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Appl Environ Microbiol. Apr 2009; 75(7): 2122–2131.
Published online Jan 30, 2009. doi:  10.1128/AEM.02052-08
PMCID: PMC2663185

Evolutionary History of the phl Gene Cluster in the Plant-Associated Bacterium Pseudomonas fluorescens[down-pointing small open triangle]

Abstract

Pseudomonas fluorescens is of agricultural and economic importance as a biological control agent largely because of its plant association and production of secondary metabolites, in particular 2,4-diacetylphloroglucinol (2,4-DAPG). This polyketide, which is encoded by the eight-gene phl cluster, has antimicrobial effects on phytopathogens, promotes amino acid exudation from plant roots, and induces systemic resistance in plants. Despite its importance, 2,4-DAPG production is limited to a subset of P. fluorescens strains. Determination of the evolution of the phl cluster and understanding the selective pressures promoting its retention or loss in lineages of P. fluorescens will help in the development of P. fluorescens as a viable and effective inoculant for application in agriculture. In this study, genomic and sequence-based approaches were integrated to reconstruct the phylogeny of P. fluorescens and the phl cluster. It was determined that 2,4-DAPG production is an ancestral trait in the species P. fluorescens but that most lineages have lost this capacity through evolution. Furthermore, intragenomic recombination has relocated the phl cluster within the P. fluorescens genome at least three times, but the integrity of the cluster has always been maintained. The possible evolutionary and functional implications for retention of the phl cluster and 2,4-DAPG production in some lineages of P. fluorescens are discussed.

The Pseudomonas genus includes species that live in close association with eukaryotic hosts such as insects, plants, and humans. Some of these, for example, P. entomophila, P. syringae, and P. aeruginosa, are pathogens, whereas others, such as P. fluorescens, may be beneficial to the host. It is emerging that there is often a fine line separating pathogenic and beneficial interactions, which makes the determination of the genes that drive interactions between different pseudomonads and their hosts fundamental to understanding microbe-host interactions (49). In this regard, gene clusters (for example, the type three secretion loci in P. syringae and the insecticidal locus in P. entomophila) appear to be very important in pathogenic pseudomonads (17, 49, 63). In contrast, the molecular basis for beneficial associations between pseudomonads and eukaryotic hosts is poorly understood. P. fluorescens is the archetypical beneficial plant-associated bacterium and has been isolated from the rhizospheres of diverse plants, making this an ideal model to study the evolution of beneficial plant-microbe interactions. Gene clusters in P. fluorescens are known, and one of the best studied is the phl locus, which encodes the biosynthetic and regulatory genes for production of 2,4-diacetylphloroglucinol (2,4-DAPG). This low-molecular-weight polyketide inhibits a variety of phytopathogens, including fungi, helminths, and nematodes, and is responsible for the biocontrol capabilities of many P. fluorescens isolates (8, 16, 22, 30, 42, 64, 65). 2,4-DAPG also directly affects plants and is reported to trigger induced systemic resistance, leading to enhanced plant protection (26, 54), and to promote exudation of amino acids from the plant root (45). Intriguingly, there are a number of reports that some plant genotypes actually select for 2,4-DAPG-producing strains, suggesting that 2,4-DAPG may be involved in mediating some type of association with plant hosts (39, 46).

Despite the potential benefits of 2,4-DAPG production, the percentage of P. fluorescens isolates that produce 2,4-DAPG is likely to be in single figures (12, 42; J. A. Moynihan and J. P. Morrissey, unpublished data). The 8-kb cluster involved in the biosynthesis, regulation, export, and degradation of 2,4-DAPG consists of eight genes, phlHGFACBDE, and is conserved at the organizational level in 2,4-DAPG-producing strains (1, 2, 5, 7, 13, 31, 57). The key biosynthetic gene is phlD, which displays interesting similarity to genes for plant chalcone synthases. This type III polyketide synthase is unusual in prokaryotes, and it was suggested that the gene may have been acquired by horizontal gene transfer (5, 6, 12). In contrast, phylogenetic analysis of phlD does not support this theory, so the provenance of 2,4-DAPG production remains uncertain (52, 53). Understanding the evolution of the phl biosynthetic genes offers a route to determining the precise role of 2,4-DAPG in beneficial plant-microbe interactions. In this study, we exploited genomic resources to reconstruct the evolutionary history of the phl cluster in P. fluorescens.

MATERIALS AND METHODS

Genome sequences.

The complete nucleotide sequences and annotations of the P. fluorescens Pf-5, P. fluorescens Pf0-1, P. putida KT2440, P. aeruginosa PAO1, P. syringae pv. tomato DC3000, P. syringae pv. phaseolicola 1448A, P. syringae pv. syringae B728a, and P. entomophila L48 genomes used in this project were retrieved and downloaded from the National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov/genomes) (9, 15, 28, 43, 44, 60, 63). Relevant sequences from P. fluorescens SBW25 were retrieved from the Sanger Institute (http://www.sanger.ac.uk/Projects/P_fluorescens/). A 170-kb bacterial artificial chromosome (BAC) library clone from P. fluorescens F113, which contained the phl cluster, was also examined. For examination of the regions flanking the phl cluster, BLAST analysis was first performed (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Approximately 10 of the open reading frames (ORFs) flanking the cluster in Pf-5 were analyzed against P. fluorescens Pf0-1, P. putida KT2440, P. aeruginosa PAO1, P. syringae pv. tomato DC3000, P. syringae pv. phaseolicola 1448A, P. syringae pv. syringae B728a, and P. entomophila L48. In addition to this, bioinformatic analysis was also performed using WebACT, an Artemis comparison tool based on BLAST scores that allows for the comparison of complete genomes (www.webact.org/WebACT/home) (3).

F113 annotation and mapping.

Annotation of an approximately 170-kb BAC clone was performed using Artemis. Predicted ORFs were identified using two gene prediction programs, Glimmer and Orpheus (14, 19). BLASTP analysis was performed to determine the putative function of the predicted gene. To search for the presence of tRNA in the F113 sequence, tRNAscan-SE was used (35). Following the identification of putative genes, mapping of the 170-kb region against other sequenced Pseudomonas species was performed to identify core regions present in P. fluorescens sequenced strains and regions present in other Pseudomonas species. BLAST identity values as well as gene numbers were noted to identify regions of interest. Genes were scored as positive if the identity values were above 70%. Genes with the identity values close to 70% were also acceptable if these genes were contiguous with other genes that had identity values of 70% or greater.

Bacterial strains.

A total of 36 bacterial strains, comprising 21 2,4-DAPG-producing and 15 nonproducing strains, were used in this study (Table (Table1).1). These strains were isolated from different plant rhizospheres in diverse geographical locations. With the exception of P. brassicacearum NFM421, all are classified as P. fluorescens. Standard Pseudomonas growth and storage conditions were used.

TABLE 1.
Strains used in this study

DNA isolation.

Chromosomal DNA was extracted from a 5-ml overnight culture of each strain in LB broth using the G-nome DNA isolation kit following the manufacturer's protocol (Bio 101, Cambridge, United Kingdom). Following extraction, DNA was resuspended in 200 μl of 10 mM Tris buffer (pH 8), quality was verified on a 0.6% agarose gel, and DNA was stored at −20°C.

Molecular analysis.

The oligonucleotide primers used in this study are listed in Table Table2.2. Standard PCR mixtures and PCR conditions were employed: typically, an initial denaturation step at 96°C for 3 min followed by 30 cycles of denaturation at 94°C for 30 s, 30 s of primer annealing with temperatures dependent on the primer pairs used, and an extension of 1 kb per min at 72°C, in a PTC-200 Peltier thermal cycler (MJ Research). PCR using the primer pair Phl2a and Phl2b (50) was used to identify phlD-positive strains. PCR assays were also performed to examine the genomic context of the phl cluster, based on primers from Pf-5 and F113, among our collection of phl-positive isolates. For two phl-positive strains, Q2-1 and Q1-87, additional analysis of the genomic context was performed using a semirandom, two-step PCR protocol (ST-PCR) (10). The phl-negative insertion site was examined using primers designed to Pf0-1 and SBW25 (Table (Table22).

TABLE 2.
Primers used in this study

Multilocus sequence analysis.

Multilocus sequence analysis was performed using primer pairs designed for PCR amplification of four housekeeping (HK) genes (gyrB, leuB, groEL, and rpoD) and one gene from the phl cluster, phlD (Table (Table2).2). Many of the phlD sequences from the phl-positive isolates were obtained through the accession numbers given in Table Table1.1. Following PCR product amplification, PCR purification was performed using the JetQuick protocol from GenoMed (Löhne, Germany) or the gel extraction kit protocol from Qiagen (West Sussex, United Kingdom), and sequencing was performed by MWG Biotech (Martinsried, Germany).

Phylogenetic analysis.

Sequences for the four HK genes and phlD were aligned using the ClustalW multiple-sequence alignment program (http://www.ebi.ac.uk/clustalw/) (62). Further sequence analysis was performed using the Molecular Evolutionary Genetics Analysis (MEGA) version 3.1 program (32). The rates per site of synonymous (KS) and nonsynonymous (KN) substitutions were calculated. The KN/KS ratios, which provide information on whether purifying (ratio, <1), neutral (ratio, 1), or diversifying (ratio, >1) selection is acting on the gene under study, were determined. Tranversion and transition changes were also examined. Total nucleotide diversity (π) was measured for each gene between the two main lineages, phl positive and phl negative. The concatenated sequence was used to measure π within the phl-positive strains and within the phl-negative lineage. Four different statistical tests to test for recombination in each gene sequence were employed. These were the maximum chi-square test (http://www.lifesci.sussex.ac.uk/CSE/test/maxchi.php) (48, 59), LD (r2 versus distance), LD (D′ versus distance) (http://www.lifesci.sussex.ac.uk/CSE/test/ld.php) (23, 34, 47), and the PHI test (SplitsTree4, version 4.8) (25). Phylogenetic trees for the phlD gene and concatenated HK genes were constructed using the neighbor-joining method with the Jukes-Cantor distance method for all sites (29, 55). Bootstrap tests using 1,000 replicates were performed to test the robustness of each phylogeny.

Nucleotide sequence accession numbers.

The nucleotide sequences determined in this study have been deposited in the GenBank database under accession numbers FJ012172 (F113 BAC clone), FJ012173 to FJ012205 (groEL), FJ012206 to FJ012238 (gyrB), FJ012239 to FJ012271 (leuB), FJ012277 to FJ012309 (rpoD), FJ012272 to FJ012276 (phlD), FJ012310 and FJ012311 (Q2-1 and Q1-87 3′ region before the 2,4-DAPG gene cluster), and FJ012312 and FJ012313 (Q2-1 and Q1-87 5′ region after the 2,4-DAPG gene cluster).

RESULTS

The phl gene cluster is located in several genomic loci.

To understand the evolution of the 2,4-DAPG biosynthetic and regulatory genes, we first looked at the genomic context of the phl cluster (PFL_5951-PFL_5958) in the sequenced strain P. fluorescens Pf-5. We compared the region flanking the phl cluster in Pf-5 with genomes of other sequenced Pseudomonas species (Fig. (Fig.1a;1a; see Table S1 in the supplemental material). This analysis showed that although these other Pseudomonas strains do not carry the phl cluster, the genes 5′ and 3′ of the cluster in Pf-5 have clear orthologues in all the sequenced Pseudomonas strains. Furthermore, with only minor variations, these orthologous genes are contiguous in all species, indicating that the phl cluster in Pf-5 is located in a region of the ancestral Pseudomonas genome. To determine whether the phl cluster genomic location was a hot spot for integration of foreign DNA in P. fluorescens, we analyzed this genomic region in phl-negative strains using primers designed from Pf0-1 and SBW25 gene sequences (Fig. (Fig.1b).1b). Some variation in the size of the product at this site was seen, but no evidence for large DNA insertions was found in Pf0-1 and SBW25 or any of the other 13 phl-negative strains examined (data not shown). This demonstrates that this genomic region is not an insertional hot spot. As expected, a band was not amplified from the phl-positive strain Pf-5 under these conditions, but unexpectedly, another phl-positive strain, P. fluorescens F113, resembles the phl-negative strain SBW25 at this locus, which therefore indicates that a large DNA insertion is not present at this locus in strain F113 (Fig. (Fig.1b).1b). This finding means that the phl cluster has different locations in the Pf-5 and F113 genomes and raised the possibility that the cluster could be on a mobile genetic element.

FIG. 1.
The P. fluorescens Pf-5 phl cluster is located within the core Pseudomonas genome. The phl cluster genes are surrounded by a box. The arrows on either side represent the genes flanking the cluster in P. fluorescens Pf-5 and indicate ORF orientation. Also ...

To determine the genomic context of the phl locus in F113, we annotated and analyzed a 170-kb F113 BAC clone that carried the phl genes to establish the genomic context of the phl cluster in strain F113. Along with the eight genes of the phl cluster, we identified 109 putative genes on this BAC clone. There were two interesting features to these genes: first, most did not have clear orthologues in other sequenced Pseudomonas species and thus constitute a large genomic region that appeared to be unique to strain F113, and second, there were several examples of gene clusters with orthologues in P. syringae or P. entomophila but not in other P. fluorescens strains (see Tables S2 and S3 in the supplemental material). The phl cluster had the same gene order in Pf-5 and F113 and showed a high level of sequence similarity, averaging 77% at the nucleotide level and 81% at the amino acid level over the eight genes. Comparison of the flanking genes showed that the phl cluster in F113 was not located in the same genomic context as in Pf-5, which led us to investigate the location of the phl cluster among a collection of 21 phl-positive isolates by PCR and Southern blotting using primer pairs specific for either the Pf-5 or F113 locations (Table (Table2).2). Employing these strain-specific primers, PCR was used to amplify the sequences between the first gene before the cluster and the phlG gene (5′ region) and between the phlD gene and a gene downstream of the cluster (3′ region). Of the 19 additional strains, 5 strains resembled Pf-5, 12 strains resembled F113, and 2 strains, Q2-1 and Q1-87, returned inconclusive results (data not shown). To further analyze strains Q2-1 and Q1-87 and to identify the regions flanking the phl cluster, we performed ST-PCR. This established that the 5′ region flanking the cluster in Q2-1/Q1-87 resembles the F113 context but the 3′ region is distinct, with the second putative gene downstream of the cluster showing most similarity to an oligopeptide transporter from P. syringae. The DNA region immediately 3′ of the cluster (downstream of the phlE gene) displayed interesting characteristics that led to further analysis. Examination of the sequence indicated that a putative gene of 921 nucleotides was present. This region was highly homologous to a putative gene that we identified in our annotation of the F113 BAC clone using Glimmer, and when identified, it had an E value of 1e−15 (see Table S3 in the supplemental material). In addition, however, the DNA showed homology to sequence within the 947-bp intergenic region found between the phlE gene (PFL_5958) and the glycine dehydrogenase gene (PFL_5959) in Pf-5. This putative coding sequence was not annotated as a gene in Pf-5; therefore, we carried out sequence alignment of the translated amino acid sequences of this putative gene from the three strains (Fig. (Fig.2).2). The alignment clearly showed that there is a highly conserved sequence in this location with levels of identity and similarity comparable to those for the genes in the phl cluster. The presence of this putative gene in all three strains, coupled with the lack of a homologue in other sequenced strains of Pseudomonas (see Table S3 in the supplemental material) or in other sequenced bacteria, indicates that this putative gene may constitute an additional gene in the phl cluster, which is involved in some way in 2,4-DAPG production. In conclusion, based on the location of the phl locus, the strains divided into three groups: those similar to Pf-5, those similar to F113, and two strains, Q2-1 and Q1-87, that resemble F113 at the 5′ end of the cluster but differ at the 3′ end (Fig. (Fig.33).

FIG. 2.
Alignment of the protein sequences of the putative gene identified in the Pf-5, F113, and Q2-1 lineages. This gene is located in the region following the phlE gene from the phl cluster in all lineages and may constitute an additional phl cluster gene. ...
FIG. 3.
The genomic locations of the phl gene cluster differ between P. fluorescens strains. Shown here are representatives of the main genomic location groups, F113, Q2-1/Q1-87, and Pf-5. The white arrows indicate genes flanking the phl cluster. The black arrows ...

Sequence analysis of HK genes and the phlD gene.

The low frequency of phloroglucinol-producing strains within the species P. fluorescens and the presence of the phl cluster in different genomic loci is reminiscent of horizontally transferred genetic elements. We addressed this possibility by comparing the phl cluster phylogeny with the species phylogeny of our strains. Four HK loci, gyrB, leuB, groEL, and rpoD, were partially sequenced from 21 phl-positive and 15 phl-negative isolates and used to reconstruct the phylogenetic history of the species. The number of polymorphic sites in each gene among the isolates was determined. A total of 623 polymorphic sites were present, with leuB (124 sites) being the least polymorphic gene and rpoD (195 sites) the most variable gene. The 623 polymorphic nucleotide sites resulted in 112 amino acid replacements, which ranged from 17 replacements for gyrB and leuB to 42 replacements for rpoD (see Table S4 in the supplemental material). To determine the phl cluster phylogeny, we obtained sequences for the phlD genes from all phl-positive strains. At the nucleotide level, phlD had 187 polymorphic sites, resulting in 36 amino acid replacements (see Table S4 in the supplemental material). The KN/KS ratio was <1 for all five loci, indicating that each of these genes is subject to purifying selection. We also analyzed the sequences for recombination using four statistical tests and found no evidence for recombination in the phlD, groEL, or leuB gene but inconclusive data for gyrB and rpoD (data not shown).

The phl cluster is ancestral in P. fluorescens.

To determine relationships among the 36 P. fluorescens isolates, we first constructed a phylogenetic tree using the 2.3-kb concatenated sequence of our four HK genes (gyrB, leuB, groEL, and rpoD) (Fig. (Fig.4a).4a). From the concatenated HK tree, two major lineages were observed, which correspond precisely to the 21 phl-positive and the 15 phl-negative isolates (Fig. (Fig.4a).4a). It was unexpected that the phl-positive and -negative isolates should have such distinct divergent lineages and indicates an evolutionary split soon after the species emerged. We examined nucleotide polymorphism and diversity within each lineage and found that the phl-negative lineage is more diverse (0.051) than the phl-positive lineage (0.014) (see Table S5 in the supplemental material). This suggests that the phl-positive lineage may be under selective constraints. Next, we constructed a phylogenetic tree using the phlD gene sequences of our 21 phl-positive isolates (Fig. (Fig.4b).4b). Both trees have an almost identical branching pattern, showing that the trees are highly congruent. These data demonstrate that the phlD gene has an evolutionary history identical to that of the species and is ancestral in phl-positive P. fluorescens. Close examination of the phl-positive strains on both trees shows three identical subgroupings (clades). The first comprises 12 strains related to F113; the second comprises two strains, Q2-1 and Q1-87; and the third comprises five isolates related to Pf-5. Crucially, these three distinct clades correlate to the three genomic locations groups of the phl cluster (F113, Pf-5, and Q2-1). Integrating the molecular phylogeny and distribution data, it is now established that the entire phl cluster is ancestral in phl-positive strains. The final question was whether the last common ancestor to give rise to P. fluorescens strains contained the phl cluster. To answer this, we reconstructed the phylogeny of the genus using the same four HK fragments as for the species tree (Fig. (Fig.5).5). This tree included two phl-positive strains, F113 and Pf-5; two phl-negative strains, Pf0-1 and SBW25; and representatives of P. syringae, P. putida, P. entomophila, and P. aeruginosa. Within the P. fluorescens lineage, Pf-5 and F113 formed the most divergent branches, suggesting that the phl cluster was present in the last common ancestor to give rise to P. fluorescens.

FIG. 4.
Evolutionary relationships using all sites in the concatenated HK tree consisting of 21 phl-positive strains (shown in color) and 15 phl-negative strains (in black) (a) and in the phlD gene tree among our collection of 21 phl-positive strains (shown in ...
FIG. 5.
Genus tree demonstrating the phylogenetic relationships among Pseudomonas species. Shown here are representatives of Pseudomonas species, i.e., P. syringae, P. fluorescens, P. putida, P. entomophila, and P. aeruginosa. Highlighted by a box are the P. ...

DISCUSSION

Although it has been known for some time that 2,4-DAPG production is an important trait in plant-associated strains of P. fluorescens, this is the first study to look at the evolution of the phl cluster. Most previous work focused on phlD, which encodes the key biosynthetic enzyme, a polyketide synthase. Those data were somewhat conflicting, with sequence homologies suggesting that PhlD may have a relationship to plant chalcone synthases but molecular phylogeny indicating that phlD was an ancestral gene. By comparing phlD phylogeny to a robust strain phylogeny, we confirmed the ancestral nature of phlD in P. fluorescens. Furthermore, we showed that phlD is part of a gene cluster that is also ancestral in the species and is not a recent acquisition. One of the most striking findings was that there was a clear bifurcation in the species phylogeny, with only one major branch retaining the phl cluster. Within the phl-positive lineage, our data show that the cluster has relocated within the genome at least three times. This is perfectly plausible since it is known that intragenomic recombination and rearrangements are common in Pseudomonas, and it will not be surprising if further lineages with additional phl locations are found. Indeed, another recent study of phlD-positive strains proposed that six subgroups of strains could be identified, though these data were based exclusively on gene sequences and did not consider genomic data (18). The identification of the putative gene at the 3′ end of the cluster in all strains is also intriguing. Even though the genomic context of the cluster varies among the strains, the phl cluster and this putative gene are retained in the same order each time, and like the phl genes, this sequence is also not present in the other Pseudomonas species. This supports an idea that this putative gene may be a bona fide member of the phl cluster, but this requires experimental testing and alternative explanations are also plausible. Analysis of the particular locations for the phl cluster indicates that they do not show any of the typical signatures for targeted insertion (e.g., tRNA genes or insertion sequence elements) and are likely to arise from random recombination (56). The number of different locations for phl and the finding that in the F113 lineage it is situated in a large lineage-specific genomic region that also carries genes that are ancestral in the genus support the premise that the cluster dates at least to the origin of the species. These data also make it clear that F113 and Pf-5 are representatives of major lineages of P. fluorescens and illustrate the value of examining genomic organization as well as sequences of individual genes when reconstructing species phylogenies. Evolutionary relationships and species identification are sometimes problematic for the fluorescent pseudomonads (as seen by the location of P. brassicacearum NFM421 in a P. fluorescens lineage), and this genomic approach may help resolve some of these conflicts.

Detailed analysis of the HK and the phlD gene sequences and the cluster structure identify some interesting points. First, the phlD locus is subject to purifying selection, indicating that it is functionally important. Second, whereas the strains that we selected for this study were chosen simply on the basis that they were phlD positive (typically by PCR), all 21 strains carried the entire cluster in one of the three locations. This suggests a selective pressure to maintain all the genes in the cluster together even in times when the genome as a whole underwent extensive rearrangement. Third, there is less diversity in the phl-positive lineage, indicating that these strains are evolving under some constraint. As outlined earlier, there are separate data that point to plant selection of 2,4-DAPG-producing strains of P. fluorescens in the rhizosphere (39, 46). Since Pseudomonas is predominantly a plant-associated bacterium, it is tempting to speculate that 2,4-DAPG plays a role in an intimate interaction between P. fluorescens and (some) plants that confers benefits to the plant (e.g., protection against phytopathogens). Although this would explain the selective pressure to retain the phl locus, this idea remains a speculative hypothesis at this stage. It must also be noted that some phl-negative strains also intimately associate with plant hosts.

Despite the widespread belief that intimate signaling and regulated gene expression underpin interactions between plants and associated bacteria, this has proved very difficult to establish. Specifically for Pseudomonas, some success has been obtained using in vitro expression technology (21, 27) and transcriptome analysis (37) to identify niche (plant)-specific or plant-induced regulatory genes. The data from the study reported here identify another strategy that may prove useful in dissecting intimate plant microbe interactions. By focusing on the phylogenetic and evolutionary aspects, we have identified specific P. fluorescens lineages that appear to have been selected to maintain 2,4-DAPG production and possibly plant association. The advent of inexpensive bacterial genome sequencing opens the possibility of sequencing entire genomes of strains from different lineages and undertaking global comparative genomics to identify the loci that are lineage specific and therefore likely to be of functional importance. The phl cluster itself is one such locus, and the ongoing challenge is to understand the full biological role of 2,4-DAPG in interactions between P. fluorescens and plant hosts.

Supplementary Material

[Supplemental material]

Acknowledgments

This work was supported by an Embark Initiative Postgraduate Research Scholarship from the Irish Research Council for Science and Engineering Technology (IRCSET) to J.A.M. Research in this area in J.P.M.'s and F.O.G.'s groups is supported in part by grants awarded by the Irish Department of Agriculture, Fisheries and Food (RSF grants 06-321 and 06-377 and FIRM grants 06RDC506 and 06RDC459); the Irish Marine Institute (Beaufort Award); the European Science Foundation (05/EDIV/FP107/INTERPAM); and the European Commission (O36314/MICROMAIZE; QLRT-2001-00914/PSEUDOMICS).

We thank Pat Higgins for technical support; Stephen Giddens for help with construction of the F113 BAC library; and Linda Thomashow, Christoph Keel, and Brian McSpadden-Gardener for providing strains.

Footnotes

[down-pointing small open triangle]Published ahead of print on 30 January 2009.

Supplemental material for this article may be found at http://aem.asm.org/.

REFERENCES

1. Abbas, A., J. E. McGuire, D. Crowley, C. Bayesse, M. Fow, and F. O'Gara. 2004. The putative permease PhlE of Pseudomonas fluorescens F113 has a role in 2,4-diacetylphloroglucinol resistance and in general stress tolerance. Microbiology 150:2443-2450. [PubMed]
2. Abbas, A., J. P. Morrissey, P. C. Marquez, M. M. Sheehan, I. R. Delany, and F. O'Gara. 2002. Characterisation of interactions between the transcriptional repressor PhlF and its binding site at the phlA promoter in Pseudomonas fluorescens F113. J. Bacteriol. 184:3008-3016. [PMC free article] [PubMed]
3. Abbott, J. C., D. M. Aanensen, K. Rutherford, S. Butcher, and B. G. Spratt. 2005. WebACT—an online companion for the artemis comparison tool. Bioinformatics 21:3665-3666. [PubMed]
4. Achouak, W., L. Sutra, T. Heulin, J. M. Meyer, N. Fromin, S. Degraeve, R. Christen, and L. Gardan. 2000. Pseudomonas brassicacearum sp. nov. and Pseudomonas thivervalensis sp. nov., two root-associated bacteria isolated from Brassica napus and Arabidopsis thaliana. Int. J. Syst. Evol. Microbiol. 50:9-18. [PubMed]
5. Bangera, M. G., and L. S. Thomashow. 1999. Identification and characterization of a gene cluster for synthesis of the polyketide antibiotic 2,4-diacetylphloroglucinol from Pseudomonas fluorescens Q2-87. J. Bacteriol. 181:3155-3163. [PMC free article] [PubMed]
6. Bode, H. B., and R. Müller. 2003. Possibility of bacterial recruitment of plant genes associated with the biosynthesis of secondary metabolites. Plant Physiol. 132:1153-1161. [PMC free article] [PubMed]
7. Bottiglieri, M., and C. Keel. 2006. Characterization of PhlG, a hydrolase that specifically degrades the antifungal compound 2,4-diacetylphloroglucinol in the biocontrol agent Pseudomonas fluorescens CHA0. Appl. Environ. Microbiol. 72:418-427. [PMC free article] [PubMed]
8. Brodhagen, M., M. D. Henkels, and J. E. Loper. 2004. Positive autoregulation and signaling properties of pyoluteorin, an antibiotic produced by the biological control organism Pseudomonas fluorescens Pf-5. Appl. Environ. Microbiol. 70:1758-1766. [PMC free article] [PubMed]
9. Buell, C. R., V. Joardar, M. Lindeberg, J. Selengut, I. T. Paulsen, M. L. Gwinn, R. J. Dodson, R. T. Deboy, A. S. Durkin, J. F. Kolonay, R. Madupu, S. Daugherty, L. Brinkac, M. J. Beanan, D. H. Haft, W. C. Nelson, T. Davidsen, N. Zafar, L. Zhou, J. Liu, Q. Yuan, H. Khouri, N. Fedorova, B. Tran, D. Russel, K. Berry, T. Utterback, S. E. Van Aken, T. V. Feldblyum, M. D'Ascenzo, W. L. Deng, A. R. Ramos, J. R. Alfano, S. Cartinhour, A. K. Chatterjee, T. P. Delaney, S. G. Lazarowitz, G. B. Martin, D. J. Schneider, X. Tang, C. L. Bender, O. White, C. M. Fraser, and A. Collmer. 2003. The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv. tomato DC3000. Proc. Natl. Acad. Sci. USA 100:10181-10186. [PMC free article] [PubMed]
10. Chun, K. T., H. J. Edenberg, M. R. Kelley, and M. G. Goebl. 1997. Rapid amplification of uncharacterized transposon-tagged DNA sequences from genomic DNA. Yeast 13:233-240. [PubMed]
11. Compeau, G., B. J. Al-Achi, E. Platsouka, and S. B. Levy. 1988. Survival of rifampin-resistant mutants of Pseudomonas fluorescens and Pseudomonas putida in soil systems. Appl. Environ. Microbiol. 54:2432-2438. [PMC free article] [PubMed]
12. Cook, R. J., L. S. Thomashow, D. M. Weller, D. Fujimoto, M. Mazzola, G. Bangera, and D. S. Kim. 1995. Molecular mechanisms of defense by rhizobacteria against root disease. Proc. Natl. Acad. Sci. USA 92:4197-4201. [PMC free article] [PubMed]
13. Delany, I., M. M. Sheehan, A. Fenton, S. Bardin, S. Aarons, and F. O'Gara. 2000. Regulation of production of the antifungal metabolite 2,4-diacetylphloroglucinol in Pseudomonas fluorescens F113: genetic analysis of phlF as a transcriptional repressor. Microbiology 146:537-546. [PubMed]
14. Delcher, A. L., D. Harmon, S. Kasif, O. White, and S. L. Salzberg. 1999. Improved microbial gene identification with GLIMMER. Nucleic Acids Res. 27:4636-4641. [PMC free article] [PubMed]
15. Feil, H., W. S. Feil, P. Chain, F. Larimer, G. DiBartolo, A. Copeland, A. Lykidis, S. Trong, M. Nolan, E. Goltsman, J. Thiel, S. Malfatti, J. E. Loper, A. Lapidus, J. C. Detter, M. Land, P. M. Richardson, N. C. Kyrpides, N. Ivanova, and S. E. Lindow. 2005. Comparison of the complete genome sequences of Pseudomonas syringae pv. syringae B728a and pv. tomato DC3000. Proc. Natl. Acad. Sci. USA 102:11064-11069. [PMC free article] [PubMed]
16. Fenton, A. M., P. M. Stephens, J. Crowley, M. O' Callaghan, and F. O'Gara. 1992. Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain. Appl. Environ. Microbiol. 58:3873-3878. [PMC free article] [PubMed]
17. ffrench-Constant, R. H., and N. R. Waterfield. 2006. Ground control for insect pests. Nat. Biotechnol. 24:660-661. [PubMed]
18. Frapolli, M., G. Défago, and Y. Moënne-Loccoz. 2007. Multilocus sequence analysis of biocontrol fluorescent Pseudomonas spp. producing the antifungal compound 2,4-diacetylphloroglucinol. Environ. Microbiol. 9:1939-1955. [PubMed]
19. Frishman, D., A. Mironov, H. W. Mewes, and M. Gelfand. 1998. Combining diverse evidence for gene recognition in completely sequenced bacterial genomes. Nucleic Acids Res. 26:2941-2947. [PMC free article] [PubMed]
20. Fuchs, J., and G. Défago. 1991. Protection of tomatoes against Fusarium oxysporum f. sp. lycopersici by combining non-pathogenic Fusarium with different bacteria in untreated soil, p. 51-56. In C. Keel, B. Koller, and G. Défago (ed.), Plant growth-promoting rhizobacteria: progress and prospects, 2nd Workshop on PGPR. IOBC/WPRS, Interlaken, Switzerland.
21. Giddens, S. R., R. W. Jackson, C. D. Moon, M. A. Jacobs, X. X. Zhang, S. M. Gehrig, and P. B. Rainey. 2007. Mutational activation of niche-specific genes provides insight into regulatory networks and bacterial function in a complex environment. Proc. Natl. Acad. Sci. USA 104:18247-18252. [PMC free article] [PubMed]
22. Harrison, L. A., L. Letendre, P. Kovacevich, E. Pierson, and D. M. Weller. 1993. Purification of an antibiotic effective against Gaeumannomyces graminis var. tritici produced by a biocontrol agent, Pseudomonas aureofaciens. Soil Biol. Biochem. 25:215-221.
23. Hill, W. G., and A. Robertson. 1968. Linkage disequilibrium in finite populations. Theor. Appl. Genet. 38:226-231. [PubMed]
24. Howell, C. R., and R. D. Stipanovic. 1979. Control of Rhizoctonia solani in cotton seedlings with Pseudomonas fluorescens and with an antibiotic produced by the bacterium. Phytopathology 69:480-482.
25. Huson, D. H., and D. Bryant. 2006. Application of phylogenetic networks in evolutionary studies. Mol. Biol. Evol. 23:254-267. [PubMed]
26. Iavicoli, A., E. Boutet, A. Buchala, and J. P. Métraux. 2003. Induced systemic resistance in Arabidopsis thaliana in response to root inoculation with Pseudomonas fluorescens CHAO. Mol. Plant-Microbe Interact. 16:851-858. [PubMed]
27. Jackson, R. W., M. G. Preston, and P. B. Rainey. 2005. Genetic characterization of Pseudomonas fluorescens SBW25 rsp gene expression in the phytosphere and in vitro. J. Bacteriol. 187:8477-8488. [PMC free article] [PubMed]
28. Joardar, V., M. Lindeberg, R. W. Jackson, J. Selengut, R. Dodson, L. M. Brinkac, S. C. Daugherty, R. Deboy, A. S. Durkin, M. G. Giglio, R. Madupu, W. C. Nelson, M. J. Rosovitz, S. Sullivan, J. Crabtree, T. Creasy, T. Davidsen, D. H. Haft, N. Zafar, L. Zhou, R. Halpin, T. Holley, H. Khouri, T. Feldblyum, O. White, C. M. Fraser, A. K. Chatterjee, S. Cartinhour, D. J. Schneider, J. Mansfield, A. Collmer, and C. R. Buell. 2005. Whole-genome sequence analysis of Pseudomonas syringae pv. phaseolicola 1448A reveals divergence among pathovars in genes involved in virulence and transposition. J. Bacteriol. 187:6488-6498. [PMC free article] [PubMed]
29. Jukes, T. H., and C. R. Cantor. 1969. Evolution of protein molecules, p. 21-132. In H. N. Munro (ed.), Mammalian protein metabolism. Academic Press, New York, NY.
30. Keel, C., U. Schnider, M. Maurhofer, C. Voisard, J. Laville, U. Burger, P. Wirthner, D. Haas, and G. Défago. 1992. Suppression of root diseases by Pseudomonas fluorescens CHA0: importance of the bacterial secondary metabolite 2,4-diacetylphloroglucinol. Mol. Plant-Microbe Interact. 5:4-13.
31. Keel, C., D. M. Weller, A. Natsch, G. Défago, R. J. Cook, and L. S. Thomashow. 1996. Conservation of the 2,4-diacetylphloroglucinol biosynthesis locus among fluorescent Pseudomonas strains from diverse geographic locations. Appl. Environ. Microbiol. 62:552-563. [PMC free article] [PubMed]
32. Kumar, S., K. Tamura, and M. Nei. 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief. Bioinform. 5:150-163. [PubMed]
33. Landa, B. B., O. V. Mavrodi, J. M. Raaijmakers, B. B. McSpadden Gardener, L. S. Thomashow, and D. M. Weller. 2002. Differential ability of genotypes of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains to colonize the roots of pea plants. Appl. Environ. Microbiol. 68:3226-3237. [PMC free article] [PubMed]
34. Lewontin, R. C. 1964. The interaction of selection and linkage. I. General considerations; heterotic models. Genetics 49:49-67. [PMC free article] [PubMed]
35. Lowe, T. M., and S. R. Eddy. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25:955-964. [PMC free article] [PubMed]
36. Manoil, C. 2000. Tagged exported proteins using Escherichia coli alkaline phosphatase gene fusions. Methods Enzymol. 326:35-47. [PubMed]
37. Mark, G. L., J. M. Dow, P. D. Kiely, H. Higgins, J. Haynes, C. Baysse, A. Abbas, T. Foley, A. Franks, J. Morrissey, and F. O'Gara. 2005. Transcriptome profiling of bacterial responses to root exudates identifies genes involved in microbe-plant interactions. Proc. Natl. Acad. Sci. USA 102:17454-17459. [PMC free article] [PubMed]
38. Mavrodi, O. V., B. B. McSpadden-Gardener, D. V. Mavrodi, R. F. Bonsall, D. M. Weller, and L. S. Thomashow. 2001. Genetic diversity of phlD from 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas spp. Phytopathology 91:35-43. [PubMed]
39. Mazzola, M., D. L. Funnell, and J. M. Raaijmakers. 2004. Wheat cultivar-specific selection of 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas species from resident soil populations. Microb. Ecol. 48:338-348. [PubMed]
40. McSpadden Gardener, B. B., K. L. Schroeder, S. E. Kalloger, J. M. Raaijmakers, L. S. Thomashow, and D. M. Weller. 2000. Genotypic and phenotypic diversity of phlD-containing Pseudomonas strains isolated from the rhizosphere of wheat. Appl. Environ. Microbiol. 66:1939-1946. [PMC free article] [PubMed]
41. McSpadden Gardener, B. B., and D. M. Weller. 2001. Changes in populations of rhizosphere bacteria associated with take-all disease of wheat. Appl. Environ. Microbiol. 67:4414-4425. [PMC free article] [PubMed]
42. Morrissey, J. P., M. Cullinane, A. Abbas, G. L. Mark, and F. O'Gara. 2004. Biosynthesis and regulation of antifungal metabolites by pseudomonads, p. 637-669. In J. Ramos (ed.), The pseudomonads, vol. III. Biosynthesis of macromolecules and secondary metabolites. Kluyver Press, Dordrecht, The Netherlands.
43. Nelson, K. E., C. Weinel, I. T. Paulsen, R. J. Dodson, H. Hilbert, V. A. Martins dos Santos, D. E. Fouts, S. R. Gil, M. Pop, M. Holmes, L. Brinkac, M. Beanan, R. T. DeBoy, S. Daugherty, J. Kolonay, R. Madupu, W. Nelson, O. White, J. Peterson, H. Khouri, I. Hance, P. Chris Lee, E. Holtzapple, D. Scanlan, K. Tran, A. Moazzez, T. Utterback, M. Rizzo, K. Lee, D. Kosack, D. Moestl, H. Wedler, J. Lauber, D. Stjepandic, J. Hoheisel, M. Straetz, S. Heim, C. Kiewitz, J. A. Eisen, K. N. Timmis, A. Düsterhöft, B. Tümmler, and C. M. Fraser. 2002. Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ. Microbiol. 4:799-808. [PubMed]
44. Paulsen, I. T., M. C. Press, J. Ravel, D. Y. Kobayashi, G. S. Myers, D. V. Mavrodi, R. T. DeBoy, R. Seshadri, Q. Ren, R. Madupu, R. J. Dodson, A. S. Durkin, L. M. Brinkac, S. C. Daugherty, S. A. Sullivan, M. J. Rosovitz, M. L. Gwinn, L. Zhou, D. J. Schneider, S. W. Cartinhour, W. C. Nelson, J. Weidman, K. Watkins, K. Tran, H. Khouri, E. A. Pierson, L. S. Pierson III, L. S. Thomashow, and J. E. Loper. 2005. Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5. Nat. Biotechnol. 23:873-878. [PubMed]
45. Phillips, D. A., T. C. Fox, M. D. King, T. V. Bhuvaneswari, and L. R. Teuber. 2004. Microbial products trigger amino acid exudation from plant roots. Plant Physiol. 136:2887-2894. [PMC free article] [PubMed]
46. Picard, C., and M. Bosco. 2006. Heterozygosis drives maize hybrids to select elite 2,4-diacetylphloroglucinol-producing Pseudomonas strains among resident soil populations. FEMS Microb. Ecol. 58:193-204. [PubMed]
47. Piganeau, G., and A. Eyre-Walker. 2004. A reanalysis of the indirect evidence for recombination in human mitochondrial DNA. Heredity 92:282-288. [PubMed]
48. Piganeau, G., M. Gardner, and A. Eyre-Walker. 2004. A broad survey of recombination in animal mitochondrial. Mol. Biol. Evol. 21:2319-2325. [PubMed]
49. Puhler, A., M. Artlat, A. Becker, M. Gottfert, J. P. Morrissey, and F. O'Gara. 2004. What can bacterial genome research teach us about bacteria-plant interactions? Curr. Opin. Plant Biol. 7:137-147. [PubMed]
50. Raaijmakers, J. M., D. M. Weller, and L. S. Thomashow. 1997. Frequency of antibiotic-producing Pseudomonas spp. in natural environments. Appl. Environ. Microbiol. 63:881-887. [PMC free article] [PubMed]
51. Rainey, P. B., and M. J. Bailey. 1996. Physical and genetic map of the Pseudomonas fluorescens SBW25 chromosome. Mol. Microbiol. 19:521-533. [PubMed]
52. Ramette, A., Y. Moënne-Loccoz, and G. Défago. 2001. Polymorphism of the polyketide synthase gene phlD in biocontrol fluorescent pseudomonads producing 2,4-diacetylphloroglucinol and comparison of PhlD with plant polyketide synthases. Mol. Plant-Microbe Interact. 14:639-652. [PubMed]
53. Ramette, A., Y. Moënne-Loccoz, and G. Défago. 2006. Genetic diversity and biocontrol potential of fluorescent pseudomonads producing phloroglucinols and hydrogen cyanide from Swiss soils naturally suppressive or conducive to Thielaviopsis basicola-mediated black root rot of tobacco. FEMS Microbiol. Ecol. 55:369-381. [PubMed]
54. Rezzonico, F., M. Zala, C. Keel, B. Duffy, Y. Moënne-Loccoz, and G. Défago. 2007. Is the ability of biocontrol fluorescent pseudomonads to produce the antifungal metabolite 2,4-diacetylphloroglucinol really synonymous with higher plant protection? New Phytol. 173:861-872. [PubMed]
55. Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425. [PubMed]
56. Schmidt, H., and M. Hensel. 2004. Pathogenicity islands in bacterial pathogenesis. Clin. Microbiol. Rev. 17:14-56. [PMC free article] [PubMed]
57. Schnider-Keel, U., A. Seematter, M. Maurhofer, C. Blumer, B. Duffy, C. Gigot-Bonnefoy, C. Reimmann, R. Notz, G. Défago, D. Haas, and C. Keel. 2000. Autoinduction of 2,4-diacetylphloroglucinol biosynthesis in the biocontrol agent Pseudomonas fluorescens CHA0 and repression by the bacterial metabolites salicylate and pyoluteorin. J. Bacteriol. 182:1215-1225. [PMC free article] [PubMed]
58. Shanahan, P., D. J. O'Sullivan, P. Simpson, J. D. Glennon, and F. O'Gara. 1992. Isolation of 2,4-diacetylphloroglucinol from a fluorescent pseudomonad and investigation of physiological parameters influencing its production. Appl. Environ. Microbiol. 58:353-358. [PMC free article] [PubMed]
59. Smith, J. M. 1992. Analyzing the mosaic structure of genes. J. Mol. Evol. 34:126-129. [PubMed]
60. Stover, C. K., X. Q. Pham, A. L. Erwin, S. D. Mizoguchi, P. Warrener, M. J. Hickey, F. S. Brinkman, W. O. Hufnagle, D. J. Kowalik, M. Lagrou, R. L. Garber, L. Goltry, E. Tolentino, S. Westbrock-Wadman, Y. Yuan, L. L. Brody, S. N. Coulter, K. R. Folger, A. Kas, K. Larbig, R. Lim, K. Smith, D. Spencer, G. K. Wong, Z. Wu, I. T. Paulsen, J. Reizer, M. H. Saier, R. E. Hancock, S. Lory, and M. V. Olson. 2000. Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406:959-964. [PubMed]
61. Stutz, E. W., G. Défago, and H. Kern. 1986. Naturally occurring fluorescent pseudomonads involved in suppression of black root rot of tobacco. Phytopathology 76:181-185.
62. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680. [PMC free article] [PubMed]
63. Vodovar, N., D. Vallenet, S. Cruveiller, Z. Rouy, V. Barbe, C. Acosta, L. Cattolico, C. Jubin, A. Lajus, B. Segurens, B. Vacherie, P. Wincker, J. Weissenbach, B. Lemaitre, C. Médigue, and F. Boccard. 2006. Complete genome sequence of the entomopathogenic and metabolically versatile soil bacterium Pseudomonas entomophila. Nat. Biotechnol. 24:673-679. [PubMed]
64. Walsh, U. F., J. P. Morrissey, and F. O'Gara. 2001. Pseudomonas for biocontrol of phytopathogens: from functional genomics to commercial exploitation. Curr. Opin. Biotech. 12:289-295. [PubMed]
65. Weller, D. M., B. B. Landa, O. V. Mavrodi, K. L. Schroeder, L. De La Fuente, S. Blouin Bankhead, R. Allende Molar, R. F. Bonsall, D. V. Mavrodi, and L. S. Thomashow. 2007. Role of 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas spp. in the defense of plant roots. Plant Biol. (Stuttgart) 9:4-20. [PubMed]

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