Huasong Peng

Characterization of a Phenazine-Producing Strain Pseudomonas chlororaphis GP72 with Broad-Spectrum Antifungal Activity from Green Pepper Rhizosphere

A new Pseudomonas strain, designated GP72, was isolated from green pepper rhizosphere and identified as a member of species Pseudomonas chlororaphis based on morphology; conventional biochemical and physiologic tests; Biolog GN system (Biolog Inc., Hayward, CA); and 16S rDNA sequence analysis. The secondary metabolites produced by this strain have shown broad-spectrum antifungal activity against various phytopathogens of agricultural importance in vitro. Two main antifungal substances produced by this strain proved to be phenazine-1-carboxylic acid and 2-hydroxyphenazine with further purification and structure elucidation based on ultraviolet-absorbent spectrum scanning, atmospheric pressure chemical ionization–mass spectrometry (APCI-MS) spectrum, and 1H,13C nuclear magnetic resonance spectrums. Strain GP72 could produce quorum-sensing signaling molecules of N-butanoyl-L-homoserine lactone and N-hexanoyl-L-homoserine lactone, which were found to accumulate with different quantities in King’s medium B and pigment producing medium, respectively.

Comparative genomic analysis of four representative plant growth-promoting rhizobacteria in Pseudomonas

BackgroundSome Pseudomonas strains function as predominant plant growth-promoting rhizobacteria (PGPR). Within this group, Pseudomonas chlororaphis and Pseudomonas fluorescens are non-pathogenic biocontrol agents, and some Pseudomonas aeruginosa and Pseudomonas stutzeri strains are PGPR. P. chlororaphis GP72 is a plant growth-promoting rhizobacterium with a fully sequenced genome. We conducted a genomic analysis comparing GP72 with three other pseudomonad PGPR: P. fluorescens Pf-5, P. aeruginosa M18, and the nitrogen-fixing strain P. stutzeri A1501. Our aim was to identify the similarities and differences among these strains using a comparative genomic approach to clarify the mechanisms of plant growth-promoting activity.ResultsThe genome sizes of GP72, Pf-5, M18, and A1501 ranged from 4.6 to 7.1 M, and the number of protein-coding genes varied among the four species. Clusters of Orthologous Groups (COGs) analysis assigned functions to predicted proteins. The COGs distributions were similar among the four species. However, the percentage of genes encoding transposases and their inactivated derivatives (COG L) was 1.33% of the total genes with COGs classifications in A1501, 0.21% in GP72, 0.02% in Pf-5, and 0.11% in M18. A phylogenetic analysis indicated that GP72 and Pf-5 were the most closely related strains, consistent with the genome alignment results. Comparisons of predicted coding sequences (CDSs) between GP72 and Pf-5 revealed 3544 conserved genes. There were fewer conserved genes when GP72 CDSs were compared with those of A1501 and M18. Comparisons among the four Pseudomonas species revealed 603 conserved genes in GP72, illustrating common plant growth-promoting traits shared among these PGPR. Conserved genes were related to catabolism, transport of plant-derived compounds, stress resistance, and rhizosphere colonization. Some strain-specific CDSs were related to different kinds of biocontrol activities or plant growth promotion. The GP72 genome contained the cus operon (related to heavy metal resistance) and a gene cluster involved in type IV pilus biosynthesis, which confers adhesion ability.ConclusionsComparative genomic analysis of four representative PGPR revealed some conserved regions, indicating common characteristics (metabolism of plant-derived compounds, heavy metal resistance, and rhizosphere colonization) among these pseudomonad PGPR. Genomic regions specific to each strain provide clues to its lifestyle, ecological adaptation, and physiological role in the rhizosphere.

Comparative genomic analysis and phenazine production of Pseudomonas chlororaphis, a plant growth-promoting rhizobacterium.

Pseudomonas chlororaphis HT66, a plant growth-promoting rhizobacterium that produces phenazine-1-carboxamide with high yield, was compared with three genomic sequenced P. chlororaphis strains, GP72, 30–84 and O6. The genome sizes of four strains vary from 6.66 to 7.30 Mb. Comparisons of predicted coding sequences indicated 4833 conserved genes in 5869–6455 protein-encoding genes. Phylogenetic analysis showed that the four strains are closely related to each other. Its competitive colonization indicates that P. chlororaphis can adapt well to its environment. No virulence or virulence-related factor was found in P. chlororaphis. All of the four strains could synthesize antimicrobial metabolites including different phenazines and insecticidal protein FitD. Some genes related to the regulation of phenazine biosynthesis were detected among the four strains. It was shown that P. chlororaphis is a safe PGPR in agricultural application and could also be used to produce some phenazine antibiotics with high-yield.

Genetic diversity of phenazine- and pyoluteorin-producing pseudomonads isolated from green pepper rhizosphere

The genetic diversity among indigenous phenazine-1-carboxylic acid (PCA)-producing and pyoluteorin (Plt)-producing isolates of pseudomonads screened from green pepper rhizosphere was exploited in this study. A total of 48 bacterium isolates producing one or both of these antibiotics were screened from green pepper rhizosphere in diverse regions in China. Among these isolates, 45 could produce PCA, 3 could produce both PCA and Plt, and none could produce Plt only. Based on the restriction patterns of partial 16S and 16S-23S internal transcribed spacer (ITS) PCR fragments generated by enzyme HaeIII or HinfI, these isolates fell into 19 or 17 distinct groups respectively, indicating that there was a significant diversity among them. Polygenetic analysis of the partial 16S rDNA and 16S-23S ITS sequence from the representative in each group in the context of similar sequence from previously described bacterial species indicated that most isolates were closely related to the species of Pseudomonas fluorescens, P. putida, and Stenotrophomonas maltophilia. Some of these representatives of these isolates, then, are likely to be novel strains or species in these two genera.

Genome Sequence of Pseudomonas chlororaphis GP72, a Root-Colonizing Biocontrol Strain

Pseudomonas chlororaphis GP72 is a root-colonizing biocontrol strain isolated from a green pepper rhizosphere. It can produce several secondary metabolites to suppress phytopathogens. Here we present a 6.6-Mb assembly of its genome, which is the first genome sequence of the P. chlororaphis group and may provide insights into its antifungal activities.

iTRAQ-based quantitative proteomic analysis reveals potential factors associated with the enhancement of phenazine-1-carboxamide production in Pseudomonas chlororaphis P3.

Phenazine-1-carboxamide (PCN), a phenazine derivative, is strongly antagonistic to fungal phytopathogens. Pseudomonas chlororaphis HT66 is a PCN-producing, non-pathogenic biocontrol strain, and we obtained the mutant P. chlororaphis P3, which produces 4.7 times more PCN than the wild-type HT66 strain. To reveal the cause of PCN production enhancement in P3 and find potential factors related to PCN biosynthesis, an iTRAQ-based quantitative proteomic analysis was used to study the expression changes between the two strains. Of the 452 differentially expressed proteins, most were functionally mapped into PCN biosynthesis pathway or other related metabolisms. The upregulation of proteins, including PhzA/B, PhzD, PhzF, PhzG, and PhzH, involved in PCN biosynthesis was in agreement with the efficient production of PCN in P3. A number of proteins that function primarily in energy production, amino acid metabolism, and secondary metabolism played important roles in PCN biosynthesis. Notably, proteins involved in the uptake and conversion of phosphate, inorganic nitrogen sources, and iron improved the PCN production. Furthermore, the type VI secretion system may participate in the secretion or/and indirect biosynthetic regulation of PCN in P. chlororaphis. This study provides valuable clues to better understand the biosynthesis, excretion and regulation of PCN in Pseudomonas and also provides potential gene targets for further engineering high-yield strains.

Genome Sequence of Sphingomonas wittichii DP58, the First Reported Phenazine-1-Carboxylic Acid-Degrading Strain

Sphingomonas wittichii DP58 (CCTCC M 2012027), the first reported phenazine-1-carboxylic acid (PCA)-degrading strain, was isolated from pimiento rhizosphere soils. Here we present a 5.6-Mb assembly of its genome. This sequence would contribute to the elucidation of the molecular mechanism of PCA degradation to improve the antifungals effectiveness or remove superfluous PCA.

Determination of a Novel Fungicide Phenazine-1-Carboxylic Acid in Soil Samples Using Sample Stacking Capillary Electrophoresis Combined with Solid Phase Extraction

Solid-phase extraction and stacking capillary electrophoresis were used for the determination of phenazine-1-carboxylic acid, a novel fungicide, in soil samples. A 10 mmol L−1 phosphate buffer (pH = 6.6) was adequate for the separation and stacking of PCA dissolved in methanol. Optimization of SPE resulted in high recoveries ranging from 92.35% to 107.04% at spiking levels from 0.02 to 0.30 mg kg−1. Limits of quantification were 0.09 μg mL−1 in methanol solution and 0.02 mg kg−1 in soil samples, which greatly improved the sensitivity compared to previous methods. This method allowed dynamic monitoring of PCA residues during its decomposition in soil.

Genome Sequence of Sphingobium yanoikuyae B1, a Polycyclic Aromatic Hydrocarbon-Degrading Strain

ABSTRACT Sphingobium yanoikuyae B1 can utilize biphenyl, naphthalene, phenanthrene, toluene, and m-/p-xylene as sole sources of carbon and energy. Here, we present a 5.2-Mb assembly of its genome. An analysis of the genome can provide insights into the mechanisms of polycyclic aromatic hydrocarbon (PAH) degradation and potentially aid in bioremediation applications.

Reaction kinetics for the biocatalytic conversion of phenazine-1-carboxylic acid to 2-hydroxyphenazine.

The phenazine derivative 2-hydroxyphenazine (2-OH-PHZ) plays an important role in the biocontrol of plant diseases, and exhibits stronger bacteriostatic and fungistatic activity than phenazine-1-carboxylic acid (PCA) toward some pathogens. PhzO has been shown to be responsible for the conversion of PCA to 2-OH-PHZ, however the kinetics of the reaction have not been systematically studied. Further, the yield of 2-OH-PHZ in fermentation culture is quite low and enhancement in our understanding of the reaction kinetics may contribute to improvements in large-scale, high-yield production of 2-OH-PHZ for biological control and other applications. In this study we confirmed previous reports that free PCA is converted to 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA) by the action of a single enzyme PhzO, and particularly demonstrate that this reaction is dependent on NADP(H) and Fe3+. Fe3+ enhanced the conversion from PCA to 2-OH-PHZ and 28°C was a optimum temperature for the conversion. However, PCA added in excess to the culture inhibited the production of 2-OH-PHZ. 2-OH-PCA was extracted and purified from the broth, and it was confirmed that the decarboxylation of 2-OH-PCA could occur without the involvement of any enzyme. A kinetic analysis of the conversion of 2-OH-PCA to 2-OH-PHZ in the absence of enzyme and under different temperatures and pHs in vitro, revealed that the conversion followed first-order reaction kinetics. In the fermentation, the concentration of 2-OH-PCA increased to about 90 mg/L within a red precipitate fraction, as compared to 37 mg/L within the supernatant. The results of this study elucidate the reaction kinetics involved in the biosynthesis of 2-OH-PHZ and provide insights into in vitro methods to enhance yields of 2-OH-PHZ.