Xylose Transport by the Anaerobic Thermophile Thermoanaerobacter
ethanolicus and the Characterization of a
D
-Xylose-Binding Protein
Milutin Erbeznik, Myrna Ray, Karl A. Dawson, Herbert J. Strobel
Department of Animal Sciences, 212 W.P. Garrigus Building, University of Kentucky, Lexington, Kentucky 40546-0215, USA
Received: 22 April 1998 / Accepted: 21 May 1998
Abstract. Thermoanaerobacter ethanolicus is a xylose-utilizing thermophilic anaerobe that produces
considerable amounts of ethanol. A protein in xylose-growing cells was solubilized from cell membranes
by extraction with octyl--glucoside. Internal peptide sequencing revealed that the protein was the
product of a gene, xylF, encoding a putative
D
-xylose-binding protein. Metabolic labeling with
14
C
palmitic acid suggested that this is a lipoprotein that is anchored to the cell membrane via a cysteine
residue. Binding was highly specific for xylose as evident by the lack of competition by sugars with
structures similar to xylose. The apparent K
d
of the protein for xylose was approximately 1.5 µ
M
, and this
value was very similar to the affinity constant determined for xylose transport by whole cells at low
substrate concentrations. Uptake experiments with cells also suggested the presence of a separate
low-affinity system. Binding activity varied less than 20% over a pH range of 4–8, and the level of activity
was virtually unaffected when temperature was varied between 40°C and 80°C. This is the first
biochemical characterization of a
D
-xylose-binding protein from a thermophilic organism.
Much of the recent interest in thermophilic bacteria is the
result of the novel metabolic and enzymatic activities that
many of these organisms possess. The use of thermo-
philic anaerobes for the conversion of fibrous biomass to
vendable chemicals, including ethanol, has attracted
considerable attention [19]. Yet, compared with meso-
philes, there is still a significant lack of metabolic and
genetic information for many of the organisms that carry
out these potentially useful transformations. Thermoan-
aerobacter ethanolicus is an anaerobic bacterium that is
xylanolytic, produces considerable amounts of ethanol
from various polymeric carbohydrate fractions found in
potential biomass feedstocks [2, 16, 29], and has been
suggested as an organism with biotechnological applica-
tions [28]. A xylose utilization operon was recently
identified, and the expression of enzymes responsible for
xylose use was found to be regulated at the level of
transcription [7].
The uptake of nutrients is a basic requirement of
bacterial growth, but little is known regarding the trans-
port mechanisms of thermophilic bacteria, including
T. ethanolicus. However, our initial characterization of
the xylose utilization operon of T. ethanolicus revealed an
open reading frame encoding for a deduced protein with a
high degree of similarity to xylose-binding proteins from
Escherichia coli and Haemophilus influenzae [7]. Subse-
quent sequence analysis predicted that a 25-residue
N-terminal fragment of the protein has features typical of
bacterial leader peptides [8]; in addition, the C-terminal
portion of this fragment matched the cleavage consensus
for lipoproteins in Gram-positive bacteria [26]. On the
basis of this presumptive evidence, it was of interest to
determine whether T. ethanolicus possessed a
D
-xylose-
binding protein. Since most sugar-binding proteins thus
far described are found in the periplasmic space of
Gram-negative bacteria, the presence of a binding protein
in T. ethanolicus was intriguing because this organism is
Gram-positive. This report describes the biochemical
characterization of
D
-xylose-binding protein encoded by
the T. ethanolicus xylF and the potential role it serves in
the transport of xylose.
Materials and Methods
Cell growth. T. ethanolicus 39E (ATCC 33223) was obtained from the
American Type Culture Collection (Rockville, Md.) and was grown in a
medium as previously described [6]. Cell growth was monitored by
optical density at 600 nm (OD
600
).
Published with the approval of the Director of the Kentucky Agricul-
tural Experiment Station as Journal article no. 98-07-79.
Correspondence to: H.J. Strobel
C
URRENT
M
ICROBIOLOGY
Vol. 37 (1998), pp. 295–300
An International Journal
Springer-Verlag New York Inc. 1998