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. 2014 Jul;80(13):3972–3981. doi: 10.1128/AEM.00455-14

Posttranscriptional Regulation of 2,4-Diacetylphloroglucinol Production by GidA and TrmE in Pseudomonas fluorescens 2P24

Wei Zhang a, Zhao Zhao a, Bo Zhang a, Xiao-Gang Wu a,*, Zheng-Guang Ren c, Li-Qun Zhang a,b,✉
Editor: H L Drake
PMCID: PMC4054201  PMID: 24747907

Abstract

Pseudomonas fluorescens 2P24 is a soilborne bacterium that synthesizes and excretes multiple antimicrobial metabolites. The polyketide compound 2,4-diacetylphloroglucinol (2,4-DAPG), synthesized by the phlACBD locus, is its major biocontrol determinant. This study investigated two mutants defective in antagonistic activity against Rhizoctonia solani. Deletion of the gidA (PM701) or trmE (PM702) gene from strain 2P24 completely inhibited the production of 2,4-DAPG and its precursors, monoacetylphloroglucinol (MAPG) and phloroglucinol (PG). The transcription of the phlA gene was not affected, but the translation of the phlA and phlD genes was reduced significantly. Two components of the Gac/Rsm pathway, RsmA and RsmE, were found to be regulated by gidA and trmE, whereas the other components, RsmX, RsmY, and RsmZ, were not. The regulation of 2,4-DAPG production by gidA and trmE, however, was independent of the Gac/Rsm pathway. Both the gidA and trmE mutants were unable to produce PG but could convert PG to MAPG and MAPG to 2,4-DAPG. Overexpression of PhlD in the gidA and trmE mutants could restore the production of PG and 2,4-DAPG. Taken together, these findings suggest that GidA and TrmE are positive regulatory elements that influence the biosynthesis of 2,4-DAPG posttranscriptionally.

INTRODUCTION

Many strains of Pseudomonas fluorescens are important plant growth-promoting rhizobacteria (PGPR), enhancing plant growth through various mechanisms (1, 2). Secondary metabolites produced by these bacteria play a key role in the suppression of plant diseases caused by soilborne pathogens (3, 4). One of the most extensively studied metabolites, 2,4-diacetylphloroglucinol (2,4-DAPG), has antifungal, antibacterial, antiviral, antihelminthic, and phytotoxic activities at high concentrations (5, 6, 7, 8). 2,4-DAPG can also act as a signal molecule for plants, inducing systemic resistance, promoting the exudation of amino acids from roots, and enhancing branching of the root system (9, 10, 11).

The biosynthetic locus of 2,4-DAPG has been identified in several Pseudomonas strains, including P. fluorescens Q2-87, CHA0, F113, Pf-5, and 2P24 (12, 13, 14, 15, 16). The locus contains four genes, phlA, phlC, phlB, and phlD, which are transcribed as a single operon (phlACBD) (13). The phlD gene encodes a type III polyketide synthase, which is responsible for the synthesis of phloroglucinol (PG) from three molecules of malonyl-coenzyme A (CoA) (17, 18, 19). The phlA, phlC, and phlB genes together are required for conversion of PG to monoacetylphloroglucinol (MAPG) and of MAPG to 2,4-DAPG (13, 20). The phlA gene encodes a β-ketoacyl-ACP synthase III (FabH). PhlC has an acetyl-CoA binding site that contains an active cysteine residue and a glycine-rich C-terminal region, which are typical structural features of condensing enzymes (13). PhlB is a small protein and has sequence homology to several nucleic acid-binding proteins in the GenBank database (21). The phlACBD operon is flanked on either side by the separately transcribed phlE and phlF genes (see Fig. S1 in the supplemental material), which code for a putative transmembrane permease and a TetR-like transcriptional repressor, respectively (13, 14, 22). PhlF represses phlA transcription by binding to the specific binding site pho, which is located in the phlA promoter region (23). In P. fluorescens CHA0, the phlG gene, located downstream of the phlF gene, encodes a hydrolase that specifically degrades 2,4-DAPG to MAPG (24, 25). The biosynthesis of 2,4-DAPG is highly dependent on bacterial cell density and is controlled by many regulatory elements, including the conserved regulatory system Gac/Rsm. In this regulatory cascade, the GacS/GacA two-component system activates the transcription of one or several small RNAs (sRNAs), including RsmX, RsmY, and RsmZ, which subsequently bind to the translational repressor proteins RsmA and RsmE and dissociate them from the ribosome-binding sites of target genes to relieve translational repression (26, 27). Many other molecules involved in the regulation of 2,4-DAPG biosynthesis have been identified, including the sigma factors RpoS, RpoD, and RpoN (12, 28, 29); the H-NS-related proteins MvaT and MvaV (30); the oxidoreductase DsbA (31); the resistance-nodulation-division efflux pump EmhABC (16), the RNA chaperone Hfq (32); the sigma regulator PsrA (33); and various environmental factors (34, 35).

P. fluorescens 2P24 is a rhizosphere isolate capable of producing the antibiotic 2,4-DAPG. We previously showed that 2,4-DAPG biosynthesis in this strain involved a distinct and complex regulatory network (16, 32, 33). In this study, glucose-inhibited division protein A (GidA) and tRNA modification GTPase (TrmE) were selected as two novel 2,4-DAPG-positive regulators in strain 2P24, as antifungal activity was completely absent in both gidA and trmE mutants. Previous studies showed that these two proteins form a complex that catalyzes the addition of a carboxymethylaminomethyl (cmnm) group at the 5′ position of the wobble uridine (U34) of tRNAs (36, 37, 38). This modification allows base pairing at the wobble position with a purine (A or G) and restricts the misreading of codons that end with a pyrimidine (U or C) (39, 40, 41). The GidA and TrmE proteins may regulate bacterial factors in some strains by posttranscriptionally modifying tRNAs, including tRNALys, tRNAGlu, tRNAGln, tRNALeu, and tRNAArg (36, 42). In this study, we present evidence that GidA and TrmE positively control 2,4-DAPG biosynthesis at the posttranscriptional level and that this control is independent of the Gac/Rsm pathway.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.

The bacterial strains and plasmids used in this study are listed in Table 1. P. fluorescens strains were grown in Luria-Bertani (LB) (43) or King's B (KB) medium (44) at 28°C. Escherichia coli strains were routinely grown at 37°C in LB medium. Rhizoctonia solani was cultured in PDA medium (45). Where indicated, the media were supplemented with the antibiotics ampicillin (50 μg/ml), kanamycin (50 μg/ml), gentamicin (10 μg/ml), chloramphenicol (20 μg/ml), and tetracycline 20 (μg/ml) or with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) (40 μg/ml).

TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmid Descriptiona Reference or source
P. fluorescens
    2P24 Wild type; Apr 64
    PM121 gidA::Tn5 in strain 2P24; Apr Kmr This study
    PM701 gidA gene in-frame deletion in strain 2P24; Apr This study
    PM702 trmE gene in-frame deletion in strain 2P24; Apr This study
    PM703 trmE gene in-frame deletion in mutant PM701; Apr This study
    PM915 phlA gene in-frame deletion in strain 2P24; Apr This study
    PM917 gidA gene in-frame deletion in mutant PM915; Apr This study
    PM918 trmE gene in-frame deletion in mutant PM915; Apr This study
    PM919 phlD gene in-frame deletion in mutant PM915; Apr This study
    PM920 phlD gene in-frame deletion in strain 2P24; Apr This study
    PM108 2P24 with a VSV-G epitope sequence at the C terminus of PhlA; Apr This study
    PM109 PM701 with a VSV-G epitope sequence at the C terminus of PhlA; Apr This study
    PM110 PM702 with a VSV-G epitope sequence at the C terminus of PhlA; Apr This study
    PM111 2P24 with a VSV-G epitope sequence at the C terminus of PhlD; Apr This study
    PM112 PM701 with a VSV-G epitope sequence at the C terminus of PhlD; Apr This study
    PM113 PM702 with a VSV-G epitope sequence at the C terminus of PhlD; Apr This study
    PM114 PM703 with a VSV-G epitope sequence at the C terminus of PhlD; Apr This study
    PM115 2P24 with a VSV-G epitope sequence at the C terminus of RsmA; Apr This study
    PM116 PM701 with a VSV-G epitope sequence at the C terminus of RsmA; Apr This study
    PM117 PM702 with a VSV-G epitope sequence at the C terminus of RsmA; Apr This study
    PM118 2P24 with a VSV-G epitope sequence at the C terminus of RsmE; Apr This study
    PM119 PM701 with a VSV-G epitope sequence at the C terminus of RsmE; Apr This study
    PM120 PM702 with a VSV-G epitope sequence at the C terminus of RsmE; Apr This study
E. coli
    DH5α λ− ϕ80dlacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK− mK−) supE44 thi-1 gyrA relA1 65
Fungus
    R. solani Plant pathogen that causes cotton damping off Lab stock
Plasmids
    pUTKm Delivery plasmid for Tn5; R6K replicon; Apr Kmr 47
    pRK600 Helper plasmid for triparental mating; Cmr 66
    pBluescript II SK(+) Cloning vector; Apr Stratagene
    pBSKm Suicide plasmid for Pseudomonas spp., used for homologous recombination; derivative of pBluescript II SK(+); Kmr This study
    pBSKΔgidA pBSKm carrying a deleted gidA gene; Kmr This study
    pBSKΔtrmE pBSKm carrying a deleted trmE gene; Kmr This study
    pBSKΔphlA pBSKm carrying a deleted phlA gene; Kmr This study
    pBSKΔphlD pBSKm carrying a deleted phlD gene; Kmr This study
    pBBR1MCS-5 pBBR1MCS-5 containing pBluescript II KS-lacZα; Gmr 48
    pBBR5-gidA pBBR1MCS-5 containing gidA gene; Gmr This study
    pBBR5-trmE pBBR1MCS-5 containing trmE gene; Gmr This study
    pBBR5-phlD pBBR1MCS-5 containing phlD gene; Gmr This study
    pBBR5-phlDV pBBR1MCS-5 containing phlD gene with a VSV-G epitope sequence at its C terminus; Gmr This study
    pRG970Gm Cloning vector containing promoterless lacZYA for construction of transcriptional fusion; Gmr 32
    pRG970Km Cloning vector containing promoterless lacZYA for construction of transcriptional fusion; Kmr 67
    p970Km-phlAp pRG970Km containing a phlA-lacZ transcriptional fusion; Kmr 32
    p970Km-rsmXp pRG970Km containing an rsmX-lacZ transcriptional fusion; Kmr 33
    p970Gm-rsmYp pRG970Gm containing an rsmY-lacZ transcriptional fusion; Gmr 33
    p970Km-rsmZp pRG970Km containing an rsmZ-lacZ transcriptional fusion; Kmr 33
    p970Km-rsmAp pRG970Km containing an rsmA-lacZ transcriptional fusion; Kmr 33
    p970Gm-rsmEp pRG970Gm containing an rsmE-lacZ transcriptional fusion; Gmr 33
    pBSKPhlA-VSV pBSKm with a VSV-G epitope sequence at the C terminus of PhlA; Kmr This study
    pBSKPhlD-VSV pBSKm with a VSV-G epitope sequence at the C terminus of PhlD; Kmr This study
    pBSKRsmA-VSV pBSKm with a VSV-G epitope sequence at the C terminus of RsmA; Kmr This study
    pBSKRsmE-VSV pBSKm with a VSV-G epitope sequence at the C terminus of RsmE; Kmr This study
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a

Apr, Kmr, Gmr, and Cmr indicate resistance to ampicillin, kanamycin, gentamicin, and chloramphenicol, respectively.

DNA manipulation and sequence analyses.

Standard techniques were used for plasmid and chromosomal DNA preparation, restriction enzyme digestion, agarose gel electrophoresis, purification of DNA fragments, ligation, and other molecular assays (43). Plasmid DNA was introduced into E. coli or P. fluorescens chemically or by electroporation (43). Nucleotide and deduced amino acid sequences were analyzed with the on-line BLAST search engine in GenBank (http://www.ncbi.nlm.nih.gov/BLAST) and DNAMAN software (version 5.2; Lynnon Biosoft).

Cloning of gidA and trmE genes.

To screen for novel positive regulators of 2,4-DAPG production, P. fluorescens 2P24 was subjected to random Tn5 insertional mutagenesis using the pUT-Km plasmid (46). Two out of approximately 3,000 kanamycin-resistant colonies of the 2P24 strain showed decreased levels of antagonistic activity, which is linked to the production of 2,4-DAPG. The genomic DNA fragment that flanks the kanamycin resistance gene in transposon Tn5 was cloned into the pBluescript II SK(+) (Stratagene) vector at the EcoRI site and sequenced with zhang-O and zhang-I primers (see Table S1 in the supplemental material) localized at the two ends of the Tn5 transposon. The Tn5-flanking sequences of one PM121 mutant were identified as part of a deduced gidA gene using BLAST. The upstream and downstream sequences of the entire gidA gene were then determined in the ongoing genomic sequence of strain 2P24 (see Fig. S1 in the supplemental material).

Construction of P. fluorescens 2P24 mutants and complementation.

To construct the pBSKm suicide vector with kanamycin resistance, the gene that encodes ampicillin with the vector pBluescript II SK(+) (Stratagene) was replaced by a gene encoding kanamycin. An in-frame deletion of the gidA gene was made using a two-step homologous-recombination strategy. Briefly, an upstream 959-bp fragment and a downstream 836-bp fragment were PCR amplified from the genomic DNA of strain 2P24 using the primer pairs Gid-F1/Gid-R1 and Gid-F2/Gid-R2, respectively. The upstream fragment was digested with SalI plus BamHI and the downstream fragment with BamHI plus EcoRI, and the two fragments were cloned into the suicide vector pBSKm to generate pBSKΔgidA. The same approach was used to create in-frame deletion mutants of the trmE, phlA, and phlD genes (see Table S1 in the supplemental material), yielding the plasmids pBSKΔtrmE, pBSKΔphlA, and pBSKΔphlD, respectively. Allelic exchange using these plasmids and wild-type strain 2P24 resulted in the mutants PM701 (ΔgidA), PM702 (ΔtrmE), PM915 (ΔphlA), and PM920 (ΔphlD), respectively (47). The double mutants PM917 (ΔgidA ΔphlA), PM918 (ΔtrmE ΔphlA), and PM919 (ΔphlD ΔphlA) were generated by using the plasmids pBSKΔgidA, pBSKΔtrmE and pBSKΔphlD to delete the gidA, trmE, and phlD genes, respectively, from the phlA mutant PM915 (ΔphlA). For complementation analysis, the complete gidA and trmE genes were PCR amplified and inserted into the shuttle vector pBBR1MCS-5 (48), yielding the complementation plasmids pBBR5-gidA and pBBR5-trmE, respectively. The resultant plasmids were introduced into their respective deletion mutants to generate complementation strains. For overexpression analysis, the complete phlD gene and the complete phlD gene tagged at its 3′ end with a sequence encoding the vesicular stomatitis virus glycoprotein (VSV-G) epitope 5′-TATACAGATATTGAAATGAATAGATTAGGAAAA-3′ (49) were PCR amplified and inserted into the vector pBBR1MCS-5, yielding the complementation plasmids pBBR5-phlD and pBBR5-phlDV, respectively.

Extraction and detection of 2,4-DAPG, MAPG, and PG.

The production of 2,4-DAPG and MAPG by P. fluorescens 2P24 and its mutant derivatives was measured after growth for 48 h in KB medium, and the production of PG was measured in strains grown in LB medium supplemented with 2% glucose. To analyze the conversion of PG to MAPG and of MAPG to 2,4-DAPG, synthetic PG or MAPG dissolved in methanol was added to cultures grown for 24 h to yield a final concentration of 10 μg/ml, followed by culture for an additional 24 h. 2,4-DAPG and MAPG production was quantified by analytical high-performance liquid chromatography (HPLC) (50). For HPLC analysis, the sample was dissolved in 0.05 ml methanol and analyzed using a symmetric C18 reverse-phase column (4.6 by 150 mm; Agilent TC-18). Fractions were separated isocratically by elution with 55:45 (vol/vol) acetonitrile-0.1% H3PO4 at a flow rate of 1 ml min−1. 2,4-DAPG (1 μg/ml) was added to induce PG production when the growth of strains reached an optical density at 600 nm (OD600) of 0.8. After incubation for another 36 h, PG was quantified according to the method of Kidarsa et al. (20).

Fungal-inhibition assay.

Fungal-inhibition assays were performed on one-fifth-strength PDA, with R. solani as the target pathogen. A 0.6-cm circular plug from a 48-h culture of R. solani was placed in the center of the plate. Aliquots (5 μl) of suspensions of washed bacterial cells, prepared from exponential-growth-phase LB cultures and adjusted to an OD600 of 0.8, were spotted onto the plates 2.5 cm from the edge of the fungal colony. The plates were incubated for 36 h at 28°C, and the growth situation of R. solani was determined.

β-Galactosidase assays.

P. fluorescens 2P24 and its derivatives were grown in 100-ml Erlenmeyer flasks containing 20 ml LB at 28°C with shaking at 150 rpm. Aliquots were collected after certain time periods, and β-galactosidase activity was measured as described previously (44).

Western blot analysis.

To construct a C-terminal VSV-G epitope-PhlA fused gene, a PCR-generated fragment with the VSV-G sequence inserted in frame at the 3′ end of the phlA gene was cloned into pBSKm. The resulting plasmid, pBSKPhlA-VSV, was used to transform wild-type 2P24 and its gidA and trmE mutants to generate the PhlA-marked strains PM108, PM109, and PM110, respectively. The same technique was used to construct the PhlD-marked plasmid pBSKPhlD-VSV, the RsmA-marked plasmid pBSKRsmA-VSV, and the RsmE-marked plasmid pBSKRsmE-VSV. Homologous recombination using these plasmids in strain 2P24 and the gidA and trmE mutants produced strains PM111 to PM120 (Table 1).

Then, strains with VSV-G epitope sequence tags were grown in KB at 28°C with shaking. The bacterial cells were collected by centrifugation at 12,000 rpm, resuspended in 100 μl lysis buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, 200 mM dithiothreitol [DTT]), and incubated at 100°C for 10 min. Proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were incubated with primary antibodies to VSV-G (1:2,000; Sigma) and 3-phosphoglycerate kinase (PGK) (1:5,000; Invitrogen), followed by incubation with anti-mouse horseradish peroxidase-conjugated secondary antibodies (1:5,000; Sigma). Antibody-tagged protein bands were detected using an ECL Western Blot detection kit (Cowin Biotech, Beijing, China).

Statistical analysis.

Data were analyzed and compared with Fisher's least significant difference (LSD) test (a P value of <0.001 was considered significant) using SPSS 16.0 software (SPSS Corporation, Chicago, IL, USA).

Nucleotide sequence accession numbers.

The sequences of the gidA and trmE genes from strain 2P24 have been deposited in the GenBank database under accession no. KC866364.

RESULTS

Identification and characterization of gidA and trmE genes in P. fluorescens 2P24.

In our previous study, random mutagenesis of P. fluorescens 2P24 using the transposon mini-Tn5 was performed to identify the potential regulators of 2,4-DAPG production (16). Similarly, in this study, the mutant PM121 (gidA::Tn5), which is defective in antagonistic activity to R. solani (Table 1), was selected and further investigated. The mini-Tn5-flanking region in PM121 was cloned, with sequence analysis showing that Tn5 had been inserted into the gidA gene (see Fig. S1 in the supplemental material). The deduced amino acid sequence (632 amino acids; 69 kDa) of the gidA gene product was highly similar to that of the tRNA uridine 5-carboxymethylaminomethyl modification protein, GidA, from P. fluorescens Pf0-1 (accession number YP_351465; 97% identity), Pseudomonas syringae pv. syringae B728a (YP_238197; 93% identity), Pseudomonas putida GB-1 (YP_001671659; 91% identity), and Pseudomonas aeruginosa PAO1 (NP_254252; 90% identity). Multiple-sequence alignment indicated that GidA of P. fluorescens 2P24 contains a highly conserved dinucleotide-binding motif at the N terminus (see Fig. S1 in the supplemental material), which is required for tRNA modification (36, 42). Five bases downstream of gidA, we identified a deduced gidB gene encoding a 16S rRNA methyltransferase and transcribed in the same direction (see Fig. S1 in the supplemental material). In E. coli, GidB is thought to function as an S-adenosyl-l-methionine (SAM)-dependent methyltransferase in cell division or chromosomal replication (51).

In addition to the gidA gene, the trmE (mnmE) gene was recently found to be involved in the process of tRNA modification (37, 52, 53). The trmE gene of strain 2P24 was located upstream of the gidA gene and downstream of the yidC gene, which encodes a putative inner membrane protein translocase component (see Fig. S1 in the supplemental material). Sequence analysis showed that the deduced TrmE protein (456 amino acids; 49 kDa) exhibited high levels of sequence identity to its orthologs from many other Pseudomonas strains (see Table S2 in the supplemental material). In contrast to the GidA protein, the TrmE protein of P. fluorescens 2P24 contains conserved GTP-binding motifs, labeled G-1 to G-4 (see Fig. S1 in the supplemental material), which are important for GTPase activity.

Effects of GidA and TrmE on 2,4-DAPG production and antimicrobial activity.

To confirm the ability of GidA and TrmE to regulate 2,4-DAPG production, the in-frame deletion mutants PM701 (ΔgidA) and PM702 (ΔtrmE) were constructed in P. fluorescens 2P24, and their 2,4-DAPG production was assessed by HPLC. As expected, lack of either the gidA or the trmE gene significantly decreased the 2,4-DAPG level by about 20-fold compared with wild-type 2P24. The introduction of the complementation plasmid pBBR5-gidA into PM701, or of pBBR5-trmE into PM702, restored the production of 2,4-DAPG and reached levels that were higher than those of the wild type (Fig. 1A). Furthermore, the production of the precursors of 2,4-DAPG, MAPG and PG, was drastically reduced in PM701 and PM702 compared with the wild-type and complementation strains (Fig. 1B and C). Although strain 2P24 displayed strong antifungal activity against R. solani in a dual-culture test, this activity was completely absent in PM701 and PM702 (Fig. 1D). These results indicate that GidA and TrmE act as positive regulatory elements in the synthesis of 2,4-DAPG, MAPG, and PG.

FIG 1.

FIG 1

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Effects of gidA and trmE mutations on 2,4-DAPG, MAPG, and PG production and on antifungal activity toward R. solani. (A and B) HPLC analysis of 2,4-DAPG (A) and MAPG (B) production by strain 2P24 and its derivatives in KB medium. (C) PG production of strains in LB medium supplemented with 2% glucose using a colorimetric method (17). Each value is the average of three independent cultures grown for 2 days, with the error bars denoting standard deviations (SD) and the asterisks indicating significantly different results (P < 0.001). (D) Measurement of antifungal activity in one-fifth-strength PDA medium, with R. solani as the target pathogen.

Regulation of 2,4-DAPG biosynthesis genes by GidA and TrmE at the posttranscriptional level.

In the 2,4-DAPG biosynthesis pathway, PhlD is responsible for the production of PG, and PhlACB is required for the acetylation of PG to MAPG and of MAPG to 2,4-DAPG (13, 17, 19, 20, 21). To determine the mechanism of action by which GidA and TrmE regulate the expression of 2,4-DAPG biosynthesis genes, the expression of a construct of lacZ fused to the phlA gene was tested in cultures grown in LB medium. There were no evident differences in the levels of phlA gene transcription between the 2P24 strains and the two mutants (Fig. 2A), indicating that GidA and TrmE did not regulate the transcription of the phlA gene.

FIG 2.

FIG 2

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Regulation of antibiotic-biosynthetic genes phlA and phlD by GidA and TrmE. (A) The transcriptional levels of the phlA gene in P. fluorescens 2P24 and its variants were measured using the β-galactosidase assay. Growth is indicated by the dashed lines. (B) Levels of PhlA-VSV and PhlD-VSV proteins were evaluated by Western blotting using an anti-VSV antibody. An antibody directed against 3-phosphoglycerate kinase α (α-PGK) was used as a loading control. All experiments were performed in triplicate, with the means ± SD shown.

We also assayed the expression of the PhlA-VSV and PhlD-VSV proteins in the PM108 (2P24-phlAVSV), PM109 (ΔgidA-phlAVSV), PM110 (ΔtrmE-phlAVSV), PM111 (2P24-phlDVSV), PM112 (ΔgidA-phlDVSV), and PM113 (ΔtrmE-phlDVSV) strains by Western blotting using an anti-VSV antibody. We found that the expression levels of the PhlA-VSV and PhlD-VSV proteins were obviously lower in PM109, PM110, PM112, and PM113 than in PM108 and PM111 throughout cell growth (Fig. 2B). These findings confirmed that GidA and TrmE modulated the expression of the 2,4-DAPG biosynthesis genes phlA and phlD at the posttranscriptional level.

The effects of GidA and TrmE on 2,4-DAPG biosynthesis are independent of the Gac/Rsm pathway.

The Gac/Rsm pathway in P. fluorescens is a conserved signal transduction pathway that tightly controls 2,4-DAPG expression at the posttranscriptional level (26, 27). The system contains two paralogous RNA-binding proteins, RsmA and RsmE, and three sequestering sRNAs, RsmX, RsmY, and RsmZ, all of which are present in the draft genome sequence of P. fluorescens 2P24 (33). To determine whether GidA and TrmE affect the Gac/Rsm pathway, the transcription of GacA-dependent sRNA genes and that of their binding proteins were assayed in the wild-type strain and the PM701 (ΔgidA) and PM702 (ΔtrmE) mutants. There were no significant differences between the mutants and the wild type in the levels of rsmX, rsmY, and rsmZ mRNAs (Fig. 3A to C). In contrast, the levels of rsmA-lacZ and rsmE-lacZ transcripts were significantly lower in the two mutants than in the wild type (Fig. 3D and E). Western blotting results were consistent with those of transcriptional expression analysis, further confirming that GidA and TrmE positively control the expression of the rsmA and rsmE genes (Fig. 3F). Although RsmA and RsmE in P. fluorescens 2P24 have been identified as translational repressors of 2,4-DAPG production (33), the reduced expression of RsmA and RsmE in the mutants did not increase 2,4-DAPG production (Fig. 1A), suggesting that GidA and TrmE posttranscriptionally affect 2,4-DAPG biosynthesis independently of the Gac/Rsm pathway.

FIG 3.

FIG 3

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Regulation of the small noncoding RNA genes rsmX, rsmY, and rsmZ and their cognate regulator genes, rsmA and rsmE, by GidA and TrmE. (A to E) Transcriptional expression profiles of rsmX (A), rsmY (B), rsmZ (C), rsmA (D), and rsmE (E) were measured in P. fluorescens 2P24 and its gidA and trmE mutants. (F) Measurement of RsmA-VSV and RsmE-VSV proteins by Western blotting using an anti-VSV antibody. An anti-3-phosphoglycerate kinase α (α-PGK) antibody was used as a loading control. All experiments were performed in triplicate. The means ± SD are shown, and the asterisks indicate significantly different results (P < 0.001). Growth is indicated by the dashed lines.

The activity of PhlD is defective in PM701 (ΔgidA) and PM702 (ΔtrmE) mutants.

To understand the function of GidA and TrmE in the 2,4-DAPG-biosynthetic pathway, we first evaluated the activities of PhlACB in wild-type 2P24 and its mutants by chemical complementation. Neither 2,4-DAPG nor MAPG was detectable in the phlA mutant PM915 (ΔphlA), regardless of whether their respective substrates, MAPG and PG, were present. However, addition of their precursors significantly enhanced the production of 2,4-DAPG and MAPG in the PM701 and PM702 mutants (Fig. 4A and B), suggesting that the remaining PhlACB could convert PG to MAPG and MAPG to 2,4-DAPG. To avoid the conversion of PG to MAPG and 2,4-DAPG, PhlD activity was assayed in the phlA mutant PM915 (ΔphlA), and the synthetic 2,4-DAPG (1 μg/ml) was added to stimulate phl autoinduction. A large amount of PG accumulated in the PM915 mutant, whereas no PG could be detected in the double mutants PM917 (ΔgidA ΔphlA) and PM918 (ΔtrmE ΔphlA) (Fig. 4C). Since PhlD is responsible for PG production in P. fluorescens (17), as shown by the failure of the phlD mutant PM920 (ΔphlD) to produce PG (Fig. 4C), it is likely that PhlD function was absent from the PM701 and PM702 mutants. Taken together, these findings suggest that the activity of PhlD, rather than PhlACB, is defective in the PM701 and PM702 mutants.

FIG 4.

FIG 4

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Effects of GidA and TrmE on PhlA and PhlD functions. (A) Production of 2,4-DAPG in the absence and presence of 10 μg/ml MAPG. (B) Production of MAPG in the absence and presence of 10 μg/ml PG. (C) Production of PG was determined in strain 2P24 and its derivatives. 2,4-DAPG was added to a final concentration of 1 μg/ml. All experiments were repeated three times. The means ± SD are shown, and the asterisks indicate significantly different results (P < 0.001).

Overexpression of PhlD restores the wild-type phenotypes in PM701 (ΔgidA) and PM702 (ΔtrmE) mutants.

To further explore the mechanism underlying the reduced production of 2,4-DAPG, MAPG, and PG in the PM701 and PM702 mutants, the plasmid pBBR1MCS-5, carrying phlD (pBBR5-phlD), was introduced into strain 2P24 and its variants. Overexpression of PhlD in the PM701, PM702, and PM703 (ΔgidA ΔtrmE) double mutants restored the production of PG, MAPG, and 2,4-DAPG to the level observed in the wild-type strain containing pBBR5-phlD (Fig. 5A to C). However, the amounts of PhlD produced by the three mutants carrying pBBR5-phlDV were smaller than that of the wild type carrying pBBR5-phlDV (Fig. 5D), further indicating that the gidA and trmE genes affect the translation efficiency of the PhlD protein.

FIG 5.

FIG 5

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Overexpression of PhlD restores the wild-type phenotypes in gidA, trmE, and their double mutants. (A to C) 2,4-DAPG (A), MAPG (B), and PG (C) production was detected in P. fluorescens 2P24 and its derivatives containing the plasmid pBBR1MCS-5 or pBBR5-phlD. All experiments were repeated three times. The means ± SD are shown, and the asterisks indicate significantly different results (P < 0.001). (D) Western blot analysis of the amount of PhlD-VSV present in the indicated strains using an anti-VSV antibody. The numbers 1 to 8 represent strains PM111(pBBR1MCS-5), PM111(pBBR5-phlDV), PM112(pBBR1MCS-5), PM112(pBBR5-phlDV), PM113(pBBR1MCS-5), PM113(pBBR5-phlDV), PM114(pBBR1MCS-5), and PM114(pBBR5-phlDV), respectively. The blots were incubated with anti-3-phosphoglycerate kinase α (α-PGK) antibody as a loading control.

DISCUSSION

This study provides genetic evidence demonstrating that, in the biocontrol agent P. fluorescens 2P24, the GidA and TrmE proteins regulate 2,4-DAPG biosynthesis at a posttranscriptional level, independently of the Gac/Rsm signal transduction pathway. Mutations in gidA have been found to cause morphological changes in some bacteria, including E. coli and Salmonella enterica serovar Typhimurium (54). These changes are likely due to a defect in chromosome segregation and cell division in the presence of glucose. We found that PM701 (ΔgidA) mutant cells were filamentous, whereas wild-type 2P24 had a normal rod shape in LB medium supplemented with 0.2% glucose (see Fig. S2 in the supplemental material). Despite this difference, however, the growth of the gidA mutant was not markedly deficient. GidA has also been shown to regulate the expression of virulence genes in a wide range of bacteria, including Aeromonas hydrophila, Myxococcus xanthus, P. syringae, S. enterica, Shigella flexneri, and Streptococcus pyogenes (42, 55, 56, 57, 58). In P. aeruginosa, GidA selectively regulates the expression of the rhl-dependent quorum-sensing gene by modulating RhlR posttranscriptionally (59). In addition, TrmE has been reported to be involved in bacterial adaptation to cold temperatures (60), resistance to acidic pH (61), and oxidation of certain heterocyclic substrates, such as thiophene and furan (62). We found that, in addition to affecting the production of 2,4-DAPG, MAPG, and PG, mutations in the gidA and trmE genes affected many other phenotypic traits in P. fluorescens 2P24, including quorum-sensing gene expression, fluorescent-pigment production, and biofilm formation (data not shown). These findings indicated that GidA and TrmE are likely global regulators in a great variety of bacteria.

The phlACBD gene cluster has been shown to be essential for 2,4-DAPG biosynthesis, with PhlD catalyzing the iterative condensation of three malonyl-CoA units to form 3,5-diketoheptanedioate (17). This polyketide product is then cyclized to form PG (19) and subsequently acetylated to MAPG and 2,4-DAPG by PhlACB (13, 19). Downregulation of the synthesis of PG, MAPG, and 2,4-DAPG as a result of gidA and trmE mutations may be associated with insufficient production levels of proteins encoded by the phl locus. This was supported by the results, which showed that the production of PhlA-VSV and PhlD-VSV proteins was lower in the PM109 (ΔgidA-phlAVSV), PM110 (ΔtrmE-phlAVSV), PM112 (ΔgidA-phlDVSV), and PM113 (ΔtrmE-phlDVSV) mutants than in PM108 (2P24-phlAVSV) and PM111 (2P24-phlDVSV). However, 2,4-DAPG and MAPG could be detected in the mutants after their substrates were added, suggesting that the residual PhlA remained functional. The severely impaired production of PG in the PM701 and PM702 mutants may have been due to the absence of PG substrates or defective PhlD biosynthesis. Overexpression of PhlD in these mutants resulted in the recovery of PG, MAPG, and 2,4-DAPG production, suggesting that the impaired 2,4-DAPG production in the PM701 and PM702 mutants was likely due to the presence of a defective PhlD. In addition, the expression of PhlD at 36 h was always lower than that recorded at 24 h, regardless of whether the gidA and trmE genes were mutated. This phenomenon could be explained by the fact that the production of the PhlD protein might be inhibited when the yield of PG reaches a threshold level, or the instability of PhlD (17) might make PhlD sensitive to an unknown substance when the secondary metabolite is largely synthesized by 36 h. However, the exact reason remains to be further investigated.

The gidA and trmE genes are conserved in both prokaryotes and eukaryotes (42). Their encoded products, the GidA and TrmE proteins, respectively, could regulate cell motility and growth in some strains by posttranscriptionally modifying tRNAs (36, 42). Although we compared the codon usage of PhlA and PhlD, we observed no significant differences in codon types and numbers (data not shown). These findings cannot exclude the possibility, however, that amino acid residues that depend on GidA and TrmE for their synthesis play different roles in the functions of the PhlA and PhlD proteins. Although mutations of the gidA and trmE genes in strain 2P24 obviously decreased PhlD expression without abolishing it, the residual PhlD was insufficient for the production of PG. Introduction of a plasmid overexpressing the phlD gene into these mutants also showed the effects of GidA and TrmE on PhlD. Although these bacteria produced PG, the level of PhlD expression was lower than in the wild type. As GidA and TrmE modify tRNAs, thus contributing to the fidelity of mRNA translation, the amount of correctly translated PhlD in mutant strains that contain the plasmid-borne phlD gene, but not the chromosome-borne gene, was sufficient to perform its biochemical function. To further measure the differences in PhlD expression between pBBR5-phlD-containing mutants and the wild type, the purified PhlD proteins from these strains were subjected to electrospray ionization-mass spectrometry (ESI-MS) and two-dimensional electrophoresis. However, there were no significant differences in the molecular masses and isoelectric points (pI) of these PhlD proteins (data not shown). Further research is required to evaluate the effects of GidA/TrmE on important amino acids in the catalytic center of PhlD.

The Gac/Rsm signal transduction pathway in Pseudomonas has been found to regulate the production of many secondary metabolites and exoenzymes at the posttranscriptional level (27, 63). In P. syringae B728a, GidA showed some phenotypic overlap with the GacS/GacA two-component system, but they appear to be separate regulons, although SalA, a downstream regulator within the gac regulon, is a member of both (61). Similarly, we found that the regulation of 2,4-DAPG by GidA and TrmE was independent of the Gac/Rsm signal transduction pathway, although the expression of the regulatory proteins RsmA and RsmE was markedly affected by GidA and TrmE mutations. Therefore, the roles of RsmA/E in the GidA/TrmE regulatory system and the exact relationship between GidA/TrmE and the Gac/Rsm pathway remain to be determined.

In conclusion, the results of this study demonstrated that GidA and TrmE are potential regulators of 2,4-DAPG production in P. fluorescens 2P24 by the posttranscriptional regulation of genes involved in the antibiotic biosynthesis pathway but independently of the Gac/Rsm signal transduction pathway. Further research is needed to determine the precise molecular functions of GidA and TrmE relative to their target proteins in P. fluorescens 2P24.

Supplementary Material

Supplemental material
supp_80_13_3972__index.html (1.2KB, html)

ACKNOWLEDGMENTS

This work was funded by the National Programs for High Technology Research and Development of China (2011AA10A205) and the National Natural Science Foundation of China (31071725 and 31272082).

Footnotes

Published ahead of print 18 April 2014

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.00455-14.

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