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Journal of Bacteriology, July 2003, p. 3718-3725, Vol. 185, No. 13
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.13.3718-3725.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Repression of Phenazine Antibiotic Production in Pseudomonas aureofaciens Strain 30-84 by RpeA

Cheryl A. Whistler and Leland S. Pierson III^*

Department of Plant Pathology, The University of Arizona, Tucson, Arizona 85721

Received 15 January 2003/ Accepted 9 April 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
Pseudomonas aureofaciens strain 30-84 is a biological control bacterium that utilizes a two-component GacS/GacA regulatory system interconnected with the PhzR/PhzI quorum sensing system to positively regulate biosynthesis of phenazine antibiotics that contribute to its association with plant hosts. To date, no negative regulators of phenazine production have been identified, nor has the role of repression been studied. Here we describe a novel repressor of secondary metabolism in P. aureofaciens strain 30-84, RpeA, whose deduced amino acid sequence is similar to those of a group of putative two-component regulatory systems of unknown function found in several animal and plant-pathogenic bacteria. In minimal medium where phenazine production is very low, inactivation of the rpeA gene enhanced phenazine biosynthetic gene expression and increased phenazine production but did not increase quorum sensing signal accumulation. Furthermore, RpeA functioned to block phenazine biosynthetic gene transcription in minimal medium even when quorum-sensing signals were at a level that was sufficient for induction of phenazine gene expression in rich medium. Additionally, in the absence of rpeA, the quorum sensor PhzR was not required for phenazine production. Although repression plays a critical role in phenazine regulation, the rpeA mutation could not bypass the requirement for a functional GacS/GacA system, demonstrating that activation is required even in the absence of the RpeA repressor. This study reinforces that multiple signals, including nutrition and population density, are integrated to control the appropriate expression of phenazine antibiotics.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial secondary metabolites play critical roles in many aspects of bacterium-host interactions. Secondary metabolites that function as virulence factors play a central role in disease by altering host tissues (17, 29). Other secondary metabolites produced by beneficial bacteria can function to prevent infection by pathogens by altering the environment and improving the bacterium's ability to compete with pathogens, by inhibiting the activity of pathogens, or by triggering host defenses (4, 28). Therefore, understanding the regulation of secondary metabolites is critical for ameliorating disease or enhancing disease suppression. The regulation of bacterial secondary metabolites, whether they are beneficial or harmful, is remarkably similar. For example, activation of gene expression by quorum sensing via LuxI/R homologs (19) and two-component regulators homologous to GacS/GacA (13) as well as regulation of translation via the RsmA/RsmB system (14, 18) are highly conserved in function in both pathogens and beneficial bacteria. Along with pathway-specific activators and repressors, these global regulators function to control the appropriate expression of secondary metabolites.

Pseudomonas aureofaciens strain 30-84 is a biological control bacterium able to suppress take-all disease of wheat caused by the fungal pathogen Gaeumannomyces graminis var. tritici through the production of phenazine antibiotics (27). The ability of 30-84 to produce phenazine correlates directly with its capacity to inhibit growth of G. graminis var. tritici (27). In addition to its role in pathogen inhibition, phenazine production contributes to the competitive fitness of strain 30-84 in the wheat rhizosphere (20). Since phenazines are involved directly in successful disease control, understanding the regulatory systems that contribute to phenazine production may result in more consistent disease control under various field conditions.

P. aureofaciens also serves as a useful model organism for the study of secondary metabolite regulation. Multiple regulators control phenazine production by strain 30-84. The quorum sensing genes phzI and phzR (26, 37) are directly responsible for the activation of the phenazine biosynthetic operon (phzXYFABCD) (25) in response to the cell density-dependent accumulation of N-hexanoyl homoserine lactone (HHL) produced by PhzI (38). Analogous to the situation of TraR for Agrobacterium tumefaciens (42), HHL association with PhzR is believed to cause it to fold into a structure enabling it to bind to a sequence (phz box) upstream of the biosynthetic operon activating phenazine gene expression. A second quorum sensing system, CsaI/CsaR, also contributes to the accumulation of total N-acyl-homoserine lactone (AHL) signals by strain 30-84 but only influences phenazine production when present in multiple copies or when grown on specific medium (41). The global two-component regulatory system, GacS/GacA, is required for phenazine production and influences both quorum-sensing circuits (5, 6, 41). Although GacS/GacA regulates phenazine biosynthetic gene transcription in part by controlling phzI but not phzR expression, addition of exogenous HHL does not restore phenazine production to GacA mutants (5), suggesting that while GacS/GacA activates phenazine expression by controlling quorum sensing, it also activates expression by an additional, yet-uncharacterized mechanism.

The presence of multiple regulatory circuits that control phenazine biosynthesis demonstrates the importance of positive regulation of phenazine production. However, to date no negative phenazine regulators have been characterized from strain 30-84. To identify potential negative regulators, we randomly mutagenized strain 30-84Z, a derivative of 30-84 with a transcriptional fusion of lacZ to the phzB biosynthetic gene (38) with Tn5 and screened for enhanced ß-galactosidase activity indicative of enhanced phzB expression. By this approach, we identified rpeA (repressor of phenazine expression A), a gene encoding a novel negative regulator of secondary metabolite production. The deduced protein sequence of RpeA is similar to that of a group of putative two-component sensor kinases of uncharacterized function in several animal and plant pathogens. The rpeA mutation increased pathogen inhibition in vitro. However, the rpeA mutation did not increase phenazine biosynthetic gene expression via increased AHL accumulation. Although the mechanism of RpeA repression is still unknown, lack of RpeA cannot override the need for activation by GacS/GacA, demonstrating that while repression may play an important role in phenazine production under some conditions, the requirement for activation dominates regulation.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacteria, plasmids, and culture conditions. Bacterial strains and plasmids used in this study are listed in Table 1. P. aureofaciens strain 30-84 and derivatives were routinely grown at 28°C on agar plates or in broth with shaking at 200 rpm in pigment production medium (PPMD) (38), Luria-Bertani medium (LB) (33), Kings medium B (KMB) (27), or M9 minimal medium supplemented with 0.4% glucose (M9) (33). Escherichia coli was grown at 37°C in LB. Antibiotics were used where appropriate at the following concentrations: for E. coli in all media, kanamycin sulfate (KM) at 50 µg/ml, tetracycline (TC) at 25 µg/ml, and ampicillin at 100 µg/ml; for P. aureofaciens, KM at 50 µg/ml and TC at 50 µg/ml in LB and PPMD or 30 µg/ml in M9. Rifampin (RIF) was used at 100 µg/ml to counterselect against donor and helper E. coli in triparental matings. ß-Galactosidase activity was visualized qualitatively on agar plates supplemented with 40 mg of 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranosidase (X-Gal)/ml. Pathogen inhibition assays were performed using KMPE agar plates (27).

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TABLE 1. Bacterial strains and plasmids used in this study

Recombinant DNA techniques. Standard methods were used for transformations, restriction enzyme digestions, and gel electrophoresis (0.7% wt/vol) in Tris-phosphate EDTA buffer (33). Restriction enzymes were from GIBCO-BRL Life Technologies (Gaithersburg, Md.). The Master Taq kit (Eppendorf Scientific Inc., Westbury, N.Y.) was used for PCR (33). Plasmids were purified by an alkaline lysis procedure (33) or using a Perfectprep plasmid minikit (Eppendorf Scientific Inc.). Genomic DNA was isolated by a cetyltrimethylammonium bromide method (2). Digoxigenin-11-dUTP-labeled probes for Southern blots were generated by PCR with gene-specific primers or random-primed labeling using materials and protocols supplied by the manufacturer of the Genius system (Genius system user's guide for membrane hybridization, version 3.0; Boehringer Mannheim corporation, Indianapolis, Ind.). DNA restriction fragments from agarose gels were purified using a QIAquick gel extraction kit (Qiagen Inc., Valencia, Calif.).

Triparental matings. Triparental matings into strain 30-84 and derivatives were performed with E. coli DH5 as the donor and HB101(pRK2013) as the helper as described previously (27). Approximately equal numbers of washed donor, helper, and recipient cells were spotted onto sterile nitrocellulose filters on an LB agar plate and incubated at 28°C for 24 h. The resulting cells were suspended in 2 ml of sterile water, and 50 µl was spread onto LB plates supplemented with RIF to select against E. coli and an appropriate antibiotic (KM or TC) to select for recipient cells harboring plasmid DNA.

Transposon mutagenesis. P. aureofaciens strain 30-84Z (38), a derivative of strain 30-84 containing a phzB::lacZ fusion, was mutagenized by Tn5 using pSUP1021 as described previously (27). Putative transconjugants were selected on LB plates supplemented with KM and RIF. Transconjugants were patched onto M9 plates supplemented with X-Gal and were screened visually for a darker blue color than shown by the parental strain, reflective of enhanced phenazine biosynthetic gene expression.

Cloning and sequence analysis. Genomic DNA was digested with various restriction enzymes, and the DNA fragments containing the transposon were visualized via Southern hybridization. Southern blots probed with Tn5 revealed that the transposon was contained on a 9-kb KpnI fragment, a 10.5-kb SstI fragment, or an 8.6-kb SstI-KpnI fragment. These fragment sizes correspond to the genomic DNA plus an additional 4.5 kb of Tn5 DNA. The SstI-KpnI fragment was cloned into pUC18, creating pCW2A1, and the KpnI fragment was cloned into pRK415, creating pCW2B1. To simplify sequence analysis, the chromosomal fragment containing Tn5 was digested with BamHI and religated, creating pCW2D1.

The original strain 30-84 genomic library did not include the wild-type region corresponding to the mutated region (5). The region containing the wild-type gene was cloned by creating and screening a sublibrary of 6-kb SstI fragments (10.5 kb minus 4.5 kb of Tn5 DNA) from strain 30-84 in pUC18. Genomic DNA from strain 30-84 was digested with SstI, fragments were separated on an agarose gel, and 6-kb fragments were purified and ligated into SstI-digested pUC18. The SstI sublibrary was transformed into E. coli DH5 and plated on LB supplemented with ampicillin and X-Gal. Plasmid pCW2E4 was identified by screening white colonies by PCR using DNA primers specific to the region adjacent to the transposon (forward primer, 5'-ATGGCGGTGCAGATGGTGGTGC-3'; reverse primer, 5'-AAACTCGTCCAGCGACACCTCC-3'), which generated a 580-bp fragment.

DNA sequencing of the region containing rpeA was performed at the University of Arizona Biotechnology Center using an Applied Biosystems automatic DNA sequencer (model 373A, version 1.2.1). DNA sequence upstream of the transposon was obtained from pCW2A1 using the M13 forward primer specific to pUC18, whereas the sequence downstream of the transposon was obtain from pCW2D1 using a primer specific to bases 37 to 18 in the inverted repeat of Tn5 (31). From the sequence obtained, primers specific to the mutated region were designed and synthesized by GIBCO-BRL. These primers were used for additional sequence analysis, generation of gene-specific probes, and PCR amplification. The DNA sequence of the wild-type rpeA region in pCW2E4 was obtained using gene-specific primers. Additional sequence was obtained from pCW2C8 using primers to EZ::TN<KAN-2> (Epicentre, Madison Wis.) and from subclones of that fragment in pUC18 (pUC18-5.8, pUC18-2.4F, pUC18-2.3F, and pUC18-1.9R) using M13 Forward, M13 Reverse, gene-specific primers, and primers specific to EZ::TN<KAN-2>. DNA sequence analyses and deduced protein sequence analyses and comparisons with GenBank sequences were accomplished with software from the Genetics Computer Group, Inc., (Madison Wis.) (8), with the BLAST software package (1), with Motif Scanner software (39), and using the protein families database of alignments web site (http://pfam.wustl.edu) (3).

Generation of rpeA derivatives. To confirm that phenazine overexpression in 30-84ZrpeA was associated with the transposon, the mutation was reintroduced into the genome of 30-84Z by marker-exchange mutagenesis using pCW2B1, generating 30-84ZrpeA2. To generate a phenazine-producing derivative, 30-84rpeA, the phzB::lacZ mutation was replaced with the wild-type phzB gene contained in the cosmid pLSP259 (27). Replacement of phzB::lacZ with phzB was confirmed by loss of lacZ expression on X-Gal and concomitant gain of phenazine production on PPMD agar plates. A 30-84RrpeA double mutant was generated by replacing the phzR gene in strain 30-84rpeA with a phzR::Tn5lacZ transcriptional fusion by marker-exchange mutagenesis using pLSP259Tn5lacZ#42 (26). Colonies were screened for Km^r, Tc^s, and ß-galactosidase activity on LB agar plates. Replacement of phzR was confirmed by Southern analysis using a PCR-generated probe to phzR (data not shown).

Plasmid pCW2A6, which contained the wild-type rpeA region, was mutagenized using the EZ::TN<KAN-2> insertion kit (Epicentre, Madison, Wis.). Seventeen independent insertions in the SstI genomic fragment were generated and screened for loss of complementation, assessed as no reduction in phzB::lacZ expression in 30-84ZrpeA by the mutagenized plasmid. One such plasmid that exhibited loss of complementation, pCW2C8, was marker exchanged into the genome of strain 30-84Z, creating 30-84ZrpeA3.

Quantification of gene expression and antibiotic production. Phenazine antibiotics were extracted from strain 30-84 and derivatives and quantified by UV-visible light spectroscopy as described previously (5, 27). Briefly, triplicate 5-ml cultures grown overnight at 28°C with shaking in PPM broth were centrifuged, and the supernatants were acidified to ca. pH 2 with concentrated HCl. Phenazines were extracted twice with an equal volume of benzene for 1 h. Following evaporation of the benzene under air, phenazines were resuspended in 0.5 ml of 0.1 N NaOH, and serial dilutions were quantified via absorbance at 367 nm. The absorbance for each sample was normalized to total absorbance per initial 5-ml culture. The extractions were repeated, and the data from both experiments were combined (n = 6) and reported.

Expression of phzB was assessed from duplicate cultures of the phzB::lacZ reporter strain 30-84Z containing different plasmid constructs. The assay was repeated, and one representative experiment was reported. Expression of phzR was determined from duplicate cultures using a genomic phzR::Tn5lacZ fusion. The assay was repeated. ß-galactosidase assays were performed by a standard method (21).

AHL extraction and biological assays. Total AHL extractions were prepared from cell-free supernatants of strain 30-84Z derivatives as described previously (24). Briefly, triplicate 5-ml cultures were grown overnight at 28°C with shaking in PPM broth. The cultures were centrifuged, and supernatants were extracted with an equal volume of acidified ethyl acetate. The ethyl acetate phase was evaporated under anhydrous nitrogen gas, and the dried extracts containing AHLs were suspended in a volume of PPMD equal to the original culture, which was subsequently filter sterilized. AHL production was quantified by inoculating the extracted AHLs with the AHL-specific reporter P. aureofaciens strain 30-84I/Z (phzI phzB::lacZ) (37). ß-Galactosidase activity was determined on cultures grown with shaking at 28°C after 24 h as described previously (5). The assays were repeated, and one representative experiment was presented.

Statistical analysis. Effects of treatment between strains 30-84R and 30-84RrpeA were determined by analysis of variance (ANOVA) using SAS software (version 8.0; SAS Institute Inc., Cary N.C.). Mean ß-galactosidase activities (expressed as Miller units) and phenazine accumulation were compared among treatments by two-way factorial analysis of variance with bacterial strain and growth medium as factors. A significant interaction between factors (P > 0.0001) was found, so data were reanalyzed using separate one-way ANOVA by bacterial strain and by medium. Least-significant-difference comparison tests were used when treatment effects were significant.

Pathogen inhibition assays. To measure the ability of strains to inhibit the pathogen G. graminis var. tritic, overnight cultures (15 µl each) of strain 30-84 and derivatives were spotted onto triplicate KMPE plates. After 2 days of growth at 28°C, a 6-mm plug of G. graminis var. tritici was placed in the center of the plates. After 4 days, zones of inhibition, the distance between the edge of the bacterial colony and the fungal mycelium, were measured. The assays were repeated, and one representative experiment was presented.

Nucleotide sequence accession number. The nucleotide sequence of the rpeA gene has been deposited in GenBank (accession no. AY212250).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolation of a derivative of P. aureofaciens with enhanced phenazine biosynthetic gene expression. To identify repressors of phenazine biosynthetic gene expression, we mutagenized strain 30-84Z, a derivative of P. aureofaciens strain 30-84 containing a phzB::lacZ fusion, with Tn5 and screened mutants visually for enhanced phzB expression as indicated by darker blue colonies on M9 agar plates supplemented with X-Gal (data not shown). Strain 30-84Z was used in place of strain 30-84 due to the diffusibility of phenazines, which complicates visual screening of colonies. We identified a single derivative of strain 30-84Z, 30-84ZrpeA, that appeared to express higher ß-galactosidase activity. A quantitative ß-galactosidase time course assay of strains 30-84Z and 30-84rpeA in M9 minimal medium (Fig. 1) demonstrated that unlike the case with strain 30-84Z, phzB expression by 30-84ZrpeA was detectable at lower cell densities and was expressed at higher levels.

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FIG. 1. Expression of phenazine biosynthetic genes using a phzB::lacZ transcriptional reporter was determined at various time points during the growth cycle in M9 for duplicate cultures of strain 30-84Z () and strain 30-84ZrpeA (). Standard errors typically were less than 10%; error bars not shown.

To confirm that overexpression of phzB was linked to the Tn5 insertion, we cloned the genomic DNA containing Tn5 and reintroduced the mutated region into the chromosome of strain 30-84Z, creating 30-84ZrpeA2. The 30-84ZrpeA2 derivatives also appeared darker blue than 30-84Z (data not shown), confirming that the Tn5-disrupted rpeA locus enhanced phzB::lacZ expression.

Overexpression of the phenazine biosynthetic operon in strains 30-84ZrpeA and 30-84ZrpeA2 was assessed quantitatively via ß-galactosidase assays in comparison with strain 30-84Z. In M9 minimal medium, where phenazine expression by strain 30-84 is very low, both rpeA derivatives overexpressed phzB by up to 20-fold compared to results with strain 30-84Z (Table 2). In LB, where the level of phenazine expression by strain 30-84Z is relatively high, overexpression of phzB by the rpeA derivatives was increased by less than twofold compared to results for strain 30-84Z (Table 2). A spontaneous gacS mutant of 30-84ZrpeA did not express phzB in either medium (Table 2), but expression in M9 was fully restored, to 410 ± 95 Miller U, by multiple copies of gacS in pRKgacSB03 (6), whereas multiple copies of gacA did not complement the mutation. Additionally, phzB::lacZ expression was unchanged (below the detection limit of ≥1 Miller unit) in the 30-84ZgacS/rpeA mutant supplemented with exogenous AHL extract.

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TABLE 2. Phenazine biosynthetic gene expressiona,b

Identification of the rpeA locus. The Tn5-containing region responsible for increased phenazine expression and the corresponding wild-type region, isolated from an SstI sublibrary of 30-84 genomic DNA, were sequenced. Sequence analysis indicated that Tn5 was located within the codon for amino acid T112, disrupting a putative open reading frame (ORF) that we named RpeA (Fig. 2). To confirm that disruption of RpeA and not a linked ORF influenced expression of phzB, the wild-type region corresponding to rpeA was cloned (pCW2A6) and was shown to reduce phzB::lacZ expression by 30-84ZrpeA when carried in trans in multiple copies (Table 3). However, multiple copies of rpeA did not decrease phzB expression further in strain 30-84Z (Table 3). To verify that inactivation of the RpeA ORF repressed phzB::lacZ expression, pCW2A6 was mutagenized and loss of RpeA repression was assessed. Of 17 independent random TN::EZ<KAN> insertions generated in the genomic fragment, only one insertion, located in pCW2C8, caused loss of complementation in 30-84ZrpeA (Table 3). The same mutation enhanced phzB::lacZ expression when it replaced the wild-type gene in the chromosome of strain 30-84Z, creating 30-84ZrepA3 (Table 2). Sequencing of the pCW2C8 plasmid identified that the insertion was located in rpeA within the codon for S221 and not in an adjacent ORF (Fig. 2).

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FIG. 2. Predicted RpeA amino acid sequence. The 2,040-bp region that contains the RpeA ORF is given. The deduced single-letter amino acid sequence of RpeA is shown below the nucleotide sequence. The location of the original Tn5 insertion and the TN::EZ<KAN> insertion are indicated by black and gray arrows, respectively. A putative ribosomal binding site is double underlined, and potential start and stop codons are indicated in bold. Selected restriction enzyme recognition sites are underlined. Potential transmembrane domains are indicated by double dashed underlines (15). The underlined amino acids represent sequence similarity (E-value, 2.1 x 10-30) to bacterial histidine kinase domains, and the dotted underlined amino acids represent sequence similarity (E-value, 3.7 x 10-34) to histidine kinase ATPases. The putative histidine residue involved in phosphorelay is indicated in bold with an asterisk (11).

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TABLE 3. Extrachromosomal complementation of rpeA mutationa,b

The predicted product of the rpeA gene was similar to conserved but uncharacterized histidine kinases from putative two-component regulatory systems from several bacterial species, including Pseudomonas fluorescens (National Center for Biotechnology Information protein accession number ZP_00087977, 87% identical; and ZP_00087652, 35% identical), Pseudomonas putida (NP_745591, 58% identical) (22), Pseudomonas aeruginosa (NP_250489, 38% identical) (36), and Ralstonia solanacearum (NP_521184, 35% identical) (32). The predicted amino acid sequence of RpeA was 34% identical to that of RstB from E. coli (NP_288044) (23), and the partial sequence of an ORF overlapping and upstream of RpeA was also similar to that of rstA, which is located upstream of rstB (data not shown). Comparison of RpeA with similar uncharacterized ORFs revealed several regions of homology corresponding to functional domains and included a histidine kinase-like ATPase domain from amino acids 324-430 and a dimerization and phospho-acceptor domain of histidine kinases from amino acids 212 to 277 (Fig. 2).

Loss of rpeA enhanced phenazine production. To determine the effect of the rpeA mutation on phenazine production, an isogenic derivative of strain 30-84ZrpeA was constructed by replacing phzB::lacZ with an intact phzB gene (30-84rpeA), and total phenazine production was quantified. In each of the three media routinely used in our laboratory for culturing strain 30-84 and in which strain 30-84 yields relatively different amounts of phenazine, strain 30-84rpeA produced larger amounts of phenazine than wild-type strain 30-84 (Fig. 3).

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FIG. 3. Phenazine extractions from strains 30-84 (black bars) and 30-84rpeA (gray bars) from six 5-ml overnight cultures in PPM, LB, and M9 media were diluted serially and quantified by UV-visible light spectroscopy. The reported value is the absorbance divided by the dilution factor and therefore represents the total absorbance of undiluted sample at 367 nm. Error bars represent standard errors.

Loss of rpeA did not increase AHL levels. We demonstrated that phenazine expression in M9 minimal medium was detectable at a low cell density in an rpeA derivative and was higher than in the parental strain (Fig. 1). These data could indicate that loss of rpeA resulted in higher levels of AHL signal. This hypothesis further suggests that repression of phenazine under conditions where phenazine production is not favored, such as in M9, functions by preventing activation of biosynthetic gene transcription via PhzR. We tested this hypothesis by measuring the amount of AHL produced by strains 30-84Z and 30-84ZrpeA in various media using strain 30-84I/Z (phzI phzB::lacZ) as a reporter. AHL levels produced by strains 30-84Z and 30-84ZrpeA did not differ from each other in any of the media tested (Fig. 4), indicating that enhanced phzB::lacZ expression in strain 30-84ZrpeA was not due to an increase in AHL accumulation. Furthermore, AHL accumulation for strain 30-84Z did not differ between LB and M9 (Fig. 4), indicating that low production of phenazine by strain 30-84 in M9 (Fig. 3) was not due to reduced AHL accumulation.

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FIG. 4. Total AHL accumulation was quantified from triplicate overnight cultures of strain 30-84Z (black bars) and strain 30-84ZrpeA (gray bars) using the AHL-specific reporter strain 30-84I/Z (phzI phzB::lacZ). The amount of AHL was assessed by ß-galactosidase assays and is reported in Miller units. Error bars represent standard errors.

PhzR was not required for phenazine production in the absence of RpeA. Although it was apparent that the rpeA mutation did not enhance phenazine production by increasing AHL accumulation, it was still possible that rpeA functioned by regulating the expression of the AHL sensor PhzR. To test if the increased phenazine production was due in part to an increase in PhzR expression, we measured phzR expression in LB, PPMD, and M9 medium in both the presence and the absence of functional RpeA using a chromosomal phzR::Tn5lacZ fusion. The expression of phzR did not vary among these media in the presence of RpeA. In the absence of RpeA, phzR expression did not change in LB or PPMD medium, but it was increased significantly in M9 medium (from 321.4 Miller U in strain 30-84R to 1237.0 Miller U in strain 30-84RrpeA). However, it is important to note that antibiotic quantified from these cultures indicated that in all three media, the phzR mutant produced significantly less phenazine than the wild-type strain, whereas phenazine production by the phzR/rpeA double mutant did not differ from that of the wild-type strain in any of the media tested.

Strain 30-84rpeA enhanced pathogen inhibition in vitro. The ability of strain 30-84rpeA and strain 30-84 to inhibit growth of the plant pathogen G. graminis var. tritici was measured using an in vitro plate inhibition assay. The zone of fungal growth inhibition for strain 30-84 was 9.5 ± 0.2 mm. No zone was seen for the non-phenazine-producing 30-84.gacA mutant, as expected. Multiple copies of the entire biosynthetic region in pLSP259 or of phzI and phzR in pLSP2.7#20 when present in strain 30-84 increased the zone to 12.4 ± 0.3 mm and 10.3 ± 0.3 mm, respectively. However, the rpeA mutant had a 13.4 ± 0.1 mm zone, 37% larger than the zone produced by strain 30-84.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization of phenazine regulation by strain 30-84 has revealed many complexities in the activation of phenazine production, but prior to this study, genetic screens had not identified any negative regulators. The possibility that repression turns off phenazine gene expression is supported by the observation that in the wheat rhizosphere, exogenous carbon addition enhances gene expression assessed with phzB transcriptional fusions, but the increase in expression is not sustained and levels sharply diminish (F. Gong and L. S. Pierson III, unpublished data). One explanation for the observed decrease in phenazine expression is the elimination of AHL-mediated activation, for which there are examples in other organisms. In the opportunistic animal pathogen P. aeruginosa, production of a related phenazine antibiotic, pyocyanin, decreases in the presence of multiple copies of qscR, which represses transcription of lasI, encoding an AHL synthase (7). This serves to reduce the production of 3-oxo-C12-HSL required for expression of pyocyanin and other virulence factors (7). Furthermore, production of pyocyanin is suppressed in P. aeruginosa by multiple copies of aiiA (10), a gene encoding an AHL-lactonase isolated from Bacillus sp., demonstrating that directed degradation of AHLs, albeit from an enzyme that is not known to be produced by pseudomonads, could influence pyocyanin production (30). One such mechanism was demonstrated in the plant pathogen A. tumefaciens, where degradation of the AHL N-3-oxo-octanoyl homoserine lactone and concomitant loss of activation of the AHL-controlled products (40) is through an endogenous AHL-lactonase similar to that encoded by aiiA. Recently an additional negative regulator of pyocyanin, MvaT, was identified for P. aeruginosa (9). A mutation in mvaT led to both enhanced production of pyocyanin and increased production of AHL signals (9). In contrast to negative regulation functioning in other quorum-sensing cascades, RpeA repression in P. aureofaciens does not appear to correlate with a change in AHL signal accumulation. Interestingly, RpeA did influence phzR expression in minimal medium, implying that RpeA may act via increasing PhzR levels. However, while phzR expression in the phzR/rpeA double mutant was greatest in M9 medium, phenazine production was not as high as in complex medium. Also, PhzR was not required for phenazine production in the absence of the RpeA repressor in complex media. These results clearly indicate that RpeA influences phenazine by a mechanism other than the regulation of quorum sensing activation.

Although the mechanism of action of RpeA is still unclear, this study does elucidate the role of negative regulation in phenazine production. First, we observed that the rpeA mutation had a greater impact on phzB expression in minimal medium where phenazine production is normally low but had little impact on expression in rich media where phenazine production is normally high (Table 2 and Fig. 3). These data indicate that under nutrient conditions conducive for phenazine production, RpeA has little impact on phzB gene expression, whereas under conditions where phenazine production is not favored, repression through RpeA may play a major role in determining expression levels. Consistent with this interpretation, previous research correlates phenazine production in rich media with the accumulation of AHL (26), but the lack of phenazine production in M9 did not correlate with a reduction in AHL signal (Fig. 3 and 4). Indeed, the levels of AHL signal produced by strain 30-84Z in LB and M9 were not different (Fig. 4); nonetheless, phenazine accumulation was nearly undetectable from 30-84 in M9 and differed from the amount produced in LB (Fig. 3). These data suggest that lack of production of phenazine under some conditions is due not to the absence of sufficient AHL but to repression, such as through the activity of RpeA. Second, we observed that a 30-84ZgacS/rpeA mutant lacked phzB expression (Table 2) even in the presence of exogenous AHL. The observation that bypassing the need for activation of AHL synthesis via GacS/GacA combined with derepression of expression via an rpeA mutation cannot restore production of phenazine to a GacS mutant indicates that GacS/GacA controls phenazine production by a still-unknown mechanism. These data also reinforce the importance of GacS/GacA activation of phenazine production.

The data from this study suggests that RpeA may function as a sensor of the metabolic state of the cell, transducing information that is important in determining whether expression of secondary metabolites, such as phenazines, is appropriate. Under certain nutrient conditions, RpeA effectively blocks phzB expression even when quorum-sensing signals have accumulated. The uncoupling of AHL signal accumulation and phenazine production in minimal medium in the presence of a functional rpeA gene reinforces that multiple signals, including unknown signals that function through GacS/GacA, are integrated during the process of host association to trigger phenazine expression appropriately.

Sequence analysis of the rpeA gene identified that it is a member of a group of putative two-component regulators with unknown function from various bacteria including plant and human pathogens. Although two-component regulators function predominantly by activating gene expression, a role as a repressor is not unprecedented. For example, the two-component regulator GacA has a dual function, since gacA mutants have decreased phenazine expression and also have enhanced fluorescence, indicating an apparent increase in siderophore production (5). The mechanism(s) of repression by GacA and RpeA is not known, but it may involve the positive regulation of repressors or the antagonism of activation. RpeA repression of phenazine production, a secondary metabolite important in competitive fitness during host association, is the first phenotype to be associated with this family of regulators. The deduced RpeA sequence was similar to those of the products of two paralogous genes identified in the genome of P. fluorescens; however, for the completed genomes of other bacteria, it was similar to only one homolog. This may indicate that duplication led to specialization of RpeA as a regulator of secondary metabolite production in these biocontrol bacteria alone, but it is equally plausible that other members with lower sequence identity also function in host association. The conservation of several motifs within the deduced protein sequence that are involved in phosphorelay supports their assignment as signal transducers. The conserved domains include an ATPase domain and a dimerization and phosphoacceptor domain. Conservation of RpeA within a group of organisms all of which are known to associate with eukaryotic hosts suggests that future studies involving RpeA could provide insight into general regulation of secondary metabolites that contribute directly to host association by both pathogens and beneficial bacteria.

Because phenazine production by strain 30-84 contributes to its capacity in biological control, we tested the ability of 30-84rpeA to inhibit G. graminis var. tritici. In in vitro plate assays, strain 30-84rpeA was better at inhibiting mycelial growth of the fungus than wild-type strain 30-84 (Table 4). These data support the hypothesis that an rpeA mutation that enhances phenazine production may improve biological control in the wheat rhizosphere. Based on our observations that the effect of rpeA is dependent on culture conditions and that an rpeA mutation had a greater impact on phenazine production under conditions where the antibiotic is not normally produced, improving biological control success by a mutation in rpeA may depend on whether the wheat rhizosphere represents a condition that is repressive or inductive for phenazine production. Future studies will determine the mechanism of RpeA regulation of phenazine production and evaluate the long-term effect of the rpeA mutation on bacterial colonization, persistence, and take-all disease suppression on wheat.

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TABLE 4. Pathogen inhibition

 
We thank Robin Horowitz for generating the original rpeA::Tn5 mutation, Baomin Wang for construction and screening of plasmid libraries evaluated for use in this study, Patricia Figuli for constructing subclones used for sequence analysis, and Elizabeth Pierson for statistical analysis. We also thank Scott Chancey and Elizabeth Pierson for helpful conversations during the design and completion of these studies and E. G. Ruby for his generous support for the completion of these studies.

Part of this work was supported by U.S. Department of Agriculture NRICGP grant 2001-02684 (L.S.P.).

 
* Corresponding author. Mailing address: Department of Plant Pathology, P.O. Box 210036, Forbes Building, Room 204, The University of Arizona, Tucson, AZ 85721. Phone: (520) 621-9419. Fax: (520) 621-9290. E-mail: lsp{at}u.arizona.edu.

Present address: Pacific Biomedical Research Center, Kewalo Marine Laboratory, University of Hawaii, Honolulu, HI 96813.

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Journal of Bacteriology, July 2003, p. 3718-3725, Vol. 185, No. 13
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.13.3718-3725.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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