JOURNAL OF BACTERIOLOGY, May 1978, p. 476-482 Vol. 134, No. 2 0021-9193/78/0134-0476$02.00/0 Copyright © 1978 American Society for Microbiology Printed in U.S.A. of in Bacillus subtilis: Function and Genetic Control of the Riboflavin Synthase Complex A. BACHERt* AND B. MAILANDERtt Institut fir Mikrobiologie, Universitat Hohenheim, 7000 Stuttgart 70, German Federal Republic Received for publication 19 September 1977 Two riboflavin synthase activities (heavy and light) have been observed in earlier studies with Bacillus subtilis. The heavy is a complex of one molecule of light enzyme (consisting of three a subunits) and approximately 60 fi subunits (A. Bacher, R. Baur, U. Eggers, H. Harders, and H. Schnepple, p. 729-732, in T. P. Singer (ed.), Flavins and Flavoproteins, Elsevier, Amsterdam, 1976). The formation of a and f8 subunits is coordinately controlled. Mutants apparently deficient in ,B subunits were isolated as riboflavin requires after mutagenesis of B. subtilis with ICR 191. The mutants could grow with diacetyl instead of riboflavin. Growth with diacetyl was associated with the accumulation of substantial amounts of the riboflavin precursor, 6,7-dimethyl-8-(D- ribityl)lumazine. It follows that the mutants are deficient in an enzyme activity required for the formation of the lumazine from the pyrimidine precursor. We conclude that heavy riboflavin synthase is a bifunctional enzyme. The riboflavin synthase activity is mediated by the a subunits, whereas the ,B subunits are necessary for an earlier biosynthetic step. Riboflavin synthase catalyzes the formation of light enzyme (i.e., three a subunits) and ap- of one molecule each of riboflavin and 5-amino- proximately 60 identical ,B subunits (3; A. 2,6-dihydroxy-4-(D-ribitylamino) pyrimidine Bacher, M. K. Otto, and H. Schnepple, unpub- from two molecules of 6,7-dimethyl-8-(D-ribi- lished data). Isolated ,B subunits had no ribo- tyl)lumazine. The enzyme has been found in flavin synthase activity, and the low specific various microorganisms and plants (for review, activity of the heavy enzyme as compared to see references 13, 25, and 29). Riboflavin syn- that of the light enzyme supports the hypothesis thase from yeast has been highly purified, and that only the a subunits are catalytically in- the specificity and the stereospecific volved in the conversion of 6,7-dimethyl-8-(D- mode ofaction have been studied in considerable ribityl)lumazine to riboflavin. detail. On the basis of studies with isotopically The early steps of riboflavin biosynthesis are labeled substrates, a mechanism of reaction has incompletely understood. The pathway starts at been proposed (25, 26). Little is known about the level of guanosine or a respective nucleotide, the physical properties and the structure of the as shown by isotope incorporation studies (7, 9, yeast enzyme. 20). The ribose moiety of the purine precursor is The concentration of riboflavin synthase in directly converted to the ribityl moiety of the cell extracts of most microorganisms studied is vitamin. It has been suggested that the first rather low (25). However, high enzyme levels committed step of the biosynthesis is catalyzed have been found in flavinogenic mutants of Ba- by GTP cyclohydrolase II in . cillus subtilis (4, 10). It has been shown that the The enzyme catalyzes the simultaneous release biosynthesis of the enzyme is controlled by of carbon-8 and of pyrophosphate from GTP repression in this microorganism. Studies with (14). Several pyrimidine-type intermediates cell extracts from a genetically derepressed mu- have been isolated in studies with riboflavin- tant of B. subtilis showed the presence of two deficient mutants of Saccharomyces cerevisiae riboflavin synthase activities of greatly different (Fig. 1) (5, 6, 19). The conversion of 5-amino-2,6- sizes and molecular weights (3). The light en- dihydroxy-4-(D-ribitylamino)pyrimidine to 6,7- zyme, which accounts for more than 80% of the dimethyl-8-(D-ribityl)lumazine requires the ad- total activity, is a trimer of identical a subunits. dition of a four-carbon moiety ofunknown struc- The heavy enzyme is a complex of one molecule ture. Several studies suggested the involvement of acetoin or a biogenetically related compound t Present address: Fachbereich Biologie, Universitit Frankfurt, 6000 Frankfurt/Main, German Federal Republic. (12, 16, 18). The hypothesis has been criticized tt Present address: Pfizer Laboratories, 75 Karlsruhe, Ger- by other authors (1, 15). Recent studies sug- man Federal Republic. gested the involvement of a pentose or tetrose 476 VOL. 134, 1978 BIOSYNTHESIS OF RIBOFLAVIN 477

OH OH OH

H2N N rib 1 H2N N-NNH2 HN NH2 HN N NH2

C H2 (P3)OH2C I P) OH2CLo0 H-C-OH H -C-OH H v H-C-OH OH OH OH OH C H20H

II I

OH 0 0 ~H H rib 3 H3C N / H 3C\/,/N rib 2 2N N rib 4 N rib 5

HN N OH H3C N NAO H3C N NAO

C H2 CH2 CH2

H-C-OH H-C-OH H-C-OH H-C-OH H-C-OH H-C-OH H-C-OH H-C-OH H-C-OH CHH2OH CH20H CH20H

IV v VI FIG. 1. Biosynthesis ofriboflavin (14,20). Genes involved in S. cerevisiae are indicated according to studies by Oltnanns et al. (22,23). I, guanosine triphosphate; II, 2,5-diamino-6-hydroxy-4-(D-ribosylamino)pyrimidine 5'-phosphate; III, 2,5-diamino-6-hydroxy-4-(D-ribitylamino)pyrimidine; IV, 5-amino-2,6-dihydroxy-4-(D-ribi- tylamino)pyrimidine; V, 6,7-dimethyl-8-(D-ribityl)lumazine; VI, riboflavin.

(1, 2). The involvement of 6-methyl-7-(1',2'-di- TABLE 1. Mutants ofB. subtilis used0 hydroxyethyl)-8-ribityliumazine as a precursor Strain Phenotype Origin of 6,7-dimethyl-8-(D-ribityl)lumazine has been proposed on the basis of studies with mutants of H78 Rib- NGb H94 Flavinogenicc (4) B. subtilis (11). H175 Rib- NG Several biosynthetic reactions in the pathway H322 Flavinogenic (4) of riboflavin biosynthesis are not yet accessible CR2 Rib- ICRd to direct enzymatic studies in spite of consider- CR6 Rib- ICR able efforts. This paper presents evidence that CR9 Rib- ICR the heavy riboflavin synthase of B. subtilis is a 'All mutants were derived from strain 168M and bifunctional enzyme. Whereas the a subunits require tryptophan. mediate the known riboflavin synthase activity b N-Methyl-N'-nitro-N-nitrosoguanidine mutagen- of the protein, the ,B subunits appear to be esis. necessary for an earlier reaction in the biosyn- 'Genetically derepressed mutant accumulating thesis of the vitamin. riboflavin. d ICR 191 mutagenesis. MATERIALS AND METHODS Bacterial strains. B. subtilis 168M trp2C was pg/ml) ovemight (21). A portion was transferred to kindly provided by C. Anagnostopoulos, Centre de fresh complete medium, and the culture was incubated Recherches Scientifiques, Gif-sur-Yvette, France. The overnight. Riboflavin-deficient mutants were isolated other mutants used are listed in Table 1. as described (4). Only one mutant of each respective Media. The basic medium was Spizizen minimal phenotype was collected from each mutagenized cul- medium (30) supplemented with tryptophan (50 ture. mg/liter). Vitamin-free Cmino Acids (Difco), ribo- Growth of bacteria and preparation of cell flavin, and diacetyl were added as required. extracts. Bacteria were grown in 0.5-liter batches of Chemicals. 6,7-Dimethyl4-8(D-ribityl)lumazine medium supplemented with vitamin-free Casamino was prepared by published procedures (28). ICR 191 Acids (5 g/liter). Riboflavin was added as required. was a gift of Badische Anilin- und Soda-Fabrik, Lud- The cultures were incubated with shaking overnight. wigshafen. Riboflavin was purchased from Merck AG, The cells were harvested by centrifugation and stored Darmstadt, W. Germany, and diacetyl was from Fluka at -20°C. For the preparation of cell extracts, frozen AG, Buchs, Switzerland. bacterial cells were thawed in buffer containing 0.1 M Isolation of mutants. B. subtilis 168M was grown phosphate (pH 6.9), 10 mM ethylenediaminetetraace- in complete medium supplemented with ICR 191 (20 tic acid, and 10 mM sodium sulfite. The suspension 478 BACHER AND MAILANDER J. BACTERIOL. was ultrasonically treated and centrifuged. TABLE 2. Concentration of light and heavy Assay of riboflavin synthase. Assay mixtures riboflavin synthase in cell extracts ofB. subtilis contained 0.1 M phosphate (pH 7.4), 10 mM ethylene- mutants diaminetetraacetic acid, 10 mM sodium sulfite, and 0.6 Riboflavin synthase mM 6,7-dimethyl-8-(D-ribityl)lumazine. Assays were performed at 37°C as described by Plaut and Harvey Strain Riboflavin'(mg/liter) (U/mg of protein) (28). One unit of enzyme activity catalyzes the for- Total Lightb Heavyb Heavy' mation of 1 nmol of riboflavin per h. Sucrose gradient centrifugation. Sucrose gra- H94 0 107 89 18 18 dients (5 to 20%) contained 0.1 M phosphate (pH 6.9), H322 0 80 68 12 15 10 mM ethylenediaminetetraacetic acid and 10 mM H175 0.03 59 51 8 5 sodium sulfite. Samples of 0.5 ml were layered on top H175 10 3 NDd ND 0.7 of the gradients. They were centrifuged in an SW27.1 CR9 0.03 67 60 7 9 rotor (Spinco) at 23,000 rpm and 4°C for 20 h. Frac- CR9 10 1.4 ND ND 0.2 tions were collected and analyzed. CR2 0.03 118 118 <0.1 <0.1 Preparation ofantisera. Rabbits were immunized CR2 10 0.6 ND ND <0.1 with 0.2- to 0.5-mg samples oflight or heavy riboflavin a Concentration of riboflavin added to the culture synthase ofB. subtilis (3) in 0.5 ml ofFreund complete medium. adjuvant (Difco). Serum obtained after immunization b Determined by enzyme assay of sucrose gradient with heavy enzyme was made monospecific for ,8 sub- fractions. units by addition of light riboflavin synthase. 'Determined by simple radial immunodiffusion us- Immunodiffusion. Double-diffusion and simple ing anti-f, serum. radial diffusion experiments were performed according d ND, Not determined. to published procedures (17, 24). Plates contained 0.1 M phosphate (pH 6.9), 10 mM ethylenediaminetetra- acetic acid, 0.01% sodium azide, and 1.2% agar (Difco). Light and heavy riboflavin synthase, respectively, were used as standards in quantitative measurements of a and ,B subunits. Results were expressed in terms of enzyme units. Isolation of6,7-dimethyl-8-(D-ribityl)lumazine. B. subtilis mutant CR2 was grown in 1-liter batches of medium supplemented with vitamin-free Casamino Acids (5 g/liter) and diacetyl (50 id/liter). The cultures were incubated at 37°C for 36 h. The cells were removed by centrifugation. 6,7-Dimethyl-8-(D-ri- bityl)lumazine was isolated from the culture medium as described (4). Miscellaneous methods. Protein concentration was estimated by the biuret method. Thin-layer chromatography was performed with plates coated with cellulose MN 300 (Macherey & Nagel, Diiren, W. Germany). The systems were 1-butanol-acetic acid-water, 50:15:35 (vol/ vol/vol), and 3% ammonium chloride, respectively. RESULTS Regulation of enzyme subunit biosyn- thesis. The level of light and heavy riboflavin synthase in cell extracts of B. subtilis mutants FRACTION was analyzed by sucrose gradient centrifugation FIG. 2. Sucrose gradient sedinentation. Top, mu- and by simple radial immunodiffusion using tant H94 (geneticaly derepressed). Bottom, mutant monospecific antisera (Table 2). In the flavino- CR2 (Rib-, grown with 30 pg ofriboflavin per liter of genic mutants H94 and H322, the heavy enzyme culture medium). 0, Riboflavin synthase activity; 0, accounted for approximately 15% of the total concentration of,B subunits (determined by immuno- riboflavin synthase activity, as shown by sucrose diffusion). Sedimentation was from right to left. gradient centrifugation. The values obtained by immunochemical determination of ,B subunits that free I? subunits sediment faster than heavy are in agreement within the experimental limits. riboflavin synthase due to aggregate formation. Figure 2 shows the sucrose gradient centrifuga- The free ,B subunits forned precipitates with tion profile of mutant H94. The peak of f8 sub- anti-,B serum and showed a reaction of identity units coincides with the peak ofheavy riboflavin with heavy riboflavin synthase in double-diffu- synthase activity. Control experiments showed sion experiments with anti-,B serum. These data VOL. 134, 1978 BIOSYNTHESIS OF RIBOFLAVIN 479 show conclusively that the cell extracts of the per liter. The cells were harvested, washed, and mutants under study contained no free ,8 sub- suspended in riboflavin-free medium. Samples units. were retrieved at intervals and analyzed. Figure Mutants CR9 and H175 require riboflavin and, 3 shows that the concentration of a and ,B sub- when grown with limiting amounts of the vi- units increased at a similar rate. tamin, formed high levels ofboth light and heavy Characterization of ,B subunit-deficient enzyme. The pattern was similar to the flavino- mutants. Riboflavin-deficient mutants of B. genic mutants described above. Again the subtilis isolated after ICR 191 mutagenesis were amounts of heavy enzyme observed by gradient grown with low riboflavin concentrations, and centrifugation and by immunochemical analysis the cell extracts were analyzed by immunodif- were in agreement within the experimental lim- fusion using monospecific anti-a and anti-,8 se- its. When the mutants were grown with a high rum (Fig. 4). Three mutants obtained from in- concentration of riboflavin, both the total ribo- dividually mutagenized cultures formed no pre- flavin synthase activity and the level of fi sub- cipitates with anti-,f serum. These mutants units dropped to low values, indicating that the formed normal amounts of a subunits, as shown formation of a and f subunits is coordinately by enzyme assay and immunodiffusion. Mutant controlled by repression. This was confirmed by CR2 was selected for further studies. Under the following experiment. Mutant H78 was conditions of maximum derepression, this mu- grown in medium containing 10 mg of riboflavin tant formed less than 0.1 U of heavy riboflavin

A B 30 30- 20 20-

,,,0 / 10-

TIME (HOURS) FIG. 3. Metabolic derepression of riboflavin synthase. Cells of mutant H78 grown with riboflavin (10 mg/liter) were transferred to riboflavin-free medium, and samples were analyzed at intervals. (A) Total riboflavin synthase activity; (B) light riboflavin synthase (determined by immunodiffusion with anti-a serum); (C) heavy riboflavin synthase (determined by immunodiffusion with anti-,8 serum).

FIG. 4. Immuno-double-diffusion. The center well contained anti-a serum (left) or anti-fl serum (right); (1) mutant CR 2 (Rib-, grown with 30 pg of riboflavin per liter of medium); (2) light riboflavin synthase; (3) mutant H94 (constitutive synthesis ofriboflavin synthase); (4) mutant CR6 (Rib-, grown with 30pg ofriboflavin per liter ofmedium); (5) heavy riboflavin synthase. 480 BACHER AND MAILANDER J. BACTERIOL. synthase per mg of protein, as shown by immu- tein represents only a minor fraction of the total nodiffusion (Table 2). Sucrose gradient centrif- riboflavin synthase activity in all Bacillus ugation showed a normal peak oflight riboflavin strains studied (3). This could be due to disso- synthase but no trace of the heavy enzyme (Fig. ciation ofthe major amount ofthe protein under 2). It follows that the level of/1 subunits was less the conditions of cell disruption. However, we than 1% as compared to derepressed standard have shown that the cell extracts contain no free strains. ,/ subunits. It follows that the cells form /8 sub- The ,B-deficient mutants could grow with di- units in amounts insufficient for formation of acetyl instead of riboflavin. Optimal growth was complexes with all available a subunit timers. obtained with 0.2 to 1 mM diacetyl or 1 ,uM We have also shown that the formation of a and riboflavin. Higher diacetyl concentrations were /1 subunits is coordinately controlled. inhibitory (Fig. 5). When grown with diacetyl, Three mutants isolated as riboflavin requirers the mutants accumulated a green fluorescent from cultures individually mutagenized with substance. The compound was isolated from cul- ICR 191 contained no detectable amounts of/ tures of mutant CR2 grown with diacetyl and subunits. In maximally derepressed mutant cells, was identified as 6,7-dimethyl-8-(D-ribityl)lu- the concentration of the peptide was less than mazine on the basis of UV spectra, thin-layer 1% as compared to derepressed reference strains, chromatography, and enzymatic conversion to whereas the level of a subunits and, hence, ribo- riboflavin by light riboflavin synthase ofB. sub- flavin synthase activity was high. It is not known tilis. The yield was 1 mg/liter ofculture medium. whether the mutant phenotype is due to a mu- Growth with diacetyl was also observed with tation of the /B subunit structural gene or of a several mutants which produce apparently nor- regulatory gene. The possibility remains that mal /B subunits as judged by immunodiffusion the mutants produce a small amount of intact experiments. ,/ subunits that is below the level of sensitivity of the immunochemical methods used for detec- tion. DISCUSSION The /1 subunit-deficient mutants could grow Previous studies indicated that the/, subunits with diacetyl instead of riboflavin and accumu- of the riboflavin synthase complex of B. subtilis lated a substantial amount of the riboflavin pre- are not catalytically involved in the conversion cursor, 6,7-dimethyl-8-(D-ribityl)lumazine, when of 6,7-dimethyl-8-(D-ribityl)lumazine to ribo- grown with diacetyl. We conclude that the mu- flavin (3). This raised the question of whether tants produce 5-amino-2,6-dihydroxy-4-(D-ribi- they catalyze an earlier step in the biosynthesis tylamino)pyrimidine, which reacts nonenzymat- of the vitamin or represent discrete membrane ically with exogenous diacetyl resulting in for- fragments bound to the a subunit trimer (8). mation of the lumazine. A fraction of the nonen- The heavy riboflavin synthase appears to be zymatically formed lumazine is further con- a taxonomic peculiarity ofBacillaceae. The pro- verted to riboflavin by riboflavin synthase, thus

0 2

CONCENTRATKIN (M) FIG. 5. Growth ofB. subtilis mutant CR2. Erlenmeyer flasks supplemented with riboflavin (0) or diacetyl (-) were inoculated and incubated with shaking overnight. VOL. 134, 1978 BIOSYNTHESIS OF RIBOFLAVIN 481 permitting the mutants to grow in the absence LITERATURE CITED of exogenous riboflavin. Analogous results have 1. Ali, S. N., and U. A. S. Al-Khalidi. 1966. The precursors been obtained in studies with mutants of S. of the xylene ring in riboflavine. Biochem. J. cerevisiae (19). 98:182-188. 2. Alworth, W. 1., ML F. Dove, and H. N. Baker. 1977. These data suggest that the ,B subunits are Biosynthesis of the dimethylbenzene moiety of ribo- necessary for the enzymatic conversion of the flavin and dimethylbenzimidazole: evidence for the in- pyrimidine to the lumazine. However, we have volvement of C-1 of a pentose as a precursor. Biochem- also observed mutants which grow with diacetyl istry 16:526-631. 3. Bacher, A., R. Baur, U. Eggers, H. Harders, and H. instead of riboflavin but contain apparently nor- Schnepple. 1976. Riboflavin synthases ofBacillus sub- mal ,B subunits as judged by immunodiffusion. tilis, p. 729-732. In T. P. Singer (ed.), Flavins and The following hypotheses should be considered. flavoproteins. Elsevier, Amsterdam. (i) The diacetyl requirers carry a mu- 4. Bacher, A., U. Eggers, and F. Lingens. 1973. Genetic ,l-positive control of riboflavin synthetase in Bacillus subtilis. tation of the ,B subunit structural gene which Arch. Microbiol. 89:73-77. impairs the function but does not affect the 5. Bacher, A., and F. Lingens. 1970. Biosynthesis of ribo- overall structure significantly. Thus, nonfunc- flavin. Formation of 2,5-diamino-6-hydroxy-4-(1'-D-ri- tional subunits could produce apparently nor- bitylamino)pyrimidine in a riboflavin auxotroph. J. ,8 Biol. Chem. 246:4647-4652. mal precipitates in immunodiffusion experi- 6. Bacher, A., and F. Lingens. 1971. Biosynthesis of ribo- ments. (ii) Studies with S. cerevisiae have shown flavin. Formation of 6-hydroxy-2,4,5-triaminopyrimi- that the products of two unlinked genes, rib3 dine in rib7 mutants of Saccharomyces cerevisiae. J. and rib4, are necessary for the enzymatic Biol. Chem. 246:7018-7022. 7. Bacher, A., and B. Mailander. 1973. Biosynthesis of formation of 6,7-dixnethyl-8-(D-ribityl)luma- riboflavin. The structure of the purine precursor. J. zine from 5-amino-2,6-dihydroxy-4-(D-ribityl- Biol. Chem. 248:6227-6231. amino)pyrimidine. With both types of mutants, 8. Bacher, A., B. Malinder, R. Baur, U. Eggers, H. the requirement for riboflavin could be satisfied Harders, and H. Schnepple. 1975. Studies on the biosynthesis of riboflavin, p. 285-290. In W. Pfleiderer by the addition of diacetyl to the growth me- (ed.), Chemistry and biology of pteridines. Walter de dium. In analogy, the li-positive and the -neg- Gruyter, Berlin. ative diacetyl requirers of B. subtilis may lack 9. Baugh, C. ML, and C. L Krumdieck. 1969. Biosynthesis two different proteins which are both required of riboflavine in Corynebacterium species: the purine precursor. J. Bacteriol. 98:1114-1119. for the formation of the lumazine from the py- 10. Bresler, S. E., E. A. Glazunov, and D. A. Perumov. rimidine. (iii) The absence of 11 subunits and the 1972. Study ofthe riboflavin operon in Bacillus subtili. diacetyl requirement of the li-negative mutants IV. Regulation of biosynthesis of riboflavin synthase. are unrelated phenomena due to two different Study of riboflavin transport through the cell mem- A definite decision is not brane. Genetika 8(2):109-118. mutations. possible. 11. Bresler, S. E., D. A. Perumov, T. P. Chernik, and E. Experiments to demonstrate directly the en- A. Glazunov. 1976. Riboflavin operon in Bacillus sub- zymatic activity of the /B subunits have been tilis. X. Genetic and biochemical study of mutants unsuccessful so far. The heavy enzyme does not accumulating 6-methyl-7-(1',2'-dioxyethyl)-8-ribityllu- accelerate the spontaneous reaction between 5- mazine. Genetika 12(4):83-91. 12. Bryn, K., and F. C. Strmer. 1976. Decreased riboflavin amino-2,6-dihydroxy-4-(D-ribitylamino)pyrimi- formation in mutants ofAerobacter (Enterobacter) aer- dine and diacetyl to a dgnificant extent. How- ogenes deficient in the butanediol pathway. Biochim. ever, in view of the recent isotope incorporation Biophys. Acta 428:257-259. and the related com- 13. Demain, A. L 1972. Riboflavin oversynthesis. Annu. Rev. studies (1, 2), diacetyl Microbiol. 26:369-388. pounds are not likely four-carbon precursors 14. Foor, F., and G. M. Brown. 1975. Purification and prop- of 6,7-dimethyl-8-(D-ribityl)lumazine. Enzyme erties of guanosine triphosphate cyclohydrolase II from studies concerned with the role ofthe lB subunits Escherichia coli. J. Biol. Chem. 250:3545-351. are severely hampered by the lack of definite 15. Goodwin, T. W., and A. A. Horton. 1961. Biosynthesis of riboflavin in cell-free systems. Nature (London) information on the structure of the four-carbon 191:772-774. precursor. 16. Goodwin, T. W., and D. H. Treble. 1958. The incorpo- The data reported in this paper show conclu- ration of (2-'4C)acetylmethylcarbinol (acetoin) into ring sively that the l8 subunit is an obligatory com- A of riboflavin by Eremothecium ashbyii; a new route for the biosynthesis of an aromatic ring. Biochem. J. ponent of the enzyme machinery for the biosyn- 70:14P-15P. thesis of riboflavin. The definite assignment to 17. Heremans, F. J. 1971. Antigen titration by simple radial a specific biosynthetic step has to await further immunodiffusion in plates, p. 213-224. In C. A. Williams evidence. and M. W. Chase (ed.), Methods in immunology and experimental immunochemistry. Academic Press, New York. 18. Katagiri, H., I. Takeda, and K. Imai. 1959. Synthesis ofriboflavin by microorganisms. VII. The enzymic ribo- ACKNOWLEDGMENT1 flavin synthesis from 4-(N-ribitylamino)-5-aminouracil. This work was supported by the Deutsche Forschungsge- J. Vitaminol. 6:287-297. meinschaft, grant Ba 574. 19. Lingens, F., 0. Oltmanns, and A. Bacher. 1967. Ober The skillful technical assitance of Edeltraut Gerbershagen Zwischenprodukte der Riboflavinbiosynthese bei Sac- is gratefully acknowledged. charomyces cerevisiae. Z. Naturforsch. 22b:755-758. 482 BACHER AND MAILANDER J. BACTERIOL.

20. Mailander, B., and A. Bacher. 1976. Biosynthesis of sive biochemistry, vol. 1. Elsevier, Amsterdam. riboflavin. Structure of the purine precursor and origin 26. Plaut, G. W. E., and R. L Beach. 1976. Substrate of the ribityl side chain. J. Biol. Chem. 251:3623-3628. specificity and stereospecific mode of action of ribo- 21. Oeschger, N. S., and P. E. Hartman. 1970. ICR-induced flavin synthase, p. 737-746. In T. P. Singer (ed.), Flavins frameshift mutations in the histidine operon of Salmo- and flavoproteins. Elsevier, Amsterdam. nella. J. Bacteriol. 101:490-504. 27. Plaut, G. W. E., R. L Beach, and T. Aogaichi. 1970. 22. Oltmanns, O., and A. Bacher. 1972. Biosynthesis of Studies on the mechanism of elimination of protons riboflavine in Saccharomyces cerevisiae: the role of from the methyl groups of 6,7-dimethyl-8-ribitylluma- genes rib, and rib7. J. Bacteriol. 110:818-822. zine by riboflavin synthetase. Biochemistry 9:771-785. 23. Oltmanns, O., A. Bacher, F. Lingens, and F. K. Zim- 28. Plaut, G. W. E., and R. A. Harvey. 1971. The enzymatic mermann. 1969. Biochemical and genetic classification synthesis of riboflavin. Methods Enzymol. 18:515-538. of riboflavine deficient mutants of Saccharomyces cer- 29. Plaut, G. W. E., C. M. Smith, and W. L Alworth. 1974. evisiae. Mol. Gen. Genet. 105:306-313. Biosynthesis ofwater-soluble vitamins. Annu. Rev. Bio- 24. Ouchterlony, 0. 1962. Diffusion-in-gel methods for im- chem. 43:899-922. munological analysis. Prog. Allergy 6:30-154. 30. Spizizen, J. 1958. Transformation of biochemically defi- 25. Plaut, G. W. E. 1971. The biosynthesis of riboflavin, p. cient strains of Bacillus subtilis by deoxyribonucleate. 11-45. In M. Florkin and E. H. Stotz (ed.), Comprehen- Proc. Natl. Acad. Sci. U.S.A. 44:1072-1078.