基酸残基）亚基分别与真核细胞内膜系统和古细菌细胞膜中发现的各种空泡型H^+-ATP酶的A（大或a）和B（小或B）亚基高度同源（48- 60%相同）。Na^+-ATPase的A和B亚基与E的B和a亚基的同源性为23- 28%. HispensF_1-ATPase和EscherichiacoliF_1-ATPase。这些结果表明E. hi-Na ^+-ATP酶属于液泡型ATP酶。这是第一次证明V型和F型ATP酶的基因在一个细菌细胞中功能性表达。