INTRODUCTION

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Certain members of the genus Pseudomonas produce diverse low-molecular-weight ("secondary") metabolites including nitrogen-containing heterocyclic pigments known as phenazine compounds (5, 19). Phenazines are synthesized by a limited number of bacterial genera including Pseudomonas, Burkholderia, Brevibacterium, and Streptomyces (38). Almost all phenazines exhibit broad-spectrum activity against various species of bacteria and fungi (32). This activity is connected with the ability of phenazine compounds to undergo oxidation-reduction transformations and thus cause the accumulation of toxic superoxide radicals in the target cells (15). Some phenazine compounds can act as bacterial virulence factors. For example, pyocyanin, produced by the opportunistic pathogen Pseudomonas aeruginosa during cystic fibrosis, has been shown to inhibit the ciliary function of respiratory epithelial cells (40). Phenazine antibiotics produced by the biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84 are major factors in the ability of these strains to inhibit the growth of fungal root pathogens. Moreover, studies involving phenazine-deficient mutants have clearly demonstrated that antibiotic production in natural habitats plays an important role in the ecological competence and long-term survival of these strains in the environment (21). Over 50 naturally occurring phenazine compounds have been described, and certain bacterial producers are able to synthesize mixtures of as many as 10 different phenazine derivatives at one time (32, 38). Growth conditions also may influence the number and types of phenazines synthesized by an individual strain (38).

Early studies with radiolabeled precursors revealed tight links in several microorganisms between biosynthesis of phenazine compounds and the shikimic acid pathway (38). Phenazine-1,6-dicarboxylic acid is believed to be the first phenazine formed and to be the one from which others are derived (19). It also was proposed that the phenazine nucleus is formed by the symmetrical condensation of two molecules of chorismic acid and that enzymes involved in this conversion must have many features in common with anthranilate synthases (17). Despite intensive biochemical studies, the biosynthetic intermediates have not been identified and little is known about the genetics of phenazine synthesis (38). To date, the best-studied phenazine genes are those cloned from P. aureofaciens 30-84 (26, 27). The products of the phz structural genes from strain 30-84 are similar to enzymes from the shikimic acid and tryptophan biosynthetic pathways, confirming predictions from earlier biochemical analyses. Two other genes, phzI and phzR, encode parts of a quorum-sensing circuit that regulates phenazine production in P. aureofaciens 30-84 in a cell density-dependent manner (26).

In this paper, we present organizational and functional analyses of the complete genetic locus for phenazine-1-carboxylic acid biosynthesis from P. fluorescens 2-79. Two new genes from the homologous locus of P. aureofaciens 30-84 also are described, and the structure and function of the biosynthetic gene clusters from the two strains are compared. Results of this study suggest that the mechanism of phenazine biosynthesis is highly conserved among fluorescent Pseudomonas species.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are described in Table 1. A spontaneous rifampin-resistant derivative of P. fluorescens 2-79 was used in all studies. P. fluorescens 2-79 and P. aureofaciens 30-84 were grown at 28°C in Luria-Bertani (LB) broth (1). E. coli strains were grown at 28 or 37°C in LB or M9 minimal medium (1). Antibiotic supplements were used at the following concentrations: ampicillin, 80 µg/ml; carbenicillin, 80 µg/ml; rifampin, 75 µg/ml; kanamycin, 30 or 150 µg/ml; tetracycline, 12.5 µg/ml; gentamicin, 10 µg/ml.

Transposon Tn3-lacZ mutagenesis. Tn3-lacZ insertions were made by using the transposon system described by Stachel et al. (34). The target cosmid, pPHZ108A, was introduced into E. coli HB101(pHoHo1, pSShe). To select pPHZ108A::Tn3-lacZ derivatives, the strain harboring pPHZ108A, pHoHo1, and pSShe was mated with recipient strain E. coli C2110, using E. coli HB101(pRK2013) as a helper. The site and orientation of insertions into pPHZ108A were analyzed by restriction mapping. The plasmids were then introduced into the phenazine-1-carboxylic acid (PCA)-nonproducing Tn5 mutant P. fluorescens 2-79.8A by triparental matings to test the effect of each Tn3-lacZ insertion on PCA production. Expression in transconjugants of the lacZ reporter gene, an indicator of transcriptional activity, was detected on LB agar containing 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal).

DNA manipulations. Standard methods were used for DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, ligation, and transformation (1). Total DNA from P. fluorescens 2-79RN₁₀ was isolated and purified by a cetyltrimethylammonium bromide miniprep procedure (1). For Southern blotting and hybridization, total DNA samples were digested with restriction endonucleases, separated by electrophoresis in a 0.8% agarose gel, transferred onto a BrightStar-Plus nylon membrane (Ambion, Inc., Austin, Tex.), and hybridized with a specific DNA probe labeled with the DECAprime-Biotin random priming kit (Ambion, Inc.). DNA-DNA hybrids were detected with the BrightStar nonisotopic detection kit (Ambion, Inc.) as specified by the manufacturer.

Cloning of phzX and phzY genes from P. aureofaciens 30-84. A DNA fragment containing phzX and phzY genes was amplified with P. aureofaciens 30-84 genomic DNA as a template and with oligonucleotide primers 30-84/1 (5'-CAGTTCATCCGGCGGGCTGCAG-3') and 30-84/2 (5'-CCCGTTTCAGTAAGTCTTCCATGATGCG-3'). Target DNA was amplified with Vent DNA polymerase (New England Biolabs, Beverly, Mass.) and the following cycling program: 94°C for 1 min, 64°C for 45 s, and 72°C for 1 min (30 cycles). The 1.2-kb PCR product was cloned into the SmaI site of pBluescript II KS, and the resulting plasmid was used to determine the nucleotide sequence.

DNA sequencing and analysis. DNA was sequenced by the dideoxy chain termination method with Sequenase 2.0 (Amersham International, Little Chalfont, United Kingdom). Exonuclease III deletion derivatives were constructed with enzymes supplied by Amersham, as specified by the manufacturer. Sequence data were compiled and analyzed with the GCG package (13). DNA sequences were compiled with GELASSEMBLE, and open reading frames (ORFs) and codon usage were analyzed with MAP and FRAMES. A database search for similar protein sequences was carried out with the BLAST and FASTA network servers at the National Center for Biotechnology Information and the European Molecular Biology Laboratory, respectively. The probable domain homology search was performed with the PROSITE (European Molecular Biology Laboratory, Heidelberg, Germany) and SBASE (International Center for Genetic Engineering and Biotechnology, Trieste, Italy) computer servers (3, 12). Pairwise alignments were obtained with the GCG GAP program (gap weight = 4).

Analyses of polypeptide gene products. The P. fluorescens 2-79 structural phz genes were expressed under the control of the T7 promoter in plasmid vectors pT7-5 and pT7-6 in E. coli BL21 with pGP1-2 as a source of T7 RNA polymerase and with [³⁵S]methionine for selective labeling of target proteins (1). Labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by autoradiography.

Gene replacement experiments in P. fluorescens 2-79. A 1.4-kb Km^r cassette (Pharmacia Biotech, Inc., Uppsala, Sweden) containing the aminoglycoside 3'-phosphotransferase gene was used in gene replacement experiments. Plasmids containing individual phz genes that were insertionally inactivated by the antibiotic resistance gene were constructed. Target DNA fragments were then subcloned into pJQ200SK, a gene replacement vector harboring the sacB gene as a counterselectable marker (29). The resulting plasmids were transferred into P. fluorescens 2-79 via biparental matings with E. coli S17-1, followed by selection for the plasmid resistance marker. Subsequent selection was performed on LB agar containing 5% sucrose and kanamycin. Introduced mutations were verified at the DNA level by PCR screening and Southern hybridization.

Extraction and detection of phenazine compounds. Prior to extraction of phenazine compounds, Pseudomonas strains were cultivated in LB medium for 3 days at 28°C. E. coli BL21(DE3) clones harboring phz genes under control of the T7 promoter were grown in LB medium at 28°C without addition of isopropyl--D-thiogalactopyranoside (IPTG). The "leaky" lac promoter of the DE3 lysogen maintained high enough levels of T7 RNA polymerase in the cell to enable the production of detectable amounts of phenazines.

Nucleotide sequence accession numbers. The GenBank accession numbers for the nucleotide sequence data for phz genes from P. fluorescens 2-79 and P. aureofaciens 30-84 are L48616 and AF007801, respectively.

	RESULTS

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Mutagenesis of P. fluorescens 2-79 with Tn3-lacZ. The phenazine biosynthetic locus from P. fluorescens 2-79 was cloned previously as a 12-kb HindIII-BamHI DNA fragment within plasmid pPHZ108A and characterized by restriction mapping (37). Mutagenesis of pPHZ108A with Tn3-lacZ, which can generate fusions in which expression of the lacZ gene is regulated by the promoter of the gene bearing the insertion, yielded 57 unique transposon insertions that interfered with phenazine production. These insertions identified two adjacent, divergently transcribed units of approximately 6 and 0.75 kb that were strongly and weakly expressed, respectively, under conditions favorable to the production of PCA (data not shown). Most of the large transcriptional unit was contained within a 5.4-kb BglII fragment, with the remainder localized to the adjacent 2.0-kb BglII-XbaI DNA fragment (Fig. 1). Consistent with results from Tn3-lacZ mutagenesis, the BglII fragment alone did not enable PCA production in P. fluorescens 2-79.8A, suggesting a requirement for additional downstream sequences.

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Physical maps of cosmid clone pPHZ108 (A), the DNA region from P. fluorescens 2-79 encoding genes involved in the production of PCA (B), and a portion of the homologous genetic locus from P. aureofaciens 30-84 (C). Open boxes indicate genes encoding phenazine biosynthesis enzymes. The direction of the gene transcription is shown by an arrow. The symbols PA and PX represent the position and orientation of corresponding promoters. Insertions of Tn3-lacZ that interfered with phenazine production are marked on the map of pPHZ108A as | (Lac+ phenotype) and | (Lac phenotype).

DNA sequence analysis. The overlapping 5.7-kb EcoRI-XbaI and 5.4-kb BglII fragments and the adjacent 1.8-kb BglII fragment were subcloned into pBluescript II KS and SK cloning vectors and used to determine the nucleotide sequence of an 8,505-bp DNA segment from pPHZ108A. Computer analysis of the DNA sequence within the large transcriptional unit revealed seven ORFs with high coding probability, designated phzABCDEFG. Each of these genes is preceded by a well-conserved ribosome binding site. In the phzD-phzE and phzF-phzG pairs, the stop (UGA) and start (AUG) codons of the adjacent genes overlap, possibly reflecting their translational coupling. Two additional genes homologous to phzI and phzR also were identified upstream of the phzABCDEFG cluster (20).

View larger version (51K): [in this window] [in a new window]   FIG. 2.   Alignment of the deduced amino acid sequences of the PhzA (P. fluorescens 2-79), PhzX (P. aureofaciens 30-84), PhzB (P. fluorescens 2-79), and PhzY (P. aureofaciens 30-84) proteins. Identical (*) and similar (·) amino acid sequences are indicated. Dashes represent gaps inserted in the sequences to improve the alignment.

Identification of proteins encoded by phzABCDEFG, phzX, and phzY. Using a pair of pT7-5 and pT7-6 transcriptional vectors and the two-plasmid expression system of Tabor, we identified products for all of the phzABCDEFG genes (Fig. 3). The rather high level of expression of all seven genes reflects efficient utilization of the Pseudomonas translational signals by the E. coli protein-synthesizing machinery.

View larger version (73K): [in this window] [in a new window]   FIG. 3.   Autoradiograph of polypeptides labeled with [35S]methionine and resolved by electrophoresis on an SDS-12% polyacrylamide gel. Samples were prepared from E. coli BL21 containing pGP1-2 plus the indicated plasmids. The location of DNA fragments inserted in pT7-5 and pT7-6 vectors for gene expression is shown in Fig. 4. Lanes: 1, pT7-6; 2, pT7-6ABCD; 3, pT7-6AB; 4, pT7-5CDE; 5, pT7-6CDEFG; 6, pT7-5CD; 7, pT7-5G. An intense 16-kDa band in lane 4 is a product of a truncated phzF gene, and an intense protein band almost overlapping with PhzC in lane 2 is a product of a truncated phzE gene. Positions of molecular size markers are given on the left in kilodaltons.

Functional analysis of the phz genes. To determine which genes in the phenazine biosynthetic cluster are essential for the production of PCA, the Kan^r cassette was inserted into phzC, phzD, phzE, and phzG and each disrupted gene was introduced into the genome of strain 2-79 by homologous recombination. For unknown reasons, it was not possible to recover phzA, phzB, or phzF recombinants. Insertions of the Kan^r cassette in phzC, phzD, and phzE interfered with phenazine production, and the corresponding mutant strains were completely deficient in PCA biosynthesis (Fig. 4A). P. fluorescens 2-79 MXG, bearing a Kan^r insertion within phzG, was able to produce PCA, although the yield was only about 1.3% of that of wild-type strain 2-79. 

View larger version (24K): [in this window] [in a new window]   FIG. 4.   Analysis of phenazine biosynthesis in P. fluorescens 2-79 gene replacement mutants (A) and by E. coli BL21(DE3) clones harboring plasmids with different sets of phz genes from P. fluorescens 2-79 (B) or P. aureofaciens 30-84 (C) cloned under the control of a T7 promoter. Restriction maps, locations of individual phz genes, and DNA fragments contained within plasmids used in the study are shown. Arrows indicate the position and orientation of the T7 promoter. Solid triangles denote locations of the Kanr cassette insertions in the chromosome of P. fluorescens 2-79 mutants. N.D., not detected. OH-PCA, 2-hydroxyphenazine-1-carboxylic acid; OH-PHZ, 2-hydroxyphenazine.

	DISCUSSION

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Results of structural and functional analyses presented here show that products of the phzABCDEFG gene cluster are responsible for synthesis of PCA by P. fluorescens 2-79. Phenazines are products of the bacterial common aromatic amino acid pathway, with chorismate as the probable branch point intermediate (38), but the precise mechanism of synthesis and the identity of the biosynthetic intermediates remain unknown. Our evidence that the products of phzC, phzD, and phzE have significant homology to well-characterized enzymes of the shikimate and tryptophan biosynthetic pathways is consistent with previous findings (19, 38) and provides new insight into the phenazine biosynthetic pathway in fluorescent Pseudomonas spp.

PhzC shows a high degree of similarity to plant DAHP synthases that catalyze the first step of the shikimate pathway. In bacteria, DAHP synthase isoenzymes are regulated transcriptionally as well as through feedback inhibition by specific amino acids or pathway intermediates and thus represent a key control point regulating carbon flow into the shikimate pathway (16). All known DAHP synthases recently were grouped into two distinct classes based on protein sequence similarity. Our sequence data indicate that the PhzC protein from P. fluorescens 2-79 is a typical type II enzyme, together with P. aureofaciens PhzF and the recently studied DAHP synthases from Streptomyces coelicolor and S. rimosus (39). Being expressed late in growth, PhzC could function to divert common carbon metabolites into the shikimate pathway, providing the high levels of chorismic acid needed to support the synthesis of PCA, which can accumulate in culture media at concentrations of up to 1 g/liter. The remainder of the enzymatic activities needed for chorismate biosynthesis probably are provided by shikimic acid pathway enzymes encoded by aroD, aroB, aroE, aroL, aroA, and aroC, since these genes are known to be expressed constitutively in pseudomonads (24).

Based on their sequence, it is likely that products of the phzD and phzE genes act to modify chorismate prior to the condensation reaction resulting in formation of the phenazine nucleus. The first two-thirds of the PhzE protein display relatively weak similarity to component I of bacterial anthranilate synthases. The C-terminal part (amino acid residues 438 to 637) exhibits strong homology to members of class I GATase enzymes, which comprise component II of anthranilate synthases. The PhzE protein is most closely related to a small subset of anthranilate synthases of unusual structure (TrpE from Streptomyces venezualae, TrpE from Rhizobium meliloti, and TrpE from Azospirillum brasilense) that have evolved as a fusion of genes encoding anthranilate synthase components I and II (2, 8). Component I of anthranilate synthase is a bifunctional enzyme that catalyzes the formation of the aromatic product anthranilate from chorismic acid in two discrete steps (22). The first step is catalyzed by aminodeoxyisochorismate (ADIC) synthase and is thought to involve amination of chorismic acid to ADIC. The second step, which is catalyzed by ADIC lyase, involves elimination of pyruvate and aromatization to form anthranilate. ADIC remains enzyme bound, but it has been demonstrated that a substitution of a single amino acid residue within component I of anthranilate synthase is sufficient to uncouple ADIC synthase and ADIC lyase activity (22). Interestingly, it previously was postulated that ADIC could be a potential precursor for the phenazine compounds iodinin and aminophenoxazinone, produced by cultures of Brevibacterium iodinum (30). Collectively, these observations and the rather weak similarity between the PhzE protein and most bacterial anthranilate synthases may indicate that PhzE functions specifically as an ADIC synthase (Fig. 5). The GATase domain might confer upon PhzE the ability to use glutamine as a source for the amination of chorismate. This is consistent with the data of Römer and Herbert, who demonstrated that the amide nitrogen of glutamine serves as the immediate source of nitrogen in the heterocyclic nucleus of phenazine compounds (30).

View larger version (12K): [in this window] [in a new window]   FIG. 5.   Proposed action of PhzC, PhzD, PhzE, PhzF, and PhzG in the biosynthesis of PCA.

The product of the phzD gene shows a high degree of similarity to bacterial 2,3-dihydro-2,3-dihydroxybenzoate synthases (isochorismatases). The best-studied isochorismatase, EntB from E. coli, has a predicted M_r of 32,500 and is active as a pentamer in the isochorismatase reaction, catalyzing the hydrolysis of pyruvate from isochorismic acid (11). The presence of an isochorismatase analog in the phz locus might reflect the need for pyruvate hydrolase activity, rather than an ADIC lyase activity (which could be provided by anthranilate synthase), for modification of the immediate precursor of the phenazine nucleus. EntB actually is a bifunctional enzyme (often called EntB/G), with EntG activity encoded by the entB 3' terminus (33). EntG functions as a part of the EntDEFG enterobactin synthetase multienzyme complex that catalyzes the last step of enterobactin biosynthesis. Interestingly, the PhzD protein resembles a truncated EntB, which lacks 78 amino acid residues at the C terminus. It therefore is probable that PhzD has only isochorismatase activity, which could function to remove the pyruvate side chain from ADIC to yield the putative phenazine precursor 3-hydroxyanthranilate (Fig. 5).

It seems likely that the products of phzF and phzG function in the condensation of two molecules of 3-hydroxyanthranilate, or a similar precursor, to generate the phenazine nucleus. Sequence analysis of PhzF revealed no motifs or similarities to other proteins of known function. However, PhzG resembles bacterial pyridoxamine-5'-phosphate oxidases that function in the de novo synthesis of pyridoxine (vitamin B₆) and in the conversion of pyridoxamine-5'-phosphate to pyridoxal phosphate, a cofactor for numerous transamination reactions (18). Interestingly, pyridoxal phosphate is required for aminodeoxychorismate lyase (PabC) activity in E. coli (14), where the cofactor is proposed to bind via an imine linkage to the 4-amino position of 4-amino-4-deoxychorismate during the synthesis of p-aminobenzoic acid (22). While it is tempting to speculate that pyridoxal phosphate may play a similar role in phenazine synthesis, binding at the 2-amino position of the hypothetical precursor 3-hydroxyanthranilate (Fig. 5), no conserved pyridoxal phosphate-binding motifs were identified in PhzF or, indeed, in any of the other phz gene products. Thus, the step(s) involved in this final condensation reaction remains obscure.

We also were unable to identify any motifs or similarity between products of phzA and phzB and protein sequences listed in various databases. However, PhzA and PhzB are remarkably similar to each other (Fig. 2), which strongly suggests that their cognate genes evolved as the result of a duplication event. Homologs of phzA and phzB also were identified in the phenazine biosynthetic locus from P. aureofaciens 30-84. Functional analysis indicated that in both 2-79 and 30-84, these genes influence the kinds and relative amounts of aromatic and heterocyclic compounds synthesized as a result of phz gene expression. Thus, E. coli expressing the complete phz locus synthesized large amounts exclusively of PCA whereas the same host expressing the phzCDEFG genes produced large quantities of a mixture of aromatic and heterocyclic nitrogen-containing compounds that included only minor amounts of PCA. PhzA and PhzB may stabilize a multienzyme phenazine biosynthetic complex, whose existence has been postulated previously (6). In their absence, the biosynthetic system still functions, but the specificity and perhaps also the efficiency decrease dramatically.

Results of this study indicate that the phenazine biosynthetic gene cluster from P. fluorescens 2-79 is remarkably conserved relative to that previously described from P. aureofaciens. In related work, we recently have shown that a portion of the pyocyanin biosynthetic locus from P. aureofaciens PAO1 retains certain of these same organizational and structural features (20). It thus appears that different fluorescent Pseudomonas species may have a common pathway, which confers upon them the ability to synthesize the phenazine nucleus.

How, then, can the specific array of phenazine compounds produced by individual species of fluorescent pseudomonads be explained? P. fluorescens 2-79 produces only PCA, whereas P. aureofaciens 30-84 produces, in addition to PCA, lesser amounts of 2-hydroxyphenazine-1-carboxylic acid and (probably by spontaneous, nonenzymatic decarboxylation) small quantities of 2-hydroxyphenazine (25, 27). The conversion of PCA to 2-hydroxyphenazine-1-carboxylic acid in strain 30-84 previously was attributed to the product of phzC (27), which has 93.5% identity and 95.3% similarity to the corresponding PhzF protein from P. fluorescens 2-79. Although small differences in sequence between these proteins cannot formally be ruled out as contributing to product specificity, the sequence data do not provide a useful insight into how phzC might function in derivatization of the phenazine nucleus, nor is the location of the gene within a cluster of core biosynthetic genes consistent with modification of the key product. Moreover, our observations that expression in E. coli of incomplete loci lacking phzAB or phzXY yielded mixtures of compounds including PCA and unsubstituted phenazine, whereas expression of the complete locus from either strain enabled the synthesis of large amounts of essentially homogeneous PCA, argue that the compounds detected in earlier expression studies with strain 30-84 that included only phzFABCD are products of inefficient or nonspecific synthesis that do not accurately reflect the biosynthetic potential of the intact locus. Our results obtained by HPLC, UV-visible spectral analyses, and mass spectrometry support the idea that the phzF product from strain 2-79 and its homolog from strain 30-84 participate directly in the condensation reactions leading to production of phenazine-1,6-dicarboxylic acid, the postulated precursor of other phenazines including PCA (19). We further hypothesize that enzymes encoded by other genes which may or may not be physically linked to the phz loci, as well as spontaneous chemical reactions, are responsible for modification of phenazine-1,6-dicarboxylic acid or PCA to form the various phenazine compounds characteristic of different species of fluorescent pseudomonads.