Thiamin and riboflavin are precursors of essential coenzymes-thiamin pyrophosphate (TPP) and flavin mononucleotide (FMN)/flavin adenine dinucleotide (FAD), respectively. In Bacillus spp, genes responsible for thiamin and riboflavin biosynthesis are organized in tightly controllable operons. Here, we demonstrate that the feedback regulation of riboflavin and thiamin genes relies on a novel transcription attenuation mechanism. A unique feature of this mechanism is the formation of specific complexes between a conserved leader region of the cognate RNA and FMN or TPP. In each case, the complex allows the termination hairpin to form and interrupt transcription prematurely. Thus, sensing small molecules by nascent RNA controls transcription elongation of riboflavin and thiamin operons and possibly other bacterial operons as well.

A 3-5 kb EcoRl-BamHI fragment of Baci//us subtilis chromosomal DNA carrying the ribR gene, involved in regulation of the B. subtilis riboflavin operon, was cloned in the B. subtilis-€scherichia coli shuttle vector pCB2O. DNA sequence analysis of this fragment revealed several ORFs, one of which encodes a polypeptide of 230 amino acids with up to 45% sequence identity with FAD synthetases from a number of micro-organisms, such as Corynebacterium ammoniagenes, E. coli and Pseudomonas fluorescens, and also to the ribC gene product of B. subtilis. The ribR gene was amplified by PCR, cloned and expressed in E. coli. Measurement of f lavokinase activity in cell extracts demonstrated that ribR encodes a monof unctional f lavokinase which converts riboflavin into FMN but not to FAD, and is specific for the reduced form of riboflavin.Keywords : Bacillus subtilis, flavinogenesis, riboflavin, operon regulation, flavokinase INTRODUCTIONThe Bacillus subtilis riboflavin operon comprises a cluster of five non-overlapping genes that encode the enzymes which catalyse the reactions for de novo riboflavin biosynthesis starting from GTP. The operon is located at 209" on the B. subtilis genetic map. The functional organization of the riboflavin operon has been described in detail in several articles (Perkins & Pero, 1993;Mironov et al., 1994). The expression of the operon is negatively regulated by the product of the ribC gene, which is situated at 147" on the B. subtilis chromosome and appears to encode a flavin-activated aporepressor. A possible repressor binding site is situated in a region of 294 bp between the promoter and the first structural gene of the operon (Kil et al., 1992). Mutations in this region designated rib0 mutations are cis-dominant and, like ribC mutations, have a phenotype of riboflavin overproduction, resulting in accumulation of the vitamin in the growth medium. While the exact mechanism of regulation is still un- The EMBL accession number for the sequence referred t o in this paper is Y09721.known (Azevedo et al., 1993) it is evident that the regulatory system of the operon is not limited to the ri6C gene and the rib0 regulatory region. We have examined B. subtilis mutants which carry the constitutive ri6Cl mutation and are partially resistant to the riboflavin analogue 7,8-dimethyl-l0-( O-methylacetoxime)-isoalloxazine (MO), in which the ribityl moiety is replaced with -CH,-CH = N-O-CH, (Fig. 1). A number I. M. S O L O V I E V A a n d OTHERSof these mutants had completely lost the ability to oversynthesize riboflavin but retained the original ri6Cl mutation in the chromosome (Kreneva & Perumov, 1994). This was interpreted as the restoration of regulatory activity due to a mutation at a previously unknown locus, resulting in expression of a product that could also regulate expression of the riboflavin operon of B. subtilis. This effect is only seen in ri6C constitutive mutants and not in the background of rib0 regulatory mutations.Two mutations which decrease the constitutive expression of the B. subtil...

Seventeen cis-dominant mutations leading to riboflavin overproduction in Bacillus subtilis were localized to the region between nucleotides +37 and +159 relative to the transcription initiation site of the riboflavin operon. This region displays an unusual structure for regulatory sequences. The main part of it represents clusters of A/T and G/C-rich sequences that symmetrically blank a short inverted repeat.

The riboflavin kinase encoding gene ribR is situated within a 12 genes locus ytmI-ytnM of the Bacillus subtilis chromosome. Here we demonstrate that ribR is transcribed as part of a 10 kb ytmI-ytnM operon. The riboflavin overproduction phenotype of B. subtilis ribC mutant strains, which is a result of the strongly reduced flavokinase activity of the riboflavin kinase/FAD synthetase RibC, was suppressed by ribR expression. Analysis of mutations with an upregulated ribR gene revealed 2 different groups of mutants. One class of mutants contained base substitutions in an 8 nucleotide sequence of the promoter region of the ytmI-ytnM operon. A second class of mutants had single point mutations within the yrzC gene or in the RBS of this gene. Dot-blot analysis of ytmI-ytnM transcription and the results of in trans complementation experiments for the yrzC mutants confirmed a role of the yrzC gene product as a negative regulator for the ytmI-ytnM operon.