Cloning and analysis of genes involved in coenzyme B12 biosynthesis in Pseudomonas denitrificans.

B Cameron, K Briggs, S Pridmore, G Brefort and J Crouzet J. Bacteriol. 1989, 171(1):547.

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JOURNAL OF BACTERIOLOGY, Jan. 1989, p. 547-557

Vol. 171, No. 1

0021-9193/89/010547-11$02.00/0
Copyright C) 1989, American Society for Microbiology

Cloning and Analysis of Genes Involved in Coenzyme B12 Biosynthesis in Pseudomonas denitrificans
BEATRICE CAMERON,t KATHLEEN BRIGGS,4 SYLVIE PRIDMORE, GEORGES BREFORT,t AND JOEL CROUZETt* Gene'tica S.A., 160 Quai de Polangis, 94340 Joinville-le-Pont, France Received 17 February 1988/Accepted 4 October 1988
Cobalamin synthesis probably requires 20 to 30 different enzymatic steps. Pseudomonas putida and Agrobacterium tumefaciens mutants deficient in cobalamin synthesis (Cob mutants) have been isolated. In P. putida, Cob mutants were identified as being unable to use ethanolamine as a source of nitrogen in the absence of added cobalamin (deamination of ethanolamine requires coenzyme B12 as a cofactor). In A. tumefaciens, Cob mutants were simply screened for their reduced cobalamin synthesis. A genomic library of Pseudomonas denitrificans was constructed on a mobilizable wide-host-range vector. Eleven plasmids from this library were able to complement most of these mutants. By complementation and restriction mapping analysis, four genomic loci of P. denitrificans were found to be responsible for complementation of the Cob mutants. By subcloning fragments from the four genomic loci, we identified at least 14 different genes involved in cobalamin synthesis.

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Cobalamins are among the most complex nonpolymeric natural products known (4). The coenzyme B12 biosynthetic pathway contains the following steps (Fig. 1): (i) formation of uroporphyrinogen III, the common intermediate for the synthesis of hemes, chlorophylls, cobalamin, sirohemes, and F430 (23); (ii) conversion of uroporphyrinogen III into cobyrinic acid; (iii) formation of cobinamide from cobyrinic acid; and (iv) conversion of cobinamide into coenzyme B12 (for reviews on cobalamin biosynthesis, see references 3, 22, 26, 41, and 46). Uroporphyrinogen III is synthesized in four distinct and well-known enzymatic steps from succinyl coenzyme A and glycine (3). In contrast, the biosynthetic pathway from uroporphyrinogen III to coenzyme B12 is not clearly understood, since only one enzymatic activity has been partially purified (38) and the precise number of enzymatic steps is not known. In addition, the intermediates between porphobilinogen and cobyrinic acid are very unstable and sensitive to oxygen (3). Cloning of genes coding for enzymes involved in adenosylcobalamin biosynthesis and the associated regulatory genes would be a valuable tool in understanding the pathway. The isolation and genetic characterization of mutants blocked in cobalamin biosynthesis in Salmonella typhimurium and Bacillus megaterium have been recently described (29, 30, 48). Three bacteria are presently used in industry for vitamin B12 production: Pseudomonas denitrificans, Propionibacterium shermanii, and Propionibacterium freudenreichii (19). We report here a genetic study of coenzyme B12 biosynthesis in a P. denitrificans strain of industrial interest (19). We have isolated mutants of Pseudomonas putida and Agrobacterium tumefaciens that are deficient in the synthesis of cobalamin (Cob mutants). These bacteria were used to isolate Cob mutants for two reasons: (i) molecular genetic techniques are
*

far more established in P. putida and A. tumefaciens than in P. denitrificans (2, 25), and (ii) these mutants are, like P. denitrificans, gram-negative aerobic rods that synthesize cobalamin. We constructed a genomic library of P. denitrificans on a mobilizable wide-host-range vector, and this library was used to complement P. putida and A. tumefaciens Cob mutants in order to clone genes from the cobalamin biosynthetic pathway. Four genomic fragments of P. denitrificans were found to be responsible for complementation of most of the mutants, which were classified into 14 complementation groups.
MATERIALS AND METHODS Bacterial strains and plasmids. Unless otherwise specified, chemicals were purchased from Sigma Chemical Co., St. Louis, Mo. Bacterial strains and plasmids used are listed, with their relevant characteristics, in Table 1. Strains related to SC510 are used for the industrial production of vitamin B12 (19). Strain SC510, which is characterized by significantly improved cobalamin productivity, was derived from strain MB580, originally defined as a strain of P. denitrificans (Rhone-Poulenc Sante, U.S. patent 3,018,225), after numerous chemical and physical mutagenesis steps. Although the taxonomic validity of the species P. denitrificans is questionable (14), we retain this taxonomic definition for strains derived from MB580, in accordance with previous publications (17, 33, 47). Rifampin-resistant isolates were obtained by spreading fresh culture (5 x 109 cells per ml) on LB agar medium supplemented with 100 mg of rifampin per liter. Resistant colonies were streaked on LB agar medium containing rifampin to obtain single-colony isolates, some of which were checked for parental characteristics. To obtain nalidixic acid-resistant isolates, a similar procedure was used at a nalidixic acid concentration of 20 mg/liter. Media, bacteriological techniques, and chemicals. For routine culture, all bacteria were grown in LB medium (36) at 37C for Escherichia coli and at 30C for P. denitrificans, P. putida, and A. tumefaciens. PS4 medium used for the production of cobalamin, derived from the medium of Lago and Demain (34), consisted of sucrose (30 g/liter), glutamic acid (5.8 g/liter), NZ case (Kraft Inc., Norwich, England) (10 g/liter), betaine (10 g/liter), MgSO4 7H20 (1.5 g/liter),
-

t Present address: Laboratoire de Gdndtique, Institut de Biotechnologie, Centre de Recherche de Vitry, Rh6ne-Poulenc Sante, 94403
Vitry sur Seine Cedex, France. t Present address: Amersham International P.L.C., Amersham, Buckinghamshire HP7 9LL, England. Present address: 52 Habsbarger Strasse, 4310 Rheinfelden, Switzerland.

Corresponding author.

547

548

CAMERON ET AL.
UROPORPHYRINOGEN III
COCH

J. BACTERIOL.
COBYRINIC ACID

SUCCINYL CoA
CONC
HOOC

CONH

HEMES
L2

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GLYCINE

"PI',)

(IV)
2 v

OC

COBALAMIN

COBINAMIDE

FIG. 1. Cobalamin biosynthetic pathway. R = CH3, Methylcobalamin; R = OH, hydroxocobalamin, R = 5'-deoxyadenosyl, adenosylcobalamin; R = CN, cyanocobalamin; X, cobalt ligands (e.g., OH-); CoA, coenzyme A. The cobalamin pathway contains the following steps: (i) formation of uroporphyrinogen III; (ii) conversion of uroporphyrinogen III to cobyrinic acid, which requires eight methylations, decarboxylation of the acetic acid side chain at C-12, loss of C-20, and insertion of cobalt(III) (3, 41, 42); (iii) formation of cobinamide from cobyrinic acid through six amidations and the addition of aminopropanol (22); and (iv) conversion of cobinamide to coenzyme B12, which includes phosphorylation of the aminopropanol residue and addition of GMP from GTP and of the lower base, 5,6-dimethylbenzimidazole, to form cobalamin phosphate, which, after dephosphorylation, gives coenzyme B12 (26).

(NH4)2HP04 (3 g/liter), MnSO4* H20 (0.02 g/liter), ZnSO4 . 7H20 (0.02 gfliter), FeSO4 * 7H20 (0.03 g/liter), MoO3Na2H20 (0.005 g/liter), CoCl2 6H20 (0.12 g/liter), KCI (0.9 g/ liter), and 5,6-dimethylbenzimidazole (0.045 g/liter). Cobalt and 5,6-dimethylbenzimidazole are incorporated into cobalamin, and betaine is known to stimulate vitamin B12 production (17). Eth medium consisted of glucose (4 g/liter), K2HPO4 (7 g/liter), KH2PO4 (3 g/liter), Na2SO4 (1 g/liter), MgSO4. 7H20 (0.25 g/liter), and ethanolamine (1 ml/liter) as a nitrogen source. Vitamin B12 or cobinamide dicyanide was
-

added at a concentration of 1 pLg/ml. For E. coli, antibiotics used at the following concentrations: kanamycin sulfate, 100 mg/liter; tetracycline hydrochloride, 12 mg/liter; and spectinomycin dihydrochloride, 50 mg/liter. For P. putida and A. tumefaciens, the concentrations used were: kanamycin sulfate, 50 mg/liter; lividomycin sulfate (RhonePoulenc Sante), 100 mg/liter; rifampin, 100 mg/liter; and nalidixic acid, 20 mg/liter. P. putida and A. tumefaciens cobalamin production was determined by using the following procedure: (i) a colony from a fresh reisolate was cultured in
were

VOL. 171, 1989

COENZYME B12 BIOSYNTHESIS IN P. DENITRIFICANS

549

TABLE 1. Bacterial strains and plasmids used

Bacterial strain or plasmid

Relevant genotype, phenotype, or properties

Source or reference

Strain E. coli MC1060 LG90 J53 113-3 113-3 Cbll

P. putida KT2440 KT2440 Rif Nalr P. denitrificans SC510
A. tumefaciens

A(lacIPOZYA)X74 galU galK strA2 hsdR A(lac-pro)XIII nal Pro Met

metE Mutant of 113-3 that cannot convert cobinamide dicyanide to cobalamin
r- m+

9 ATCC 37114 5 11 This paper

2 This paper

Spontaneous nalidixic acid- and rifampin-resistant derivative of KT2440 Rif High-cobalamin-producing strain obtained from MB580 (U.S. patent 3,018,225) by numerous random-screening mutagenesis cycles

Rh6ne-Poulenc Santd

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C58-C9 C58-C9 Rifr Nalr Plasmid pRK2013 pRK2073 (pRK2013::Tn7) pFR10 pFR237 pXL59 pKT230 pXL435 pJB4J1 pRK2013::TnS

Strain C58 cured of its Ti plasmid Spontaneous nalidixic acid- and rifampin-resistant derivative of C58-C9
ColEl Kmr Tra+ (RK2) ColE1 Sp' Tpr Tra+ (RK2) ColEl multiple-site cloning polylinker incQ Kmr Mob' incQ Kmr Mob' incQ Kmr Smr Mob' incQ Kmr Mob' multicloning site incP pPHIJ1::Mu::Tn5 ColEl Kmr Tra+ (RK2)

25 This paper

18 35 43 F. Richaud, unpublished data This paper 2 This paper 5 This paper

a 250-ml Erlenmeyer flask containing 25 ml of PS4 medium; (ii) after incubation (4 days for P. putida and 5 days for A. tumefaciens) on a rotary shaker at 30C, 1 ml of the culture was cyanurated with 1 ml of a solution of 50% (vol/vol) acetonitrile-75 mM KCN and incubated for 1 h at 56C; during this treatment, cells were lysed and cobalamins were converted to the more stable cyano forms, which could be

assayed.
General methods. Genomic digests were obtained by incubating 5 jxg of DNA with 20 U of restriction enzyme (New England BioLabs, Inc., Beverly, Mass.) for 2 h. Plasmids and DNA fragments were labeled by nick translation with [a-32P]dCTP as described elsewhere (40). E. coli strains were transformed by the standard calcium chloride procedure (36). Agarose gel electrophoresis, purification of DNA fragments, preparation of plasmid DNA, and Southern blotting were carried out as described previously (6, 36). Genomic DNA was prepared according to the method of Shepard and Polisky (44). Mutagenesis. E. coli, P. putida, and A. tumefaciens were mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine according to published procedures (37). TnS transposon mutagenesis was carried out by using either pRK2013::TnS (constructed in this laboratory by a TnS insertion into pRK2013; data not shown) or pJB4J1, each of which is a Tn5-carrying plasmid. pRK2013::Tn5 and pJB4J1 are self-transmissible plasmids (5); they do not replicate in bacteria such as P. putida and A. tumefaciens (pRK2013 has only the origin of replication of ColEl [18], and pJB4J1 contains bacteriophage Mu, which prevents the establishment in non-Enterobacteriaceae of the carrier plasmid [5]). These plasmids were introduced into the bacteria to be mutagenized by mating the donor organism, HB1O1(pRK2013::TnS) or J53(pJB4J1), and the recipient organism, KT2440 Rif Nalr or C58-C9 Rif' Nalr and selecting rifampin-, nalidixic acid-, and kanamycin-

resistant clones.

Screening for Cob mutants of A. tumefaciens and P. putida. In the case of P. putida mutants, each single colony obtained after mutagenesis was transferred to 100 ,u of 10 mM MgSO4 in a 96-well microdilution plate. A drop of each suspension was transferred, by using a replicating device, from the microdilution plate onto a square plate of Eth medium with or without vitamin B12 in order to identify clones that were auxotrophs for vitamin B12. For A. tumefaciens, each colony obtained after mutagenesis was individually transferred to 100 RI of PS4 medium in a 96-well microdilution plate. After incubation and cyanuration, the Cob mutants were detected by microbiological assay or enzyme-linked immunosorbent assay (ELISA). Mobilization of plasmid DNA from E. coli to other gramnegative bacteria. Triparental mating (13) was carried out by mixing the recipient strain (P. putida or A. tumefaciens) with HB1O1(pRK2073) and the E. coli strain carrying the mobilizable plasmid to be transferred. Clones of the genomic library stored in Hogness freezing medium were mobilized as described elsewhere (21). The transconjugants were selected on LB medium supplemented with rifampin and nalidixic acid for selection of the recipient bacteria and with kanamycin or lividomycin for determination of the presence of the plasmid. Lividomycin was used for selection of pXL59 derivatives in Tn5 mutants of A. tumefaciens and P. putida since the enzyme, aminoglycoside-O-phosphotransferase (3')I, encoded by pXL59 (originating from Tn9O3) confers lividomycin resistance, contrary to aminoglycoside-Ophosphotransferase(3')II from TnS (20). Complementation of cobalamin-deficient mutants of P. putida and A. tumefaciens. After transfer of plasmids from the genomic library into mutants, we screened for complementing plasmids. In the case of P. putida, transconjugants were replicated on Eth medium with or without vitamin B12; after 3 days of incubation, clones that were able to grow on both media were reisolated and their cobalamin production was

550

CAMERON ET AL.
BE

J. BACTERIOL.

studied. In the case of A. tumefaciens, transconjugants were inoculated 88 by 88 into a 96-well microdilution plate, each well containing 100 ,ul of PS4 medium supplemented with kanamycin or lividomycin. After incubation and cyanuration, vitamin B12 production was measured by the microbiological assay. Cobalamin production by positive clones in a 250-ml Erlenmeyer flask was compared with the level of cobalamin synthesized by the mutant alone or the mutant containing pXL59. Mutants were considered complemented for the Cob mutation when the cobalamin level was comparable with that of the parent strain. Vitamin B12 assays. Three types of assays were alternatively used to determine cobalamin concentrations: (i) a microbiological assay for which the indicator strain is an E. coli vitamin B12 auxotroph (113-3 or 113-3 Cbll); (ii) a radiochemical assay derived from a method previously published (24) that uses intrinsic factor (IF) or nonintrinsic factor (NIF) (from porcine gastric mucosa); and (iii) an ELISA that involves IF and an anti-B12-KLH goat serum. These assays are sensitive in the range of 10 to 80, 0.02 to 0.08, and 1 to 5 ,ug of vitamin B12 per liter, respectively. The standard deviation was 5% for the radiochemical assay and around 15% for the microbiological assay and ELISA. The microbiological assay was carried out as described previously (J. Crouzet, these de docteur ingenieur, Institut National Agronomique de Paris-Grignon, Paris, 1987). In the radiochemical assay, IF and NIF bind 57Co-labeled, 3H-labeled, and nonradioactive cyanocobalamins indiscriminately but display different specificities, since vitamin B12 and cobinamide bind to NIF with similar affinities whereas cobinamide does not bind to IF, which is more specific for the assay of vitamin B12 and related products (39). To assay 400 ,ul of a vitamin B12 sample, 325 pul of a solution of 350 mM Tris hydrochloride (pH 10), 75 mM NaCl, and 1.5 mg of bovine serum albumin per ml, containing 10 nCi of cyano[G3H]cobalamin (4.4 Ci/mmol; Amersham, United Kingdom) and 15 U of IF or NIF, was added. After incubation for 1 h at 37C, 250 p.l of a suspension containing 2.5% activated charcoal and 0.5% bovine serum albumin was added to the solution. After a 5-min incubation at room temperature, the charcoal was removed by centrifugation (the free forms of cobalamins and cobinamides bind to activated charcoal, whereas IF or NIF complexes do not), thus allowing elimination of the unbound corrinoids; the amount of radioactivity bound to IF or NIF was then measured by counting the radioactivity present in the supernatant. If cyano[57Co] cobalamin (180 to 300 puCi/,ug) was used, the sensitivity of the assay could be improved by a factor of 200, thus allowing determination of concentrations ranging from 100 to 400 pg/ liter. The ELISA was a sandwich-type assay derived from a previously described procedure (15) and adapted to vitamin B12. The solution to be assayed was added to the IF-coated plates and incubated in the presence of an anti-B12-KLH goat serum (Biospecia Ltd., Wembley, United Kingdom). A sheep anti-goat serum conjugated with alkaline phosphatase (Biosys S.A., Compiegne, France) was then added, and alkaline phosphatase activity was assayed as described elsewhere (16). Construction of pXL59, pXL435, and plasmids carrying P. deniifi cans DNA fragments. A vector was constructed which (i) has unique restriction sites in the coding region of a testable genetic marker and (ii) carries a wide-host-range replicon that can be mobilized in many gram-negative bacteria. The replicon is of the IncQ type and contains part of plasmid pFR237 (a generous gift of F. Richaud) (Fig. 2).

pFR237
mb
17 kb

c X

s4ri
P

I19 kb

Ric

x
H

C.Ss,P,H*C.X,Xb.S
x

xc H P
p

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mb

17 kb sic sic~~~~~~~~~~~~~~i

pXL59

pXL59

~~~~pXL435 iO 1.6 kcb

k

sri

sri

ob
I

Xb

M 2 M 3E

4 M

FIG. 2. Physical maps of plasmids pFR237, pKT230, pXL59, and pXL435. B, BamHI; C, CMaI; E, EcoRI; H, HindlIl; P, PstI; S, SstI; Sa, Sall; X, XhoI; Xb, XbaI. Key to symbols: 1, PstI-SstI fragment of RSF 1010, which contains the origin of vegetative replication (ori) (12), the relaxation nick site (nic), and a determinant (mob) that is essential for plasmid mobilization (2); 2, PstI-BamHI fragment of pACYC177 (2); 3, BamHI-SstI fragment containing the E. coli lactose operon without the promoter, the operator, the translation initiation sites, and the first eight nonessential codons of lacZ (9); 4, Sau3AI fragment of P. putida chromosomal DNA.

P-Galactosidase activity was not detected in strain MC1060(pFR237). We inserted Sau3AI fragments of P. putida KT2440 genomic DNA into the BamHI-linearized vector pFR237 and searched for recombinant plasmids that allowed expression of P-galactosidase in E. coli MC1060. One of the plasmids was shown to carry a 75-base-pair (bp) fragment that reconstituted the BamHI site on its right boundary, toward the lacZ gene, and was named pXLS9 (Fig. 2). Moreover, it was confirmed that pXL59 could be mobilized with the same efficiency as could pFR237 or pKT230 from E. coli to gram-negative bacteria such as A. tumefaciens and P. putida (data not shown). pXL435 was derived from pKT230 by substituting the BamHI-SstI fragment for the multiple-cloning fragment from pFR10 (43). The three wide-host-range vectors (pKT230, pXL435, and pXL59) were chosen for subcloning of P. denitrificans fragments. MC1060 was used as the recipient strain except for the cloning of EcoRI or SstI fragments into pKT230; in these cases, the streptomycin-sensitive strain LG90 was used and screened for streptomycin-sensitive recombinants. Construction of a P. denitriicans genomic DNA library. A genomic DNA library from strain SC5150 was constructed in pXL59. A partial Sau3AI digest of SC510 chromosomal DNA (200 pug) was fractionated by sucrose gradient centrifugation (36). Fractions corresponding to DNA fragments with sizes ranging from 10 to 15 kbp were pooled. The size-fractionated genomic DNA fragments (100 pug/ml each) were ligated with BamHI-digested pXL59, using T4 DNA ligase (New England BioLabs) at a concentration of 300 U/ ml. A fraction of the reaction mixture was used to transform competent MC1060(pRK2073) cells. Transformed cells were

VOL. 171, 1989

COENZYME B12 BIOSYNTHESIS IN P. DENITRIFICANS

551

plated on LB agar medium containing kanamycin, spectinomycin, and X-Gal (5-bromo-4-chloro-3-indolyl-,-t-galactopyranoside). A total of 3,200 white colonies on X-Galsupplemented medium were picked and individually inoculated in a 96-well microdilution plates containing 200 ,ud of Hogness freezing medium per well for conservation (21). Analysis of 24 clones showed that the average size of the inserted DNA fragments in pXL59 was around 13 kbp. Assuming that the P. denitrificans genome has a molecular size of about 5,000 kbp, this library is large enough to represent any DNA sequence of the genome with a probability greater than 99% (10). Isolation of an E. coli mutant deficient in the transformation of cobinamide dicyanide into methylcobalamin. It has been reported that E. coli 113-3, a metE mutant, requires vitamin B12 to grow without an exogenous supply of methionine (11). It has been also reported that E. coli is able to catalyze the transformation of cobinamide into methylcobalamin (39). Therefore, we searched for a mutant blocked in methylcobalamin biosynthesis from cobinamide dicyanide. After Nmethyl-N'-nitro-N-nitrosguanidine mutagenesis, 800 colonies of E. coli 113-3 plated on LB medium were replica plated on M9 medium supplemented with cobinamide dicyanide. We found one mutant, named 133-3 Cbll, that was complemented by vitamin B12 or methionine but not by cobinamide dicyanide or 5,6-dimethylbenzimidazole. E. coli 113-3 Cbll is apparently blocked in the synthesis of methylcobalamin from cobinamide dicyanide, since in E. coli cobinamide dicyanide and vitamin B12 use the same transport system (7). This strain was used as an indicator strain for the microbiological assay of compounds that are formed between cobinamide and methylcobalamin. Since all samples assayed for cobalamin content were cyanurated, mutant 113-3 Cbll is an indicator strain that should give a more specific response for the assay of the end products in the biosynthesis of cobalamin.
RESULTS Isolation of mutants deficient in cobalamin synthesis. Since cobalamin synthesis is a biochemical pathway composed of many different steps, two mutagenesis strategies were used to obtain as many different mutants as possible: transposon mutagenesis with TnS, which is known to have a low translocation specificity (32), and N-methyl-N'-nitro-Nnitrosoguanidine mutagenesis. Two gram-negative bacteria that produce cobalamin under aerobic growth conditions were mutagenized: P. putida KT2440 (2) and A. tumefaciens C58-C9 (25). These two bacteria synthesize 50 and 500 pug of cobalamin per liter, respectively, under our culture conditions (see Materials and Methods). It is known that bacterial ethanolamine ammonia-lyase (EC 4.3.1.7), which catalyzes the deamination of ethanolamine to acetaldehyde and ammonia, requires adenosylcobalamin as a cofactor; this property has been described for enzymes of various bacteria, such as E. coli, Klebsiella aerogenes, S. typhimurium, and Clostridium sp. (1). If ethanolamine is the sole source of nitrogen, then the bacterium requires adenosylcobalamin to grow; therefore, E. coli and K. aerogenes, which do not produce cobalamin aerobically, grow aerobically on ethanolamine as a nitrogen source only in the presence of added cobalamin (1). Since P. putida grows on minimal medium with ethanolamine as the sole source of nitrogen, we hypothesized that in P. putida this pathway of nitrogen assimilation was also dependent on endogenous adenosylcobalamin synthesis. P.

TABLE 2. Cobalamin biosynthesis-deficient mutants of P. putidaa

Strain

Phenotype

Cobalamin Cofncn
53

Mutant class

KT2440 Rif' Nalr

G547 G548 G549 G550 G551

G552 G553 G554 G555 G556 G557

G558 G559 G560

G561 G562 G563

Cobl Cob2 Cob3 Cob4 CobS Cob6 Cob7 Cob8 Cob9 CoblO Cobll Cobl2

2 0.3 0.2 0.2 0.2 0.3

0.1 0.1 0.2 8 0.2 0.06 0.1 0.6 0.01 0.06 0.06 0.04 0.04

6 4 1

5
4 2 3 1 6 2 2 5 4 2 4 1 3 5 3

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Cobl3 Cobl4
CoblS Cobl6 Cobl7 Cobl8 Cobl9 Cob2O Cob2l Cob22 Cob23 Cob24 Cob25 Cob26

G566
G567

G568 G570
G571 G572

0.06 0.08 0.8 3

0.1 0.04 S

G573
G576 G577

a Mutants G568, G570, G571, G572, G573, G576, and G577 were obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. Cobalamin corlcentrations were determined by IF, as described in Materials and Methods, when the strains were cultivated under standard conditions for cobalamin production. Data are means of two assays. Mutant class refers to the six classes defined according to the size of the mutant genomie EcoRI DNA fragment hybridizing with a 32P-labeled Tn5 probe.

putida KT2440 Rif' Nalr was chosen as the recipient in the transposon-mediated mutagenesis experiment; 6,000 mutagenized colonies were selected at a frequency of 10-5 per recipient cell as described in Materials and Methods. After replica plating on ethanolamine medium with or without cyanocobalamin, mutants that required cyanocobalamin for growth were further investigated by confirming their phenotype and studying cobalamin production in PS4 liquid culture. A total of 19 transposon-mediated mutants were found to produce reduced levels of cobalamin (Table 2). These transposon-nmediated mutants were also studied by Southern blot analysis on total EcoRI-digested chromosomal DNA (there is no EcoRI restriction site in TnS [31]) by using 32P-labeled TnS as a probe. This analysis revealed that for each mutant a single fragment of genomic DNA hybridized with the probe; in each case, therefore, insertion of the transposon was likely to be unique in the chromosome of the strain. There was no detectable level of hybridization with chromosomal DNA of the parent strain (data not shown). The Cob mutants were then classified according to the estimated size of the EcoRI fragment hybridizing to 32p_ labeled TnS. This classification defined six classes of mutants (1 to 6; Table 2), which corresponded to estimated EcoRI chromosomnal fragments of 17, 14.5, 13, 12, 10.5, and 6 kbp, respectively (data not shown). P. putida mutants were also selected after treatment with N-methyl-N'-nitro-N-nitrosoguanidine as described in Materials and Methods. The screening procedure described above was applied to 1,400 mutagenized colonies. Seven selected

552

CAMERON ET AL.

J. BACTERIOL.

TABLE 3. A. tumefaciens Cob mutantsa

Strain
C58-C9 Rifr Nair G159 G160 G161 G162 G164 G165 G166 G168 G169 G170 G171 G172 G256 G258 G260 G261 G262 G609-617

Phenotype

Mutant class

Cobalamin concn

(,ug/liter)
500

Cob'
Cobl Cob2 Cob3 Cob4 CobS Cob6 Cob7 Cob8 Cob9 CoblO Cobll Cobl2 Cobl3 Cobl4 CoblS Cobl6 Cobl7 Cobl8-26 Cob27-52 Cob53-54 Cob55-146 Cobl47-148
VI II

V
IV I II I I VI III V I IV VI III I III

<10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10

G619-644
G646, G648 G1%3-2054 G2056, G2057

<1ob <1ob <1ob <job

<10

a The cobalamin assay was performed with E. coli 113-3 Cbll. Cobalamin concentrations were not significantly different when tested with E. coli 113-3 except for mutants G642 and G2038 to G2043. Mutants G609 to G617, G619 to G644, G646, G648, G1963 to G2054, G2056, and G2057 were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. b Most of the mutants produced cobalamin at levels of lower than 10 p.g/ liter. Exceptions (with production [in micrograms per liter] indicated in parentheses) were G614(80), G615 (30), G616(30), G617 (30), G640(40), G643 (70), G644 (70), G646 (200), G1663 (60), G1965 (120), G1975 (260), G1983 (30), G1991 (260), G1992 (200), G2008 (20), G2010 (150), G2012 (90), G2018 (220), G2026 (40), G2034 (40), G2045 (280), and G2054 (20). Data are means of two assays. Mutant class refers to the six classes defined according to the size of the mutant genomic EcoRI DNA fragment hybridizing with a 32P-labeled Tn5 probe.

mutants were found to exhibit the same properties as did the Cobl to Cobl9 mutants (Table 2) and to produce a low level of cobalamin. We noticed that all mutants except G547, G556, G572, and G577 produced 50 times less cobalamin than did the wildtype strain as determined by the radiochemical assay. A. tumefaciens C58-C9 Rif' Nalr was also mutagenized to obtain Cob mutants. Unfortunately, we have not been able to determine a procedure for screening vitamin B12-negative mutants of A. tumefaciens, as has been done for P. putida and for S. typhimurium and B. megaterium (29, 48). Therefore, each mutagenized colony was screened for cobalamin production (see Materials and Methods). Mutants deficient in adenosylcobalamin synthesis were kept on the basis of at least a 20-fold-reduced production compared with that of the wild-type strain. Transposon-mediated mutagenesis in A. tumefaciens was performed as described in Materials and Methods. Each colony was studied for ability to produce cyanocobalamin as determined by ELISA. A total of 17 clones from 5,000 mutants were deficient in production of cobalamin. The mutants were analyzed as described above. Six classes, related to the estimated size of the EcoRI chromosomal fragment of the mutants hybridizing with the 32P-labeled TnS probe, were found. Fragments of 17, 15.5, 14.5, 13.5, 8.5, and 7.5 kbp corresponded to classes I, II, III, IV, V, and VI, respectively (data not shown). Characteristics of and cobalamin production by these mutants are

shown in Table 3. In addition, 17,000 colonies mutagenized by N-methyl-N'-nitro-N-nitrosoguanidine were screened for the absence of cobalamin production, and 131 cobalaminnegative mutants were obtained. Cob mutants were detected by ELISA for the first 39 mutants; the other mutants were analyzed by the microbiological assay, using E. coli 133-3 Cbll. Except for 22 mutants, cobalamin production as determined by the microbiological assay was at least 50 times lower than the parental level (Table 3). Since the Cob mutants were obtained in two obligate aerobes, it is probable that they are not blocked in the part of the cobalamin synthetic pathway common with heme biosynthesis; otherwise, they should have been somehow deficient in heme synthesis and characterized by an impaired respiration mechanism leading to a lethal phenotype. For instance, Rhizobium meliloti mutants blocked in the first enzymatic step of the heme pathway (corresponding to b-aminolevulinic acid synthetase, which catalyzes the condensation of succinyl coenzyme A and glycine into 8aminolevulinic acid) require b-aminolevulinic acid for growth (35). Since 5,6-dimethylbeizimidazole is a component of PS4 medium, it is likely that all of the mutants that have been obtained are not blocked in 5,6-dimethylbenzimidazole synthesis. In S. typhimurium, cysG mutants have been described; these mutants, which do not synthesize sirohemes, are also blocked in vitamin B12 synthesis (29). We investigated whether such mutants were isolated in our study. Since the A. tumefaciens mutants were first plated on LB medium and tested for cobalamin production in PS4 medium (containing NZ case), cysteine-requiring mutants would not have been eliminated. This procedure should therefore have allowed us to isolate mutants blocked in sirohemes and vitamin B12 synthesis if such mutants could be found in A. tumefaciens. However, among all of the A. tumefaciens mutants studied, none were cysteine auxotrophs; this finding indicated an absence of these mutants or a different genetic organization than exists in S. typhimurium (30). Since P. putida Cob mutants were screened on Eth medium without cysteine, it is probable that mutants defective in sirohemes and vitamin B12 synthesis were not isolated. Complementation of Cob mutants of P. puida and A. tumefaciens by a genomic library of P. denitficans. Each clone of the genomic library of P. denitrificans was mobilized individually into Cob mutants of P. putida as described in Materials and Methods. The resulting transconjugants were tested for ability to grow on Eth medium. Clones that were no longer vitamin B12 auxotrophs were identified. It was verified that they were also complemented for cobalamin synthesis in 25-ml PS4 medium cultures. Because the plasmids were introduced individually, by replica mating, it was possible to identify plasmids that complemented the same mutants. Plasmids pXL151, pXL152, pXL156 to pXL158, pXL160, and pXL161 were shown to complement 16 P. putida Cob mutants (Table 4). Among the other 10 mutants, 9 (G547, G550, G553, G555, G563, G566, G567, G568, and G571) were not complemented by the genomic library of P. denitrificans and 1, G576, was partially complemented (5% of the wild-type level) by pXL159. The genomic library was also used to complement Cob mutants of A. tumefaciens. Cobalamin synthesis by each transconjugant was studied as described in Materials and Methods. Mutants were defined as complemented when transconjugants produced the wild-type level of cobalamin. pXL151 through 154, pXL156 to 161, and pXLS19 complemented A. tumefaciens Cob mutants (Table 4). pXL151,

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TABLE 4. Cob mutants of P. putida and A. tumefaciens and the corresponding plasmids from the genomic library of P. denitrificans that allow complementationa
that complement Complemented Cob mutants Group Group Plasmids mutants.v ~Cob A. tumefaciens P. putida

pXL151

G558,b G570

pXL151, pXL152
B
C

G548,b G551,b G552,b G557,b

G559/b G560/b G556,b G561b

G164/' G261,/ G609, G610, G638, G1985G1998, G2056, G2057 G166,b G611-617, G619, G620, G1963-1984 G159,b G169,/ G258,b G2035 G161,/ G171,b G2034, G2037

pXL153, pXL154 pXL154 pXL156

G549, .c G562,b G573c

G554/' G572, G577

pXL156, pXL157 pXL157 pXL157, pXL158, pXL160, pXL161 pXL157-pXL161

D
a

G170,b G260,' G262b, G622-630, G642,c G2021-G2033, G2036, G2043C G632, G633, G2018-G2020 G631, G2005-G2017 G160/' G165,b G634, G644, G1999-02004, G2054 G162,b G256,b G635-637, 6639, 6640, 6643, G646, G648

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pXL519

G2038,c G2039,c G20WO,c G2041,c G2042C

Uncomplemented mutants for P. putida: G547, G550, G553, G555, G563, G566, G567, G568, and G571 (G576 is partially complemented by pXL159). Uncomplemented mutants for A. tumefaciens: G168, G172, G621, G641, and G2044 to G2053. b Cob mutants obtained by insertion of transposon TnS. c Mutants positively assayed with E. coli 133-3.

pXL152, pXL156 to pXL158, and pXL161 were therefore able to complement Cob mutants of both A. tumefaciens and P. putida. The complementation data allowed us to classify the plasmids into four complementation groups, designated A, B, C, and D. Plasmids in each group complemented a specific set of mutants not complemented by plasmids from the other groups (Table 4). Group A consisted of pXL151 and pXL152, group B contained pXL153 and pXL154, group C included pXL156 to pXL161, and group D contained only pXL519. The restriction maps of the inserts carried by these various plasmids were established (Fig. 3). The maps are in agreement with complementation groups, since overlapping inserts always correspond to plasmids belonging to the same complementation group. Any physical linkage deduced from the restriction pattern was checked by Southern blot analysis. For instance, the 2.6-kbp EcoRI-SstI fragment of pXL156, which overlaps with pXL1574 was purified and radiolabeled with 32p; hybridization with EcoRI-SstI digests of pXL156 and pXL157 showed that the same fragment was present on both pXL156 and pXL157 (data not shown). The sizes of cloned DNA corresponding to groups A to D were 15.5, 25.5, 29, and 8 kbp, respectively. No correlation could be found among the restriction maps of the four groups (Fig. 3), which indicated that there is no overlap between the four different fragments. Therefore, at least four genomic loci of P. denitrificans are involved in cobalamin synthesis and can complement Cob mutants of P. putida and A. tumefaciens. We do not yet know whether those loci are tightly clustered or scattered on the chromosome. Genetic organization of the Cob loci. A more precise complementation map of the Cob loci was established to gain deeper insight into the genetic organization of the pathway. Smaller fragments representative of the four genomic loci, in the range of 2 to 4 kbp, were subcloned into pKT230, pXL435, or pXL59, depending on the available restriction sites (Fig. 2). We thus identified 14 different plasmids that specifically complemented most of the mutants investigated

(Table 5): pXL220, pXL227, pXL239, pXL436, pXL444, pXL452, pXL556, pXL617, pXL622, pXL678, pXL684, pXL698, pXL735, and pXL837 (Fig. 3). All of these plasmids complemented mutants that were not complemented by the others. All of the plasmids except pXL220 complemented A. tumefaciens Cob mutants. Complementation data with subclones are in agreement with the classification of P. putida and A. tumefaciens TnS mutants. For instance, a subclone complemented TnS mutants of P. putida or A. tumefaciens belonging to the same hybridization class (i.e., pXL684 complemented the three P. putida mutants of class 2; Table 5); this finding suggests that in complemented Tn5 mutants the transposon has inserted into Cob genes. Complemented mutants of a hybridization class were all complemented by the same subclone (see example cited above) or by subclones of the same cluster (pXL617, pXL622, and pXL239 complemented the P. putida mutants G562, G549, and G554, respectively, which constitute class 1; Table 5). This result may indicate that some hybridization classes represent a genomic EcoRI fragment that carries more than one gene involved in coenzyme B12 synthesis. In addition, uncomplemented P. putida TnS mutants belonged to the same hybridization classes (classes 3 and 6). Although some inserts of these 14 plasmids overlap, we can deduce that each insert carries at least one different gene involved in cobalamin biosynthesis, since each plasmid defines a specific complementation group (Table 5). A total of at least 14 P. denitrificans genes involved in the transformation of uroporphyrinogen III to coenzyme B12 have thus been cloned. Complementation by pXL189, pXL190, pXL191, or pXL300 (Fig. 3) was not specific because several mutants were complemented by these plasmids and not by their subclones (Table 5). For instance, pXL190 complemented G162, which was not complemented by pXL239 or pXL556 (Fig. 3). pXL189, pXL190, pXL191, and pXL300 should carry additional genes that are not revealed by the comple-

554

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J. BACTERIOL.

GROUP A

HE

B e

1 kb

pXL 151
pXL 152

so
I

xa
I

8
I

8
I
-

E
pXL452

pXL444 pXL67S 8
Sg

pXLSS8

B9
pXL436

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GROUP B

pXLSOO

IX ES E

HS Hl~~~~IS B H I I I I 1111 -1 I I 11I

XHB BH

XX

E ES X aXs

11

I I
pXL 154

I1

pXL1 53

pXL735 E

pXLS37

GROUP C

H S

I - I I 1 I I
pXLI 56

ESSS8

XSB

SB

I II

SIX

SH H

IIpXL157 pXLI58

e E X S

I I

pXLI 59
pAL
J

VU

pXL6IS
S
I-

S S pXL622
P

S
I

C
I

EcoRV

pXL61 7
Sau

pXL227
C
a

pXL556 H
I I

S.u
I

pXL239
E I

pXL189
C

B I

pXL220 Sau
pXLl90

II

Sou
pXL1 91

GROUP D
S S

BE

pXL519
Bg

Bg

pXL6SS FIG. 3. Physical map of inserts of the 11 plasmids from the genomic library of P. denitrificans that complement Cob mutants of P. putida and A. tumefaciens. Shown are inserts of each plasmid as well as inserts of the 14 subclones and plasmids pXL300, pXL189, pXL190, and pXL191. The 11 plasmids and their subclones were introduced into mutants by triparental mating; the transconjugants were studied for cobalamin synthesis in a 25-ml PS4 culture. The mutants complemented by these are listed in Tables 4 and 5. B, BamHI; Bg, BgIII; C, ClaI; E, EcoRI; H, HindlIl; P, PstI; Sa, Sall; Sau, Sau3AI; S, SstI; X, XhoI.

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TABLE 5. Relevant Cob mutants of A. tumefaciens and P. putida and the subclones that complement them
Subclones that

Group
A

Complemented mutantsa
P.

complement mutants
pXL444 pXL678 pXL452 pXL684

putida

A. tumefaciens

pXL436 pXL300b
B

G551 (4), G559 (4), G561 (4) G558 (5), G570 G552 (2), G556 (2), G557 (2), G560 (2) G548 (4)

G612 G614, G616, G617 G609, G610 G166 (1), G620 G615 G164 (1), G261 (1), G613, G611, G619, G638
G159 (VI), G169 (VI), G258 (VI), G2035 G161 (V), G171 (V), G2034, G2037

pXL735 pXL837
pXL617 pXL622 pXL227 pXL556 pXL239 pXL220

G562 (1) G549 (1), G573

G554 (1), G577 G572

G170 (III), G260 (III), G262 (III), G624, G629 G642,c G2043C G632, G633 G637, G648 G643 G631 G162 (IV), G256 (IV), G635, G636, G639 G160 (II), G165 (II), G634

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pXL189d
pXL190e

pXL191f
D

pXL698

G2038,C G2039,c G2040,c G2041,c, G2042C

a Arabic and roman numerals in parentheses indicate genetic classes to which the TnS mutants belong. b Complements all mutants complemented by pXL452, pXL678, pXL684. c Mutants positively assayed with E. coli 113-3. d Complements all mutants complemented by pXL227. e Complements all mutants complemented by pXL556 or pXL239. f Complements all mutants complemented by pXL556, pXL239, pXL220, or pXL190.

mentation data obtained with the 14 subclones mentioned above. Analysis of mutants blocked in the conversion of cobinamide into cobalamin. Among the various Cob mutants isolated, some exhibited the same levels of cobalamin production as did the wild type when assayed with E. coli 113-3 as the indicator strain (Table 5); in contrast, when production of cobalamin was assayed by using E. coli 113-3 Cbll (which is blocked in the conversion of dicyanocobinamide into cobalamin), synthesis of cobalamin by these mutants was at least 50 times lower than that of the wild type. Among these Cob mutants were two mutants of P. putida (G549 and G573) and seven mutants of A. tumefaciens (G642 and G2038 to G2043). In addition, cobalamin production was comparable with that of the wild type when assayed with NIF but was significantly reduced when assayed with IF (data not shown). These results suggest that such mutants are blocked in the conversion of cobinamide to cobalamin. They were complemented by two subclones, pXL622 and pXL698, defining two classes of mutants (Table 5 and Fig. 3). pXL622 and pXL698 should each carry at least one gene whose product is involved in the conversion of cobinamide to cobalamin. Among the 14 genes cloned, two are thus implicated in the last part of the cobalamin biosynthetic pathway and are carried by plasmids pXL622 and pXL698.
DISCUSSION The selection of mutants unable to grow on ethanolamine as the sole source of nitrogen has enabled us to isolate P. putida mutants deficient in cobalamin biosynthesis. This result supports the idea that in P. putida, ethanolamine ammonia-lyase requires adenosylcobalamin to be active. Similar results have been obtained with B. megaterium (48), from which cobalamin biosynthesis-deficient mutants were

isolated by screening for mutants auxotrophic for vitamin B12 in the presence of ethanolamine as the sole source of nitrogen. It is likely that this procedure can be generalized to many cobalamin-producing bacteria that are able to metabolize ethanolamine. However, attempts to find screening conditions for the detection of A. tumefaciens Cob mutants have failed, and most A. tumefaciens Cob mutants described in this report grow on Eth medium. A similar approach, based on the vitamin B12 requirement in anaerobiosis for the growth of an S. typhimurium metE mutant, has been described to screen Cob mutants (29). This type of screening can probably be used for bacteria such as R. meliloti, the growth of which depends on the presence of vitamin B12 in the medium when cobalt ions are absent (27, 28). In this case, the vitamin B12-dependent enzymes involved are a ribonucleotide reductase (28) and a methionine synthetase (27). R. meliloti Cob mutants could probably be detected by the absence of growth on a minimal medium and correction of this defect by vitamin B12. Such a screening procedure did not allow the isolation of Cob mutants in A. tumefaciens. At least 14 genes involved in cobalamin synthesis in P. denitrificans have been cloned; 12 are implicated in the conversion of uroporphyrinogen III to cobinamide, and 2 are implicated in the conversion of cobinamide to coenzyme B12. These genes have been identified by heterologous complementation of P. denitrificans or A. tumefaciens Cob mutants with amplified P. denitrificans DNA. We do not know whether the absence of complementation for 10 of 26 P. denitrificans Cob mutants and for 15 of 148 A. tumefaciens Cob mutants is due to the lack of the desired fragments in the genomic library or to problems related to either the absence of heterologous gene expression or differences in genetic organization between P. denitrificans and A. tumefaciens or P. putida. Another possibility is that at least some enzymes involved in such a metabolic pathway are arranged

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J. BACTERIOL.
2. Bagdasarhn, M., R. Lurz, B. Riekert, F. C. Franklin, M. M. Bagdasarian, J. Frey, and K. TImmis. 1981. Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host vector system for gene cloning in Pseudomonas. Gene 16:237-247. 3. Battersby, A. R., and E. MacDonald. 1982. Biosynthesis of the corrin macrocycle, p. 107-144. In D. Dolphin (ed.), B12, vol. 1. John Wiley & Sons, Inc., New York. 4. Beck, W. S. 1982. Biological and medical aspects of vitamin B12, p. 1-30. In D. Dolphin (ed.), B12, vol. 1. John Wiley & Sons, Inc., New York. 5. Beringer, J. E., J. L. Beynon, A. V. Buchanan-Wolaston, and A. W. B. Johnston. 1978. Transfer of the drug-resistance transposon TnS to Rhizobium meliloti. Nature (London) 276:633634. 6. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 7. Bradbeer, C. 1982. Cobalamin transport in microorganisms, p. 145-167. In D. Dolphin (ed.), B12, vol. 2. John Wiley & Sons, Inc., New York. 8. Brey, R. N., C. D. B. Banner, and J. B. Wolf. 1986. Cloning of multiple genes involved with cobalamin (vitamin B12) biosyn-

in multienzymatic complexes (45), which would make it less probable that mutants in the corresponding genes would be complemented by heterologous DNA. On the basis of the percentage of complemented mutants with P. denitrificans amplified DNA, it appears that complementation of A. tumefaciens mutants was more successful than was that of P. putida mutants. It can be deduced from several publications (3, 22, 26, 41, 42, 46) that more than 20 intermediates should be required in the pathway from uroporphyrinogen III to cobalamin. However, the number of enzymes involved in this pathway could be smaller, since (i) during the building of the corrin macrocycle, one could carry out more than one methylation when methyl groups are transferred at equivalent positions (42) (it seems, for instance, that one enzyme in Propionibacterium shermanii catalyzes the transfer of two methyl groups to uroporphyrinogen III [38]); and (ii) during the conversion of cobyrinic acid to cobinamide, the six amidations of the propionic and acetic groups in the corrin macrocycle may not require six different enzymes. Analyses of mutants and their products could establish this point. Moreover, the number of cloned genes is likely to be underestimated because more than one gene could be present on each subclone defining a complementation group. Although we cannot yet determine whether all of the cloned genes are structural or whether some of them play only a regulatory role, our results illustrate the genetic and biochemical complexity of this pathway. The cloned genes are grouped into four genomic regions, and we do not yet know whether those Cob loci are clustered or located at distant positions on the chromosome of P. denitrificans. Clustering of Cob genes is not peculiar to P. denitrificans, having already been reported for B. megaterium and S. typhimurium (8, 29). In B. megaterium, all of the Cob mutations found are linked by cotransduction (48); in S. typhimurium, the same results have been found except for the cysG mutation, which indicates that most of the Cob genes are located in the same region on the chromosome. For P. denitrificans, the cluster would cover at least 78 kbp if all of the Cob genes were linked on the chromosome. Furthermore, the inserts of the 14 subclones complementing Cob mutants (Fig. 3) together represent 32 kbp, which is much more than the total length of 12 kbp for the cloned DNA fragments of B. megaterium containing 11 Cob genes (8); this fact may suggest that P. denitrificans Cob genes are more spread out than are the corresponding genes from B. megaterium.
ACKNOWLEDGMENTS We acknowledge A. Rambach, former president of Gdnetica S.A., for his continued interest and support during this work. We express our gratitude to M. Knapp for his help during the initial part of the work and for his advice. We thank P. E. Bost, G. Bourat, and J. Lunel (Rh6ne-Poulenc Sante) for their constant interest in our work. We express our gratitude to G. Ditta, D. Helinski, F. Richaud, and K. Timmis for the gift of plasmids or strains; J. Davies (Institut Pasteur) and J.-F. Mayaux for critical reading of the manuscript and helpful discussions; and F. Blanche, D. Thibaut, and S. Schil. We also thank S. Bonnamy, L. Cauchois, A. Driver, N. Faulconnier, S. Rigault, and M.-C. Rouyez for their excellent technical assistance. This work was supported by Rhone-Poulenc Sante.
LITERATURE CITED
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thesis in Bacillus megaterium. J. Bacteriol. 167:623-630. 9. Casadaban, M. J., A. Martinez-Arias, S. T. Shapira, and J. Chou. 1983. ,B-Galactosidase gene fusion for analysing gene expression in Escherichia coli and yeast. Methods Enzymol. 100:293-308. 10. Clarke, L., and J. Carbon. 1976. A colony bank containing synthetic ColE1 hybrid plasmids representative of the entire E. coli genome. Cell 9:91-99. 11. Davis, B. D., and E. Mingioli. 1950. Mutants of Escherichia coli requiring methionine or vitamin B12. J. Bacteriol. 60:17-28. 12. De Graff, J., J. H. Crosa, F. Heffron, and S. Falkow. 1978. Replication of the nonconjugative plasmid RSF1010 in Escherichia coli K-12. J. Bacteriol. 146:117-122. 13. Ditta, G., S. Stanfield, D. Corbin, and D. R. Helnski. 1980. Broad host range DNA cloning system for Gram-negative bacteria: construction of a gene library of Rhizobium meliloti. Proc. Natl. Acad. Sci. USA 77:7347-7351. 14. Doudoroff, M., R. Contopoulou, R. Kunisaw, and N. J. PaLeroni. 1974. Taxonomic validity of Pseudomonas denitrificans (Christensen) Bergey et al. Int. J. Syst. Bacteriol. 24:294-300. 15. Doyen, N., A. J. Pesce, and C. Lapresle. 1981. Specificity of two anti-human albumin monoclonal antibodies. Immunol. Lett. 3: 365-370. 16. Engvall, E. 1980. Enzyme immunoassay ELISA and EMIT. Methods Enzymol. 70:419439. 17. Fa, Y. H., J. P. Kusel, and A. L. Demain. 1984. Dependence of betaine stimulation of vitamin B12 overproduction on protein synthesis. Appl. Environ. Microbiol. 47:1067-1069. 18. Figurskl, D. H., and D. R. HeUInskl. 1979. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl. Acad. Sci. USA 76:1648-652. 19. Florent, J. 1986. Vitamins, p115-158. In H.-J. Rehm and G. Reed (ed.), Biotechnology, vol. 4. VCH Verlagsgesellschaft mbH, Weinheim, Federal Republic of Germany. 20. Foster, T. J. 1983. Plasmid-determined resistance to antimicrobial drugs and toxic metal ions in bacteria. Microbiol. Rev. 47:

361409.
21. Frey, J., M. Bagdaan, D. Feiss, F. C. H. Franklin, and J. Dehusses. 1983. Stable cosmid vectors that enable the introduction of cloned fragments into a wide range of Gram-negative bacteria. Gene 24:299-308. 22. Friedmann, H. C. 1975. Biosynthesis of corrinoids, p. 75-109. In B. Babior (ed.), Cobalamin biochemistry and pathophysiology. John Wiley & Sons, Inc., New York. 23. Gilles, H., and R. K. Thauer. 1983. Uroporphyrinogen III, an intermediate in the biosynthesis of the nickel-containing F430 in Methanobacterium thermoautotrophicum. Eur. J. Biochem.

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135:109-112. 24. Gotlieb, C., K. S. Lau, and L. R. Wasserman. 1965. Rapid

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25.

26.

27. 28.
29. 30.
31.

32.
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