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The puf operon of the photosynthetic bacterium Rhodobacter sphaeroides encodes reaction centre and B875 (LH1) antenna polypeptides of the photosynthetic apparatus. This region of the genome was used to establish the applicability of random transposon Tn5 mutagenesis in this bacterium. Four Tn5 insertions have been mapped and one of the mutants characterized using a variety of techniques in order to establish that the fluorescence properties and polypeptide composition were consistent with the absence of reaction centre polypeptides. Following the ‘rescue’ of the transposon along with flanking regions of the puf operon, re-introduction of this construction into wild-type Rb. sphaeroides yielded the original mutation. This demonstrates that following homologous recombination, localized mutagenesis can direct Tn5 into a predetermined region of the Rb. sphaeroides chromosome. Accordingly, puf genes borne on plasmid pSRC2 were mutagenized with Tn5 in Escherichia coli, and the sites of insertion mapped physically. pSRC2 derivatives containing Tn5 were transferred to wild-type Rb. sphaeroides. puf genes have been mapped by correlating the photosynthetic properties of resulting strains with the sites of Tn5 insertion into pSRC2.
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