Figure 1.
Schematic of the physical location of the psrA gene in Pseudomonas fluorescens 2P24.
lexA, gene encoding LexA repressor protein; psrA, Pseudomonas sigma regulator; nagZ, gene encoding β-N-acetyl-D-glucosaminidase; other gene names refer to the gene symbols as annotated in the Pseudomonas fluorescens Pf0-1 genome (GenBank accession no. CP000094). The bars indicate the fragments cloned into the vector pHSG299 to obtain p299DpsrA. The fragment inserted into pRK415 was used to complement the psrA mutant. Two putative PsrA binding sites are indicated with inverted arrows. Δ, the region deleted in the mutant PM113 and in plasmid p229DpsrA. Artificial restriction sites are marked with asterisks.
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.
Figure 3.
The transcriptional fusions psrA-lacZ and phlF-lacZ were introduced into P. fluorescens 2P24, respectively.
Bacteria were grown in LB medium, and absorbance was measured at 600 nm (solid circles, psrA-lacZ; open circles, phlF-lacZ). Expression of the fusions was assessed by measuring levels of β-galactosidase. Black shading represents psrA-lacZ expression, and grey shading represents phlF-lacZ expression. Triplicate cultures were assayed and the standard deviations are presented with error bars.
Figure 4.
Transcription of small non-coding RNA genes rsmX (A), rsmY (B), and rsmZ (C) and their cognate regulator genes rsmE (D) and rsmA (E) in P. fluorescens 2P24, its psrA mutant and its gacA mutants.
(F) HPLC analysis of 2,4-DAPG production by strain 2P24 and its rsmA mutant. All experiments were performed in triplicate, and the mean values ±SD are indicated. Growth is indicated by the dotted line.
Figure 5.
RpoS regulates the 2,4-DAPG production via RsmA in P. fluorescens 2P24.
Biosynthesis of 2,4-DAPG in strains 2P24 and its rpoS mutant was assayed by HPLC (A). The expression of the rpoS gene is activated by PsrA in strain 2P24 (B). Expression of the rsmA gene in the wild type strain 2P24 and the rpoS mutant PM303 (C). Binding assay of PsrA to the rpoS promoter. 30 ng DNA probe was incubated with increasing amounts of PsrA. Lane 1, DNA probe alone; lanes 2–5, DNA probe incubated with 25, 50, 75, or 100 ng PsrA, respectively; lane 6, the mutated DNA probe from p399rpoSp derivative (a 3-bp substitution [GGG for TTT] in the rpoS promoter) incubated with 100 ng PsrA (D). Western blot analysis of RsmA-V in strain 2P24 and the rpoS mutant (E).
Figure 6.
PsrA is not regulated by the GacS/GacA two-component system in P. fluorescens 2P24.
Transcriptional fusion assay (A) and Western blot analysis (B) demonstrating that the expression of PsrA is not altered in the gacA mutant or in the gacS mutant.
Figure 7.
Model for the regulation of 2,4-DAPG biosynthesis in P. fluorescens 2P24.
In this complex cascade, the sensor GacS is activated by a putative environmental factor. Subsequently, GacS stimulates its cognate regulator GacA. GacA activates small non-coding RNAs, RsmX/Y/Z and sigma factor RpoS, which negatively regulate 2,4-DAPG production at posttranscriptional level through RsmA titration. In addition, PsrA negatively controls transcription of phlA and positively regulates rpoS by binding to their promoter regions. RpoS has a positive effect on rsmA expression. Thus, effect of PsrA on 2,4-DAPG production exerted at both transcriptional and posttranscriptional levels.