TrmB, a Sugar-specific Transcriptional Regulator of the
Trehalose/Maltose ABC Transporter from the Hyperthermophilic
Archaeon Thermococcus litoralis*
Sung-Jae
Lee
,
Afra
Engelmann§,
Reinhold
Horlacher
,
Qiuhao
Qu
,
Gudrun
Vierke§,
Carina
Hebbeln§,
Michael
Thomm§, and
Winfried
Boos
¶
From the
Department of Biology, University of
Konstanz, 78457 Konstanz, Germany, and § Institut
für Allgemeine Mikrobiologie, Universität Kiel, 24118 Kiel,
Germany
Received for publication, October 7, 2002, and in revised form, November 6, 2002
 |
ABSTRACT |
We report the characterization of TrmB, a protein
of 38,800 apparent molecular weight, that is involved in the
maltose-specific regulation of a gene cluster in Thermococcus
litoralis, malE malF malG orf trmB malK, encoding a
binding protein-dependent ABC transporter for trehalose and
maltose. TrmB binds maltose and trehalose half-maximally at 20 µM and 0.5 mM sugar concentration,
respectively. Binding of maltose but not of trehalose showed
indications of sigmoidality and quenched the intrinsic tryptophan
fluorescence by 15%, indicating a conformational change on maltose
binding. TrmB causes a shift in electrophoretic mobility of DNA
fragments harboring the promoter and upstream regulatory motif
identified by footprinting. Band shifting by TrmB can be prevented by
maltose. In vitro transcription assays with purified
components from Pyrococcus furiosus have been established
to show pmalE promoter-dependent transcription at 80 °C. TrmB specifically inhibits transcription, and this
inhibition is counteracted by maltose and trehalose. These data
characterize TrmB as a maltose-specific repressor for the
trehalose/maltose transport operon of Thermococcus
litoralis.
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INTRODUCTION |
The hyperthermophilic Archaeon Thermococcus litoralis
belongs to the order Thermococcales of euryarchaeota. It
grows optimally at 85 °C under anaerobic conditions and was
originally isolated from deep submarine hydrothermal vents (1). This
organism can use starch, maltose, cellobiose, or sucrose as carbon
sources (2). The enzymology of maltose utilization is known (3). Trehalose and maltose are transported by a high affinity and binding protein-dependent ABC transporter with a
Km of ~20 nM (4, 5). As a result of
lateral gene transfer, the same system with nearly identical sequence
is present in Pyrococcus furiosus (6). The latter organism
has a second, very similar maltodextrin-specific transporter that is
absent in T. litoralis. This ABC transporter does not
recognize trehalose (6-9). The genes encoding the trehalose/maltose transporter in T. litoralis are clustered, most likely
forming an operon, and are positioned divergently to another gene that shows homology to genes encoding sugar kinases (Fig. 1). The
trehalose/maltose transport genes are induced by the presence of
maltose or trehalose in the growth medium (4, 5), as are the identical
genes in P. furiosus (6). The components of the transporter
have been purified (5, 10, 11), and the cognate binding protein as well
as the ABC subunit have been crystallized and their structures solved
(7, 12). Thus far, nothing was known about the mechanism of induction.
In contrast, the regulation of the homologous system in
Escherichia coli, one of the most thoroughly studied binding protein-dependent ABC transporters, is well understood
(13). The central feature is the function of MalT, a transcriptional activator that needs the cAMP/CAP complex, the inducer maltotriose and
ATP for the specific activation of
70-programmed RNA
polymerase (14-16). In addition, MalK, the ATP-hydrolyzing subunit of
the ABC transporter, is closely connected to the control of MalT
activity (17, 18).
The basic transcriptional machinery in Archaea appears to be more
similar to eukaryotes than to prokaryotes (19, 20). Thus, transcription
initiation requires three promoter elements, the TATA box centered at
26/
27 from the start point of transcription, a transcription factor
B recognition element (BRE)1
comprising two adenines upstream of the TATA box, and an initiation element around the transcription initiation site.
Correspondingly, the minimal factors needed to facilitate binding of
the eukaryotic polymerase II-like archaeal RNA polymerase to the
promoter are TATA box-binding protein (TBP) and transcription factor
binding protein (21-23). One would therefore expect
that regulation of transcription in Archaea would follow eukaryotic
rather than prokaryotic schemes (24). Yet, the few examples of Archaeal
transcriptional regulation point to a bacterial-like regulation. MDR1
(metal-dependent repressor) was found to inhibit RNA
polymerase function in Archaeoglobus fulgidus (25), and
Lrs14 regulates its own expression by preventing the binding of TBP at
the corresponding BRE-TATA box in Sulfolobus sp. (26).
Homologues to the bacterial Lrp/AsnC family of transcriptional regulators were found in Methanococcus jannaschii and
P. furiosus (27-30). In contrast to the growing
understanding of the basic machinery of transcription initiation, the
understanding of the specific regulation of this process, especially
for genes involved in sugar uptake and metabolism, is not well
understood (31).
Here we report the characterization of TrmB (transcriptional
regulator of mal operon) as a
maltose/trehalose-specific gene regulator (repressor) for a cluster of
genes encoding a high affinity ABC transporter as well as TrmB itself.
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EXPERIMENTAL PROCEDURES |
Purification of the Transcriptional Regulator
TrmB--
Chromosomal DNA from T. litoralis was prepared as
previously described (35). PCR was performed using chromosomal DNA as template and primers encompassing trmB. 5' ends carried
NcoI and BamHI restriction sites for convenient
cloning. After digestion with the corresponding restriction enzyme, the
fragment was ligated into plasmid pCS19 (36) containing a C-terminal
His6 tag. The resulting plasmid was named pSL152. It is
ampicillin-resistant, contained the lacIq gene
and expressed trmB under
isopropyl-
-D-thiogalactoside-inducible promoter control.
E. coli strain SF120 (37) was transformed with pSL152
selecting for ampicillin resistance. For TrmB expression, cells were
grown in NZA medium [10 g NZ-amine A (Sheffield Product Inc), 5 g
yeast extract, 7.5 g NaCl per liter] at 30 °C. 0.2 mM isopropyl-
-D-thiogalactoside was added at
OD 0.5-0.7, and cells were grown for 5 more h at 37 °C and
harvested by centrifugation. The pellet was resuspended in 20 ml Ni-NTA
affinity solution (buffer I; 50 mM sodium phosphate, 300 mM NaCl, pH 7.5), ruptured in a French pressure cell at
16,000 p.s.i., and centrifuged for 30 min at 17,300 × g. The
supernatant was heated to 80 °C for 10 min. After centrifugation of
the precipitated proteins, the supernatant was loaded onto a Ni-NTA
superflow column from Qiagen equilibrated with buffer I. The column was
washed with 50 mM imidazole and eluted with 250 mM imidazole, both in the same buffer. TrmB-containing fractions were pooled and dialyzed against 50 mM Tris, 200 mM sodium chloride, pH 7.5, and subsequently against 200 mM sodium phosphate buffer, pH 7.5. The final concentration
of purified TrmB was about 1.0 mg/ml. It was kept frozen at
70 °C.
EMSA (Electrophoretic Mobility-shift Assay)--
Labeled DNA was
obtained from appropriate PCR products and end-labeled with T4
polynucleotide kinase (MBI Fermentas) according to the instructions of
the manufacturer. The labeled DNA was purified using mini Quick Spin
Columns (Roche). The binding buffer was 25 mM HEPES, 150 mM potassium glutamate, 10% glycerin, 1 mM
dithiothreitol, pH 7.5. All samples contained about 40 nCi
labeled DNA (0.5-1 µM) and 1 µg of
poly(dI·dC)·poly(dI·dC) competitor DNA (Roche, Germany) per 10 µl. TrmB and sugar were added in the concentrations indicated in the
legends to the figures. The reaction samples were incubated at 70 °C
for 10 min, mixed with 3 µl of loading buffer (25 mM
HEPES, 150 mM potassium-glutamate, pH 7.5 containing 50%
glycerol and 0.05% bromphenol blue), loaded directly onto 8% native
PAGE gels, and run at room temperature under a constant voltage of 200 V.
Footprint and Primer Extension Assays--
For footprinting, the
DNA fragment containing the target promoter region was produced using
two primers in the PCR. The first, starting in the putative sugar
kinase, is 5'-CCCAAGCCTTCTCAGACCAACTACA-3' (coding strand); the second,
starting in malE, is 5'-ATGTCTGTTGGCCACCAATG-3' (noncoding
strand). The primers were 5'-labeled with [
-32P]dATP
and polynucleotide kinase. The labeled PCR product (~20,000 cpm) was
incubated with TrmB at 70 °C for 10 min in binding buffer and
digested with 0.005 units of DNaseI for 5 min at 37 °C. The reactions were stopped with Stop buffer containing 20 mM
EDTA, 0.6 M sodium acetate, pH 5.2, and put on liquid
nitrogen immediately. The reaction samples were extracted with
phenol/chloroform and chloroform/isoamylalcohol, precipitated with 0.1 volume of 3 M sodium actetate and 2.5 volume 96% ethanol,
and centrifuged. The pellet was washed once with 70% ethanol. It was
dried and resuspended in loading solution (0.3% bromphenol blue and
xylene cyanol FF, 10 mM EDTA, pH 7.5, 97.5%
deionized formamide) and applied directly onto a 6% polyacrylamide
sequencing gel containing 7 M urea.
For primer-extension analysis, total RNA was isolated (38)
from T. litoralis cells at different growth phases (6 and 12 h), with and without maltose induction (0.2%). Growth was
in modified marine culture medium (6). The start point for the 5'-end
labeled primer was 90 nucleotides downstream of the malE
translation start site. The sequence ladder was prepared from
sequencing reactions with the same labeled primer. 50 µg of RNA
together with the labeled primer (150,000 cpm) was precipitated with
0.1 volume of 3 M sodium acetate and 3 volumes of 96%
ethanol. The precipitate was centrifuged at 14,000 × g for 30 min
in an Eppendorf centrifuge and washed with 70% ethanol. After
resuspension in 10 µl of annealing buffer (50 mM Tris
HCl, pH 7.5, 200 mM KCl, 2.5 mM EDTA) the
sample was denatured at 85 °C for 3 min and hybridized for 10 min at
55 °C. The primer-extension reaction was started by adding prewarmed avian myeloblastosis virus reverse transcriptase mixture
(Promega; 1 unit). After 30 min at 37 °C, the sample was
precipitated with sodium acetate and ethanol as before and resuspended
in 10 µl of loading solution containing formamide.
Sugar-binding Assay--
100 µl of 50 mM potassium
phosphate buffer, pH 7.5, containing 2.5 µM or 5 µM TrmB were incubated with 0.1 µCi of
[14C]maltose (0.65 µM) or
[14C]trehalose (1.1 µM). Prior to the
addition, the sugars had been mixed with unlabeled maltose or trehalose
to reach a final chemical concentration between 2 and 100 µM for maltose and 2 µM and 10 mM for trehalose. Incubation was for 5 min at 80 °C. The
assay was stopped with 2 ml of ice-cold saturated ammonium sulfate in 50 mM potassium phosphate buffer, pH 7.5, and kept on ice
for 10 min. The suspension was then filtered through cellulose nitrate membrane filters (Schleicher und Schüll; pore size 0.45 µm) and washed with 2 ml of 95% saturated ammonium sulfate in 50 mM potassium phosphate buffer, pH 7.5. Bound radioactivity
was determined in a scintillation counter.
In Vitro Transcription Assay--
DNA was obtained
from pMLP. This plasmid is derived from pUC19 and carries an
EcoRI-BamHI fragment starting in the putative sugar kinase gene and ending in malE (see
Fig.1A). The experimental conditions for the in
vitro transcription assay (39) are as follows. 300 ng of
linearized pMLP DNA harboring the intergenic region (digested with
EcoRI) in 40 mM HEPES buffer, pH 7.3, 250 mM KCl, 2.5 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 0.44 mM of each ATP, GTP, CTP, 0.002 mM UTP, and 2 µCi [
-32P]UTP were mixed with transcriptional
components from P. furiosus, i.e. 25 ng of
transcription factor binding protein (recombinant), 200 ng of TBP,
(recombinant) and 110 ng of RNA polymerase. Maltose and trehalose were
added to a final concentration of 25 µM to 100 mM, each. TrmB was added to final concentrations as
indicated in the legend to Fig. 8.
Fluorescence-based Analysis of Conformational Change--
We
used 25 mM Hepes, 300 mM potassium glutamate,
pH 8.0, as buffer and 20 µg of TrmB in a 1-ml sample at 50 °C.
Measurements were done with a PerkinElmer Model 650-40 instrument at
284 nm excitation and 336 nm emission (40). Sugars were added in
10-µl portions to a final concentration of 0.2 mM maltose
and 2 mM trehalose, respectively.
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RESULTS |
Purification of the Transcriptional Regulator
TrmB--
The search for the specific gene regulator of the
trehalose/maltose transport gene cluster was initiated by the
observation that a lateral gene transfer between T. litoralis and P. furiosus had taken place: a 16-kb DNA
fragment harboring this gene cluster had been transferred, probably in
recent times (6). Because the trehalose-specific induction of the
transporter had been maintained after the transfer, it seemed
likely that the elements of induction were also present on the 16-kb
DNA fragment. Therefore, we cloned all of the genes contained on it as
His-tag variants, which, as judged from their sequence, might be
candidates to encode transcriptional regulators. We expressed these
genes in E. coli, purified the corresponding proteins, and
tested them in a gel-shift assay using a DNA fragment spanning the
putative regulatory elements of the operon (see below). We obtained a
positive signal with the protein encoded by the gene directly proximal
to malK located within the cluster of transport genes (Fig.
1). We named the gene trmB
(transcriptional regulator of the
mal operon).

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Fig. 1.
Genetic scheme of the trehalose/maltose ABC
transport operon of T. litoralis. A,
gene cluster encoding the binding protein-dependent ABC
transporter for trehalose/maltose and the divergent promoter region
between malE and the putative sugar kinase. B,
sequence of the intergenic region. Bold letters
indicate binding sites for the transcriptional regulator TrmB as seen
by footprinting (see Fig. 4); BRE, transcription factor B recognition
element; RBS, ribosome-binding site; +1, transcription start site of
malE; `cat' and `atg' indicate the
methionine start codon for the putative sugar kinase and
maltose-binding protein, MalE, respectively.
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Sequence homology searches revealed that TrmB (338 amino acids and
38,838 calculated molecular weight) belongs to the DUF118 gene family
of Pfam (Sanger Institute, UK). Weak homology (30% identity over 159 consecutive amino acids) could be recognized with CymJ of
Klebsiella oxytoca, YrhO of Bacillus subtilis
(26% identity over 165 consecutive amino acids), and AF1232 from
Archaeoglobus fulgidus (27% identity over 194 consecutive
amino acids). High homology was recognized with PH1034 of
Pyrococcus horikoshii (71% identity over the full size
protein) as shown in Fig. 2. These proteins exhibit sequence similarity mostly at the N terminus and
contain a distinct helix-turn-helix motif. CymJ of K. oxytoca is also positioned within an operon encoding a binding
protein-dependent ABC transporter specific for
cyclodextrins (32). The function of CymJ has not been elucidated except
that it is dispensable for transport.

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Fig. 2.
Multiple protein sequence alignment of
T. litoralis TrmB. T. litoralis
TrmB is shown together with its related homologs: P. horikoshii PH1034, Archaeoglobus fulgidus AF1232,
K. oxytoca CymJ, and Bacillus subtilis YrhO. An
asterisk indicates amino acid identity; colon,
high conservation; dots, low conservation. The putative
helix-turn-helix motif was predicted by the PHD program.
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TrmB, as a C-terminal His-tag version, was overexpressed (from plasmid
pSL152) in E. coli SF120 that is characterized by the lack
of several proteases. Growth was in rich medium (NZA) at 30 °C and
using 0.2 mM iso-propyl-
-D-thio-galactoside
as inducer. Prior to Ni-NTA affinity chromatography, the cell-free
extract was heated to 80 °C for 10 min to remove most of the
E. coli proteins. After Ni-NTA chromatography, the protein
exhibited one major band in SDS-PAGE with an apparent molecular weight
of 38,800. The protein easily precipitated after dialysis overnight in
Tris buffer, pH 7.6 containing 200 mM NaCl. The precipitate
could be solubilized in 200 mM sodium phosphate, pH 7.5, even without the addition of NaCl, and revealed a pure protein
preparation. From 1 liter of culture, 4 mg of pure protein was
routinely obtained.
EMSA of TrmB--
The intergenic region, upstream of the
trehalose/maltose transport operon, contained an inverted repeat
(TTTACTTTAXGXA), BRE (transcription factor B
recognition element), a potential TATA box, and a putative RBS
(Fig. 1B). To determine the DNA elements that can be
recognized by TrmB, we used EMSA. Fig.
3B shows EMSA with different
concentrations of TrmB and DNA encompassing the entire intergenic
region (region 1). 1.5 µM TrmB was sufficient to completely shift the DNA. We noticed that after binding TrmB, a
minor portion of the bound DNA never entered the gel, possibly indicating the formation of higher oligomeric forms of TrmB. We prepared three different size fragments of the promoter region. The
region 1 fragment contained the entire intergenic region, region 2 the
BRE-TATA box but not the inverted repeat sequence, and region 3 only
the inverted repeat sequence as well as the proximal region leading
into the gene encoding the putative sugar kinase (Fig. 3A).
When these three different labeled DNAs were used as EMSA probes, all
were retarded by TrmB binding, the least effective one being region 3 (Fig. 3C). Thus, the intergenic region must contain at least
two binding sites for TrmB.

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Fig. 3.
EMSA with TrmB. A, DNA fragments
used in this study. The open box indicates the
inverted repeat sequence, the filled box the BRE-TATA box of
malE. B, EMSA with labeled DNA consisting of the
entire intergenic region (Reg1); the final concentrations of
TrmB were 0, 0.5, 1.25, 1.5, 2.0, and 2.5 µM.
C, comparison of EMSA with fragments of different sizes of
the intergenic region (Reg1, Reg2,
Reg3). Purified TrmB was added to each fragment at 0, 1.25, and 2.5 µM final concentration, respectively. 1 µg
poly(dI·dC)·poly(dI·dC) was present in these reactions as
competitor DNA.
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Detection of TrmB-binding Sites by Footprinting--
To define the
correct binding sites for TrmB, we used protection from DNaseI
digestion in the presence of TrmB (Fig.
4). We used two samples labeled at one
end: the coding and noncoding sequence (left and
right in Fig. 4, respectively). We could observe two binding
sites for TrmB. One is the promoter-proximal part of the inverted
repeat sequence, 5'-CTTTAGGGATGTTTGTACTTAAAGTAAATAAATA-3', and
the other is the promoter itself, the BRE-TATA box,
5'-CAAAATATATATACTTTTAGTATATACA-3' (Fig. 1B).
Therefore, TrmB binding to the promoter region will inhibit the binding
of transcriptional components TBP and transcription factor binding
protein and will thus prevent recruitment of RNA polymerase,
essentially by repressing the initiation of transcription of the
malE operon. When 100 µM maltose was added,
TrmB no longer protected the DNA from DNaseI digestion (Fig. 4). The
addition of 2.5 mM trehalose gave the same effect (data not
shown). Thus, TrmB binding to the promoter elements is prevented by
these sugars acting as inducers.

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Fig. 4.
Footprint analysis of the intergenic promoter
region (encompassing Reg1). The DNaseI protection assay was done
on the coding strand (left) and noncoding strand
(right). TrmB protected the BRE-TATA box (a and
d) and part of the inverted repeat sequence (b
and c) (compare with Fig. 1B). Final
concentrations of added TrmB were 0, 1.25, 2.5, and 5 µM.
C, G, and A indicate the sequence
ladder. M, 100 µM maltose was added to the
assay containing 5 µM TrmB.
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TrmB Binds Maltose and Trehalose with Different
Affinities--
Sugar-binding assays were done by ammonium sulfate
precipitation in the presence of 14C-labeled sugar (33).
Maltose-binding was linearly dependent on TrmB concentration (Fig.
5A), and TrmB was
half-maximally saturated at 20 µM maltose (Fig.
5B). The binding of maltose showed indications of sigmoidal
behavior at two concentrations of TrmB (2.5 and 5 µM;
only the data for 5 µM are shown in Fig. 5, B
and C). This can hardly be recognized in the
Michaelis-Menten plot shown in Fig. 5B. However, it can
easily be seen when only the amount of TrmB-bound radioactivity (but
not the chemical amount of bound maltose) is plotted against the total
maltose concentration (labeled plus unlabeled) as seen in Fig.
5C. Because the amount of radioactive maltose in all assays
is the same (61,000 cpm) the expected behavior for a noncooperative
(Michaelis-Menten type) maltose-binding would be such that the number
of TrmB-bound radioactivity would only decline when increasing amounts
of unlabeled maltose are present in the assay. However, as seen in Fig.
5C, the amount of labeled maltose bound to TrmB increased
with increasing total maltose concentration before declining,
suggesting positive cooperative binding. To better visualize this
cooperativity in the calculated binding data of Fig. 5B and
to show the difference to a purely noncooperative, Michaelis-Menten
type binding behavior, we added a theroretical Michaelis-Menten type
binding curve using the concentration of half-maximal binding (20 µM) as the "Kd" and 1.8 µM as maximally TrmB-bound maltose. Now the difference of
maltose binding to TrmB and a purely Michaelis-Menten type binding is obvious.

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Fig. 5.
Sugar binding by TrmB. A, 1.6 µM labeled sugars (maltose and trehalose) were incubated
at 80 °C with increasing amounts of TrmB as indicated. The protein
was precipitated with ammonium sulfate in the cold, filtered, and
counted. B, maltose-binding. 20 µg of purified TrmB (5 µM) was incubated at 80 °C with 0.65 µM
radioactive maltose and increasing amounts of unlabeled maltose. The
concentration of TrmB-bound maltose was calculated and is shown as
filled circles. Maximally, 1.8 µM maltose was
bound to TrmB. Half-maximal binding occurs at 20 µM
maltose. For comparison, a theoretical Michaelis-Menten type binding
curve with 20 µM as Kd and 1.8 µM maximally bound TrmB is added.
C, the cpm's of maltose bound to TrmB derived from the
experiment shown in Fig. 5B are plotted against the total
maltose concentration (filled squares). In
contrast to Michaelis-Menten type binding behavior, the bound cpm's
increase with unlabeled maltose concentration. The same experiment with
0.65 µM radioactive maltose was done with increasing
concentrations of unlabeled trehalose (open
squares).
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When radioactive trehalose was used in the binding assay, half-maximal
binding was only observed at concentrations higher than 0.5 mM. But in contrast to maltose as substrate, the TrmB-bound radioactivity only declined with the addition of increasing amounts of
unlabeled trehalose giving no indication of cooperative binding for
trehalose (data not shown). Binding of labeled trehalose was abolished
in the presence of unlabeled maltose (data not shown). Also, binding of
maltose (at 0.65 µM concentration) could be chased by
increasing amounts of trehalose (Fig. 5C), showing initially (at lower trehalose concentration) the same phenomenon of cooperative binding of maltose with increasing amounts of unlabeled trehalose. Using the intrinsic tryptophan fluorescence of TrmB (284 nm excitation and 336 nm emission), we observed 15% reduction in fluorescence at
80 °C upon addition of 200 µM maltose but no effect
with trehalose (data not shown).
Maltose and Trehalose Affect TrmB-mediated EMSA
Differently--
When maltose was included in the TrmB-mediated EMSA,
it prevented the retardation of labeled region 1 DNA. The concentration of maltose that elicited the half-maximal effect was comparable with
that which is needed to half-maximally saturate TrmB in the in
vitro binding assay (20-50 µM). Surprisingly,
trehalose showed the opposite effect. At concentrations comparable with
half-maximal saturation of binding to TrmB (0.5 mM), there
was no discernible effect in the EMSA. However, at 5-10 mM
trehalose, the stability of the TrmB-DNA complex was clearly increased
and EMSA of region 1 became more effective (Fig.
6).

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Fig. 6.
Trehalose and maltose affect TrmB-mediated
EMSA with labeled region 1. 1.25 µM purified TrmB
was used in this experiment. Trehalose and maltose were added at
concentrations of 1, 2.5, 5, 10 mM, and 5, 10, 20, 50 µM, respectively. 0, no sugar added.
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Transcription Start Site by Primer Extension--
To determine the
transcriptional start point in the direction of malE, we
prepared total RNA of cells harvested at early (6 h) and mid-log (12 h)
phase grown in peptone (uninduced conditions) and peptone plus maltose
(induced conditions). As primer we used DNA starting 90 base pairs
downstream of the translational start site of malE. Also, we
used a primer starting within gdh (encoding glutamate
dehydrogenase) as control for a constitutively expressed gene. Fig.
7 shows the two transcription start sites
in the malE direction. The major start site is 26 nucleotides downstream of the BRE-TATA box (AAATATA). The transcript is
stronger when RNA was isolated from early log cultures than from
mid-log cultures (Fig. 7). The transcript is not detectable when cells
were grown under noninducing conditions (data not shown).

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Fig. 7.
Determination of the transcription start site
of malE. Primer-extension analysis was done with
50 µg T. litoralis total RNA. Lanes 1 and
2 show the minor (a) and major (b)
transcription start site of malE; lanes 3-6 show
the transcription start site of the gene for glutamate dehydrogenase as
positive control (c). Total RNAs were prepared from
different carbon sources, i.e. peptone (lanes 3 and 4) and peptone plus maltose (lanes 1,
2, 5, and 6), and growth phases,
i.e. early log phase (lanes 1, 3, and
5) and mid-log phase (lanes 2, 4, and
6). G, A, T, and C contain dideoxy sequencing reactions
primed with labeled malE primer. The sequences of the two
transcriptional start sites, a and b, are
indicated.
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In Vitro Transcription Assay of malE--
To determine the
function of TrmB in the transcription of the malE promoter,
we used the established in vitro transcription assay with
purified components (RNA polymerase, TBP, and transcription factor
binding protein) from P. furiosus. As a control, we also used a constitutively transcribed gene encoding glutamate dehydrogenase of P. furiosus. The templates used produced a transcript of
110 bases for the malE operon and of 89 bases for the gene
encoding glutamate dehydrogenase (Fig.
8). When TrmB was present in increasing concentrations, the transcript of malE was reduced
concomitantly. The presence of maltose prevented this inhibition at a
concentration of 100 µM (Fig. 8A).
Surprisingly, trehalose had the same effect as maltose albeit at
25-fold higher concentration (above 2.5 mM; Fig.
8B). At this and even higher concentrations, trehalose had not prevented the retardation of region 1 DNA during EMSA.

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Fig. 8.
In vitro transcription assay of
malE. malE transcription was performed
at 80 °C using basic transcriptional components of P. furiosus. As template, linearized pMLP DNA containing the T. litoralis malE promoter was used. Lane 1, control assay
with plasmid containing the gene for glutamate dehydrogenase
(gdh) of P. furiosus; lanes 2 and 3,
malE transcription in the presence of maltose (A)
or trehalose (B) at 5 mM concentration each, but
in the absence of TrmB; lanes 4-6, malE
transcription in the presence of 0.8-3.2 µg TrmB; lanes
7-14, malE transcription in the presence of 3.2 µg
TrmB and 25, 100, 250 µM, 1, 2.5, 10, 25, and 100 mM maltose (A) or trehalose (B).
Inhibition of transcription by TrmB is overcome with 100 µM maltose (A, lane 8) and 2.5 mM trehalose (B, lane 11),
respectively. The malE (110 bases) and gdh (89 bases) transcripts are indicated by arrows.
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DISCUSSION |
In this publication we report the characterization of TrmB, a
novel Archaeal substrate-specific transcriptional regulator controlling
the maltose/trehalose-specific induction of the binding protein-dependent ABC transporter in T. litoralis. TrmB binds maltose and trehalose with 20 µM and >500 µM half-saturating
concentration, respectively and is able to interfere with in
vitro transcription of the malE promoter. In line with
the binding properties of TrmB, maltose (100 µM) and
trehalose (2.5 mM) were shown to prevent TrmB-mediated
transcription inhibition. Thus, TrmB could be characterized as a
transcriptional repressor. Like other Archaeal repressors, TrmB
protected the TATA box as well as the adjacent BRE site in DNaseI
protection assays. Thus, substrate-specific gene regulation appears to
be based on the prevention of TBP binding and subsequent polymerase
recruitment. The role of the inducer is to prevent TrmB from binding to
the TATA box. In this function, maltose is much more effective (50-100
µM) than trehalose (2.5 mM). This appears
physiologically reasonable because maltose is metabolized rather
efficiently by T. litoralis (3) and will never reach significant concentrations in vivo, whereas trehalose is
accumulated, preferentially at high osmolarity, to high internal
concentrations (34).
In E. coli, MalT, the central gene activator of the maltose
system, interacts with the C-terminal extension of MalK, the
ATP-hydrolyzing subunit of the ABC transporter, whose expression is
controlled by MalT (17, 18). T. litoralis MalK exhibits the
same C-terminal regulatory extension as E. coli MalK.
However, our attempts to demonstrate an interaction between isolated
MalK of T. litoralis and TrmB were unsuccessful. Possibly,
an interaction between these two proteins, if it occurs at all, is
restricted to MalK when complexed with the membrane components of the
transporter and occurs only in the process of actively transporting substrate.
There is something curious about the effect of trehalose on TrmB and
its interaction with the intergenic DNA region. Aside from the
observation that binding of TrmB to trehalose is much weaker than to
maltose, trehalose binding, in contrast to maltose binding, does not
show signs of cooperativity and does not induce a conformational change
that is detectable by fluorescence measurements. TrmB-mediated EMSA of
the entire intergenic region (region 1 in Fig. 1) is
prevented by maltose at concentrations that reflect its binding
characteristics to TrmB but not by trehalose, even at concentrations
exceeding the saturation of trehalose binding by TrmB; it even makes
TrmB/DNA complex formation seemingly stronger even though trehalose
binding to TrmB prevents footprinting and interferes with TrmB-mediated
malE transcription. Presently, we have no sensible
explanation for this curious effect of trehalose binding.
TrmB elicits incomplete band shift of the BRE-TATA box alone. The same
is seen with the inverted repeat motif. Thus, binding of TrmB to region
1 containing both the BRE-TATA box as well as the inverted repeat must
be enhanced by cooperativity between the two sites.
TrmB, when complexed with maltose, cannot recognize a potential binding
site in the entire fragment of region 1. Because DNaseI protection
assays with TrmB showed not only protection of the TATA box but also of
the upstream inverted repeat, we conclude that binding of TrmB to the
inverted repeat and BRE-TATA box is affected by maltose and trehalose.
It remains to be seen whether binding of TrmB to both elements is
involved in the regulation of malE transcription.
Alternatively, TrmB binding to the inverted repeat could be involved in
the transcriptional regulation of the divergently oriented gene
encoding the putative sugar kinase.
 |
ACKNOWLEDGEMENT |
We thank Eva-Maria Ladenburger for her advice
in the footprint technique.
 |
FOOTNOTES |
*
This work was supported by the Deutsche
Forschungsgemeinschaft (Schwerpunktprogramm "Genomfunktion und
Genregulation in Archaea," SPP 1112) and the Fonds der Chemischen
Industrie.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Biology, University of Konstanz, 78457 Konstanz, Germany.
Tel.: 49-7531- 882658; Fax: 49-7531-883356; E-mail:
Winfried.Boos@uni-konstanz.de.
Published, JBC Papers in Press, November 7, 2002, DOI 10.1074/jbc.M210236200
 |
ABBREVIATIONS |
The abbreviations used are:
BRE, transcription
factor B recognition element;
TBP, TATA box-binding protein;
TrmB, transcriptional regulator of mal operon;
Ni-NTA, nickel-nitrilotriacetic acid;
EMSA, electrophoretic mobility-shift
assay.
 |
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