Use of Transposon Tn5367 Mutagenesis and a Nitroimidazopyran-Based Selection System To Demonstrate a Requirement for fbiA and fbiB in Coenzyme F420 Biosynthesis by Mycobacterium bovis BCG

doi:10.1128/JB.183.24.7058-7066.2001

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Journal of Bacteriology, December 2001, p. 7058-7066, Vol. 183, No. 24
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.24.7058-7066.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Use of Transposon Tn5367 Mutagenesis and a Nitroimidazopyran-Based Selection System To Demonstrate a Requirement for fbiA and fbiB in Coenzyme F₄₂₀ Biosynthesis by Mycobacterium bovis BCG

Kwang-Pil Choi, Thomas B. Bair, Young-Min Bae, and Lacy Daniels^*

Department of Microbiology, University of Iowa, Iowa City, Iowa 52242

Received 12 April 2001/Accepted 21 September 2001

Three transposon Tn5367 mutagenesis vectors (phAE94, pPR28, and pPR29) were used to create a collection of insertion mutantsof Mycobacterium bovis strain BCG. A strategy to select for transposon-generatedmutants that cannot make coenzyme F₄₂₀ was developed using thenitroimidazopyran-based antituberculosis drug PA-824. One-thirdof 134 PA-824-resistant mutants were defective in F₄₂₀ accumulation.Two mutants that could not make F₄₂₀-5,6 but which made the biosynthesisintermediate FO were examined more closely. These mutants containedtransposons inserted in two adjacent homologues of Mycobacteriumtuberculosis genes, which we have named fbiA and fbiB for F₄₂₀biosynthesis. Homologues of fbiA were found in all seven microorganismsthat have been fully sequenced and annotated and that are knownto make F₄₂₀. fbiB homologues were found in all but one such organism.Complementation of the fbiA mutant with fbiAB and complementationof the fbiB mutant with fbiB both restored the F₄₂₀-5,6 phenotype.Complementation of the fbiA mutant with fbiA or fbiB alone didnot restore the F₄₂₀-5,6 phenotype, but the fbiA mutant complementedwith fbiA produced F₄₂₀-2,3,4 at levels similar to F₄₂₀-5,6 madeby the wild-type strain, but produced much less F₄₂₀-5. Thesedata demonstrate that both genes are essential for normal F₄₂₀-5,6production and suggest that the fbiA mutation has a partial polareffect on fbiB. Reverse transcription-PCR data demonstrated thatfbiA and fbiB constitute an operon. However, very low levels offbiB mRNA are produced by the fbiA mutant, suggesting that a low-levelalternative start site is located upstream of fbiB. The specificreactions catalyzed by FbiA and FbiB are unknown, but both functionbetween FO and F₄₂₀-5,6, since FO is made by bothmutants.

^* Corresponding author. Mailing address: Department of Microbiology, University of Iowa, Iowa City, IA 52242. Phone: (319) 335-7780.Fax: (319) 335-9006. E-mail: lacy-daniels{at}uiowa.edu.

Present address: Department of Microbiology, Changwon University, Changwon, Kyungnam 641-773, SouthKorea.

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