Vol. 131, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Aug. 1977, p. 379-381 Copyright C 1977 American Society for Microbiology
Porphyrin and Corrinoid Mutants of Bacillus subtilis A. MICZAK Institute of Microbiology, University Medical School, 6720 Szeged, D6m ter 10, Hungary Received for publication 14 March 1977
Porphyrin auxotrophs of Bacillus subtilis can be divided into two groups. Strains belonging to the first group (hemA, hemB, or hemC) are not able to synthesize or metabolize porphobilinogen. These strains require cysteine, cystine, and methionine, respectively. Traces of aminolevulinic acid, in a hemincontaining medium, can replace the cysteine requirement in a mutant lacking aminolevulinic acid synthetase. In bacteria belonging to the second group (hemE, hemF, or hemG), porphyrin biosynthesis is blocked at later steps, and the amino acids mentioned above are not required. It is of interest that both the activity of ribonucleotide reductase and the amount of vitamin B12 were significantly lower in the first group. The addition of vitamin B,2 to the medium did not promote the growth of strains examined. We assume that porphobilinogen deaminase is essential for the synthesis of corrinoids.
Porphyrin-deficient mutants have been isolated from different bacteria (1, 11, 12) by aminoglycoside selection. There are six types of Bacillus subtilis heme-deficient mutants and previous papers from this laboratory dealt with their enzyme defects (3) and the chromosomal location of the genes involved (2, 8, 10). The corrinoids are side products of porphyrin biosynthesis. Several hypotheses have been. proposed dealing with the question of whether the con-in cycle arises from uroporphyrinogen III or porphobilinogen, its precursor. There is some evidence suggesting that porphobilinogen is the precursor (4), while other evidence (7) indicates that uroporphyrinogen III, or a tetrapyrrole (6), is the precursor common to uroporphyrinogens I and III and the corrin ring structure. The present paper describes experiments carried out to determine whether there are any differences in the synthesis of vitamin B12 among the six groups of porphyrin auxotrophs of B. subtilis.
Assay of vitamin B12. The vitamin B12 determination was carried out by a microbiological method, using a vitamin B12-dependent mutant strain of Escherichia coli. The strain was kindly provided by E. Cserey (Richter Pharmaceutical Works, Budapest, Hungary).
RESULTS Among the strains listed in Table 1, Sz3, Sz15, and Sz16 grow on HCA medium; the others (Sz26, Sz27, and Sz28) do not require cysteine for growth on HA medium. Cysteine could be substituted with cystine or methionine. Syntropism tests. Linear streaks of inoculum were made with strains Sz3, Sz15, and Sz16 on HA agar plates. Strain Szl, Sz26, Sz27, or Sz28 was streak-inoculated near these bacteria. After 24 h of incubation, growth was observed only in the case of strain Sz3. Strains Sz15 and Sz16 could not be cross-fed by strain Szl, Sz26, Sz27, or Sz28. Growth of strain Sz3. gGM medium supplemented with tryptophan and aminolevulinic acid (ALA) (2 ,ug/ml) is an ideal medium for MATERIALS AND METHODS mutant Sz3. Reducing the level of ALA in this Bacterial strains. The porphyrin mutants of B. medium to 0.0025 jig/ml did not provide sufficient ALA for growth of strain Sz3. However, subtilis 168 used in this work are listed in Table 1. Growth media. A chemically defined medium HA medium supplemented with 0.0025 jig of (gGM) (1) was supplemented with tryptophan (50 ALA per ml allowed growth of strain Sz3 (Fig. 1 ,ug/ml). In the case of porphyrin auxotroph, HCA and 2). (gGM supplemented with tryptophan, 2.5 zg of Ribonucleotide reductase activity. Table 2 hemin per ml, 50 Ag of cysteine per ml, and 1 to 2 shows the ribonucleotide reductase activities of mg of bovine serum albumin per ml) or HA (HCA group (Sz3, Sz15, and minus cysteine) medium was used (9). Agar media the mutants. In the first were observed activities limited Sz16), very contained 1.5% agar (Difco). Ribonucleotide reductase. The activity of ribonu- compared with those of the second group (Sz26, cleotide reductase was measured by the method of Sz27, and Sz28). Amount of vitamin B12 in the mutants. TaCowles et al. (5). ,79
380
J. BACTERIOL.
MICZAK
TABLE 1. Strains used Strain
Previous nationdesig-
defect in porphyrin Enzymebiosynthesis
Genotypea
Supplied by
P. Schaeffer T. Anderson I. Berek I. Berek
None ALA synthetase ALA dehydrase Porphobilinogen deaminase Urogen decarboxtrpC2 hemE64 V/64 Sz26 ylase Coprogen oxidase trpC2 hemF180 VI/180 Sz27 Ferrochelatase trpC2 hemG321 III/321 Sz28 a hem, Genes responsible for heme synthesis in bacteria (13).
trpC2 trpC2 hemAl trpC2 hemBI trpC2 hemC33
RI2 R15 I/1 II/33
Szl Sz3 Sz15 Sz16
I. Berek
I. Berek I. Berek
1.1
a9
c C c 0
*_1
0.71-
a a IC
N
%. 40
;:
CL
a
0.3p[
1
2 Time
3
4
5
(hours)
FIG. 1. Growth of strain Sz3 in HCA (0) or HA (A) medium or in gGM medium containing 2 pg of ALA per ml (-).
ble 3 summarizes the results of experiments in which the amounts of vitamin B.2 synthesized by porphyrin mutants were determined. Group II strains, Sz26, Sz27, and Sz28, contained a much higher level of vitamin B12 than did strains in group I. The addition of vitamin B12 to the medium did not support the growth ofthe mutants.
DISCUSSION From the results of syntropism tests, we suppose that strains Szl, Sz26, Sz27, and Sz28 secrete a certain amount of ALA into the medium. The presence of ALA supported the growth of strain Sz3. Strains Sz15 and Sz16 were unable to utilize the ALA because their
1
2
3
4
5
(hours) FIG. 2. Growth of strain Sz3 in HA medium with 0.0025 pg ofALA per ml (O)or in gGM medium with tryptophan and 0.0025 pg of ALA per ml (0). Tim.
enzyme defects are located after the point of ALA synthesis in the biosynthetic pathway (3). In the presence of a minimal concentration of ALA, strain Sz3 could not grow on gGM medium. In HA medium supplemented with this amount of ALA, growth was normal. We assume that in this case a minimal concentration of ALA was sufficient for vitamin B12 synthesis and that the hemin found in the HA medium served as a porphyrin source. The other results show that in the first group of strains vitamin B12 is not synthesiied, or to only a very limited extent. Addition of vitamin B12 to the medium did not promote growth. We think that this is a transport problem. The branch in the porphyrin and corrinoid
PORPHYRIN AND CORRINOID MUTANTS
VOL. 131 ? 1977
TABLE 3. Amount of vitamin B12 in the mutants" Amt of B,2b Strain 1 Sz3 1 Sz16 23 Sz26 30 Sz27 28 Sz28 a The strains were grown in HCA medium. Cultures in the log phase were centrifuged; the bacteria were sonically treated, and the solutions were concentrated. The quantity of vitamin B,2 in this concentrate was determined with the vitamin B,2-dependent mutant of E. coli. b Nanograms per gram (wet weight) of bacteria.
TABLE 2. Ribonucleotide reductase activity Strain Sz3
Enzyme actiVitya 3 2 3 42 34 32
Sz15 Sz16 Sz26 Sz27 Sz28 a Nanomoles of deoxyguanosine triphosphate produced per hour per milligram of protein.
,I
, /UROGEN Ill cosynthetose
hem C
'%
PUG
rnppnr.oJ I
UROGEN I
deominase
381
' UROGEN
-
'decarboxylose g COPROGENIUoLOGEN Ill , I I
hem E
Nb CORRINOIOS
FIG. 3. Branch in the pathway ofporphyrin and corrinoid synthesis. A mutant bearing the hemC locus can not synthesize corrinoids.
synthetic pathway should occur after the synthesis of porphobilinogen. Porphobilinogen deaminase is necessary for the synthesis of both. In Fig. 3 we have summarized our hypothesis, deduced from the results presented above. ACKNOWLEDGMENTS I thank I. Beladi for her very useful suggestions and current interest, and I am also greatly indebted to I. Roszt6czy for his help in the preparation of this manuscript.
LITERATURE CITED 1. Anderson, T., and G. Ivdnovics. 1967. Isolation and some characteristics of haemin dependent mutants of
Bacillus subtilis. J. Gen. Microbiol. 49:31-40.
2. Berek, I., A. Miczik, and G. Ivdnovics. 1974. Mapping the 8-aminolaevulinic acid dehydrase and porphobili-
nogen deaminase loci in Bacillus subtilis. Mol. Gen. Genet. 132:233-239. 3. Berek, I., A. MiczAk, I. Kiss, G. Ivtinovics, and I. Durk6. 1975. Genetic and biochemical analysis of haemin dependent mutants of Bacillus subtilis. Acta Microbiol. Acad. Sci. Hung. 22:157-167. 4. Brown, C. E., J. J. Katz, and D. Shemin. 1972. The biosynthesis of vitamin B,2: a study by C13 magnetic resonance spectroscopy. Proc. Natl. Acad. Sci. U.S.A. 69:2585-2588. 5. Cowles, J. R., H. J. Evans, and S. A. Russell. 1969. B,2 coenzyme-dependent ribonucleotide reductase in Rhizobium species and the effects of cobalt deficiency on
the activity of the enzyme. J. Bacteriol. 97:1460-1465. 6. Dalton, J., and R. C. Dougherty. 1969. Formation of the macrocyclic ring of the tetrapyrrole biosynthesis. Nature (London) 223:1151-1153. 7. Frydman, B., R. B. Frydman, A. Valasinos, E. S. Levy, and G. Feinstein. 1976. Biosynthesis of uroporphyrinogens from porphobilinogen: mechanism and the nature of the process. Philos. Trans. R. Soc. London Ser. B 273:137-160. 8. Kiss, I., I. Berek, and G. Ivinovics. 1971. Mapping the 8-aminolaevulinic acid synthetase locus in Bacillus subtilis. J. Gen. Microbiol. 66:153-159. 9. Mariai, E., I. Kiss, and G. Ivinovics. 1970. Auxotrophic mutation associated with low streptomycin resistance and slow growth in Bacillus subtilis. Acta Microbiol. Acad. Sci. Hung. 17:133-145. 10. Micz&k, A., I. Berek, and G. Ivinovics. 1976. Mapping the uroporphyrinogen decarboxylase, coproporphyrinogen oxidase and ferrochelatase loci in Bacillus subtilis. Mol. Gen. Genet. 146:85-87. 11. Sisirman, A., P. Chartraud, R. Proschek, M. Desrochers, D. Tardif, and C. Lapointe. 1975. Uroporphyrinaccumulating mutant of Escherichia coli K-12. J. Bacteriol. 124:1205-1212. 12. Tien, W., and D. C. White. 1968. Linear sequential arrangement of genes for the biosynthetic pathway of protoheme in Staphylococcus aureus. Proc. Natl. Acad. Sci. U.S.A. 61:1392-1398. 13. Young, F. E., and G. A. Wilson. 1976. Revision of the linkage map of Bacillus subtilis, p. 686-703. In Handbook of biochemistry and molecular biology. C.R.C. Press, Cleveland, Ohio.
JOURNAL OF BACTERIOLOGY, Aug. 1977, p. 379-381 Copyright C 1977 American Society for Microbiology
Porphyrin and Corrinoid Mutants of Bacillus subtilis A. MICZAK Institute of Microbiology, University Medical School, 6720 Szeged, D6m ter 10, Hungary Received for publication 14 March 1977
Porphyrin auxotrophs of Bacillus subtilis can be divided into two groups. Strains belonging to the first group (hemA, hemB, or hemC) are not able to synthesize or metabolize porphobilinogen. These strains require cysteine, cystine, and methionine, respectively. Traces of aminolevulinic acid, in a hemincontaining medium, can replace the cysteine requirement in a mutant lacking aminolevulinic acid synthetase. In bacteria belonging to the second group (hemE, hemF, or hemG), porphyrin biosynthesis is blocked at later steps, and the amino acids mentioned above are not required. It is of interest that both the activity of ribonucleotide reductase and the amount of vitamin B12 were significantly lower in the first group. The addition of vitamin B,2 to the medium did not promote the growth of strains examined. We assume that porphobilinogen deaminase is essential for the synthesis of corrinoids.
Porphyrin-deficient mutants have been isolated from different bacteria (1, 11, 12) by aminoglycoside selection. There are six types of Bacillus subtilis heme-deficient mutants and previous papers from this laboratory dealt with their enzyme defects (3) and the chromosomal location of the genes involved (2, 8, 10). The corrinoids are side products of porphyrin biosynthesis. Several hypotheses have been. proposed dealing with the question of whether the con-in cycle arises from uroporphyrinogen III or porphobilinogen, its precursor. There is some evidence suggesting that porphobilinogen is the precursor (4), while other evidence (7) indicates that uroporphyrinogen III, or a tetrapyrrole (6), is the precursor common to uroporphyrinogens I and III and the corrin ring structure. The present paper describes experiments carried out to determine whether there are any differences in the synthesis of vitamin B12 among the six groups of porphyrin auxotrophs of B. subtilis.
Assay of vitamin B12. The vitamin B12 determination was carried out by a microbiological method, using a vitamin B12-dependent mutant strain of Escherichia coli. The strain was kindly provided by E. Cserey (Richter Pharmaceutical Works, Budapest, Hungary).
RESULTS Among the strains listed in Table 1, Sz3, Sz15, and Sz16 grow on HCA medium; the others (Sz26, Sz27, and Sz28) do not require cysteine for growth on HA medium. Cysteine could be substituted with cystine or methionine. Syntropism tests. Linear streaks of inoculum were made with strains Sz3, Sz15, and Sz16 on HA agar plates. Strain Szl, Sz26, Sz27, or Sz28 was streak-inoculated near these bacteria. After 24 h of incubation, growth was observed only in the case of strain Sz3. Strains Sz15 and Sz16 could not be cross-fed by strain Szl, Sz26, Sz27, or Sz28. Growth of strain Sz3. gGM medium supplemented with tryptophan and aminolevulinic acid (ALA) (2 ,ug/ml) is an ideal medium for MATERIALS AND METHODS mutant Sz3. Reducing the level of ALA in this Bacterial strains. The porphyrin mutants of B. medium to 0.0025 jig/ml did not provide sufficient ALA for growth of strain Sz3. However, subtilis 168 used in this work are listed in Table 1. Growth media. A chemically defined medium HA medium supplemented with 0.0025 jig of (gGM) (1) was supplemented with tryptophan (50 ALA per ml allowed growth of strain Sz3 (Fig. 1 ,ug/ml). In the case of porphyrin auxotroph, HCA and 2). (gGM supplemented with tryptophan, 2.5 zg of Ribonucleotide reductase activity. Table 2 hemin per ml, 50 Ag of cysteine per ml, and 1 to 2 shows the ribonucleotide reductase activities of mg of bovine serum albumin per ml) or HA (HCA group (Sz3, Sz15, and minus cysteine) medium was used (9). Agar media the mutants. In the first were observed activities limited Sz16), very contained 1.5% agar (Difco). Ribonucleotide reductase. The activity of ribonu- compared with those of the second group (Sz26, cleotide reductase was measured by the method of Sz27, and Sz28). Amount of vitamin B12 in the mutants. TaCowles et al. (5). ,79
380
J. BACTERIOL.
MICZAK
TABLE 1. Strains used Strain
Previous nationdesig-
defect in porphyrin Enzymebiosynthesis
Genotypea
Supplied by
P. Schaeffer T. Anderson I. Berek I. Berek
None ALA synthetase ALA dehydrase Porphobilinogen deaminase Urogen decarboxtrpC2 hemE64 V/64 Sz26 ylase Coprogen oxidase trpC2 hemF180 VI/180 Sz27 Ferrochelatase trpC2 hemG321 III/321 Sz28 a hem, Genes responsible for heme synthesis in bacteria (13).
trpC2 trpC2 hemAl trpC2 hemBI trpC2 hemC33
RI2 R15 I/1 II/33
Szl Sz3 Sz15 Sz16
I. Berek
I. Berek I. Berek
1.1
a9
c C c 0
*_1
0.71-
a a IC
N
%. 40
;:
CL
a
0.3p[
1
2 Time
3
4
5
(hours)
FIG. 1. Growth of strain Sz3 in HCA (0) or HA (A) medium or in gGM medium containing 2 pg of ALA per ml (-).
ble 3 summarizes the results of experiments in which the amounts of vitamin B.2 synthesized by porphyrin mutants were determined. Group II strains, Sz26, Sz27, and Sz28, contained a much higher level of vitamin B12 than did strains in group I. The addition of vitamin B12 to the medium did not support the growth ofthe mutants.
DISCUSSION From the results of syntropism tests, we suppose that strains Szl, Sz26, Sz27, and Sz28 secrete a certain amount of ALA into the medium. The presence of ALA supported the growth of strain Sz3. Strains Sz15 and Sz16 were unable to utilize the ALA because their
1
2
3
4
5
(hours) FIG. 2. Growth of strain Sz3 in HA medium with 0.0025 pg ofALA per ml (O)or in gGM medium with tryptophan and 0.0025 pg of ALA per ml (0). Tim.
enzyme defects are located after the point of ALA synthesis in the biosynthetic pathway (3). In the presence of a minimal concentration of ALA, strain Sz3 could not grow on gGM medium. In HA medium supplemented with this amount of ALA, growth was normal. We assume that in this case a minimal concentration of ALA was sufficient for vitamin B12 synthesis and that the hemin found in the HA medium served as a porphyrin source. The other results show that in the first group of strains vitamin B12 is not synthesiied, or to only a very limited extent. Addition of vitamin B12 to the medium did not promote growth. We think that this is a transport problem. The branch in the porphyrin and corrinoid
PORPHYRIN AND CORRINOID MUTANTS
VOL. 131 ? 1977
TABLE 3. Amount of vitamin B12 in the mutants" Amt of B,2b Strain 1 Sz3 1 Sz16 23 Sz26 30 Sz27 28 Sz28 a The strains were grown in HCA medium. Cultures in the log phase were centrifuged; the bacteria were sonically treated, and the solutions were concentrated. The quantity of vitamin B,2 in this concentrate was determined with the vitamin B,2-dependent mutant of E. coli. b Nanograms per gram (wet weight) of bacteria.
TABLE 2. Ribonucleotide reductase activity Strain Sz3
Enzyme actiVitya 3 2 3 42 34 32
Sz15 Sz16 Sz26 Sz27 Sz28 a Nanomoles of deoxyguanosine triphosphate produced per hour per milligram of protein.
,I
, /UROGEN Ill cosynthetose
hem C
'%
PUG
rnppnr.oJ I
UROGEN I
deominase
381
' UROGEN
-
'decarboxylose g COPROGENIUoLOGEN Ill , I I
hem E
Nb CORRINOIOS
FIG. 3. Branch in the pathway ofporphyrin and corrinoid synthesis. A mutant bearing the hemC locus can not synthesize corrinoids.
synthetic pathway should occur after the synthesis of porphobilinogen. Porphobilinogen deaminase is necessary for the synthesis of both. In Fig. 3 we have summarized our hypothesis, deduced from the results presented above. ACKNOWLEDGMENTS I thank I. Beladi for her very useful suggestions and current interest, and I am also greatly indebted to I. Roszt6czy for his help in the preparation of this manuscript.
LITERATURE CITED 1. Anderson, T., and G. Ivdnovics. 1967. Isolation and some characteristics of haemin dependent mutants of
Bacillus subtilis. J. Gen. Microbiol. 49:31-40.
2. Berek, I., A. Miczik, and G. Ivdnovics. 1974. Mapping the 8-aminolaevulinic acid dehydrase and porphobili-
nogen deaminase loci in Bacillus subtilis. Mol. Gen. Genet. 132:233-239. 3. Berek, I., A. MiczAk, I. Kiss, G. Ivtinovics, and I. Durk6. 1975. Genetic and biochemical analysis of haemin dependent mutants of Bacillus subtilis. Acta Microbiol. Acad. Sci. Hung. 22:157-167. 4. Brown, C. E., J. J. Katz, and D. Shemin. 1972. The biosynthesis of vitamin B,2: a study by C13 magnetic resonance spectroscopy. Proc. Natl. Acad. Sci. U.S.A. 69:2585-2588. 5. Cowles, J. R., H. J. Evans, and S. A. Russell. 1969. B,2 coenzyme-dependent ribonucleotide reductase in Rhizobium species and the effects of cobalt deficiency on
the activity of the enzyme. J. Bacteriol. 97:1460-1465. 6. Dalton, J., and R. C. Dougherty. 1969. Formation of the macrocyclic ring of the tetrapyrrole biosynthesis. Nature (London) 223:1151-1153. 7. Frydman, B., R. B. Frydman, A. Valasinos, E. S. Levy, and G. Feinstein. 1976. Biosynthesis of uroporphyrinogens from porphobilinogen: mechanism and the nature of the process. Philos. Trans. R. Soc. London Ser. B 273:137-160. 8. Kiss, I., I. Berek, and G. Ivinovics. 1971. Mapping the 8-aminolaevulinic acid synthetase locus in Bacillus subtilis. J. Gen. Microbiol. 66:153-159. 9. Mariai, E., I. Kiss, and G. Ivinovics. 1970. Auxotrophic mutation associated with low streptomycin resistance and slow growth in Bacillus subtilis. Acta Microbiol. Acad. Sci. Hung. 17:133-145. 10. Micz&k, A., I. Berek, and G. Ivinovics. 1976. Mapping the uroporphyrinogen decarboxylase, coproporphyrinogen oxidase and ferrochelatase loci in Bacillus subtilis. Mol. Gen. Genet. 146:85-87. 11. Sisirman, A., P. Chartraud, R. Proschek, M. Desrochers, D. Tardif, and C. Lapointe. 1975. Uroporphyrinaccumulating mutant of Escherichia coli K-12. J. Bacteriol. 124:1205-1212. 12. Tien, W., and D. C. White. 1968. Linear sequential arrangement of genes for the biosynthetic pathway of protoheme in Staphylococcus aureus. Proc. Natl. Acad. Sci. U.S.A. 61:1392-1398. 13. Young, F. E., and G. A. Wilson. 1976. Revision of the linkage map of Bacillus subtilis, p. 686-703. In Handbook of biochemistry and molecular biology. C.R.C. Press, Cleveland, Ohio.
