Autoinduction of 2,4-diacetylphloroglucinol biosynthesis in the biocontrol agent Pseudomonas fluorescens CHA0 and repression by the bacterial metabolites salicylate and pyoluteorin.
The antimicrobial metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) contributes to the capacity of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soilborne pathogens. A 2, 4-DAPG-negative Tn5 insertion mutant of strain CHA0 was isolated, and the nucleotide sequence of the 4-kb genomic DNA region adjacent to the Tn5 insertion site was determined. Four open reading frames were identified, two of which were homologous to phlA, the first gene of the 2,4-DAPG biosynthetic operon, and to the phlF gene encoding a pathway-specific transcriptional repressor. The Tn5 insertion was located in an open reading frame, tentatively named phlH, which is not related to known phl genes. In wild-type CHA0, 2, 4-DAPG production paralleled expression of a phlA'-'lacZ translational fusion, reaching a maximum in the late exponential growth phase. Thereafter, the compound appeared to be degraded to monoacetylphloroglucinol by the bacterium. 2,4-DAPG was identified as the active compound in extracts from culture supernatants of strain CHA0 specifically inducing phlA'-'lacZ expression about sixfold during exponential growth. Induction by exogenous 2,4-DAPG was most conspicuous in a phlA mutant, which was unable to produce 2, 4-DAPG. In a phlF mutant, 2,4-DAPG production was enhanced severalfold and phlA'-'lacZ was expressed at a level corresponding to that in the wild type with 2,4-DAPG added. The phlF mutant was insensitive to 2,4-DAPG addition. A transcriptional phlA-lacZ fusion was used to demonstrate that the repressor PhlF acts at the level of transcription. Expression of phlA'-'lacZ and 2,4-DAPG synthesis in strain CHA0 was strongly repressed by the bacterial extracellular metabolites salicylate and pyoluteorin as well as by fusaric acid, a toxin produced by the pythopathogenic fungus Fusarium. In the phlF mutant, these compounds did not affect phlA'-'lacZ expression and 2, 4-DAPG production. PhlF-mediated induction by 2,4-DAPG and repression by salicylate of phlA'-'lacZ expression was confirmed by using Escherichia coli as a heterologous host. In conclusion, our results show that autoinduction of 2,4-DAPG biosynthesis can be countered by certain bacterial (and fungal) metabolites. This mechanism, which depends on phlF function, may help P. fluorescens to produce homeostatically balanced amounts of extracellular metabolites.
Schnider-Keel
U
U
Laboratoire de Biologie Microbienne, Université de Lausanne, CH-1015 Lausanne, Switzerland.
Seematter
A
A
Maurhofer
M
M
Blumer
C
C
Duffy
B
B
Gigot-Bonnefoy
C
C
Reimmann
C
C
Notz
R
R
Défago
G
G
Haas
D
D
Keel
C
C
D000900:Anti-Bacterial Agents / Q000378:metabolism* / Q000494:pharmacology; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005659:Fungicides, Industrial / Q000378:metabolism; D005669:Fusaric Acid / Q000494:pharmacology; D005670:Fusarium / Q000737:chemistry / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005798:Genes, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010636:Phenols; D010696:Phloroglucinol / Q000031:analogs & derivatives / Q000378:metabolism; D010935:Plant Diseases / Q000382:microbiology; D011551:Pseudomonas fluorescens / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011758:Pyrroles; D012459:Salicylates / Q000378:metabolism* / Q000494:pharmacology; D017422:Sequence Analysis, DNA
D000900:Anti-Bacterial Agents; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D005659:Fungicides, Industrial; D010636:Phenols; D011758:Pyrroles; D012459:Salicylates; C095503:pyoluteorin; C059817:2,4-diacetylphloroglucinol; D010696:Phloroglucinol; D005669:Fusaric Acid