Identification of a New Gene Required for the Biosynthesis of Rhodoquinone in Rhodospirillum rubrum

Rhodoquinone (RQ) is a required cofactor for anaerobic respiration in Rhodospirillum rubrum, and it is also found in several helminth parasites that utilize a fumarate reductase pathway. RQ is an aminoquinone that is structurally similar to ubiquinone (Q), a polyprenylated benzoquinone used in the aerobic respiratory chain. RQ is not found in humans or other mammals, and therefore, the inhibition of its biosynthesis may provide a novel antiparasitic drug target. To identify a gene specifically required for RQ biosynthesis, we determined the complete genome sequence of a mutant strain of R. rubrum (F11), which cannot grow anaerobically and does not synthesize RQ, and compared it with that of a spontaneous revertant (RF111). RF111 can grow anaer-obically and has recovered the ability to synthesize RQ. The two strains differ by a single base pair, which causes a nonsense mu-tation in the putative methyltransferase gene rquA. To test whether this mutation is important for the F11 phenotype, the wild-type rquA gene was cloned into the pRK404E1 vector and conjugated into F11. Complementation of the anaerobic growth defect in F11 was observed, and liquid chromatography-time of flight mass spectrometry (LC-TOF-MS) analysis of lipid extracts con-firmed that plasmid-complemented F11 was able to synthesize RQ. To further validate the requirement of rquA for RQ biosyn-thesis, we generated a deletion mutant from wild-type R. rubrum by the targeted replacement of rquAwith a gentamicin resis-tance cassette. TherquAmutant exhibited the same phenotype as that of F11. These results are significant because rquA is the first gene to be discovered that is required for RQ biosynthesis. Rhodoquinone (RQ) (Fig. 1, compound 1) is found in the mi-tochondrial membrane of parasitic helminths (43, 46) an