Factors that influence the response of the LysR type transcriptional regulators to aromatic compounds

Abstract

Background: The transcriptional regulators DntR, NagR and NtdR have a high sequence identity and belong to the large family of LysR type transcriptional regulators (LTTRs). These three regulators are all involved in regulation of genes identified in pathways for degradation of aromatic compounds. They activate the transcription of these genes in the presence of an inducer, but the inducer specificity profiles are different.

Results: The results from this study show that NtdR has the broadest inducer specificity, responding to several nitro-aromatic compounds. Mutational studies of residues that differ between DntR, NagR and NtdR suggest that a number of specific residues are involved in the broader inducer specificity of NtdR when compared to DntR and NagR. The inducer response was also investigated as a function of the experimental conditions and a number of parameters such as the growth media, plasmid arrangement of the LTTR-encoding genes, promoter and gfp reporter gene, and the presence of a His6-tag were shown to affect the inducer response in E.coli DH5α. Furthermore, the response upon addition of both salicylate and 4-nitrobenzoate to the growth media was larger than the sum of responses upon addition of each of the compounds, which suggests the presence of a secondary binding site, as previously reported for other LTTRs.

Conclusions: Optimization of the growth conditions and gene arrangement resulted in improved responses to nitro-aromatic inducers. The data also suggests the presence of a previously unknown secondary binding site in DntR, analogous to that of BenM.

Figure 1

(a) Overview of one monomer of DntR with the seven residues that are replaced in this study marked in red, with the number of the residue next to it. Model (see Smirnova et al. [8]) of the DNA-binding domain (DBD) and linker connected to the structure of the inducer-binding domain (IBD) are shown as grey ribbons. The previously identified inducer-binding pocket is situated in the region between subdomains 1 (SD1) and 2 (SD2) and its position is encircled (see b). One monomer is packed with another monomer in a head-to-tail orientation so that the β-strands seen to the right under the inducer-binding pocket in SD2 forms a parallel β-sheet together with the β-strands seen to the upper left in SD1. (b) The inducer-binding pocket shown in detail, with the position of a salicylate (shown in yellow, oxygens marked in red) modelled based on the position of the acetate ion found in the crystal structure. The side-chains of all residues within 4Å of the modelled salicylate molecule are displayed (with oxygens in red and nitrogens in blue, the carbons of His 169 are marked in red). In this model, the hydroxyl group points away from His 169. The His169 residue is the only substituteded residue in this study within 5 Å from the modelled salicylate. Below the pictures, the differences in sequence for NtdR and NagR, compared to DntR, are indicated. In bold are the amino-acid residues in the DBD, while the other five amino-acid residue substitutions are found in the IBD.

Figure 2

Histograms showing the cell populations expressing DntR (a), NagR (b) and NtdR (c). The fluorescence from the gfp reporter gene under the control of the P_DNT promoter is measured for 10000 cells. The background level of fluorescence where only the solvent DMSO has been added to a cell population harbouring the one-plasmid system (solid red) and DMSO + IPTG added to cells harbouring the two-plasmid system (dashed dark red). The fluorescence level for the cell population with salicylate added for the one-plasmid system (solid green) and for the two-plasmid system (dashed green).

Figure 3

Example of cell populations with non-filamentous and filamentous cell-growth. (a)-(c) shows dotplots for the side scattering and forward scattering for cell populations harbouring the dntR gene. The more filamentous cell growth, the more scattering of light is observed, both in the forward scatter (FSC-H) and side scatter (SSC-H) channels. In (a) E.coli DH5α harbouring the one-plasmid system and in (b), E.coli DH5α harbouring the two-plasmid system with addition of only DMSO are shown. Here, the cell population divided normally, and no filamentous cell growth was observed. For the cell population in (c), E.coli DH5α harbouring the two-plasmid system with addition of DMSO and IPTG, a large subpopulation shows filamentous cell-growth. In (d) are shown fluorescence microscope images of the E.coli DH5α cells harbouring the two-plasmid system without addition of IPTG (upper picture) and with IPTG (lower picture).

Figure 4

The inducer response in E.coli DH5α for DntR (black bars), NagR (light grey bars) and NtdR (dark grey bars) for three experimental conditions. (a) E.coli DH5α harbouring the two-plasmid system for DntR, NagR and NtdR grown in LB. (b) E.coli DH5α harbouring the one-plasmid system for DntR, NagR and NtdR grown in LB (c) E.coli DH5α harbouring the one-plasmid system for DntR, NagR and NtdR grown in modified M9 medium. For all conditions the measurements were made 15 hours after addition of the potential inducers. Shown in the diagrams is the increase in fluorescence after addition of the potential inducer, compared to the control culture where only the solvent DMSO (or DMSO+IPTG in A) was added. The mean fluorescence was measured for 10 000 cells in each measurement. All data are based on three independent analyses and shown in error bars are the standard deviations based on these measurements.

Figure 5

The response, measured as the fold of induction (mean fluorescence when salicylate is added/mean fluorescence for DMSO control) for salicylate concentrations ranging from 1-2000 μM added to E. coli cells harbouring the one-plasmid system grown in LB overnight. Filled diamonds are the data for wild-type DntR without the His₆-tag, and open circles are the data for wild-type DntR with the His₆-tag at its C-terminal.

Figure 6

The effect of addition of 100 μM salicylate (SAL), 100 μM 4-nitrobenzoate (4-NB)or both 100 μM salicylate and 100 μM 4-nitrobenzoate simultaneously to E. coli DH5α harbouring WT DntR in the one-plasmid system grown in LB in (a) and grown in M9 in (b). In (c) the effect is shown for cells grown in LB at 0-500 μM salicylate in the presence of 0 (black filled diamonds), 100 μM (grey squares) and 250 μM (black open triangles) 4-nitrobenzoate respectively. The fold of induction was measured as the mean fluorescence for 10 000 cells with inducer(s) added, divided by the mean fluorescence of 10 000 cells from the control culture with only the solvent added after overnight induction. Data are presented as the mean values based on three independent experiments and shown as error bars are the standard deviations.