JOURNAL OF BACTERIOLOGY, Jan. 2004, p. 411–418
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 186, No. 2
Molecular and Functional Characterization of a Unique Sucrose
Hydrolase from Xanthomonas axonopodis pv. glycines
Hong-Suk Kim,1Hyoung-Joon Park,1Sunggi Heu,2and Jin Jung1*
School of Agricultural Biotechnology, Seoul National University, Seoul 151-742,1and Plant Pathology Division,
National Institute of Agricultural Science and Technology, RDA, Suwon 441-707,2South Korea
Received 11 August 2003/Accepted 20 October 2003
A novel sucrose hydrolase (SUH) from Xanthomonas axonopodis pv. glycines, a causative agent of bacterial
pustule disease on soybeans, was studied at the functional and molecular levels. SUH was shown to act rather
specifically on sucrose (Km? 2.5 mM) but not on sucrose-6-phosphate. Protein analysis of purified SUH
revealed that, in this monomeric enzyme with an estimated molecular mass of 70,223 ? 12 Da, amino acid
sequences determined for several segments have corresponding nucleotide sequences in XAC3490, a protein-
coding gene found in the genome of X. axonopodis pv. citri. Based on this information, the SUH gene, consisting
of an open reading frame of 1,935 bp, was cloned by screening a genomic library of X. axonopodis pv. glycines
8ra. Database searches and sequence comparison revealed that SUH has significant homology to some family
13 enzymes, with all of the crucial invariant residues involved in the catalytic mechanism conserved, but it
shows no similarity to known invertases belonging to family 32. suh expression in X. axonopodis pv. glycines
requires sucrose induction, and insertional mutagenesis resulted in an absence of sucrose-inducible sucrose
hydrolase activity in crude protein extracts and a sucrose-negative phenotype. Recombinant SUH, overpro-
duced in Escherichia coli and purified, was shown to have the same enzymatic characteristics in terms of kinetic
Plant-pathogenic bacteria grow in the intercellular spaces of
plant tissues, relying on nutrients available there. In higher
plants, sucrose (?-D-glucopyranosyl ?-D-fructofuranoside) is
the major transportable product of photosynthesis that flows
from the source organs to the sink organs. The process of the
source-sink flow involves phloem loading, for which sucrose
has to exit from the mesophyll cell, and from the apoplasm, it
enters the phloem. Sucrose is naturally the predominant form
of carbohydrate found in the intercellular spaces of photosyn-
thetically active tissues. Pathogens with their habitats in ma-
ture leaves, therefore, may utilize sucrose as the main, if not
the only, source for carbon and energy and possess systems for
a sucrose utilization pathway. Nevertheless, information on
such systems in plant-associated bacteria is rather scanty. To
our knowledge, Erwinia amylovora, the causative agent for fire
blight of rosaceous plants, is the only phytopathogen that has
been studied at the molecular level in relation to sucrose uti-
Many sucrose-positive bacteria have a phosphoenolpyru-
vate-dependent, sucrose-specific phosphotransferase system
(PTS) that promotes sucrose translocation across the cytoplas-
mic membrane with concomitant phosphorylation (27, 28).
The resulting intracellular sucrose-6-phosphate (S-6-P) is then
hydrolyzed by S-6-P hydrolase to yield D-glucose-6-phosphate
and D-fructose. In some bacteria, however, sucrose itself can be
transported into the cytoplasm without phosphorylation via a
pathway independent of PTS. For instance, the Escherichia coli
strain EC3132, which is able to grow on sucrose, bears a chro-
mosomally located regulon with structural genes for a sucrose
hydrolase, a fructokinase, and a transporter, regulated by a
sucrose-specific repressor (3, 13). Similar pathways involving
facilitated diffusion or active sucrose-ion symport and intracel-
lular invertase have also been invoked for several other bacte-
ria (11, 14, 35, 38). Certain bacteria secrete enzymes acting on
sucrose, such as hexosyltransferase, levansucrase, and levan-
ase, which split sucrose in the culture medium (22, 25, 40):
these microbes utilize sucrose as an energy source for growth,
as well as a substrate for oligosaccharide synthesis. Some bac-
teria show more than one sucrose hydrolysis activity (18, 25).
The genus Xanthomonas is arguably one of the most ubiq-
uitous groups of plant-associated bacteria. Members of this
group have been shown to infect at least 124 monocotyledon-
ous and 268 dicotyledonous plants (6). To gain insight into
sucrose utilization by Xanthomonas pathogens, a novel sucrose
hydrolase from Xanthomonas axonopodis was isolated and
characterized at the functional and molecular levels in the
present study. This protein, designated SUH, appears to be
essential for sucrose metabolism in X. axonopodis pv. glycines,
a causative agent of bacterial pustule disease on soybean. As a
sucrose-specific enzyme, SUH is unique in that not only does it
have no structural similarity to invertases (EC 184.108.40.206), the
typical sucrose hydrolases, but it also shows no significant se-
quence homology to ?-glucosidases (EC 220.127.116.11) with known
MATERIALS AND METHODS
Bacterial strains and culture conditions. Two strains of X. axonopodis pv.
glycines, the rifampin-resistant wild-type strain 8ra, obtained from E. J. Braun of
the University of Illinois at Urbana-Champaign, and a mutant constructed from
strain 8ra, were cultured at 28°C in either Luria-Bertani (LB) broth or minimal
medium. The basal composition of minimal medium was 20 mM NaCl, 10 mM
(NH4)2SO4, 5 mM MgSO4, 1 mM CaCl2, 0.32 mM K2HPO4, 0.16 mM KH2PO4,
* Corresponding author. Mailing address: School of Agricultural
Biotechnology, Seoul National University, Kwanak-ku, Seoul 151-742,
South Korea. Phone: (82) 2 880 4648. Fax: (82) 2 873 3112. E-mail:
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418KIM ET AL. J. BACTERIOL.