Cloning and comparison of third β-glucoside utilization (bglEFIA) operon with two operons of Pectobacterium carotovorum subsp. carotovorum LY34

Article abstract of DOI:10.1271/bbb.70.798

A third bgl operon containing bglE, bglF, bglI, and bglA was isolated from Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34). The sequences of BglE, BglF, and Bgll were similar to those of the phosphotransferase system (PTS) components IIB, II

Full text of DOI:10.1271/bbb.70.798

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Cloning and Comparison of Third β-Glucoside
Utilization (bglEFIA) Operon with Two Operons of
Pectobacterium carotovorum subsp. carotovorum
LY34
Su Young HONGa, Chang Long ANa, Kye Man CHOa, Sun Mi LEEa, Yong Hee KIMa, Min Keun
KIMa, Soo Jeong CHOa, Yong Pyo LIMc, Hoon KIMd & Han Dae YUNab
a Division of Applied Life Science, Gyeongsang National University
b Research Institute of Life Science, Gyeongsang National University
c Department of Horticulture, Chungnam National University
d Department of Agricultural Chemistry, Sunchon National University
Published online: 22 May 2014.
To cite this article: Su Young HONG, Chang Long AN, Kye Man CHO, Sun Mi LEE, Yong Hee KIM, Min Keun KIM, Soo Jeong
CHO, Yong Pyo LIM, Hoon KIM & Han Dae YUN (2006) Cloning and Comparison of Third β-Glucoside Utilization (bglEFIA)
Operon with Two Operons of Pectobacterium carotovorum subsp. carotovorum LY34, Bioscience, Biotechnology, and
Biochemistry, 70:4, 798-807, DOI: 10.1271/bbb.70.798
To link to this article: http://dx.doi.org/10.1271/bbb.70.798
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Biosci. Biotechnol. Biochem., 70 (4), 798–807, 2006
Cloning and Comparison of Third ꢀ-Glucoside Utilization (bglEFIA) Operon
with Two Operons of Pectobacterium carotovorum subsp. carotovorum LY34
1;
1
1
1
Su Young HONG,1; Chang Long AN, Kye Man CHO, Sun Mi LEE, Yong Hee KIM,
*
*
y
Min Keun KIM,1 Soo Jeong CHO,1 Yong Pyo LIM,3 Hoon KIM,4 and Han Dae YUN
1;2;
1Division of Applied Life Science, Gyeongsang National University, Chinju 660-701, Korea
2Research Institute of Life Science, Gyeongsang National University, Chinju 660-101, Korea
3Department of Horticulture, Chungnam National University, Daejeon 305-764, Korea
4Department of Agricultural Chemistry, Sunchon National University, Sunchon 540-742, Korea
Received July 28, 2005; Accepted December 7, 2005
A third bgl operon containing bglE, bglF, bglI, and
bglA was isolated from Pectobacterium carotovorum
subsp. carotovorum LY34 (Pcc LY34). The sequences of
BglE, BglF, and Bgll were similar to those of the
phosphotransferase system (PTS) components IIB, IIC,
and IIA respectively. BglF contains important residues
for the phosphotransferase system. The amino acid
sequence of BglA showed high similarity to various 6-
phospho-ꢀ-glucosidases and to a member of glycosyl
hydrolase family 1. Sequence and structural analysis
also revealed that these four genes were organized in a
putative operon that differed from two operons pre-
viously isolated from Pcc LY34, bglTPB (accession
no. AY542524) and ascGFB (accession no. AY622309).
The transcription regulator for this operon was not
found, and the EII complexes for PTS were encoded
separately by three genes (bglE, bglF, and bglI). The
BglA enzyme had a molecular weight estimated to be
57,350 Da by SDS–PAGE. The purified ꢀ-glucosidase
hydrolyzed salicin, arbutin, ꢁNPG, ꢁNPꢀG6P, and
MUG, exhibited maximal activity at pH 7.0 and 40 ꢀC,
and displayed enhanced activity in the presence of Mg2þ
cellulases, and ꢀ-glucosidases, all of which occur in
multiple enzymatic forms. Bacterial synthesis of these
isozymes might ensure a more efficient degradation of
polysaccharides present in the plant cell wall.1)
ꢀ-glucosidase is involved in the final step of cellulose
degradation, in which cellobiose is degraded to glu-
cose.2) ꢀ-glucosidases are widespread in bacteria, where
they are involved in the metabolism of various carbo-
hydrate substrates, including aromatic glycosides like
arbutin and salicin.2–6)
The phosphoenolpyruvate:phosphotransferase system
(PEP:PTS) is important for carbohydrate uptake, and is
largely used for bacterial utilization of ꢀ-glucoside.7–9)
PTS contains the general proteins enzyme I (EI) and HPr
(histidine containing protein), as well as the substrate-
specific enzyme II (EII).10–12) Reporting the sequence
data on the carbohydrate transport proteins, Saier13)
distinguished six families of sugar transporters of
bacterial PEP:PTS. Representing the sugar-specific
permease, EII complexes usually possess cytoplasmic
IIA and IIB and a membrane-spanning IIC.9,14)
The Pcc strain LY34 used in the present study was
originally isolated from Chinese cabbage with soft-rot
symptoms. To analyze their importance in phytopatho-
genicity, three different kinds of cellulases were pre-
viously isolated: CelA, CelB, and CelC of Pcc LY34.15)
Also, bglTPB and ascGFB operons, which regulate the
utilization of ꢀ-glucoside, were isolated and analyzed in
our previous studies.16,17) These two operons from Pcc
LY34 contain genes involved in sugar transport and
metabolism. The bglTPB operon possesses a positive
regulator (operon-specific antiterminator: BglT) for the
transcription of this operon, while the ascGFB operon
possesses a negative regulator (operon-specific repress-
or: AscG). Furthermore, BglP comprises three domains
(EIIB, EIIC, and EIIA) that are encoded by a single
ORF, whereas AscF consists of two domains (EIIB and
and Ca2þ. Two glutamate residues (Glu178 and Glu378
)
were found to be essential for enzyme activity.
Key words: Pectobacterium carotovorum subsp. caroto-
vorum LY34; bgl operon; PTS system; 6-
phospho-ꢀ-glucosidase
Pectobacterium carotovorum subsp. carotovorum
(Pcc), classified previously as Erwinia carotovora
subsp. carotovora (Ecc), is a pathogenic enterobacteria
that causes soft-rot disease in plants. This phytopath-
ogen degrades macromolecules that compose the plant
cell wall, resulting in maceration of plant tissue. This
macerating capacity comes from the secretion of a set of
extracellular enzymes including pectinases, proteases,
*
The first two authors contributed equally to this work.
To whom correspondence should be addressed. Tel: +82-55-751-5469; Fax: +82-55-757-0178; E-mail: hdyun@nongae.gsnu.ac.kr
y

Third bgl Operon of Pectobacterium sp. LY34
799
EIIC) in a single polypeptide. BglP and AscF proteins
have high similarities to those of E. coli BglF and AscF
respectively. The bglB and ascB genes encode 6-
phospho-ꢀ-glucosidase, which belongs to glycosyl hy-
drolase family 1.
ends. The resulting DNA fragments were ligated with
cloning-ready pCC1FOS vector and then packaged
using a lambda DNA packing kit (Epicentre, Wisconsin,
USA). The library was screened on M9 media,18)
containing 1 mM 4-methylumbelliferyl ꢀ-D-glucoside
(MUG) for ꢀ-glucosidase activity (a positive colony
has a fluorescent halo). A cosmid clone (pAY3) bearing
ꢀ-glucosidase activity was isolated. For subcloning,
pAY3 was partially digested with Sau3AI. Three-to six-
kb fragments of the cosmid DNA from this partial
digestion were ligated into the BamHI site of pBlue-
script II SK+ vector and transformants in E. coli DH5ꢁ
were screened as above. The positive subclone was
obtained (pAY300).
In this report, we describe the putative bglEFIA
operon genes from Pcc LY34, the third operon of ꢀ-
glucoside sugar utilization genes that are functionally
expressed in E. coli from a Pcc LY34 genomic library.
Sequence analysis of the active clone revealed a group
of linked genes involved in sugar transport and
metabolism, specifically, a putative bglEFIA operon
encoding a 6-phospho-ꢀ-glucosidase (bglA). The struc-
ture of this operon differed from those of bglTPB and
ascGFB operons isolated from the same strain. The
operon lacked a regulator gene for the transcription of
this operon, and the EII complex was composed of three
polypeptide chains encoded by three separate genes. The
biochemical properties of the BglA protein were also
examined.
Cloning of the bglE, bglF, bglI, and bglA genes. To
amplify bglE, bglF, bglI, and bglA from the Pcc LY34
chromosome, specific oligonucleotide primers were
designed based on the DNA sequence data. The primers
for bglE, bglF, bglI, and bglA were 50-TTCCCCGATT-
GCGCTATTTATG-30 (sense), 50-GATGGAGCGCAG-
ATGAGCAGAAC-30 (antisense); 50-CGCTGTTGAG-
ATAAAAGCCGTAG-30 (sense), 50-CACAGAAGCC-
GCGTTCAGCAGTTC-30 (antisense); 50-CTGATTGT-
CGTCTCATCCTTAATC-30 (sense), 50-CTCCAGCTC-
ATCGCCATTGGGGAAAATACG-30 (antisense); and
Materials and Methods
Bacterial strains and growth conditions. Tryptone-
yeast extract medium was used for the routine culti-
vation of Pectobacterium carotovorum subsp. carotovo-
rum LY34 (Pcc LY34) strain. Escherichia coli DH5ꢁ,
BL21 (DE3), and recombinant E. coli harboring the
bglA gene were cultured in LB medium containing
50 mg/ml ampicillin when appropriate.
50-ATCCAGACTGCTTTGATTGGTGCC-30
(sense),
50-AACCCCGCTTACACACTACATTCT-30 (antisense)
respectively. The purified PCR products of the 1.4-, 1.7-,
0.8-, and 1.8-kb fragments respectively were cloned into
pGEM-T Easy vector (Promega, Wisconsin, USA) and
sequenced. The resulting plasmids were designated
pAY310, pAY320, pAY330, and pAY340. For high
expression of bglA, the PCR product generated with
primers 50-GGATCCATGTCTGTTCAACAATTACC-
G-30 (sense, containing a BamHI site as underlined)
and 50-AAGCTTGTAGCTCGGCACCGTTGCT-30 (an-
tisense, containing a HindIII site as underlined) was
cloned into expression vector pET-21a (+) (Novagen,
Darmstadt, Germany) using BamHI and HindIII sites,
resulting in the addition of a C-terminal (His)6 tag. The
resulting plasmid was designated pET-21a(+)/BglA
(pAY510). The absence of mutations within the coding
region of BglA was verified by DNA sequencing.
Recombinant DNA techniques and DNA sequencing.
Standard procedures for restriction endonuclease diges-
tions, agarose gel electrophoresis, purification of DNA
from agarose gels, DNA ligation, and other cloning-
related techniques were followed.18) Restriction en-
zymes and DNA modifying enzymes were purchased
from Gibco-BRL (Gaithersburg, Maryland, USA) and
Boehringer Mannheim (Indianapolis, Indiana, USA).
Other chemicals were purchased from Sigma Chemical
(St. Louis, Missouri, USA). Nucleotide sequences were
determined by the dideoxy-chain termination method
using the PRISM Ready Reaction Dye terminator/
primer cycle sequencing kit (Perkin-Elmer., Norwalk,
Connecticut, USA). The samples were analyzed with an
automated DNA sequencer (Applied Biosystems, Foster
City, California, USA). The BLAST program was used
to find the protein coding regions. The nucleotide
sequence data reported are available in the GenBank
database under accession no. AY769096.
Site-directed mutagenesis. Site-directed mutagenesis
of the bglA gene to create the E178A and E378A
mutations was performed using the following oligonu-
cleotide primers: E178A, 50-TGGATGACTTTCAAT-
GCGATCAACAACCAGC-30 (sense) and 50-GCT-
GGTTGTTGATCGCATTGAAAGTCATCCA-30 (anti-
sense); E378A, 50-CAATGTTCATCGTCGCAAACGG-
TTTTGGCGC-30 (sense) and 50-GCGCCAAAAC-
CGTTTGCGACGATGAACATTG-30 (antisense). The
50 ml of reaction mixtures contained 1 ml of pET-
21a(+)/BglA DNA (80 ng/ml), 100 ꢂmol of each pri-
mer, 5 ml of 2 mM dNTP mixture, 5 ml of 10 ꢀ pfu DNA
polymerase buffer containing 20 mM MgSO4, and 2.5 U
Construction of cosmid library and cloning of the
bglEFIA operon. A genomic library was constructed in
the cosmid vector pCC1FOS as previously described.19)
Total genomic DNA from Pcc LY34 was sheared into
approximately 40-kb fragments using a syringe needle,
size-fractionated on a 5 to 40% linear sucrose gradient,
and then end-repaired to yield blunt, 50-phosphorylated

800
S. Y. HONG et al.
of cloned Pfu DNA polymerase purchased from Stra-
tagene (La Jolla, California, USA). PCR products were
incubated on ice for 5 min, and then 1 ml of DpnI
restriction enzyme (10 U/ml) was added for 1 h incuba-
tion at 37 ꢁC. DpnI-treated plasmids were then trans-
formed into E. coli DH5ꢁ according to the manufac-
turer’s specifications (Site-directed mutagenesis kit,
Stratagene, La Jolla, California, USA). The resulting
plasmids were designated pAY520 and pAY530 respec-
tively.
glucosidase activity was assayed using ꢂ-nitrophenyl
ꢀ-D-glucopyranoside-6-PO4 (ꢂNPꢀG6P). Preparation of
ꢂNPꢀG6P was by the procedure described earlier for the
synthesis of ꢂNPꢁG6P.17,28,29)
Expression and purification of the enzyme. E. coli
strain BL21 (DE3) carrying pET-21a(+)/BglA was
grown at 37 ꢁC to mid-log phase in LB medium
containing 50 mg/ml ampicillin. Expression was then
induced by adding IPTG to a final concentration of
0.5 mM, and growth was continued for 6 h. The cells
were harvested by centrifugation (6,000 rpm, 10 min)
and washed twice with 10 mM Tris–HCl buffer (pH 7.0).
The cells were resuspended in the same buffer disrupted
by sonication at 4 ꢁC, and centrifuged (6,000 rpm, 30
min) to remove cell debris. The solubilized recombinant
BglA with His-tag (BglA-His) was applied on a HisTrap
kit (Amersham Pharmacia Biotech, Pennsylvania, USA).
BglA was eluted with 100 mM imidazole. The enzyme
samples were analyzed by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS–PAGE). The
active fractions were combined and purified by ion
exchange chromatography on a Q-Sepharose column
(Amersham Pharmacia Biotech, Pennsylvania, USA).30)
For the final purification step, the dialyzed sample was
loaded onto an anion exchange Mono Q HR (Amersham
Pharmacia Biotech, Pennsylvania, USA) column pre-
equilibrated with 20 mM MOPS buffer, pH 6.5. The
fractions with ꢀ-glucosidase activity eluted as a single
protein peak and the purity of the enzyme was assessed
by SDS–PAGE. The protein concentration was deter-
mined by the method of Bradford.31)
Enzyme assay. ꢀ-glucosidase activity was determined
using ꢂ-nitrophenyl ꢀ-D-glucopyranoside (ꢂNPG),20,21)
4-hydroxyphenyl ꢀ-D-glucopyranoside (arbutin), 2-(hy-
droxymethyl) phenyl ꢀ-D-glucopyranoside (salicin), and
4-methylumbelliferyl ꢀ-D-glucoside (MUG)22) as sub-
strates. The standard assay consisted of incubating
enzyme with 5 mM ꢂNPG in 50 mM sodium phosphate
buffer (pH 7.0) for 10 min in a total volume of 0.5 ml.
The reaction was stopped by the addition of 0.5 ml of
0.2 M glycine–NaOH (pH 10.5), and the amount of ꢂ-
nitrophenol released from the ꢂNPG was determined by
measuring absorbance at 405 nm.23,24) One unit of ꢀ-
glucosidase was defined as the amount of enzyme
required to release 1 mole of ꢂ-nitrophenol per min
under the assay conditions. All assays were carried out
in 50 mM sodium phosphate buffer (pH 7.0) at 40 ꢁC,
unless otherwise noted. In order to detect the ꢀ-
glucosidase activity for salicin as substrate, an appro-
priate aliquot of cell suspension of the bglA clone was
added to 800 ml of 30 mM salicin in 50 mM phosphate
buffer (pH 7.0). After 30 min of incubation, the enzy-
matic reaction was stopped by adding 0.5 ml of 1 M
Na2CO3. The production of saligenin from salicin was
detected as described previously.5,25) Enzyme activity
for the arbutin substrate was measured by washing the
culture and resuspending it in 0.8 ml of 50 mM phosphate
buffer (pH 7.0). The reaction was started by adding
0.1 ml of 10 mM MgCl2 and 0.1 ml of 30 mM arbutin, and
was stopped by 0.5 ml of 1 M Na2CO3, as described
previously.26)
The effects of pH and temperature on ꢀ-glucosidase
activity were examined with the purified recombinant
enzyme. The effect of pH on ꢀ-glucosidase activity was
determined in the standard assay, but with pH values
ranging from pH 4.0 to 9.0; all assays were performed at
40 ꢁC. To determine the effect of temperature on the
enzymatic activity, samples were incubated at temper-
atures from 20 to 60 ꢁC for 30 min. Thermostability data
were obtained by pre-incubating BglA at various
temperatures and then measuring residual activity under
the standard assay condition. The effects of various
metal ions classified by a concentration of 10 mM on ꢀ-
glucosidase activity were also examined. The kinetic
constants, Km and Vmax, corresponding to the ꢀ-D-
glucosidase activity of purified mature BglA for the ꢂ-
nitrophenyl ꢀ-D-glucopyranoside were examined by
using the Michaelis-Menten equation.27) Phospho-ꢀ-
Results
Cloning and nucleotide sequence analysis of the
putative bglEFIA operon
A cosmid library of Pcc LY34 genomic DNA was
screened for clones expressing ꢀ-glucosidase activity on
M9 media containing 1 mM MUG. A cosmid clone
expressing ꢀ-glucosidase activity was isolated and
designated pAY3. Sau3AI subclones of pAY3 were
then screened using the same assay, yielding the positive
clone pAY300 containing a 3.7-kb insert (Fig. 1). A
3,738-bp DNA fragment was sequenced and analyzed
(accession no. AY769096). Sequence analysis identified
the presence of four open reading frames, bglE, bglF,
bglI, and bglA, putatively encoding proteins of 101-,
437-, 108-, and 480-aa respectively (Fig. 2). The bgl
genes were cloned separately, as described in ‘‘Materials
and Methods.’’
Structure of the putative bglEFIA operon of Pcc LY34
and comparison to other bgl operons
The National Center for Biotechnology Information’s
BLAST e-mail server (USA) was used to search the
peptide sequence databases for proteins homologous to
the Bgl proteins. The Pcc LY34 BglE, BglF, BglI, and

Third bgl Operon of Pectobacterium sp. LY34
801
bglB
bglT
bglP
ascF
pAY100
pAY200
pAY300
1
5557
ascB
ascG
1
1
5618
3738
bglE
bglF
BglI
bglA
Enzyme activity for substrate
Salicin
+
pNPG
Arbutin
+
MUG
+
pNPβGlu6P
pAY340
bglA
pET-21(a)/bglA
E178A
+
+
+
-
-
+
-
-
+
-
-
+
-
-
+
-
-
pAY510
pAY520
pAY530
E378A
Fig. 1. Comparison of the Putative ꢀ-Glucoside Utilization Operons from Pectobacterium carotovorum subsp. carotovorum LY34.
Physical map of recombinant DNA fragments. pAY100 (accession no. AY542524), pAY200 (accession no. AY622309), and pAY300
(accession no. AY769096) were constructed by cloning 5.5-, 5.6-, and 3.7-kb fragments of cosmid DNA (pAY1, pAY2, and pAY3) into
pBluescript II SK+ vector. pAY340 was derived by cloning the PCR products from pAY300 into pGEM-T Easy vector. pAY510 was derived by
cloning into pET-21a(+) expression vector. pAY520 and pAY530 were derived by site-direct mutation (E178A and E378A) from pAY510. ꢀ-
glucosidase activity (BglA/pAY500) was determined using ꢂ-nitrophenyl ꢀ-D-glucopyranoside (ꢂNPG), 4-hydroxyphenyl ꢀ-D-glucopyranoside
(arbutin), 2-(hydroxymethyl) phenyl ꢀ-D-glucopyranoside (salicin), 4-methylumbelliferyl ꢀ-D-glucoside (MUG), and ꢂ-nitrophenyl ꢀ-D-
glucopyranoside-6-PO4 (ꢂNPꢀGlu6P).
BglA proteins are associated with transport and utiliza-
tion of ꢀ-glucosides (Table 1). Based on these similar-
ities, we hypothesized that the Pcc LY34 proteins could
functionally replace CelA and CelB in Photorhabdus
luminescens subsp. laumondii TTO1 and BglA in E. coli
(Table 2). The putative operon bglEFIA differs from
two putative operons, bglTPB and ascGFB, previously
isolated from Pcc LY34 (Fig. 1).
The first ORF (bglE) spans 306 nucleotides and
encodes a deduced protein of 101 amino acids. There is
a potential ribosome-binding site, GAGG, located 6
bases upstream from the start codon of this ORF
(Fig. 2). The Pcc LY34 BglE protein exhibits 55.0,
54.0, and 51.5% similarity with P. luminescens subsp.
laumondii TTO1 CelA, V. cholerae phosphotransferase
system IIB component, and Clostridium acetobutylicum
phosphotransferase system IIB component respectively
(Table 1).
The second ORF (bglF) is 1,314-bp long, and the
deduced protein is 437-aa long. The start site of this
ORF is 16-bp downstream from the end of bglE. There
is a putative ribosome-binding site, GGAGGA, located 3
bases upstream from the start of this ORF (Fig. 2). The
deduced protein from this ORF displayed similarity to
several GenBank entries, with very high similarity with
the phosphotransferase system components IIC from
V. vulnificus YJ016, CelB from P. luminescens subsp.
laumondii TTO1, and diacetylchitobiose-specific IIC
from Bacillus cereus ATCC 14579 (Table 1, 2). Lai32)
compared PTS-dependent EII permease homologies and
observed patterns of hydrophobicity, suggesting a
central membrane-spanning IIC domain flanked by
hydrophilic IIB and IIA domains located in the N-
terminus and C-terminus respectively of CelA. But as a
single protein, BglF did not have a central membrane-
spanning IIC domain flanked by hydrophilic IIB and IIA
domains as in BglP and AscF of Pcc LY34 (Fig. 3A, B).
The third ORF (bglI) started 11-bp upstream of the
terminator of bglF. bglI was 327-bp long and was
deduced to be a protein of 108 amino acids (Fig. 2).
Preceding this ORF, no ribosome-binding site was
found. The Pcc LY34 BglI protein has 52.9, 51.5, and
49.5% similarity with the phosphotransferase system
components IIA from E. coli CFT073, CelC from
Yersinia pestis KIM, and CelC from P. luminescens
subsp. laumondii TTO1 (Table 1, 2).
The fourth ORF (bglA) begins 348-bp downstream
from bglF and is 1,443 nucleotides long. It is predicted
to encode a protein of 480 amino acids. A putative
ribosome-binding site, TAAGAAG, is positioned 8
bases from the start of the coding region (Fig. 2). The
predicted BglA protein, which has homology to ꢀ-
glucosidase from a variety of organisms, is classified in
family 1 of the glycosyl hydrolases.33) BglA also
contained two family 1 glycosyl hydrolase signature
regions. We found a high level of identity (approx-
imately 76%) among the Y. pestis KIM BglA,
E. coli BglA, and S. typhimurium LT2 BglA proteins
(Table 1).
Purification and characterization of BglA
Using an E. coli strain that overexpressed BglA, the
protein was purified using column filtration techniques,
as described in ‘‘Materials and Methods.’’ Protein
fractions from the purification steps were analyzed by
SDS–PAGE, and only one protein band (approximately
57 kDa) was present after the final purification step
(Fig. 4). The effect of pH on the activity of BglA against
ꢂNPG was determined at 40 ꢁC in various buffers
ranging from pH 4.0 to 9.0 (Fig. 5A). Maximal activity
was observed at pH 7.0. The temperature dependence of
BglA activity toward ꢂNPG was determined by meas-
uring activity at various temperatures at pH 7.0. Max-
imal activity was observed at 40 ꢁC (Fig. 5B). Thermo-

802
S. Y. HONG et al.
Fig. 2. Nucleotide Sequence of Pectobacterium carotovorum subsp. carotovorum LY34 bgl Genes and the Flanking Regions.
The deduced amino acid sequences for each ORF are placed below the nucleotide of the corresponding codon. Putative Shine-Dalgarno
sequences for ribosomal binding site (RBS) are underlined and labeled. Proteins are labeled at their respective start codons. The signature
sequence for disaccharide binding site is underlined and labeled. The family 1 hydrolase signature sequences are also underlined and labeled.
stability data were obtained by pre-incubating BglA at
various temperatures and then measuring residual ꢂNPG
hydrolyzing activity under the standard assay condition.
After 10 min of incubation at 60 ꢁC, the activity dropped
by 38% (Fig. 5C). ꢀ-glucosidase activity was also
measured at pH 7.0 and 40 ꢁC in the presence of various
The Km value of BglA was 0.23 mM and the Vmax value
was 36 mmol/min/mg of protein. The values of Km and
Vmax were not detected in single-point mutants E178A
and E378A (Table 4).
Identification of essential residues for ꢀ-glucosidase
activity
metal ions. Divalent cations such as Zn2þ, Cu2þ, Hg2þ
,
Mn2þ, and Co2þ inhibited enzyme activity while Mg2þ
and Ca2þ had positive effects (Table 3). The addition of
15 mM Mg2þ yielded the maximum activity on ꢂNPG
(Fig. 5D). The kinetic parameters of ꢀ-glucosidase (Km
and Vmax) were determined using ꢂNPG as the substrate.
In vitro site-directed mutagenesis can be invaluable in
studying protein structure-function relationships. In the
present study, we used site-directed mutagenesis to
replace two Glu residues with Ala at sites in BglA that
might serve catalytic functions. Mutant plasmids were

Third bgl Operon of Pectobacterium sp. LY34
803
sequenced on both strands to confirm that only the
Discussion
intended mutation was introduced. Enzyme activity
assays were then performed with BglA mutants carrying
Glu178Ala and Glu378Ala. These mutations abolished
enzyme activity for the ꢀ-glucoside (Table 4). These
results indicate that Glu178 and Glu378, which are both
conserved in the ꢀ-glucosidase sequences of family 1,
are essential for BglA activity (Table 2).
There have been several physiological and genetic
studies of the ꢀ-sugar metabolic system. Many bacteria
contain more than one ꢀ-sugar catabolic pathway, with
E. coli containing four ꢀ-glucoside sugar systems.34)
Previously, bglTPB and ascGFB operons for uptake and
utilization of ꢀ-sugars were isolated from Pcc LY34. In
the present study, we isolated four genes (bglE, bglF,
bglI, and bglA) that form the genetic organization of a
putative operon. The respective genes encode (I) the
putative IIB component of PTS system, BglE; (II) PTS
permease BglF, a phosphotransferase system component
IIC protein; (III) the putative IIA component of PTS
system, BglI; and (IV) the ꢀ-glucosidase, BglA, that
contains two conserved regions of glycosyl hydrolase
family 1 (Fig. 2). This operon differed from the two
operons above (bglTPB and ascGFB) in that it lacked an
operon-specific antiterminator (BglT) and an operon-
specific repressor (AscG). The bglEFIA system might at
least specify a transport system and a hydrolase that acts
on salicin, arbutin, ꢂNPG, and MUG.
The molecular weight of ꢀ-glucosidase (BglA) was
estimated by SDS–PAGE to be 57,350 Da. ꢀ-glucosi-
dase exhibited maximal activity at pH 7.0 and 40 ꢁC,
with enhanced activity in the presence of Mg2þ and
Ca2þ (Table 3, Fig. 5D). The optimal activity of BglA
showed similarly optimal activity at the same pH and
temperature to that previously reported for BglB and
AscB, with Mg2þ also enhancing these enzymes.16,17)
Furthermore, BglA showed 52.7 and 52.2% identity with
BglB and AscB respectively. Three ꢀ-glucosidases,
BglA, BglB, and AscB, clearly belong to glycosyl
hydrolase family 1, containing a conserved enzyme that
employs a double-displacement mechanism of cataly-
sis.35) In view of two glutamic acid residues, acting as an
acid/base and a nucleophile respectively in the hydrol-
ysis of ꢀ-glucosidic bonds in the ꢀ-glucosidase from
Table 1. Pairwise Similarity (%) of BglE, BglF, BglI, and BglA of
Pcc LY34
Similarity (%) with related amino acid sequencesa
Sequence
BglE
1
2
3
4
100.0
55.0
100.0
54.0
47.5
51.5
42.6
CAE15128
D82219
A96947
100.0
43.6
100.0
BglF
100.0
100.0
100.0
53.6
100.0
51.1
55.3
41.2
38.0
BAC95664
CAE15129
AAP09357
100.0
36.9
100.0
BglI
52.9
100.0
51.5
49.5
49.5
50.0
AAN80414
AAM84827
CAE15130
100.0
42.5
100.0
BglA
80.7
100.0
76.2
79.0
76.1
79.5
AAM86483
Q46829
AAL21926
100.0
95.4
100.0
a
Calculated with CLUSTAL W and the PAM250 residue weight table.
The sequences are from the following sources: BglE, BglF, BglI, and BglA
from Pcc LY34 (1); CAE15128 (2), CAE15129 (3), and CAE15130 (4) from
Photorhabdus luminescens subsp. laumondii TTO1; D82219 (3) from Vibrio
cholerae; A96947 (4) from Clostridium acetobutylicum; BAC95664 (2)
from Vibrio vulnificus YJ016; AAP09357 (4) from Bacillus cereus ATCC
14579; AAN80414 (2) from E. coli CFT073; AAM84827 (3) and
AAM86483 (2) from Yersinia pestis KIM; Q46829 (3) from E. coli;
AAL21926 (4) from Salmonella typhimurium LT2.
Table 2. Alignment of the Regions Surrounding the Homologous Region of BglE and BglI, Conserved Regions of BglF, and Putative Acid/Base
and Nucleophile of BglA and Some Typical Representatives of Glycosyl Hydrolase Family 1
BglE
BglF
BglI
BglA (ꢀ-glucosidase)
EIIB
EIIC
EIIA
Acid/base
Nucleophile
BglE
CCAAGMSTS BglF
FNINEPVIFGSPV BglI
VHAQDHLMN BglA
TFNEINNQ MFIVENGFG
CAE15128 CCAAGMSTS BAC95664 FQINEPVIFGSPV AAN80414 IHAQDHLMN AAM86483 TFNEINNQ LFIVENGFG
D82219
A96947
CCSAGMSTS CAE15129 FNINEPLLFGSPI AAM84827 VHAQDHLMN Q46829
FCSAGMSTS AAP09357 FNINEPVIFGLPI CAE15130 VHAQDHLMN AAL21926 TFNEINNQ LFIVENGFG
TFNEINNQ LFIVENGFG
AAL00638 VCNAGMSTS AAO81318 FNINEPVIFGVPI CAG21150 THIQDHIMT AAN44371 TFNEINNQ LFIVENGFG
AAP29103 CCAAGMSSS H70216 FNINEPIMFGAPI AAL20239 VHAQDHLMT H82185 TFNEINNQ IFVVENGLG
CAD00451 VCAAGMSTS BAC12654 FNISEPTIFGAPV AAF94442 VHAQDHLMT CAE14566 TFNEINNQ IFIVENGLG
AAC66322 VCGAGMSTS AAM23871 FNINEPIIFGAPI AAN43082 VHAQDHLMT P42973 TFNEINNQ LFIVENGFG
* * * * . * * . * * * . * * * . * * * . * . * * * * * * * * . * . * ** *
*
*
*
The sequences are from the following sources: BglE, BglF, BglI, and BglA from Pcc LY34; CAE15128, CAE15129, CAE15130, and CAE14566 from Photorhabdus
luminescens subsp. laumondii TTO1; D82219 from Vibrio cholerae; A96947 from Clostridium acetobutylicum; AAL00638 from Streptococcus pneumoniae R6;
AAP29103 from Bacillus anthracis str. Ames; CAD00451 from Listeria monocytogenes; AAC66322 from Borrelia burgdorferi B31; BAC95664 from Vibrio
vulnificus YJ016; AAP09357 from Bacillus cereus ATCC 14579; AAO81318 from Enterococcus faecalis V583; H70216 from Borrelia burgdorferi; BAC95664 from
Vibrio vulnificus YJ016; AAM23871 from Thermoanaerobacter tengcongensis; AAN80414 from E. coli CFT073; Q46829 from E. coli; AAM84827 and AAM86483
from Yersinia pestis KIM; CAG21150 from Photobacterium profundum; AAL20239 and AAL21926 from Salmonella typhimurium LT2; AAF94442 from Vibrio
cholerae 01 biovar eltor str. N16961; AAN43082 and AAN44371 from Shigella flexneri 2a str. 301; H82185 from Vibrio cholerae; P42973 from Bacillus subtilis.

804
S. Y. HONG et al.
A
19NVISVMHCATRLRFKLKD26
110
370FGITEPAVYGVTL382
549GAEVLIHVGIDTV561
487 610
4
390
EIIB81
EIIC
EIIA
BglP
1
633
21NIAAVTHCMTRLRFVLKD38
82
372AGISEPALYGVAL384
9
113
33
397
AscF
EIIC
EIIB
486
1
3
326FNINEPVIFGSPV338
352
4
99
110
BglEFI
EIIC
EIIA
EIIB
108
1
101
1
437
B
4
81
110
390
487
610
EIIC
EIIB
EIIA
BglP
82
113
397
9
EIIB
EIIC
AscF
33
352
EIIC
BglF
Fig. 3. Comparison of Phosphotransferase EII Domains from Pectobacterium carotovorum subsp. carotovorum LY34.
A, PTS EII Domains of BglP, AscF, and BglEFI. Three EII complexes of BglP, AscF, and BglEFI respectively are exhibited. The numbers at
the extremes of the putative EII domain represent the first and last amino acid residues of the putative EIIA, EIIB, and EIIC regions respectively.
Disaccharide-binding sites in EIIC domains are designated above the regions of the polypeptide chains. Residues corresponding by homology to
those important for phosphoryl transfer (EIIA and EIIB) are indicated. B, Maps of the putative transmembrane proteins across membranes by
SOSUI of proteomics tool (http://sosui.proteome.bio.tuat.ac.jp/sosui subunit.html). The respective domains of EII complexes are shown.
Designated numbers indicate the positions of the corresponding amino acids.
Agrobacterium faecalis,35,36) site-directed mutagenesis
was performed on BglA (Table 2). Based on the results,
we propose that Glu178 and Glu378 are catalytic sites in
BglA.
The PTS proteins encoded by bglE, bglF, and bglI
were similar to those from the IIB, IIC, and IIA
components respectively of PTS (Table 1, 2). These four
genes (bglE, bglF, bglI, and bglA) are also contiguously
transcribed in the same direction of the reading frame
(Fig. 1). This further suggests that this gene cluster
might form an operon involved in the utilization of
carbon sources.
specific PTS transporters have been identified.39) The EII
complex (sugar-specific permease) of the putative bgl
operon from Pcc LY34 contained BglE, BglF, and BglI,
which correspond to EIIB, EIIC, and EIIA respectively.
BglF, BglP, and AscF belong to the glucose-glucoside
(Glc) PTS system which transports glucose, N-acetyl-
glucosamine, and various ꢁ- and ꢀ-glucosides of the six
PTS family. Hydropathy analysis using GCG-ALOM
has indicated that BglF contains 7 transmembrane
helices,40) and thus differs from BglP and AscF trans-
membrane patterns. BglF separates with BglE (EIIB)
and BglI (EIIA), whereas BglP and AscF contain the
EIIA or EIIB domain (Fig. 3B). Hence, we propose that
this putative EII complex might belong to one of the
currently known six PTS families and might function as
a ꢀ-sugar transporter in the Pcc bgl operon. Addition-
ally, it is assumed that different compositions of these
PTS EII typically consists of two hydrophilic domains
(EIIA and EIIB) that bind to the sugar substrate and a
membrane spanning domain for transport (EIIC)14,32,37)
whereas the IIA, IIB, and IIC components can occur as
individual proteins.13,38) Currently, six families of sugar-

Third bgl Operon of Pectobacterium sp. LY34
805
three proteins might exist to serve different roles in
various bacterial environments. This implies that there
are various complex catabolic mechanisms of ꢀ-oligo-
sugar in Pcc LY34. Further experimentation is needed to
further clarify the PTS system.
In summary, we found that Pcc LY34 possesses at
least three sets of genes with potential for ꢀ-glucoside
utilization. It is believed that cellulolytic enzymes serve
as cell wall-modifying enzymes, since their action
renders other polysaccharide components in plant cell
walls more susceptible to hydrolysis. It might be
advantageous for Pcc LY34 to have a variety of
mechanisms for ꢀ-glucoside utilization. The cloning of
additional ꢀ-glucosidases of Pcc LY34 should help
towards a better understanding of this complex system.
1 2 3 4
116
Table 3. Effect of Metal Ions on BglA
Ion (10 mM)
Relative activitya
Mg2þ
Ca2þ
Cu2þ
Mn2þ
Co2þ
Hg2þ
Zn2þ
110
109
90
75
69
0
66
45
56
a
35
ꢀ-glucosidase (0.05 unit in 200 ml of 50 mM phosphate buffer, pH 7.0) was
exposed for 30 min at room temperature to various concentrations of metal
ions. The activity was then measured under standard conditions and
expressed relative to the activity measured on ꢂ-nitrophenyl ꢀ-D-glucopyr-
anoside (100) without added ions.
25
(kDa)
Table 4. Specific Activity and Kinetic Parameters for Hydrolysis of
ꢂNPG by the BglA, E178A, and E378A Mutants
Fig. 4. Electrophoretic Analysis of Purified ꢀ-Glucosidase.
Separation was performed on a 12.5% (W/V) SDS–polyacryl-
amide gel. Lane 1, marker; lane 2, crude extract from un-induced
BL21 (DE3) containing pET-21a(+)/BglA; lane 3, crude extract
from IPTG-induced BL21 (DE3) containing pET-21a(+)/BglA;
lane 4, purified ꢀ-glucosidase from HiTrap kit (Amersham). The gel
was stained with 0.025% Coomassie blue R-250. The molecular
weight markers used were ꢀ-galactosidase (116,000 Da), bovine
serum albumin (66,200 Da), ovalbumin (45,000 Da), lactate dehy-
drogenase (35,000 Da), restriction endonuclease Bsp 981 (25,000
Da), ꢀ-lactoglobulin (18,400 Da), and lysozyme (14,400 Da).
Kinetic parametera
Km (mM)
Vmax (mmol/min/mg protein)
BglA
E178A
E378A
0.23
ND
ND
36
ND
ND
a
The parameters were determined at 40 ꢁC (pH 7.0).
ND, not detectable.
A
B
100
90
80
70
60
50
40
30
20
100
90
80
70
60
50
40
30
4
5
6
7
8
9
20
30
40
50
60
pH
Temperature (°C)
C
D
140
100
90
80
120
100
70
60
50
40
30
20
10
0
80
60
40
20
0
0
10
20
30
40
50
0
5
10
15
20
25
Time (min)
Concentration (mM)
Fig. 5. Effect of pH, Reaction Temperature, Pre-Incubation Temperature, and Magnesium/Calcium Ions on the Activity of BglA.
A, Effect of pH on the relative activity of BglA. Enzyme activity was assayed at 40 ꢁC for 30 min in sodium phosphate buffers of indicated pH.
B, Effect of temperature on the relative activity of BglA. Enzyme activity was assayed at pH 7.0 for 30 min at the indicated temperatures. C,
Effect of time and temperature on the relative activity of BglA. Enzyme activity was assayed at 40 ꢁC ( ), 50 ꢁC ( ), and 60 ꢁC ( ), pH 7.0 for
60 min at the indicated reaction times. D, Effect of concentration of Mg2þ
was assayed at 40 ꢁC.
( ( ) on the relative activity of BglA. Enzyme activity
) and Ca2þ

806
S. Y. HONG et al.
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Acknowledgments
This work was supported by a grant from BioGreen
21 and ARPC (Ministry of Agriculture and Forestry of
Korea) and KRF (2005-042-F00013), Republic of
Korea. S.Y.H and C.L.A. are recipients of a BK21
fellowship from the Ministry of Education of Korea.
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