Regulation of riboflavin biosynthesis in Bacillus subtilis is affected by the activity of the flavokinase/flavin adenine dinucleotide synthetase encoded by ribC - PubMed (original) (raw)

Regulation of riboflavin biosynthesis in Bacillus subtilis is affected by the activity of the flavokinase/flavin adenine dinucleotide synthetase encoded by ribC

M Mack et al. J Bacteriol. 1998 Feb.

Abstract

This work shows that the ribC wild-type gene product has both flavokinase and flavin adenine dinucleotide synthetase (FAD-synthetase) activities. RibC plays an essential role in the flavin metabolism of Bacillus subtilis, as growth of a ribC deletion mutant strain was dependent on exogenous supply of FMN and the presence of a heterologous FAD-synthetase gene in its chromosome. Upon cultivation with growth-limiting amounts of FMN, this ribC deletion mutant strain overproduced riboflavin, while with elevated amounts of FMN in the culture medium, no riboflavin overproduction was observed. In a B. subtilis ribC820 mutant strain, the corresponding ribC820 gene product has reduced flavokinase/FAD-synthetase activity. In this strain, riboflavin overproduction was also repressed by exogenous FMN but not by riboflavin. Thus, flavin nucleotides, but not riboflavin, have an effector function for regulation of riboflavin biosynthesis in B. subtilis, and RibC seemingly is not directly involved in the riboflavin regulatory system. The mutation ribC820 leads to deregulation of riboflavin biosynthesis in B. subtilis, most likely by preventing the accumulation of the effector molecule FMN or FAD.

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Figures

FIG. 1

FIG. 1

Overproduction and purification of B. subtilis wild-type RibC and mutant RibC820. SDS-PAGE of cell extracts of IPTG-induced E. coli strains harboring the B. subtilis ribC wild-type (E. coli BL21MM01; lane 2) and ribC820 mutant (E. coli BL21MM02; lane 3) genes. As a control, lane 1 shows a cell extract of an IPTG-induced E. coli BL21 strain harboring the expression vector pJF119HE without insert. Lanes 4 and 5 show RibC wild type and mutant, respectively, after (NH4)2SO4 purification. Cation-exchange eluates of RibC wild type and mutant are shown in lanes 6 and 7. Lanes marked M are molecular weight markers (in kilodaltons).

FIG. 2

FIG. 2

HPLC chromatograms of the products of flavokinase/FAD-synthetase assays. Assay mixtures containing 50 μM riboflavin, 3 mM ATP, 15 mM MgCl2, and 10 mM sodium sulfite (Na2SO3) were preincubated at 37°C for 5 min. Pure wild-type RibC (A and B) or mutant RibC820 (C and D) (3 μg of each) was added, and the mixtures were incubated for another 5 (A and C) or 30 (B and D) min. An aliquot was removed and separated on an HPLC column (Nucleosil 10 C18; 4.6 by 250 mm; Macherey & Nagel). The following solvent system was used at a flow rate of 2.5 ml/min: 25% (vol/vol) methanol–100 mM formic acid–100 mM ammonium formate (pH 3.7). The reaction was monitored with a fluorescence detector (excitation, 470 nm; emission, 530 nm; Waters Associates). The chromatograms show three clearly resolved peaks of riboflavin (8 min), FMN (5 min), and FAD (4 min). Numbers in parentheses indicate decrease of riboflavin and increase of flavin nucleotides during the enzyme assay.

FIG. 3

FIG. 3

FMN-dependent riboflavin overproduction in B. subtilis 1012MM006. The FMN auxothropic ribC deletion mutant B. subtilis 1012MM006 was cultivated for 16 h in the presence of the indicated amounts of FMN. Riboflavin in the culture medium at the end of fermentation was determined by HPLC analysis as described for Fig. 2. Cell optical density at 600 nm (OD600) at the end of fermentation was estimated photometrically.

FIG. 4

FIG. 4

Exogenuous FMN represses riboflavin overproduction in a B. subtilis ribC820 (triangles) but not in a B. subtilis 1012mro87 (ribO mutant, circles) strain. A B. subtilis ribC820 strain and strain 1012mro87 (ribO mutant) were cultivated for 16 h in growth medium supplemented with indicated amounts of FMN. Riboflavin in the supernatant was determined by HPLC as described for Fig. 2. Cell growth was estimated photometrically at 600 nm.

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