Posttranscriptional regulation of 2,4-diacetylphloroglucinol production by GidA and TrmE in Pseudomonas fluorescens 2P24

Abstract

Pseudomonas fluorescens 2P24 is a soilborne bacterium that synthesizes and excretes multiple antimicrobial metabolites. The polyketide compound 2,4-diacetylphloroglucinol (2,4-DAPG), synthesized by the phlACBD locus, is its major biocontrol determinant. This study investigated two mutants defective in antagonistic activity against Rhizoctonia solani. Deletion of the gidA (PM701) or trmE (PM702) gene from strain 2P24 completely inhibited the production of 2,4-DAPG and its precursors, monoacetylphloroglucinol (MAPG) and phloroglucinol (PG). The transcription of the phlA gene was not affected, but the translation of the phlA and phlD genes was reduced significantly. Two components of the Gac/Rsm pathway, RsmA and RsmE, were found to be regulated by gidA and trmE, whereas the other components, RsmX, RsmY, and RsmZ, were not. The regulation of 2,4-DAPG production by gidA and trmE, however, was independent of the Gac/Rsm pathway. Both the gidA and trmE mutants were unable to produce PG but could convert PG to MAPG and MAPG to 2,4-DAPG. Overexpression of PhlD in the gidA and trmE mutants could restore the production of PG and 2,4-DAPG. Taken together, these findings suggest that GidA and TrmE are positive regulatory elements that influence the biosynthesis of 2,4-DAPG posttranscriptionally.

FIG 1

Effects of gidA and trmE mutations on 2,4-DAPG, MAPG, and PG production and on antifungal activity toward R. solani. (A and B) HPLC analysis of 2,4-DAPG (A) and MAPG (B) production by strain 2P24 and its derivatives in KB medium. (C) PG production of strains in LB medium supplemented with 2% glucose using a colorimetric method (17). Each value is the average of three independent cultures grown for 2 days, with the error bars denoting standard deviations (SD) and the asterisks indicating significantly different results (P < 0.001). (D) Measurement of antifungal activity in one-fifth-strength PDA medium, with R. solani as the target pathogen.

FIG 2

Regulation of antibiotic-biosynthetic genes phlA and phlD by GidA and TrmE. (A) The transcriptional levels of the phlA gene in P. fluorescens 2P24 and its variants were measured using the β-galactosidase assay. Growth is indicated by the dashed lines. (B) Levels of PhlA-VSV and PhlD-VSV proteins were evaluated by Western blotting using an anti-VSV antibody. An antibody directed against 3-phosphoglycerate kinase α (α-PGK) was used as a loading control. All experiments were performed in triplicate, with the means ± SD shown.

FIG 3

Regulation of the small noncoding RNA genes rsmX, rsmY, and rsmZ and their cognate regulator genes, rsmA and rsmE, by GidA and TrmE. (A to E) Transcriptional expression profiles of rsmX (A), rsmY (B), rsmZ (C), rsmA (D), and rsmE (E) were measured in P. fluorescens 2P24 and its gidA and trmE mutants. (F) Measurement of RsmA-VSV and RsmE-VSV proteins by Western blotting using an anti-VSV antibody. An anti-3-phosphoglycerate kinase α (α-PGK) antibody was used as a loading control. All experiments were performed in triplicate. The means ± SD are shown, and the asterisks indicate significantly different results (P < 0.001). Growth is indicated by the dashed lines.

FIG 4

Effects of GidA and TrmE on PhlA and PhlD functions. (A) Production of 2,4-DAPG in the absence and presence of 10 μg/ml MAPG. (B) Production of MAPG in the absence and presence of 10 μg/ml PG. (C) Production of PG was determined in strain 2P24 and its derivatives. 2,4-DAPG was added to a final concentration of 1 μg/ml. All experiments were repeated three times. The means ± SD are shown, and the asterisks indicate significantly different results (P < 0.001).

FIG 5

Overexpression of PhlD restores the wild-type phenotypes in gidA, trmE, and their double mutants. (A to C) 2,4-DAPG (A), MAPG (B), and PG (C) production was detected in P. fluorescens 2P24 and its derivatives containing the plasmid pBBR1MCS-5 or pBBR5-phlD. All experiments were repeated three times. The means ± SD are shown, and the asterisks indicate significantly different results (P < 0.001). (D) Western blot analysis of the amount of PhlD-VSV present in the indicated strains using an anti-VSV antibody. The numbers 1 to 8 represent strains PM111(pBBR1MCS-5), PM111(pBBR5-phlDV), PM112(pBBR1MCS-5), PM112(pBBR5-phlDV), PM113(pBBR1MCS-5), PM113(pBBR5-phlDV), PM114(pBBR1MCS-5), and PM114(pBBR5-phlDV), respectively. The blots were incubated with anti-3-phosphoglycerate kinase α (α-PGK) antibody as a loading control.