Use of transposon Tn5367 mutagenesis and a nitroimidazopyran-based selection system to demonstrate a requirement for fbiA and fbiB in coenzyme F-420 biosynthesis by Mycobacterium bovis BCG

Three transposon Tn5367 mutagenesis vectors (phAE94, pPR28, and pPR29) were used to create a collection of insertion mutants of Mycobacterium bovis st rain BCG. A strategy to select for transposon-generated mutants that cannot make coenzyme F-420 was developed using the nitroimidazopyran-based antitu berculosis drug PA-824. One-third of 134 PA-824-resistant mutants were defe ctive in F-420 accumulation. Two mutants that could not make F-420-5,6 but which made the biosynthesis intermediate FO were examined more closely. The se mutants contained transposons inserted in two adjacent homologues of Myc obacterium tuberculosis genes, which we have named fbiA and fbiB for F-420 biosynthesis. Homologues of fbiA were found in all seven microorganisms tha t have been fully sequenced and annotated and that are known to make F-420. fbiB homologues were found in all but one such organism. Complementation of the fbiA mutant with fbiAB and complementation of the fbiB mutant with fbi B both restored the F-420-5,6 phenotype. Complementation of the fbiA mutant with fbiA or fbiB alone did not restore the F-420-5,6 phenotype, but the f biA mutant complemented with fbiA produced F-420-2,3,4 at levels similar to F-420-5,6 made by the wiLd-type strain, bat produced much less F420-5. The se data demonstrate that both genes are essential for normal F-420-5,6 prod uction and suggest that the fbiA mutation has a partial polar effect on fbi B. Reverse transcription-PCR data demonstrated that fbiA and fbiB constitut e an operon. However, very low levels of fbiB mRNA are produced by the fbiA mutant, suggesting that a low-level alternative start site is located upst ream of fbiB. The specific reactions catalyzed by FbiA and FbiB are unknown , but both function between FO and F-420-5,6, since FO is made by both muta nts.