Characterization of a genomic locus required for synthesis of the antibiotic 2,4-diacetylphloroglucinol by the biological control agent Pseudomonas fluorescens Q2-87.

The antibiotic 2,4-diacetylphloroglucinol (Phl) is an important factor in the biological control by fluorescent Pseudomonas spp. of many soilborne diseases including take-all disease of wheat. A 6.5-kb genomic DNA fragment from Pseudomonas fluorescens Q2-87 conferred production of Phl and of a red pigment distinct from Phl, but which typically is present when Phl is produced, upon all of 13 Phl-nonproducing recipient Pseudomonas strains into which it was introduced. Larger fragments that included flanking DNA sequences did not transfer this capability, suggesting that they contain negative regulatory element(s). Analysis of the 6.5-kb fragment by Tn3HoHo1 mutagenesis further localized the sequences required for Phl production to a segment of approximately 5 kb and revealed the presence of at least two divergently oriented transcriptional units. Insertions within the smaller unit or within about 3 kb of the 5' end of the larger unit caused loss of production of both Phl and the red pigment. Other insertions within the distal 1.5 kb of the larger transcriptional unit abolished production of only the red pigment. Pleiotropic changes in secondary metabolism or colony morphology were not observed in Pseudomonas strains containing the 6.5-kb fragment, although some Phl-producing derivatives grew more slowly and gave rise to smaller colonies than did the wild-type parental strains. The size of the genomic region involved in Phl production, and the consistency and specificity with which these sequences transferred Phl biosynthetic capability, support the conclusion that the 6.5-kb fragment contains the Phl biosynthetic locus.

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

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.

beta-Ketoacyl-[acyl carrier protein] synthase I of Escherichia coli: aspects of the condensation mechanism revealed by analyses of mutations in the active site pocket.

beta-Ketoacyl-[acyl carrier protein (ACP)] synthase forms new carbon-carbon bonds in three steps: transfer of an acyl primer from ACP to the enzyme, decarboxylation of the elongating substrate and its condensation with the acyl primer substrate. Six residues of Escherichia coli beta-ketoacyl-ACP synthase I (KAS I) implicated in these reactions were subjected to site-directed mutagenesis. Analyses of the abilities of C163A, C163S, H298A, D306A, E309A, K328A, and H333A to carry out the three reactions lead to the following conclusions. The active site Cys-163 is not required for decarboxylation, whereas His-298 and His-333 are indispensable. Neither of the histidines is essential for increasing the nucleophilicity of Cys-163 to enable transfer of the acyl primer substrate. Maintenance of the structural integrity of the active site by Asp-306 and Glu-309 is required for decarboxylation but not for transfer. One function of Lys-328 occurs very early in catalysis, potentially before transfer. These results in conjunction with structural analyses of substrate complexes have led to a model for KAS I catalysis [Olsen, J. G., Kadziola, A., von Wettstein-Knowles, P., Siggaard-Andersen, M., and Larsen, S. (2001) Structure 9, 233-243]. Another facet of catalysis revealed by the mutant analyses is that the acyl primer transfer activity of beta-ketoacyl-ACP synthase I is inhibited by free ACP at physiological concentrations. Differences in the inhibitory response by individual mutant proteins indicate that interaction of free ACP with Cys-163, Asp-306, Glu-309, Lys-328, and His-333 might form a sensitive regulatory mechanism for the transfer of acyl primers.

Gene expression in Pseudomonas aeruginosa biofilms.

Bacteria often adopt a sessile biofilm lifestyle that is resistant to antimicrobial treatment. Opportunistic pathogenic bacteria like Pseudomonas aeruginosa can develop persistent infections. To gain insights into the differences between free-living P. aeruginosa cells and those in biofilms, and into the mechanisms underlying the resistance of biofilms to antibiotics, we used DNA microarrays. Here we show that, despite the striking differences in lifestyles, only about 1% of genes showed differential expression in the two growth modes; about 0.5% of genes were activated and about 0.5% were repressed in biofilms. Some of the regulated genes are known to affect antibiotic sensitivity of free-living P. aeruginosa. Exposure of biofilms to high levels of the antibiotic tobramycin caused differential expression of 20 genes. We propose that this response is critical for the development of biofilm resistance to tobramycin. Our results show that gene expression in biofilm cells is similar to that in free-living cells but there are a small number of significant differences. Our identification of biofilm-regulated genes points to mechanisms of biofilm resistance to antibiotics.

Interaction of pseudomonas aeruginosa with epithelial cells: identification of differentially regulated genes by expression microarray analysis of human cDNAs.

Pseudomonas aeruginosa is an opportunistic pathogen that plays a major role in lung function deterioration in cystic fibrosis patients. To identify critical host responses during infection, we have used high-density DNA microarrays, consisting of 1,506 human cDNA clones, to monitor gene expression in the A549 lung pneumocyte cell line during exposure to P. aeruginosa. We have identified host genes that are differentially expressed upon infection, several of which require interaction with P. aeruginosa and the expression of the major subunit of type IV pili, PilA. Differential expression of genes involved in various cellular functions was identified, and we selected the gene encoding the transcription factor interferon regulatory factor 1 (IRF-1) for further analysis. The levels of the IRF-1 transcript increased 3- to 4-fold in A549 cells after adherence by P. aeruginosa. A similar increase of IRF-1 mRNA was observed in A549 cells exposed to wild-type P. aeruginosa when compared to an isogenic, nonpiliated strain. However, this difference was abolished when serum was present during the incubation of bacteria. Exposure of A549 cells to purified P. aeruginosa lipopolysaccharide did not result in a significant increase in IRF-1 mRNA. Although the P. aeruginosa-induced increased IRF-1 expression depends on the presence of bacterial adhesin, our findings do not preclude the possibility that other bacterial products are responsible for IRF-1 activation, which is enhanced by bacterial adherence to cells. These data show that microarray technology can be an important tool for studying the complex interplay between bacterial pathogens and host.