Cloning and Genetic Analysis of Six Pyrroloquinoline Quinone Biosynthesis Genes in Methylobacterium organophilum DSM 760

Abstract

After EMS mutagenesis, mutants of Methylobacterium organophilum DSM 760 unable to synthesize pyrroloquinoline quinone (PQQ) were selected among mutants which did not utilize methanol but were still able to use methylamine as growth substrate. Six different pqq genes (pqqA to pqqF) were identified by complementation analysis. The genes pqqA to pqqD, cloned in a single R′ plasmid, were grouped in a 3·9 kb DNA fragment. The genes pqqA and pqqB belonged to a single transcription unit independent from the adjacent gene pqqC. The gene pqqD was contained in a short DNA segment of approximately 0·1 kb, separated from pqqC by a region with no apparent role in PQQ biosynthesis. Two other genes were identified:pqqE, which was closely linked to pqqD; and pqqF, located approximately 19 kb from the other genes. Directed mutagenesis by marker exchange provided chromosomal insertion mutations of these genes in M. organophilum. Attempts to express the pqq genes in two heterologous hosts, Escherichia coli and Pseudomonas testosteroni, were unsuccessful, and no plasmid containing all of the pqq genes was isolated.