Isolation and characterization of Rhodobacter capsulatus mutants defective in oxygen regulation of the puf operon

Abstract

cis-acting mutations that affect regulation of the Rhodobacter capsulatus puf operon by oxygen were isolated by placing the mutagenized puf regulatory region 5' to a promoterless Tn5 neo gene, which encodes resistance to kanamycin (Kmr). R. capsulatus mutants that failed to show wild-type repression of KMr by oxygen were selected and analyzed. Four independent clones contained point mutations, three of which were identical, in a region of dyad symmetry located between puf operon nucleotide positions 177 and 207, approximately 45 base pairs 5' to the site of initiation of puf transcripts. The phenotypic effects of the aerobically selected mutations were duplicated by single and double point mutations introduced site specifically into the region of dyad symmetry by oligonucleotide-directed mutagenesis. Determinations of the bacterial 50% lethal dose of kanamycin, of aminoglycoside phosphotransferase activity in cell sonicates, and of neo-specific mRNA confirmed the diminished responsiveness of the mutants to oxygen and consequently implicated the mutated region in O2-mediated transcriptional regulation.