Identification and characterization of a gene cluster for synthesis of the polyketide antibiotic 2,4-diacetylphloroglucinol from Pseudomonas fluorescens Q2-87

Abstract

The polyketide metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) is produced by many strains of fluorescent Pseudomonas spp. with biocontrol activity against soilborne fungal plant pathogens. Genes required for 2,4-DAPG synthesis by P. fluorescens Q2-87 are encoded by a 6.5-kb fragment of genomic DNA that can transfer production of 2,4-DAPG to 2,4-DAPG-nonproducing recipient Pseudomonas strains. In this study the nucleotide sequence was determined for the 6.5-kb fragment and flanking regions of genomic DNA from strain Q2-87. Six open reading frames were identified, four of which (phlACBD) comprise an operon that includes a set of three genes (phlACB) conserved between eubacteria and archaebacteria and a gene (phlD) encoding a polyketide synthase with homology to chalcone and stilbene synthases from plants. The biosynthetic operon is flanked on either side by phlE and phlF, which code respectively for putative efflux and regulatory (repressor) proteins. Expression in Escherichia coli of phlA, phlC, phlB, and phlD, individually or in combination, identified a novel polyketide biosynthetic pathway in which PhlD is responsible for the production of monoacetylphloroglucinol (MAPG). PhlA, PhlC, and PhlB are necessary to convert MAPG to 2,4-DAPG, and they also may function in the synthesis of MAPG.

FIG. 1

Genes identified in the 2,4-DAPG biosynthetic locus are indicated by black horizontal arrows. The vertical arrow indicates the site of Tn5 insertion in Q2-87::Tn5-1. Key plasmids used in assigning functions to the phl genes are shown. Construction of these plasmids is described in Materials and Methods. Restriction sites: A, AccI, B, BamHI; E, EcoRV; Eg, EagI; P, PstI; R, EcoRI; S, SalI; Sp, SphI; St, StuI.

FIG. 2

Important residues conserved in PhlC. (A) Alignment of cysteine 88 (∗) of PhlC with the catalytic active site cysteine of thiolases. (B) Alignment of histidine 349 (▾) and neighboring glycine residues of PhlC. Cysteine 378 (●), a key active site residue in type II thiolases, is absent from PhlC, SCPx, and AcaB. In both panels, the compared proteins are chrom_aat, Chromatium vinosum acetoacetyl-CoA thiolase (accession. no. L01112); alcal_aat, Alcaligenes eutrophus acetoacetyl-CoA thiolase (accession no. J04987); ratmit_aat, rat mitochondrial acetoacetyl-CoA thiolase (accession no. D13921); ratmit_thiol, rat mitochondrial oxoacyl-CoA thiolase (accession no. X05341); rat_pota, rat peroxisomal oxoacyl-CoA thiolase A (accession no. M32801); rat_potb, rat peroxisomal oxoacyl-CoA thiolase B (accession no. J02749); ecfada, E. coli 3-ketoacyl-CoA thiolase (accession no. P21151); caen_aat, Caenorhabditis elegans acetyl-CoA acetyltransferase (accession no. M77697); scpx, mouse sterol carrier protein 2 precursor (accession no. P32020); and acab, P. furiosus acetyl-CoA synthase. Numbers to the right of the alignment are amino acid residue numbers.

FIG. 3

Kyte-Doolittle hydropathy plots of PhlE and S. aureus NorA protein. Both plots show central hydrophilic loops flanked by twelve hydrophobic, potential membrane spanning regions (window size = 19).

FIG. 4

Northern blot analysis of phl mRNA. Total RNA was isolated at 3 h (lanes 1 and 3) and 24 h (lanes 2 and 4) from P. fluorescens Q69c-80 (lanes 1), P. fluorescens Q69c-80::miniTn5Phl (lanes 2), and P. fluorescens Q2-87 (lanes 3 and 4). The probes used were a 0.7-kb EcoRV fragment specific for phlC (A) and a 1.1-kb AccI-BamHI fragment containing the entire phlD region (B). Positions of the 23S and 16S RNAs are shown. ∗, transcript detected from wild-type Q2-87 strains; ∗∗, smaller transcript detected in Q69c-80::miniTn5Phl.

FIG. 5

Autoradiograph of polypeptides labeled with [³⁵S]methionine and resolved by electrophoresis in SDS-polyacrylamide gels. Samples were prepared from E. coli BL21(DE3) containing the indicated plasmids. The DNA fragments inserted in pT7-5 for gene expression are illustrated in Fig. 1. Top panel, 10% acrylamide gel; bottom panel, 12% acrylamide gel. Positions of phl gene products are indicated on the right; D∗ indicates the truncated PhlD product produced by strains expressing pPHL5141 and its derivatives. Positions of molecular size markers are given on the left in kilodaltons. Lanes: 1, pT7-5; 2, pPHL5140; 3, pPHL5141; 4, pPHL5142A⁻; 5, pPHL5142B⁻; 6, pPHL5142C⁻.

FIG. 6

Proposed roles of PhlA, PhlB, PhlC, and PhlD in the biosynthesis of MAPG and 2,4-DAPG. Enz, enzyme.