CONSTRUCTION, CHARACTERIZATION, AND COMPLEMENTATION OF A PUF- MUTANT OF RHODOBACTER-SPHAEROIDES
CONSTRUCTION, CHARACTERIZATION, AND COMPLEMENTATION OF A PUF- MUTANT OF RHODOBACTER-SPHAEROIDES
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DOI：
10.1128/jb.170.1.320-329.1988
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发表时间：
1988-01-01
影响因子：
3.2
通讯作者：
KAPLAN, S
中科院分区：
文献类型：
--
作者：
DAVIS, J;DONOHUE, TJ;KAPLAN, S
通过删除Puf操纵子近端部分并插入卡那霉素抗性基因盒，构建了球形红杆菌Puf-菌株（PUFB1）。Southern blot分析表明，在PUFB1中，puf操纵子的缺陷拷贝通过同源重组取代了正常的染色体拷贝。Puf-表型的特征是PUFB1不能光异养生长（PS-），缺乏可检测的Puf特异性转录物，缺乏光收集I复合物，由此推断，反应中心光谱复合物，以及光收集II复合物的水平大大降低。将含有PUFB1整个操纵子和侧翼区域的13万碱基BamHI限制性内切酶片段克隆到广宿主质粒载体RK2衍生物pRK404中，偶联导入PUFB1后，PS+表型恢复为PUFB1。在这些互补菌株中，由于质粒拷贝数的增加，puf操纵子的拷贝数增加（每个缺陷染色体拷贝4到6个拷贝）。然而，当这些菌株进行光异养培养时，特异性细菌叶绿素含量或puf特异性转录物水平均未同时增加。
A Puf- strain of Rhodobacter sphaeroides (PUFB1) was constructed by deleting a portion of the proximal region of the puf operon and inserting a kanamycin resistance gene cartridge. Southern blot analysis demonstrated that in PUFB1, the defective copy of the puf operon had replaced, through homologous recombination, the normal chromosomal copy. The Puf- phenotype was characterized by the inability of PUFB1 to grow photoheterotrophically (PS-), the lack of detectable puf-specific transcripts, the absence of the light-harvesting I complex and, by inference, the reaction center spectral complex, and greatly reduced levels of the light-harvesting II complex. The PS+ phenotype was restored to PUFB1 when a 13-kilobase BamHI restriction endonuclease fragment containing the entire puf operon and flanking regions was cloned into the broad-host-range plasmid vector RK2 derivative pRK404 and introduced by conjugation into PUFB1. In these complemented strains, there was an increased number of copies of the puf operon (four to six copies per defective chromosomal copy) as the result of plasmid copy number. However, there was no concomitant increase in either the specific bacteriochlorophyll content or the level of puf-specific transcripts when these strains were grown photoheterotrophically.