Carrie Selin

Phenazines are not essential for Pseudomonas chlororaphis PA23 biocontrol of Sclerotinia sclerotiorum, but do play a role in biofilm formation

Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of suppressing disease caused by the fungal pathogen Sclerotinia sclerotiorum. This bacterium produces the diffusible antibiotics phenazine-1-carboxylic acid, 2-hydroxyphenazine and pyrrolnitrin (PRN). Because the individual contribution of these antibiotics to PA23 biocontrol has not been defined, mutants deficient in the production of phenazine (PHZ), PRN or both antibiotics were created. Analysis of the PHZ mutant revealed enhanced antifungal activity in vitro and wild-type levels of Sclerotinia disease suppression. Conversely, the PRN- and the PRN/PHZ-deficient strains exhibited decreased antifungal activity in vitro and markedly reduced the ability to control Sclerotinia infection of canola in the greenhouse. These findings suggest that PRN is the primary antibiotic mediating biocontrol of this pathogen. Analysis of prnA-lacZ and phzA-lacZ transcriptional fusions revealed that PRN and PHZ are not subject to autoregulation; moreover, they do not cross-regulate each other. However, HPLC showed a twofold increase in PRN levels in the PHZ(-) background. Finally, PHZ, but not PRN production, is involved in biofilm development in P. chlororaphis PA23.

The PhzI/PhzR quorum-sensing system is required for pyrrolnitrin and phenazine production, and exhibits cross-regulation with RpoS in Pseudomonas chlororaphis PA23

The aim of the current study was to determine how quorum sensing (QS) affects the production of secondary metabolites in Pseudomonas chlororaphis strain PA23. A phzR mutant (PA23phzR) and an N-acylhomoserine lactone (AHL)-deficient strain (PA23-6863) were generated that no longer inhibited the fungal pathogen Sclerotinia sclerotiorum in vitro. Both strains exhibited reduced pyrrolnitrin (PRN), phenazine (PHZ) and protease production. Moreover, phzA-lacZ and prnA-lacZ transcription was significantly reduced in PA23phzR and PA23-6863. As the majority of secondary metabolites are produced at the onset of stationary phase, we investigated whether cross-regulation occurs between QS and RpoS. Analysis of transcriptional fusions revealed that RpoS has a positive and negative effect on phzI and phzR, respectively. In a reciprocal manner, RpoS is positively regulated by QS. Characterization of a phzRrpoS double mutant showed reduced antifungal activity as well as PRN and PHZ production, similar to the QS-deficient strains. Furthermore, phzR but not rpoS was able to complement the phzRrpoS double mutant for the aforementioned traits, indicating that the Phz QS system is a central regulator of PA23-mediated antagonism. Finally, we discovered that QS and RpoS have opposing effects on PA23 biofilm formation. While both QS-deficient strains produced little biofilm, the rpoS mutant showed enhanced biofilm production compared with PA23. Collectively, our findings indicate that QS controls diverse aspects of PA23 physiology, including secondary metabolism, RpoS and biofilm formation. As such, QS is expected to play a crucial role in PA23 biocontrol and persistence in the environment.

Elucidating the Role of Effectors in Plant-Fungal Interactions: Progress and Challenges

Pathogenic fungi have diverse growth lifestyles that support fungal colonization on plants. Successful colonization and infection for all lifestyles depends upon the ability to modify living host plants to sequester the necessary nutrients required for growth and reproduction. Secretion of virulence determinants referred to as “effectors” is assumed to be the key governing factor that determines host infection and colonization. Effector proteins are capable of suppressing plant defense responses and alter plant physiology to accommodate fungal invaders. This review focuses on effector molecules of biotrophic and hemibiotrophic plant pathogenic fungi, and the mechanism required for the release and uptake of effector molecules by the fungi and plant cells, respectively. We also place emphasis on the discovery of effectors, difficulties associated with predicting the effector repertoire, and fungal genomic features that have helped promote effector diversity leading to fungal evolution. We discuss the role of specific effectors found in biotrophic and hemibiotrophic fungi and examine how CRISPR/Cas9 technology may provide a new avenue for accelerating our ability in the discovery of fungal effector function.

Pyrrolnitrin and Hydrogen Cyanide Production by Pseudomonas chlororaphis Strain PA23 Exhibits Nematicidal and Repellent Activity against Caenorhabditis elegans

Pseudomonas chlororaphis strain PA23 is a biocontrol agent able to suppress growth of the fungal pathogen Sclerotinia sclerotiorum. This bacterium produces an arsenal of exometabolites including pyrrolnitrin (PRN), phenazine (PHZ), hydrogen cyanide (HCN), and degradative enzymes. Production of these compounds is controlled at both the transcriptional and posttranscriptional levels by the Gac-Rsm system, RpoS, PsrA, and the Phz quorum-sensing system. Beyond pathogen-suppression, the success of a biocontrol agent is dependent upon its ability to establish itself in the environment where predation by bacterivorous organisms, including nematodes, may threaten persistence. The focus of this study was to investigate whether PA23 is able to resist grazing by Caenorhabditis elegans and to define the role played by exoproducts in the bacterial-nematode interaction. We discovered that both PRN and HCN contribute to fast- and slow-killing of C. elegans. HCN is well-established as having lethal effects on C. elegans; however, PRN has not been reported to be nematicidal. Exposure of L4 stage nematodes to purified PRN reduced nematode viability in a dose-dependent fashion and led to reduced hatching of eggs laid by gravid adults. Because bacterial metabolites can act as chemoattractants or repellents, we analyzed whether PA23 exhibited attractant or repulsive properties towards C. elegans. Both PRN and HCN were found to be potent repellents. Next we investigated whether the presence of C. elegans would elicit changes in PA23 gene activity. Co-culturing the two organisms increased expression of a number of genes associated with biocontrol, including phzA, hcnA, phzR, phzI, rpoS and gacS. Exoproduct analysis showed that PHZ and autoinducer signals were upregulated, consistent with the gene expression profiles. Collectively, these findings indicate that PA23 is able to sense the presence of C. elegans and it is able to both repel and kill the nematodes, which should facilitate environmental persistence and ultimately biocontrol.

Stringent response mutants of Pseudomonas chlororaphis PA23 exhibit enhanced antifungal activity against Sclerotinia sclerotiorum in vitro

The stringent response (SR) is a regulatory mechanism that enables bacteria to adapt to nutrient stress through the production of the alarmone (p)ppGpp. The aim of the current study was to understand how the SR affects the antifungal (AF) activity of Pseudomonas chlororaphis PA23. Two SR mutants were generated, PA23relA and PA23relAspoT, that no longer produced (p)ppGpp. Both mutants exhibited increased inhibition of Sclerotinia sclerotiorum in vitro and elevated pyrrolnitrin (PRN), lipase and protease production. Phenazine (PHZ) levels, on the other hand, remained unchanged. Through transcriptional fusion analysis we discovered that prnA-lacZ (PRN) activity was increased in the SR mutants, whereas phzA-lacZ (PHZ) activity was equal to that of the wild-type. We also examined how the sigma factor RpoS impacts PA23-mediated antagonism. Similar to the SR mutants, an rpoS mutant of PA23, called PA23rpoS, exhibited enhanced AF activity in vitro and increased expression of PRN, protease and lipase. However, PHZ production and expression of phzA-lacZ were dramatically reduced. Consistent with what has been reported for other bacteria, the SR exerted positive control over rpoS expression. In addition, providing rpoS in trans restored the SR phenotype to that of the wild-type. Collectively, our findings indicate that this global stress response impacts production of PA23 AF compounds via regulation of rpoS transcription and has an overall negative influence on S. sclerotiorum antagonism.

A Pipeline for Screening Small Molecules with Growth Inhibitory Activity against Burkholderia cenocepacia

Infections with the bacteria Burkholderia cepacia complex (Bcc) are very difficult to eradicate in cystic fibrosis patients due the intrinsic resistance of Bcc to most available antibiotics and the emergence of multiple antibiotic resistant strains during antibiotic treatment. In this work, we used a whole-cell based assay to screen a diverse collection of small molecules for growth inhibitors of a relevant strain of Bcc, B. cenocepacia K56-2. The primary screen used bacterial growth in 96-well plate format and identified 206 primary actives among 30,259 compounds. From 100 compounds with no previous record of antibacterial activity secondary screening and data mining selected a total of Bce bioactives that were further analyzed. An experimental pipeline, evaluating in vitro antibacterial and antibiofilm activity, toxicity and in vivo antibacterial activity using C. elegans was used for prioritizing compounds with better chances to be further investigated as potential Bcc antibacterial drugs. This high throughput screen, along with the in vitro and in vivo analysis highlights the utility of this experimental method to quickly identify bioactives as a starting point of antibacterial drug discovery.

Repression of the Antifungal Activity of Pseudomonas sp. Strain DF41 by the Stringent Response

ABSTRACT The stringent response (SR) enables bacteria to adapt to nutrient limitation through production of the nucleotides guanosine tetraphosphate and guanosine pentaphosphate, collectively known as (p)ppGpp. Two enzymes are responsible for the intracellular pools of (p)ppGpp: RelA acts as a synthetase, while SpoT can function as either a synthetase or a hydrolase. We investigated how the SR affects the ability of the biological control agent Pseudomonas sp. strain DF41 to inhibit the fungal pathogen Sclerotinia sclerotiorum (Lib.) de Bary. Strain DF41 relA and relA spoT mutants were generated and found to exhibit increased antifungal activity. Strain DF41 produces a lipopeptide (LP) molecule that is essential for Sclerotinia biocontrol. LP production and protease activity were both elevated in the relA and relA spoT mutants. Addition of relA but not spoT in trans restored the mutant phenotype to that of the parent. Next, we investigated whether an association exists between the SR and known regulators of biocontrol, including the Gac system and RpoS. A gacS mutant of strain DF41 produced less (p)ppGpp and exhibited a 1.7-fold decrease in relA expression compared to the wild type, suggesting that relA forms part of the Gac regulon. We discovered that rpoS transcription was reduced significantly in the SR mutants. Furthermore, rpoS provided in trans restored protease activity to wild-type levels but did not attenuate antifungal activity. Finally, relA expression was decreased in the mutants, indicating that the SR is required for maximum expression of relA.

Draft Genome Sequences of Burkholderia contaminans, a Burkholderia cepacia Complex Species That Is Increasingly Recovered from Cystic Fibrosis Patients

ABSTRACT Burkholderia contaminans belongs to the Burkholderia cepacia complex (BCC), a group of bacteria that are ubiquitous in the environment and capable of infecting the immunocompromised and people with cystic fibrosis. We report here draft genome sequences for the B. contaminans type strain LMG 23361 and an Argentinian cystic fibrosis sputum isolate.

The requirement for the LysR-type regulator PtrA for Pseudomonas chlororaphis PA23 biocontrol revealed through proteomic and phenotypic analysis

BackgroundPseudomonas chlororaphis strain PA23 is a biocontrol agent capable of suppressing the fungal pathogen Sclerotinia sclerotiorum. This bacterium produces the antibiotics phenazine and pyrrolnitrin together with other metabolites believed to contribute to biocontrol. A mutant no longer capable of inhibiting fungal growth was identified harboring a transposon insertion in a gene encoding a LysR-type transcriptional regulator (LTTR), designated ptrA (Pseudomonas transcriptional regulator). Isobaric tag for relative and absolute quantitation (iTRAQ) based protein analysis was used to reveal changes in protein expression patterns in the ptrA mutant compared to the PA23 wild type.ResultsRelative abundance profiles showed 59 differentially-expressed proteins in the ptrA mutant, which could be classified into 16 clusters of orthologous groups (COGs) based on their predicted functions. The largest COG category was the unknown function group, suggesting that many yet-to-be identified proteins are involved in the loss of fungal activity. In the secondary metabolite biosynthesis, transport and catabolism COG, seven proteins associated with phenazine biosynthesis and chitinase production were downregulated in the mutant. Phenotypic assays confirmed the loss of phenazines and chitinase activity. Upregulated proteins included a lipoprotein involved in iron transport, a flagellin and hook-associated protein and four proteins categorized into the translation, ribosome structure and biogenesis COG. Phenotypic analysis revealed that the mutant exhibited increased siderophore production and flagellar motility and an altered growth profile, supporting the proteomic findings.ConclusionPtrA is a novel LTTR that is essential for PA23 fungal antagonism. Differential protein expression was observed across 16 COG categories suggesting PtrA is functioning as a global transcriptional regulator. Changes in protein expression were confirmed by phenotypic assays that showed reduced phenazine and chitinase expression, elevated flagellar motility and siderophore production, as well as early entrance into log phase growth.

The global regulator ANR is essential for Pseudomonas chlororaphis strain PA23 biocontrol.

Pseudomonas chlororaphis PA23 is a biocontrol agent capable of protecting canola from stem rot disease caused by the fungus Sclerotinia sclerotiorum. The focus of the current study was to elucidate the role of the transcriptional regulator ANR in the biocontrol capabilities of this bacterium. An anr mutant was created, PA23anr, that was devoid antifungal activity. In other pseudomonads, ANR is essential for regulating HCN production. Characterization of PA23anr revealed that, in addition to HCN, ANR controls phenazine (PHZ), pyrrolnitrin (PRN), protease and autoinducer (AHL) signal molecule production. In gene expression studies, hcnA, phzA, prnA and phzI were found to be downregulated, consistent with our endproduct analysis. Because the phenotype of PA23anr closely resembles that of quorum sensing (QS)-deficient strains, we explored whether there is a connection between ANR and the PhzRI QS system. Both phzI and phzR are positively regulated by ANR, whereas PhzR represses anr transcription. Complementation of PA23anr with pUCP-phzR, C6-HSL or both yielded no change in phenotype. Conversely, PA23phzR harbouring pUCP23-anr exhibited partial-to-full restoration of antifungal activity, HCN, PRN and AHL production together with hcnA, prnA, phzI and rpoS expression. PHZ and protease production remained unchanged indicating that ANR can complement the QS-deficient phenotype with respect to some but not all traits. Our experiments were conducted at atmospheric O2 levels underscoring the fact that ANR has a profound effect on PA23 physiology under aerobic conditions.