Table 1.
List of strains and plasmids used in this study.
Table 2.
Oligonucleotides used in this study.
Fig 1.
Genomic arrangement of znu gene clusters identified in L. asiaticus.
Regulon #1 possesses the genomic arrangement znuACB (A), regulon #2 has an arrangement znuACBB (B), whereas regulon #3 displays the arrangement znuCBA (C). The components of each system are colored as follows: znuA (blue), znuB (green), and znuC (orange). Homologs from Liberibacter species, as well as S. meliloti, E. coli, A. tumefaciens, Afiphia massiliensis, Rhodopseudomonas palustris, Bradyrhizobiaceae bacterium, Desulfovibrio sp., Rhizobium leguminosarum, and Pseudomonas syringae were included in the analysis. The arrows represent each homologue (drawn to scale) and describe the direction of their transcription.
Fig 2.
LdtR binds to PznuA2 but not to PznuA1.
EMSAs were performed using znuA promoters from L. asiaticus. PznuA1 and PznuA2 probes were incubated with increasing concentration of LdtR (0–1000 nM), as indicated on top of each panel. Protein was not added to the first lane.
Fig 3.
Effect of metal cofactors in the interaction between LdtR and PldtP.
EMSAs were carried out using 400 nM LdtR with the addition of 100 μM metal chlorides, as indicated on top of each panel. Protein was not added to the first lane.
Fig 4.
Zinc and EDTA have antagonistic effects on the LdtR interaction with DNA.
EMSAs were carried out using 400 nM LdtR on PldtP (A) or 1000 nM LdtR on PznuA2 (B) in the absence or presence of different concentrations of Zn2+, Fe2+, or EDTA. The ligand:LdtR molar ratio is indicated on top of each panel. Protein was not added to the first lane.
Fig 5.
Identification of amino acids in LdtR involved in Zn2+ interaction.
(A) Close view of the Benz1 pocket identified in LdtR [28]. The model is shown in cartoon representation with monomers A and B colored in brown and green, respectively. The identified amino acids are depicted as sticks and colored in orange (Benz1) or blue for residues C28 and E33. (B) Titration of LdtR binding with PldtP and PznuA2 probes by EMSAs. (C) Titration of LdtR(C28S) binding with PldtP and PznuA2 probes by EMSAs. (D) Titration of LdtR(E33A) binding with PldtP and PznuA2 probes by EMSAs. The concentration of LdtR used in the binding assay is shown on top of each panel. Protein was not added to the first lane.
Fig 6.
Mutations in residues C28 and T43 and L61 affect the interaction of LdtR with Zn2+.
The PldtP probe was incubated with LdtR WT or the LdtR mutants T43A, L61A, F64A, C28S, and E33A in the absence or presence of increasing concentrations of zinc. Since some LdtR mutants have different affinities for PldtP [28] and the Zn2+:LdtR molar ratio was kept constant, as indicated on top of each panel. Protein was not added to the first lane.
Table 3.
Thermodynamic parameters of for the calorimetric titration of LdtR with Zn2+.
Fig 7.
Zn2+ and benzbromarone disrupt the binding of LdtR to DNA.
The combined effect of both ligands was tested in EMSA. LdtR was preincubated with 0.6 μM Zn2+ (A) or benzbromarone (B), in a 1:1 ligand:protein molar ratio. Subsequently, the protein was either incubated with increasing concentrations of either Zn2+ or benzbromarone (0.3–15 μM), as indicated on top of each panel. Protein was not added to the first lane.
Table 4.
The effect of Zn2+, phloretin, and benzbromarone in the growth parameters of L. crescens.