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MICROBIOLOGY
LEITERS
FEMS Microbiology
Letters 136 (1996) 123- 127
The regulation of thiomethylgalactoside transport in
Clostridium acetobutylicum P262 by inducer exclusion and
inducer expulsion mechanisms ’
Francisco Diez-Gonzalez a, James B. Russell b,c3
*
a Department of Food Science, Cornell l/nice&y, Ithaca, NY 14853, USA
b Section of Microbiology, Wing Hall, Cornell Unicersity, Ithaca, NY 14853, USA
’ Agricultural Research Service, USDA, Ithaca, NY 14853, USA
Received 20 September
1995; revised 8 November
1995; accepted
10 November
1995
Abstract
Clostridium acetobutylicum
P262 had phosphotransferase systems for glucose and lactose, and the lactose system was
inducible. When C. acetobutylicum P262 was provided with glucose and lactose, the cultures grew in a diauxic fashion, and
glucose was used preferentially. Cells grown on lactose took up thiomethylgalactoside, and retained this non-metabolizable
lactose analog for long periods of time. Because glucose inhibited thiomethylgalactoside uptake and caused the efflux of
thiomethylgalactoside that had already been taken up, it appeared that C. acetobutylicum P262 had inducer exclusion and
inducer expulsion mechanisms similar to those found in lactic acid bacteria.
Keywords: Clostridium acetoburylicum; Inducer expulsion;
Catabolite
1. Introduction
It has long been recognized that bacteria can
utilize certain energy sources preferentially and grow
diauxically [l]. Sequential patterns of substrate utilization minimize the synthesis of unneeded protein
and in many cases enhance the growth rate. In
enteric bacteria, substrate preferences are mediated
* Corresponding
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(607) 255 3904.
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Microbiological
regulation
by two different types of regulation. CAMP-dependent regulation of transcription prevents protein synthesis [2], and phosphotransferase system @‘IS)mediated inducer exclusion inhibits the uptake of
non-preferred substrates [3].
Reizer and Panos [4] reported that Streptococcus
pyogenes was able to regulate lactose catabolism via
a different mechanism that they called ‘inducer expulsion’. Inducer expulsion is an energy-dependent
process that is mediated by an increase in fructose
1,6-phosphate @BP) and a cascade of phosphorylated proteins [5]. When glucose is added to the
culture, PBP accumulates, and an ATP-dependent
protein kinase is activated. The protein kinase phosphorylates the histidine-containing protein, HPr, a
non-sugar specific phospho-carrier, at serine 46. HPr
Societies, All rights reserved
C. acetohu~vlicwn strain P262 was provided by
Prof. D.R. Woods. University of Cape Town, South
Africa. Spores of this organism were maintained at
4°C in anaerobic 20 mM phosphate buffer and were
used to inoculate a basal medium [9] supplemented
with glucose and lactose (up to 10 mM). The cultures were incubated at 37°C.
formation was monitored spectrophotometrically
at
340 nm. Assays were performed in 300-p.1 cuvettes
at 38°C. The assay mixture contained per ml: 50
prnol Tris. HCI (pH 7.5). 10 prnol
phosphoenolpyruvate (PEP), 5 prnol MgCl,. 5 kmol dithiothreitol (DTT). 2 pmol NADP, 20 prnol glucose. 5
units glucose-6-phosphate
dehydrogenase
and I-3
mg cell protein. /?-Galactosidase
was measured by
assaying
the release of o-nitrophenol
from onitrophenyl-P-D-galactoside
(ONPG) in 0.5 ml reaction mixture containing (per ml): 50 prnol Tris HCI
(pH 7.5), 5 pmol MgCl?. 5 prnol DTT, 5 pmol
ONPG and l-2 mg cell protein. At measured times
the reaction was stopped with 250 ~1 1 M Na,CO,
and absorbance was measured at 420 nm. One unit
of absorbance was equivalent to 0.52 pmol of onitrophenol [ 1I]. Lactose PTS activity was estimated
by measuring n-nitrophenol
formation in the presence of PEP after subtracting the P-galactosidase
activity. The reaction mixture was the same as for
,&galactosidase
but 10 mM PEP were included.
Phospho-P-galactosidase
activity was determined as
described for @-galactosidase except that 5 pmol
ONPG-6-P was substituted for ONPG.
2.2. Chemical umz1y.w.~
2.4. Transport of TMG
(ser-P) then activates a sugar-phosphate phosphatase.
and the dephosphorylated
sugar exits the cell.
Inducer expulsion has been demonstrated
in a
variety of lactic acid bacteria [6], but to our knowledge it has never been demonstrated in other groups
of bacteria. Preliminary experiments indicated that
Clostridium ~~crtohu~licwn P262. a strain selected
for its ability to utilize lactose [7,8], had a lactose-PTS
and was able to expel the non-metabolizable
lactose
analog,
methyl-P-D-thiogalactopyranoside
(TMG),
when glucose was added.
2. Materials and methods
Fluid samples were centrifuged (13000 >( s. 2
min, 22°C) and sugars in the supematant were determined by HPLC (BioRad HPX-87H column, 0.17 N
H,SO,, 0.5 ml mm ‘. 50°C; refractive index detector). Protein was determined by the method of Lowry
[IO]. Cell concentration
was estimated by optical
density at 600 nm.
Cultures were harvested by centrifugation ( 10 000
5°C 10 min) and washed twice in 50 mM
Tris HCI buffer (5 mM MgCl?, 5 mM DTT, pH
7.5). Cells were disrupted by passage through a
French press (room temperature,
1000 psi). Cell
debris was eliminated by centrifugation (IO 000 X g,
5°C. 10 min) and cell-free extracts were stored at
4°C before assays.
PEP-dependent
phosphorylation
of glucose of
cell-free extracts was determined by a coupled assay
with glucose-6-phosphate
dehydrogenase.
NADPH
x g,
Exponentially
growing
cells were harvested
anaerobically
by centrifugation
(3500 X g, IO min,
4°C) and resuspended in anaerobic 50 mM Tris HCI
buffer (pH 7.0) containing 5 mM MgCl?. Aliquots of
200 ~1 of cell suspension (approx. 200 mg protein
I ‘) were incubated
under N, tlushing
with
[I-( C]TMG (8 PM: 50 mCi mmol- ’ ). For expulsion
experiments. 5 mM glucose or 2-deoxy-glucose were
added. Transport was stopped by the addition of 2 ml
ice-cold 0. I M LiCl to the reaction mixture and cells
were collected by filtration through 0.45 ,um pore
size cellulose nitrate membrane filters. Filters were
washed with 2 ml of 0.1 M LiCl. dried for 15 min at
12O”C, and radioactivity was determined by liquid
scintillation counting. The transport had first-order
kinetics (protein versus activity was linear).
3. Results
When C. acetobu~licum
P262 was provided with
glucose and lactose. the cultures grew diauxically
F. Die:-Gonzalez, J.B. Russell/ FEMS Microbiology Letters 136 (1996) 123-127
125
Table 2
P-Galactosidase
and phospho-fi-galactosidase
activities of cell-free
extracts of Clostridium acetobutylicum P262 grown on glucose
and lactose
Growth
substrate
P-Galactosidase
activity (nmol (mg
protein)-’
min- ’ )
Phospho-/3-galactosidase
activity fnmol fmg
protein)- ’ mitt- ‘)
Glucose
Lactose
< 0.5
30.0
600.0
100.0
All values are the means of duplicate
measurements.
C. acetobutylicum P262 cells that had been grown
on lactose transported the non-metabolizable
lactose
analog, TMG, at a rapid rate, but no TMG uptake
0
1
2
3
4
5
Time (h)
Fig. I. Growth of CIosfridiunt trcetobufy[icum P262 on mixed
glucose and lactose. (A) Diauxic curve. (B) Utilization of substrates.
(Fig. IA) and glucose was utilized preferentially to
lactose (Fig. 1B). Toluene-treated
cells had virtually
no detectable PTS activity (less than 0.5 nmol (mg
protein)- I min- ’ 1, but PEP-dependent
glucose and
lactose phosphorylations
could be measured with
cell-free extracts (Table I>. The lactose PTS activity
was inducible. Cells grown on lactose were able to
hydrolyze the chromogenic
lactose analog, ONPG,
but much higher activity was observed when ONPG
6-phosphate was provided (Table 2).
-10
ii
Table 1
PEP-dependent sugar phosporylation activities of cell-free extracts
of CIostridium acefobutylicum P262 grown on glucose and lactose
Growth
substrate
Glucose-PTS activity
(nmol (mg protein)min-’ )
Glucose
Lactose
23.5
28.3
’
All values are the means of duplicate
Lactose-PTS activity
(nmol (mg protein)min-‘)
< 0.5
88.0
measurements
’
1
I
0-j
7.5
10
12.5
15
17.5
20
Time(min)
Fig. 2. Accumulation of thiomethylgalactoside
(TMG) by cells of
Clostridium acetobutylicum P262 grown on lactose and glucose.
(A) TMG uptake in the presence of glucose and 2-deoxyglucose
(2.DG). Glucose or 2-DG (5 n&l) were added before TMG
addition. (B) Retention of TMG after addition of glucose or 2-DG
by cells grown on lactose and previously incubated with TMG (10
min). Addition of sugar is indicated by the arrow. Values are the
means of at least two separate experiments.
was observed when the glucose was added to the
transport assay (Fig. 2A). Cells retained TMG for at
least 10 min, but rapid TMG expulsion was observed
when glucose was added (Fig. 2B). The nonmetabolizable
glucose analog, 2-deoxyglucose,
did
not cause TMG expulsion.
4. Discussion
PTS activity has traditionally been measured in
toluene-treated
cells [ 121. Booth and Morris [I 31
demonstrated
PTS in toluene-treated
C. pasteuriunum cells, but Mitchell et al. [14] only detected
PTS activity with cell extracts of C. crcetobu~licum
NCIMB 8052. Cell-free
extracts of C. untobutylicum
P262 had PTS activity, but no activity was
detected
in toluene-treated
cells. Because
the
toluene-treated cells had butyryl-CoA dehydrogenase
and NAD-independent
lactic acid dehydrogenase activities (data not shown), the lack of PEP-dependent
glucose phosphorylation
could not be explained by
permeabilization
per se. Because even the cell extracts had relatively low PTS activity, it appeared
that the PTS of P262 was sensitive to standard
methods of cell disruption.
Many species of clostridia use lactose [ 151, but to
our knowledge a PTS for lactose had never been
reported in clostridia. C. ucetobutylicum
P262 had
more PTS activity for lactose than glucose, and this
activity was induced by lactose. The idea that strain
P262 was using a lactose-PTS to take up lactose was
supported by the observation that the cells had high
phospho-P-galactosidase
activity and little P-galactosidase activity. When lactose is transported by the
PTS, lactose is converted to lactose 6-phosphate, a
phosphorylated
derivative
that is hydrolyzed
by
phospho-/3-galactosidase
but not P-galactosidase
[161.
C. acetobutylicum
P262
accumulated
the nonmetabolizable lactose analog, TMG, and was able to
retain it for long periods of time. Based on the
observation that cells provided with glucose could no
longer transport TMG and expelled TMG that had
already accumulated, it appeared that the cells had
mechanisms of inducer exclusion and expulsion. The
independence of these mechanisms was supported by
the observation that 2-deoxyglucose,
a non-metabo-
lizable glucose analog, could promote inducer exclusion but not inducer expulsion.
Clostridia were traditionally classified as ‘sporeforming fermentative rods’ [ 151, but recent work has
indicated that spores are not a definitive taxonomic
trait for clostridia [17]. Further work is needed to
define the phylogeny of the clostridia more precisely,
but it should be noted that the clostridia and lactic
acid bacteria share many common characteristics: (1)
cell wall structure; (2) low mol.% G + C DNA; (3)
PTS systems; (4) FBP-activated lactic acid dehydrogenases [9,18]; and (5), as shown here, glucose-dependent mechanisms of inducer expulsion.
Acknowledgements
This research was supported by the US Dairy
Forage Research Center, Madison, WI. F.D.-G. is
grateful to Consejo National de Ciencia y Tecnologia. Mexico, for their support.
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