Construction, characterization, and complementation of a Puf- mutant of Rhodobacter sphaeroides

作者：John Gorton Davis, Timothy J. Donohue, Samuel Kaplan · 发表于：Journal of Bacteriology · 年份：1988 · DOI：10.1128/jb.170.1.320-329.1988 · 被引用次数：192 · 研究领域：Photosynthetic Processes and Mechanisms、Bacterial Genetics and Biotechnology、Protist diversity and phylogeny

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.