- Research article
- Open Access
Transcriptional control by two leucine-responsive regulatory proteins in Halobacterium salinarum R1
© Schwaiger et al; licensee BioMed Central Ltd. 2010
- Received: 21 January 2010
- Accepted: 28 May 2010
- Published: 28 May 2010
Archaea combine bacterial-as well as eukaryotic-like features to regulate cellular processes. Halobacterium salinarum R1 encodes eight leucine-responsive regulatory protein (Lrp)-homologues. The function of two of them, Irp (OE3923F) and lrpA1 (OE2621R), were analyzed by gene deletion and overexpression, including genome scale impacts using microarrays.
It was shown that Lrp affects the transcription of multiple target genes, including those encoding enzymes involved in amino acid synthesis, central metabolism, transport processes and other regulators of transcription. In contrast, LrpA1 regulates transcription in a more specific manner. The aspB3 gene, coding for an aspartate transaminase, was repressed by LrpA1 in the presence of L-aspartate. Analytical DNA-affinity chromatography was adapted to high salt, and demonstrated binding of LrpA1 to its own promoter, as well as L-aspartate dependent binding to the aspB3 promoter.
The gene expression profiles of two archaeal Lrp-homologues report in detail their role in H. salinarum R1. LrpA1 and Lrp show similar functions to those already described in bacteria, but in addition they play a key role in regulatory networks, such as controlling the transcription of other regulators. In a more detailed analysis ligand dependent binding of LrpA1 was demonstrated to its target gene aspB3.
- Amino Acid Metabolism
- Deletion Strain
- Aspartate Transaminase
- Overexpressing Strain
- Inverted Repeat Sequence
The basal transcription apparatus in Archaea shows similarity to the eukaryotic RNA polymerase (RNAP) II system [1–4]. Archaeal promoter sequences and the core proteins RNA polymerase (RNAP), TATA-binding protein (TBP), and the transcription factor IIB homologue (TFB) are structurally and functionally related to their eukaryotic counterparts [2, 5]. Although the basal transcriptional complex is composed of eukaryotic-like components, archaeal regulatory proteins are often homologous to bacterial regulators . One group of bacterial regulators which have been found in all archaeal genomes belongs to the Lrp/AsnC family (leucine-responsive regulatory protein (Lrp), asparagine synthase C (AsnC)). Escherichia coli Lrp is the most extensively studied member in bacteria [7, 8] and controls the expression of up to 75 target genes. As a global regulator of transcription, Lrp is believed to coordinate cellular metabolism in response to nutritional and environmental alterations . Most of these genes are involved in amino acid metabolism. Lrp can bind to DNA in its homodimeric form and either represses or activates transcription, modulated by the effector molecule L-leucine. Negative autoregulation of Lrp, however, occurs in a leucine independent way .
Genes encoding putative Lrp/AsnC-homologues have been studied in several archaea , including Methanocaldococcus jannaschii[12–14], Sulfolobus species [15–17] and Pyrococcus species [18, 19]. As demonstrated for the Sulfolobus solfataricus Ss-Lrp and Lrs14, those Lrp/AsnC-homologues were shown to bind to their own promoter regions, thereby repressing transcription [20–22]. Besides controlling its own gene expression, Sulfolobus solfataricus Ss-LrpB positively regulates the pyruvate ferredoxin oxidoreductase (POR) encoding operon and two permease genes [23, 24]. Another Lrp-like protein, LysM from S. solfataricus, regulates the expression of the lysWXJK operon encoding lysine biosynthetic enzymes. In fact, in vitro binding of LysM to the lysW promoter takes place only if lysine is absent . Footprint analysis of the Sa-Lrp gene from Sulfolobus acidocaldarius revealed multiple binding sites in the promoter region , a pattern that had been described earlier for bacterial Lrp proteins . Leucine has been suggested as a possible cofactor for Sa-Lrp under certain physiological conditions .
In M. jannaschii, not only do the Lrp-like proteins Ptrl and Ptr2 regulate their own transcription, but Ptr2 can activate transcription of the ferredoxin (fdx) and the rubredoxin (rb2) genes by facilitating recruitment of TBP to their promoters . In a Pyrococcus furiosus cell-free transcription system, LrpA exerts negative autoregulation of its own transcription  by interfering with the recruitment of RNA polymerase . Crystal structures determined for several bacterial and archaeal Lrp-like proteins (for an overview, see  show that they contain a N-terminal helix-turn-helix DNA-binding domain (HTH). A flexible hinge connects this domain with the C-terminal oligomerization and effector binding domain [30–32]. The latter forms a so-called RAM-domain (regulation of amino acid metabolism-domain) , designed to bind an effector molecule in the interface between the two dimers. A structure alignment of archaeal and bacterial Lrp-homologues is shown in additional file 1.
In halophilic archaea relatively little is known about Lrp-like-regulators. The current study focuses on the Lrp-like-regulators, LrpA1 and Lrp in H. salinarum R1. To identify Lrp-targets, deletion mutants (Δlrp, ΔlrpA1) as well as strains upregulated in these genes (↑lrp, ↑lrpA1) were compared pairwise against the parental strain R1 by DNA-microarrays. These results demonstrated that Lrp exerts a global transcriptional control in this organism. On the other hand, LrpA1 was shown to possess a specific regulatory function targeting the aspartate transaminase gene (aspB3). We demonstrated effector molecule dependent binding of LrpA1 to the aspB3 promoter by DNA-affinity chromatography, as well as effector molecule dependent gene expression of aspB3 by northern analysis.
Specific transcriptional control by LrpAl in H. salinarum R1
Differentially expressed genes in ΔlrpA1 and ↑lrpA1
Transcriptional regulators (REG)
transcription regulator LrpA1
Amino acid metabolism (AA)
transcription initiation factor TFB
conserved hypothetical protein
As expected, transcription of aspB3, the gene adjacent to lrpA1, was affected by the absence or overexpression of lrpA1. In the ↑lrpA1 background (Table 1), aspB3 showed slight repression, while deletion of lrpA1 led to strong induction. Another LrpA1 target gene identified by the microarray analysis was tfbB, the basal transcriptional regulator gene. This gene was repressed by LrpA1-overexpression. Additionally, strong repression was found for OE6130F, the gene encoding a hypothetical protein of unknown function. OE6130F is located on the plasmid PHS2. Its adjacent genes are OE6128R encoding a conserved hypothetical protein and OE6133R encoding a transposase.
In the ΔlrpA1 mutant, induction of aspB3 and OE6130F was confirmed by RT-qPCR, which showed reductions in transcript levels of 19- and 28-fold, respectively (additional file 5)
Organisation of lrpA1 and aspB3 operons of H. salinarum Rl
In contrast to lrpA1, the aspB3 transcript has a 5'UTR leader sequence of 31 bp, without a Shine-Dalgarno sequence upstream of the AUG start codon (Fig. 2). Inspection upstream of the aspB3 transcription start site revealed, that the aspB3 promoter does not contain a consensus TATA-box at the expected position (-24 to -27). The 3'UTR of aspB3 included a 127 bp terminal sequence that is complementary to the 3'end of lrpA1. No characteristic termination signal was detected in this region (Fig. 2).
LrpAl binds to the aspB3 and to its own promoter
Relative binding efficiencies of LrpA1 to sepharose-bound DNA fragments
846 ± 280
201 ± 33
313 ± 70
aspB3-Pincl (+ 5 mM asp)
1404 ± 228
473 ± 220
control-DNA (+ 5 mM asp) (C)
92 ± 33
aspB3-Pincl (+5 mM arg)
175 ± 94
52 ± 22
control-DNA (+ 5 mM arg) (C)
135 ± 69
LrpA1 regulates transcription of aspB3 in an aspartate dependent manner
H. salinarum possesses three different aspartate transaminases, AspB1, AspB2 and AspB3 (see sequence comparison in additional file 7). All belong to subgroup Ib of the aspartate transaminases , and share 35, 37 and 32% sequence identity, respectively, with the Thermus thermophilus enzyme . Aspartate transaminases catalyze the reversible conversion of aspartate and oxoglutarate to oxaloacetate and glutamate.
In synthetic medium, H. salinarum showed similar growth behaviour whether or not aspartate was present (Fig. 6D). These results show that aspB3 transcription is repressed in the presence of aspartate. If there is no aspartate in the synthetic medium (Fig. 6B-3,4), or it has been metabolized in the early stationary phase, repression is released, and synthesis of aspartate from glutamate ensues.
At cell densities of ≥ 109 cells/ml (OD600 ≥ 1.0) H. salinarum R1 has been reported to rapidly metabolize aspartate . Previously reported growth studies of H. salinarum R1 have shown that when media have both aspartate and glutamate present, the former amino acid is metabolized rapidly while the levels of the latter remains constant . To test the regulatory effect of LrpA1 on the aspB3 gene transcription, mRNA levels of aspB3 were analyzed in ΔlrpA1 cells grown with or without aspartate or glutamate (Fig. 6C). We observed an increased and constitutive transcription, independent of the added amino acids. This demonstrates unambiguously the involvement of LrpA1 in the regulation of the aspB3 gene expression.
Multiple transcriptional control by Lrp in H. salinarum
Differentially expressed genes in Δlrp and ↑lrp
transcription initiation factor TFB
Transport processes (TP)
ABC-phosphate transporter permease
ABC-phosphate transporter ATP binding protein
ABC-peptide transport ATP binding protein
ABC-peptide transport ATP binding protein
ABC-peptide transport permease
ABC-peptide transport permease
Amino acid metabolism (AA)
Central intermediary processes (CIM)
oxoglutarate ferredoxin oxidoreductase β-subunit
oxoglutarate ferredoxin oxidoreductase α-subunit
Signal transduction (SIG)
transducer protein Car
Lrp-homologues have been described for several bacterial and archaeal organisms, but not yet for halophilic archaea. Here we investigated the function of a halophilic Lrp-homologue, LrpA1. Additional file 1 shows an alignment of LrpA1 with other archaeal and bacterial Lrp-homologues. High sequence similarity was observed with LrpA (PF1601) from P. furiosus. The low sequence identity in the ligand binding pocket, named RAM-domain (additional file 1; β3β4) between LrpA1 and Lrp suggests different regulatory mechanisms (additional file 1). The H. salinarum LrpA1 binding pocket belongs to a subgroup of the Lrp-like proteins, which includes some that might be effector-independent  and some for which the effector regulation is unknown (additional file 1). The Lrp ligand binding site shows high amino acid conservation with S. solfataricus LysM which probably binds lysine  (additional file 1). We therefore expect the H. salinarum Lrp to be a ligand dependent regulator, which is a subject of future investigation.
In contrast to LrpA1, another Lrp-homologue named Lrp affects the transcription of genes encoding proteins involved not only in amino acid but also in central metabolism. For many organisms, Lrp acts as both an activator and a repressor of transcription. Like E. coli Lrp, the Lrp of H. salinarum R1 affects the regulation of amino acid metabolism and genes encoding peptide transporter dpp (Fig. 7; Table 3). Lrp binding sites in H. salinarum NRC-1, a strain that shows a 99.9% sequence identity to H. salinarum R1 , have been previously reported . In NRC-1 Lrp-homologues are designated as Trh and nine of them are annotated in the genome of H. salinarum NRC-1. H. salinarum R1 LrpA1 (OE2621R) and Lrp (OE3923F) are 100% identical with the H. salinarum NRC-1 Trh7 and Trh4, respectively, the latter one was previously analyzed . Comparison of our data with the published NRC-1 data revealed that, out of all the affected genes, only three were showing the same trends in both: the glutamine synthetase gene, glnA, which is located adjacent to lrp; the glycerol dehydrogenase gene, gldA1 and the transducer gene, car (Table 3). GldA1 was repressed by Lrp in H. salinarum R1. The metabolism of glycerol is complex. It can either be converted to dihydroxyacetone (DHA) by glycerol dehydrogenase GldA1 , or phosphorylated by glycerol kinase to glycerol-3-phosphate. The latter can be fed into glycolysis as dihydroxyacetone phosphate (DHAP), or is converted to glycerol-1-phosphate which is used as a substrate for the production of archaeal phospholipids. However, the fate of DHA remains unclear because the corresponding kinase for the subsequent conversion of DHA to DHAP is not yet known . The repression of gldA1 might favour glycerol phosphorylation by reducing the flow of glycerol to dihydroxyacetone (DHA). Besides the three affected genes that were common between NRC-1 and R1, there were distinct targets of Lrp in strain R1. For example, activation of korAB, encoding the oxoglutarate oxidoreductase complex, a TCA cycle enzyme (Fig. 7; Table 3). KorAB belongs to the family of two oxoacid:ferredoxin oxidoreductases (OR) and catalyzes the oxidative decarboxylation of oxoglutarate and is part of the following conversion together with CoA to succinyl-CoA. For S. solfataricus Ss-LrpB, activation of the pyruvate ferredoxin oxidoreductases por-operon has been reported by Peeters; 2009 . The OR-enzymes act on various substrates that play key roles in amino acid metabolism .
In H. salinarum R1, korAB induction by Lrp suggests that KorAB catalyzes the rate-determining step of the TCA-cycle. This might influence the oxoglutarate/glutamate balance and shift carbon flow towards glutamate synthesis or degradation. In the Lrp-overexpression strain, a slight induction was observed for the glutamate dehydrogenase gene, gdhA2. Glutamate is incorporated into the TCA-cycle by GdhA2 and metabolized by KorAB to generate further metabolites or provide reducing equivalents. As already mentioned, Lrp regulates the synthesis of glutamine from glutamate by induction of glnA. In H. salinarum, glutamate is accumulated as a carbon storage compound and as a compatible osmolyte, and reaches concentrations of 50-100 mM [45, 46]. If needed, glutamate can be converted into other metabolites, e.g. amino acids.
Both regulators, LrpA1 and Lrp, influence the expression of tfb's. It has been proposed earlier that different combinations of TFBs and TBPs may act in an analogous way to bacterial sigma factors in order to control global gene expression in H. salinarum NRC-1 [46–49]. Lrp activates tfbF, whereas LrpA1 represses tfbB. In strain NRC-1, TfbF is thought to control either directly or indirectly the transcription of target genes .
The transcriptional regulator sirR, a homologue of the staphylococcal iron regulator repressor, was found to be induced by Lrp (Fig. 7; Table 3) . SirR is described as a repressor of a putative Mn-dependent ABC-transporter in H. salinarum NRC-1 . In R1, induction of the putative Mn-dependent ABC-transport operon (OE5144R, OE5146R, OE5147R) in a ΔsirR deletion strain was shown (Schwaiger, unpublished data). In the current study slight repression of the ABC-transporter gene, OE5147R was detected in the Lrp-overexpression strain, where sirR is induced. This is consistent with SirR acting as a repressor of the ABC-transport operon. The data also showed induction of pstC2 and phnC, which belong to phosphate and phosphonate transport operons (Fig. 7; Table 3). In NRC-1, SirR is thought to take part in the regulation of phosphate transport processes . Lrp might then indirectly influence phosphate metabolism by controlling sirR expression.
In summary, these studies on Lrp-like homologues in the halophilic branch of archaea have clearly demonstrated that they share a similar general function to their homologues in bacteria, i.e. they are transcriptional regulators that may have narrow or global regulatory actions. Lrp activates the gene expression of the glutamine synthetase gene glnA, influences peptide- and phosphate transport, as well as the central intermediary metabolism, and activates the expression of the transcriptional regulator sirR. By the control of sirR gene expression through Lrp correlation between amino acid metabolism and metal dependent processes could be demonstrated. In contrast to Lrp, LrpA1 regulates gene expression of fewer genes, amongst them the aspartate transaminase gene aspB3. LrpA1 was shown to bind to the lrpA1 promoter region, as well as an aspartate dependent binding to the aspB3 promoter region. To gain more insights into the LrpA1 and L-aspartate dependent aspB3 gene expression, northern blot analysis were performed, that showed an induction of the aspB3 transcription in the absence of L- aspartate. This occurs either in a medium lacking aspartate or after aspartate is metabolized in the stationary phase. At the same time, an induction of the lrpA1 gene expression was observed. This can be illustrated in a model that postulates a reciprocal regulation of the lrpA1 and aspB3 gene expression. Much remains to be understood, but the current work provides a solid foundation for further investigations of the haloarchaeal Lrp protein family and their regulatory networks.
Strains and growth conditions
H. salinarum R1 (DSM 671) and the deletion strains (Δlrp, ΔlrpA1) were grown in either complex or synthetic medium, as described previously [51, 52]. The E. coli strains DH5α and BL21(DE3), used for cloning and protein expression, were grown in Luria-Bertani (LB) medium, supplemented with antibiotics when necessary .
Construction of deletion and overexpression mutants in H. salinarum
The construction of lrpA1 and lrp deletion mutants was performed according to . Briefly, oligonucleotides were used to amplify the adjacent region downstream and upstream of the gene of interest (additional file 10). The obtained PCR-products were digested with PstI, fused by ligation, reamplified and cloned into pMKK100  using BamHI and XbaI restriction sites. The lrp↑ and lrpA1↑ strains were constructed by insertion of pKF203 and pKF204 plasmids into the lrp and lrpA1 region of H. salinarum, respectively. The plasmids were constructed as described in additional file 11. Deletion plasmids and overexpression plasmids were introduced into H. salinarum by the PEG-mediated method according to . Deletion mutants were generated by a two-step procedure of selecting separate single cross over events using red-blue screening as described by . The correct genotype was verified by PCR and Southern blot hybridization (additional file 3). The presence of the overexpression plasmid in each transformant was determined by PCR followed by sequencing of the amplified fragments.
Isolation of total RNA
H. salinarum cells were harvested at an OD600 of 0.2-0.8 by centrifugation for 5 min at 12000 g (4°C). The pellet was resuspended in peqGold RNAPure extraction solution (Peqlab
Biotechnology, Erlangen) and total RNA was extracted following the manufacturer's instructions. Finally, the RNA was dissolved in DEPC (diethylpyrocarbonate)-H2O and stored at -80°C until further use. After incubation with DNase (Promega-Kit RQ1) a DNA-free RNA sample was obtained. To confirm the absence of remaining DNA in the DNase digested RNA samples, a PCR-reaction was performed using HotStarTaq (Qiagen, Hilden) and selected gene specific oligonucleotide primers (see additional file 10 probes for southern blotting primers lrp). Only RNA, which did not yield any product after amplification (40 cycles) was used in subsequent RT-PCR's. RNA integrity was proven by using the 2100 Bioanalyzer (Agilent Technologies, Waldbronn) or alternatively with denaturating 1% TBE-agarose-gels containing 20 mM guanidinium thiocyanate.
Wild type R1 and the deletion strains (Δlrp, ΔlrpA1) utilized for the microarray approach were grown in complex medium. Total RNA (5 μg), isolated from cells having an OD600 of 0.4 (4 × 108 cells/ml), was reverse transcribed into Cy3/Cy5-labeled cDNA using CyScribe First-Strand cDNA Synthesis Kit with enclosed random nonamer primers and Cy3-/Cy5-dUTP (both Amersham Biosciences, Freiburg). Labelled cDNA was hybridized to in-house fabricated whole genome DNA-microarrays  at 64°C overnight. To determine the fluorescence ratios the slides were scanned (GenePix 4000 B, Axon Instruments) and the data were extracted using the GenePix Pro 6 software. After background substraction, pin-wise normalization and data evaluation by a Student's T-test, those transcripts displaying a p-value equal or lower than 5.10-5 and a ratio of +/- 1.3 were selected as significantly regulated. A detailed description of the microarray design, experimental procedure and data-evaluation is described in . We considered ratios with a p-value equal or lower than 5 × 10-5 as significant. This reflects a stringent interpretation of data as a two times less stringent p-value results in 4.8% false positives . The data obtained from the microarray experiment were deposited at http://www.ebi.ac.uk/miamexpress under the accession number (E-MEXP-1447).
Reverse transcription-quantitative PCR and RACE
5 μg DNA-free total RNA was reverse transcribed using 0.5 μg random hexamer primers (Promega, Mannheim) and Superscript III reverse transcriptase (Invitrogen, Karlsruhe) according to the manufacturer's instructions. 1 μ1 of the cDNA reaction mixture was quantified by using the SYBR Green PCR Master Mix Kit (Applied Biosystems, Darmstadt) in a GeneAmp 5700 Sequence Detection System (Applied Biosystems, Darmstadt) in a final reaction volume of 25 μl. The primer pairs (additional file 10) for amplification were designed with Primer Express 2.0 (Applied Biosystems, Darmstadt) and were added to a final concentration of 0.2 μM. The data were analyzed via the 2ΔΔCt-method using the mean-Ct-value of 3 replicate reactions per primer pair. The constitutively expressed gene OE4759F, encoding a S-layer glycoprotein, was chosen as internal standard. RACE (rapid amplification of cDNA ends) was essentially performed by an RNA circularization mediated method according to  to determine the 5'ends and the 3'-ends of transcripts.
Northern blot hybridizations
15 μg total RNA was separated by electrophoresis on 1% TBE-agarose-gel, containing 20 mM guanidinium thiocyanate. Gels were then equilibrated in alkaline buffer  and transferred onto Hybond N+ membrane (GE-Healthcare, München), by vacuum blotting for 3 hours. For generation of DIG-dUTP-labelled RNA-probes a PCR amplified DNA-fragment (additional file 10), including the T7-promoter sequence was generated in order to function as a template for in vitro transcription with T7-RNA-polymerase (DIG RNA labelling kit (SP6/T7), Roche Applied Science, Mannheim). Furthermore, hybridization and chemiluminiscent detection were carried out using the "DIG Wash and Block Buffer Set" (Roche Applied Science, Mannheim) according to "DIG system user's guide for filter hybridization" (Boehringer, Mannheim). All oligonucleotides used for generating the probes are mentioned in additional file 10.
Expression and purification of LrpA1
LrpA1 was amplified by PCR using oligonucleotides (additional file 10) including the sequence for the restriction sites of NdeI and XhoI. After digestion with these enzymes the PCR fragment was cloned into the vector pET26b (Novagen, Darmstadt). The obtained LrpA1-expression vector was transformed into E. coli BL21(DE3). The expressed polypeptide contained a C-terminal His6-tag. A single colony was picked in order to inoculate 30 ml Luria-Bertani (LB) medium supplemented with 50 μg/ml kanamycin. The culture was grown overnight in a rotary shaker at 37°C. On the next day the culture was used to inoculate 1 liter of the identical medium also supplemented with 50 μg/ml kanamycin. Protein expression was induced with 0.6 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at an OD600 of 0.8. The cells were harvested after 3 hours of growth by centrifugation for 10 min at 5000 g (4°C). The pellet was resuspended in 30 ml buffer A (8 M urea; 100 mM NH2PO4; 10 mM Tris-HCl, pH 8.0) with 10 mM imidazole and disrupted by sonification (3 × 15 sec; 50% duty cycle; Branson Sonifier). The lysate was centrifuged for 80 min at 50000 g. Subsequently, 1.5 ml of Ni-NTA fast flow matrix (Qiagen, Hilden) was added to the supernatant and was incubated on a rotary wheel for 2 h (4°C). Afterwards the Ni-NTA matrix was packed on a column, washed three times with 7.5 ml of buffer A plus 20 mM imidazole and eluted in three times 1.5 ml fractions with buffer A plus 150 mM imidazole. To restore the native conditions the purified LrpA1-His6 was dialyzed against cell-free extract (CFE) buffer (3 M KCl; 1 M NaCl; 10 mM HEPES, pH 7.1) over night at room temperature. To prove successful refolding we performed CD spectroscopy (method see below). For the determination of multimerisation, we performed size exclusion chromatography by using a 3.2/3 Superdex 200 column (GE-Healthcare, München) on a SMART chromatography device (GE-Healthcare, München) (flow-rate 50 μl per min CFE buffer). Presence of protein was detected at 280 nm.
Circular dichroism spectroscopy
To determine the secondary structures of LrpA1 after renaturation (see Expression and purification of LrpA1 (Methods)) we used circular dichroism in the far-UV (190-250 nm). CD-Spectra were monitored using a JASCO-J-810 spectrometer. After renaturation against the high salt CFE buffer (3 M KCl; 1 M NaCl; 5 mM MgCl2; 10 mM HEPES, pH 7.1) LrpA1 had a final protein concentration of 1.2 mg/ml and was measured in 0.01 mm quartz cuvettes (Helma). All measurements were performed in CFE buffer at 21°C. The spectra were calculated from the average of 12 scans and repeated in two independent measurements, followed by the subtraction of spectra measured only with CFE buffer. Percentages of secondary structure were determined with the CDNN-program . For secondary structure prediction we used the program "Scratch Protein Predictor"(Expasy).
Protein-DNA-binding assay for halophilic proteins
Analytical DNA-affinity chromatography was performed by a modification of the method described by . For each experiment, 150 μl of streptavidin sepharose high performance (GE-Healthcare, München) suspension was spun down at 340 g in a column (MoBiTec M1002S, Göttingen) to remove ethanol. After five consecutive washing steps (500 μl of binding buffer: 0.15 M NaCl; 20 mM Na2PO4, pH 7.5) streptavidin sepharose was incubated with biotin-labelled DNA probes (61 pmol) for 2 h at room temperature with gentle shaking. Biotinylated DNA was prepared by PCR using biotin-labelled primers (Metabion, Martinsried) for amplification of the lrpA1 and the aspartate transaminase (aspB3) promoter, as well as a fragment of the flgB1 gene as negative-control. For binding of LrpA1 to the aspB3 promoter, reactions were performed in CFE buffer (3 M KCl; 1 M NaCl; 5 mM MgCl2; 10 mM HEPES, pH 7.1) in the presence of either 5 mM L-aspartate or 5 mM L-arginine. Columns were washed three times with CFE buffer. Unbound DNA was monitored spectrophotometrically. Protein-DNA-binding reactions were carried out by incubation of LrpA1 with the DNA-affinity matrix at room temperature for 4 h with shaking. We applied a stoichiometric excess of protein (2 nmol) relative to the molar amount of DNA (61 pmol). The mixture was then transferred to a column, centrifuged and washed twice with 200 μl CFE. LrpA1 was eluted from the DNA-Sepharose with 100 μl of 1% SDS and analyzed by SDS-PAGE (NuPAGE®Pre-Cast Gel System, Invitrogen) with subsequent silver staining. We performed three independent binding experiments. From these data we quantified band intensities in each single gel using densitometry (Total Lab Version 1.11). Both, monomeric as well as dimeric bands account for the depicted final values. The band with the highest intensity in each gel represents 100% (see Fig 4A lane 2, Fig 4B lane 3, Fig 4C lane 3). The two other bands with weaker binding intensity were calculated in relation to 100% highest intensity. Oligonucleotides used for the amplification of the DNA fragments are mentioned in additional file 10.
We thank Mike Dyall-Smith, Martin Grininger and Friedhelm Pfeiffer for carefully reading the manuscript and for discussions. We also than Patrik Johannson for making the structure alignment.
- Langer D, Hain J, Thuriaux P, Zillig W: Transcription in archaea: similarity to that in eucarya. Proc Natl Acad Sci USA 1995,92(13):5768-5772. 10.1073/pnas.92.13.5768 10.1073/pnas.92.13.5768View ArticlePubMedPubMed CentralGoogle Scholar
- Thomm M: Archaeal transcription factors and their role in transcription initiation. FEMS Microbiol Rev 1996,18(2-3):159-171. 10.1111/j.1574-6976.1996.tb00234.x 10.1111/j.1574-6976.1996.tb00234.xView ArticlePubMedGoogle Scholar
- Soppa J: Transcription initiation in Archaea: facts, factors and future aspects. Mol Microbiol 1999,31(5):1295-1305. 10.1046/j.1365-2958.1999.01273.x 10.1046/j.1365-2958.1999.01273.xView ArticlePubMedGoogle Scholar
- Bell SD, Magill CP, Jackson SP: Basal and regulated transcription in Archaea. Biochem Soc Trans 2001,29(Pt 4):392-395. 10.1042/BST0290392 10.1042/BST0290392View ArticlePubMedGoogle Scholar
- Reeve JN, Sandman K, Daniels CJ: Archaeal histones, nucleosomes, and transcription initiation. Cell 1997,89(7):999-1002. 10.1016/S0092-8674(00)80286-X 10.1016/S0092-8674(00)80286-XView ArticlePubMedGoogle Scholar
- Kruger K, Hermann T, Armbruster V, Pfeifer F: The transcriptional activator GvpE for the halobacterial gas vesicle genes resembles a basic region leucine-zipper regulatory protein. J Mol Biol 1998,279(4):761-771. 10.1006/jmbi.1998.1795 10.1006/jmbi.1998.1795View ArticlePubMedGoogle Scholar
- Calvo JM, Matthews RG: The leucine-responsive regulatory protein, a global regulator of metabolism in Escherichia coli . Microbiol Rev 1994,58(3):466-490.PubMedPubMed CentralGoogle Scholar
- Newman EB, Lin R: Leucine-responsive regulatory protein: a global regulator of gene expression in E. coli. Annu Rev Microbiol 1995, 49: 747-775. 10.1146/annurev.mi.49.100195.003531 10.1146/annurev.mi.49.100195.003531View ArticlePubMedGoogle Scholar
- Tani TH, Khodursky A, Blumenthal RM, Brown PO, Matthews RG: Adaptation to famine: a family of stationary-phase genes revealed by microarray analysis. Proc Natl Acad Sci USA 2002,99(21):13471-13476. 10.1073/pnas.212510999 10.1073/pnas.212510999View ArticlePubMedPubMed CentralGoogle Scholar
- Wang Q, Wu J, Friedberg D, Plakto J, Calvo JM: Regulation of the Escherichia coli lrp gene . J Bacteriol 1994,176(7):1831-1839.PubMedPubMed CentralGoogle Scholar
- Brinkman AB, Ettema TJ, de Vos WM, Oost J: The Lrp family of transcriptional regulators. Mol Microbiol 2003,48(2):287-294. 10.1046/j.1365-2958.2003.03442.x 10.1046/j.1365-2958.2003.03442.xView ArticlePubMedGoogle Scholar
- Ouhammouch M, Geiduschek EP: A thermostable platform for transcriptional regulation: the DNA-binding properties of two Lrp homologs from the hyperthermophilic archaeon Methanococcus jannaschii . EMBO Journal 2001,20(1-2):146-156. 10.1093/emboj/20.1.146 10.1093/emboj/20.1.146View ArticlePubMedPubMed CentralGoogle Scholar
- Ouhammouch M, Dewhurst RE, Hausner W, Thomm M, Geiduschek EP: Activation of archaeal transcription by recruitment of the TATA-binding protein. Proc Natl Acad Sci USA 2003,100(9):5097-5102. 10.1073/pnas.0837150100 10.1073/pnas.0837150100View ArticlePubMedPubMed CentralGoogle Scholar
- Bult CJ, White O, Olsen GJ, Zhou L, Fleischmann RD, Sutton GG, Blake JA, FitzGerald LM, Clayton RA, Gocayne JD, et al.: Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii . Science 1996,273(5278):1058-1073. 10.1126/science.273.5278.1058 10.1126/science.273.5278.1058View ArticlePubMedGoogle Scholar
- Charlier D, Roovers M, Thia-Toong TL, Durbecq V, Glansdorff N: Cloning and identification of the Sulfolobus solfataricus lrp gene encoding an archaeal homologue of the eubacterial leucine-responsive global transcriptional regulator Lrp. Gene 1997,201(1-2):63-68. 10.1016/S0378-1119(97)00428-9 10.1016/S0378-1119(97)00428-9View ArticlePubMedGoogle Scholar
- Peeters E, Wartel C, Maes D, Charlier D: Analysis of the DNA-binding sequence specificity of the archaeal transcriptional regulator Ss-LrpB from Sulfolobus solfataricus by systematic mutagenesis and high resolution contact probing. Nucleic Acids Res 2007,35(2):623-633. 10.1093/nar/gkl1095 10.1093/nar/gkl1095View ArticlePubMedPubMed CentralGoogle Scholar
- Bell SD, Jackson SP: The role of transcription factor B in transcription initiation and promoter clearance in the archaeon Sulfolobus acidocaldarius . J Biol Chem 2000,275(17):12934-12940. 10.1074/jbc.275.17.12934 10.1074/jbc.275.17.12934View ArticlePubMedGoogle Scholar
- Kyrpides NC, Olsen GJ, Klenk HP, White O, Woese CR: Methanococcus jannaschii genome: revisited. Microb Comp Genomics 1996,1(4):329-338.PubMedGoogle Scholar
- Brinkman AB, Dahlke I, Tuininga JE, Lammers T, Dumay V, de Heus E, Lebbink JH, Thomm M, de Vos WM, Oost J: An Lrp-like transcriptional regulator from the archaeon Pyrococcus furiosus is negatively autoregulated. J Biol Chem 2000,275(49):38160-38169. 10.1074/jbc.M005916200 10.1074/jbc.M005916200View ArticlePubMedGoogle Scholar
- Napoli A, Oost J, Sensen CW, Charlebois RL, Rossi M, Ciaramella M: An Lrp-like protein of the hyperthermophilic archaeon Sulfolobus solfataricus which binds to its own promoter. J Bacteriol 1999,181(5):1474-1480.PubMedPubMed CentralGoogle Scholar
- Bell SD, Jackson SP: Mechanism of autoregulation by an archaeal transcriptional repressor. J Biol Chem 2000,275(41):31624-31629. 10.1074/jbc.M005422200 10.1074/jbc.M005422200View ArticlePubMedGoogle Scholar
- Enoru-Eta J, Gigot D, Glansdorff N, Charlier D: High resolution contact probing of the Lrp-like DNA-binding protein Ss-Lrp from the hyperthermoacidophilic crenarchaeote Sulfolobus solfataricus P2. Mol Microbiol 2002,45(6):1541-1555. 10.1046/j.1365-2958.2002.03136.x 10.1046/j.1365-2958.2002.03136.xView ArticlePubMedGoogle Scholar
- Peeters E, Willaert R, Maes D, Charlier D: Ss-LrpB from Sulfolobus solfataricus condenses about 100 base pairs of its own operator DNA into globular nucleoprotein complexes. J Biol Chem 2006,281(17):11721-11728. 10.1074/jbc.M600383200 10.1074/jbc.M600383200View ArticlePubMedGoogle Scholar
- Peeters E, Albers SV, Vassart A, Driessen AJ, Charlier D: Ss-LrpB, a transcriptional regulator from Sulfolobus solfataricus , regulates a gene cluster with a pyruvate ferredoxin oxidoreductase-encoding operon and permease genes. Mol Microbiol 2009,71(4):972-988. 10.1111/j.1365-2958.2008.06578.x 10.1111/j.1365-2958.2008.06578.xView ArticlePubMedGoogle Scholar
- Brinkman AB, Bell SD, Lebbink RJ, de Vos WM, Oost J: The Sulfolobus solfataricus Lrp-like protein LysM regulates lysine biosynthesis in response to lysine availability. J Biol Chem 2002,277(33):29537-29549. 10.1074/jbc.M203528200 10.1074/jbc.M203528200View ArticlePubMedGoogle Scholar
- Enoru-Eta J, Gigot D, Thia-Toong TL, Glansdorff N, Charlier D: Purification and characterization of Sa-lrp, a DNA-binding protein from the extreme thermoacidophilic archaeon Sulfolobus acidocaldarius homologous to the bacterial global transcriptional regulator Lrp. J Bacteriol 2000,182(13):3661-3672. 10.1128/JB.182.13.3661-3672.2000 10.1128/JB.182.13.3661-3672.2000View ArticlePubMedPubMed CentralGoogle Scholar
- Wang Q, Sacco M, Ricca E, Lago CT, De Felice M, Calvo JM: Organization of Lrp-binding sites upstream of ilvIH in Salmonella typhimurium . Mol Microbiol 1993,7(6):883-891. 10.1111/j.1365-2958.1993.tb01179.x 10.1111/j.1365-2958.1993.tb01179.xView ArticlePubMedGoogle Scholar
- Dahlke I, Thomm M: A Pyrococcus homolog of the leucine-responsive regulatory protein, LrpA, inhibits transcription by abrogating RNA polymerase recruitment. Nucleic Acids Res 2002,30(3):701-710. 10.1093/nar/30.3.701 10.1093/nar/30.3.701View ArticlePubMedPubMed CentralGoogle Scholar
- Kawashima T, Aramaki H, Oyamada T, Makino K, Yamada M, Okamura H, Yokoyama K, Ishijima SA, Suzuki M: Transcription regulation by feast/famine regulatory proteins, FFRPs, in archaea and eubacteria. Biol Pharm Bull 2008,31(2):173-186. 10.1248/bpb.31.173 10.1248/bpb.31.173View ArticlePubMedGoogle Scholar
- Platko JV, Calvo JM: Mutations affecting the ability of Escherichia coli Lrp to bind DNA, activate transcription, or respond to leucine. J Bacteriol 1993,175(4):1110-1117.PubMedPubMed CentralGoogle Scholar
- Chen S, Rosner MH, Calvo JM: Leucine-regulated self-association of leucine-responsive regulatory protein (Lrp) from Escherichia coli . J Mol Biol 2001,312(4):625-635. 10.1006/jmbi.2001.4955 10.1006/jmbi.2001.4955View ArticlePubMedGoogle Scholar
- Leonard PM, Smits SH, Sedelnikova SE, Brinkman AB, de Vos WM, Oost J, Rice DW, Rafferty JB: Crystal structure of the Lrp-like transcriptional regulator from the archaeon Pyrococcus furiosus . Embo J 2001,20(5):990-997. 10.1093/emboj/20.5.990 10.1093/emboj/20.5.990View ArticlePubMedPubMed CentralGoogle Scholar
- Ettema TJ, Brinkman AB, Tani TH, Rafferty JB, Oost J: A novel ligand-binding domain involved in regulation of amino acid metabolism in prokaryotes. J Biol Chem 2002,277(40):37464-37468. 10.1074/jbc.M206063200 10.1074/jbc.M206063200View ArticlePubMedGoogle Scholar
- Brenneis M, Hering O, Lange C, Soppa J: Experimental Characterization of Cis-Acting Elements Important for Translation and Transcription in Halophilic Archaea. PLoS Genet 2007,3(12):e229. 10.1371/journal.pgen.0030229 10.1371/journal.pgen.0030229View ArticlePubMedPubMed CentralGoogle Scholar
- Geiduschek EP, Ouhammouch M: Archaeal transcription and its regulators. Mol Microbiol 2005,56(6):1397-1407. 10.1111/j.1365-2958.2005.04627.x 10.1111/j.1365-2958.2005.04627.xView ArticlePubMedGoogle Scholar
- Okamoto A, Kato R, Masui R, Yamagishi A, Oshima T, Kuramitsu S: An aspartate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8. J Biochem 1996,119(1):135-144.View ArticlePubMedGoogle Scholar
- Nakai T, Okada K, Akutsu S, Miyahara I, Kawaguchi S, Kato R, Kuramitsu S, Hirotsu K: Structure of Thermus thermophilus HB8 aspartate aminotransferase and its complex with maleate. Biochemistry 1999,38(8):2413-2424. 10.1021/bi9819881 10.1021/bi9819881View ArticlePubMedGoogle Scholar
- Gonzalez O, Gronau S, Falb M, Pfeiffer F, Mendoza E, Zimmer R, Oesterhelt D: Reconstruction, Modeling & Analysis of Halobacterium salinarum R-1 metabolism. Molecular Biosystems 2007, 3: 1-13. 10.1039/b617447g 10.1039/b617447gView ArticleGoogle Scholar
- Hill PJ, Cockayne A, Landers P, Morrissey JA, Sims CM, Williams P: SirR, a novel iron-dependent repressor in Staphylococcus epidermidis . Infect Immun 1998,66(9):4123-4129.PubMedPubMed CentralGoogle Scholar
- Pfeiffer F, Schuster SC, Broicher A, Falb M, Palm P, Rodewald K, Ruepp A, Soppa J, Tittor J, Oesterhelt D: Evolution in the laboratory: The genome of Halobacterium salinarum strain R1 compared to that of strain NRC-1. Genomics 2008,91(4):335-346. Newman EB, Lin R: Leucine-responsive regulatory protein: a global regulator of gene expression in E. coli. Annu Rev Microbiol 1995, 49:747-775 10.1016/j.ygeno.2008.01.001 10.1016/j.ygeno.2008.01.001View ArticlePubMedGoogle Scholar
- Bonneau R, Facciotti MT, Reiss DJ, Schmid AK, Pan M, Kaur A, Thorsson V, Shannon P, Johnson MH, Bare JC, et al.: A predictive model for transcriptional control of physiology in a free living cell. Cell 2007,131(7):1354-1365. 10.1016/j.cell.2007.10.053 10.1016/j.cell.2007.10.053View ArticlePubMedGoogle Scholar
- Rawal N, Kelkar SM, Altekar W: Alternative routes of carbohydrate metabolism in halophilic archaebacteria. Indian J Biochem Biophys 1988,25(6):674-686.PubMedGoogle Scholar
- Falb M, Muller K, Konigsmaier L, Oberwinkler T, Horn P, von Gronau S, Gonzalez O, Pfeiffer F, Bornberg-Bauer E, Oesterhelt D: Metabolism of halophilic archaea. Extremophiles 2008,12(2):177-196. 10.1007/s00792-008-0138-x 10.1007/s00792-008-0138-xView ArticlePubMedPubMed CentralGoogle Scholar
- Schut GJ, Menon AL, Adams MW: 2-keto acid oxidoreductases from Pyrococcus furiosus and Thermococcus litoralis . Methods Enzymol 2001, 331: 144-158. full_text full_textView ArticlePubMedGoogle Scholar
- Desmarais D, Jablonski PE, Fedarko NS, Roberts MF: 2-Sulfotrehalose, a novel osmolyte in haloalkaliphilic archaea. J Bacteriol 1997,179(10):3146-3153.PubMedPubMed CentralGoogle Scholar
- Kokoeva MV, Storch KF, Klein C, Oesterhelt D: A novel mode of sensory transduction in archaea: binding protein-mediated chemotaxis towards osmoprotectants and amino acids. Embo J 2002,21(10):2312-2322. 10.1093/emboj/21.10.2312 10.1093/emboj/21.10.2312View ArticlePubMedPubMed CentralGoogle Scholar
- Baliga NS, Goo YA, Ng WV, Hood L, Daniels CJ, DasSarma S: Is gene expression in Halobacterium NRC-1 regulated by multiple TBP and TFB transcription factors? Mol Microbiol 2000,36(5):1184-1185. 10.1046/j.1365-2958.2000.01916.x 10.1046/j.1365-2958.2000.01916.xView ArticlePubMedGoogle Scholar
- Dennis PP, Shimmin LC: Evolutionary divergence and salinity-mediated selection in halophilic archaea. Microbiol Mol Biol Rev 1997,61(1):90-104.PubMedPubMed CentralGoogle Scholar
- Coker JA, DasSarma S: Genetic and transcriptomic analysis of transcription factor genes in the model halophilic Archaeon: coordinate action of TbpD and TfbA. BMC Genet 2007, 8: 61. 10.1186/1471-2156-8-61 10.1186/1471-2156-8-61View ArticlePubMedPubMed CentralGoogle Scholar
- Kaur A, Pan M, Meislin M, Facciotti MT, El-Gewely R, Baliga NS: A systems view of haloarchaeal strategies to withstand stress from transition metals. Genome Res 2006,16(7):841-854. 10.1101/gr.5189606 10.1101/gr.5189606View ArticlePubMedPubMed CentralGoogle Scholar
- Cline SW, Doolittle WF: Efficient transfection of the archaebacterium Halobacterium halobium . J Bacteriol 1987,169(3):1341-1344.PubMedPubMed CentralGoogle Scholar
- Oesterhelt D, Krippahl G: Phototrophic growth of halobacteria and its use for isolation of photosynthetically-deficient mutants. Ann Microbiol (Paris) 1983,134B(1):137-150.Google Scholar
- Sambrook J, Fritsch E, Maniatis T: Molecular Cloning. A Laboratory Manual. In Cold Spring Harbor. 2nd edition. NY: Cold Spring Harbor Laboratory Press; 1989.Google Scholar
- Koch MK, Oesterhelt D: MpcT is the transducer for membrane potential changes in Halobacterium salinarum . Molecular Microbiology 2005,55(6):1681-1694. 10.1111/j.1365-2958.2005.04516.x 10.1111/j.1365-2958.2005.04516.xView ArticlePubMedGoogle Scholar
- Twellmeyer J, Wende A, Wolfertz J, Pfeiffer F, Panhuysen M, Zaigler A, Soppa J, Welzl G, Oesterhelt D: Microarray Analysis in the Archaeon Halobacterium salinarum Strain R1. PLoS ONE 2007,2(10):e1064. 10.1371/journal.pone.0001064 10.1371/journal.pone.0001064View ArticlePubMedPubMed CentralGoogle Scholar
- Hung SP, Baldi P, Hatfield GW: Global gene expression profiling in Escherichia coli K12. The effects of leucine-responsive regulatory protein. J Biol Chem 2002,277(43):40309-40323. 10.1074/jbc.M204044200 10.1074/jbc.M204044200View ArticlePubMedGoogle Scholar
- Chomczynski P, Mackey K: One-hour downward capillary blotting of RNA at neutral pH. Analytical Biochemistry 1994,221(2):303-305. 10.1006/abio.1994.1416 10.1006/abio.1994.1416View ArticlePubMedGoogle Scholar
- Bohm G, Muhr R, Jaenicke R: Quantitative-Analysis of Protein Far Uv Circular-Dichroism Spectra by Neural Networks. Protein Eng 1992,5(3):191-195. 10.1093/protein/5.3.191 10.1093/protein/5.3.191View ArticlePubMedGoogle Scholar
- Kumar NV, Bernstein LR: A new analytical scale DNA affinity binding assay for analyses of specific protein-DNA interactions. Anal Biochem 2001,299(2):203-210. 10.1006/abio.2001.5433 10.1006/abio.2001.5433View ArticlePubMedGoogle Scholar
- Eckhoff: Kristallisation und NMR-spektroskopische Untersuchungen an dem Twisted Dwarf Protein aus Arabidopsis thliana. 2003.Google Scholar
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