Affiliation:
1. EC Slater Institute, BioCentrum, University of Amsterdam, 1018 TV Amsterdam,1 and
2. Department of Molecular Genetics and Gene Technology, TNO Nutrition and Food Research Institute, 3700 AJ Zeist,2The Netherlands, and
3. Department of Food Science, Cornell University, Ithaca, New York 148533
Abstract
ABSTRACT
A 3-kb region, located downstream of the
Lactobacillus brevis xylA
gene (encoding
d
-xylose isomerase), was cloned in
Escherichia coli
TG1. The sequence revealed two open reading frames which could code for the
d
-xylulose kinase gene (
xylB
) and another gene (
xylT
) encoding a protein of 457 amino acids with significant similarity to the
d
-xylose–H
+
symporters of
E. coli
, XylE (57%), and
Bacillus megaterium
, XylT (58%), to the
d
-xylose–Na
+
symporter of
Tetragenococcus halophila
, XylE (57%), and to the
l
-arabinose–H
+
symporter of
E. coli
, AraE (60%). The
L. brevis xylABT
genes showed an arrangement similar to that of the
B. megaterium xylABT
operon and the
T. halophila xylABE
operon. Southern hybridization performed with the
Lactobacillus pentosus xylR
gene (encoding the
d
-xylose repressor protein) as a probe revealed the existence of a
xylR
homologue in
L. brevis
which is not located with the
xyABT
locus. The existence of a functional XylR was further suggested by the presence of
xylO
sequences upstream of
xylA
and
xylT
and by the requirement of
d
-xylose for the induction of
d
-xylose isomerase,
d
-xylulose kinase, and
d
-xylose transport activities in
L. brevis
. When
L. brevis
was cultivated in a mixture of
d
-glucose and
d
-xylose, the
d
-xylose isomerase and
d
-xylulose kinase activities were reduced fourfold and the
d
-xylose transport activity was reduced by sixfold, suggesting catabolite repression by
d
-glucose of
d
-xylose assimilation. The
xylT
gene was functionally expressed in
Lactobacillus plantarum
80, a strain which lacks proton motive force-linked
d
-xylose transport activity. The role of the XylT protein was confirmed by the accumulation of
d
-xylose in
L. plantarum
80 cells, and this accumulation was dependent on the proton motive force generated by either malolactic fermentation or by the metabolism of
d
-glucose. The apparent affinity constant of XylT for
d
-xylose was approximately 215 μM, and the maximal initial velocity of transport was 35 nmol/min per mg (dry weight). Furthermore, of a number of sugars tested, only 6-deoxy-
d
-glucose inhibited the transport of
d
-xylose by XylT competitively, with a
K
i
of 220 μM.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
56 articles.
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