A CHROMOSOMAL LOCUS REQUIRED FOR COPPER RESISTANCE, COMPETITIVE FITNESS, AND CYTOCHROME-C BIOGENESIS IN PSEUDOMONAS-FLUORESCENS

A chromosomal locus required for copper resistance and competitive fit ness was cloned from a strain of Pseudomonas fluorescens isolated from copper-contaminated agricultural soil. Sequence analysis of this locu s revealed six open reading frames with homology to genes involved in cytochrome c biogenesis in other bacteria, helC, cycJ, cycK, tipB, cyc L, and cycH, with the closest similarity being to the aeg-46.5(yej) re gion of the Escherichia coli chromosome, The proposed functions of the se genes in other bacteria include the binding, transport, and couplin g of heme to apocytochrome c in the periplasm of these Gram-negative b acteria, Putative heme-binding motifs were present in the predicted pr oducts of cycK and cycL, and TipB contained a putative disulfide oxido reductase active site proposed to maintain the heme-binding site of th e apocytochrome in a reduced state for ligation of heme. Tn3-gus mutag enesis showed that expression of the genes was constitutive but enhanc ed by copper, and confirmed that the genes function both in copper res istance and production of active cytochrome c, However, two mutants in cycH were copper-sensitive and oxidase-positive, suggesting that the functions of these genes, rather than cytochrome c oxidase itself, wer e required for resistance to copper.