Molecular basis for coordinating secondary metabolite production by bacterial and plant signaling molecules

Abstract

The production of secondary metabolites is a major mechanism used by beneficial rhizobacteria to antagonize plant pathogens. These bacteria have evolved to coordinate the production of different secondary metabolites due to the heavy metabolic burden imposed by secondary metabolism. However, for most secondary metabolites produced by bacteria, it is not known how their biosynthesis is coordinated. Here, we showed that PhlH from the rhizobacterium Pseudomonas fluorescens is a TetR-family regulator coordinating the expression of enzymes related to the biosynthesis of several secondary metabolites, including 2,4-diacetylphloroglucinol (2,4-DAPG), mupirocin, and pyoverdine. We present structures of PhlH in both its apo form and 2,4-DAPG-bound form and elucidate its ligand-recognizing and allosteric switching mechanisms. Moreover, we found that dissociation of 2,4-DAPG from the ligand-binding domain of PhlH was sufficient to allosterically trigger a pendulum-like movement of the DNA-binding domains within the PhlH dimer, leading to a closed-to-open conformational transition. Finally, molecular dynamics simulations confirmed that two distinct conformational states were stabilized by specific hydrogen bonding interactions and that disruption of these hydrogen bonds had profound effects on the conformational transition. Our findings not only reveal a well-conserved route of allosteric signal transduction in TetR-family regulators but also provide novel mechanistic insights into bacterial metabolic coregulation.

Keywords: 2,4-diacetylphloroglucinol; TetR-family transcriptional regulator; allosteric switching mechanism; bacterial metabolic coregulation; secondary metabolite.

Figure 1

Pseudomonas fluorescens PhlH as a TetR-family regulator controls the transcriptional regulation of biosynthesis of the secondary metabolites 2,4-DAPG, mupirocin, and pyoverdine. A, comparative proteomic analysis of the WT and ΔphlH strains. A volcano plot showing the differentially expressed proteins in the ΔphlH strain compared with the WT strain. The red and blue spots indicate upregulated and downregulated proteins, respectively. B, GO enrichment analysis of the differentially expressed proteins in the ΔphlH strain. The enriched GO terms with p< 0.05 were displayed. 2,4-DAPG, 2,4-diacetylphloroglucinol; GO, Gene Ontology.

Figure 2

Structure of PhlH in complex with 2,4-DAPG. A, cartoon representation of PhlH in complex with 2,4-DAPG. The two protomers are colored in cyan and light cyan, respectively, with the bound 2,4-DAPG shown in sticks. The ligand-binding tunnel is the hydrophobic tunnel in ligands recognition and colored in orange. B, a close-up view of the 2,4-DAPG binding site and residues in the vicinity of 2,4-DAPG (colored in cyan) are shown in sticks. The electron density around 2,4-DAPG is colored gray and contoured at 1.0 σ. C, a closed-up view of the interaction mode between PhlH and the docked phloretin (colored in green). 2,4-DAPG, 2,4-diacetylphloroglucinol.

Figure 3

Ligand-induced dissociation of the PhlH–DNA complex results in an open to closed conformational transition of PhlH. Electrophoretic mobility shift assays of the WT PhlH and PhlH mutants (V80A, L85A, L148A, F173A, F178A, L186A, and V190A) with the upstream region of phlG (free DNA) in the presence of increasing concentrations (0–80 μM) of 2,4-DAPG (upper row) or increasing concentrations (0–800 μM) of phloretin (bottom row). The interaction modes between PhlH and 2,4-DAPG/phloretin are shown on the right of separate rows. 2,4-DAPG, 2,4-diacetylphloroglucinol.

Figure 4

Superposition of apo-PhlH and 2,4-DAPG-bound-PhlH. A, comparison of dimer structures of apo-PhlH (yellow) and 2,4-DAPG-bound-PhlH (cyan). The distances between the DNA recognition helices α3 within the dimer are displayed. B, separate superposition of the DBDs and LBDs from the apo-PhlH and 2,4-DAPG-bound-PhlH protomers. The DBDs are well superimposed, whereas the corresponding LBDs show large conformational changes. C, changes of the hydrogen bonding interactions in the LBDs from the apo-PhlH and 2,4-DAPG-bound-PhlH. The hydrogen bonds are represented by black dotted lines. 2,4-DAPG, 2,4-diacetylphloroglucinol; DBD, DNA-binding domain; LBD, ligand-binding domain.

Figure 5

Key residues involved in the conformational switching. 2D PMF profiles of the H76-K144 distance versus the DBD-DBD distance from 1000 ns conventional MD (cMD) simulation of the 2,4-DAPG-bound-PhlH dimer (A), 1000 ns cMD (B), and GaMD (C) simulations of the PhlH dimer with 2,4-DAPG removed. D, 2D PMF profiles of the R124-A34 distance versus the DBD-DBD distance from 1000 ns cMD simulation of the apo-PhlH dimer. 2D PMF profiles of the R124-A34 distance versus the R124-F33 distance from 1000 ns cMD simulation of the apo-PhlH dimer (E) and the 2,4-DAPG-bound-PhlH dimer (F). 2,4-DAPG, 2,4-diacetylphloroglucinol; MD, molecular dynamics; PMF, potential of mean force.

Figure 6

Binding affinity assays of key residues of PhlH involved in the conformational switching. A, the binding affinities of PhlH and the mutant proteins (H76A and R124A) with 2,4-DAPG were evaluated using ITC analysis. The binding curves corrected for the dilution effects were fit to a one-site binding model and the Kd values were calculated by the NanoAnalyze software. B, electrophoretic mobility shift assays of the WT PhlH (from the same WT-PhlH EMSA assay in upper row of Fig. 3) and PhlH mutants (H76A and R124A) with fluorescently labeled DNA in the presence of increasing concentrations (0–80 μM) of 2,4-DAPG. 2,4-DAPG, 2,4-diacetylphloroglucinol; EMSA, electrophoretic mobility shift assay; ITC, isothermal titration calorimetry.

Figure 7

PhlH homologues reveal a conserved conformational switching mechanism of TetR-family regulators. A, SSN of 1000 PhlH homologs. The Markov clustering method was used to classify these PhlH homologs into several isofunctional sequence clusters. Nodes from different clusters were colored differently. Different node shapes denote different bacterial phyla. Nodes of characterized PhlH (in cluster 7) and PltZ (in cluster 4) protein sequences are colored in black. B, Genomic Neighborhood Network with the SSN clusters of PhlH (cluster 1–13) as the hub nodes and Pfam families of the neighboring genes as the spoke nodes. SSN, sequence similarity network.

Figure 8

A putative transcriptional regulation mechanism of PhlH. A, a schematic representation illustrating the allosteric mechanism of closed to open conformational transition for PhlH. B, a conserved basic residue (Arg or Lys) also exists in α6 helix in other TetR-family regulators such as PaDesT (PDB code: 3LSJ and 3LSP), BhFadR (PDB code: 5GPC and 5GP9), and SaFadR (PDB code: 6EL2 and 6EN8) and bridges the C-terminal end of the α1 helix via hydrogen bonding in the DNA-bound conformation (green, orange, and magenta), yet in the ligand-bound conformation (grays), these interactions are disrupted. 2,4-DAPG, 2,4-diacetylphloroglucinol.