Multiple-Level Regulation of 2,4-Diacetylphloroglucinol Production by the Sigma Regulator PsrA in Pseudomonas fluorescens 2P24
Figure 2
EMSA of PsrA with the phlA (30 ng) promoter fragment that contains PsrA-binding sequence showing formation of a PsrA-DNA complex.
Lane 1, DNA probe alone; lanes 2–5, DNA probe incubated with 50, 75, 100, or 150 ng PsrA, respectively; lane 6, the mutagenized DNA probe from p399phlAp derivative (a 3-bp substitution [GGG for TTT] in the phlA promoter) incubated with 150 ng PsrA (A). Biosynthesis of 2,4-DAPG in strain 2P24 and its psrA and phlF mutants was assayed by HPLC (B). For transcriptional assay, strain 2P24 and its psrA mutant carrying p970Gm-phlAp (wild type phlA-lacZ), p970Gm-phlApM3G (PsrA box mutTTT phlA-lacZ) or p970Gm-phlAD3T (PsrA box ΔTTT phlA-lacZ) were grown in LB, and β-galactosidase activities were determined (C). Analysis of PhlA-V levels in strain 2P24 and the psrA mutant by immunoblotting. An antibody directed against 3-phosphoglycerate kinase α (α-PGK) is used as a loading control in this and later blots (D). All experiments were performed in triplicate, and the mean values ±SD are indicated. Growth is indicated by the dotted line.