when Roth and co-workers, showing de novo synthes cobalamin under ...... Hogenkamp for the gift of analogs and John Roth for strains and information before ...
JOURNAL OF BACTERIOLOGY, Apr. 1986, p. 51-58
Vol. 166, No. 1
0021-9193/86/040051-08$02.00/0 Copyright © 1986, American Society for Microbiology
Isolation and Genetic Characterizations of Bacillus megaterium Cobalamin Biosynthesis-Deficient Mutants JULIE B. WOLFt AND ROBERT N. BREY* Genex Corporation, Gaithersburg, Maryland 20877 Received 1 November 1985/Accepted 23 January 1986 Ethanolamine is deaminated by the action of ethanolamine ammonia-lyase (EC 4.3.1.7), an adenosylcobalamin-dependent enzyme. Consequently, to grow on ethanolamine as a sole nitrogen source, Bacillus megaterium requires vitamin B12. Identification of B. megaterium mutants deficient for growth on ethanolamine as the sole nitrogen source yielded a total of 34 vitamin Bi2 auxotrophs. The vitamin B12 auxotrophs were divided into two major phenotypic groups: Cob mutants, which could use cobinamide or vitamin B12 to grow on ethanolamine, and Cbl mutants, which could be supplemented only by vitamin B12. The Cob mutants were resolved into six classes and the Cbl mutants were resolved into three, based on the spectrum of cobalt-labeled corrinoid compounds which they accumulated. Although some radiolabeled cobalamin was detected in the wild type, little or none was evident in the auxotrophs. The results indicate that Cob mutants contain lesions in biosynthetic steps before the synthesis of combinamide, while Cbl mutants are defective in the conversion of cobinamide to cobalamin. Analysis of phage-mediated transduction experiments revealed tight genetic linkage within the Cob class and within the Cbl class. Similar transduction analysis indicated the Cob and Cbl classes are weakly linked. In addition, cross-feeding experiments in which extracts prepared from mutants were examined for their effect on growth of various other mutants allowed a partial ordering of mutations within the cobalamin biosynthetic pathway. The basic corrin ring structure (cobyrinic acid; Fig. 1) which. gives rise to vitamin B12 is biosynthetically derived from utoporphyrinogen III (also a precursor to heme and siroheme) by a series of reductive methylations, a decarboxylation at C-12, ring scission between C-19 and C-20, and insertion of cobalt (Fig. 1). Although it is. known that four melthylations of the uroporphyrinogen III ring probably occur first in the biosynthesis of cobalamin, the order of further steps in cobyrinic acid formation is not known. Beyond cobyrinic acid there are six aniidations of carboxyl side groups and an addition of aminopropanol (biosynthetically derived from threonine) to yield cobinamide. Cobinamide is phosphorylated by ATP to yield cobinamide P04, which reacts with GTP to yield GDPcobinamide. GMP is displaced by the 5' nucleotide of dimethylbenzimidazole (DMBI; biosynthetically derived from riboflavin) to produce cobalamin P04, which is dephosphorylated to yield cobalamin (vitamin B12). Adenylation of the corrin macrocycle, which is necessary for the formation of the cofactor form of cobalamin (adenosylcobalamin; also known as coenzyme B12), may occur very early in the biosynthetic sequence but can occur after the formation of cobalamin. For more detailed description of the cobalamin biosynthetic pathway, the reader is referred to recent reviews (2, 11, 13). The correlation of a particular biosynthetic step with a particular enzyme has not been accomplished. With the possible exception of thb partiai purification of an enzyme(s) involved with the methylation of uroporphyrinogen III, none of the biosynthetic enzymes has been well characterized (17). Thus, knowledge of enzymology is insufficient even to conclude how many enzymes are *
involved with cobalmin biosynthesis. Virtually nothing is known about the genetic control of this pathway. To begin a genetic study of a complex biosynthetic pathway, it is first desirable to establish conditions under which an organism requires the pathway end product for growth. A logical way to approach a study of cobalamin biosynthesis is to take advantage of the microbial enzymes that are known to require either methylcobalamin or adenosylcobalamin to act, forcing an organism to utilize a substrate whose metabolism is strictly dependent on vitamin B12. There are a number of such enzymes known (1). Ethanolamine ammonia-lyase (EC 4.3.1.7) catalyzes the deamination of ethanolamine to acetaldehyde and ammonia (1, 16). Bradbeer first discovered this reaction in a choline-fermenting Clostridium sp. and demonstrated that the enzyme required adenosylcobalamin (6, 7). Subsequently, this enzyme has been detected in a number of bacterial species, including Klebsiella aerogenes, Escherichia coli, and Salmonella typhimurium, in which exogenous vitamin B12 is required for growth on ethanolamine (3-5, 10, 19). Ethanolamine ammonia-lyase is also present in Bacillus megaterium, an obligate aerobe which is capable of vitamin B12 synthesis under aerobic growth conditions. B. megaterium can use ethanolamine as a sole source of nitrogen. Growth of B. megaterium on ethanolamine, but not on ammonia, is inhibited by adeninylalkyl analogs (12, 18) of adenosylcobalamin, suggesting that mutant bacteria unable to synthesize cobalamin would require, as do wild-type E. coli and S. typhimurium cells, exogenous cobalamin to utilize ethanolamine as a sole source of nitrogen (unpublished data). The establishment of growth conditions under which B. megaterium requires vitamin 1312 is analogous to the behavior of metE mutants of S. typhimurium and E. coli. Retaining the methylcobalamin-dependent tetrahydropteroylglutamate methyltransferase (EC 2.1.1.13), metE mutants can utilize exogenous vitamin B12 to bypass their requirement for
Corresponding author.
t Present address: Cell Biology and Metabolism Branch, National
Institute of Child Health and Human Developmerit, Bethesda, MD 20892. 51
52
WOLF AND BREY Succinyl CoA
+
Glycine
2
J. BACTERIOL. to analyze linkage of mutations with respect to each other, and to define a biosynthetic ordering of mutant phenotypes.
d Aminolevulinic Acid -
MATERIALS AND METHODS
Porphobilinogen
Uroporphyrinogen
-~
III
_ 2CH3
--
Heme
Sirohydrochlorin
Siroheme CO2 aH CO H
(Sulfite Reductase)
r
5CH3
Co
+
+
Cobyrin iC Acid
cob
_.-6NHd4 ,--aminopropanol
Cobinamide ATP
Riboflavin
Cobinamide P04 GTP
\
cbl
GDP-Cobinamide DMBI
Vitamin B12 t FIG. 1. Cobalamin biosynthetic pathway according to cu understanding (2, 10). The region bracketed by cob indicate-s the portion of the pathway affected in Cob mutants; region bracketiSed by cbl indicates the portion of the pathway affected in Cbl mutarnts. exogenous methionine. Because E. coli and S. typhimurrium metE mutants require exogenous vitamin B12 or methioinine, it had been thought that these organisms were incapab le of synthesizing vitamin B12. Hence, genetic studiers on cobalamin biosynthesis in general have lagged until recgently when Roth and co-workers, showing de novo synthes,is of cobalamin under anaerobic growth conditions, characteirized a series of cobalamin auxotrophs of S. typhimurium (15). The basis for their work stemmed from the observation that metE mutants of S. typhimurium were able to grow anae robically in the absence of vitamin B12 or methionine. We initiated a genetic study of the cobalamin biosynt hetic pathway in B. megaterium to attempt to correlate bioclhemical steps with specific genetic lesions. B. megateriutm is especially amenable to studies on cobalamin biosynti hesis because it synthesizes the vitamin aerobically and can Igrow with ethanolamine as the sole nitrogen source. In addiition, B. megaterium can be used for genetic studies since the-re is a known transducing bacteriophage for it and its protop)lasts can be transformed with several plasmids (9, 21). In this study we isolated 34 mutants deficient in cobalamin bic)synthesis by their failure to grow on ethanolamine as a sour*ce of nitrogen in the absence of vitamin B12, deriving auxotrc)phic recipients for cloning genes which may be rate limititng in cobalamin biosynthesis, a tactic for the construction of a strain for the industrial production of vitamin B12. We describe the experiments to differentiate mutant phenot3ypes,
Bacterial strains and bacteriophage. Bacterial strains used in this study are shown in Table 1. All B. megaterium strains were derived from the prototroph B. megaterium ATCC 10778. S. typhimurium LT2 strains used as vitamin B12 indicator strains were TT8723 (metE205 metP760 ara-9 cob-il::TnlO), which utilizes vitamin B12 or cobinamide to satisfy requirements for methionine, or TT7573 (metE205 metP760 ara-9 cob4::TnlO), which can only utilize vitamin B12 for growth in the absence of methionine (15). Bacteriophage MP13, a B. megaterium generalized transducing phage, was obtained from P. Vary (21). Media and culture conditions. Minimal media consisted of 14.0 g of K2HPO4 per liter, 6 g of KH2PO4 per liter, 1 g of sodium citrate, 1 mM MgCl2, and 0.5% D-glucose with either 10 mM NH4SO4 or 0.2% (vol/vol) ethanolamine as the nitrogen source. When ethanolamine was used as the nitrogen source, the medium was also supplemented with 10 mM Na2SO4. MGY medium contained 4.18 g of MOPS [3-(Nmorphilino)ethanesulfonic acid] per liter, 0.375 g of Ntris(hydroxymethyl)methyl glycine (Tricine), 10 mM NH4Cl, 0.27 mM K2HPO4, 0.1 mM MgCl2, 5.0 g of D-glucose per liter,
and
10
g
of
yeast
extract
(Difco) per liter, adjusted
to
pH 7.3 with KOH. All minimal media and MGY medium were supplemented with the following micronutrients (per liter): 27 mg of FeCl3 * 6H20, 7.2 mg of ZnSO4- 7H20, 5 mg of MnCl2 * 4H2O, 1.25 mg of CuSO4 * 5H20, and 0.3 mg of H3BO3. When cells were grown in minimal medium, 1.25 mg of CoCl2 per liter was also added. Concentrations of nutritional supplements, as required, were 15 nM cyanocobalamin (vitamin B12), 20 ,ug of dicyanocobinamide per liter, 5 mg of DMBI per liter, and 20 mg of cysteine per liter. All incubations were performed at 37°C. Routine culturing of B. megaterium was done in L broth. Mutagenesis and enrichment for vitamin B12 auxotrophs. Early-exponential-phase cultures of B. megaterium ATCC 10778 (2 x 107 cells per ml) in L broth were treated with N-methyl-N'-nitro-nitrosoguanidine at a final concentration of 100 ,ug/ml for 20 min at 37°C, washed, and grown in LB for 1 h to allow expression of mutations. Cultures were washed and suspended in minimal ethanolamine medium supplemented with 15 nM vitamin B12 and 20 mg of cysteine per liter to allow growth of mutants also blocked in the bipsynthesis of siroheme. After overnight growth, the cells were TABLE 1. Bacterial strains used in this study Strain aGntp hntp GenOtYPe Phenotype designationa
ATCC 10778 GX5101 GX5139 GX5141 GX5151 GX5137 GX5134 GX5127 GX5157 GX5160 GX5143
Wild type cob-i cob-39
cob4i cob-51 cob-37 cys-i cob-34 cob-27 cbl-57 cbl-60 cbl43
CobI Cbl1 Cob I Cob I Cob II Cob III Cob IV CysCob V Cob VI Cbl X Cbl XI Cbl XII
a Strain ATCC 10778 was obtained from the American Type Culture Collection, Rockville, Md. All other strains originated from this study.
VOL. 166, 1986
washed and suspended in minimal ethanolamine without vitamin B12 supplementation. After the cell population had doubled, 1 g of carbenicillin per liter and 2 g of cycloserine per liter were added, and cultures were incubated until cell lysis had occurred. The survivors of the antibiotic treatment were plated on minimal ethanolamine medium supplemented with cysteine and vitamin B12. Bacterial colonies were replica plated on ethanolamine minimal medium without supplementation, and auxotrophs were identified as those isolates which required vitamin B12 for growth. 57CoC12 labeling of vitamin B12 and corrinoids. The wildtype strain and each of the vitamin B12 auxotrophs were grown for 16 h at 37°C in 5 ml of MGY medium containing 0.2 ,uCi of 57CoC12 per ml until cultures had attained approximately 108 cells per ml (late-logarithmic-growth phase). Corrin compounds labeled with 57Co were extracted by boiling these cultures for 15 minutes in the presence of 0.2 M acetic acid. Corrin compounds were concentrated further from boiled cultures by extraction into m-cresol-CCL4 (4:1) as described by Bray and Shemin (8). The corrinoid compounds were back extracted into 0.2 N NH40H in the presence of enough 2-butanol to allow phase separation. The total water-soluble corrinoid fraction was subjected to lyophilization and was finally redissolved in 50 ,ul of 0.01 N NH40H containing traces of KCN to convert corrins into their dicyano forms. Samples (5 RI) of the extracts were analyzed on high-performance silica gel thin-layer chromatography plates by developing them in a solvent consisting of 2-butanol-NH40H (2:1). The 57Co-labeled species were detected by autoradiography. Bacteriophage transductions. Lysates of several vitamin B12 auxotrophs were prepared b using B. megaterium bacteriophage MP13. Reciprocal tv )-factor transductional crosses were carried out as described by Vary et al. (21). Recipient bacteria were grown in 10 ml of SNB broth (21) at 37°C to mid-log phase (approximately 5 x 107 cells per ml), washed twice, and suspended in 5 ml of minimal ethanolamine medium. Minimal ethanolamine plates were spread with 2 x 107 to 2 x 108 phage particles inactivated by irradiation for 30 s. After irradiation, plates were spread with 3 x 107 recipient bacteria and incubated for 48 h at 37°C. The results of two-factor transductional crosses were calculated as the number of prototrophic transductants per 2 x 107 PFU. Cotransduction studies were carried out by transducing Cbl mutants to Cbl+ with MP13 phage grown on mutants requiring B12 or cobinamide (Cob-) and selecting for growth on ethanolamine minimal medium supplemented with cobinamide. Among the Cbl+ transductants, those that were Cob- were identified as those colonies unable to grow on ethanolamine minimal medium without cobinamide. Bioassay for vitamin B12. To determine the amount of vitamin B12 produced by a particular strain, the appropriate B. megaterium strains were cultured in 50 ml of minimal ammonia medium until they had reached a density of approximately 7 x 107 cells per ml. The cells were harvested, washed twice in 100 mM potassium phosphate (pH 7.0), and resuspended in a final volume of 2 ml in the same buffer and boiled for 20 min to release vitamin B12 (and other corrinoid compounds). Portions (20 ,u) of the boiled extracts were placed on sterile antibiotic sensitivity disk blanks on minimal ammonia plates superimposed with soft agar lawns of approximately 5 x 106 cells of the S. typhimurium vitamin B12-requiring strains TT7573 or TT8723. After 16 h of incubation at 37°C, the growth zone surrounding the indicator strain was measured and compared to a vitamin B12 standard curve. The bioassay was sensitive in the range of 1
VITAMIN B12 IN B. MEGATERIUM
53
ng to 10 ,ug of vitamin B12. A standard curve of cobinamide was identical with the curve for vitamin B12 (data not shown). In cross-feeding experiments with vitamin B12 auxotrophs of B. megaterium, the same procedure was used to extract corrinoid compounds from the mutants. The biologic activity was determined on soft agar lawns of B. megaterium mutants containing approximately 5 x 105 cells on minimal ethanolamine medium. Materials, chemicals, and radiochemicals. High-performance silica gel thin-layer chromatography plates (5 by 10 cm) were obtained from EM Science, Gibbstown, N.J. Sterile antibiotic sensitivity disk blanks were obtained from BBL Microbiology Systems, Cockeysville, Md. Carrier-free 57CoC12 (4 mCi/mg) was purchased from New England Nuclear Corp., Boston, Mass. Cyanocobalamin, dicyanocobinamide, DMBI, ethanolamine, and other nutritional biochemicals were from Sigma Chemical Co., St. Louis, Mo. RESULTS Isolation and characterization of vitamin B12 auxotrophs. Because ethanolamine ammonia lyase requires adenosylcobalamin (coenzyme B12) as a cofactor, mutant B. megaterium cells that are incapable of utilizing ethanolamine as a sole source of nitrogen should be defective in production of ethanolamine ammonia lyase, the biosynthesis of vitamin B12, or the adenylation of vitamin B12. Mutagenized cultures of B. megaterium 10778 were enriched for vitamin B12 auxotrophs by counterselecting in an ethanolamine minimal medium in the presence of carbenicillin and cycloserine. Among the survivors, cobalamin auxotrophs were identified as those strains unable to use ethanolamine as a nitrogen source except when supplied with exogenous vitamin B12. Thirty-four such mutants were identified. All of the mutants isolated required vitamin B12 for growth on ethanolamine as the sole nitrogen or carbon source. All strains grew normally on minimal ammonia medium, except GX5137, which also required cysteine. To distinguish different classes of mutants, the auxotrophs were tested for their ability to use cobinamide, a vitamin B12 biosynthetic precursor, instead of vitamin B12 for growth on ethanolamine. Six of the mutants could not use cobinamide and were designated Cbl mutants. Twenty-eight of the auxotrophs were able to use cobinamide or vitamin B12 for growth on ethanolamine and were designated Cob mutants. None of the auxotrophs was capable of utilizing DMBI instead of vitamin B12, indicating that none had a biosynthetic block in DMBI synthesis. In addition, no mutants blocked in sirohydrochlorin synthesis were obtained. These mutants would evince a simultaneous requirement for reduced sulfur (cysteine) and vitamin B12 to grow on ethanolamine minimal medium, or a requirement for reduced sulfur alone to grow on minimal ammonia medium. All revertants of GX5137 to vitamin B12 independence still required cysteine, indicating an unrelated mutation in cysteine biosynthesis in that strain (data not shown). Cobalt incorporation into corrinoids in Cob and Cbl mutants. Phenotypic classification of the auxotrophs based on feeding of vitamin B12 precursors was limited to the use of cobinamide, the only commercially available cobalamin biosynthetic precursor. Hence, further subdivision of the Cob and Cbl mutant classes depended on developing criteria other than growth properties. When B. megaterium was cultured in the presence of 57CoC12, the cobalt was incorporated exclusively into vita-
54
WOLF AND BREY
J. BACTERIOL.
2, lane 6). Indeed, when
a concentrated cell extract of a mutant of the Cob V class was made, it was found to contain -cobinamide enough active cobalamin to allow growth of other B. megaterium vitamin B12 auxotrophs as well as S. typhimurium vitamin B12 auxotrophs (Tables 2 and 3). Although the amount of the vitamin that the Cob V strain -cobalamin v synthesized was approximately 20% of the wild-type level, it required exogenous vitamin for growth on ethanolamine. The six mutants with the Cbl phenotype exhibited three characteristic 57Co-labeling patterns (Fig. 3). Three of these mutants were designated Cbl X, two were designated Cbl XI, and one was designated Cbl XII. Two of the Cbl mutants accumulated a 57Co-labeled compound which migrated on a thin-layer chromatogram in a manner identical to that of cobinamide. Cbl mutants also accumulated labeled compounds which had mobilities identical with compounds that accumulated in some of the Cob mutants. 3, wPresence of vitamin B12 in concentrated extracts of Cob and Cbl mutants. To confirm that the auxotrophic phenotype was due to an inability to synthesize active cobalamin, the ; > t_ amount of intracellular vitamin B12 was determined for each o a> 8 0 mutant. When concentrated extracts prepared from each of the Cob and Cbl mutants were used to feed lawns of the S. FIG. 2. Separation of 57Co-labeleed corrinoid compounds obtyphimurium vitamin B12 auxotrophs TT8723 and TT7573, tained from representative Cob mutarnts. Lane 1, ATCC 10778; lane no growth was detected, indicating that no active cobalamin 2, GX5101; lane 3, GX5141; lane 4, G:X5151; lane 5, GX5137; lane 6, was present in any of the extracts with the exception of the GX5134; lane 7, GX5127. The approp] riate phenotypic designation of extract from GX5134 (Cob V) which synthesized reduced each mutant is noted below each lan( amounts of the vitamin. Extracts of the six Cbl mutants were e* able to support the growth of S. typhimurium TT8723, a min B12 and related corrinoids (dlata not shown). When the mutant whose auxotrophy can be satisfied by exogenously corrinoid compounds were extraicted from cells of 57CoC12supplied cobinamide. This observation indicates that B. labeled cultures of the wild type , the major corrin species megaterium Cbl auxotrophs are defective in steps which migrated on thin-layer chromatogIrams identically to vitamin occur late in the vitamin B12 biosynthetic pathway, after the B12. To subdivide the Cob and (2bl mutant classes further, synthesis of cobinamide (Table 2). similar cobalt labeling experime-nts were performed with Biosynthetic order of Cob and Cbl mutations. It was each of the auxotrophs (Fig. 2 anid 3). As expected, most of possible to deduce an order for the biosynthetic steps the strains classified as vitamin B12 auxotrophs did not accumulate significant amounts of 57Co-labeled vitamin B12. The mutants (e.g., GX5134; see below) that did appear to 1 2 3 incorporate 57Co into vitamin B12 produced reduced levels as compared with the wild type. In addition, many of the Cob -cobinamide and Cbl mutants exhibited characteristic, albeit different,
1
2
3
4
5
6
7
9
labeling patterns of cobalt-containing compounds. Analysis of these labeling patterns resulted in a subclassification of mutants. Mutants of the Cob phenotype showed at least six different patterns and were classified as Cob I, II, III, IV, V, and VI (Fig. 2); three different patterns were observed in the Cbl mutants, and these were designated Cbl X, XI, and XII (Fig. 3). Specifically, Cob mutants classified as Cob I did not accumulate any cobalt-containing corrinoid. Ten mutant strains were classified as Cob I based on this phenotype. Cob II, III, IV, V, and VI strains did not synthesize any vitamin B12 or cobinamide but did accumulate other cobaltcontaining corrinoids that were not found in detectable quantities in the wild-type strain. Based on particular labeling patterns, five separate mutants were classified as Cob II, whereas two were classified as Cob III (e.g., GX5151), with one each classified as Cob IV (GX5137), Cob V (GX5134), and Cob VI (GX5127). The remaining eight mutants having the Cob phenotype could not be classified definitively into any of the above categories and were not further characterized in this study. A mutant of the Cob V class (GX5134), in addition to accumulating cobalt-containing corrinoids, also appeared to synthesize significant amounts of a compound that migrated in a manner similar to that of vitamin B12 (Fig.
-cobalamin
n .. ._S _. _L
_.
~L
FIG. 3. Separation of 57Co-labeled corrinoid compounds isolated from Cbl mutants. Lane 1, GX5157; lane 2, GX5160; lane 3, GX5143.
VITAMIN B12 IN B. MEGATERIUM
VOL. 166, 1986
TABLE 2. Presence of vitamin B12 or bioactive corrinoids in concentrated extracts of Cob and Cbl mutants Phenotypic class
Cob I Cob I Cob II Cob III Cob IV Cob V Cob VI Cbl X Cbl XI Cbl XII Wild type
Vitamin B12 activity
assayed ona:
Extract source
GX5101 GX5139 GX5141 GX5151 GX5137 GX5134 GX5127 GX5157 GX5160 GX5143 ATCC 10778
TT8723
TT7573
0 0 0 0 0 0.4 0 3.6 2.0 1.6 1.6
0 0 0 0 0 0.3 0 0 0 0 1.6
a Vitamin B12 activity is expressed in nanograms of vitamin B12 or equivalent (in the case of cobinamide) per 10' cells. Cultures were grown to a density of 7 x 10i cells per ml; 1.6 ng of vitamin B12 is thus equivalent to 12 .g/liter.
affected by some of the Cob and Cbl mutants by crossfeeding experiments in which extracts prepared from the mutants strains were tested in all possible combinations for the ability to support growth (Table 3). The results were that extracts prepared from each of the Cbl mutants were capable of forming compounds which supplemented each of the mutants of the Cob phenotype. None of the extracts prepared from the Cob mutants was capable of supporting growth of any of the Cbl mutants. Because the extracts of GX5157 (Cbl X) was capable of feeding both GX5160 (Cbl XI) and GX5134 (Cbl XII), the lesion in GX5157 may likely occur in a biosynthetic step(s) after steps determined by lesions in GX5160 and GX5143. Further support for this order is evidenced by the fact that extracts prepared from neither GX5143 nor GX5160 could support the growth of GX5157. Within the Cob phenotypic grouping, each of the members of the Cob II class, typified by GX5141, was capable of supporting growth of all other mutants of the Cob phenotype, indicating a biosynthetic lesion(s) possibly just before the synthesis of cobinamide. Extracts prepared from mutants of the Cob III, IV, V, and VI classes were incapable of cross-feeding each of the others of those classes, but extracts of each of those supported the growth of some of the Cob I mutants, exemplified by GX5139. Of 10 mutants of the Cob I phenotype, 2 (e.g., GX5101) failed to be supplemented
55
by extracts of Cob III, IV, V, or VI mutants, while the remaining 8 behaved identically with GX5139 (data not shown). The failure of extracts of Cob III, IV, V, and VI mutants to feed GX5101 indicated that the effect of the cob-i mutation might be pleiotropic or that GX5101 harbors multiple mutations. As expected, none of the extracts from the 10 Cob I strains could support the growth of Cob II, III, IV, V, or VI mutants. Further, none of the Cob I mutants was capable of cross-feeding any of the other Cob I mutants. The results of the cross-feeding experiments permitted the ordering of mutations or mutant classes (Fig. 4). Transductional linkage of Cob and Cbl mutations. To determine whether the mutations that define the Cob and Cbl mutants are genetically linked, reciprocal two-factor transductional crosses were performed on representative strains of several of the Cob and Cbl subclasses. The transductions were performed with the B. megaterium generalized transducing bacteriophage MP13 (21). Phage lysates prepared from five of the Cob strains and from three of the Cbl strains were tested in all possible combinations for the ability to transduce recipient strains to vitamin B12 independence on ethanolamine. The Cob I mutants having the phenotype of GX5139 were not included in this analysis. The results of the reciprocal crosses are shown in Table 4. In general, a low number of prototrophic recombinants indicated the probability of few crossovers between defective allele(s) and, hence, tight linkage between markers. Because reversion of each of the recipient strains was less than 0.33 x 107, values which are low but not background might indicate some crossing over between markers or multiple mutations in some of the strains. These data suggest that the mutations fall into two linkage groups in that those mutations that characterize the Cob class form one linkage group and the mutations that characterize the Cbl class form another. In independent experiments with GX5141 (Cob II) in two factor crosses, weak linkage between cob-41 and the other cob and cbl loci was inferred, suggesting that the Cob II phenotype belongs in yet another linkage group (data not shown). To determine whether the Cob group is linked to the Cbl group, Cbl- strains were transduced to growth on ethanolamine in the presence of cobinamide with phage lysates prepared from Cob- strains. Cbl+ transductants were then scored for inheritance of the defective Cob allele by growth on ethanolamine in the absence of cobinamide or vitamin B12. Only several Cob- colonies were detected when each of the Cob mutants was transduced into each of the Cbl
TABLE 3. Cross-feeding of Cob and Cbl mutants Indicator strain (Phenotype)
GX5101 (Cob I) GX5139 (Cob I) GX5141 (Cob II) GX5151 (Cob III) GX5137 (Cob IV) GX5134 (Cob V) GX5127 (Cob VI) GX5157 (Cbl X) GX5160 (Cbl XI) GX5143 (Cbl XII)
Growth stimulation by extract from the following source (phenotype)a:
GX5101 (Cob I)
GX5139 (Cob I)
GX5141 (Cob II)
GX5151 (Cob III)
GX5137 (Cob IV)
GX5134 (Cob V)
GX5127 (Cob VI)
GX5157 (Cbl X)
GX5160 (Cbl XI)
GX5143 (Cbl XII)
-
-
+ + + + + + -
-
-
+
-
-
+ + +
-
+ -
+ + + + + + +
+ + + + + + + -
-
-
-
+ + + + + + + +
+ -
+ + + + +
-
+ +
-
+
-
-
a Extracts of the strains listed horizontally were fed to lawns of the strains listed vertically. Symbols: +, significant growth stimulation of the lawns; -, no growth stimulation observed; ±, poor but significant growth observed.
56
WOLF AND BREY
J. BACTERIOL.
Cobl I
I,
Cobl V, CobV,
CobV]
(cob-i9) j
Cobil j---i-----C-CbNH2--5bm{CbIXI,CbIXII
{CbIX{ C-X---
---B12
FIG. 4. Positioning of biosynthetic defects in the cobalamin biosynthetic pathway. Abbreviations: SHC, sironydrochlorin; CbNH2, cobinamide. Phenotypic classes or particular mutations within a class are surrounded by brackets indicating a single grouping within the pathway.
mutants (Table 5). This result indicates between the two mutant classes.
a
weak linkage
none of the Cob I mutants required reduced sulfur for growth they must be able to synthesize sirohydrochlorin and hence siroheme, the prosthetic group for sulfite and nitrite reductases (14, 20; Fig. 1). The exact point at which cobalt is inserted into the ring structure is unknown. Thus, members of the Cob I class could be deficient in any of a number of biosynthetic steps in the conversion of sirohydrochlorin to cobyrinic acid. Cobalt insertion could occur at any point in that series of steps, possibly by enzymatic means; it could also occur nonspecifically or nonenzymatically at multiple points. Failure to accumulate cobalt ion could also result in the inability to synthesize cobalt-containing compounds. Among the 20 Cob mutants, six different cobalt-labeling patterns were obtained; three different patterns were obtained in the Cbl mutants. An interpretation for these results is that lesions in particular biosynthetic enzymes, presumably caused by mutations in separate genes, caused accumulation of pathway intermediates before the lesions. Each of the 57Co-labeled compounds could be intermediates in the cobalamin pathway, but some of the compounds could be side products. Biosynthetic defects present in Cob II, III, IV, V, and VI mutants must occur at points after the insertion of cobalt and, thus, after those of the Cob I mutants. Defects in the five Cob II mutants could be positioned after the biosynthetic defects of the Cob III, IV, V, and VI mutants because extracts prepared from Cob II mutants were able to feed all other Cob mutants. The Cob III, IV, V, and VI mutants formed a group in that none of those four mutants
DISCUSSION The work that we have reported in this paper should provide a rational framework for further studies in cobalamin biosynthesis and regulation. The biosynthetic pathway for cobalamin surely involves the construction of the most complex biological molecules other than proteins and involves an unknown number of gene products, the method of regulation of which is also unknown. The isolation of mutants provides a first discreet step in understanding genetic regulation of cobalamin biosynthesis in an aerobic organism in which regulation of trace quantity synthesis of vitamin B12 might have evolved differently from the pathway in facultative organisms. The isolation of mutants will eventually permit the correlation of a particular enzyme with a defined biosynthetic conversion and will facilitate the cloning and further definition of the genes. The mutations that result in the Cob or Cbl phenotypes cannot be assigned to defects in any known biosynthetic enzymes. However, some genetic defects could be assigned to narrow regions of the cobalamin biosynthetic pathway based on the combined results of cross-feeding and cobaltlabeling experiments. For example, mutants of the Cob I class did not accumulate any cobalt-containing corrinoids; hence, some of those mutants were probably defective in the conversion of sirohydrochlorin to cobyrinic acid, at points before the insertion of cobalt into the ring structure. Because
Recipient (genotype)
GX5157 (cbl-57) GX5160 (cbl-60) GX5143 (cbl43) GX5137 (cob-37) GX5134 (cob-34) GX5151 (cob-SI) GX5127 (cob-27) GX5101 (cob-i)
TABLE 4. Transductional linkage of B. megaterium cob and cbl mutations No. of transductants obtained with the following donor (phenotype)a: GX5157 GX5160 GX5143 GX5137 GX5134 GX5151 GX5127 (Cbl XII) (Cob IV) (Cbl XI) (Cob V) (Cbl X) (Cob III) (Cob VI) 0 5 1 68 108 45 15 ob 0 28 840 52 0 0 7 1 2 0 16 51 106 10 3 9 6 30
18 12 39 20 153
36 140 48 30 126
0 0 0 1 6
6 0 9 3 41
1 3 0 1 12
2 5 0 0 7
GX5101 (Cob I) 39 783 81
9 12 8 3 0
10778 (WT)
16 136 30 11 38 39
9 50
a Values represent the number of prototrophic transductants obtained per 2 x 107 PFU. In each of these crosses, a single lysate of each of the donor strains was used to transduce each of the recipients; values in a column are therefore comparable. The values for the wild-type (10778) control were compiled from several experiments with different phage lysates. Reversion of each of the recipients was
