Biosynthesis of UDP-glucuronic acid and UDP-galacturonic acid in Bacillus cereus subsp. cytotoxis NVH 391-98

Article abstract of DOI:10.1111/j.1742-4658.2011.08402.x

The food borne pathogen Bacillus cereus produces uronic acid-containing glycans that are secreted in a shielding biofilm environment, and certain alkaliphilic Bacillus deposit uronate-glycan polymers in the cell wall when adapting to alkaline environments. The source of these acidic sugars is unknown and, in the present study, we describe the functional identification of an operon in Bacillus cerues subsp. cytotoxis NVH 391-98 that comprises genes involved in the synthesis of UDP-uronic acids in Bacillus spp. Within the operon, a UDP-glucose 6-dehydrogenase converts UDP-glucose in the presence of NAD⁺ to UDP-glucuronic acid and NADH, and a UDP-GlcA 4-epimerase (UGlcAE) converts UDP-glucuronic acid to UDP-galacturonic acid. Interestingly, in vitro, both enzymes can utilize the TDP-sugar forms as well, albeit at lower catalytic efficiency. Unlike most of the very few bacterial 4-epimerases that have been characterized, which are promiscuous, the B. cereus UGlcAE enzyme is very specific and cannot use UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylglucosaminuronic acid or UDP-xylose as substrates. Size exclusion chromatography suggests that UGlcAE is active as a monomer, unlike the dimeric form of plant enzymes; the Bacillus UDP-glucose 6-dehydrogenase is also found as a monomer. Phylogenic analysis further suggests that the Bacillus UGlcAE may have evolved separately from other bacterial and plant epimerases. Our results provide insight into the formation and function of uronic acid-containing glycans in the lifecycle of B. cereus and related species containing homologous operons, as well as a basis for determining the importance of these acidic glycans. We also discuss the ability to target UGlcAE as a drug candidate.

Full text of DOI:10.1111/j.1742-4658.2011.08402.x

Biosynthesis of UDP-glucuronic acid and UDP-galacturonic
acid in Bacillus cereus subsp. cytotoxis NVH 391-98
Bryan Broach1,*, Xiaogang Gu1,* and Maor Bar-Peled1,2
1 Complex Carbohydrate Research Center (CCRC), University of Georgia, Athens, GA, USA
2 Department of Plant Biology, University of Georgia, Athens, GA, USA
Keywords
The food borne pathogen Bacillus cereus produces uronic acid-containing
glycans that are secreted in a shielding biofilm environment, and certain
alkalinity; Bacillus; biofilm; UDP-galacturonic
acid; UDP-glucuronic acid
alkaliphilic Bacillus deposit uronate-glycan polymers in the cell wall when
adapting to alkaline environments. The source of these acidic sugars is
unknown and, in the present study, we describe the functional identifica-
Correspondence
M. Bar-Peled, CCRC, 315 Riverbend Road,
tion of an operon in Bacillus cerues subsp. cytotoxis NVH 391-98 that
comprises genes involved in the synthesis of UDP-uronic acids in Bacillus
spp. Within the operon, a UDP-glucose 6-dehydrogenase converts UDP-
glucose in the presence of NAD+ to UDP-glucuronic acid and NADH,
and a UDP-GlcA 4-epimerase (UGlcAE) converts UDP-glucuronic acid to
UDP-galacturonic acid. Interestingly, in vitro, both enzymes can utilize the
TDP-sugar forms as well, albeit at lower catalytic efficiency. Unlike most
of the very few bacterial 4-epimerases that have been characterized, which
are promiscuous, the B. cereus UGlcAE enzyme is very specific and cannot
use UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylglucosaminu-
ronic acid or UDP-xylose as substrates. Size exclusion chromatography
suggests that UGlcAE is active as a monomer, unlike the dimeric form of
plant enzymes; the Bacillus UDP-glucose 6-dehydrogenase is also found as
a monomer. Phylogenic analysis further suggests that the Bacillus UGlcAE
may have evolved separately from other bacterial and plant epimerases.
Our results provide insight into the formation and function of uronic acid-
containing glycans in the lifecycle of B. cereus and related species contain-
ing homologous operons, as well as a basis for determining the importance
of these acidic glycans. We also discuss the ability to target UGlcAE as a
drug candidate.
Athens, GA 30602, USA
Fax: +1 706 542 4412
Tel: +1 706 542 2062
E-mail: peled@ccrc.uga.edu
*These authors contributed equally to this
work
(Received 21 June 2011, revised 15
September 2011, accepted 10 October
2011)
doi:10.1111/j.1742-4658.2011.08402.x
Database
l
Nucleotide sequence data have been deposited in the Genbank database with the accession
numbers HM581979 and HM581980.
Introduction
Bacillus cereus has garnered much notoriety as a food-
poisoning bacterium and, similar to its close relatives,
the human pathogen Bacillus anthracis and the insecti-
cidal Bacillus thuringiensis, B. cereus has several life
forms. For example, it can survive as a protective
endospore in harsh conditions, in a shielding biofilm
Abbreviations
Gal, galactose; GalA, galacturonic acid; Glc, glucose; GlcA, glucuronic acid; GlcNAc, N-acetylglucosamine; GlcNAcA,
N-acetylglucosaminuronic acid; Rha, rhamnose; UGlcAE, UDP-glucuronic acid 4-epimerase; UGlcDH, UDP-glucose 6-dehydrogenase.
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B. Broach et al.
UDP-uronic acids in Bacillus spp.
environment, or as a free-living cell in soil and water.
An explanation of how changes in surface glycans con-
fer advantages to Bacillus species during different
stages of its life cycle is of great interest for under-
standing the pathogenesis of the organism. However,
as a result of the infectious nature B. cereus and
related Bacillus spp., it is understandable that many
glycans have yet to be characterized, particularly those
that may be made by the pathogen within the human
body. Recently, polysaccharides isolated from spores
of B. anthracis were shown to be antigenic and similar
to cross-reactive epitopes found only in pathogenic
strains of B. cereus, highlighting the role of carbohy-
drates in Bacillus spp. infections [1]. To identify new
metabolic pathways involved in the formation of sur-
face glycans in these pathogens, we aimed to identify
putative genes encoding enzymes involved in the syn-
thesis of glycan precursors (i.e. nucleotide sugars).
These nucleotide sugars are used by specific glyco-
syltransferases to make the different polysaccharide
structures in an organism.
growing in biofilm have indicated that it secretes exo-
polysaccharides consisting of the glycuronosyl residues
glucuronic acid (GlcA) and GalA [7]. GlcA and GalA
residues, however, were not reported in B. cereus
glycans isolated from spores or cultures grown in the
laboratory [8–10].
Synthesis of UDP-galacturonic acid (UDP-GalA)
[11,12] has been studied extensively in plants, where
UDP-glucose (UDP-Glc) is first converted to UDP-
glucuronic acid (UDP-GlcA) by UDP-glucose 6-dehy-
drogenase (annotated as UGDH, UGD, UDP-GlcDH
or UGlcDH). Subsequently, a very specific membrane-
bound 4-epimerase, UDP-GlcA 4-epimerase (UGlcAE)
[13,14] interconverts UDP-GlcA and UDP-GalA.
Fewer bacterial UGlcAEs have been characterized,
and questions still remain concerning how the specific-
ity for UDP-GlcA of these epimerases varies across
bacterial species. Studies of the Streptococcus pneumo-
coccus type I epimerase [15] and Glakp from Klebsiella
pneumoniae have shown that the enzymes are capable
of interconverting several UDP-sugars, including
UDP-GlcA, UDP-Glc and UDP-N-acetylglucosamine
(GlcNAc) [5]. On the other hand, the characterization
of the type I S. pneumococcus Cap1J 4-epimerase dem-
onstrated that the enzyme had specificity for the uronic
acid moiety because it was unable to interconvert
UDP-Glc or UDP-Gal [16]. In the present study, we
report the identification and characterization of two
genes (BcUGlcDH and BcUGlcAE) involved in the bio-
synthesis of UDP-GlcA and UDP-GalA (Fig. 1A)
from Bacillus cereus subsp. cytotoxis NVH 391-98.
Interestingly, the operon for these two genes is not
common in all Bacillus spp., which may provide insight
into the roles of these charged-glycans within the life-
cycle of this pathogen.
The occurrence of the sugar residue galacturonic
acid (GalA) in cell surface glycans varies across differ-
ent bacterial species. In Gram-negative bacteria, for
example, the amount and distribution of GalA in lipo-
polysaccharides, the major glycan molecule in the
outer cell surface membrane [2], shows significant vari-
ations. For example, the core oligosaccharide portion
of the lipopolysaccharides in the symbiotic nitrogen-
fixing bacteria Rhizobium leguminosarum [3] and
Bradyrhizobium japonicum [4], and in the pathogen
Klebsiella pneumonia [5], consists of several GalA resi-
dues. However, the core oligosaccharides of Escherichia
coli and Salmonella typhimurium lack GalA and,
instead, consist of glucosamine residues. One reason
for the variations in surface glycans could be that
organisms form specific glycans to facilitate exclusive
interactions with a particular host. Alternatively, spe-
cific glycans may display a mimicry-like structure to
facilitate entry into the host, or perhaps to allow the
bacteria to survive under different environmental con-
ditions. It is also a possibility that the negative charge
of acidic sugars (glycuronosyl residues) functions as an
ionic barrier or to sequester metals required for normal
organism function.
Results
Bioinformatic analyses of UGlcDH and UGlcAE in
Bacillus
A BLAST analysis with characterized proteins from
plants and bacteria was performed to identify enzymes
involved in UDP-GalA production in B. cereus; and
those with the greatest homology to known UGlcAE
and UGlcDH were selected for further study. How-
ever, the validation of the selected enzymes based on
the homology alone was challenging as a result of rela-
tive low amino-acid sequence identity with the known
enzymes. For example, the potential dehydrogenase
from B. cereus, Bcer98_2076, shares only 35%, 32%,
31% and 32% amino-sequence identity with func-
tional UGlcAE from Drosophila (sugarless, UDP-Glc
GalA-containing glycans have also been reported in
Gram-positive bacteria. For example, GalA has been
observed in the capsule of Streptococcus pneumoniae
serotype 1 [6]. Interestingly, the bacterial capsular sur-
face in S. pneumoniae was suggested to comprise the
first barrier against the innate immune system, as well
as being involved in mediating attachment to cells ⁄ sur-
faces to resist clearance. Recent analyses of B. cereus
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UDP-uronic acids in Bacillus spp.
B. Broach et al.
dehydrogenase), plants (AtUGlcDH), Cryptococcus
(Ugd1) and Gram-negative Sinorizobium (rkpK,
SMc02641), respectively (Fig. S1A). In addition, the
Bcer98_2076 also shares 38% sequence identity with
functional GDP-mannose dehydrogenase from Pseudo-
monas aeruginosa [17]. Similarly, the potential epimerase
from B. cereus, Bcer98_2077, shares 36% and 32%
sequence identity with functional UGlcAE (AtUGl-
cAE3) from plants, and LpsL from Sinorizobium
(SMc02640), respectively (Fig. S2B). Having such rela-
tive low sequence identities, it is impossible to predict
the function for these putative Bacillus spp. UGlcDH
and UGlcAE homologs without further biochemical
characterization. Interestingly, the putative dehydroge-
nase, Bcer98_2076, and epimerase, Bcer98_2077, from
B. cereus are found in the same operon along with a
putative glycosyltransferase, Bcer98_2078, that shares
sequence similarity to an annotated lipopolysaccha-
ride N-acetylglucosaminyltransferase. Similar operons
(Fig. 1B) with homologous genes are found in Bacil-
lus subtilis subsp. 168, Bacillus weihenstephanensis
KBAB4, Bacillus thuringiensis serovar berliner ATCC
10792 and Geobacillus sp. G11MC16, whereas such
operon organization is not found in the genomes of
other Bacillaceae, such as B. anthracis strains Ames,
A0248 or CDC 684. A putative nucleotidyl transferase,
Bcer98_2075, is also found in proximity to the operon
containing the putative dehydrogenase and epimerase,
and homologous genes are found alongside the operon
in B. subtilis subsp. 168, B. weihenstephanensis KBAB4,
and B. thuringiensis serovar berliner ATCC 10792 but
absent from Geobacillus sp. G11MC16. In B. subtilis
subsp. 168, the homologous gene (ytdA BSU30850) is
annotated as a putative UDP-glucose pyrophosphory-
lase, suggesting that Bcer98_2075 and its homologs may
play a role in the synthesis of UDP-Glc (Fig. 1A)
for subsequent interconversion to UDP-GlcA and
UDP-GalA. To determine and elaborate upon the
functions of the putative B. cereus UGlcDH and UGl-
cAE, we have cloned and expressed the genes as recom-
binant proteins in E. coli and examined their enzyme
activities.
Fig. 1. The biosyntheses of acidic sugar nucleotides in Bacillus. (A)
In Bacillus, based on the present report, UDP-Glc pyrophosphory-
lase (UDP-Glc PPase) converts Glc-1-P and UTP to UDP-Glc. In the
presence of NAD+, UDP-Glc is interconverted by UDP-glucose
dehydrogenase (UGlcDH) into UDP-GlcA and NADH. UDP-GlcA can
then be interconverted by the 4-epimerase, UGlcAE, into UDP-
GalA. (B) Organization of the three genes within the UGalA operon
and the flanking regions across selective members of Bacillus spp.
The locus number for each gene in the operon across the different
species shown is indicated. Based on this report, Bcer98_2076
functions as a UGlcDH and Bcer98_2077 as a UGlcAE. The operon
Biochemical characterization of B. cereus UGlcDH
and UGlcAE
A distinct protein band migrating slightly lower than
the 50 kDa standard, was detected by SDS ⁄ PAGE
analysis of the extracts from E. coli cells expressing
recombinant BcUGlcDH (Fig. 2A, lane 2) but was
absent in E. coli cells expressing the control vector.
The expressed UGlcDH protein (theoretical mass
49,792) was purified (Fig. 2A, lane 4) and shown,
also comprises
a putative glycosyltransferase (Bcer98_2078 in
B. cereus), which may be involved in the synthesis of GlcA or
GalA-containing glycans. Note: except for Geobacillus sp.
G11MC16, in the 5¢ region flanking the UGalA operon, there is a
single gene in the opposite direction of the operon encoding a puta-
tive UDP-Glc PPase (Bcer98_2075 in B. cereus, putative ytdA in
B. subtilis).
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B. Broach et al.
UDP-uronic acids in Bacillus spp.
Fig. 2. Expression and characterization of recombinant of BcUGlcDH.
(A) SDS ⁄ PAGE of total soluble protein isolated from E. coli cells
expressing UGlcDH (lane 2), negative vector control (lane 3) and of
column-purified UGlcDH or control (lanes 4 and 5, respectively). An
arrow points to the purified protein. (B) High-performance anion-
exchange chromatography of the products formed by UGlcDH. Puri-
fied recombinant UGlcDH was reacted with UDP-Glc for 30 min in
the presence (panel 4) or absence (panel 6) of NAD+. The corre-
sponding column-purified protein isolated from cells expressing the
control vector was reacted with UDP-Glc and NAD+ for 30 min
(panel 5) as a control. The reaction products were separated on a
Q15 anion-exchange column. The distinct UDP-sugar peak marked
by an asterisk (panels 2 and 4 with a retention time of 18.8 min)
was collected and characterized by mass spectrometry and 1H-
NMR spectroscopy. The activity of total soluble protein (denoted
S20) isolated from cells expressing recombinant BcUGlcDH (panel
2) or vector control (panel 3) is also shown. (C) Analysis of
BcUGlcDH enzymatic product by MS operated in the negative ion
mode. The HPLC peak (panel 2B or 4B, *) was collected and
directly infused to ESI-MS. The spectrum of the full MS molecular
ions (panel 1, m ⁄ z of 579.05 for deprotonated [M-H])) and of the
derived CID-fragments (panel 2, m ⁄ z of 323.06 and 403.05) are
shown. (D) The peak in panel 4B marked by a double asterisk (**)
has major UV absorbance peaks at 259 and 340 nm, the character-
istic absorbance signature for NADH.
decrease of the peak corresponding to the anomeric
proton (peak G1¢¢, 5.62 p.p.m.) of UDP-Glc was
observed alongside a simultaneous increase of the
peaks corresponding to the anomeric and H-5¢¢ pro-
tons of UDP-GlcA (peaks A1¢¢ and A5¢¢, 5.64 and
4.16 p.p.m., respectively). Additionally, the signal from
the protons added to the NAD+ nicotinamide ring
after enzymatic reduction to NADH (peak H1a,b)
appeared in the spectrum in the range 2.7–2.9 p.p.m.
as time advanced. Peaks from other diagnostic protons
of NAD+ (N3, N5, N6, N7) were also diminished con-
comitantly with an increase of diagnostic peaks of
NADH (H2, H3, H4a,b, H5, H6, H7). The ratio of
the change in the diagnostic NADH proton signals
compared to those of UDP-GlcA was found to be
2 : 1, validating that two moles of NAD+ are reduced
per mole of UDP-GlcA made in the enzymatic reac-
tion. Hence, we named Bcer98_2076 as UDP-Glc
6-dehydrogenase (i.e. UGlcDH, or dehydrogenase).
To determine substrate specificity, other nucleotide-
glucose were tested. TDP-Glc was a substrate for the
dehydrogenase as well, albeit with lower efficiency
compared to UDP-Glc (Table 1). The dehydrogenase,
however, could not utilize ADP-Glc, GDP-Glc or
GDP-mannose as substrates. In addition, NADP+
could not substitute NAD+ as the cofactor. After the
reaction with TDP-Glc, the enzyme product was vali-
dated as TDP-GlcA as determined by MALDI-TOF
using a HPLC-based assay, to convert UDP-Glc to a
new UDP-sugar (Fig. 2B, panels 2 and 4, asterisk) in
the presence of NAD+ and to a product that migrated
as NADH (Fig. 2B, panel 4, double asterisk) with a
characteristic dual UV absorption maximum at 259
and 340 nm (Fig. 2D). The newly formed UDP-sugar
eluted from the Q15 ion-exchange column with a
retention time (18.8 min), comparable to UDP-GlcA
standard (Fig. 2B, panel 1). Analysis of the reaction
product peak by ESI-MS operated in the negative ion
mode identified an ion product (Fig. 2C, panel 1) of
m ⁄z 579.05, as expected for a UDP-hexuronic acid.
Collision-induced ion fragmentation yielded two major
ions at m ⁄ z 323.06 and 403.03 (Fig. 2C, panel 2), sug-
gesting the formation of UMP and UDP ions, as
expected. The dehydrogenase enzymatic reaction was
also followed with 1H-NMR (Fig. 3). As time
advanced during the real-time NMR assay, a clear
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Table 1. Steady-state kinetics parameters of recombinant BcUGl-
cAE and BcUGlcDH. Kinetics of UGlcAE were measured with var-
ied concentrations of UDP-GlcA (0.1–1.0 mM) or TDP-GlcA (0.08–
0.5 mM) and 1 mM NAD+ after 8 min at standard conditions. The
reciprocal initial velocity was plotted against the reciprocal UDP-
GlcA or TDP-GlcA concentration according to Lineweaver and Burk
to calculate the corresponding Km values. The data presented are
the mean Km values from three experiments. Kinetics of BcUGlcDH
were measured with 2 mM NAD+ and varied concentrations of
UDP-Glc (0.1–1.0 mM) after 15 min or with varied concentrations of
TDP-Glc (0.1–1.0 mM) after 25 min under standard conditions.
Kinetics of BcUGlcDH were also measured with varied concentra-
tions of NAD+ (0.1–2 mM) with 1 mM UDP-Glc after 15 min or
1 mM TDP-Glc after 25 min under standard conditions. The data
presented are the average Km values from two experiments.
kcat ⁄ Km
Km (mM)
kcat (s)1
)
(mM)1Æs)1
)
UDP-GlcAE activity
TDP-GlcAE activity
UDP-GlcDH
0.12 0.01
0.18 0.01
3.2 0.1
2.1 0.1
26 1.4
12 0.4
0.137 0.019 0.974 0.031 7.1 1.0
0.042 0.006 0.898 0.014 21.4 3.1
2.07 0.39 0.646 0.091 0.31 0.073
0.664 0.050 0.307 0.012 0.46 0.039
activity – (UDP-Glc)
UDP-GlcDH
activity – (NAD+)
TDP-GlcDH
activity – (TDP-Glc)a
TDP-GlcDH
Fig. 3. Analysis of BcUGlcDH enzymatic reaction in time-resolved
1H-NMR. Selected regions of the 600 mHz 1H-NMR spectrum
show the interconversion of UDP-a-D-Glc and NAD+ to UDP-a-D-
GlcA and NADH at 5, 15 and 45 min after the reaction was initi-
ated. Diagnostic peaks of NAD+ and NADH are labeled N and H,
respectively, whereas UDP-Glc peaks are labeled G and UDP-GlcA
peaks are labeled A. Peaks are arbitrarily numbered to denote cor-
responding protons numbered as indicated in the molecular struc-
tures.
activity – (NAD+)
a
The kinetic values for TDP-Glc and NAD are an estimate and likely
qualitative rather than quantitative because the reaction with TDP-
Glc could not be carried out at saturation. This in turn may affect
the Kcat value for NAD. Nonetheless, the data provide sufficient
information showing that the dehydrogenase is more efficient with
UDP-Glc as the substrate.
MS in the negative mode with a major m ⁄z ion at
577.6 and by H-NMR (Fig. 4A,B). To determine the
marker as expected from the theoretical mass (36 734)
(Fig. 5A, lane 3). The enzyme was tested against vari-
ous commercially available ADP-, UDP- and GDP-
sugars in the presence or absence of co-factors. The
1
preferred in vivo substrate of dehydrogenase, E. coli
harboring the expression plasmid was grown to mid-
log phase and, 2 h after induction, the cells were
extracted and the amount of UDP-sugar produced by
E. coli determined by HPLC and ESI-MS⁄ MS. HPLC
analyses reveal a major peak at 18.3 min (comparable
to the UDP-GlcA standard peak) that is absent from
NDP-sugars extracted from E. coli control (data not
shown). MS analyses confirm the in vivo production of
UDP-GlcA as the major ion as well. Interestingly,
in vitro the dehydrogenase was unable to fully convert
UDP-Glc to UDP-GlcA; this was later confirmed (see
below) as a result of strong inhibition of the enzyme
by its product NADH.
recombinant Bcer98_2077 converted
a portion of
UDP-GlcA to a new peak migrating at 22.5 min when
separated on the HPLC column (Fig. 5B, panels 2 and
3, arrow). The peak was collected from the column
1
and H-NMR confirmed the enzyme product as UDP-
GalA because it had identical proton assignments as
described previously [14]. Thus, the enzyme is a true
4-epimerase because it is capable of interconverting
UDP-GalA to UDP-GlcA (not shown) as well. Hence,
we named Bcer98_2077 as UDP-GlcA 4-epimerase,
BcUGlcAE. Based on the sensitivity of our assay, this
B. cereus 4-epimerase was unable to convert UDP-
glucose or UDP-xylose. The characterization of a
4-epimerase in P. aeruginosa capable of epimerizing
UDP-GlcNAc and UDP-GlcNacA [18] and the recent
identification of a UDP-GlcNAc 6-dehydrogenase in
We next proceeded to characterize the function of
the second gene in the operon, Bcer98_2077. Expres-
sion of this recombinant protein in E. coli yielded a
highly expressed protein migrating alongside a 37 kDa
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B. Broach et al.
UDP-uronic acids in Bacillus spp.
Fig. 4. BcUGlcDH can convert TDP-Glc to TDP-GlcA as determined by MALDI and NMR. (A) MALDI-MS analysis: negative-ion MALDI mass
spectra were recorded using a Microflex LT mass spectrometer (Bruker Daltonik, Bremen, Germany). Aqueous samples (1 lL) of 1 mM
TDP-GlcA (produced from the dehydrogenase reaction with TDP-Glc ⁄ NAD+ and collected by HPLC) were mixed with an equal volume of
matrix solution (1 lgÆlL)1 2,5-dihydroxybenzoic acid in 50% methanol) and dried on the plate. Spectra from 500 laser (N2; 337 nm) shots
were summed to generate a mass spectrum. The ion mass, calculated (abbr. Cal.) or observed (abbr. Obs.), are listed below the spectrum.
(B) 1H-NMR spectroscopy at 600 MHz of the HPLC collected peak after reaction of BcUGlcDH with TDP-Glc and NAD+. The assignments of
each proton are indicated on the spectrum: peaks from protons on the thymidine (T) ring are indicated by H, the ribose (R) protons are indi-
cated by H¢, and the GlcA (G) protons protons are indicated by H¢¢. The peaks marked by an asterisk (*) are resonances from impurities.
B. cereus subsp. cytotoxis NVH 391-98 [19] motivated
us to test the epimerase with UDP-GlcNAc and UDP-
GlcNAcA as substrates, although no conversion was
observed. However, the fact that the recombinant
dehydrogenase (described above) was capable of utiliz-
ing TDP-Glc prompted us to test whether TDP-GlcA
could be utilized as a substrate. Indeed, the recombi-
nant BcUGlcAE converted a portion of TDP-GlcA to
a new product as observed after separation by HPLC
(Fig. 5C, panels 2 and 3, arrow). To further validate
the nature of this new TDP-sugar, the peak was col-
lected and its mass was analyzed by MALDI-TOF MS
and the structure by NMR. The major mass ion at
m ⁄z 577.6 suggested a TDP-hexuronic acid (Fig. 6A)
Relatively little UDP-GlcA was produced in enzymatic
assays conducted at pH 4 or lower. Optimal epimerase
activity was observed at 55 ꢀC and, similar to the
dehydrogenase, it was also most active above pH 7.
Epimerase assays conducted at pH 8.2 in sodium phos-
phate buffer yielded the highest levels of activity.
Kinetic analyses of the enzymes are summarized in
Table 1. The data from kinetic experiments of dehy-
drogenase with UDP-Glc as the variable substrate and
NAD+ as the fixed substrate (or vice versa) fit well to
the Michaelis–Menten model. The apparent Km value
for the dehydrogenase utilizing UDP-Glc was
137.2 lM for UDP-Glc and 42.3 lM for NAD+, with
Vmax values of 27.1 pmolÆs)1. The kcat ⁄ Km (mM)1Æs)1
)
1
and H-NMR (Fig. 6B) confirmed the epimerase prod-
values for the dehydrogenase were found to be 7.1
(UDP-Glc) and 21.4 (NAD+). When TDP-Glc was
used as the substrate, the apparent Km values for the
dehydrogenase were 2067 lM (TDP-Glc) and 644 lM
(NAD+). The Vmax was found to be 19.5 pmolÆs)1
when TDP-Glc was the variable substrate and
9.26 pmolÆs)1 when variable concentrations of NAD+
were used, and the kcat ⁄ Km (mM)1Æs)1) values were 0.31
for TDP-Glc and 0.46 for NAD+. The epimerase had
apparent Km values of 120 lM (UDP-GlcA) and
180 lM when TDP-GlcA was used as the substrate.
uct as TDP-GalA.
Enzymatic characterization of UGlcDH and
UGlcAE
The B. cereus UDP-glucose 6-dehydrogenase (BcUGlcDH)
had the highest activity at a temperature of 42 ꢀC
(Table S1). Additionally, the dehydrogenase was found
to have substantial activity at high pH with its optimal
pH at 9.0 in sodium phosphate buffer (Table S1).
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B. Broach et al.
Fig. 5. Expression and characterization of recombinant BcUGlcAE. (A) SDS ⁄ PAGE of total soluble protein isolated from E. coli cells express-
ing BcUGlcAE (lane 1), control vector (lane 2) and of column-purified BcUGlcAE or control (lanes 3 and 4, respectively). (B) HPLC analysis of
the products formed by BcUGlcAE reacting with UDP-GlcA. Total soluble proteins (denoted S20) or purified recombinant UGlcAE was
reacted with UDP-GlcA for 30 min in the presence (panels 2 and 3), or absence (panel 4) of NAD+. The crude or purified protein isolated
from cells expressing control vector was incubated with UDP-GlcA for 60 min (panels 5 and 6) as a control. The distinct UDP-sugar peak
marked by the arrow (panels 2–4 with the same retention time) was collected and analyzed by 1H-NMR spectroscopy. (C) HPLC analysis of
BcUGlcAE reacted with TDP-GlcA to yield a product (marked by an arrow in panels 2–4) that was collected and analyzed by NMR and
MALDI-TOF.
The kcat ⁄ Km (mM)1Æs)1) values were 26 (UDP-GlcA)
and 12 (TDP-GlcA). Inhibition studies (Tables S2 and
S3) have shown that the Bacillus dehydrogenase is
strongly inhibited by its product NADH, whereas, on
the other hand, NADH had only a marginal inhibitory
effect on the epimerase activity. However, the epimer-
ase was highly inhibited by UDP and to a lesser extent
by UDP-xylose.
with two distinctive peaks of activity: the first (with
ꢀ 20% of total activity) corresponded to a molecular
mass of 152.1 kDa and the second corresponded to a
molecular mass of 80.8 kDa. Considering the theoreti-
cal molecular mass of the dehydrogenase (49 kDa) and
the 2 : 1 ratio of the estimated masses for the two
active forms of the enzyme, size-exclusion chromatog-
raphy suggested that the enzyme was most likely active
as a monomer. Performing the size-exclusion chroma-
tography of the dehydrogenase in the presence of
UDP, UDP-Glc and NAD+, or only with NADH,
yielded the same elution pattern (data not shown). The
elution behavior of the dehydrogenase from the
Size-exclusion chromatography analysis of the epim-
erase gave an estimated molecular mass of 46.5 kDa,
suggesting that the BcUGlcAE is active in a mono-
meric form (Table S1). Active dehydrogenase eluted
from the size-exclusion column (Table S1) consistently
106
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B. Broach et al.
UDP-uronic acids in Bacillus spp.
Fig. 6. 1H-NMR spectroscopic and MALDI-TOF analyses of BcUGlcAE reaction products indicates the formation of TDP-a-D-galacturonic acid.
(A) The HPLC peaks (Fig. 5C, panels 2–4) corresponding to the product formed by BcUGlcAE were collected and analyzed by MALDI-TOF.
(B) 1H-NMR spectroscopy at 600 MHz of the HPLC peak from Fig. 5C (panel 3). The assignments of each proton are indicated on the spec-
trum: peaks from protons on the thymidine (T) ring are indicated by H, the ribose (R) protons are indicated by H¢, and the GalA (G) protons
protons are indicated by H¢¢. The peaks marked by an asterisk (*) are resonances from impurities.
column is not sufficient to draw conclusion for its size.
It could be function as a monomer or as a dimer and
efforts to crystalize the dehydrogenase are in progress
aiming to determine its form(s) in solution.
the cell walls of alkaliphilic Bacillus species B. halodu-
rans C-125 and Oceanobacillus iheyensis. The amount
of polymers containing these acidic sugars increases
when such Bacillus spp. are grown in an alkaline envi-
ronment. It has been demonstrated that alkaline condi-
tions (up to pH 9) do not substantially inhibit the
growth of B. cereus [20,21], and certain Bacillus spp.,
such as the deep sea-dwelling O. iheyensis, can grow at
pH 10 [22]. It has been hypothesized that the presence
of these acidic sugars in the outer layers of the cell
wall serves to permit growth in an alkaline pH [23,24].
The enzymatic activity of the dehydrogenase (Fig. 1A)
could also help to promote survival in high pH envi-
ronments because three moles protons are released per
mole of UDP-GlcA formed: two protons are released
to form NADH, and, at a pH of the cytosolic environ-
ment above 3.8, the carboxylic moiety of UDP-GlcA is
deprotonated. Oxidation of the produced NADH
could further generate free protons for the cell. An
additional mechanism that may facilitate growth at
high pH is the fact that a number of Bacillus spp. pro-
duce ‘pectinases’ and ‘lyases’ under alkaline conditions.
Such hydrolytic enzymes are capable of hydrolyzing
polygalacturonan [25] and perhaps the cell’s own uron-
ic-acid glycan. Such enzymes from B. cereus, B. subtilis
and Bacillus sp. BP-23, for example, have been found
Discussion
In the present study, we have demonstrated that the
food borne pathogenic bacterium B. cereus subsp.
cytotoxis NVH 391-98 contains an operon that func-
tions to generate acidic sugar- precursors (UDP-GlcA,
UDP-GalA and possibly their TDP-analogs). The
operon organization (Bcer98_2076, Bcer98_2077,
Bcer98_2079) is conserved in few terrestrial Bacillus
spp. including B. weihenstephanensis KBAB4, B. thur-
ingiensis IBL 200, Geobacillus sp. G11MC16, and
B. subtilis subsp. subtilis str. 168. Similar genes are
also found in sea-dwelling bacteria such as Bacil-
lus halodurans C-125 and others, although the operon
organization varies slightly.
The UGalA operon also contains a putative glyco-
syltransferase, which may utilize the UDP-GlcA
and ⁄ or UDP-GalA as substrates for the biosynthesis
of acidic sugar-containing glycans (e.g. the acidic gly-
can secreted by B. cereus living in biofilm). Glycans
containing GlcA and GalA have been also noted in
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107

UDP-uronic acids in Bacillus spp.
B. Broach et al.
to be optimally active at pH 8.5, 9.5 and 10, respec-
tively [26,27]. Genes for the transport and catabolism
of GlcA and GalA have been identified in Bacillus
spp., and the release of these free acidic sugars may
help to permit Bacillus spp. growth in alkaline environ-
ments [28]. At high pH, Bacillus spp. can hydrolyze
polyanionic oligosaccharide structures (i.e. GlcA- and
GalA-containing glycans) and the free acidic sugars
can be transported to the cell where GlcA and GalA
metabolites can serve as a source of protons to acidify
the cytoplasm despite the high external pH.
UDP-GlcNAcA or other nucleotide sugars has not been
tested [16]. We have tried to address the promiscuity of
some 4-epimerases by analyzing the origin of UGlcAE
proteins (from the known UGlcAE and homologs that
share high amino acid sequence identity). Phylogenic
analysis of eukaryotic and bacterial UGlcAE clearly
separates the Bacillus spp. UGlcAE into a separate and
distinct clade (Fig. S2A). Although plant and promis-
cuous bacterial UGlcAE are related (e.g. AtUGlcAE2,
Cap1J and Glakp), the Bacillus spp. appear to belong to
a different group that is well separated from other bacte-
rial and eukaryotic UGlcAE. The phylogeny suggests
that UGlcAE enzymes might share the same ancestor
because those enzymes are well divided from UDP-
glucose-4-epimerases (UGEs) and decarboxylases. How-
ever, currently we cannot predict the evolution of
Bacillus UGlcAE.
The balance between the oxidized and reduced forms
of NAD+ is assumed to reflect the metabolic activities
of the cell, and the fact that the recombinant B. cereus
dehydrogenase is strongly inhibited in the presence of
NADH may point to the role of the UGalA operon in
the B. cereus lifecycle. It is possible that the dehydroge-
nase serves as a checkpoint in the regulation of down-
stream metabolic pathways utilizing UDP-GlcA or
UDP-GalA because high NADH levels will likely
strongly inhibit UDP-GlcA formation and therefore
also hinder UDP-GalA production in vivo. This suggests
that B. cereus, in a state of abundant intracellular energy
(i.e. a high NADH ⁄NAD+ ratio), may produce less
GlcA and ⁄ or GalA-containing glycans. On the other
hand, one function of the dehydrogenase could be to
support the formation of NADH for various metabolic
pathways, especially under conditions when the bacte-
rium is deprived of essential nutrients and required to
form a protective spore. This may reflect a possible role
of the operon in the biosynthesis of GlcA and GalA-
containing glycans in a nutrient-starved cell (e.g. during
sporulation). The intracellular concentration of NADH
may act to modulate UDP-GlcA and UDP-GalA pro-
duction in vegetative cells. Indeed, NADH inhibition
may be a conserved feature of some UGlcDHs and has
been reported in spore and nonspore forming bacteria
alike. For example, NADH has been shown to inhibit a
UGlcDH (TuaD BSU35580) of B. subtilis subsp. 168
that is distinct from the BcUGlcDH homolog, as well as
UGlcDHs from P. aeruginosa PAO1 [29] and Crypto-
coccus neoformans [30].
The ability of the Bacillus dehydrogenase and epim-
erase to utilize TDP-Glc and TDP-GlcA, respectively,
may point to a separate or additional role of the
operon in the B. cereus life cycle. TDP-Glc is a product
formed in B. anthracis for the synthesis of TDP-rham-
nose (Rha) [31], and homologous proteins for TDP-
Rha production are also found in the B. cereus genome
[32]. In B. anthracis, Rha residues are incorporated into
the spore glycoprotein bclA, and Rha residues have
been shown to be major components of the glycans in
the exosporium of B. cereus [33]. Rha is also a minor
constituent of the biofilm exo-polysaccharides of
B. cereus [7]. However, the capability of the enzymes to
interconvert TDP-Glc and TDP-GlcA may be the result
of the structural similarity between the thymine and
uracil moieties of the TDP- and UDP-sugars.
At present, the function of glucuronic and galact-
uronic acid at different points of the B. cereus lifecycle
remains to be determined, and the identification of the
UGalA operon will help to allow the elucidation of
their importance. Our laboratory is unfortunately not
licensed to work with this human pathogen, as is nec-
essary for genetic deletion of the operon; however,
gene knockouts in nonpathogenic Bacillus spp. also
possessing the UGalA operon (Fig. 1B) could be used
to reveal its role. Efforts are underway to examine the
function of these genes and perhaps their utilization in
adapting to high pH in different Bacillus spp. Determi-
nation of the expression of the genes of the UGalA
operon in response to various environment stimuli
(e.g. alkaline pH, nutrient limitation or temperature
extremes) may also be advantageous in developing an
understanding of the biological role of the operon.
If the GalA-containing glycans are shown to be
necessary for survival of the organism, the Bacillus
UGlcAE may prove to be a potential target for drugs
The enzyme specificity and the nature of their evolu-
tion are fundamental questions with respect to bacterial
UGlcAE. For example, the K. pneumoniae UGlcAE can
4-epimerize UDP-GlcA, UDP-Glc and UDP-GlcNAc,
whereas the Bacillus enzyme has a strict specificity for
the uronic acid moiety. The Cap1J UGlcAE from
S. pneumoniae has also been shown to have specificity
for the uronic acid moiety, and is capable of 4-epimeriz-
ing UDP-GlcA and UDP-GalA at the same time as
being unable to interconvert UDP-Glc and UDP-Gal;
unfortunately, whether Cap1J can utilize UDP-GlcNAc,
108
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B. Broach et al.
UDP-uronic acids in Bacillus spp.
centrifugation (6000 g for 10 min at 4 ꢀC) and suspended in
20 mL of lysis buffer. [For the dehydrogenase, the buffer
was 50 mM Tris–HCl, pH 8, 20% glycerol (v ⁄ v), 1 mM
EDTA, 100 mM NaCl, 25 mM KCl, 25 mM (NH4)2SO4 sup-
plemented with fresh 5 mM dithiothreitol and 0.5 mM phen-
ylmethylsulfonyl fluoride; for BcUGlcAE, the lysis buffer
was 50 mM sodium phosphate, pH 7.6, containing 10%
(v ⁄ v) glycerol, 1 mM EDTA, and fresh 1 mM dithiothreitol
and 0.5 mM phenylmethylsulfonyl fluoride] Cells were lysed
by sonication [34] and centrifuged (6000 g for 10 min at
4 ꢀC). The supernatant was supplemented with fresh dith-
iothreitol (1 mM for BcUGlcAE, 10 mM for BcUGlcDH)
and centrifuged again (30 min at 20 000 g). The resulting
supernatant (termed S20) was kept at –20 ꢀC. His-tagged
proteins were purified on a Ni-Sepharose fast-flow column
(GE Healthcare Life Sciences, Piscataway, NJ, USA; 2 mL
of resin packed in a polypropylene column; inner diameter
1 cm · 15 cm). The column was pre-equilibrated with load-
ing buffer [For BcUGlcAE, 50 mM sodium phosphate (pH
7.6), 0.1 M NaCl; for BcUGlcDH, 50 mM sodium phos-
phate (pH 8), 0.3 M NaCl]. The bound BcUGlcAE-6His
was eluted with the same buffer containing an increased
amount of imidazole (0–250 mM). Similarly, the bound
BcUGlcDH-6His was eluted from Ni-column with solution
adjusted to pH 8, which was composed of 0.3 M NaCl,
50 mM sodium phosphate and imidazole (0–250 mM). The
fraction containing BcUGlcAE activity, typically eluted
with 250 mM imidazole, was supplemented with 1 mM dith-
iothreitol and 10% glycerol, and was dialyzed (6000–8000
molecular weight cut-off; Spectrum Laboratories, Inc.,
Rancho Dominguez, CA, USA) three times (twice for
30 min, and once for ꢀ 1 h), each time against 800 mL of
cold dialysis buffer [50 mM sodium phosphate, pH 7.6, con-
taining 0.1 M NaCl, 10% (v ⁄ v) glycerol, 1 mM dithiothrei-
tol]. The protein after dialysis was divided into small
aliquots, flash frozen in liquid nitrogen and stored at –
80 ꢀC. The Ni-column fraction containing UDP-GlcDH
activity, typically eluted with 250 mM imidazole, was sup-
plemented with 20% glycerol, divided into aliquots, flash
frozen and kept at )80 ꢀC. Proteins extracted from E. coli
cells expressing the control vector were obtained using the
same purification protocol and were used as controls in
enzyme assays and SDS ⁄ PAGE analyses. Proteins concen-
tration was determined using the Bradford reagent with
BSA as standard. The native molecular weight of the dehy-
drogenase and the epimerase was estimated by size-exclu-
sion chromatography using a Superdex-75 column (GE
Healthcare) as described previously [34].
combating this pathogen. As far as we are aware,
humans do not synthesize GalA-containing glycans
and are not considered to possess UGlcAEs. Indeed,
the specificity of the Bacillus enzyme may permit the
development of inhibitors restricted to BcUGlcAE that
do not affect similar enzymes necessary for normal
human metabolism.
Materials and methods
Cloning of BcUGlcDH and BcUGlcAE
The Bcer98_2076 and Bcer98_2077 genes from Bacil-
lus cereus subsp. cytotoxis NVH 391-98, herein named,
UDP-Glc 6-dehydrogenase (BcUGlcDH) and UDP-glucu-
ronic acid 4-epimerase (BcUGlcAE), respectively, were
cloned by high-fidelity PCR from genomic DNA. PCR
primers were: sense 5¢-Ccatggtgaatatatgcattataggatctg-3¢ and
antisense 5¢-AAGCTTtgagcgaccaactcctacatagc-3¢ for BcUG-
lcDH; and sense 5¢-Tcatgaaaatacttgttactggagcag-3¢ and anti-
sense 5¢-AAGCTTatataattgcttcatatactc-3¢ for BcUGlcAE.
Genomic DNA was kindly provided by the laboratory of
A. Sorokin (Genetique Microbienne, INRA, France). The
´ ´
agarose gel-purified PCR fragments were cloned to yield the
plasmids: pCR4-gmdh#2 and pCR4-gmad#1, respectively.
The corresponding DNA sequences were deposited in Gen-
bank (with respective accession numbers HM581980 and
HM581979). The Nco1-HindIII fragment of BcUGlcDH
(1289 bp) and BspH1-HindIII fragment of BcUGlcAE
(944 bp) were cloned into an E. coli expression vector to
form pET28b:BcUGlcDH#1 and pET28b:BcUGlcAE#1,
respectively. The recombinant enzymes were designed to
have a six-histidine extension at their C-terminus to facilitate
affinity-purification.
Expression and purification of recombinant
proteins
E. coli cells harboring pET28b:BcUGlcAE#1 or a control
vector (pET28) were cultured for 16 h at 37 ꢀC (or 30 ꢀC
for pET28b:BcUGlcDH#1 or a control vector) in 20 mL of
LB medium supplemented with kanamycin (50 lgÆmL)1
)
and chloramphenicol (34 lgÆmL)1). A portion (5–7 mL) of
the cultured cells was transferred into fresh LB liquid med-
ium (250 mL) supplemented with the same antibiotics, and
the cells were grown on a rotary shaker at 37 ꢀC (BcUGl-
cAE) or 30 ꢀC (BcUGlcDH) at 250 r.p.m. until D600 of 0.8
(BcUGlcAE) or 0.6 (BcUGlcDH) was reached. Isopropyl
thio-b-D-galactoside (0.5 mM) was added to induce gene
expression. The cells harboring pET28b:BcUGlcDH#1 were
transferred to an incubator at 18 ꢀC and grown for 20–24 h
with shaking at 250 r.p.m.; cell harboring pET28b:BcUGl-
cAE#1 were incubated at 30 ꢀC and grown for 4 h at
250 r.p.m. After induction, the cells were harvested by
UGlcDH and UGlcAE enzyme assays, HPLC and
product analyses by NMR
Standard UGlcDH reactions (final volume of 50 lL) con-
tained 100 mM sodium phosphate (pH 8), 20 mM KCl,
1 mM NAD+, 0.5 mM UDP-Glc and 1.1 lg of purified
FEBS Journal 279 (2012) 100–112 ª 2011 The Authors Journal compilation ª 2011 FEBS
109

UDP-uronic acids in Bacillus spp.
B. Broach et al.
recombinant BcUGlcDH. UGlcAE reactions (final volume
of 50 lL) contained 50 mM sodium phosphate (pH 7.6),
1 mM NAD+, 1 mM UDP-GlcA and 0.2 lg of purified
recombinant BcUGlcAE. UGlcAE or UGlcDH reactions
were kept for up to 15 or 30 min, respectively, at 37 ꢀC,
and then terminated (100 ꢀC water bath for 45 s). Reaction
products were extracted with chloroform and chromato-
graphed [34] on a Q15 anion-exchange column (1 · 250
mm; Amersham Pharmacia, Piscataway, NJ, USA) using
an Agilent 1200 Series HPLC system equipped with an
autosampler, diode-array detector and CHEMSTATION soft-
ware (Agilent Technologies Inc., Santa Clara, CA, USA).
Nucleotides were detected by their UV absorbance and the
maximum absorbance for UDP-sugars, NAD+ and NADH
was 261, 259 and 259 ⁄ 340 nm, respectively. The amount of
product formed was determined using calibration curves of
standard UDP-GlcA. The product formed by the specific
reaction (eluted from Q-column) was collected, lyophilized,
dissolved in 99.9% D2O, and analyzed by 1H-NMR spec-
troscopy [34]. TDP-Glc, UDP-Glc, NADH, NAD+ were
obtained from Sigma (St Louis, MO, USA).
37 ꢀC for 15 min in 0.1 M sodium phosphate buffer (pH 8),
20 mM KCl, and 1.5 lg of protein. When calculating the Km
for UDP-Glc or TDP-Glc, UGlcDH assays were carried out
with fixed amount of NAD+ (2 mM) and variable concen-
trations of UDP-Glc or TDP-Glc (0.1–1.0 mM); for the Km
value for NAD+, BcUGlcDH was incubated with variable
concentration of NAD+ (0.1–2.0 mM) and a fixed amount
of UDP-Glc or TDP-Glc (1 mM). DELTAGRAPH, version 5
(Red Rock Software, Inc., Salt Lake City, UT, USA) was
used to generate a best-fit curve calculated by nonlinear
regression analyses. The reciprocal initial velocity was plot-
ted against the reciprocal nucleotide sugar concentration
according to Lineweaver and Burk to calculate Km values.
Acknowledgements
We thank Sung Lee and James A. Smith for technical
help. This research was supported in part by NSF:
IOB-0453664 (M.B.-P.) and by the BioEnergy Science
Center (Grant DE-PS02-06ER64304), which is sup-
ported by the Office of Biological and Environmental
Research in the DOE Office of Science. This research
also benefited from activities at the Southeast Collabo-
ratory for High-Field Biomolecular NMR, a research
resource at the University of Georgia, funded by the
National Institute of General Medical Sciences (NIG-
MS grant number GM66340) and the Georgia
Research Alliance.
Characterization of the recombinant enzymes
UGlcDH and UGlcAE activities were determined using dif-
ferent buffers, at different temperatures, and in the presence
of selected cations or potential inhibitors. For optimal pH
studies, solutions of recombinant BcUGlcDH or BcUGl-
cAE were first mixed in 100 mM of each individual buffer.
Subsequently, NAD+ (1 mM) and UDP-Glc (0.5 mM) were
added to BcUGlcDH reactions, whereas 1 mM NAD+ and
UDP-GlcA were added to BcUGlcAE reactions. Assays
were then carried out for 15 min (UGlcAE) or 30 min
(UGlcDH) at 37 ꢀC. Assays to determine the optimal tem-
perature of recombinant BcUGlcDH or BcUGlcAE were
similarly run under standard UGlcDH or UGlcAE reaction
conditions and incubated at variable temperatures for
15 min (UGlcAE) or 30 min (UGlcDH) before termination.
UGlcDH or UGlcAE inhibition assays were performed by
first mixing the enzyme with 100 mM phosphate buffer (pH
8) or 50 mM phosphate buffer (pH 7.6), respectively, with
various additives (e.g. nucleotides) on ice for 10 min. UDP-
Glc and NAD+ (0.5 and 1 mM, respectively) were then
added to UGlcDH assays, whereas 1 mM UDP-GlcA and
NAD+ were added to UGlcAE reactions. After 15 min
(UGlcAE) or 30 min (UGlcDH) at 37 ꢀC, the reactions
were terminated and the amount of UDP-sugar formed was
determined by quantitative-HPLC.
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Fig. S1. Sequence comparison and phylogenic analysis
of Bacillus UglcDH.
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