Simply enhancing throughput of free-flow electrophoresis via organic-aqueous environment for purification of weak polarity solute of phenazine-1-carboxylic acid in fermentation of Pseudomonas sp. M18.

Herein, a simple novel free-flow electrophoresis (FFE) method was developed via introduction of organic solvent into the electrolyte system, increasing the solute solubility and throughput of the sample. As a proof of concept, phenazine-1-carboxylic acid (PCA) from Pseudomonas sp. M18 was selected as a model solute for the demonstration on feasibility of novel FFE method on account of its faint solubility in aqueous circumstance. In the developed method, the organic solvent was added into not only the sample buffer to improve the solubility of the solute, but also the background buffer to construct a uniform aqueous-organic circumstance. These factors of organic solvent percentage and types as well as pH value of background buffer were investigated for the purification of PCA in the FFE device via CE. The experiments revealed that the percentage and the types of organic solvent exerted major influence on the purification of PCA. Under the optimized conditions (30 mM phosphate buffer in 60:40 (v/v) water-methanol at an apparent pH 7.0, 3.26 mL/min background flux, 10-min residence time of injected sample, and 400 V), PCA could be continuously purified from its impurities. The flux of sample injection was 10.05 µL/min, and the recovery was up to 93.7%. An 11.9-fold improvement of throughput was found with a carrier buffer containing 40% (v/v) methanol, compared with the pure aqueous phase. The developed procedure is of evident significance for the purification of weak polarity solute via FFE.

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.

Novel antiphytopathogenic compound 2-heptyl-5-hexylfuran-3-carboxylic acid, produced by newly isolated Pseudomonas sp. strain SJT25.

Pseudomonas sp. strain SJT25, which strongly antagonizes plant pathogens, was isolated from rice rhizosphere soil by a bioactivity-guided approach. A novel antiphytopathogenic compound was isolated from the fermentation broth of Pseudomonas sp. SJT25 and identified as 2-heptyl-5-hexylfuran-3-carboxylic acid. This compound showed antimicrobial activities both in vitro and in vivo.

Enhanced production of 2-hydroxyphenazine in Pseudomonas chlororaphis GP72.

Pseudomonas chlororaphis GP72 is a root-colonizing biocontrol strain isolated from the green pepper rhizosphere that synthesizes two phenazine derivatives: phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH-PHZ). The 2-OH-PHZ derivative shows somewhat stronger broad-spectrum antifungal activity than PCA, but its conversion mechanism has not yet been clearly revealed. The aim of this study was to clone and analyze the phenazine biosynthesis gene cluster in this newly found strain and to improve the production of 2-OH-PHZ by gene disruption and precursor addition. The conserved phenazine biosynthesis core operon in GP72 was cloned by PCR, and the unknown sequences located upstream and downstream of the core operon were detected by random PCR gene walking. This led to a complete isolation of the phenazine biosynthesis gene cluster phzIRABCDEFG and phzO in GP72. Gene rpeA and phzO were insertionally mutated to construct GP72AN and GP72ON, respectively, and GP72ANON collectively. The inactivation of rpeA resulted in a fivefold increase in the production of PCA, as well as 2-OH-PHZ. The addition of exogenous precursor PCA to the broth culture, to determine the conversion efficiency of PCA to 2-OH-PHZ under current culture conditions, revealed that PCA had a positive feedback effect on its own accumulation, leading to enhanced synthesis of both PCA and 2-OH-PHZ. The production of 2-OH-PHZ by GP72AN increased to about 170 µg ml(-1), compared with just 5 µg ml(-1) for the wild type. The hypothesis of biosynthetic pathway for 2-OH-PHZ from PCA was confirmed by identification of 2-hydroxyphenazine-1-carboxylic acid as an intermediate in the culture medium of the high-phenazine producing GP72AN mutant.

Stacking and determination of phenazine-1-carboxylic acid with low pKa in soil via moving reaction boundary formed by alkaline and double acidic buffers in capillary electrophoresis.

As shown herein, a normal moving reaction boundary (MRB) formed by an alkaline buffer and a single acidic buffer had poor stacking to the new important plant growth promoter of phenazine-1-carboxylic acid (PCA) in soil due to the leak induced by its low pK(a). To stack the PCA with low pK(a) efficiently, a novel stacking system of MRB was developed, which was formed by an alkaline buffer and double acidic buffers (viz., acidic sample and blank buffers). With the novel system, the PCA leaking into the blank buffer from the sample buffer could be well stacked by the prolonged MRB formed between the alkaline buffer and blank buffer. The relevant mechanism of stacking was discussed briefly. The stacking system, coupled with sample pretreatment, could achieve a 214-fold increase of PCA sensitivity under the optimal conditions (15 mM (pH 11.5) Gly-NaOH as the alkaline buffer, 15 mM (pH 3.0) Gly-HCl-acetonitrile (20%, v/v) as the acidic sample buffer, 15 mM (pH 3.0) Gly-HCl as the blank buffer, 3 min 13 mbar injection of double acidic buffers, benzoic acid as the internal standard, 75 µm i.d. × 53 cm (44 cm effective length) capillary, 25 kV and 248 nm). The limit of detection of PCA in soil was decreased to 17 ng/g, the intra-day and inter-day precision values (expressed as relative standard deviations) were 3.17-4.24% and 4.17-4.87%, respectively, and the recoveries of PCA at three concentration levels changed from 52.20% to 102.61%. The developed method could be used for the detection of PCA in soil at trace level.

Purification of low-concentration phenazine-1-carboxylic acid from fermentation broth of Pseudomonas sp. M18 via free flow electrophoresis with gratis gravity.

The low-concentration phenazine-1-carboxylic acid (PCA) ( = 0.3 mM) extracted from fermentation broth of Pseudomonas sp. M18 was selected to be purified with a newly facile free flow electrophoresis (FFE) device with gratis gravity. Three factors of pH value and concentration of background buffer, and the cooling circle of FFE device were investigated for the purification of PCA in the FFE device. It was found that the pH value and concentration of background buffer had mild influences on the separation of PCA whether with cooling circle or not. However, the cooling circle had a much greater impact on the separation of PCA. The controlling of the band zone of PCA in FFE chamber would be difficult if without cooling circle, while the controlling would become easy if with cooling circle. Under the optimal conditions (10 mM pH 5.5 phosphate as background buffer, 30 mM pH 5.5 phosphate buffer as electrode solution, 5.46 mL/min background flux, 10 min residence time of injected sample, and 500 V), PCA could be continuously prepared from its impurities with relative high purity. The flux of sample injection was 115 µL/min, viz. 7 mL sample throughput per hour, and the recovery was up to 85%. All of the experiments indicated that the FFE technique was a good alternative tool for the study on natural biological control agents.

Medium optimization for phenazine-1-carboxylic acid production by a gacA qscR double mutant of Pseudomonas sp. M18 using response surface methodology.

Statistics based experimental designs were applied to optimize the culture medium components for enhancing phenazine-1-carboxylic acid (PCA) production by Pseudomonas sp. strain M18GQ, a gacA qscR double mutant of Pseudomonas sp. strain M18. The medium components, including soybean meal, glucose and corn steep liquor, had significant effects on PCA production based on a 2(5-1) fractional factorial design. The concentrations of these three significant factors were subsequently optimized using a central composite design. An optimum concentration of soybean meal 73.3g/L, corn steep liquor 18.1g/L, glucose 17.9g/L, and ethanol 18.0ml/L was obtained by response surface analysis. The predicted maximum PCA yield was as high as 4011.5mg/L, and was confirmed by validation experiments. The double mutant M18GQ produced 4032.2mg/L PCA, which was almost 2 to 3.3-fold that produced by either single mutant M18G and M18Q. The achieved production level is economically useful for industrial application.

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.

Optimization of critical medium components using response surface methodology for phenazine-1-carboxylic acid production by Pseudomonas sp. M-18Q.

The optimal flask-shaking batch fermentation medium for phenazine-1-carboxylic acid (PCA) production by Pseudomonas sp. M-18Q, a qscR chromosomal inactivated mutant of the strain M18 was studied using statistical experimental design and analysis. The Plackett-Burman design (PBD) was used to evaluate the effects of eight medium components on the production of PCA, which showed that glucose and soytone were the most significant ingredients (P<0.05). The steepest ascent experiment was adopted to determine the optimal region of the medium composition. The optimum composition of the fermentation medium for maximum PCA yield, as determined on the basis of a five-level two-factor central composite design (CCD), was obtained by response surface methodology (RSM). The high correlation between the predicted and observed values indicated the validity of the model. A maximum PCA yield of 1240 mg/l was obtained at 17.81 g/l glucose and 11.47 g/l soytone, and the production was increased by 65.3% compared with that using the original medium, which was at 750 mg/l.

Medium factor optimization and fermentation kinetics for phenazine-1-carboxylic acid production by Pseudomonas sp. M18G.

We investigated the production of biofungicide phenazine-1-carboxlic (PCA) by Pseudomonas sp. M18G, a gacA-deficient mutant of M18 for PCA high-production. Glucose was chosen as the optimal carbon source and soy peptone as the nitrogen source. A Plackett-Burman design revealed that glucose, soy peptone and NaCl were the most significant factors in PCA fermentation. Response surface methodology (RSM) and artificial neural network (ANN) models involving the significant factors were developed using common data. The prediction accuracy of ANN was slightly higher compared to RSM. The genetic algorithm (GA) was used to search the optimal input space of the trained ANN model and find the corresponding PCA yield. The optimum composition was found to be: glucose 34.3 g L(-1), soy peptone 43.2 g L(-1), NaCl 5.7 g L(-1), and the predictive maximum PCA yield reached 980.1 microg mL(-1). The optimized medium allowed PCA yield to be increased from 673.3 to 966.7 microg mL(-1) after verification experiment tests. Additionally, PCA fermentation kinetics was investigated. Kinetic models based on the modified Logistic and Luedeking-Piret equations were developed, providing a good description of temporal variations of biomass (X), product (P), and substrate (S) in PCA fermentation.

Isolation, identification, and degradation characteristics of phenazine-1-carboxylic acid-degrading strain Sphingomonas sp. DP58.

A phenazine-1-carboxylic acid (PCA)-degrading bacterium, strain DP58, was isolated from pimiento rhizosoil. Based on morphology, physiologic tests, 16S rDNA sequence, and phylogenetic characteristics, it was identified as Sphingomonas sp. The PCA-degradation experiments were conducted both in Luria-Bertani and inorganic salt medium at 28 degrees C. The relationship between bacterium growth and PCA degradation suggested that strain DP58 could use PCA as the sole source of carbon and nitrogen and was able to completely degrade PCA in 40 hours. Newly isolated strain DP58 represents the first bacterium that can degrade PCA.

[Construction of Pseudomonas sp. M18 qscR- mutant and its regulation on biosynthesis of PCA and Plt].

In Gram-negative bacteria, global regulator QscR controls the expression of many virulence determinants, secondary metabolites, stationary phase genes and genes involved in biofilm formation through quorum sensing (QS) systems. QscR binds the promoter region of target genes and regulates the gene expression at the transcriptional level. Using homologous recombination technique a chromosomal qscR inactivated mutant strain M-18Q was constructed in Pseudomonas sp. M-18, a strain of plant-growth-promoting rhizobacteria, which could inhibit several soilborn phytopathogens by producing secondary metabolites including phenazine-1-carboxylic acid (PCA) and pyoluteorin (Plt) in one single strain. To further study the effect of QscR on the synthesis of Plt and PCA in the wild type strain M-18, the dynamic curves of Plt and PCA produced respectively by M-18 and M-18Q strains were measured in both KMB and PPM mediums. The synthesis of PCA was much more activated in the mutant than in the wild type both in KMB and PPM mediums. The PCA production in the mutant strain is four-to-six fold over that in the wild type in the PPM medium, reaching 480 pg/mL, and three-to-five fold in the KMB medium, reaching 140 microg/mL. The synthesis of Plt, however, was not detected in PPM medium and was nearly not influenced by the QscR protein in KMB medium. PCA production was inhibited but Plt biosynthesis was not altered after complementation with qscR gene in trans in the strain of M-18Q. The regulation of qscR gene on PCA production was further confirmed by the analysis of beta-galactosidase activities from the translational phzA '-' lacZ fusion, in which phzA is the first enzyme gene of the phenazine biosynthesis pathway. These results indicate that QscR can control PCA production negatively but not Plt production in M-18, and show that QscR functions as a global regulator to differently regulate the synthesis of PCA and Plt on the gene expression level.

Correlation between antifungal agent phenazine-1-carboxylic acid and pyoluteorin biosynthesis in Pseudomonas sp. M18.

Biosynthesis and secretion of two different types of antifungal compound [phenazine-1-carboxylic acid (PCA) and pyoluteorin (Plt) in Pseudomonas sp. M18] contribute to its suppression of soil-borne root pathogens. To better understand the correlation between two antifungal agents in secondary metabolism, a DNA fragment covering partial pltC and pltD coding sequences was obtained by screening the genomic library of Pseudomonas sp. M18. A mutant, M18T, was then constructed by insertion of the aacC1 gene cassette (encoding gentamycin resistance). With the same methods, one PCA biosynthetic gene cluster was insertionally inactivated and a mutant M18Z1 was created. The mutant strain M18T produces no Plt and the same amount of PCA in comparison with the wild-type strain M18. The mutant M18Z1, however, produces less PCA but more Plt than the wild-type strain M18. According to the documented data on strain M18, it is suggested that production of PCA is not influenced by Plt yield, but Plt biosynthesis is influenced by an alteration of PCA production.

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, sigma(S), or sigma(38)) 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 beta-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 beta-galactosidase expression level was enhanced and almost restored to that in strain M18 (pMERS). Similarly, expression of beta-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.

[Construction and identification of rpoS gene inserted and fused in frame with the promoterless lacZ in Pseudomonas sp. M18].

With hybridization in situ and Southern blots, an Eco RI- Xho I DNA fragment of 3.1 kb in length containing an rpoS gene and its flanking sequence was first cloned into pBluescript SK to generate pBLS by screening the genomic DNA library of Pseudomonas sp. M18. In order to identify the potential factors involved in rpoS gene expression and the regulatory mechanism of RpoS in strain M18, the rpoS gene was inserted and fused in frame with a promoterless and truncated lacZ gene, and a mutant named as M18SZ was then constructed through homologous recombination. Growth curves in KMB medium indicated that loss of RpoS made the mutant strain M18SZ more sensitive to alteration of some environmental factors. With detection and comparison of beta-galactosidase activities from both the wild type strain M18 and its derivative M18SZ cultivated in KMB medium respectively, it was found that the expression level of beta-galactosidase activities in the mutant M18SZ was high and could come to 480U. Expression of beta-galactosidase activities of the wild type strain M18 in KMB medium was not almost detected during its whole growth phase. With these results, it was confirmed that the rpoS gene did be fused in frame with the truncated lacZ gene in chromosome of the mutant M18SZ. Meanwhile, it is suggested that construction of a mutation, which is made with fusion in frame with the truncated lacZ gene, may be verified by detecting its beta-galactosidase activity, not using Southern blot or PCR.

[Isolation and characterization of a new Pseudomonas strain against Phytophthora capsici].

One Pseudomonas strain GP72, which was against Phytophthora capsici, was isolated from green pepper rhizosphere in Jiangsu province. It had distinctively inhibitive effect on several kinds of pathogenic fungi; mostly of them are soilborne pathogens. Therefore, this strain may be used for an effective biocontrol strain in the crop protection. The morphological, biochemical and physiological characteristics, Biolog GN, G + C mol% content and 16S rDNA sequence analysis of this strain were studied. In comparison and conclusion of all the experimental data, GP72 is identified as Pseudomonas chlororaphis. The strain is single-cellular and motile by means of single polar flagellum. It was not able to accumulate ploy-beta-hydroxybutyrate. Compared to P. aureofaciens 30-84, the strain was able to survive at the concentration of 5% NaCl. It could strongly utilize 45 of 95 carbon-substrates; weakly utilize 6 of the whole carbon-substrates and never utilize 43 of the whole carbon-substrates resulting from analysis of Biolog GN, bearing the similarity probability of 98% with Pseudomonas chlororaphis and with the similarity index 0.72. The G + C content of the strain DNA was 65.1 mol% using the thermal denaturation method. A phylogenetic tree was constructed by comparing with the validly published 16S rDNA sequences of the related type strains from GenBank, using the Neighbor-Joining method of Saitou and Nei and the Clustal X program to do the multiple alignments. The tree topology was tested by a bootstrap analysis of 1000 samplings. The overall similarity value between strain GP72 and typical is the closest in the phylogenetic tree. For the latest taxonomical development has put genus Pseudomonas aureofaciens to the genus Pseudomonas chlororaphis, then it is appropriate to say that GP72 belongs to the genus Pseudomonas chlororaphis. This is the first time in China to report that a strain of Pseudomonas chlororaphis was isolated from green pepper rhizosphere, having a strong inhibitive effect on Phytophthora capsici and other soilborne pathogenic fungus. The other characteristics and the biocontrol mechanism are yet to be further studied.

[Analysis of mechanism and relationship of GacA and RsmA, two regulators of antibiotics production in Pseudomonas sp. M18].

In previous study, it has already been confirmed that the wild type strain of Pseudomonas sp. M18 isolated from the agricultural soil can produce two antifungal agents phenazine-1-carboxylic acid (PCA) and pyoluteorin (Plt). Biosynthesis and secretion of these secondary metabolites contribute to its biological control and suppression of soilborne pathogenic fungi. As main regulators, GacA and RsmA differentially exert global regulation on production of PCA and Plt, respectively. In order to study the regulatory mechanism of secondary metabolites production in Pseudomonas sp. M18, a gacArsmA double mutant, designated as M18GR, was constructed with insertional mutation. Then, the mutant M18G, M18R, M18GR and the wild type strain M18 were inoculated into PPM or King's medium B (KMB), respectively. During cultivation of strain M18 and its derivatives, their PCA and Pit were respectively detected with High Performance Liquid Chromatography (HPLC). The results showed that PCA production in the mutant M18GR was lower than that in the mutant M18G and higher than that in the mutant M18R. Plt production in the mutant M18GR was, however, much less than that in the mutant M18R and much more than that in the strain M18 and the mutant M18G. With these observations, it is tempting to suggest that biosynthesis of PCA and Plt regulated by GacA or RsmA seem to occur at posttranscriptional level, not at transcriptional level. This regulation on secondary metabolites seems to be indirectly mediated by other unknown factors. Meanwhile, based on the construction of two translational fusions, gacA'-'lacZ and rsmA'-'lacZ, the assay of beta-galactosidase activities in KMB medium indicated that both GacA and RsmA did not have autoinduction of their own gene expression, respectively. Although GacA did not influence expression of the rsmA gene, RsmA could exert some positive influence on the gacA gene expression in Pseudomonas sp. M18.

[Negative regulation on PCA production is not correlated with positive regulation on BHL and HHL synthesis by gacA in Pseudomonas sp. M18].

At least 5 kinds of N-acyl-homoserine lactones(AHLs) were identified in Pseudomonas sp. M18. They were: N-butyryl-L-homoserine lactone(C4-HSL, BHL), N-hexanoyl-L-homoserine lactone(C6-HSL, HHL), N-(3-oxohexanoyl)-L-homoserine lactone(3-Oxo-C6-HSL, OHHL), N-(3-oxooctanoyl)-L-homoserine lactone (3-Oxo-C8-HSL, OOHL) and N-(3-oxodecanoyl)-L-homoserine lactone(3-Oxo-C10-HSL, ODHL). Compared with the wide-type strain M18, the variety of the AHLs in the gacA mutant strain M18G reduced to 4 species with the decreased quantity. But the phenazine-1-carboxylic acid (PCA) production was increased by about 2-fold. The product of rhll plays an important role in synthesizing BHL and HHL. The rhll'-'lacZ translational fusion expression plasmid pMEIZ was constructed in vector pME6015 and then was introduced into the wild-type strain M18 and the gacA mutant strain M18G. The galactosidase activity in chromosomal gacA disruption mutant was only 60% of that in the wild-type strain M18. This result suggested that GacA could regulate the rhll expression positively. There was no influence on PCA production by adding exogenous BHL and HHL and both together to the culture of strains M18 and M18G. This result suggested that there was no relation between GacA's negative regulation on PCA production and positive regulation on BHL and HHL synthesis.

[Effects of sigma38 subunit deletion of RNA polymerase on antibiotics biosynthesis in pseudomonas].

With the designed primers, PCR was carried out using the genomic DNA of Pseudomonas sp. M18 as a template and a 378bp DNA fragment of the rpoS gene was amplified. Then, a 3. 1kb EcoR I -Xho I fragment containing the rpoS gene and its flanking sequence was obtained by screening the genomic DNA library of Pseudomonas sp. M18.A sigma38-subunit-deficient mutant M18S was constructed with insertional gentamycin gene cassette. In PPM medium, the mutant M18S produced 20.4 microg/mL of PCA and 75 microg/mL of Plt. In KMB medium, the mutant M18S produced no PCA and 185.6 microg/mL of Pit. It is obvious that the deficiency of sigma38 subunit in the mutant M18S leads less or no PCA production and much more Plt production than those in the wild type strain M18. PCA and Plt production were restored to the levels in wild type strain after complementation with rpoS gene in trans in strain M18S. Moreover, beta-galactosidase activities of the translational fusions phzA'-'lacZ and pltA'-'lacZ in strain M18S confirmed the effects of sigma38 subunit on PCA and Plt biosynthetic operons. With these results, it is suggested that sigma38 subunit gives a differential impacts on PCA and Plt biosynthesis, i. e, PCA production is positively regulated but Plt production is negatively influenced by sigma38 subunit in Pseudomonas sp. M18.

[Transcriptional repression of pltZ on pyoluteorin ABC transporter of Pseudomonas sp. M18].

A pltZ gene involved in negatively regulating pyoluteorin (Plt) biosynthesis, and an ABC (ATP-binding cassette) transporter involved in pyoluteorin secretion and itself resistance, were identified downstream of Plt biosynthesis gene cluster in Pseudomonas sp. M18. The ABC transporter gene pltH'-lacZ translational and transcriptional fusion expression plasmids, pHZLF and pHZCF, were constructed using promoter probe vector pME6015 and pME6522 respectively, and then were introduced into the wild-type strain of Pseudomonas sp. M18 and the pltZ mutant strain M18Z. The chromosomal pltZ disruption mutant gave rise to 3.7-8.4-fold enhancement of translational pltH'-'lacZ fusion expression and 2.8-7.4-fold enhancement of transcriptional pltH'-'lacZ fusion expression compared with those in the wild-type strain M18. These results suggested that pltZ can repress transcription of Plt ABC transporter, and it can indirectly regulate Plt biosynthesis negatively through the Plt ABC transporter system.