Use of Transposon Tn 5367 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.

Identification of a nitroimidazo-oxazine-specific protein involved in PA-824 resistance in Mycobacterium tuberculosis

“…2B, lanes 4 and 5) and PA-824 sensitivity (Table 1). It has also been reported that inactivation of the fbiA (Rv3261) or fbiB (Rv3262) genes in M. bovis lead to an accumulation of FO with concomitant loss of F 420 (11). Therefore, mutant 7A2 was complemented with the fbiAB operon, restoring normal levels of F 420 biosynthesis ( Fig.…”

“…Studies had established that loss of FGD1 or the loss of biosynthetic potential for F 420 individually resulted in resistance to PA-824 (6,10,11). To establish whether FGD1 or reduced F 420 interacted with PA-824 directly, we partially purified recombinant Mtb-FGD1 from Escherichia coli and incubated this with oxidized F 420 purified from Mycobacterium smegmatis (see Supporting Text, which is published as supporting information on the PNAS web site).…”

The methanogenic redox cofactor F420 is widely synthesized by aerobic soil bacteria

“…In the first, a riboflavin precursor (5-amino-6-(D-ribitylamino)uracil) is condensed with tyrosine to form 8-hydroxy-5-deazaflavin, also known as F o ; this step is catalyzed by the radical S-adenosylmethionine enzymes CofG and CofH that are fused into a single protein in some bacteria (known as CofGH or FbiC) (Choi et al, 2002;Philmus et al, 2015). Subsequently, LPPG (L-lactyl-2-diphospho-5 0 -guanosine) is proposed to be synthesized from 2-phospho-L-lactate by CofC (Grochowski et al, 2008) and transferred to F o by CofD (also known as FbiA) (Choi et al, 2001;Graupner and White, 2001;Graupner et al, 2002). The resulting LPPG sidechain is finally elongated with glutamate residues by the F 420 :γ-L-glutamyl ligase CofE (also known as FbiB) (Choi et al, 2001;Li et al, 2003;Nocek et al, 2007) that is fused with an FMNdependent oxidoreductase in Actinobacteria (Bashiri et al, 2016).…”

“…Subsequently, LPPG (L-lactyl-2-diphospho-5 0 -guanosine) is proposed to be synthesized from 2-phospho-L-lactate by CofC (Grochowski et al, 2008) and transferred to F o by CofD (also known as FbiA) (Choi et al, 2001;Graupner and White, 2001;Graupner et al, 2002). The resulting LPPG sidechain is finally elongated with glutamate residues by the F 420 :γ-L-glutamyl ligase CofE (also known as FbiB) (Choi et al, 2001;Li et al, 2003;Nocek et al, 2007) that is fused with an FMNdependent oxidoreductase in Actinobacteria (Bashiri et al, 2016). For reasons still not understood, the number of glutamate residues added varies between organisms, ranging from two to three in most methanogens (Gorris and van der Drift, 1994), four to five in Methanosarcina (Gorris and van der Drift, 1994) and five to seven in Mycobacterium .…”

Critical Role of 7,8-Didemethyl-8-hydroxy-5-deazariboflavin for Photoreactivation in Chlamydomonas reinhardtii

“…FbiA and FbiB are enzymes that catalyze steps in the biosynthesis of F 420 downstream of FO synthase (48). Searches of the Chlamydomonas genome revealed no genes encoding putative homologs of FbiA and FbiB.…”