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J Bacteriol. Apr 1999; 181(7): 2166–2174.
PMCID: PMC93630

Characterization of the Pyoluteorin Biosynthetic Gene Cluster of Pseudomonas fluorescens Pf-5

Abstract

Ten genes (plt) required for the biosynthesis of pyoluteorin, an antifungal compound composed of a bichlorinated pyrrole linked to a resorcinol moiety, were identified within a 24-kb genomic region of Pseudomonas fluorescens Pf-5. The deduced amino acid sequences of eight plt genes were similar to the amino acid sequences of genes with known biosynthetic functions, including type I polyketide synthases (pltB, pltC), an acyl coenzyme A (acyl-CoA) dehydrogenase (pltE), an acyl-CoA synthetase (pltF), a thioesterase (pltG), and three halogenases (pltA, pltD, and pltM). Insertions of the transposon Tn5 or Tn3-nice or a kanamycin resistance gene in each of these genes abolished pyoluteorin production by Pf-5. The presumed functions of the eight plt products are consistent with biochemical transformations involved in pyoluteorin biosynthesis from proline and acetate precursors. Isotope labeling studies demonstrated that proline is the primary precursor to the dichloropyrrole moiety of pyoluteorin. The deduced amino acid sequence of the product of another plt gene, pltR, is similar to those of members of the LysR family of transcriptional activators. pltR and pltM are transcribed divergently from the pltLABCDEFG gene cluster, and a sequence with the characteristics of a LysR binding site was identified within the 486-bp intergenic region separating pltRM from pltLABCDEFG. Transcription of the pyoluteorin biosynthesis genes pltB, pltE, and pltF, assessed with transcriptional fusions to an ice nucleation reporter gene, was significantly greater in Pf-5 than in a pltR mutant of Pf-5. Therefore, PltR is proposed to be a transcriptional activator of linked pyoluteorin biosynthesis genes.

Certain strains of Pseudomonas fluorescens produce secondary metabolites that are toxic to plant-pathogenic fungi. It is not surprising, therefore, that the production of antifungal compounds enhances the ability of these bacteria to suppress a variety of plant diseases and in some instances contributes to the ecological competence of the producing strain within the rhizosphere (16, 54). The mechanisms that regulate antifungal metabolite production in P. fluorescens include global regulatory factors that simultaneously affect multiple biosynthetic pathways (9, 18, 31, 46) and pathway-specific regulators that control linked biosynthetic genes (4, 41).

Pyoluteorin is an antibiotic that inhibits Oomycete fungi, including the plant pathogen Pythium ultimum, and suppresses plant diseases caused by this fungus (25). Pyoluteorin is composed of a resorcinol ring, derived through polyketide biosynthesis (10, 40), which is linked to a bichlorinated pyrrole moiety whose biosynthesis remains uncharacterized. Because halogenation can increase the pharmacological effects of many compounds (38), considerable effort has been directed toward the isolation and characterization of haloperoxidases, enzymes that are capable of forming carbon-halogen bonds in the presence of halide ions and hydrogen peroxide (56). It has yet to be demonstrated, however, that any of the haloperoxidases thus far characterized are responsible for the in vivo halogenation of known natural products. Like halogenation, little is known about pyrrole formation in secondary metabolic pathways. Whereas the pyrrole ring within porphobilinogen, a precursor to heme ring systems, originates from 5-aminolevulinic acid (26), the specific incorporation of [1,2-13C2]acetate into the pyoluteorin dichloropyrrole moiety indicates that this functionality is derived directly from a tricarboxylic acid cycle intermediate (10). Therefore, in addition to its ecological importance, pyoluteorin provides an opportunity to understand novel biochemical transformations involved in chlorinated pyrrole synthesis.

A genomic region of P. fluorescens Pf-5 that is required for pyoluteorin biosynthesis was identified previously by Tn5 mutagenesis and cosmid cloning (30). We recently described the DNA sequence analysis of two open reading frames (ORFs) (pltB and pltC) within this genomic region that encode a type I polyketide synthase (40). PltB and PltC contain several discrete functional domains similar to those required for polyketide and fatty acid biosynthesis. These domains are organized into three distinct modules, two in PltB and one in PltC, that are thought to catalyze the formation of the resorcinol ring within pyoluteorin. By analogy to other type I polyketide synthases, each module is likely to incorporate and modify a single malonate extender unit into the resorcinol moiety of pyoluteorin. The pyoluteorin polyketide synthase is unusual, however, because it does not possess either a loading module or a thioesterase domain responsible for the initiation and the termination, respectively, of polyketide biosynthesis. This paper describes the further characterization of the pyoluteorin biosynthesis region. We report the nucleotide sequence of an additional eight ORFs within the pyoluteorin gene cluster, including seven putative biosynthetic loci and a regulatory gene encoding a transcriptional activator of linked pyoluteorin biosynthesis genes. In addition, we describe isotopic labeling studies that unequivocally demonstrate that proline is the primary precursor to the dichloropyrrole moiety of pyoluteorin. Although pathway intermediates have yet to be identified, the predicted functions of proteins encoded by pyoluteorin biosynthetic genes are consistent with a model specifying the biochemical transformations required for pyoluteorin biosynthesis.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.

The bacterial strains and plasmids used in this study are listed in Table Table1.1. Escherichia coli was cultured in Luria-Bertani medium (44) at 37°C. P. fluorescens was cultured routinely in King’s medium B (28) at 27°C. Antibiotic concentrations were as follows unless otherwise specified: 100 μg of ampicillin per ml, 10 μg gentamicin per ml, 50 μg of kanamycin per ml, and 20 μg of tetracycline per ml.

TABLE 1
Strains and plasmids used in this study

DNA manipulations.

Plasmids were isolated from E. coli and P. fluorescens by an alkali lysis method (44). Genomic DNA was isolated by a cetyltrimethylammonium bromide (CTAB) method with isopropanol precipitation (3). Electrophoresis was done in 0.7% (wt/vol) agarose gels with Tris-phosphate-EDTA buffer (44). Restriction digestions and ligation procedures were performed by standard methods (44).

Nucleotide sequence analysis.

Sequence analysis was done on restriction fragments from pJEL1938 and pJEL1939, two members of a P. fluorescens Pf-5 genomic library that hybridized to DNA flanking Tn5 insertions of Plt mutants (30) (Fig. (Fig.1).1). Restriction fragments were cloned into pUC19, and sequencing was initiated from primers complementary to pUC19 and subsequently completed by “primer walking” in each direction across the length of the plasmid insert. Automated DNA sequence analysis and primer synthesis were performed by the Center for Gene Research Central Services Laboratory, Oregon State University, and by Macromolecular Resources Sequi-net Division, Colorado State University, by using dideoxynucleotide chain termination (45) on Applied Biosystems model 373A and 377 sequencers. Compilation, manual editing, and analysis of the sequence data were done with the University of Wisconsin Genetics Computer Group programs (19). Open reading frames were identified within the DNA sequence by codon usage analysis with the codon preference frequencies compiled for P. aeruginosa (60). The precise locations of Tn5 and Tn3-nice insertions in the plt region (30) were determined from restriction fragments containing the transposon and flanking DNA of each Plt mutant. Restriction fragments were cloned and DNA flanking transposon insertions was sequenced from primers complementary to a terminal region of Tn5 (5′-GGTTCCGTTCAGGACGCTAC-3′) or Tn3-nice (5′-AGACCATTAAAAGAGGCGTCAGAG-3′).

FIG. 1
Pyoluteorin biosynthesis gene cluster of P. fluorescens Pf-5. ORFs (pltA-G, pltR, and pltM) identified in the gene cluster are indicated by large arrows. An external file that holds a picture, illustration, etc.
Object name is jb0791464gr1.gif, Tn5 insertion site. An external file that holds a picture, illustration, etc.
Object name is jb0791464gr2.gifand An external file that holds a picture, illustration, etc.
Object name is jb0791464gr3.gif, Tn3-nice insertion site (the flag indicates the direction of transcription ...

Deduced peptide sequence analysis.

Deduced protein sequences encoded by ORFs in the pyoluteorin gene cluster were compared to those in databases available through the National Center for Biotechnology Information (U.S. National Institutes of Health, Bethesda, Md.) with the BLAST algorithm and standard parameters of Altschul et al. (2). Functional domains and catalytic residues within the pyoluteorin biosynthetic enzymes were identified from protein sequence alignments performed with PILEUP (GCG version 8.0), using standard parameters.

PltA, PltD, and PltM were aligned with Cts4 of Streptomyces aureofaciens (11) and PrnC of P. fluorescens (20). The 5′ regions of pltA, pltD, and pltM were similar to nucleotides upstream of the assigned translational start site of cts4, and codon preference analysis of the chlortetracycline biosynthesis gene cluster suggested that the translational start site of cts4 was likely to be located 306 nucleotides (nt) upstream of that assigned (39). Sequence alignments to pltA, pltD, pltM (39), and prnC (20) indicated that the cts4 sequence contained a frame shift error between bases 2856 and 2859. Therefore, an additional 102 amino acid (aa) residues encoded by nucleotides 2634 to 2858 and 2860 to 2940 of the reported cts4 locus were added to the N terminus of Cts4 for our analysis.

Domains within PltR were identified by comparison with a LysR profile sequence by using standard parameters for the ProfileGap algorithm. The LysR profile was compiled by using ProfileMake (GCG version 8.0) from the following representative LysR-type regulators: PtxR (GenBank accession no. U35068) and TrpI (X51868) of P. aeruginosa, TcbR of an unidentified Pseudomonas species (M80212), RcbR of Chromatium vinosum (M64032), NahR (J04233) and CatR (U12557) of P. putida, LysR (J01614), LeuO (J03862), and IlvY (M14492) of E. coli, and GltC of Bacillus subtilis (M28509). The presence of a helix-turn-helix motif was confirmed with the program Helix-turn-helix version 1.0.5 (21a). The putative PltR-binding promoter sequence was identified manually by using the search routine in the sequence editor SeqEd (GCG version 8.0).

Insertional inactivation of pltM and pltR.

The 2-kb BamHI fragment containing aacC1, which confers gentamicin resistance, was isolated from pMGm and inserted into the EcoRI site internal to pltR, which had been cloned previously into the vector pUC18ΔEcoRI as a 2.2-kb HindIII-KpnI fragment. The 4.4-kb HindIII-KpnI fragment containing pltR::aacC1 was then cloned into pRK415 to construct pJEL6051.

pltM was mutagenized by insertion of an aphI cassette from pUC4K, which causes nonpolar mutations in P. fluorescens (29). The 2.7-kb EcoRI fragment containing pltM was cloned from the cosmid pJEL1939 into pUC19. The 1.2-kb AccI fragment containing aphI was isolated from pUC4K and cloned into the NspV site internal to pltM. The 4.5-kb EcoRI fragment containing pltM::aphI was then cloned into the suicide vector pME3087 to construct pJEL6049.

The pltR::aacC1 and pltM::aphI fusions were introduced into the genome of Pf-5 or derivatives by marker exchange mutagenesis, exploiting the instability of pRK415 or the ColE1 replicon of pME3087, which does not replicate in Pseudomonas spp. pJEL6049 and pJEL6051 were mobilized from E. coli S17-1 to Pf-5 in conjugation experiments done as described previously (30), selecting for colonies that were resistant to ampicillin, to counterselect against the E. coli donor, and either kanamycin or 40 μg of gentamicin per ml, to select for mutants containing genomic integrations. Selected colonies were scored for growth on King’s medium B containing 200 μg of tetracycline per ml, to determine if the plasmid vector was still present in the cell. Putative pltR::aacCI mutants (i.e., gentamicin-resistant, tetracycline-sensitive colonies selected following the introduction of pJEL6051 into Pf-5, JL4365, JL4389, or JL4390) were confirmed as such by Southern blot analysis. Putative pltM::aphI mutants (i.e., kanamycin-resistant, tetracycline-sensitive colonies selected following the introduction of pJEL6049 into Pf-5) were confirmed as such by PCR with primers flanking the NspV site internal to pltM.

Transcription of plt genes assessed with an ice nucleation reporter gene.

The transposon Tn3-nice contains a promoterless inaZ gene that, when inserted into a gene in the appropriate orientation, generates a transcriptional fusion that confers ice nucleation activity (INA) on its bacterial host (30, 33). The effect of pltR on the transcription of the pyoluteorin biosynthesis genes was determined by comparing INA expressed by derivatives of Pf-5 containing insertions of Tn3-nice in genomic plt genes (JL4365, JL4389, and JL4390) (Fig. (Fig.1)1) to INA expressed by near-isogenic strains with pltR::aacCI mutations (JL4564, JL4565, and JL4566, respectively). INA was quantified by a droplet-freezing assay at −5°C as described previously (34) from cultures grown for 2 days with shaking at 20°C in nutrient broth (Difco Laboratories, Detroit, Mich.) amended with 2% glycerol. Data were analyzed with the general linear models program of Statistical Analysis Systems (SAS Institute Inc., Cary, N.C.). Treatments were replicated three times, the experiment was repeated, and the results of a representative experiment are presented.

Assessment of pyoluteorin production.

Triplicate cultures of Pf-5 and derivative strains were shaken for 2 days at 20°C in nutrient broth amended with 2% glycerol. Pyoluteorin was extracted from culture supernatants and analyzed by high-pressure liquid chromatography (Waters Nova-Pak C18 reversed-phase column eluted with acetonitrile-methanol-water [30:25:45] at a flow rate of 1.5 ml/min, and photodiode array detection), as described previously (46). Pyoluteorin was detected by UV spectroscopy (λ = 310 nm, retention time of 3.4 min) and quantified with authentic pyoluteorin as a standard. Authentic pyoluteorin was purified from culture supernatants of Pf-5 and structurally characterized by nuclear magnetic resonance spectroscopy (NMR) as described below. The detection limit was 0.02 mg per liter of culture.

Isolation and isotopic labeling of pyoluteorin.

For the biosynthetic studies, P. fluorescens Pf-5 was grown in a modified King’s medium B composed of 2.0% phytone peptone (Becton-Dickinson, Cockeysville, Md.), 0.5% (wt/vol) glycerol, 0.15% K2HPO4, and 0.15% MgSO4 · 7H2O adjusted to pH 7.0 to 7.2. All cultures were grown at 20°C with stirring at 150 rpm. A 1-ml volume of a 24-h seed culture (40 ml in a 250-ml Erlenmeyer flask) was used to inoculate 100-ml cultures in 1-liter Erylenmeyer flasks.

Culture supernatants were extracted three times with 1/10 volume of ethyl acetate, and the combined organic phases were back-extracted three times with water (1/20 volume). The organic phase was dried over anhydrous MgSO4, and the solvent was removed by rotary evaporation. The crude extract was fractionated by flash chromatography on silica gel (Silica Gel 60, 40 to 63 μm; EM Science, Gibbstown, N.J.) equilibrated and eluted with either 3:1 toluene-acetone or 4:1 CHCl3-acetone. Pyoluteorin was detected as an orange-brown diazosulfanilic acid derivative when the column fractions were analyzed on thin-layer chromatography plates sprayed with a 1:2:3 mixture of 5% NaNO2 and 0.9% sulfanilate in 1 M HCl–20% K2CO3 (43). Pyoluteorin was further purified from the combined fractions by recrystallization from hot CHCl3.

The isolated pyoluteorin was identical to an authentic sample and exhibited the following 1H and 13C NMR resonances. 1H NMR (300 MHz; d6-acetone) δ 9.02 (br s, exchangeable), 7.15 (t, 1H, J = 8.2 Hz), 6.80 (s, 1H), 6.47 (d, 2H, J = 8.2 Hz), and 3.00 (br s, exchangeable). 13C NMR (75 MHz; d6-acetone) δ 183.1, 157.2, 132.5, 130.9, 119.5, 117.3, 113.0, 110.5, and 107.5.

Incorporation of proline into pyoluteorin was demonstrated initially by the following radioisotope feeding study. Equal portions of an aqueous solution containing 9.99 μCi (2.20 × 107 dpm) of l-[U-14C]proline (ICN, Irvine, Calif.) were aseptically transferred to two 1-liter flasks, each containing 100 ml of 16-h-old cultures of the pyoluteorin-overproducing mutant JL4239. At 28 h later, the cultures were combined, the cells were removed by centrifugation, and pyoluteorin was extracted from the culture supernatant. A sample of unlabeled pyoluteorin (20 mg) was added to the crude extract, and pyoluteorin was recovered from the combined samples (yield, 56 mg or 207 μmol). The sample then was recrystallized to a constant specific radioactivity.

To demonstrate the specific incorporation of proline into pyoluteorin, equal portions of an aqueous solution containing 16 mg of l-[1-13C]proline (CIL, Cambridge, Mass.) were added to each of 10 1-liter flasks containing 100-ml cultures of Pf-5, 13 h after inoculation. The cultures were grown until 48 h after inoculation, at which point the cells were removed from the combined cultures and 47 mg (173 μmol) of pyoluteorin was isolated from the culture supernatant. Isotopic enrichment was determined by comparing the peak height observed for the carbonyl resonance (75 MHz, d6-acetone; δ 183.0) in the 13C NMR spectra of the enriched sample with the peak height of the same resonance observed in an unenriched sample.

Nucleotide sequence accession number.

The accession number of the nucleotide sequence of the pyoluteorin gene cluster from Pf-5 is AF081920.

RESULTS

Identification of coding regions within the pyoluteorin biosynthetic gene cluster.

We detected 10 ORFs within the pyoluteorin biosynthesis gene cluster of P. fluorescens Pf-5 (Fig. (Fig.1).1). Except for a 486-bp gap between the divergent coding regions of pltL and pltR, contiguous plt genes are separated by less than 50 bp. Putative ribosomal binding sites were identified for pltLABCDEFG and pltR but not for pltM. We presume that pltM and pltR are translationally coupled, because no ribosomal binding site could be identified for pltM and the ATG initiation codon of pltM overlaps the 3′ end of pltR by 4 bp. Whereas other genes within the pyoluteorin cluster exhibit a G+C bias of the third codon position in the range of 75 to 85%, the G+C bias of the first 500 nt of the pltR coding region is approximately 35 to 45%. Consequently, this region has a higher frequency of rare codons than do the other genes within the pyoluteorin gene cluster. An alternative start codon and ribosomal binding site for PltR exist approximately 60 nt upstream of the identified translation initiation site, but it is unlikely that this is the site of translation initiation, because of the presence of an in-frame stop codon 12 nt downstream. No alternative start sites are present within 100 nt downstream of the identified PltR initiation site.

Deduced protein sequences of pyoluteorin biosynthesis genes.

Putative functions for each of the translated proteins encoded within the pyoluteorin gene cluster initially were assigned from database search results. Subsequently, motifs within the deduced amino acid sequence of each ORF (except pltE and pltL) were identified by sequence alignments with proteins of known function.

(i) PltA, PltD, PltM.

The deduced amino acid sequences of PltA, PltD, and PltM are similar to the halogenating enzymes required for chlortetracycline biosynthesis by Streptomyces aureofaciens (Cts4) (11) and for pyrrolnitrin biosynthesis by P. fluorescens BL915 (PrnC) (20). Protein sequence alignment detected considerable similarity among the N-terminal and central regions for each of these five halogenases (Fig. (Fig.2).2). PltA and PltM contain the characteristic motif sequence GxGx2(G/A)x3(G/A)x6G, which is believed to form the β-α-β structure required for NADH cofactor binding (49), whereas PltD does not contain this sequence motif.

FIG. 2
Multiple sequence alignment for PltA, PltD, PltM, Cts4, and PrnC. Residues in capital letters signify conserved substitutions. Invariant residues are indicated in bold capital letters. The consensus sequence was derived by using a plurality of four. Residues ...

(ii) PltE.

The deduced amino acid sequence of PltE is similar to many flavin-dependent acyl coenzyme A (acyl-CoA) dehydrogenases, which catalyze the α,β-dehydrogenation of acyl-CoA thioesters involved in fatty acid and amino acid degradation. A butyryl-CoA dehydrogenase of Megasphaera elsdenii, whose three-dimensional structure has been determined (14), is the most thoroughly characterized acyl-CoA dehydrogenase to which PltE has sequence similarity. Sequence comparison of PltE to the butyryl-CoA dehydrogenase from M. elsdenii identified a 291-aa sequence overlap that contains 95 identical (32%) and 160 similar (54%) amino acid residues. The identified active-site Glu residue of the M. elsdenii butyryl-CoA dehydrogenase, located 17 residues from the C terminus, is conspicuously absent from PltE. Instead, PltE possesses a Glu residue at aa 243 that aligns with the catalytic Glu residues identified within the human isovaleryl-CoA (35) and long-chain acyl-CoA (13) dehydrogenases. It is thought that the location of this Glu residue determines, in part, the substrate specificity of these enzymes (32, 37). Therefore, it is possible that the identified Glu residue within PltE may function as a catalytic base. A Ser residue (shown in bold type) at position 132 in PltE lies within the consensus sequence TEPxAGSD, which is conserved in several short- and medium-chain CoA dehydrogenases. The X-ray crystal structure of the M. elsdenii butyryl-CoA dehydrogenase shows that this Ser residue forms a hydrogen bond to the pantetheine moiety of the substrate (14). Moreover, the Thr residue within this same consensus sequence is involved in binding of the flavin adenine dinucleotide cofactor required for catalytic function. Therefore, it is very likely that PltE catalyzes a dehydrogenation within the pyoluteorin pathway. PltE also has 175 identical (46%) and 227 similar (60%) amino acid residues of a total of 377 aa residues within three identified high-scoring segment pairs of RedW from Streptomyces coelicolor (AL021530), an acyl-CoA dehydrogenase involved in the biosynthesis of the proline-derived secondary metabolite undecylprodigiosin (5).

(iii) PltF.

The deduced protein sequence of PltF is similar to several peptide synthetases including GrsB from Bacillus brevis (X61658), SnbC from Streptomyces pristinaespiralis (X98690), and PvdD from P. aeruginosa (U07359). PltF also contains core sequence motifs present in all adenylate-forming enzymes. Within PltF, the core C, D, and G sequences possess the most striking identity to conserved sequence motifs found within the peptide synthetases (Fig. (Fig.3)3) (42). Core sequences C and G are involved in adenylate formation and bind AMP and ATP, respectively, whereas core sequence F is an ATPase motif that binds ATP (42). A single Gly residue that is essential for amino acid activation by the gramicidin S synthase and that is invariant among other amino acid-adenylating enzymes (55) is also present within the core G sequence of PltF. PltF does not contain the core sequence motif involved in 4′-phosphopantetheine cofactor binding, whose absence is a defining characteristic for coumarate-CoA ligases and acetyl-CoA synthetases (12, 42). Nevertheless, the region of PltF surrounding core sequences E (involved in adenine binding [42]) and G appeared to be more highly conserved among the peptide synthetases than among the CoA synthetases or ligases (data not shown). In addition, PltF contains core sequence H (involved in adenine binding [42]), which is not found in the coumarate-CoA ligase and acetyl-CoA synthetase subfamily of adenylating enzymes (12).

FIG. 3
Organization and sequence motifs identified within PltF. Shaded areas represent the locations of regions present within peptide synthetases that are involved in substrate adenylation. A consensus sequence for each of the regions is aligned above the corresponding ...

(iv) PltG.

The deduced protein sequence of PltG is similar to several thioesterases involved in secondary-metabolite production. PltG contains the motif GxSxG, which is found approximately 100 aa residues from the N terminus of all known thioesterases, and a second diagnostic motif, GxH, near the carboxy terminus. These motifs contain the catalytic Ser and His residues, respectively, that are essential for thioesterase activity (52, 61). In PltG, these motifs are separated by 131 aa residues, which is similar to the spatial arrangement observed in known thioesterases. Consensus sequences of amino acid residues surrounding the GxSxG and GxH motifs within rat thioesterase II (Y00311), GrsT of B. brevis (X15577), CmaT of P. syringae (B55543), and the thioesterase domain of the chicken fatty acid synthase (J04485) were identified as F1xGHSF2GAxIA and PGxHFF, respectively. PltG differs from the former motif by F2-to-M and I-to-L substitutions and from the latter motif by a P-to-A substitution.

(v) PltL.

Of the sequences currently in the National Center for Biotechnology Information databases, PltL shows the greatest similarity (30% identity and 56% similarity over a 78-aa overlap) to the hypothetical protein SC3F7.09 of Streptomyces coelicolor (AL021409), an 87-aa peptide encoded by a member of the red gene cluster (5). PltL shows less similarity to a 69-aa acyl carrier protein (ACP) involved in fatty acid biosynthesis in Rhodobacter sphaeroides (7) and to presumed ACPs in Borrelia burgdorferi (AE001170) and Haemophilus influenzae (P43709). Although PltL does possess a conserved S residue which is required for 4′-phosphopantetheine binding in the ACP of R. sphaeroides (7), flanking amino acid residues deviate significantly from the ADSLD sequence motif present in the ACPs from R. sphaeroides, B. burgdorferi, and H. influenzae. Therefore, we refrain from assigning even a tentative function to PltL.

Insertional inactivation of pltM.

The pyoluteorin biosynthesis region was defined initially by the location of Tn5 insertions that abolished pyoluteorin production by Pf-5 (Fig. (Fig.1)1) (30). The pyoluteorin biosynthesis genes pltLABCDEFG are located within the defined region, whereas pltM falls outside of the region known previously to be required for pyoluteorin biosynthesis. To determine if pltM is required for pyoluteorin biosynthesis, we constructed a mutant of Pf-5 by inserting an aphI cassette into the genomic pltM gene. The resultant pltM mutant JL4562 did not produce detectable levels of pyoluteorin (<0.02 mg/liter) in experiments in which parallel cultures of Pf-5 produced 8.2 ± 0.2 mg of pyoluteorin/liter.

Deduced protein sequence of pltR.

The deduced peptide sequence of PltR exhibits significant similarity to amino acid sequences of more than 20 members of the LysR family of transcriptional regulators (22, 47). For example, the deduced amino acid sequence of PltR was similar to those of GstR of Rhizobium japonicum (58% similarity over 293 aa) (AF007569) and Rhizobium leguminosarum (52% similarity over 281 aa) (53), CitB of Klebsiella pneumoniae (49% similarity over 296 aa) (6), and PtxR of P. aeruginosa (49% similarity over 300 aa) (21). The highly conserved N-terminal domain that is characteristic of all LysR-type proteins was readily apparent within PltR (Fig. (Fig.4).4). Further analysis with a calibrated weight matrix (15) predicted that a helix-turn-helix motif exists between residues 20 and 41 within the N-terminal region of PltR. In addition to the N-terminal domain, a coinducer-binding domain and a C-terminal domain were assigned by comparison of the PltR sequence with a 70% consensus sequence for each of the three domains common to LysR-type proteins (Fig. (Fig.4)4) (47). Furthermore, many amino acid residues along the entire length of a profile sequence compiled from 10 LysR-type proteins representing several phylogenetic groupings (48) were also conserved within PltR.

FIG. 4
Regions of PltR exhibiting homology to a LysR profile sequence, obtained by aligning the amino acid sequences of 10 LysR proteins. Solid and cross-hatched regions indicate sequence similarity of >50% and 30 to 49%, respectively, ...

Identification of a putative PltR-binding promoter sequence.

An inverted repeat sequence (TGTAA-N7-TTACA), which conforms to an Ebright box motif (TNTNA-N7-TNANA) conserved among many promoters regulated by LysR-type proteins (17, 47), is centered 45 nt 5′ to the putative translational start codon of pltR. In addition, sequences flanking the identified Ebright box are particularly A+T rich, an attribute of some LysR-regulated promoters (reference 57 and references therein).

Influence of pltR on pyoluteorin production and transcription of pyoluteorin biosynthesis genes.

pltR mutants, constructed by introducing a gentamicin resistance cartridge into pltR (Fig. (Fig.1),1), did not produce detectable concentrations of pyoluteorin, whereas parallel cultures of Pf-5 produced 4.3 mg of pyoluteorin per liter. Disruption of pltR in strains containing Tn3-nice insertions in pltB, pltE, and pltF allowed us to assess the influence of pltR on the transcription of pyoluteorin biosynthesis genes. INA expressed from plt-inaZ fusions in pltR mutants was 7 orders of magnitude lower than in analogous strains with a wild-type pltR gene (Table (Table2).2). Because INA is related to the square root of InaZ protein content in Pseudomonas spp. (33), these data indicate that pltR+ strains had approximately 5,000 times more InaZ than did near-isogenic pltR mutants.

TABLE 2
Influence of pltR on transcription of plt biosynthesis genes assessed with an ice nucleation reporter gene (inaZ)

Incorporation of proline into pyoluteorin.

It had been suggested previously that the dichloropyrrole ring of pyoluteorin is derived from a tricarboxylic acid cycle intermediate, presumably through proline (10). Initial evidence for the incorporation of proline into pyoluteorin was obtained when l-[U-14C]proline was added to a culture of P. fluorescens Pf-5. Following isolation and purification of pyoluteorin from a culture extract that also contained a known quantity of pyoluteorin to serve as an unlabeled carrier, the final specific radioactivity of the pyoluteorin produced in culture was calculated to be 1.18 × 105 ± 0.01 × 105 dpm/mg. This corresponds to 19% of the total added radioactivity being present within the pyoluteorin sample. Subsequently, l-[1-13C]proline was added to cultures of Pf-5 cultures and a 13C-enrichment of 5.7% over and above the natural abundance signal of the 13C NMR carbonyl resonance for the purified pyoluteorin sample was observed, confirming the specificity of the proline incorporation.

DISCUSSION

The specific enrichment of the pyoluteorin 13C NMR carbonyl resonance demonstrates that [1-13C] proline was incorporated intact into the dichloropyrrole ring and establishes unequivocally that proline is the primary precursor to the dichloropyrrole ring in the pyoluteorin biosynthetic pathway of P. fluorescens Pf-5. Our labeling study corroborates and extends the conclusions of Cuppels et al. (10), who demonstrated that [1,2-13C2]acetate specifically labeled the pyoluteorin dichloropyrrole moiety in a manner consistent with the incorporation of a tricarboxylic acid cycle intermediate into the pyrrole ring of pyoluteorin. Several hypothetical pathways leading from proline to pyoluteorin are possible (Fig. (Fig.5).5). Despite extensive efforts to determine which of these pathways is responsible for pyoluteorin production in P. fluorescens Pf-5, we were not successful in demonstrating that any of several tested intermediates were incorporated into pyoluteorin (39). While we can only speculate on the identity of pathway intermediates, DNA sequence analysis of the pyoluteorin gene cluster has identified enzyme activities that logically correlate with the proposed biochemical transformations required for pyoluteorin synthesis (Fig. (Fig.5).5).

FIG. 5
Hypothetical pyoluteorin biosynthetic pathways. Arrows represent the predicted biochemical transformations catalyzed by the products encoded by the pyoluteorin gene cluster. The designator X indicates covalent attachment of proline either to CoA or to ...

Characteristic domains for the initiation or termination of polyketide assembly are not present in the pyoluteorin polyketide synthase composed of PltB and PltC (40). Typically, starter units used in polyketide assembly are activated as acyl-CoA derivatives and then are transferred to a ketosynthase domain within the polyketide synthase by way of an enzyme-bound 4′-phosphopantotheinyl cofactor (24). The presence of adenylation domains in PltF suggests that this protein activates an early pathway intermediate of pyoluteorin biosynthesis, such as proline, and that the activated intermediate is utilized to initiate polyketide assembly by PltB and PltC. It is not certain from sequence analysis, however, whether PltF is an acyl-CoA synthetase, as indicated by the presence of the core H sequence, or whether it is an AMP-ligase and simply catalyzes adenylate formation. If PltF does not generate an acyl-CoA intermediate, initiation of polyketide assembly may involve an ACP, perhaps PltL, in transferring the starter unit to the ketosynthase domain within module 1 of PltB. This mechanism is analogous to that proposed for initiating nonribosomal peptide biosynthesis by the pristinamycin I peptide synthetase in S. pristinaespiralis (12). Initiation of polyketide biosynthesis by using an ACP-bound starter unit may serve to channel amino acid starter units away from nonribosomal peptide pathways competing for activated substrates.

The putative thioesterase encoded by pltG is most probably responsible for termination of polyketide assembly. The identified motifs within PltG are predicted to hydrolyze the thioester formed between the fully extended polyketide substrate and the ACP domain of PltC. Considering the favorable transition state for formation of the resorcinol ring following polyketide assembly, it is likely that the required cyclization occurs concurrently with thioester hydrolysis. Nevertheless, we have yet to identify the outer limits of the pyoluteorin biosynthetic gene cluster, and other loci may exist that encode proteins required for cyclization and/or aromatization of the extended polyketide intermediate.

The formation of the pyrrole ring in pyoluteorin formally requires an oxidation of the proline-derived carbon ring, a transformation that most probably involves the catalytic activity of PltE. The overall similarity of PltE to many acyl-CoA dehydrogenases suggests that this protein introduces a double bond adjacent to a thioester linkage within the substrate and implies that PltF is an acyl-CoA synthetase despite the core H sequence motif therein. It is likely, therefore, that PltE catalyzes the formation of a Δ2,3-dehydroproline derivative before assembly of the pyoluteorin resorcinol ring but after the transformation catalyzed by PltF. Aromatization of the resulting pyrroline ring to the pyrrole may occur spontaneously, a reaction with precedence in the formation of pyrrole-2-carboxylate from a pyrroline intermediate of a 4-hydroxyproline catabolic pathway in a Pseudomonas sp. (1). The similarity of deduced peptide sequences of pltE and redW, a gene in the undecylprodigiosin biosynthesis locus of S. coelicolor, may reflect a comparable mechanism for the proline-to-pyrrole transformation in the pyoluteorin and undecylprodigiosin biosynthesis pathways.

PltA, PltD, and PltM belong to a new class of halogenase enzymes that chlorinate secondary metabolites (23, 56). Cts4 of the chlortetracycline biosynthetic gene cluster from Streptomyces aureofaciens (11) and PrnA and PrnC of the pyrrolnitrin biosynthetic gene cluster from P. fluorescens BL915 (20) are the only other previously described halogenases. PltA, PltD, and PltM exhibit sequence similarity only to PrnC, which also shares their proposed function in chlorination of a pyrrole derivative (29). With the exception of PltD, each of the halogenases possesses a putative NADH cofactor-binding site. The lack of a catalytic function for PltD, which could be inferred from the lack of an NADH-binding site, provides a rational explanation for the presence of three halogenase genes within the gene cluster of a dichloro-substituted product. Nevertheless, Tn5 or aphI insertions in pltA, pltD, and pltM abolish pyoluteorin production, indicating a role for each of the genes in production of the antibiotic. Definitive evidence for the involvement of the three halogenases in pyoluteorin production will require the generation of nonpolar mutations in each of these genes.

The similarity of the deduced peptide encoded by pltR to other transcriptional activators, the presence of a LysR-type protein binding site (the Ebright box) 5′ to pltR, and the loss of pyoluteorin production in pltR mutants suggest that PltR is a positive transcriptional activator of linked pyoluteorin biosynthesis genes in P. fluorescens Pf-5. This conclusion is supported by studies demonstrating that transcription of pltB, pltE, and pltF genes, assessed from transcriptional fusions to an ice nucleation reporter gene, was lower in pltR mutants than in near-isogenic pltR+ derivatives of Pf-5. Previously, we reported the presence of at least two promoters in the plt region, based on ice nucleation activities expressed by Pf-5 harboring plasmids with transcriptional fusions of inaZ to pltB or pltE (30), but the specific locations of these promoters are not known. The Ebright box located 5′ to pltR was the only unambiguous LysR-type binding site detected in the plt gene cluster, but functional sites that lack the characteristic structure could be present. Future studies characterizing promoters within the plt region are needed to understand the structural and functional relationships involved in transcriptional regulation of pyoluteorin biosynthesis genes by PltR. pltR provides the second example of a gene encoding a positive transcriptional activator linked to loci encoding the biosynthesis of an antifungal metabolite in Pseudomonas spp. The other example is the luxR homolog phzR, which, in concert with the luxI homolog phzI, regulates the transcription of linked phenazine biosynthesis genes in Pseudomonas aureofaciens (41). Due to the positive self-regulation characteristic of the luxR and lysR homologs, both types of regulators provide a mechanism for amplifying an environmental or physiological signal that controls the expression of antibiotic biosynthesis genes. The phzI-phzR gene pair activates the transcription of phenazine biosynthesis genes in response to increased cell density (41), but signals required for the transcription of pltR and production of a putative PltR coinducer, which is typically required for optimal activity of LysR regulators (47), are yet to be identified.

ACKNOWLEDGMENTS

We extend our gratitude to Marcella Henkels for assistance in preparing the figures and to Carol Bender, Mary Hagen, Phil Proteau, and Mark Schell for their critical reviews of the manuscript.

Portions of this work were funded by a Tartar fellowship to BNT from the Department of Chemistry, Oregon State University.

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