and AW is currently with the NUTECH

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.

We determined the DNA sequence of the Rhizobium meliloti gene encoding anthranilate synthase, the first enzynie of the tryptophan pathway. Sequences similar to those seen for the two subunits of the enzyme as found in all other procaryotic species studied are present in a single open reading frame of 729 codons. This apparent gene fusion joins the C terminus of the large subunit (TrpE) to the N terminus of the small subunit (TrpG) through a short connecting segment. We designate the fused gene trpE(G). The gene is flanked by a typical rho-independent terminator at the 3' end and a complex regulatory region at the 5' end resembling those of operons under transcriptional attenuation control. The location of the promoter was determined by Sl nuclease protection, using Rhizobium mRNA. Although this promoter was inactive in Escherichia coli, mutations eliciting activity were easily obtained. One of these was a C-*T change at position -9 in the -10 region. The + 1 position of the mRNA is the first base of the initiation codon of the leader peptide, implying that unlike trpE(G), which has a normal Shine-Dalgarno sequence, the leader peptide gene lacks a ribosome-binding site.

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