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

Abstract

Three transposon Tn5367 mutagenesis vectors (phAE94, pPR28, and pPR29) were used to create a collection of insertion mutants of Mycobacterium bovis strain BCG. A strategy to select for transposon-generated mutants that cannot make coenzyme F(420) was developed using the nitroimidazopyran-based antituberculosis drug PA-824. One-third of 134 PA-824-resistant mutants were defective 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. These mutants contained transposons inserted in two adjacent homologues of Mycobacterium tuberculosis genes, which we have named fbiA and fbiB for F(420) biosynthesis. Homologues of fbiA were found in all seven microorganisms that 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 fbiB 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 fbiA mutant complemented with fbiA produced F(420)-2,3,4 at levels similar to F(420)-5,6 made by the wild-type strain, but produced much less F(420)-5. These data demonstrate that both genes are essential for normal F(420)-5,6 production and suggest that the fbiA mutation has a partial polar effect on fbiB. Reverse transcription-PCR data demonstrated that fbiA and fbiB constitute 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 upstream 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 mutants.

FIG. 1

(A) Structure of coenzyme F₄₂₀-5 from Mycobacterium spp.(4). (B) Overview of the hypothesized pathway for F₄₂₀ biosynthesis, which is based on our modifications of the reactions proposed by Bacher and Thauer and their colleagues (13, 22, 38), and including the role of 2-phospholactate proposed by Graupner and White (17).

FIG. 2

Hypothesis for the role of F₄₂₀ in PA-824 activation. Protein X is a hypothetical enzyme that transfers electrons from F₄₂₀H₂ to PA-824.

FIG. 3

Sequence characteristics of the Tn5367 transposon insertion sites. In the large graph, percent A+T at each position to the right (positive values) and to the left (negative values) of the insertion site is given for nine bases on both sides of the 31 gene insertion sites observed in our experiments and the 8 duplicated bases (positive values 1 to 8) for an additional 19 insertion sites reported by others (5, 30, 34). The horizontal dotted line indicates the A+T content of the M. tuberculosis genome (34.4%). The inset graph provides a sequence logo plot to graphically emphasize the incidence of individual base types, if they predominate at positions 1 to 9.

FIG. 4

Gene arrangement of the fbiAB cluster in M. bovis BCG, corresponding transposon insertion sites, and alignment of these genes with homologs from other microorganisms. Triangles indicate transposon insertion sites. CAB, Streptomyces coelicolor; MTH, Methanobacterium thermoautotrophicum; AF, Archaeoglobus fulgidus; Vng, Halobacterium sp; SLL, Synechocystis sp.; AAC, Nostoc sp. Numbers refer to gene designations in the corresponding genome sequences. Rv3259 and Rv3263 are used to indicate the M. bovis homologs of these M. tuberculosis H37Rv genes, since the M. bovis sequence is not yet annotated, and thus M. bovis genes have not been assigned unique designations. The lines below the M. bovis genes indicate the sizes and locations of RT-PCR products expected; a solid line indicates that a product was seen, and a dotted line indicates that no product was observed. The primer pairs used were (from left to right) F1-R2, F3-R4, and F5-R6. Boxes with solid lines indicate genes identified with default NCBI Blast settings. Boxes with dashed lines indicate genes identified with ψ-Blast iterations. Boxes connected by bars indicate the genes are adjacent, and those not connected are not adjacent. Figure is approximately to scale, with boxes indicating the sizes of all putative homologous genes.

FIG. 5

HPLC elution profiles of extracts made from M. bovis BCG wild-type, the fbiA mutant (Rv3261 homolog interrupted), the fbiB mutant (Rv3262 homolog interrupted) that was complemented with p3262, and the fbiA mutant that was complemented with p3261.

FIG. 6

HPLC elution profiles of extracts made from the fbiA mutant and the fbiB mutant. Compare the scale to the HPLC data in Fig. 5; the scale here is enhanced by 13.3-fold.