The requirement for the LysR-type regulator PtrA for Pseudomonas chlororaphis PA23 biocontrol revealed through proteomic and phenotypic analysis
© Klaponski et al.; licensee BioMed Central Ltd. 2014
Received: 16 December 2013
Accepted: 9 April 2014
Published: 17 April 2014
Pseudomonas 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.
Relative 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.
PtrA 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.
KeywordsAntifungal Biocontrol Chitinase Motility Phenazine Pseudomonas Siderophore Transcriptional regulator
Pseudomonas chlororaphis strain PA23 is a biocontrol agent able to protect canola from stem rot disease caused by the fungus Sclerotinia sclerotiorum (Lib.) de Bary [1, 2]. This bacterium produces a number of compounds including phenazine 1-carboxylic acid (PCA), 2-hydroxyphenazine (2-OH-PHZ), pyrrolnitrin, protease, lipase, chitinase and siderophores, some of which have been shown to contribute to fungal antagonism [3–5]. Public concern over the use of chemical pesticides together with the potential for acquiring resistance to these compounds has led to renewed interest in bacterial antagonists, such as PA23, for biocontrol. Despite demonstrating excellent disease control in the greenhouse, many biocontrol agents suffer from inconsistent performance in the field [6–8]. Poor field performance is likely due, at least in part, to variable expression of genes and gene products required for disease suppression. It is essential, therefore, to elucidate the molecular mechanisms mediating PA23 biocontrol so that production of the pathogen-suppressing factor(s) can be optimized in the environment.
In Pseudomonas spp. that act as biocontrol agents, expression of disease-suppressive metabolites is controlled by a multi-tiered network of regulation. One of the key regulatory elements is the GacS/GacA two-component signal transduction system, comprised of the sensor kinase GacS and its cognate response regulator GacA . In many pseudomonads, including PA23, a mutation in gacS or gacA leads to a loss of fungal antagonism [4, 9]. Working in concert with GacS/GacA is the Rsm system which consists of RsmA-like repressor proteins and untranslated regulatory RNAs. The repressor proteins act post-transcriptionally by binding to the ribosome-binding site (RBS) in target mRNA . The regulatory RNAs antagonize repression by titrating out the RsmA-like proteins, rendering the RBS of target genes accessible to the translational machinery . Additional regulatory elements that oversee production of PA23 antifungal metabolites include the PhzR/PhzI quorum-sensing (QS) circuit , the stationary phase sigma factor RpoS , a regulator of RpoS called PsrA , and a global stress response system known as the stringent response . Substantial interaction occurs between the regulators themselves, which adds to the complexity of the regulatory hierarchy [11–13].
Through transposon mutagenesis, a PA23 mutant was identified that exhibited a complete loss of antifungal activity, similar to what is observed for a gac mutant [4, 13]. Sequence analysis revealed that the interrupted gene, designated ptrA (Pseudomonas transcriptional regulator), encodes a protein belonging to the LysR-type transcriptional regulator (LTTR) family. LTTRs can act as either activators or repressors and are known to control a diverse range of metabolic functions including cell invasion and virulence, QS, oxidative stress, and amino acid metabolism . Given the remarkably complex regulatory network that oversees the production of antifungal compounds, the aim of the current study was to understand the global impact of the ptrA mutation on PA23 protein expression. Using the isobaric tag for relative and absolute quantitation (iTRAQ) technique, 59 proteins were found to be differentially expressed in the ptrA mutant compared to the wild type. 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 the logarithmic growth phase.
Results and discussion
Isolation of a Pseudomonas chlororaphis PA23 mutant deficient in antifungal activity
Sequence analysis revealed that the site of Tn insertion lies 803 bp downstream of the PtrA translational start (data not shown), which is predicted to disrupt the co-inducer recognition/response domain . Previous studies of the LTTRs NodD and NahR revealed that mutations in this region result in a co-inducer-independent phenotype which affects DNA binding and thus the activation/repression properties of the proteins [14, 15]. Directly downstream of ptrA but in the opposite orientation lies a gene encoding a protein that is 99% identical at the amino acid level to a DoxX-family protein found in P. chlororaphis subsp. aurantiaca PB-St2 [Genbank accession #WP_023968058]. Based on sequence similarity, DoxX could be involved in pathways related to elemental sulfur oxidation . Immediately upstream of ptrA, in the opposite orientation, lies a gene encoding a short-chain dehydrogenase (scd). Short-chain dehydrogenases are part of a superfamily of enzymes designated as the NAD(H)- or NADP(H)-dependent short-chain dehydrogenases/reductases (SDRs). The SDRs comprise a very large grouping of biologically important proteins found in virtually all forms of life . At present, it is unclear whether the genes upstream and downstream of ptrA play a role in regulation.
Through blastn analysis, ptrA homologs were found within the genomes of several Pseudomonas species, with the highest degree of nucleotide identity exhibited by Pseudomonas sp. UW4 (85%), followed by Pseudomonas protegens strains Pf-5 (84.7%) and CHA0 (84.7%), Pseudomonas fluorescens strains Pf0-1 (84.5%) and F113 (82.5%), Pseudomonas brassicacearum subsp. brassicacearum NFM421 (82.4%), Pseudomonas poae RE*1-1-14 (79.3%), and Pseudomonas resinovorans NBRC 106553 (76.1%) . Collectively, our findings indicate that PtrA is a newly identified regulator of PA23 biocontrol, and homologs of this regulator are present in a number of Pseudomonas species.
Differential protein expression between the PA23 wild type and the ptrA mutant
Differentially expressed proteins in mutant PA23-443 compared to the PA23 wild type
Amino acid transport and metabolism
4-aminobutyrate aminotransferase and related aminotransferases
Nucleotide transport and metabolism
Carbohydrate transport and metabolism
Lipid transport and metabolism
Translation, ribosomal structure and biogenesis
Translation elongation factor P (EF-P)/translation initiation factor 5A (eIF-5A)
ribosomal protein L32
aspartyl/glutamyl-tRNA(Asn/Gln) amidotransferase, C subunit
ribosomal protein S19, bacterial/organelle
cold shock domain protein CspD
Cold shock proteins
Cold shock proteins
Replication, recombination and repair
competence protein ComEA helix-hairpin-helix repeat region
Cell wall, membrane and envelope biogenesis
Outer membrane protein and related peptidoglycan-associated (lipo)proteins
Flagellin and related hook-associated proteins
Post-translational modification, protein turnover and chaperones
monothiol glutaredoxin, Grx4 family
peroxiredoxin, OsmC subfamily
Peptidyl-prolyl cis-trans isomerase (rotamase) - cyclophilin family
Inorganic ion transport and metabolism
Predicted periplasmic lipoprotein involved in iron transport
Secondary metabolites biosynthesis, transport and catabolism
Phenazine biosynthesis protein A/B.
Phenazine biosynthesis protein A/B.
phenazine biosynthesis protein PhzF family
Aromatic ring hydroxylase
General function prediction only
Predicted periplasmic or secreted lipoprotein
intracellular protease, PfpI family
Serine protease inhibitor ecotin
Protein of unknown function (DUF3313).
Protein of unknown function (DUF1161).
Sigma 54 modulation protein/S30EA ribosomal protein.
poly(hydroxyalkanoate) granule-associated protein
poly(hydroxyalkanoate) granule-associated protein
Uncharacterized protein conserved in bacteria
type VI secretion protein, VC_A0107 family
type VI secretion protein, EvpB/VC_A0108 family
outer membrane porin, OprD family.
Uncharacterized protein conserved in bacteria
Protein of unknown function (DUF3613).
Predicted integral membrane protein
Putative phospholipid-binding domain./LysM domain.
Uncharacterized protein conserved in bacteria
Iron-sulfur cluster assembly accessory protein
Signal transduction mechanisms
Putative Ser protein kinase
PtrA regulates phenazine production in PA23
The secondary metabolite biosynthesis, transport and catabolism COG category represented the next largest grouping (Table 1). Initially, two of the proteins (MOK_01048, MOK_01053) were classified under the general function category and one protein (MOK_01054) was categorized under the transport and metabolism grouping. Upon further investigation, the locus tags indicated that they are part of the phenazine biosynthetic operon, leading to their reclassification into the secondary metabolite biosynthesis COG.
The phenazine operon has been well characterized in many pseudomonads, with phzABCDEFG comprising the core biosynthetic locus . In this study, proteins with locus tags MOK_01048 and MOK_01049, identified as phenazine biosynthesis protein A/B, were significantly downregulated (Table 1). All phenazine-producing pseudomonads have an adjacent and nearly identical copy of the phzB gene, termed phzA. PhzA catalyzes the condensation reaction of two ketone molecules in the phenazine biosynthesis pathway . PhzF (identified as MOK_01053 in this study) works as an isomerase, converting trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) into 6-amino-5-oxocyclohex-2-ene-1-carboxylic acid prior to the condensation reaction catalyzed by the PhzA/B proteins . phzG encodes an FMN-dependent pyridoxamine oxidase (identified as MOK_01054 in this study), which is hypothesized to catalyze the conversion of DHHA to 5,10-Dihydro-PCA . In some pseudomonads, genes downstream of the core biosynthetic operon are required for generation of phenazine derivatives [22–24]. In P. chlororaphis 30–84, for example, phzO lies downstream of the core operon; PhzO is an aromatic hydroxylase that catalyzes the conversion of PCA into 2-OH-PHZ . More recently, in P. chlororaphis gp72, the phzO gene was shown to convert PCA into 2-OH-PHZ through a 2-OH-PCA intermediate . Like other P. chlororaphis strains, PA23 produces 2-OH-PHZ and we believe the downregulated aromatic ring hydroxylase (MOK_01055) is PhzO. Therefore, in the absence of a functional ptrA gene, four of the core phenazine biosynthetic enzymes (PhzA, PhzB, PhzF, PhzG) and one aromatic ring hydroxylase (PhzO) are significantly downregulated. The fact that PtrA plays a critical role in regulating phz expression was not surprising considering the lack of orange pigment produced by the ptrA mutant (Figures 1 and 2A). Reduced phenazine expression was further substantiated by quantitative assays. As illustrated in Figure 2B, there is a 15-fold decrease in phenazine production in PA23-443 compared to the PA23 wild type. When ptrA was expressed in trans, some restoration of phenazine production was achieved.
Chitinase production is under PtrA control
Chitinase activity of P. chlororaphis strain PA23 and derivative strains
Chitinase Activity (A550*min−1*mg total protein−1)
Early stationary phasea
Late stationary phasea
Siderophore production is upregulated in PA23-443 compared to the PA23 wild type
Siderophore production by P. chlororaphis PA23, PA23-443 and PA23-443 harboring ptrA in trans
Zone of orange haloa
Loss of ptrA results in early entry into stationary phase
PtrA negatively affects motility
Motility analysis of P. chlororaphis strain PA23, PA23-443 and PA23-443 harboring ptrA in trans
Motility zone diameter (mm) at 48 ha
PtrA regulates pyrrolnitrin production in PA23
Based on iTRAQ analysis, a tryptophan halogenase (MOK_04031) was identified under the amino acid transport and metabolism COG category, but was not significantly differentially expressed in the ptrA mutant (Vdiff = −0.24). At locus tag MOK_04033, another chlorinating halogenase was identified in the P. chlororaphis gp72 genome, but was not differentially expressed in the ptrA mutant. These enzymes are likely prnA and prnC, forming part of the prnABCD pyrrolnitrin biosynthetic operon . Subsequent pyrrolnitrin quantification via HPLC analysis revealed that wild type PA23 produced an average of 3.48 (±0.45) μg of pyrrolnitrin, whereas in the ptrA mutant, no pyrrolnitrin was detected. However, when ptrA was expressed in trans in PA23-443, pyrrolnitrin production was restored to wild-type levels (3.90 ± 0.20 μg). Significant downregulation of pyrrolnitrin expression may not have been identified through iTRAQ analysis as cell samples were taken at the onset of stationary phase. To obtain enough pyrrolnitrin for quantification, cell culture extracts are routinely performed after five days of growth . Thus, there may have been differences in protein expression in late stationary phase that were not detected in our iTRAQ analysis. As pyrrolnitrin has previously been reported as essential for PA23 biocontrol , the lack of pyrrolnitrin production by the ptrA mutant is likely a major contributor to the loss of antifungal activity.
In the present study, we describe the characterization of a PA23 derivative with a mutation in a gene encoding a novel transcriptional regulator, designated PtrA. As the mutant is no longer capable of suppressing the fungal pathogen S. sclerotiorum, PtrA is essential for PA23 biocontrol. It is apparent that PtrA affects many facets of PA23 physiology. Differential protein expression was observed across 16 different COG categories, indicating that PtrA is likely acting as a global transcriptional regulator. One of the limitations associated with this study stems from the fact that our proteomic analysis was based on the P. chlororaphis gp72 reference genome. In the future, the availability of the PA23 genome sequence may allow us to better understand the function of these differentially expressed proteins. In addition, several aspects of PtrA regulation have yet to be revealed, for example, LTTRs are frequently autoregulated and co-inducer molecules profoundly impact binding specificity . We are currently investigating the DNA targets of PtrA transcriptional regulation, including ptrA itself. Furthermore, the nature of the PtrA effector and its role in binding has yet to be discovered. It is hoped that by unraveling the complex regulatory hierarchy overseeing production of antifungal compounds, this bacterium can be used in a consistent and predictable manner for suppression of S. sclerotiorum in the field.
Bacterial strains and growth conditions
Bacterial strains, plasmids and primers used in this study
Relevant genotype or phenotype
Source or reference
P. chlororaphis PA23
Phz+RifR wild type (soybean plant isolate)
Phz− RifR ptrA::Tn5- OT182 genomic fusion
supE44 ΔlacU169 (φ80 lacZ ΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1
Mobilizing strain; RP4 tra genes integrated in chromosome; KmR TcR
pSUP102(GM)::Tn5-OT182 CmR GmR AmpR TcR
pOT182-443 (Xho I)
pOT182 containing ptrA::Tn5-OT182 genomic fusion
Cloning vector for PCR products
Broad-host-range vector; IncP OriT, AmpR GmR
pUCP22 containing ptrA from P. chlororaphis PA23
Polymerase Chain Reaction (PCR) was performed under standard conditions as suggested by Invitrogen Life Technologies data sheets supplied with their Taq polymerase.
Nucleic acid manipulation
Cloning, purification, electrophoresis, and other manipulations of nucleic acid fragments and constructs were performed using standard techniques . To clone the PA23 ptrA gene, oligonucleotide primers ptrA-F and ptrA-R were used to amplify a 2.2-kb product which was cloned into vector pCR2.1-TOPO following manufacturer’s instructions. The 2.2-kb ptrA insert was then excised with Xba I and Bam HI and cloned into the same sites of pUCP22, generating pUCP22-ptrA.
Tn5-OT182 transposon mutagenesis
Bacterial conjugations were performed to introduce Tn5-OT182 into P. chlororaphis PA23 by biparental mating following the method of Lewenza et al., . For each mating, 5–10 TcR colonies were screened by PCR to ensure that transconjugants contained a Tn5 insertion using TNP5-FORWARD and TNP5-REVERSE primers. To determine the site of Tn5-OT182 insertion, rescue cloning was performed following previously described methods .
Sequence analysis and nucleotide accession number
Plasmids isolated from TcR Xho I clones were sent for sequencing using oligonucleotide primer Tn5-ON82, which anneals to the 5′ end of Tn5-OT182. BamH I or Cla I rescue plasmids were sequenced using primer Tn5-OT182 right, which anneals to the 3′ end of the transposon. All sequencing was performed at the University of Calgary Core DNA Services facility. Sequences were analyzed using BLASTn and BLASTx databases (http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome). The GenBank accession number for the P. chlororaphis PA23 ptrA gene sequence is EF054873.
Radial diffusion assays to assess fungal inhibition against S. sclerotiorum in vitro were performed with wild-type PA23, mutant PA23-443 and PA23-443 harboring the ptrA gene in trans according to previously described methods . Five replicates were analyzed for each strain and assays were repeated three times.
Wild-type PA23 and mutant PA23-443 cells were grown as duplicate samples. At the point when cultures were just entering stationary phase (OD600 = 1.2), they were centrifuged at 10,000 × g for 10 minutes at 4°C, and pellets were washed three times in PBS buffer and frozen at −80°C. Further sample preparation and iTRAQ labelling was carried out at the Manitoba Centre for Proteomics and Systems Biology. Briefly, 100 μg protein samples were mixed with 100 mM ammonium bicarbonate, reduced with 10 mM dithiothreitol (DTT) and incubated at 56°C for 40 min. Samples were then alkylated with 50 mM iodoacetamide (IAA) for 30 min at room temperature in the dark. Addition of 17 mM DTT was used to quench excess IAA, and proteins were digested with sequencing-grade trypsin (Promega, Madison, WI, USA) overnight. Dried samples were then desalted with 0.1% trifluoroacetic acid and subjected to two-dimensional high-performance liquid chromatography (2D-HPLC)-mass spectrometry (MS) according to previously described methods .
Database search and protein identification
2D-HPLC-MS/MS spectra data from three independent runs were analyzed using ProteinPilot (v2.0.1, Applied Biosystems/MDS Sciex, Concord, ON, Canada) which employs the Paragon™ algorithm. Searches were performed against the P. chlororaphis strain gp72 reference genome. Reporter ion iTRAQ tags were labelled as follows: tags 114 and 115 to replicates of wild-type PA23 grown to early stationary phase, and tags 116 and 117 to replicates of mutant PA23-443 grown to early stationary phase. Results were reported as Z-scores, the log2 of the ratio among replicates (Z0 = tag116/tag114; Z1 = tag117/tag115; Z2 = tag115/tag114; Z3 = tag117/tag116). Peptide Z-scores values were histogrammed (Z0, Z1) to determine the overall population distribution. Further statistical analysis was performed according to the methods outlined in Rydzak et al., . Briefly, Vdiff scores were assigned to allow the determination of statistical significance of protein expression ratios between both the wild-type and mutant samples while also taking into account the variation between biological replicates. Plotted Z-scores were transformed into vector values, allowing comparison between points (Z0,Z1) and (Z2,Z3). Differences between magnitudes of the vector values from the origin to points (Z0,Z1) and (Z2,Z3) were adjusted to the widths of the peptide population distributions. Direction of the vector values (+or -) were assigned based on the angle subtended by the vector value from the origin to point (Z0,Z1). A Vdiff value greater than or equal to +1.65 and less than or equal to −1.65 corresponds to proteins expressed in the upper or lower 10% of the population distribution . Functional classification of proteins was carried out using the Integrated Microbial Genomes (IMG) database (http://img.jgi.doe.gov/cgi-bin/w/main.cgi) against the P. chlororaphis strain gp72 genome.
Growth curve analysis
Cultures of wild-type PA23 and mutant PA23-443 were inoculated at a starting optical density (OD) 600 of 0.01 and grown in M9 minimal media (1 mM MgSO4; 0.2% glucose). OD600 readings were taken at 1 hour, 5 hours and 9 hours, followed by readings every 2 hours until 27 hours of growth. Triplicate samples were analyzed.
PA23 and derivative strains were assayed for chitinase production during early stationary and late stationary phases following the methods outlined by Wirth and Wolf . Briefly, cultures were grown to the desired growth phase in M9 minimal media (1 mM MgSO4; 0.2% glucose) and 250 μL aliquots of each of cell-free supernatant, 0.1 M NaOAc, pH 5.2 and carboxymethyl-chitin-Remazol brilliant violet aqueous solution (Loewe Biochemica, Germany) were incubated for 1 hour at 37°C. The reaction was stopped by the addition of 250 μL 1 M HCL. Reaction mixtures were cooled on ice for 10 min and spun at 20,000 × g for 10 min, and the absorbances at 550 nm were recorded. Each experiment was performed in triplicate.
Flagellar motility analysis
Flagellar (swimming) was monitored according to Poritsanos et al.,. Strains were grown overnight in M9 minimal media (1 mM MgSO4; 0.2% glucose) and 5 μL was inoculated into the center of 0.3% M9 agar plates. Four replicates were analyzed and the experiment repeated three times.
Overnight cultures in M9 minimal media (1 mM MgSO4; 0.2% glucose) were subjected to phenazine extraction and quantification by UV-visible light spectroscopy at 367 nm and 490 nm for PCA and 2-OH-PHZ, respectively . Phenazine analysis was performed in triplicate.
Overnight cultures grown in M9 minimal media (1 mM MgSO4; 0.2% glucose) were spotted onto CAS media according to the methods outlined in Schwyn and Neilands  to analyze siderophore production.
Production of the antibiotic PRN was quanitified according to the methods outlined in . Briefly, 20 mL cultures of PA23 and its derivatives were grown for 5 days in M9 minimal media and PRN was extracted with an equal volume of ethyl acetate. Before extraction, toluene (5 mL) was added to each sample as an internal control. Toluene and PRN UV absorption maxima were recorded at 225 nm with a Varian 335 diode array detector. PRN peaks were detected at 4.7 mins. Samples were analyzed in duplicate.
All statistical analysis was performed using unpaired Students’s t test.
Availability of supporting data
The data sets supporting the results of this article are included within the article.
LysR-type transcriptional regulator
Pseudomonas transcriptional regulator A
Isobaric tag for relative and absolute quantitation
Phenazine 1-carboxylic acid
Clusters of orthologous groups
Chrome Azurol S
Two-dimensional high-performance liquid chromatography
The authors gratefully acknowledge financial support for this work through grants awarded to T.R. de K., W.G.D.F. and M.F.B. from the Natural Sciences and Engineering Research Council (NSERC) Discovery Grants Program and the Agri-Food Research and Development Initiative (ARDI). We thank T. Verbeke, R. Sparling, and Dr. D. Court for helpful discussions and S. Liban for critical review of the manuscript. We are indebted to the Manitoba Centre for Proteomics and Systems Biology for the proteomic analyses.
- Savchuk SC, Fernando WGD: Effect of timing of application and population dynamics on the degree of biological control of Sclerotinia sclerotiorum by bacterial antagonists. FEMS Microbiol Ecol. 2004, 49: 379-388. 10.1016/j.femsec.2004.04.014.View ArticlePubMedGoogle Scholar
- Zhang Y: Biocontrol of Sclerotinia stem rot of canola by bacterial antagonists and study of biocontrol mechanisms involved. M.Sc. Thesis. 2004, Winnipeg: University of ManitobaGoogle Scholar
- Zhang Y, Fernando WGD, de Kievit T, Berry C, Daayf F, Paulitz TC: Detection of antibiotic-related genes from bacterial biocontrol agents using polymerase chain reaction. Can J Microbiol. 2006, 52: 476-481. 10.1139/w05-152.View ArticlePubMedGoogle Scholar
- Poritsanos N, Selin C, Fernando WGD, Nakkeeran S, de Kievit TR: A GacS deficiency does not affect Pseudomonas chlororaphis PA23 fitness when growing on canola, in aged batch culture or as a biofilm. Can J Microbiol. 2006, 52 (12): 1177-1188. 10.1139/w06-079.View ArticlePubMedGoogle Scholar
- Selin C, Habibian R, Poritsanos N, Athukorala SN, Fernando D, de Kievit TR: Phenazines are not essential for Pseudomonas chlororaphis PA23 biocontrol of Sclerotinia sclerotiorum, but do play a role in biofilm formation. FEMS Microbiol Ecol. 2010, 7: 73-83.View ArticleGoogle Scholar
- Cook RJ: Making greater use of introduced microorganisms for biological control of plant pathogens. Annu Rev Phytopathol. 1993, 31: 53-80. 10.1146/annurev.py.31.090193.000413.View ArticlePubMedGoogle Scholar
- Haas D, Keel C: Regulation of antibiotic production in root-colonizing Pseudomonas spp. and relevance for biocontrol of plant disease. Annual Rev Phytopathol. 2003, 41: 117-153. 10.1146/annurev.phyto.41.052002.095656.View ArticleGoogle Scholar
- Walsh UF, Morrissey JP, O’Gara F: Pseudomonas for biocontrol of phytopathogens: from functional genomics to commercial exploitation. Curr Opin Biotechnol. 2001, 12: 289-295. 10.1016/S0958-1669(00)00212-3.View ArticlePubMedGoogle Scholar
- Heeb S, Haas D: Regulatory roles of the GacS/GacA two-component system in plant-associated and other Gram-negative bacteria. Mol Plant-Microbe Interact. 2001, 14: 1351-1363. 10.1094/MPMI.2001.14.12.1351.View ArticlePubMedGoogle Scholar
- Lapouge K, Schubert M, Allain F, Haas D: Gac/Rsm signal transduction pathway of γ-proteobacteria: from RNA recognition to regulation of social behaviour. Mol Microbiol. 2008, 67 (2): 241-253.View ArticlePubMedGoogle Scholar
- Selin C, Fernando WGD, de Kievit T: The PhzI-PhzR quorum-sensing system is required for pyrrolnitrin and phenazine production, and exhibits cross-regulation with RpoS in Pseudomonas chlororaphis PA23. Microbiol. 2012, 158: 896-907. 10.1099/mic.0.054254-0.View ArticleGoogle Scholar
- Manuel J, Selin C, Fernando WGD, de Kievit T: Stringent response mutants of Pseudomonas chlororaphis PA23 exhibits enhanced antifungal activity against Sclerotinia sclerotiorum in vitro. Microbiol. 2012, 158: 207-216. 10.1099/mic.0.053082-0.View ArticleGoogle Scholar
- Selin C, Manuel J, Fernando WGD, de Kievit T: Expression of the Pseudomonas chlororaphis strain PA23 Rsm system is under control of GacA, RpoS, PsrA, quorum sensing and the stringent response. Biol Control. 2014, 69: 24-33.View ArticleGoogle Scholar
- Maddocks E, Oyston P: Structure and function of the LysR-type transcriptional regulator (LTTR) family proteins. Microbiol. 2008, 154: 3609-3623. 10.1099/mic.0.2008/022772-0.View ArticleGoogle Scholar
- Schell MA: Molecular biology of the LysR family of transcriptional regulators. Ann Rev Microbiol. 1993, 47: 597-626. 10.1146/annurev.mi.47.100193.003121.View ArticleGoogle Scholar
- Müller FH, Bandeiras TM, Urich T, Teixeira M, Gomes CM, Kletzin A: Coupling of the pathway of sulphur oxidation to dioxygen reduction: characterization of a novel membrane-bound thiosulphate:quinone oxidoreductase. Mol Microbiol. 2004, 53 (4): 1147-1160. 10.1111/j.1365-2958.2004.04193.x.View ArticlePubMedGoogle Scholar
- Jornvall H, Hoog JO, Persson B: SDR and MDR: completed genome sequences show these protein families to be large, of old origin, and of complex nature. FEBS Lett. 1999, 445 (2–3): 261-264.View ArticlePubMedGoogle Scholar
- Windsor GL, Lam DK, Fleming L, Lo R, Whiteside MD, Yu NY, Hancock RE, Brinkman FS: Pseudomonas genome database: improved comparative analysis and population genomics capability for pseudomonas genomes. Nucleic Acids Res. 2011, 39: D596-D600. 10.1093/nar/gkq869.View ArticleGoogle Scholar
- Shen X, Chen M, Hu H, Wang W, Peng H, Xu P, Zhang X: Genome sequence of Pseudomonas chlororaphis GP72, a root-colonizing biocontrol strain. J Bacteriol. 2012, 194: 1269-1270. 10.1128/JB.06713-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Mentel M, Ahuja EG, Mavrodi DV, Breinbauer R, Thomashow LS, Blankenfeldt W: Of two make one: the biosynthesis of phenazines. Chem Bio Chem. 2009, 10: 2295-2304. 10.1002/cbic.200900323.View ArticlePubMedGoogle Scholar
- Pierson LS, Gaffney T, Lam F, Gong F: Molecular analysis of genes encoding phenazine biosynthesis in the biological control bacterium Pseudomonas aureofaciens 30–84. FEMS Microbiol Lett. 1995, 134: 299-307.PubMedGoogle Scholar
- Mavrodi DV, Bonsall RF, Delaney SM, Soule MJ, Phillips G, Thomashow LS: Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1. J Bacteriol. 2001, 183 (21): 6454-6465. 10.1128/JB.183.21.6454-6465.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Delaney SM, Mavrodi DV, Bonsall RF, Thomashow LS: phzO, a gene for biosynthesis of 2-hydrolyated phenazine compounds in Pseudomonas aureofaciens 30–84. J Bacteriol. 2001, 183: 318-27. 10.1128/JB.183.1.318-327.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Chin-A-Woeng TFC, Thomas-Oates JE, Lugtenberg BJJ, Bloemberg GV: Introduction of the phzH gene of Pseudomonas chlororaphis PCL1391 extends the range of biocontrol ability of phenazine-1-carboxylic acid-producing Pseudomonas spp. strains. Mol Plant-Microbe Interact. 2001, 14 (8): 1006-1015. 10.1094/MPMI.2001.14.8.1006.View ArticlePubMedGoogle Scholar
- Huang L, Chen M-M, Wang W, Hu H-B, Peng H-S, Xu Y-Q, Zhang X-H: Enhanced production of 2-hydroxyphenazine in Pseudomonas chlororaphis gp72. Appl Microbiol Biotechnol. 2010, 89 (1): 169-177.View ArticlePubMedGoogle Scholar
- Suzuki K, Uchiyama T, Suzuki M, Nikaidou N, Regue M, Watanabe T: LysR-type transcriptional regulator ChiR is essential for production of all chitinases and a chitin-binding protein, CBP21, in Serratia marcescens 2170. Biosci Biotechnol Biochem. 2001, 65 (2): 338-347. 10.1271/bbb.65.338.View ArticlePubMedGoogle Scholar
- Kay E, Humair B, Denervaud V, Riedel K, Spahr S, Eberl L, Valverde C, Haas D: Two GacA-dependent small RNAs modulate the quorum-sensing response in Pseudomonas aeruginosa. J Bacteriol. 2006, 188 (16): 6026-6033. 10.1128/JB.00409-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Lecompte O, Ripp R, Thierry J-C, Moras D, Poch O: Comparative analysis of ribosomal proteins in complete genomes: an example of reductive evolution at the domain scale. Nucl Acids Res. 2002, 30 (24): 5382-5390. 10.1093/nar/gkf693.PubMed CentralView ArticlePubMedGoogle Scholar
- Driscoll WW, Pepper JW, Pierson LS, Pierson EA: Spontaneous Gac mutants of Pseudomonas biological control strains: cheaters or mutualists?. Appl Environ Microbiol. 2011, 77 (20): 7227-7235. 10.1128/AEM.00679-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Wei Q, Le Minh PN, Dotsch A, Hildebrand F, Panmanee W, Elfarash A, Schultz S, Plaisance S, Charlier D, Hassett D, Haussler S, Cornelis P: Global regulation of gene expression by OxyR in an important human opportunistic pathogen. Nucl Acids Res. 2012, 40 (10): 4320-4333. 10.1093/nar/gks017.PubMed CentralView ArticlePubMedGoogle Scholar
- Vinckx T, Wei Q, Matthijs S, Cornelis P: The Pseudomonas aeruginosa oxidative stress regulator OxyR influences production of pyocyanin and rhamnolipids: protective role of pyocyanin. Microbiol. 2010, 156: 768-686.View ArticleGoogle Scholar
- Hammer PE, Burd W, Hill DS, Ligon JM, van Pée K: Conservation of the pyrrolnitrin biosynthetic gene cluster among six pyrrolnitrin-producing strains. FEMS Microbiol Lett. 1999, 180 (1): 39-44. 10.1111/j.1574-6968.1999.tb08775.x.View ArticlePubMedGoogle Scholar
- Simon R, Priefer U, Pühler A: A broad-host-range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Bio/Technology. 1983, 1: 784-791. 10.1038/nbt1183-784.View ArticleGoogle Scholar
- Merriman TR, Lamont IL: Construction and use of a self-cloning promoter probe vector for gram-negative bacteria. Gene. 1993, 126: 17-23. 10.1016/0378-1119(93)90585-Q.View ArticlePubMedGoogle Scholar
- West SE, Schweizer HP, Dall C, Sample AK, Runyen-Janecky LJ: Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene. 1994, 148: 81-86. 10.1016/0378-1119(94)90237-2.View ArticlePubMedGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: a Laboratory Manual. 1989, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory, 2Google Scholar
- Lewenza S, Conway B, Greenberg EP, Sokol PA: Quorum sensing in Burkholderia cepacia: identification of LuxRI homogs CepRI. J Bacteriol. 1999, 181: 748-756.PubMed CentralPubMedGoogle Scholar
- Rydzak T, McQueen PD, Krokhin OV, Spicer V, Ezzati P, Dwivedi RC, Shamshurin D, Levin DB, Wilkins JA, Sparling R: Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression. BMC Microbiol. 2012, 12: 214-232. 10.1186/1471-2180-12-214.PubMed CentralView ArticlePubMedGoogle Scholar
- Wirth SJ, Wolf GA: Dye-labelled substrates for the assay and detection of chitinase and lysozyme activity. J Microbiol Methods. 1990, 12: 197-205. 10.1016/0167-7012(90)90031-Z.View ArticleGoogle Scholar
- Schwyn B, Neilands JB: Universal chemical assay for the detection and determination of siderophores. Anal Biochem. 1987, 160 (1): 47-56. 10.1016/0003-2697(87)90612-9.View ArticlePubMedGoogle Scholar
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