Only <i>ail1</i>_Pl is directly regulated by PhoP.

<p><b>A</b>. RT-qPCR: <i>ail1</i>_Pl expression is PhoP-dependent. Total RNA from <i>phoP</i> mutant or TT01 wild-type strain of <i>Photorhabdus luminescens</i> was used for RT-qPCR analysis with internal primers specific for the indicated genes. mRNA levels were normalized against those of a reference gene (<i>gyrB</i>). Data are presented as a ratio of values for <i>phoP</i> mutant and TT01 wild-type strain. A ratio of 1 indicates no difference in expression level between both strains. The bars indicate standard errors calculated using Taylor's series. Significant differences (<i>p</i>-value <0.05) are indicated by asterisks (*). The relative quantification results were obtained from three independent experiments with the REST 2009 program. <b>B</b>. Gene transcription monitored by GFP quantification: <i>ail1</i>_Pl promoter region is positively regulated by PhoP. The dynamic expression of <i>ail1</i>_Pl and <i>ail2</i>_Pl promoter in TT01 and <i>phoP</i> backgrounds was monitored over time after growth in LB medium. Each histogram represents the specific fluorescence at the peak of expression for each condition. One experiment representative of more than three independent experiments is shown. Standard deviations represent technical replicates. <b>C</b>. PhoP-His binds the promoter region of <i>ail1</i>_Pl. Electrophoretic mobility shift assay was carried out to test the binding of PhoP-His protein activated <i>in vitro</i> with ACP 10 mM (P-PhoP-His) or non activated PhoP-His (PhoP-His) on <i>ail1</i>_Pl promoter. The PhoP-His concentrations indicated are in micromolar. To ensure that the fixation is specific, we used BSA proteins and poly(dI-dC) in the binding buffer. <b>D</b>. <i>ail1</i>_Pl expression is higher at low MgSO₄ concentrations. We evaluated the impact of low and high MgSO₄ concentrations on <i>ail1</i>_Pl and <i>ail2</i>_Pl expression. Cultures diluted at 1/200 were grown in M9 minimal medium supplemented with 10 µM or 10 mM MgSO₄. Each histogram represents specific fluorescence at the peak of expression for each condition. Experiments were realized at least three times.