JOURNAL OF BACTERIOLOGY, Aug. 1995, p. 4392–4401 Vol. 177, No.

15
0021-9193/95/$04.0010
Copyright 1995, American Society for Microbiology

Biochemical and Molecular Characterization of the Oxidative

Branch of Glycerol Utilization by Citrobacter freundii
ROLF DANIEL, KRISTIN STUERTZ, AND GERHARD GOTTSCHALK*
Institut für Mikrobiologie, Georg-August-Universität Göttingen, D-37077 Göttingen, Germany
Received 3 March 1995/Accepted 15 May 1995

Glycerol dehydrogenase (EC 1.1.1.6) and dihydroxyacetone kinase (EC 2.7.1.29) were purified from
Citrobacter freundii. The dehydrogenase is a hexamer of a polypeptide of 43,000 Da. The enzyme exhibited a
rather broad substrate specificity, but glycerol was the preferred substrate in the physiological direction. The
apparent Kms of the enzyme for glycerol and NAD1 were 1.27 mM and 57 mM, respectively. The kinase is a
dimer of a polypeptide of 57,000 Da. The enzyme was highly specific for the substrates dihydroxyacetone and
ATP; the apparent Kms were 30 and 70 mM, respectively. The DNA region which contained the genes encoding
glycerol dehydrogenase (dhaD) and dihydroxyacetone kinase (dhaK) was cloned and sequenced. Both genes
were identified by N-terminal sequence comparison. The deduced dhaD gene product (365 amino acids)
exhibited high degrees of homology to glycerol dehydrogenases from other organisms and less homology to type
III alcohol dehydrogenases, whereas the dhaK gene product (552 amino acids) revealed no significant homology
to any other protein in the databases. A large gene (dhaR) of 1,929 bp was found downstream from dhaD. The
deduced gene product (641 amino acids) showed significant similarities to members of the s54 bacterial
enhancer-binding protein family.

Microorganisms such as Citrobacter freundii and Klebsiella
pneumoniae are able to grow anaerobically on glycerol as the
sole carbon and energy source (19). In the absence of an
external oxidant, glycerol is fermented by a dismutation pro-
cess involving two pathways, one serving for glycerol oxidation
and the other for the consumption of the reducing equivalents
generated. The oxidation of glycerol is catalyzed by NAD1-
linked glycerol dehydrogenase, which converts the substrate to
dihydroxyacetone (DHA). This product is then phosphorylated
by DHA kinase and funneled to glycolysis (15). Generation of
NAD1 is achieved by the sequential action of coenzyme B12-
dependent glycerol dehydratase and NADH-linked 1,3-pro-
panediol dehydrogenase (15). Glycerol is first converted to
3-hydroxypropionaldehyde, which then is reduced to 1,3-pro-
panediol, accounting for about 50 to 66% of the glycerol con-
sumed. The four key enzymes of this pathway are encoded by
the dha regulon, the expression of which is induced when DHA
or glycerol is present (15, 34). The entire dha regulon was
cloned from C. freundii (10) and K. pneumoniae (48), but mo-
lecular data are available only for C. freundii.
Recently we have subcloned, sequenced, and overexpressed
the gene encoding 1,3-propanediol dehydrogenase (9). In this
report, we describe the purification of glycerol dehydrogenase
(EC 1.1.1.6) and DHA kinase (EC 2.7.1.29) from C. freundii
and the cloning, identification, and organization of the corre-
sponding genes.
MATERIALS AND METHODS
Materials. Q-Sepharose Fast Flow, Reactive Red, and Blue Sepharose CL-6B
were obtained from Pharmacia LKB GmbH (Freiburg, Germany), and hydroxy-
apatite was from Sigma Chemie (Deisenhofen, Germany). Tris, EDTA, and
sodium dodecyl sulfate were from Serva (Heidelberg, Germany). 1,3-Pro-
panediol and 1,2-propanediol were purchased from Merck (Darmstadt, Germa-
ny). All other reagents used were commercial products of the highest grade
available.
* Corresponding author. Mailing address: Institut für Mikrobiolo-
gie, Georg-August-Universität Göttingen, Grisebachstrasse 8, 37077
Göttingen, Germany. Phone: (49) 551-393781. Fax: (49) 551-393793.
Bacterial strains, plasmids, and growth conditions. C. freundii DSM 30040
was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkul-
turen GmbH, Braunschweig, Germany. The genes coding for glycerol dehydro-
genase and DHA kinase were isolated from the recombinant cosmid pRD1,
which harbors a 32-kb chromosomal DNA insert from C. freundii (10). Esche-
richia coli ECL707 (43) and JM109 (57) were used as hosts, and pBR322 (45) and
pUC18 (52) were used as the vectors for the cloning and expression experiments.
C. freundii was grown in a 300-liter fermentor under anaerobic conditions in a
medium containing the following (per liter): glycerol, 9.0 g; K2HPO4, 14.0 g;
KH2PO4, 6.0 g; (NH4)2SO4, 3.0 g; MgSO4 7H2O, 0.2 g; CoCl2 6H2O, 0.2 g;
yeast extract, 0.2 g; cysteine-HCl, 0.2 g; and trace element solution SL4 (31), 1
ml, pH 7.5. After 22 h of incubation at 378C, the cells were harvested by
centrifugation at 18,600 3 g and 48C. Cells were stored at 2208C until use. For
purification of the enzymes, the cells (20 g) were washed once with 50 mM
potassium phosphate buffer, pH 8.0 (glycerol dehydrogenase), or with 20 mM
Tris-HCl buffer, pH 7.0, containing 0.5 mM EDTA (DHA kinase) and resus-
pended in 20 or 40 ml of the same buffer, respectively. The cells were disrupted
by treatment with a French press (1.38 3 108 Pa), and the extract was cleared by
centrifugation at 119,000 3 g and 48C for 2 h.
E. coli was routinely grown at 308C in LB medium (36), which was supple-
mented with ampicillin (100 mg/ml) when necessary. Recombinant E. coli strains
used for expression of genes encoded by the dha regulon were grown at 288C in
the medium described above supplemented with sodium pyruvate (0.7 g) and
casein acid hydrolysate (3.0 g).
Recombinant E. coli cells in the stationary growth phase from 500-ml anaer-
obic cultures were harvested by centrifugation at 6,000 3 g for 20 min, washed
once with 100 mM potassium phosphate buffer (pH 8.0), and resuspended in 2 to
3 ml of the same buffer. The cells were disrupted as described above, and the
extract was cleared by centrifugation at 32,000 3 g and 48C for 35 min. All steps
were done under anaerobic conditions.
Assays. Glycerol dehydrogenase activity was determined by the method of Ruch
et al. (34), and DHA kinase activity was determined by the method of Johnson et
al. (21). Assays on glycerol dehydratase were done by the method of Toraya
et al. (49) and on 1,3-propanediol dehydrogenase as described by Boenigk et al.
(5). All enzyme activities are expressed in micromoles per minute (units).
Protein concentrations were determined by the method of Bradford (6), with
bovine serum albumin as the standard.
Purification of glycerol dehydrogenase. All purification steps were carried out
under aerobic conditions, and the buffer used was 50 mM potassium phosphate
buffer (pH 7.0) unless otherwise stated.
(i) Anion-exchange chromatography. The supernatant fraction of the cell
extract from C. freundii (160.6 mg of protein in 7 ml) was loaded onto a Q-
Sepharose Fast Flow column (22 ml) at room temperature. The column was
washed with 60 ml of buffer and then with 60 ml of buffer containing 100 and 125
mM (NH4)2SO4 sequentially (flow rate, 0.5 ml/min). The glycerol dehydrogenase
was eluted with 175 mM (NH4)2SO4. Enzymatically active fractions were pooled
and desalted by passing once through prepacked disposable Sephadex G-25
columns (Pharmacia LKB GmbH) as recommended by the manufacturer.
(ii) Affinity chromatography. The following procedure was carried out at 48C.

4392
VOL. 177, 1995 ANAEROBIC GLYCEROL UTILIZATION BY C. FREUNDII 4393

The pooled and desalted fractions (27 ml) were applied to a Blue Sepharose TABLE 1. Purification of glycerol dehydrogenase
CL-6B column (40 ml) which had been equilibrated with buffer containing 20
mM glycerol. After the column was washed with 100 ml of equilibration buffer, Total Purifi-
Protein Sp act Recovery
the glycerol dehydrogenase was eluted with a 225-ml linear gradient of 0 to 1.2 Step activity cation
(mg) (U/mg) (%)
mM NAD1 (flow rate, 0.3 ml/min). Active fractions were pooled, and the (U) (fold)
coenzyme was removed by passing through disposable Sephadex G-25 columns
as described above. Unless otherwise specified, this preparation was used for Clarified extract 350 161.60 2.2 1 100
characterization of the enzyme. Q-Sepharose Fast Flow 142 4.85 29.3 13 41
Purification of DHA kinase. All purification steps were carried out at room Blue Sepharose CL-6B 55 0.84 65.5 30 16
temperature under aerobic conditions, and all buffers used were supplemented
with 3 mM dithioerythritol (DTE) and 0.1 mM phenylmethylsulfonyl fluoride
(PMSF).
(i) Anion-exchange chromatography. The cleared cell extract (372.6 mg in 15 a Macrophor sequencing unit (Pharmacia LKB GmbH) as recommended by the
ml) was applied to a Q-Sepharose Fast Flow column (25 ml) equilibrated with 20 manufacturer.
mM Tris-HCl buffer, pH 8.2. After the column was washed with 50 ml of Computer analysis. The DNA sequence data and the deduced amino acid
equilibration buffer and 50 ml of equilibration buffer containing 200 mM NaCl, sequences were analyzed with the DNA Strider program (27) on a Macintosh
the DHA kinase was eluted with a 200-ml linear gradient of 200 to 400 mM NaCl Performa 450 computer (Apple Computer, Inc., Cupertino, Calif.). Further
(flow rate, 0.3 ml/min). Enzymatically active fractions were pooled and concen- sequence analyses were carried out on a VAX 9000 computer, with the Genetics
trated by pressure dialysis (Diaflo-YM-30-membrane; Amicon, Beverly, Mass.) Computer Group, Inc., sequence analysis software package, version 6.0 (11).
against 10 mM potassium phosphate buffer, pH 6.8. Nucleotide sequence accession number. The sequence data presented were
(ii) Hydroxyapatite chromatography. The concentrated solution (7.5 ml) was submitted to the GenBank database and assigned accession number U09771.
then applied to a column of hydroxyapatite (8 ml) equilibrated with 10 mM
potassium phosphate buffer, pH 6.8. The DHA kinase was eluted by washing with
equilibration buffer (flow rate, 0.5 ml/min). The active fractions were collected. RESULTS
(iii) Affinity chromatography. Solid ammonium sulfate was added to the en- Enzyme purification. The two-step procedure devised for
zyme solution (12 ml) to give a 100 mM final concentration. This solution was
applied to a column of Reactive Red 120 (7 ml) equilibrated with 50 mM
the purification of glycerol dehydrogenase yielded a 30-fold
potassium phosphate buffer, pH 6.5, containing 0.5 mM MgCl2 and 0.5 mM enrichment of the protein, with a 16% recovery (Table 1). The
(NH4)2SO4. After the column was washed with 15 ml of equilibration buffer, the last step involved the elution of the dehydrogenase from the
DHA kinase was eluted with a 30-ml linear gradient of 0 to 400 mM NaCl in 50 Blue Sepharose CL-6B matrix by adding the coenzyme. The
mM potassium phosphate buffer, pH 7.5 (flow rate, 0.5 ml/min). The active purified glycerol dehydrogenase was stable for several months
fractions were pooled and used for characterization of the enzyme.
Removal of metal ions. For removal of any reversibly bound metal ions, when stored at 2208C in 50 mM potassium phosphate buffer
enzyme preparations were passed twice through a prepacked disposable Seph- (pH 7.0).
adex G-25 column (Pharmacia) as recommended by the manufacturer. The three steps that led to a 111-fold purification of DHA
Determination of molecular masses. Analytical sodium dodecyl sulfate-poly- kinase with 11% recovery are summarized in Table 2. The
acrylamide gel electrophoresis was carried out in 10% polyacrylamide slab gels at
258C by the procedure of Laemmli (25). A commercial sodium dodecyl sulfate
enzyme was stable at 2708C in Tris-HCl buffer, pH 7.0, con-
molecular mass calibration kit of standard proteins (Sigma Chemie) served as the taining 3 mM DTE, 0.1 mM PSMF, and 0.5 mM EDTA for
subunit molecular mass standard. Protein bands were located by staining with several weeks.
AgNO3 (4). Molecular masses and subunit composition. The final glyc-
The sedimentation coefficient of the purified glycerol dehydrogenase was de- erol dehydrogenase preparation migrated as a single band cor-
termined by sedimentation in a linear sucrose density gradient (20 to 50%
[wt/vol] sucrose) by the method of Martin and Ames (28), with catalase (231,000
responding to a molecular mass of 43,000 Da during sodium
Da) and aldolase (158,000 Da) as the standards. Centrifugation was done for 16 dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 1).
h at 48C and 193,000 3 g. The Stokes’ radius of the enzyme was determined by Analysis of the dehydrogenase by gel filtration on a fast protein
gel filtration in a buffered Pharmacia Superose 12-prepacked 10/30 column by liquid chromatography (FPLC) column and by sedimentation
use of a computer-controlled low-pressure liquid chromatography system (Phar-
macia LKB GmbH) as recommended by the manufacturer. Ferritin (440,000
in a linear sucrose density gradient gave a Stokes’ radius of 5.73
Da), catalase (232,000 Da), and aldolase (158,000 Da) were used as standards. nm and a sedimentation coefficient of 10.42S, respectively. The
The native molecular mass of glycerol dehydrogenase was calculated from the native molecular mass of the purified glycerol dehydrogenase,
sedimentation coefficient, and the Stokes’ radius was calculated by the method of calculated by the equation of Siegel and Monty (40), was
Siegel and Monty (40).
Kinetic data. Km values for the substrate and the coenzyme were determined
246,000 Da. This result suggests that the native enzyme is a
from Lineweaver-Burk plots derived from the results of experiments in which a hexamer.
fixed concentration of substrate or coenzyme and an appropriate range of con- When the DHA kinase was subjected to gel electrophoresis
centrations of the other reactant were used. in the presence of sodium dodecyl sulfate, a protein band
Determination of N-terminal amino acid sequence. The N termini of peptides
were determined by automated Edman degradation. For this purpose, purified
corresponding to a molecular mass of 57,000 Da was observed
DHA kinase was separated by sodium dodecyl sulfate-polyacrylamide gel elec- (Fig. 1). Gradient gel polyacrylamide electrophoresis under
trophoresis and blotted onto a polyvinylidene difluoride membrane as described nondenaturing conditions gave a major band corresponding to
by Kyhse-Andersen (24). The final enzyme preparation of glycerol dehydroge- a native molecular mass of 105,000 Da (data not shown). This
nase was used directly for peptide sequencing.
Nucleic acid isolation and manipulation. Plasmid isolation from E. coli was
result suggests that the native kinase is a dimer.
performed with the Quiagen Midi kit (Diagen GmbH, Düsseldorf, Germany). Substrate specificity and kinetic properties. The glycerol
DNA manipulations were done by standard methods (36). Restriction enzymes dehydrogenase exhibited a rather broad substrate specificity
and T4 DNA ligase were obtained from GIBCO/BRL GmbH (Eggenstein, Ger- (Table 3). Glycerol, 1,2-propanediol, and 2,3-butanediol were
many) and used according to the manufacturer’s instructions.
DNA sequencing. Double-stranded plasmid DNA was sequenced by the
dideoxy chain termination method (37), with [35S]dATP (DuPont NEN Research
Products, Bad Homburg, Germany) and a Sequenase version 2.0 DNA sequenc- TABLE 2. Purification of DHA kinase
ing kit from US Biochemicals (Braunschweig, Germany) according to the pro-
tocol given by the manufacturer. The entire sequence of the C. freundii DNA Total Purifi-
Protein Sp act Recovery
insert was determined for both strands. Sequencing started within the vector Step activity cation
(mg) (U/mg) (%)
pUC18, using the commercial sequencing forward and reverse primers (US (U) (fold)
Biochemicals). Further sequencing was carried out by using sequentially synthe-
sized oligonucleotides (17-mers) fitting to the ends of the already determined Clarified extract 27.1 372.6 0.07 1 100
DNA sequences (primer walking). Oligonucleotides were synthesized on a Gene Q-Sepharose Fast Flow 17.0 33.2 0.51 7 63
Assembler Plus (Pharmacia LKB GmbH) according to the manufacturer’s in- Hydroxyapatite 16.5 8.5 1.94 28 61
structions. The dideoxy-terminated fragments were separated on 55-cm wedge- Reactive Red 3.1 0.4 7.80 111 11
shaped thickness gradient gels (0.2 to 0.4 mm, 6% [wt/vol] polyacrylamide) with
4394 DANIEL ET AL.
FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of puri-
fied glycerol dehydrogenase and DHA kinase. The purified protein was subjected
to electrophoresis on a 10% polyacrylamide slab gel in the presence of 0.1%
sodium dodecyl sulfate. The protein bands were stained with AgNO3. (A) Glyc-
erol dehydrogenase. Lanes: 1, molecular mass markers (bovine albumin, 67,000
Da; ovalbumin, 45,000 Da; carbonic anhydrase, 29,000 Da; trypsin inhibitor,
20,100 Da; and lactalbumin, 14,200 Da); 2, 1 mg of purified glycerol dehydroge-
nase. (B) DHA kinase. Lanes: 1, molecular mass markers (bovine albumin,
67,000 Da; ovalbumin, 45,000 Da; carbonic anhydrase, 29,000 Da; trypsinogen,
24,000 Da; trypsin inhibitor, 20,100 Da; and lactalbumin, 14,200 Da); 2 and 3,
0.15 and 1.5 mg of purified DHA kinase, respectively.
oxidized preferentially. Lower primary alcohols were oxidized
with rates below 10% of that with glycerol. In the reverse
reaction, DHA was the most active substrate. NADP1 was not
used as an electron acceptor for the oxidation of glycerol. ADP
and ATP had no significant effect on enzyme activity when
added to the assay mixture at 1 or 10 mM. Glycerol dehydro-
genase displayed classical Michaelis-Menten kinetics, with ap-
parent Kms for glycerol of 1.27 mM and for NAD1 of 57 mM.
DHA kinase exhibited Michaelis-Menten kinetics for DHA
and ATP, with apparent Kms of 30 and 70 mM, respectively. No
significant kinase activity was found when glycerol, glyceralde-
hyde, or hydroxyacetone was tested as the substrate or when
ATP was replaced by CTP, GTP, UTP, ITP, ADP, or AMP.
Activation of the enzymes by divalent cations. When glycerol
dehydrogenase was treated with EDTA and separated from
the chelator by gel filtration, the specific activity had dropped
TABLE 3. Substrate specificity of glycerol dehydrogenase
Relative
Substratea activityb
(%)
Oxidation
Glycerol........................................................................................ 100
DL-a-Glycerophosphate..............................................................
1,2-Propanediol ........................................................................... 66
1,3-Propanediol ........................................................................... 29
2,3-Butanediol ............................................................................. 63
1,3-Butanediol .............................................................................
Ethylene glycol ............................................................................ 33
Reduction
DHA ............................................................................................. 100
DL-Glyceraldehyde ......................................................................
Glycolaldehyde ............................................................................ 34
Propionaldehyde ......................................................................... ,1
a
Enzyme activity of the oxidation reaction was determined with 3.5 mg of
purified enzyme under the assay conditions described in Materials and Methods.
The activity of glycerol dehydrogenase in reduction reactions was determined
spectrophotometrically (E365) at 258C by the initial rate of substrate-dependent
NADH decrease. The assay mixture contained 50 mM MES (morpholineethane-
sulfonic acid) buffer (pH 6.0), 0.25 mM NADH, 0.5 mM substrate, and 2.5 mg of
purified enzyme in a 1-ml final volume.
b
Activities are expressed relative to that obtained with glycerol (5.1 U/ml) for
the oxidation reaction or with DHA (2.3 U/ml) for the reduction reaction.
J. BACTERIOL.
TABLE 4. Levels of enzymes encoded by the dha regulon in crude
extracts of E. coli ECL707/pRD18 and ECL707/pRD1
Sp act of dha-encoded enzymesa
(U/mg of protein)
Strain GLYC 1,3-PD
DHA GLYC
dehydrog- dehydrog-
kinase dehydratase
enase enase
E. coli ECL707/pRD18 6.67 0.191 ND 1.68
E. coli ECL707/pRD1 5.92 0.133 1.6 0.81
E. coli ECL707 ,0.01 ,0.009 ND ,0.01
C. freundii 5.12 0.114 0.9 0.90
a
Abbreviations: GLYC, glycerol; PD, propanediol; ND, not detectable.
from 65.5 to 27.7 U/mg. Addition of Mg21 or Zn21 had no
effect on restoring the activity, but the activity was increased
fourfold by 100 mM Mn21. The glycerol dehydrogenase activity
was inhibited 75% by addition of 100 mM Zn21 to the final
enzyme preparation, whereas 100 mM Mg21 had no effect and
100 mM Mn21 yielded a twofold stimulation.
DHA kinase activity depended on divalent cations such as
Mg21 and Ca21, as is typical of most kinases.
Cloning and sequencing of the genes encoding glycerol de-
hydrogenase and DHA kinase. The recombinant cosmid
pRD1, which contains a 32-kb insert of C. freundii genomic
DNA, harbors the entire dha regulon of C. freundii, as de-
scribed previously (10). Recombinant E. coli strains carrying
pRD1 are able to grow anaerobically with glycerol as the sole
carbon and energy source when vitamin B12 is present in the
growth medium (10). After digestion of pRD1 with several
restriction enzymes, ligation into pBR322, and transformation
into the glycerol-minus mutant E. coli ECL707, one subclone
(E. coli ECL707/pRD18) that had glycerol dehydrogenase,
DHA kinase, and 1,3-propanediol dehydrogenase activities in
cell extracts was obtained (Table 4). This clone contained a
7,969-bp Sau3AI-HindIII insert of C. freundii genomic DNA.
The origin of the cloned C. freundii DNA was established by
Southern blot analysis (data not shown). To generate more
starting points for sequencing, subclones of the insert in
pUC18 were produced (Fig. 2).
The entire cloned fragment was sequenced in both direc-
tions. The restriction map and the apparent gene organization
are shown in Fig. 2, and part of the sequence, including the
genes encoding glycerol dehydrogenase (dhaD) and DHA ki-
nase (dhaK), is given in Fig. 3. Seven potential genes were
27 identified within the sequence. Two of the genes are located at
the ends of the cloned DNA and are hence incomplete. All
presumptive genes except the incomplete ones were preceded
by a potential ribosome-binding site, appropriately spaced
8 from the start codon (38).
Identification of the dhaD and dhaK genes. The genes en-
coding glycerol dehydrogenase and DHA kinase were identi-
fied by N-terminal sequence comparison. The N-terminal
4 amino acid sequences of purified glycerol dehydrogenase (30
amino acids) and DHA kinase (13 amino acids), determined by
Edman degradation, were identical to those deduced from the
sequences of the dhaD and dhaK genes, respectively, except
that the initial methionine was not present in the mature DHA
kinase. The dhaD gene (1,098 bp) codes for 365 amino acids,
and the dhaK gene (1,659 bp) codes for 552. The predicted
molecular masses are 38,993 and 57,772 Da, respectively, and
are in good accordance with the molecular masses determined
for the purified enzymes by sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis.
VOL. 177, 1995
FIG. 2. Schematic map of the cloned chromosomal DNA region from C. freundii containing parts of the dha regulon. The restriction map of the 7,969-bp
Sau3AI-HindIII insert of pRD18 is shown. Arrows and arrowheads represent the length, location, and orientation of potential genes. Only the flanking Sau3AI
restriction site is shown. The locations of genomic C. freundii inserts in recombinant plasmids, which were used for sequencing, are given below the restriction map.
The dhaK and dhaD genes are both terminated by the same
single stop codon (UAA). A sequence that could represent a
transcriptional terminator (a punctuated palindrome that could
form a stem-loop structure in an RNA transcript) follows ap-
proximately 37 nucleotides (nt) downstream from the stop
codon of dhaD and 28 nt downstream from the stop codon of
dhaK (Fig. 3). The sequence upstream from the ribosome-
binding site of dhaD and dhaK showed no homology to the s70
promoter consensus sequence described for E. coli (18).
The amino acid sequences deduced from dhaD and dhaK
were compared with deduced amino acid sequences for pro-
teins available in the EMBL and NBRF databases. The dhaD
gene product showed a high degree of homology to the glycerol
dehydrogenase (gldA) of E. coli (3) and Bacillus stearother-
mophilus (26), the fraction of identical amino acids being 57.4
and 49.6%, respectively. Lower homology values (21.0 to
23.7% identity, 44.3 to 50.2% similarity) to a novel family of
alcohol dehydrogenases (type III) were obtained, including,
e.g., Adh2 of Zymomonas mobilis (8) and AdhE of E. coli (17;
for a review, see reference 33).
The deduced dhaK gene product revealed no significant
homology to any other protein whose sequence is available in
databases. The triad G-K-G (amino acids 70 to 72) is the
central part of the putative ATP-binding site of the glycerol
kinase from E. coli (30) and adenylate kinase from Oryctolagus
cuniculus (16).
Other genes in the region. The coding region of part of the
gene expressed from the strand opposite that of the dhaK gene
could represent the 89-amino-acid, C-terminal end of a pro-
tein. A comparison of this sequence with proteins available
in the EMBL and NBRF databases showed 70.8% identity
(80.9% similarity) with the C-terminal region of cyclopropane
fatty acid synthase (Cfa) from E. coli (54). The C. freundii gene
was therefore designated cfa.
Separated by a 90-bp intergenic region, a large gene (dhaR)
of 1,929 bp was found downstream from dhaD. The deduced
amino acid sequence (641 amino acids, 71,390 Da) showed
about 25% identity (50% similarity) to members of the s54
bacterial enhancer-binding protein family; e.g., NifA of K.
pneumoniae (1), FlbD of Caulobacter crescentus (32), AcoR of
Alcaligenes eutrophus (23), and DmpR of a Pseudomonas sp.
(39). An alignment of DhaR to DmpR and AcoR is depicted in
Fig. 4. The conserved domains of these activator proteins,
ANAEROBIC GLYCEROL UTILIZATION BY C. FREUNDII 4395
which are responsible for their interaction with the s54-depen-
dent RNA polymerases (for a review, see reference 29), exhib-
ited even higher homology to the central region of DhaR, and
the fraction of identical amino acids varied between 32.8 and
40.0%. The domain also included two conserved regions, which
represent putative ATP-binding sites (Fig. 4). The C-terminal
region contained a helix-turn-helix motif, which is characteris-
tic of DNA-binding proteins (Fig. 4). In contrast, the N-ter-
minal region of DhaR exhibited no significant homology to the
corresponding region of activator proteins from other organ-
isms or to any other gene product whose sequence is available
from the above-mentioned databases. The nucleotide se-
quence immediately downstream of the dhaR gene does not
contain any obvious terminator-like sequences. A role for
DhaR in the regulation of anaerobic glycerol utilization is
indicated by measuring the glycerol dehydrogenase activity of
subclones which were generated for sequencing. The recombi-
nant E. coli strain containing pRD13 (complete dhaR gene;
Fig. 2) showed high glycerol dehydrogenase activity, whereas
the clone harboring pRD15 (incomplete dhaR gene; Fig. 2)
lacked significant activity. This result is in accordance with the
putative function of the dhaR gene product as a transcriptional
activator.
The deduced products of the remaining three presumptive
genes, orfW, orfX, and the incomplete orfY, showed no signif-
icant homology to sequences of proteins available in the data-
bases. The dhaT gene codes for the 1,3-propanediol dehydro-
genase of C. freundii, as described previously (9).
DISCUSSION
The oxidative branch of anaerobic glycerol fermentation by
C. freundii requires the sequential action of two enzymes, glyc-
erol dehydrogenase and DHA kinase. Glycerol dehydrogenase
catalyzes the initial oxidation of glycerol. In this study, we
present biochemical and molecular data for both enzymes.
Glycerol dehydrogenases have been purified from various
microorganisms, including K. pneumoniae (35), E. coli (46), Cel-
lulomonas sp. (56), and B. stearothermophilus (42). All known
glycerol dehydrogenases consist of identical subunits, with a
molecular mass of about 40,000 Da. This in accordance with
the data on purified glycerol dehydrogenase from C. freundii.
4396 DANIEL ET AL. J. BACTERIOL.

FIG. 3. Nucleotide sequence of the cloned region. Only one strand is shown. The genes encoding glycerol dehydrogenase (dhaD), DHA kinase (dhaK), the putative
activator protein (dhaR), and the C-terminal region of cyclopropane fatty acid synthase (cfa) have been translated in the one-letter amino acid code; amino acid symbols
are written below the first nucleotide of the corresponding codon. Putative ribosome-binding sites are underlined, and putative secondary structures are marked by open
arrows, indicating the lengths and orientation of the stems. The sequence of the 7,969-bp Sau3AI-HindIII insert has been submitted in full to GenBank under accession
number U09771. The sequence of the gene encoding 1,3-propanediol dehydrogenase (dhaT) has been described by Daniel et al. (9).

The biochemical properties of the C. freundii enzyme are sim- ions, and inhibition by zinc ions. Since the gene encoding
ilar to those of the E. coli and K. pneumoniae enzymes in the glycerol dehydrogenase (dhaD) of C. freundii has been cloned
following aspects: sensitivity to chelators of divalent cations, and sequenced, a comparison on the molecular level can now
relatively low substrate specificity, activation by manganese be made. The dhaD gene codes for 365 amino acids and ex-
VOL. 177, 1995 ANAEROBIC GLYCEROL UTILIZATION BY C. FREUNDII 4397

FIG. 3—Continued.

hibits a high degree of similarity to the genes encoding glycerol
dehydrogenase from E. coli and B. stearothermophilus. In ad-
dition, sequence studies revealed homologies of the dhaD gene
product to a novel family of alcohol dehydrogenases which
include DhaT of C. freundii (9), Adh2 of Z. mobilis (8), Adh4
of Saccharomyces cerevisiae (55), Mdh of Bacillus methanolicus
C1 (12), 4Hbd of Clostridium kluyveri (41), FucO and AdhE of
E. coli (7, 17), and Adh1, BdhA, BdhB, and AdhE of Clostrid-
ium acetobutylicum (14, 53, 58). This group of enzymes, type III
alcohol dehydrogenases, is very heterogeneous and distinct
4398 DANIEL ET AL. J. BACTERIOL.

FIG. 3—Continued.

from the horse liver-type alcohol dehydrogenase (type I) and
the Drosophila-type alcohol dehydrogenase (type II).
The subunit size of the type III enzymes is approximately
40,000 Da, except for the multifunctional AdhE of E. coli (17)
and a similar enzyme from C. acetobutylicum (14), which is
approximately 95,000 Da. Bairoch (2) proposed a putative
iron-binding motif (G-X-X-H-X-X-A-H-X-X-G-X-X-X-X-X-
P-H-G) as a fingerprint pattern for type III alcohol dehydro-
genases. It is fully conserved in all reported iron-dependent
enzymes (Adh2 from Z. mobilis [50, 51], DhaT from C. freundii
[9], and FucO and AdhE from E. coli [22, 44]) and in Adh4 of
S. cerevisiae, which requires Zn21 for its catalytic activity (13,
55). Like the glycerol dehydrogenases of E. coli and B. stearo-
thermophilus and some type III alcohol dehydrogenases, the
VOL. 177, 1995 ANAEROBIC GLYCEROL UTILIZATION BY C. FREUNDII 4399

FIG. 3—Continued.

dhaD gene product of C. freundii lacks the iron-binding motif.
However, glycerol dehydrogenases show general homology
to this class of alcohol dehydrogenases and are likely a sub-
class.
DHA kinase has been purified from K. pneumoniae (21).
This enzyme is a dimer of a 53,000 6 5,000-Da polypeptide,
highly specific for DHA, and active with Ca21 or Mg21 as the
cationic cofactor. The data for the DHA kinase of C. freundii,
as determined during purification, correspond to these results.
The dhaK gene codes for 552 amino acids; the starting methi-
onine is lacking, as became apparent from the determination
of the N terminus. A molecular mass of 57,772 Da can be
calculated. Sequence homology studies revealed no significant
homologies to glycerol kinases although they exhibit DHA
kinase activity, or to any other protein whose sequence is
available in the databases. The DHA kinase of C. freundii and
glycerol kinase of E. coli have some interesting structural
similarities. Both enzymes are composed of subunits of simi-
lar size. Glycerol kinase, however, exists as a tetramer (47),
whereas DHA kinase is a dimer. Both enzymes contain the
amino acid motif G-K-G as the central part of the putative
ATP-binding site.
Separated by a 90-bp intergenic region, the dhaR gene, cod-
ing for 641 amino acids, was found downstream from dhaD.
The data obtained from amino acid sequence comparison
point to a regulatory nature of DhaR and indicate that DhaR
belongs to the s54 bacterial enhancer-binding protein family.
Homologies were encountered in the central region of approx-
imately 200 amino acids, including two putative ATP-binding
motifs, and with the C-terminal DNA-binding domain. The
central and C-terminal regions of these regulatory proteins are
conserved, whereas the N-terminal regions, which provide spe-
4400 DANIEL ET AL. J. BACTERIOL.

FIG. 4. Homology of the putative dhaR gene product to other regulatory proteins. The amino acids of AcoR (A. eutrophus), DmpR (Pseudomonas sp.), and DhaR
(C. freundii) are aligned. Matching amino acids are marked by bold letters. Dashed lines indicate gaps which were introduced to optimize the alignment. The conserved
central region of the protein, which is responsible for the interaction of the protein with the s54-dependent RNA polymerases, is boxed. The putative ATP-binding sites
are marked by shaded boxes. The putative helix-turn-helix motifs are underlined.

cific regulatory functions, are often variable (29). Only those
activators which are modified by sensor proteins interacting
with the N termini of the regulators, such as NtrC and NifA,
exhibit homologies of the N-terminal domains. Since the N-
terminal region of DhaR shows no homologies to any other
activator protein, the molecular data for dhaR provide no
evidence for the occurrence of a separate sensor protein in C.
freundii. A similar lack of homology was reported for the N-
terminal regions of XylR of Pseudomonas putida (20) and
AcoR of A. eutrophus (23). Further studies will focus on char-
acterization of the dhaR gene and the corresponding gene
product in order to examine the involvement of DhaR in the
regulation of glycerol fermentation by C. freundii. This will also
clarify which s factor is responsible for the transcription of the
dha regulon.
ACKNOWLEDGMENTS
This work was supported by the Deutsche Forschungsgemeinschaft
within the Forschungsschwerpunkt ‘‘Neuartige Reaktionen und Kata-
lysemechanismen bei anaeroben Mikroorganismen.’’
We thank Bernhard Schmidt (University of Göttingen, Göttingen,
Germany) and Hartmut Kratzin (Max-Planck-Institut, Göttingen, Ger-
many) for performing the peptide sequencing.
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