Open Access Highly Accessed Research article

Transcriptional control by two leucine-responsive regulatory proteins in Halobacterium salinarum R1

Rita Schwaiger1, Christoph Schwarz2, Katarina Furtwängler1, Valery Tarasov1, Andy Wende3 and Dieter Oesterhelt1*

Author Affiliations

1 Max Planck Institute of Biochemistry, Department of Membrane Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany

2 School of Life Sciences, Arizona State University, Tempe, Arizona 85287, USA

3 QIAGEN GmbH, Qiagen Strasse 1, 40724 Hilden, Germany

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BMC Molecular Biology 2010, 11:40  doi:10.1186/1471-2199-11-40

Published: 28 May 2010

Additional files

Additional file 1:

Structure-based sequence alignment of the H. salinarum LrpA1 (OE2621R) with other archaeal and bacterial Lrp-homologues. The percentages in parenthesis represent a sequence comparison between LrpA1 and the aligned sequences. The alignment includes P. furiosus LrpA (PF1601; 38%), M. jannaschii Ptr2 (MJ0723; 30%), H. walsbyi Lrp-like protein (HQ3354A; 76%), H. salinarum Lrp (OE3923F; 25%), S. solfataricus LysM (SSO0157; 21%), B. subtilis LrpC (BSU04250; 24%), E. coli AsnC (APECO1_2720; 26%), and E. coli Lrp (b0889; 23%). The HTH DNA-binding motif (αB-αC) and the RAM-domain (β2αDβ3β4αEβ5) are boxed, including the asparagine binding site of E. coli AsnC. Amino acids are shaded in grey according to sequence conservation. Conserved methionine/prolines of the LrpA1-subgroup are shaded in blue. LrpA1 shares highest sequence identity (76%) with the Lrp-like regulator (HQ3354A) from Haloquadratum walsbyi. A comparison between LrpA1 and other Lrp-homologues revealed 38% identity with LrpA (PF1601) from P. furiosus, 30% identity with Ptr2 (MJ0723) from M. jannaschii, 21% identity with S. solfataricus LysM (SSO0157) and 24% identity with LrpC (BSU04250) from Bacillus subtilis. E. coli Lrp (b0889) showed 23% and E. coli AsnC (APECO1_2720) 26% identity. Secondary structure elements are indicated as red α-helices and green β-strands. In both H. salinarum Lrp proteins the N-terminal helix-turn-helix (HTH) motif and the C-terminal regulation of amino acid metabolism (RAM)-domain were identified based on the structure of cristallized Lrp/AsnC homologues. The figure was made by using the INDONESIA alignment package (D. Madsen, P. Johansson and G.J. Kleywegt manuscript in preparation).

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Additional file 2:

Growth curves of Δlrp, ΔlrpA1, ↑lrp and ↑lrpA1. Growth curves of the deletion strains Δlrp and ΔlrpA1 as well as the overexpressing strains ↑lrp and ↑lrpA1 were compared to the wild type strain R1. All strains were grown in complex medium. Growth occurred aerobic in the dark for the deletion strains (A) and anaerobic in the light for the overexpression strains (B). The optical density of the cultures was determined at OD600.

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Additional file 3:

Southern blot analysis to verify the correct genotype of the deletion mutants (ΔlrpA1 Δlrp). In a first approach deletion strains were pre-selected by PCR (oligonucleotides see additional file 10; probes for southern blotting). We used two primer pairs, one, which amplifies the entire gene to be deleted (only in wild type, not in the mutant) and another, which anneals to flanking regions up-and downstream of that ORF. Typically a mutant strain does not yield the first, but the second product of the size: (wild type amlicon-gene length). As a positive control we have used chromosomal DNA of wild type cells. To ensure that the deleted gene has not been relocated by chromosomal rearrangements we subsequently confirmed the mutant genotype by southern blotting. Genomic DNA from the PCR-positive clones as well as wild type DNA was cut with the restriction enzyme BglI. Southern blot hybridization was performed using two different types of digoxygenin labelled probes generated by PCR. (see additional file 10: flanking probe (ΔlrpA1, Δlrp) and gene probe (lrpA1, lrp)). Samples were separated by denaturing agarose gel electrophoresis (1%), and vacuum blotted onto a nylon membrane. Hybridization and detection were performed with "DIG Easy Hyb" (Roche Diagnostics) according to the manufacturer's instructions. The obtained fragments are marked by an arrow (A1, 2 and B1, 2) and explained in additional table S1. Figure A shows the southern blot for the PCR-positive deletion strains ΔlrpA1. PCR-positive clones (lane 1-9), wild type DNA (lane 11) and a Dig-labelled DNA-standard (lane 12). Using two different types of probes (flanking probe A1 and gene probe A2) fragments obtained from southern blot are marked by an arrow and described in additional table S1. PCR-positive deletion strains Δlrp (lane 1-3), wild type DNA (4, 5) and a Dig-labelled DNA-standard (lane 7) were loaded to a 1% agarose gel (B). The resulted fragments after using the flanking probe (B1) and the gene probe (B2) are marked by an arrow and corresponding sizes are shown in additional table S1.

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Additional file 4:

Differentially expressed genes in ΔlrpA1. All significantly differentially expressed genes in ΔlrpA1 having a ratio higher than +/-1.7 and those between +/-1.7 and +/-1.3 are depicted in this table. The regulated genes are sorted by their Identification number (ID).

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Additional file 5:

RT-qPCR data compared to microarray data in ΔlrpA1 compared against wild type R1. Additional table S2 shows a comparison of RT-qPCR data with the microarray data. Total RNA was isolated from the deletion mutant ΔlrpA1 and wild type at a cell density OD600 0.4. We determined the transcript amount of the genes aspB3 and OE6130F, which encodes for a conserved hypothetical protein.

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Additional file 6:

Expression, oligomerization and folding of LrpA1. Heterologous expression of the His6-tagged H. salinarum LrpA1 in E. coli BL21(DE3) and protein purification analyzed by SDS-PAGE. E. coli extracts before induction (lane 1), two and three hours after induction with 0.6 mM IPTG (lane 2, 3) and purified protein, displayed by an arrow (lane 4-6) (A). After dialysis against a high salt buffer correct folding of LrpA1 was proved by CD-spectroscopy, were 56% α-helices, 11% β-sheet, 14% β-turn and 24% random coil structure was determined (B). The theoretical calculated values for LrpA1 are 42% α-helices, 27% β-sheet and 31% random coil structure was determined. Folded LrpA1 in high salt has a predominant α-helical structure. An aberrance of ~10% between the measured and the theoretical value is in the range of error and has been shown in previous studies [60] (B). The size exclusion chromatography elution profile showed dimerisation of LrpA1 after renaturation (C). Calibration standards used for this run are indicated in additional table S2 (C). LrpA1 elutes at a volume of 1.32 ml which is a corresponding molecular weight 31.1 kDa showing a LrpA1 dimer. The theoretical size of a LrpA1 monomer is 15.2 kDa.

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Additional file 7:

Sequence comparison of the three H. salinarum aspartate transaminases. AspB1, AspB2 and AspB3 were compared with aspartate transaminases subgroup Ia (having a conserved R at the position marked by an □) and aspartate transaminases Ib (having an conserved K at the position marked by an x).

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Additional file 8:

Western blot analysis to detect the overexpression of lrp on protein level. Transcription of the lrp gene was under the control of the bacteriorhodopsin (bop) promoter resulting in the KF203 (↑lrp) mutant. The bop promoter is maximally induced under light exposure and anaerobic conditions. Therefore wild type cells and cells from the Lrp-overexpression strain were grown anaerobically under light exposure and harvested at an OD600 of 0.8. For detection of the Lrp protein we used an antibody against Lrp. Proteins from strains as indicated in the figure were separated on a gradient gel (4-12%), blotted on a nitrocellulose membrane and finally subjected to an immune detection reaction with an antibody against Lrp. Low expression was observed for the wild type (lane 1), whereas significant overexpression was detected in the Lrp-overexpression strain (lane 2). Furthermore we tested the Δlrp deletion mutant, grown aerobically in the dark. Using an antibody against Lrp (lane 3) no signal could be obtained, as expected.

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Additional file 9:

Differentially expressed genes in Δlrp. All significantly differentially expressed genes in Δlrp having a ratio higher than +/-1.7 and those between +/-1.7 and +/-1.3 are depicted in this table. The regulated genes are sorted by their Identification number (ID).

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Additional file 10:

A table of oligonucleotides. Oligonucleotides used in these experiments

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Additional file 11:

Scheme of integration of pKF204 into the genome of H. salinarum resulting in KF204 (↑lrpA1) mutant. The plasmid pKF204 contains a portion of the 5'end of the lrpA1 gene. The bop promoter Pbop (violet box) was inserted upstream of the truncated lrpA1 gene (black arrow). The plasmid contains a selection marker (MevR). After integration of pKF204 into the H. salinarum genome, only the lrpA1 gene downstream the bop promoter is functionally transcribed, while transcription product under the native promoter is truncated and presumably not functional. The KF203 (↑lrp) mutant was constructed similarly using plasmid pKF203.

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