Mutants of Escherichia coli affected in respiration: the cloning and nucleotide sequence of ubiA, encoding the membrane-bound p-hydroxybenzoate: octaprenyltransferase

Abstract

A mutant of Escherichia coli has been isolated that is unable to grow aerobically on non-fermentable substrates, but able to grow anaerobically on glycerol with alternative electron acceptors such as fumarate. Nitrate as electron acceptor supports anaerobic growth on glycerol, but not on succinate or lactate. Oxygen consumption rates by cell-free extracts with succinate, lactate or glycerol 3-phosphate as substrates were low relative to activities in an isogenic control strain but were restored in vitro by adding ubiquinone-1. Transformation of the mutant with a cloned 2.6 kb ClaI-PvuII fragment of chromosomal DNA restored cellular quinone levels and growth on succinate. The plasmid also complemented a previously isolated ubiA mutant for aerobic growth on non-fermentable substrates. The nucleotide sequence of the cloned fragment revealed a fragment of plsB (91.7 min on the E. coli chromosome map) and three open reading frames (ORFs), one of which (ORF3) encodes a protein with a predicted molecular mass of 32511 Da. The hydrophobicity profile of the ORF3 protein is characteristic of a membrane protein with five hydrophobic regions and is very similar to that of the Saccharomyces cerevisiae COQ2 gene product (p-hydroxybenzoate:polyprenyltransferase, required for the second step of ubiquinone biosynthesis) and to the product of the E. coli cyoE gene. Complementation of ubi mutants with various deletion derivatives of the cloned DNA fragment confirms that ORF3 is ubiA. ORF3 is closely linked to ubiC (ORF2), which encodes chorismate lyase.