Mark D. Azevedo

4-Formylaminooxyvinylglycine, an herbicidal germination-arrest factor from Pseudomonas rhizosphere bacteria.

A new oxyvinylglycine has been identified as a naturally occurring herbicide that irreversibly arrests germination of the seeds of grassy weeds, such as annual bluegrass (Poa annua), without significantly affecting the growth of established grass seedlings and mature plants or germination of the seeds of broadleaf plant species (dicots). Previously, Pseudomonas fluorescens WH6 and over 20 other rhizosphere bacteria were isolated and selected for their ability to arrest germination of P. annua seeds. The germination-arrest factor (GAF, 1) responsible for this developmentally specific herbicidal action has now been isolated from the culture filtrate of P. fluorescens WH6. Purification of this highly polar, low molecular weight natural product allowed its structure to be assigned as 4-formylaminooxy-l-vinylglycine on the basis of NMR spectroscopic and mass spectrometric data, in combination with D/L-amino acid oxidase reactions to establish the absolute configuration. Assay results for P. annua inhibition by related compounds known to regulate plant growth are presented, and a cellular target for 1 is proposed. Furthermore, using bioassays, TLC, and capillary NMR spectroscopy, it has been shown that GAF (1) is secreted by all other herbicidally active rhizosphere bacteria in our collection.

High resolution characterization of soil biological communities by nucleic acid and fatty acid analyses

Fatty acid methyl ester (FAME) and length heterogeneity-polymerase chain reaction (LH-PCR) analyses were used to generate ‘fingerprints’ of FAMEs and eubacterial 16S rDNA sequences characteristic of agricultural soil communities. We hypothesized that pooling data from two methods that characterized different components of soil biological communities would improve the resolution of fingerprints characterizing the effects of contrasting tillage and ground cover practices. By using supervised classifications of FAME and LH-PCR, a discriminant analysis procedure distinguished soils from contrasting tillage and ground cover management and predicted the origin of soil samples. Used independently, FAME provided higher resolution of tillage, ground cover, and field location than LH-PCR, but LH-PCR was effective at identifying field location. Pooling data from both methods did not enhance the predictive power. A comparison of linear discriminant analysis, quadratic discriminant analysis, and nonparametric density estimation demonstrated that minimizing assumptions about data distribution improved the capacity of FAME analysis to resolve differences in soil types. Use of a purely statistical Bayesian method to select a subset of fatty acids (FA’s) as variables in discriminant analyses identified FA’s that represented signature FA’s for specific groups of organisms. Published by Elsevier Science Ltd.

Genetics of germination-arrest factor (GAF) production by Pseudomonas fluorescens WH6: identification of a gene cluster essential for GAF biosynthesis

The genetic basis of the biosynthesis of the germination-arrest factor (GAF) produced by Pseudomonas fluorescens WH6, and previously identified as 4-formylaminooxyvinylglycine, has been investigated here. In addition to inhibiting the germination of a wide range of grassy weeds, GAF exhibits a selective antimicrobial activity against the bacterial plant pathogen Erwinia amylovora. We utilized the in vitro response of E. amylovora to GAF as a rapid screen for loss-of-function GAF phenotypes generated by transposon mutagenesis. A Tn5 mutant library consisting of 6364 WH6 transformants was screened in this Erwinia assay, resulting in the identification of 18 non-redundant transposon insertion sites that led to loss of GAF production in WH6, as confirmed by TLC analysis. These insertions mapped to five different genes and four intergenic regions. Three of these genes, including two putative regulatory genes (gntR and iopB homologues), were clustered in a 13 kb chromosomal region containing 13 putative ORFs. A GAF mutation identified previously as affecting an aminotransferase also maps to this region. We suggest that three of the genes in this region (a carbamoyltransferase, an aminotransferase and a formyltransferase) encode the enzymes necessary to synthesize dihydroGAF, the putative immediate precursor of GAF in a proposed GAF biosynthetic pathway. RT-qPCR analyses demonstrated that mutations in the gntR and iopB regulatory genes, as well as in a prtR homologue identified earlier as controlling GAF formation, suppressed transcription of at least two of the putative GAF biosynthetic genes (encoding the aminotransferase and formyltransferase) located in this 13 kb region.

The Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid inhibits growth of Erwinia amylovora and acts as a seed germination-arrest factor.

The Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) shares biological activities with 4-formylaminooxyvinylglycine, a related molecule produced by Pseudomonas fluorescens WH6. We found that culture filtrates of a P. aeruginosa strain overproducing AMB weakly interfered with seed germination of the grassy weed Poa annua and strongly inhibited growth of Erwinia amylovora, the causal agent of the devastating orchard crop disease known as fire blight. AMB was active against a 4-formylaminooxyvinylglycine-resistant isolate of E. amylovora, suggesting that the molecular targets of the two oxyvinylglycines in Erwinia do not, or not entirely, overlap. The AMB biosynthesis and transport genes were shown to be organized in two separate transcriptional units, ambA and ambBCDE, which were successfully expressed from IPTG-inducible tac promoters in the heterologous host P. fluorescens CHA0. Engineered AMB production enabled this model biocontrol strain to become inhibitory against E. amylovora and to weakly interfere with the germination of several graminaceous seeds. We conclude that AMB production requires no additional genes besides ambABCDE and we speculate that their expression in marketed fire blight biocontrol strains could potentially contribute to disease control.

Pseudomonas fluorescens SBW25 produces furanomycin, a non-proteinogenic amino acid with selective antimicrobial properties

BackgroundPseudomonas fluorescens SBW25 has been extensively studied because of its plant growth promoting properties and potential as a biocontrol agent. The genome of SBW25 has been sequenced, and among sequenced strains of pseudomonads, SBW25 appears to be most closely related to P. fluorescens WH6. In the authors’ laboratories, WH6 was previously shown to produce and secrete 4-formylaminooxyvinylglycine (FVG), a non-proteinogenic amino acid with selective herbicidal and antimicrobial activity. Although SBW25 does not have the genetic capacity to produce FVG, we were interested in determining whether this pseudomonad might produce some other type of non-proteinogenic amino acid.ResultsP. fluorescens SBW25 was found to produce and secrete a ninhydrin-reactive compound with selective antimicrobial properties. This compound was purified from SBW25 culture filtrate and identified as the non-proteinogenic amino acid L-furanomycin [2S,2′R,5′S)-2-amino-2-(5′methyl-2′,5′-dihydrofuran-2′-yl)acetic acid].ConclusionsThe identification of furanomycin as a secondary metabolite of SBW25 is the first report of the production of furanomycin by a pseudomonad. This compound was known previously only as a natural product produced by a strain of Streptomyces. This report adds furanomycin to the small list of non-proteinogenic amino acids that have been identified as secondary products of pseudomonads. This study also extends the list of bacteria that are inhibited by furanomycin to include several plant pathogenic bacteria.

Selective inhibition of Erwinia amylovora by the herbicidally active germination-arrest factor (GAF) produced by Pseudomonas bacteria

Aims:  The germination‐arrest factor (GAF) produced by Pseudomonas fluorescens WH6, and identified as 4‐formylaminooxyvinylglycine, specifically inhibits the germination of a wide range of grassy weeds. This study was undertaken to determine whether GAF has antimicrobial activity in addition to its inhibitory effects on grass seed germination.

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.

Development of a Quantitative Assay Amenable for High-Throughput Screening to Target the Type II Secretion System for New Treatments against Plant-Pathogenic Bacteria

Plant-pathogenic bacteria are the causative agents of diseases in important agricultural crops and ornamental plants. The severe economic burden of these diseases requires seeking new approaches for their control, particularly because phytopathogenic bacteria are often resistant to available treatments. The type II secretion (T2S) system is a key virulence factor used by major groups of phytopathogenic bacteria. The T2S machinery transports many hydrolytic enzymes responsible for degradation of the plant cell wall, thus enabling successful colonization and dissemination of the bacteria in the plant host. The genetic inactivation of the T2S system leads to loss of virulence, which strongly suggests that targeting the T2S could enable new treatments against plant-pathogenic bacteria. Accordingly, we have designed and optimized an assay to identify small-molecule inhibitors of the T2S system. This assay uses a double parametric output: measurement of bacterial growth and the enzymatic activity of cellulase, which is secreted via the T2S pathway in our model organism Dickeya dadantii. The assay was evaluated by screening natural extracts, culture filtrates isolated from rhizosphere bacteria, and a collection of pharmaceutically active compounds in LOPAC1280. The calculated Z′ values of 0.63, 0.63, and 0.58, respectively, strongly suggest that the assay is applicable for a high-throughput screening platform.