TrmB, a Sugar-specific Transcriptional Regulator of the Trehalose/Maltose ABC Transporter from the Hyperthermophilic Archaeon Thermococcus litoralis*

Sung-Jae LeeDagger , Afra Engelmann§, Reinhold HorlacherDagger , Qiuhao QuDagger , Gudrun Vierke§, Carina Hebbeln§, Michael Thomm§, and Winfried BoosDagger

From the Dagger  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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 sigma 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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-beta -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-beta -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 [gamma -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 [alpha -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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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.

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-beta -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.

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.

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).

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.

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.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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