Vol. 171, No.

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

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.

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.

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
*

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),

Corresponding author.

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.

547

548

J. BACTERIOL.

CAMERON ET AL.
COBYRINIC ACID

UROPORPHYRINOGEN III
COCH

SUCCINYL CoA
CONC

HOOC

CONH

HEMES
L2

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

Relevant genotype, phenotype, or properties

Bacterial strain or plasmid

Strain
E. coli
MC1060
LG90
J53
113-3
113-3 Cbll
P. putida
KT2440
KT2440 Rif Nalr
P. denitrificans SC510

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+

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

Source or reference

9
ATCC 37114
5
11
This paper
2
This paper

Rh6ne-Poulenc Santd

A. tumefaciens

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

25
This paper

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)

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.

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).

J. BACTERIOL.
BE

pFR237

s4ri

c
X

I19 kb

17 kb

mb

Ric

x
H

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

xc H P

x
p

~~~~pXL435
iO
1.6 kcb

pXL59

pXL59

mb

sri

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

sri

Xb

ob
I

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

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.

551

TABLE 2. Cobalamin biosynthesis-deficient mutants

of P. putidaa
Strain

Phenotype

KT2440 Rif' Nalr

G547
G548
G549
G550
G551
G552
G553
G554
G555
G556
G557

G558
G559
G560
G561
G562
G563

G566
G567

G568
G570
G571
G572

G573
G576
G577

Cobalamin Cofncn

Mutant class

53

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

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

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

0.06
0.08
0.8
3
0.1
0.04
S

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

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

Phenotype

Mutant
class

Cobalamin concn

VI
II

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

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

(,ug/liter)
500

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

<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,

COENZYME B12 BIOSYNTHIESIS IN P. DENITRIFICANS

VOL. 171, 1989

553

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

pXL151

G558,b G570

pXL151, pXL152

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

G559/b G560/b G556,b G561b

G159,b G169,/ G258,b G2035

G161,/ G171,b G2034, G2037

pXL153, pXL154
pXL154

pXL156

G549, .c G562,b G573c

pXL156, pXL157
pXL157
pXL157, pXL158, pXL160, pXL161

G554/' G572, G577

pXL157-pXL161
D

G164/' G261,/ G609, G610, G638, G1985G1998,

G2056, G2057
G166,b G611-617, G619, G620, G1963-1984

pXL519

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

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.
a

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

CAMERON ET AL.

J. BACTERIOL.

HE

GROUP A

Be

pXL 151

1 kb

pXL 152
8

pXL444 pXL67S
8

xa

so

pXL452

pXLSS8

B9

Sg

pXL436

GROUP B

pXLSOO

Hl~~~~IS B HHS
I I I I 1111 -1 I I 11I

IXES
E

XHB BH

E ESaXs
X

XX

I I

11

I1

pXL1 53

pXL 154
pXL735 E

pXLS37

GROUP C

H
S

ESSS8

XSB

SB

SIX

II-

I II

I
- I I
1
I
I

SH H

Se E X

I I

pXLI 56

pXL157
pXLI58

pXLI 59
pAL

VU

pXL6IS
I

I-

pXL61 7

pXL556
H

pXL227

EcoRV

S.u

pXL622

Sau

pXL189

pXL239
EI

BI

pXL220
Sau

II

pXLl90

Sou

C
pXL1 91

GROUP D
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.

COENZYME B12 BIOSYNTHESIS IN P. DENITRIFICANS

VOL. 171, 1989

555

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

Subclones that

Group

complement mutants

pXL444
pXL678
pXL452
pXL684

pXL436
pXL300b
B

pXL735
pXL837

pXL617
pXL622
pXL227
pXL556
pXL239
pXL220

P.

putida

A. tumefaciens

G551 (4), G559 (4), G561 (4)

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

G159 (VI), G169 (VI), G258 (VI), G2035

G161 (V), G171 (V), G2034, G2037

G170 (III), G260 (III), G262 (III), G624, G629

G642,c G2043C
G632, G633
G637, G648
G643

G562 (1)
G549 (1), G573
G554 (1), G577
G572

pXL189d
pXL191f

G631
G162 (IV), G256 (IV), G635, G636, G639
G160 (II), G165 (II), G634

pXL698

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

pXL190e
D

G612
G614, G616, G617
G609, G610
G166 (1), G620
G615
G164 (1), G261 (1), G613, G611, G619, G638

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

556

CAMERON ET AL.

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.
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