FEMS Microbiology Letters 122 (1994) 217-222
© 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00
Published by Elsevier
217
FEMSLE 06172
Pentose transport by the ruminal bacterium
Butyrivibrio fibrisolvens
H e r b e r t J. S t r o b e l *
Department of Animal Sciences, 212 W.P. Garrigus Building, University of Kentucky, Lexington, KY 40517-0215, USA
(Received 25 April 1994; revision received and accepted 14 July 1994)
Abstract Butyrivibrio fibrisolvens is a fibrolytic ruminal bacterium that degrades hemicellulose and ferments the resulting pentose
sugars. Washed cells of strain D1 accumulated radiolabelled xylose (Km = 1.5 /xM) and arabinose (Km = 0.2 /xM) when the
organism was grown on xylose, arabinose, or glucose, but cultures grown on sucrose or cellobiose had little capacity to transport
pentose. Glucose and xylose inhibited transport of each other non-competitively. Both sugars were utilized preferentially over
arabinose, but since they did not inhibit transport of arabinose, it appeared that the preference was related to an internal metabolic
step. Although the protonmotive force was completely abolished by ionophores, cells retained some ability to transport pentose. In
contrast, the metabolic inhibitors iodoacetate, arsenate, and fluoride had little effect on protonmotive force but caused a large
decrease in intracellular ATP and xylose and arabinose uptake. These results suggested that high-affinity, ATP-dependent
mechanisms were responsible for pentose transport and hexose sugars affected the utilization of xylose and arabinose.
Key words: Ruminal bacteria; Xylose; Arabinose; Butyrivibrio fibrisolvens
Introduction
Considerable amounts of pentose sugars, principally xylose and arabinose, are found in feedstuffs provided to ruminant animals. Although
many ruminal bacteria ferment pentoses, there
has been relatively little study devoted to pentose
sugar utilization. Butyrivibrio fibrisolvens is a predominant ruminal organism that has also been
isolated from fecal material of non-ruminants and
from anaerobic digesters [1,2]. All characterized
strains are xylanolytic, and hemicellulose degradation may be an important niche for this bac-
* Corresponding author.
SSDI 0 3 7 8 - 1 0 9 7 ( 9 4 ) 0 0 3 2 4 - 6
terium. However, previous work indicated that
the organism prefers disaccharides over xylose [3]
and recently it has been shown that glucose was
used preferentially over arabinose [4]. Since solute transport is one possible process influencing
substrate preferences, it was of interest to examine pentose transport by B. fibrisolvens.
Materials and Methods
Growth conditions
Butyrivibrio fibrisolvens D1 (ATCC 19171)was
obtained from M. Allison, National Animal Disease Center, Iowa and grown in a defined anaerobic medium as described previously [5]. The
218
D-isomer of xylose and L-isomer of arabinose
were used in all experiments.
Transport assays
Uptake of radiolabelled [U-14C]xylose (83 mCi
mmol i), [3H]arabinos e (3 Ci mmol 1), or [UHC]glucose (320 mCi mmol-1) was performed as
described previously [6]. Cells were harvested
during exponential growth, washed anaerobically
in sodium phosphate buffer containing 10 mM
MgCI: (50 mM, pH 7.0) unless otherwise indicated, and resuspended in buffer. Preliminary
experiments established that uptake was proportional to the amount of bacterial protein in assays
( < 100 p.g protein) and all kinetic data were
based on initial rate determinations ( < 20 s). All
assays were conducted in sodium phosphate
buffer unless indicated otherwise. Radiolabelled
xylose and glucose were purchased from DuPontNEN (Boston, MA) and [3H]arabinose was synthesized by Moravek Biochemicals (Brea, CA).
Protonmotiue force determinations
lntracellular pH was determined essentially as
described previously [6]. Cells (2 ml; 100-350 /xg
protein ml i) were incubated anaerobically with
3 H 2 0 (1 /xCi), [1,2-14C]taurine (5(10 nCi; 92.1
mCi m m o l - l ) , or [7-14C]benzoate (500 nCi; 21.8
mCi mmol i) for 5 min at 39 ° C. The cells were
centrifuged through silicone oil (50:50 mixture of
Dow Corning 550 and 556; Accumetric, Inc., Elizabethtown, KY). Supernatant samples (20 /xl)
were collected, and the bottoms of the microcentrifuge tubes (containing cell pellets) were clipped
with dog nail clippers after freezing. Radioactivity
in the supernatant and cell pellets was determined by liquid scintillation after mixing with
scintillation cocktail. Membrane potential (A0)
was measured from the uptake of [U-~4C]tetraphenylphosphonium bromide (400 nCi; 19.2 mCi
mmol ~; final concentration of 10.4 /xM). lntracellular volume was estimated by the difference
in specific activities of 3 H 2 0 and [14C]taurine
and ranged from 4.1 to 5.4 p,l (mg protein) 1.
Non-specific binding of tetraphenylphosphonium
bromide was determined by exposing cells to oxygen and a mixture of nigericin and valinomyein
(10/xM each) for 15 min prior to the addition of
radioisotope. The protonmotive force (Ap) was
calculated as the summation of the membrane
electrical potential (A0) and the chemical gradient of protons (ZApH). Radiolabelled chemicals
were obtained from DuPont-NEN.
Analyses
Samples were withdrawn anaerobically from
culture vessels using a syringe, cells were immediately separated from culture fluid by centrifugation (15 000 × g, 5 min, 4°C), and cell-free supernatants were frozen at - 2 0 ° C until analysis. Glucose was measured using hexokinase and glucose
6-phosphate dehydrogenase [7]. In cultures containing only a single pentose, a colorimetric assay
was used to measure pentose [8]; hexosc was
differentiated from pentose by absorption difference at 660 nm versus 600 nm. In cultures containing both xylose and arabinose, alditol acetate
derivatives of the sugars were prepared using a
previously described method [4,9]. Intracellular
ATP was measured with a Bio-Orbit Luminometer (LKB, Gaithersburg, MD) using luciferin plus
luciferase [10], and bacterial protein was measured by the method of Lowry et al. [11] after cell
hydrolysis (0.2 N NaOH, 20 min, 100°C).
Results
Pentose transport
Washed B. fibrisoh,ens cells accumulated radiolabelled xylose and arabinose in anaerobic
potassium phosphate buffer and the inclusion of
sodium did not stimulate uptake (data not shown).
The capacity to transport pentoses was affected
by the growth substrate and cells grown on either
cellobiose or sucrose had relatively little pentose
transport activity (Table 1). However, maltosegrown cells did have appreciable levels of xylose
uptake. Optimal transport was between pH 6.5
and 7.0 with progressively less uptake observed as
pH was reduced to 5.0, at which no uptake was
detected (data not shown).
When cells were treated with iodoacetate, arsenate, or fluoride, intracellular ATP concentrations fell by more than 7(1% within 10 min but
these chemicals had little effect on the protonmo-
219
10
Table 1
a
Effect of growth substrate on xylose and arabinose transport
activity by B. fibrisolvens
Growth substrate "
Xylose
Arabinose
Glucose
Maltose
Cellobiose
Sucrose
Pentose transport activity b
(nmol min - 1 (rag protein)- l )
6
Xylose
Arabinose
4
63.4
23.7
27.4
18.4
3.6
(l.6
5.1
7.1
1.9
ND c
ND c
ND c
2
•
I
'
I
"
1
10
Cultures were provided with 11 mM monosaccharides or 5
mM disaccharides and harvested during exponential growth.
b Transport assays contained 6.9 ~M xylose or 0.17 /xM
arabinose. Values represent averages of at least duplicate
determination.
ND, not detected,
b
6
"~
4
2"
,
tive force ( T a b l e 2). A c o m b i n a t i o n of nigericin
a n d valinomycin, which results in free p e r m e a t i o n
of protons, caused a large decrease in A T P a n d
the p r o t o n m o t i v e force was nearly abolished. All
t r e a t m e n t s caused a large decline in the u p t a k e
of xylose. Similar results were n o t e d for a r a b i n o s e
transport.
•
0
i
2
.
.
4
.
6
.
i
8
,
10 12 14
Time (h)
Fig. 1. Effect of a pulse addition of glucose ( • ) or xylose (o)
to cultures growing on (a) xylose or (b) glucose, respectively.
The dotted lines represent utilization by control cultures
which did not receive sugar pulses.
Pentose utilization
Table 2
Effect of metabolic inhibitors on cellular ATP concentration,
protonmotive force, and xylose uptake in B. fibrisolvens
Inhibitor ~
ATP
(nmoI
(rag prorein)- 1)
Proton motive
force
(mV)
Xylose
uptake
(%) b
Control
400 p.M iodoacetate
10 mM Na-fluoride
10 mM Na-arsenate
10/zM nigericin plus
10 ~M valinomycin
1"t.8
3.9
2.3
2.9
1.5
110
91
98
100
3
100
20
15
26
18
a lnhibitors were added 10 rain prior to measurements. Cells
treated with Na-fluoride or Na-arsenate were washed and
incubated in 50 mM sodium piperazine-N,N'-bis(2-ethanesulfonic acid) containing 10 mM MgC12 (pH 7.0). Values
represent averages of at least duplicate determination.
b Percent uptake with 100% representing 60 nmol xylose
rain -I (rag protein) ~. Assays contained 6.9/xM xylose.
Previous results i n d i c a t e d that B. fibrisolvens
co-utilized a c o m b i n a t i o n of glucose a n d x~lose
w h e n both sugars were p r e s e n t at the b e g i n n i n g
of the i n c u b a t i o n [4]. W h e n glucose was a d d e d to
a c u l t u r e already growing o n xylose, t h e r e was an
a b r u p t decrease in the rate of p e n t o s e utilization
(Fig. la). In the converse e x p e r i m e n t , a pulse of
xylose h a d a similar effect o n the rate of glucose
d i s a p p e a r a n c e (Fig. lb), In each case, utilization
of the sugar a d d i t i o n b e g a n nearly i m m e d i a t e l y
( < 10 rain) after the pulse a n d the original substrate c o n t i n u e d to be m e t a b o l i z e d , albeit at a
r e d u c e d rate. In contrast to these results, arabinose utilization nearly ceased for 2 h after a pulse
dose of glucose a n d rapid a r a b i n o s e d i s a p p e a r ance did not r e s u m e until glucose was d e p l e t e d
(Fig. 2a). A similar, a l t h o u g h less p r o n o u n c e d ,
i n h i b i t i o n was seen w h e n xylose was a d d e d to a
220
culture growing on arabinose (Fig. 2b). Additions
of arabinose to cultures growing on glucose or
xylose had no effect on the rate of sugar utilization and little arabinose was used before depletion of the original carbohydrate (data not shown).
0.6
~.~
Inhibition of transport
Since the shifts in substrate utilization seen
after carbohydrate pulses were very rapid, it was
possible that an effect was exerted at the level of
substrate transport. Increasing concentrations of
unlabelled glucose inhibited the uptake of radiolabelled xylose in a non-competitive fashion (K i
= 2.2 /xM; Fig. 3a). Similarly, xylose non-competitively inhibited (K i = 2.5 ~ M ) glucose transport
by the organism (Fig. 3b). Since xylose and glucose displayed mutual inhibition of transport, the
specificity of the xylose transport system was investigated by adding a 100-fold excess of 22 pentoses, hexoses, and alcohol sugars to transport
assays. However, none of compounds (including
0.0
0.4
b
E
"a
0.2
©
0.0
2
i
0
•
i
1
•
1
2
•
i
3
.
i
4
.
i
5
,
1
6
•
7
a
1/S (1/gM)
Fig. 3. (a) Lineweaver-Burk(double reciprocal) plot for xylose
uptake in the presence of no glucose (-), 2.5/xM (×), 5/xM
(•), and 10 /~M glucose (+). (b) Plot for glucose uptake in
the presence of no xylose (o), 2.5/zM ( × ), and 5.0/xM ( • ).
g
t~
q~
•
-1
10
•
6"
4-
o
2-
D- and L-isomers) inhibited xylose transport by
more than 15% (data not shown).
Arabinose uptake exhibited saturation kinetics
and the affinity for the pentose was very high
( K m = 0.2 /zM; data not shown). When a 30-fold
excess of unlabelled glucose or xylose was added
simultaneously to transport assays with radiolabelied arabinose, uptake of the labelled pentose
was not affected•
0
10
b
6
•
i
0
2
4
1
6
•
, r - .
8
Discussion
10
Time (h)
Fig. 2. Effect of a pulse addition of (a) glucose ( • )
or (b)
xylose (o) to cultures growing on arabinose (•). The dotted
lines represent arabinose utilization by control cultures which
did not receive sugar pulses.
Although pentose sugars are probably an important energy source for many ruminal bacteria,
relatively little study had focused on pentose
transport and utilization. However, recent work
221
demonstrated that ruminal organisms possess a
variety of xylose and arabinose transport systems
which are regulated by different mechanisms
[5,12]. Unlike the ion-driven pentose uptake systems found in Preuotella ruminicola (sodium-dependent) and Selenomonas ruminantium (protondependent), the present results suggest that xylose and arabinose transport in B. fibrisolvens
was driven by high-affinity, A T P - d e p e n d e n t
mechanisms. This conclusion was supported by
the observations that (i) transport did not require
sodium; (ii) elimination of the protonmotive force
did not completely abolish uptake; and (iii) pentose accumulation was related to intracellular
ATP concentrations. Since iodoacetate can affect
sulfhydryl groups, it is possible that there was a
direct inhibition of the transport protein(s). However, the effects of arsenate and fluoride on ATP
and protonmotive force corroborated the hypothesis of ATP-dependent mechanisms. Recent studies have shown that another fibrolytic organism,
Ruminococcus albus, also possesses ATP-driven
pentose transport systems [13].
Previous work with B. fibrisoluens indicated
that glucose and xylose were co-utilized but utilization rates of each sugar in dual substrate
incubations were slower than rates in cultures
provided with only a single carbohydrate [4]; this
result suggested that the presence of glucose influenced xylose utilization. An interaction between xylose and glucose utilization was also evidenced by the fact that pulses of each sugar
significantly decreased the utilization of the other
sugar; a mutual non-competitive inhibition of
transport was apparently responsible for these
observations. Although similar utilization patterns were noted in R. albus, in this organism
glucose competitively inhibited xylose uptake and
it appeared that a common permease was used
for the transport of these structurally similar carbohydrates [13]. The non-competitive nature of
the inhibition in B. fibrisoluens was not unprecedented; glucose was found to inhibit xylose transport non-competitively in Candida shehatae [14].
Although an intracellular interaction might explain the results with the ruminal organism, the
rapid nature of the transport assays would largely
preclude such an effect. Further work is needed
to elucidate the molecular events causing this
inhibition.
In contrast to the interactions between xylose
and glucose utilization, arabinose use was affected more strongly by glucose. The severe and
immediate decrease in arabinose utilization when
glucose was added to the culture was reminiscent
of phosphotransferase system (PTS)-mediated
catabolite inhibition of non-preferred substrate
transport [15]. Recent studies have suggested that
there was a small amount of PEP-dependent
phosphorylation of glucose by strain CE51 [16].
However, previous work has established that several other B. fibrisolvens strains did not possess
PTS [17], and glucose-PTS was not detected in
strain D1 (data not shown). Thus, it is unlikely
that such a mechanism was responsible for the
decrease in arabinose utilization. In addition, unlike the rapid mutual inhibitions of glucose and
xylose uptake, arabinose transport was not affected by either sugar. It is possible that a subsequent intracellular process caused the inhibition,
but since pentose metabolism by B. fibrisolvens
has not been well delineated the nature of this
phenomenon remains unclear.
Transport activity of cells grown on different
substrates suggested that xylose uptake was inducible, but the exact regulatory mechanisms
controlling the expression of pentose permeases
requires further study. Even though sucrose and
cellobiose cultures had very low transport activity,
cultures provided with either disaccharide and
xylose showed at least some co-utilization of both
sugars [4]; this result further implied that inductive control regulated xylose permease expression. In contrast, the strong preference for glucose over arabinose noted in the present as well
as previous work [4] suggests that, in addition to
induction, a level of repressive control exists in
the case of arabinose transport. The overall preference for hexose sugars and xylose may be related to the observation that growth on this particular pentose is associated with a much higher
maintenance energy requirement [4].
Ruminal bacteria are presented with combinations of substrates throughout the feeding cycle
and, depending on the organism and circumstance, exhibit substrate preferences. It is evident
222
from the present studies that regulation of pent o s e t r a n s p o r t i n f l u e n c e s p r e f e r e n c e p a t t e r n s in
B. fibrisolvens. S u c h r e g u l a t o r y m e c h a n i s m s m a y
b e v e r y i m p o r t a n t in d e f i n i n g b a c t e r i a l g r o w t h
c h a r a c t e r i s t i c s a n d m e t a b o l i c a c t i v i t i e s in t h e r u men.
Acknowledgements
This work was supported by the Cooperative
State Research Service, US Department of Agriculture, under agreement No. 91-37206-6717.
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