The Ferredoxin-Like Protein FerR Regulates PrbP Activity in Liberibacter asiaticus

PrbP_Las and FerR_Las interact in a bacterial two-hybrid system. β-Galactosidase activity was determined using E. coli JM109 as a reporter strain. The genetic constructs in the strains are as follows: 2HB01 carrying pB2HΔα and pB2HΔω, 2HB02 carrying pB2HΔα and pB2HΔω_ferR_Las, 2HB03 carrying the pB2HΔα_prbP_Las and pB2HΔω, and 2HB04 carrying pB2HΔα_prbP_Las and the pB2HΔω_ferR_Las. β-Galactosidase assays were performed at different stages during the exponential-growth phase (OD₆₀₀, 0.3, 0.5, and 0.8). The growth curves of all the strains tested are shown in Fig. S2A. Enzymatic activities are shown as the average arbitrary units (AU) with the standard deviation (SD) from biological and technical triplicates. Statistical significance was determined as described in Materials and Methods. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FerR_Las increases PrbP_Las activity as a transcriptional activator. (A) In vitro transcription assays using increasing concentrations of FerR_Las (0, 1, 2, and 4 μM) and/or 2.0 μM PrbP_Las added to the RNA polymerase-containing reactions as indicated at the top. (B) ImageJ was utilized to quantify the amount of the P_rplK transcript obtained in the in vitro transcription. The fold change was calculated by the band intensity normalized to the transcript level in the reactions performed in the absence of PrbP_Las and FerR_Las. The quantification was based on observations from at least three replicates. Statistical significance was determined as described in Materials and Methods. Different letters on top of the bars denote statistical significance of at least a P value of <0.05. (C) The effects of FerR_Las on PrbP_Las-DNA binding were evaluated using electrophoresis mobility shift assays. The reaction mixture contained 1.0 ng of the biotinylated P_rplk probe and 2.5 μM PrbP_Las or 1 to 5 μM FerR_Las, as indicated at the top of each lane. The first lane has no protein added.

FerR_Las activity is not modulated by oxidizing/reducing conditions. (A) Electrophoresis mobility shift assay of PrbP_Las and FerR_Las with P_rplK; the reaction mixture contained 1.0 ng of the biotinylated P_rplk probe, 2.5 μM PrbP_Las, 2.5 μM FerR_Las, and increasing concentrations (0 to 500 μM) of DTT or diamide, as indicated at the top. The first lane has no protein added. (B) The effects of oxidizing and reducing agents were tested on FerR_Las activity using in vitro transcription assays. PrbP_Las and FerR_Las (0 to 2.5 μM) were added to the reactions, as indicated at the top. Increasing concentrations of DTT or diamide were added at the indicated concentrations. All reactions contained the same amount of RNA polymerase (0.5 μM).

In silico prediction of the PrbP_Las-interacting interface in FerR_Las. (A) Hydrophobicity analysis in the 3D model FerR_Las-SD. The color gradient from red to white illustrates hydrophobic potential to neutral, while the black dashed line circle encloses the predicted hydrophobicity patch in FerR_Las-SD. (B) Surface electrostatic potential analysis in FerR_Las-SD. The color gradient from red to blue illustrates negative to positive surface charges, while the black dashed line circle encloses the predicted hydrophobicity patch in FerR_Las-SD. (C) Cartoon representation of the FerR_Las-SD model with stick representation of the amino acid residues that form the predicted hydrophobicity patch. (D) Hydrophobic amino acid residues in the predicted patch are shown in red.

FerR_Las:PrbP_Las interactions are mediated by residues V20, V23, and C40 in FerR_Las. (A) Mutations V20A, V23A, and C40A in FerR_Las decrease interactions with PrbP_Las in a bacterial two-hybrid system. β-Galactosidase activity was performed in E. coli JM109 (β-galactosidase deficient). The reporter strains used were 2HB04 carrying pB2HΔα_prbP_Las and pB2HΔω_ferR_Las, 2HB08 carrying pB2HΔα_prbP_Las and pB2HΔω_ferR_Las V20A, 2HB12 carrying pB2HΔα and pB2HΔω_ferR_Las V23A, and 2HB16 carrying pB2HΔα_prbP_Las and pB2HΔω_ferR_Las C40A. β-Galactosidase assays were performed at different stages during the exponential-growth phase (OD₆₀₀, 0.3, 0.5, and 0.8). The growth curves of all the strains tested are shown in Fig. S2B. The activities were normalized to the highest background strains of 2HB02 carrying pB2HΔα and pB2HΔω_ferR_Las, 2HB07 carrying pB2HΔα and pB2HΔω_ferR_Las V20A, 2HB11 carrying pB2HΔα and pB2HΔω_ferR_Las V23A, and 2HB15 carrying pB2HΔα and pB2HΔω_ferR_Las C40A and are shown as arbitrary units (AU). Statistical significance was determined as described in Materials and Methods. *, P < 0.05; **, P < 0.01. (B) The mutations in V20, V23, and C40 reduced FerR_Las activity on PrbP_Las. In vitro transcription assays were performed with PrbP_Las, FerR_Las WT, or the FerR_Las V20A, V23A, and C40A mutants. Each protein was added at a concentration of 2.5 μM, as indicated at the top; all reactions contained the same amount of RNA polymerase (0.5 μM). The image has been cropped and rearranged for presentation. (C) ImageJ was utilized to quantify the amount of the P_rplK transcripts obtained in the in vitro transcription assay. The activity fold change was calculated by the band intensity normalized to the transcript level in the reactions performed in the presence of PrbP_Las and FerR_Las. The quantification was based on observations from at least three replicates. Statistical significance was determined as described in Materials and Methods. *, P < 0.05; **, P < 0.01.

Increase of osmolarity induces ferR_Lcr expression level in L. crescens. The expression levels of ferR_Lcr (B488_01730) and prbP_Lcr (B488_01720) were determined in L. crescens in the prescence or absence of increasing concentrations of sucrose (50 to 100 mM) by qRT-PCR. The relative expression value of each gene was normalized to the expression levels of gyrase subunit A gene (gyrA). In black, control (Ctrl) condition in BM7 medium; light gray, BM7 medium supplemented with 50 mM sucrose; dark gray, BM7 medium supplemented with 100 mM sucrose. L. crescens cells were collected when the OD₆₀₀ was 0.3. The experiment was performed using 4 biological replicates. The black horizontal line indicates statistical significance between connecting data bars. Statistical significance was determined as described in Materials and Methods. *, P < 0.05.