Xianqing Huang

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.

Temperature-Dependent Expression of phzM and Its Regulatory Genes lasI and ptsP in Rhizosphere Isolate Pseudomonas sp. Strain M18

ABSTRACT Pseudomonas sp. strain M18, an effective biological control agent isolated from the melon rhizosphere, has a genetic background similar to that of the opportunistic human pathogen Pseudomonas aeruginosa PAO1. However, the predominant phenazine produced by strain M18 is phenazine-1-carboxylic acid (PCA) rather than pyocyanin (PYO); the quantitative ratio of PCA to PYO is 105 to 1 at 28°C in strain M18, while the ratio is 1 to 2 at 37°C in strain PAO1. We first provided evidence that the differential production of the two phenazines in strains M18 and PAO1 is related to the temperature-dependent and strain-specific expression patterns of phzM, a gene involved in the conversion of PCA to PYO. Transcriptional levels of phzM were measured by quantitative real-time PCR, and the activities of both transcriptional and translational phzM′-′lacZ fusions were determined in strains M18 and PAO1, respectively. Using lasI::Gm and ptsP::Gm inactivation M18 mutants, we further show that expression of the phzM gene is positively regulated by the quorum-sensing protein LasI and negatively regulated by the phosphoenolpyruvate phosphotransferase protein PtsP. Surprisingly, the lasI and ptsP regulatory genes were also expressed in a temperature-dependent and strain-specific manner. The differential production of the phenazines PCA and PYO by strains M18 and PAO1 may be a consequence of selective pressure imposed on P. aeruginosa PAO1 and its relative M18 in the two different niches over a long evolutionary process.

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.

The distinct quorum sensing hierarchy of las and rhl in Pseudomonas sp. M18.

Pseudomonas sp. M18 is a rhizosphere isolate capable of producing two kinds of antifungal agents: phenazine-1-carboxylic acid (PCA) and pyoluteorin. Recently, the two well-studied quorum sensing (QS) systems of Pseudomonas aeruginosa, LasR/LasI and RhlR/RhlI, have also been identified in this strain. However, in this study, through the use of lacZ translational fusion expression analysis and acyl-homoserine lactone thin-layer chromatography (TLC) bioassays, we clearly display a more complex and distinctive hierarchy of the las and rhl QS systems in strain M18. In this QS cascade, expression of rhlI was negatively controlled by the LasR/LasI QS system. In contrast with lasI, which negatively regulated the rhlR induction, lasR exerted a positive influence on rhlR expression during the log-phase. This interrelationship indicated that the response regulators (LasR and RhlR) of the QS system are expressed independently of their cognate synthases (LasI and RhlI). Furthermore, the las system also modulated the timing and magnitude of the rhlI and rhlR maximal expression. In addition, our data imply that the lasR gene exerts its negative control on PCA production through modulation of rhlI expression. Thus, interactions between the two QS systems are strain specific.

Rapid quantitative analysis of phenazine-1-carboxylic acid and 2-hydroxyphenazine from fermentation culture of Pseudomonas chlororaphis GP72 by capillary zone electrophoresis

Natural phenazines in secondary metabolites of bacteria have been receiving increasing attention in recent years due to their potential usage as antibiotics. In the present study, a rapid and reliable capillary zone electrophoresis (CZE) method was developed and validated for monitoring for the first time dynamic phenazine-1-carboxylic acid (PCA) and the 2-hydroxyphenazine (2-OH-PHZ) production of Pseudomonas chlororaphis GP72 during the entire fermentation cycle. The paper begins with the optimization of separate conditions for 2-OH-PHZ and PCA together with phenazine (PHZ), which is used as internal standard. The optimized conditions are: 10mM, pH 7.3 phosphate buffer, a fused-silica capillary with a total length of 49 cm x 75 microm ID, 375 microm OD with an effective length of 40 cm, 25 kV, 13 mbar 10s pressure sample injection and 25 degrees C air-cooling. The three compounds could be separated within 2 min under optimized conditions. The validation of the newly developed study shows the linear response of 2-OH-PHZ and PCA ranging from 10 to 250 microg mL(-1) with high correlation coefficient (r=0.9997 and 0.9993, n=7), low limits of detection (0.47 and 0.38 microg mL(-1)) and quantification (1.56 and 1.28 microg mL(-1)), respectively. Good precision values for intra- and inter-day detection and acceptable individual recovery ranges for 2-OH-PHZ and PCA are indicated. The newly developed method was also validated through monitoring dynamic PCA and 2-OH-PHZ production of P. chlororaphis GP72 during an 84 h growth cycle.

QscR acts as an intermediate in gacA-dependent regulation of PCA biosynthesis in Pseudomonas sp. M-18.

The qscR gene, encoding a quorum sensing regulator, was cloned and the qscR-null mutant strain M-18Q derived from Pseudomonas sp. M-18 was constructed to study the effect of the qscR gene on biosynthesis of phenazine-1-carboxylic acid (PCA) and pyoluteorin (Plt) in strain M-18. Results showed that the PCA produced in the mutant increased four- to six-fold, while the synthesis of Plt was barely influenced in comparison with the wild type. The results were confirmed by complementation with the qscR gene in trans in strain M-18Q. The negative effect of the qscR gene on PCA production was further confirmed by analysis of β-galactosidase activities from the translational phzA’-lacZ’ fusion. Furthermore, by introducing a qscR-lacZ transcriptional fusion vector to strains M-18, M-18Q, and M-18G, a gacA inactivation mutant in strain M-18, respectively, it was found that β-galactosidase activity in both strain M-18G and strain M-18Q was decreased to half that in the wild type. This suggested that QscR might be involved in autoinducing its own gene expression and act as an intermediate in GacA-dependent gene regulation as well. The result was further demonstrated by the overexpression of the gacA gene in strain M-18Q.

Quantitative investigation of resolution increase of free-flow electrophoresis via simple interval sample injection and separation

Interval free‐flow zone electrophoresis (FFZE) has been used to suppress sample band broadening greatly hindering the development of free‐flow electrophoresis (FFE). However, there has been still no quantitative study on the resolution increase of interval FFZE. Herein, we tried to make a comparison between bandwidths in interval FFZE and continuous one. A commercial dye with methyl green and crystal violet was well chosen to show the bandwidth. The comparative experiments were conducted under the same sample loading of the model dye (viz. 3.49, 1.75, 1.17, and 0.88 mg/h), the same running time (viz. 5, 10, 15, and 20 min), and the same flux ratio between sample and background buffer (= 10.64 × 10−3). Under the given conditions, the experiments demonstrated that (i) the band broadening was evidently caused by hydrodynamic factor in continuous mode, and (ii) the interval mode could clearly eliminate the hydrodynamic broadening existing in continuous mode, greatly increasing the resolution of dye separation. Finally, the interval FFZE was successfully used for the complete separation of two‐model antibiotics (herein pyoluteorin and phenazine‐1‐carboxylic acid coexisting in fermentation broth of a new strain Pseudomonas aeruginosa M18), demonstrating the feasibility of interval FFZE mode for separation of biomolecules.

Autoinduction of RpoS Biosynthesis in the Biocontrol Strain Pseudomonas sp. M18

The rpoS gene from Pseudomonas sp. M18, which encodes predicted protein (an alternative sigma factor s, σS, or σ38) with 99.5% sequence identity with RpoS from Pseudomonas aeruginosa PAO1, was first cloned. In order to investigate the mechanism of rpoS expression, an rpoS null mutant, named M18S, was constructed with insertion of aacC1 cassette bearing a gentamycin resistance gene. With introduction of a plasmid containing an rpoS′–′lacZ translational fusion (pMERS) to wild-type strain M18 or M18S, it was first found that β-galactosidase activity expressed in strain M18S (pMERS) decreased to fourfold of that expressed in the strain M18 (pMERS). When strain M18S (pMERS) was introduced with another plasmid pBBS containing the wild-type rpoS gene, its β-galactosidase expression level was enhanced and almost restored to that in strain M18 (pMERS). Similarly, expression of β-galactosidase from a chromosomal fusion of the promoter of the wild-type rpoS gene with lacZ (rpoS–lacZ) was enhanced fivefold in the presence of a plasmid with the wild-type rpoS gene. With these findings, it is suggested that RpoS sigma factor may be involved in autoinducing its own gene expression in Pseudomonas sp. M18.

Novel Three-Component Phenazine-1-Carboxylic Acid 1,2-Dioxygenase in Sphingomonas wittichii DP58

ABSTRACT Phenazine-1-carboxylic acid, the main component of shenqinmycin, is widely used in southern China for the prevention of rice sheath blight. However, the fate of phenazine-1-carboxylic acid in soil remains uncertain. Sphingomonas wittichii DP58 can use phenazine-1-carboxylic acid as its sole carbon and nitrogen sources for growth. In this study, dioxygenase-encoding genes, pcaA1A2, were found using transcriptome analysis to be highly upregulated upon phenazine-1-carboxylic acid biodegradation. PcaA1 shares 68% amino acid sequence identity with the large oxygenase subunit of anthranilate 1,2-dioxygenase from Rhodococcus maanshanensis DSM 44675. The dioxygenase was coexpressed in Escherichia coli with its adjacent reductase-encoding gene, pcaA3, and ferredoxin-encoding gene, pcaA4, and showed phenazine-1-carboxylic acid consumption. The dioxygenase-, ferredoxin-, and reductase-encoding genes were expressed in Pseudomonas putida KT2440 or E. coli BL21, and the three recombinant proteins were purified. A phenazine-1-carboxylic acid conversion capability occurred in vitro only when all three components were present. However, P. putida KT2440 transformed with pcaA1A2 obtained phenazine-1-carboxylic acid degradation ability, suggesting that phenazine-1-carboxylic acid 1,2-dioxygenase has low specificities for its ferredoxin and reductase. This was verified by replacing PcaA3 with RedA2 in the in vitro enzyme assay. High-performance liquid chromatography–mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) analysis showed that phenazine-1-carboxylic acid was converted to 1,2-dihydroxyphenazine through decarboxylation and hydroxylation, indicating that PcaA1A2A3A4 constitutes the initial phenazine-1-carboxylic acid 1,2-dioxygenase. This study fills a gap in our understanding of the biodegradation of phenazine-1-carboxylic acid and illustrates a new dioxygenase for decarboxylation. IMPORTANCE Phenazine-1-carboxylic acid is widely used in southern China as a key fungicide to prevent rice sheath blight. However, the degradation characteristics of phenazine-1-carboxylic acid and the environmental consequences of the long-term application are not clear. S. wittichii DP58 can use phenazine-1-carboxylic acid as its sole carbon and nitrogen sources. In this study, a three-component dioxygenase, PcaA1A2A3A4, was determined to be the initial dioxygenase for phenazine-1-carboxylic acid degradation in S. wittichii DP58. Phenazine-1-carboxylic acid was converted to 1,2-dihydroxyphenazine through decarboxylation and hydroxylation. This finding may help us discover the pathway for phenazine-1-carboxylic acid degradation.