Negative regulation of germination-arrest factor production in Pseudomonas fluorescens WH6 by a putative extracytoplasmic function sigma factor

Pseudomonas fluorescens WH6 secretes a germination-arrest factor (GAF) that we have identified previously as 4-formylaminooxyvinylglycine. GAF irreversibly inhibits germination of the seeds of numerous grassy weeds and selectively inhibits growth of the bacterial plant pathogen Erwinia amylovora. WH6-3, a mutant that has lost the ability to produce GAF, contains a Tn5 insertion in prtR, a gene that has been described previously in some strains of P. fluorescens as encoding a transmembrane regulator. As in these other pseudomonads, in WH6, prtR occurs immediately downstream of prtI, which encodes a protein homologous to extracytoplasmic function (ECF) sigma factors. These two genes have been proposed to function as a dicistronic operon. In this study, we demonstrated that deletion of prtI in WT WH6 had no effect on GAF production. However, deletion of prtI in the WH6-3 mutant overcame the effects of the Tn5 insertion in prtR and restored GAF production in the resulting double mutant. Complementation of the double prtIR mutant with prtI suppressed GAF production. This overall pattern of prtIR regulation was also observed for the activity of an AprX protease. Furthermore, reverse transcription quantitative real-time PCR analysis demonstrated that alterations in GAF production were mirrored by changes in the transcription of two putative GAF biosynthetic genes. Thus, we concluded that PrtI exerted a negative regulatory effect on GAF production, although the mechanism has not yet been determined. In addition, evidence was obtained that the transcription of prtI and prtR in WH6 may be more complex than predicted by existing models

Preference of Arabidopsis thaliana GH3.5 acyl amido synthetase for growth versus defense hormone acyl substrates is dictated by concentration of amino acid substrate aspartate.

The GH3 family of adenylating enzymes conjugate acyl substrates such as the growth hormone indole-3-acetic acid (IAA) to amino acids via a two-step reaction of acyl substrate adenylation followed by amino acid conjugation. Arabidopsis thaliana GH3.5 was previously shown to be unusual in that it could adenylate both IAA and the defense hormone salicylic acid (SA, 2-hydroxybenzoate). Our detailed studies of the kinetics of GH3.5 on a variety of auxin and benzoate substrates provides insight into the acyl preference and reaction mechanism of GH3.5. For example, we found GH3.5 activity on substituted benzoates is not defined by the substitution position as it is for GH3.12/PBS3. Most importantly, we show that GH3.5 strongly prefers Asp as the amino acid conjugate and that the concentration of Asp dictates the functional activity of GH3.5 on IAA vs. SA. Not only is Asp used in amino acid biosynthesis, but it also plays an important role in nitrogen mobilization and in the production of downstream metabolites, including pipecolic acid which propagates defense systemically. During active growth, [IAA] and [Asp] are high and the catalytic efficiency (kcat/Km) of GH3.5 for IAA is 360-fold higher than with SA. GH3.5 is expressed under these conditions and conversion of IAA to inactive IAA-Asp would provide fine spatial and temporal control over local auxin developmental responses. By contrast, [SA] is dramatically elevated in response to (hemi)-biotrophic pathogens which also induce GH3.5 expression. Under these conditions, [Asp] is low and GH3.5 has equal affinity (Km) for SA and IAA with similar catalytic efficiencies. However, the concentration of IAA tends to be very low, well below the Km for IAA. Therefore, GH3.5 catalyzed formation of SA-Asp would occur, fine-tuning localized defensive responses through conversion of active free SA to SA-Asp. Taken together, we show how GH3.5, with dual activity on IAA and SA, can integrate cellular metabolic status via Asp to provide fine control of growth vs. defense outcomes and hormone homeostasis

Detection of 4-formylaminooxyvinylglycine in culture filtrates of <i>Pseudomonas fluorescens</i> WH6 and <i>Pantoea ananatis</i> BRT175 by laser ablation electrospray ionization-mass spectrometry

<div><p>The oxyvinylglycine 4-formylaminooxyvinylglycine (FVG) arrests the germination of weedy grasses and inhibits the growth of the bacterial plant pathogen <i>Erwinia amylovora</i>. Both biological and analytical methods have previously been used to detect the presence of FVG in crude and extracted culture filtrates of several <i>Pseudomonas fluorescens</i> strains. Although a combination of these techniques is adequate to detect FVG, none is amenable to high-throughput analysis. Likewise, filtrates often contain complex metabolite mixtures that prevent the detection of FVG using established chromatographic techniques. Here, we report the development of a new method that directly detects FVG in crude filtrates using laser ablation electrospray ionization-mass spectrometry (LAESI-MS). This approach overcomes limitations with our existing methodology and allows for the rapid analysis of complex crude culture filtrates. To validate the utility of the LAESI-MS method, we examined crude filtrates from <i>Pantoea ananatis</i> BRT175 and found that this strain also produces FVG. These findings are consistent with the antimicrobial activity of <i>P</i>. <i>ananatis</i> BRT175 and indicate that the spectrum of bacteria that produce FVG stretches beyond rhizosphere-associated <i>Pseudomonas fluorescens</i>.</p></div

Results of biological assays and LAESI-MS detection of FVG for a dilution series of culture filtrate from wild-type <i>Pseudomonas fluorescens</i> WH6.

<p>Results of biological assays and LAESI-MS detection of FVG for a dilution series of culture filtrate from wild-type <i>Pseudomonas fluorescens</i> WH6.</p

Comparison of the <i>gvg</i> and PNP-1 clusters from <i>Pseudomonas fluorescens</i> WH6 and <i>Pantoea ananatis</i> BRT175.

<p>Gene arrows are colored based on the class of the encoded protein. Gene designations in bold indicate genes that, when mutated, result in a null-FVG phenotype and/or antibiotic activity against <i>Erwinia amylovora</i>. Gray shading indicates homologous regions of the <i>gvg</i> cluster in each strain. TM, transmembrane.</p

A scan for FVG ions in crude filtrate from WT and null-FVG mutant WH6 strains.

<p>Laser ablation electrospray ionization-mass spectrometry was used to scan crude culture filtrates for the ion corresponding to 4-formylaminooxyvinylglycine (FVG). Each duplicate well of a 96-well plate contained crude culture filtrate from wild-type <i>Pseudomonas fluorescens</i> WH6, a null FVG-mutant strain [WH6-30G (Δ<i>gvgH</i>) or WH6-31G (Δ<i>gvgI</i>)], or non-inoculated filtrate. Data were collected from 20 laser pulses per sample well, and the peak trace corresponds to the extracted ion chromatogram for sodiated FVG, <i>m/z</i> /183.0372.</p

Results of biological assays and LAESI-MS analysis for detection of FVG in wild-type and mutant strains of <i>Pseudomonas fluorescens</i> WH6 and <i>Pantoea ananatis</i> BRT175.

<p>Results of biological assays and LAESI-MS analysis for detection of FVG in wild-type and mutant strains of <i>Pseudomonas fluorescens</i> WH6 and <i>Pantoea ananatis</i> BRT175.</p

Laser ablation electrospray ionization-mass spectrometry analysis of crude culture filtrate from wild-type <i>Pseudomonas fluorescens</i> WH6.

<p>An MS spectrum from <i>m/z</i> 50 to 750 is shown with inset (A.) showing the peaks within the range <i>m/z</i> 183.00–183.09 only. The chemical structure of 4-formylaminooxyvinylglycine (FVG) is shown in inset B.</p

OkrentRachelBotanyPlantPathologyNegativeRegulationGermination.pdf

Pseudomonas fluorescens WH6 secretes a germination-arrest factor (GAF) that we have identified previously as 4-formylaminooxyvinylglycine. GAF irreversibly inhibits germination of the seeds of numerous grassy weeds and selectively inhibits growth of the bacterial plant pathogen Erwinia amylovora. WH6-3, a mutant that has lost the ability to produce GAF, contains a Tn5 insertion in prtR, a gene that has been described previously in some strains of P. fluorescens as encoding a transmembrane regulator. As in these other pseudomonads, in WH6, prtR occurs immediately downstream of prtI, which encodes a protein homologous to extracytoplasmic function (ECF) sigma factors. These two genes have been proposed to function as a dicistronic operon. In this study, we demonstrated that deletion of prtI in WT WH6 had no effect on GAF production. However, deletion of prtI in the WH6-3 mutant overcame the effects of the Tn5 insertion in prtR and restored GAF production in the resulting double mutant. Complementation of the double prtIR mutant with prtI suppressed GAF production. This overall pattern of prtIR regulation was also observed for the activity of an AprX protease. Furthermore, reverse transcription quantitative real-time PCR analysis demonstrated that alterations in GAF production were mirrored by changes in the transcription of two putative GAF biosynthetic genes. Thus, we concluded that PrtI exerted a negative regulatory effect on GAF production, although the mechanism has not yet been determined. In addition, evidence was obtained that the transcription of prtI and prtR in WH6 may be more complex than predicted by existing models

OkrentRachelBotanyPlantPathologyNegativeRegulationGermination_SupplementaryData.pdf

Pseudomonas fluorescens WH6 secretes a germination-arrest factor (GAF) that we have identified previously as 4-formylaminooxyvinylglycine. GAF irreversibly inhibits germination of the seeds of numerous grassy weeds and selectively inhibits growth of the bacterial plant pathogen Erwinia amylovora. WH6-3, a mutant that has lost the ability to produce GAF, contains a Tn5 insertion in prtR, a gene that has been described previously in some strains of P. fluorescens as encoding a transmembrane regulator. As in these other pseudomonads, in WH6, prtR occurs immediately downstream of prtI, which encodes a protein homologous to extracytoplasmic function (ECF) sigma factors. These two genes have been proposed to function as a dicistronic operon. In this study, we demonstrated that deletion of prtI in WT WH6 had no effect on GAF production. However, deletion of prtI in the WH6-3 mutant overcame the effects of the Tn5 insertion in prtR and restored GAF production in the resulting double mutant. Complementation of the double prtIR mutant with prtI suppressed GAF production. This overall pattern of prtIR regulation was also observed for the activity of an AprX protease. Furthermore, reverse transcription quantitative real-time PCR analysis demonstrated that alterations in GAF production were mirrored by changes in the transcription of two putative GAF biosynthetic genes. Thus, we concluded that PrtI exerted a negative regulatory effect on GAF production, although the mechanism has not yet been determined. In addition, evidence was obtained that the transcription of prtI and prtR in WH6 may be more complex than predicted by existing models