Control of photosynthetic membrane assembly in Rhodobacter sphaeroides mediated by puhA and flanking sequences.
Control of photosynthetic membrane assembly in Rhodobacter sphaeroides mediated by puhA and flanking sequences.
复制标题
puhA 和侧翼序列介导的球形红杆菌光合膜组装的控制。
DOI：
10.1128/jb.171.1.436-446.1989
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发表时间：
1989
影响因子：
3.2
通讯作者：
Kaplan,S
中科院分区：
文献类型：
--
作者：
Sockett,RE;Donohue,TJ;Varga,AR;Kaplan,S
球形红杆菌 PUHA1 的反应中心 H 菌株 (RCH-) 是通过体外删除 puhA 基因中的 XhoI 限制性内切核酸酶片段并插入卡那霉素抗性基因盒来制备的。将所得构建体递送至球形红球菌野生型2.4.1，其中有缺陷的puhA基因通过重组替换野生型拷贝，然后选择卡那霉素抗性。当在已知诱导胞质内膜发育的条件下生长时，PUHA1 合成了有色素的胞质内膜。该膜的光谱分析表明，它缺乏 B875 光谱复合物以及功能反应中心，并且 B800-850 光谱复合物的水平高于野生型。 RCH-菌株没有光合作用，但通过与质粒pRK404上反式携带的puhA基因的1.45-kb BamHI限制性内切核酸酶片段互补，恢复了光合作用生长。 B875 光谱复合物并未通过与含有 puhA 基因的 1.45-kb BamHI 限制性内切核酸酶片段互补而恢复，但通过与来自携带 puhA 基因的粘粒的 DNA 以及侧翼 DNA 序列互补而恢复光合能力。有趣的是，通过反式引入携带编码 DNA 的 puf 操纵子区域的基因的 13-kb BamHI 限制性内切酶片段，B875 光谱复合物（但不是光合作用能力）恢复到 PUHA1。通过检查来自 puf 和 puc 操纵子的特定 mRNA 种类的水平，以及通过免疫学方法测定与各种光谱复合物相关的多肽的相对丰度，进一步研究了 puhA 缺失的影响。讨论了 puhA 和其他遗传成分在光合基因表达和膜组装中的作用。
A reaction center H- strain (RCH-) of Rhodobacter sphaeroides, PUHA1, was made by in vitro deletion of an XhoI restriction endonuclease fragment from the puhA gene coupled with insertion of a kanamycin resistance gene cartridge. The resulting construct was delivered to R. sphaeroides wild-type 2.4.1, with the defective puhA gene replacing the wild-type copy by recombination, followed by selection for kanamycin resistance. When grown under conditions known to induce intracytoplasmic membrane development, PUHA1 synthesized a pigmented intracytoplasmic membrane. Spectral analysis of this membrane showed that it was deficient in B875 spectral complexes as well as functional reaction centers and that the level of B800-850 spectral complexes was greater than in the wild type. The RCH- strain was photosythetically incompetent, but photosynthetic growth was restored by complementation with a 1.45-kilobase (kb) BamHI restriction endonuclease fragment containing the puhA gene carried in trans on plasmid pRK404. B875 spectral complexes were not restored by complementation with the 1.45-kb BamHI restriction endonuclease fragment containing the puhA gene but were restored along with photosynthetic competence by complementation with DNA from a cosmid carrying the puhA gene, as well as a flanking DNA sequence. Interestingly, B875 spectral complexes, but not photosynthetic competence, were restored to PUHA1 by introduction in trans of a 13-kb BamHI restriction endonuclease fragment carrying genes encoding the puf operon region of the DNA. The effect of the puhA deletion was further investigated by an examination of the levels of specific mRNA species derived from the puf and puc operons, as well as by determinations of the relative abundances of polypeptides associated with various spectral complexes by immunological methods. The roles of puhA and other genetic components in photosynthetic gene expression and membrane assembly are discussed.