LysR-type transcriptional regulator FinR is required for phenazine and pyrrolnitrin biosynthesis in biocontrol Pseudomonas chlororaphis strain G05.

Phenazine-1-carboxylic acid and pyrrolnitrin, the two secondary metabolites produced by Pseudomonas chlororaphis G05, serve as biocontrol agents that mainly contribute to the growth repression of several fungal phytopathogens. Although some regulators of phenazine-1-carboxylic acid biosynthesis have been identified, the regulatory pathway involving phenazine-1-carboxylic acid synthesis is not fully understood. We isolated a white conjugant G05W03 on X-Gal-containing LB agar during our screening of novel regulator candidates using transposon mutagenesis with a fusion mutant G05Δphz::lacZ as a recipient. By cloning of DNA adjacent to the site of the transposon insertion, we revealed that a LysR-type transcriptional regulator (LTTR) gene, finR, was disrupted in the conjugant G05W03. To confirm the regulatory function of FinR, we constructed the finR-knockout mutant G05ΔfinR, G05Δphz::lacZΔfinR, and G05Δprn::lacZΔfinR, using the wild-type strain G05 and its fusion mutant derivatives as recipient strains, respectively. We found that the expressions of phz and prn operons were dramatically reduced in the finR-deleted mutant. With quantification of the production of antifungal metabolites biosynthesized by the finR-negative strain G05ΔfinR, it was shown that FinR deficiency also led to decreased yield of phenazine-1-carboxylic acid and pyrrolnitrin. In addition, the pathogen inhibition assay confirmed that the production of phenazine-1-carboxylic acid was severely reduced in the absence of FinR. Transcriptional fusions and qRT-PCR verified that FinR could positively govern the transcription of the phz and prn operons. Taken together, FinR is required for antifungal metabolite biosynthesis and crop protection against some fungal pathogens.Key points• A novel regulator FinR was identified by transposon mutagenesis.• FinR regulates antifungal metabolite production.• FinR regulates the phz and prn expression by binding to their promoter regions.

Pip serves as an intermediate in RpoS-modulated phz2 expression and pyocyanin production in Pseudomonas aeruginosa.

Pyocyanin, a main virulence factor that is produced by Pseudomonas aeruginosa, plays an important role in pathogen-host interaction during infection. Two copies of phenazine-biosynthetic operons on genome, phz1 (phzA1B1C1D1E1F1G1) and phz2 (phzA2B2C2D2E2F2G2), contribute to phenazine biosynthesis. In our previous study, we found that RpoS positively regulates expression of the phz2 operon and pyocyanin biosynthesis in P. aeruginosa PAO1. In this work, when a TetR-family regulator gene, pip, was knocked out, we found that pyocyanin production was dramatically reduced, indicating that Pip positively regulates pyocyanin biosynthesis. With further phenazines quantification and ß-galactosidase assay, we confirmed that Pip positively regulates phz2 expression, but does not regulate phz1 expression. In addition, while the rpoS gene was deleted, expression of pip was down-regulated. Expression of rpoS in the wild-type PAO1 strain, however, was similar to that in the Pip-deficient mutant PAΔpip, suggesting that expression of pip could positively be regulated by RpoS, whereas rpoS could not be regulated by Pip. Taken together, we drew a conclusion that Pip might serve as an intermediate in RpoS-modulated expression of the phz2 operon and pyocyanin biosynthesis in P. aeruginosa.

Overexpression of phzM contributes to much more production of pyocyanin converted from phenazine-1-carboxylic acid in the absence of RpoS in Pseudomonas aeruginosa.

Pyocyanin produced by Pseudomonas aeruginosa is a key virulence factor that often causes heavy damages to airway and lung in patients. Conversion of phenazine-1-carboxylic acid to pyocyanin involves an extrametabolic pathway that contains two enzymes encoded, respectively, by phzM and phzS. In this study, with construction of the rpoS-deficient mutant, we first found that although phenazine production increased, pyocyanin produced in the mutant YTΔrpoS was fourfold much higher than that in the wild-type strain YT. To investigate this issue, we constructed phzM-lacZ fusion on a vector and on the chromosome. By quantifying ß-galactosidase activities, we confirmed that expression of the phzM was up-regulated when the rpoS gene was inactivated. However, no changes occurred in the expression of phzS and phzH when the rpoS was knocked out. Taken together, overproduction of the SAM-dependent methyltransferase (PhzM) might contribute to the increased pyocyanin in the absence of RpoS in P. aeruginosa.

phz1 contributes much more to phenazine-1-carboxylic acid biosynthesis than phz2 in Pseudomonas aeruginosa rpoS mutant.

Pseudomonas aeruginosa PAO1, a common opportunistic bacterial pathogen, contains two phenazine-biosynthetic operons, phz1 (phzA1 B1 C1 D1 E1 F1 G1 ) and phz2 (phzA2 B2 C2 D2 E2 F2 G2 ). Each of two operons can independently encode a set of enzymes involving in the biosynthesis of phenazine-1-carboxylic acid. As a global transcriptional regulator, RpoS mediates a lot of genes involving secondary metabolites biosynthesis in many bacteria. In an other previous study, it was reported that RpoS deficiency caused overproduction of pyocyanin, a derivative of phenazine-1-carboxylic acid in P. aeruginosa PAO1. But it is not known how RpoS mediates the expression of each of two phz operons and modulates phenazine-1-carboxylic acid biosynthesis in detail. In this study, by deleting the rpoS gene in the mutant PNΔphz1 and the mutant PNΔphz2, we found that the phz1 operon contributes much more to phenazine-1-carboxylic acid biosynthesis than the phz2 operon in the absence of RpoS. With the construction of the translational and transcriptional fusion vectors with the truncated lacZ reporter gene, we demonstrated that RpoS negatively regulates the expression of phz1 and positively controls the expression of phz2, and the regulation of phenazine-1-carboxylic acid biosynthesis mediated by RopS occurs at the posttranscriptional level, not at the transcriptional level. Obviously, two copies of phz operons and their differential expression mediated by RpoS might help P. aeruginosa adapt to its diverse environments and establish infection in its hosts.

Reciprocal enhancement of gene expression between the phz and prn operon in Pseudomonas chlororaphis G05.

In previous studies with Pseudomonas chlororaphis G05, two operons (phzABCDEFG and prnABCD) were confirmed to respectively encode enzymes for biosynthesis of phenazine-1-carboxylic acid and pyrrolnitrin that mainly contributed to suppression of some fungal phytopathogens. Although some regulators were identified to govern their expression, it is not known how two operons coordinately interact. By constructing the phz- or/and prn- deletion mutants, we found that in comparison with the wild-type strain G05, phenazine-1-carboxylic acid production in the mutant G05Δprn obviously decreased in GA broth in the absence of prn, and pyrrolnitrin production in the mutant G05Δphz remarkably declined in the absence of phz. By generating the phzA and prnA transcriptional and translational fusions with a truncated lacZ on shuttle vector or on the chromosome, we found that expression of the phz or prn operon was correspondingly increased in the presence of the prn or phz operon at the post-transcriptional level, not at the transcriptional level. These results indicated that the presence of one operon would promote the expression of the other one operon between the phz and prn. This reciprocal enhancement would keep the strain G05 producing more different antifungal compounds coordinately and living better with growth suppression of other microorganisms.

[Regulation of pyocyanin biosynthesis by transcriptional factor sigma38 in Pseudomonas aeruginosa PAO1].

Pyocyanin, an important virulence factor, is synthesized and secreted by Pseudomonas aeruginosa PAO1and plays a critical role in pathogen-host interaction during infection. Sigma38 (σ38, σS) is a central regulator for many virulence production in pathogens. Objective: Our aim is to identify expression and regulation of two phenazine-producing operons mediated by the sigma38 factor in Pseudomonas aeruginosa PAO1. Methods: We first cloned the flanking fragments of rpoS from the chromosomal DNA of P. aeruginosa PAO1 and constructed the deletion mutant ΔrpoS with the insertion of gentamycin resistance cassette (aacC1). Complementation of rpoS was then carried out after construction and introduction of pME10S (containing the whole rpoS region). Finally, we created the mutant ΔrpoSphz1 and ΔrpoSphz2, and measured pyocyanin production by these mutants in GA medium, using the parental strain Δphz1 and Δphz2 as controls. Results: In GA medium, pyocyanin production by mutant ΔrpoS increased dramatically in comparison with the wild-type strain PAO1. Production of pyocyanin, however, was decreased to the level of the wild-type strain with complementation of the derivative ΔrpoS harboring pME10S. Mutant ΔrpoSphz2 produced much more pyocyanin than mutant Δphz2. Mutant ΔrpoSphz1, however, produced much less pyocyanin than mutant Δphz1. Conclusion: By positively regulating the expression of phz2 and negatively regulating the phz1, sigma38 factor exerts negative modulation on pyocyanin biosynthesis in P. aeruginosa PAO1.

[Isolation, identification and fermentation optimization of Bacillus tequilensis PanD37 producing L-aspartate α- decarboxylase].

OBJECTIVE: We screened bacteria producing L-aspartate α-decarboxylase from grapery soil and optimized the fermentation conditions. METHODS: L-aspartate α-decarboxylase producing bacteria were screened by color-changing circle and liquid secondary screening culture media. Combination of morphological, physiological and biochemical characteristics and 16S rRNA sequence analysis were used to identify the bacteria. Fermentation conditions were optimized by single factor test and orthogonal experiment. RESULTS: Strain PanD37 showed high L-aspartate α-decarboxylase producing property and was identified as Bacillus tequilensis. The optimum fermentation conditions of PanD37 were liquid volume of 50 mL in 500 mL flask, 220 r/min at 35 °C, inoculation amount of 5% for 28 h with a medium of 22.5 g/L sucrose, 7.5 g/L fumaric acid, 20 g/L peptone, 6 g/L L-aspartic acid, 2 g/L Triton X-100, at initial pH of 7.0. Under the optimal fermentation conditions, the highest L-aspartate α-decarboxylase activity reached 44.57 U/mL, which was 2.57 folds higher than that obtained before optimization. CONCLUSION: Strain PanD37 was identified as Bacillus tequilensiswhich was capable of highly producing L-aspartate α-decarboxylase under the optimal fermentation conditions.

[Positive regulation in expression of the phenazine-producing operon phz2 mediated by pip in Pseudomonas aeruginosa PAO1].

UNLABELLED: Pseudomonas aeruginosa PAO1, an opportunistic pathogenic bacterium, produces phenazine and its derivatives which play a critical role in pathogen-host interaction during its infection. In a biological control strain P. chlororaphis PCL1391, Pip positively regulates PCN production. OBJECTIVE: Our aim is to identify the function and regulation of an ORF of PA0243 (the homolog of Pip) in Pseudomonas aeruginosa PAO1. METHODS: We first cloned the fragment of the pip gene from the chromosomal DNA of P. aeruginosa PAO1 and constructed the pip-defect mutant PA-PG with the insertion of gentamycin resistance cassette (aacC1). With construction and introduction of pME10P (containing the whole pip gene region) , complementation of the pip was then carried out. With creation of the mutants PA-PD-Z1G and PA-PG-Z2K, phenazine-1-carboxylic acid and pyocyanin were measured in GA medium in relative mutants, respectively. RESULTS: In GA medium, production of phenazine-1-carboxylic acid and pyocyanin in the mutant PA-PG decreased dramatically in comparison with that produced in the wild type strain PAO1. The amounts of phenazine-1-carboxylic acid and pyocyanin, however, were recovered with complementation of the derivative PA-PG bearing pME10P. The production of phenazine-1-carboxylic acid and pyocyanin in mutant PA-PG-Z2K were same to those in parental strain PA-Z2K. Phenazine-1-carboxylic acid and pyocyanin produced by the mutant PA-PD-Z1G were lower than those in the original strain PA-Z1G. CONCLUSION: With these results, it is suggested that Pip exerts positively regulation in phenazine biosynthesis by specifically modulating expression of the phz2 operon, not by mediating expression of the phzl operon in P. aeruginosa PAO1.

[Differential expression of two phenazine-producing loci mediated by deficiency of the global regulator rsmA in Psedomonas aeruginosa PAO1].

UNLABELLED: In many Pseudomonas, RsmA mediates the production of a set of secondary metabolites or virulence factors. OBJECTIVE: Our aim is to evaluate the function and regulation of the rsmA gene on two phenazine-producing operons in Pseudomonas aeruginosa PAO1. METHODS: We first cloned the upstream and downstream fragments of the rsmA gene from the chromosomal DNA. With the insertion of gentamycin resistance cassette (aacC1), the deletion mutant PA-RG was created and verified with PCR. To complement and overexpress the rsmA gene, pME10R and pME32R were also constructed. By constructing the translational fusion plasmids phz1'-'lacZ pMEZ1 and phz2'-'lacZ pMEZ2, we introduced them into the wild type strain PAO1 and the mutant PA-RG, respectively. Activities of beta-galactosidase were determined with Miller method. RESULTS: In glycerol-alanine medium, overexpression of the rsmA gene results in dramatical decrease of pyocyanin production in PA-RG and PAO1 strain. In addition, beta-galactosidase activity of phz1'-'lacZ in the mutant PA-RG was much higher than that in the wild type strain. However, beta-galactosidase activity of phz2'-'lacZ in the wild type strain was 2fold more than that in the mutant PA-RG. CONCLUSION: The regulation mediated by RsmA on two phenazine loci is specific and differential.

EppR, a new LysR-family transcription regulator, positively influences phenazine biosynthesis in the plant growth-promoting rhizobacterium Pseudomonas chlororaphis G05.

Pseudomonas chlororaphis G05 has the capability to repress the mycelial growth of many phytopathogenic fungi by producing and secreting certain antifungal compounds, including phenazines and pyrrolnitrin. Although some regulatory genes have been identified to be involved in antifungal metabolite production, the regulatory mechanism and pathway of phenazine-1-carboxylic acid biosynthesis remain poorly defined. To identify more new regulatory genes, we applied transposon mutagenesis with the chromosomal lacZ fusion strain G05Δphz::lacZ as an acceptor. In the white conjugant colony G05W05, a novel transcriptional regulator gene, eppR, was verified to be interrupted by the transposon mini-Tn5Kan. To evaluate the specific function of eppR, we created a set of eppR-deletion mutants, including G05ΔeppR, G05Δphz::lacZΔeppR and G05Δprn::lacZΔeppR. By quantifying the production of antifungal compounds and ß-galactosidase expression, we found that the expression of the phenazine biosynthetic gene cluster (phz) and the production of phenazine-1-carboxylic acid were markedly reduced in the absence of EppR. Moreover, the pathogen suppression test verified that the yield of phenazine-1-carboxylic acid was significantly decreased when eppR was deleted in frame. At the same time, no changes in the expression of the phzI/phzR quorum-sensing (QS) system and the production of N-acyl homoserine lactones (AHLs) and pyrrolnitrin were found in the EppR-deficient mutant. In addition, chromosomal fusion analyses and quantitative real-time polymerase chain reaction (qRT-PCR) results also showed that EppR could positively mediate the expression of the phz cluster at the posttranscriptional level. In summary, EppR is specifically essential for phenazine biosynthesis but not for pyrrolnitrin biosynthesis in P. chlororaphis.

[Effect of rpoS mutation on two gene clusters of phenazine in Psedomonas aeruginosa PAO1].

OBJECTIVE: As an opportunistic pathogen, Pseudomonas aeruginosa PAO1 can produce phenazine and its derivatives, which play a critical role in their pathogenesis. In many bacteria, RpoS, the product of rpoS gene, mediates biosynthesis of a set of secondary metabolites. OBJECTIVE: This study aims to elucidate rpoS gene's function and regulation on two phenazine gene clusters in Pseudomonas aeruginosa PAO1. METHODS: The rpoS gene and its upstream and downstream fragments were cloned from the chromosome of Pseudomonas aeruginosa. With the insertion of gentamycin resistance cassette (aacC1), the mutant PA-SG has been created by homologous recombination. Translational fusion plasmids phz1'-'lacZ (pMEZ1) and phz2'-'lacZ (pMEZ2) were constructed, and then were introduced into the wild type strain PAO1 and the mutant PA-SG, respectively. Activities of beta-galactosidase in them were determined with Miller method. RESULTS: In KMB or PPM medium, beta-galactosidase activity of phzl'-'lacZ in the mutant PA-SG is much more than that in the wild type strain. However, beta-galactosidase activity of phz2'-'lacZ in the wild type strain is 2 -3 folds more than that in the mutant PA-SG. CONCLUSION: With these results, it is suggested that regulation mediated by rpoS gene on two phenazine loci is specific and different.

Extracellular Expression of L-Aspartate-α-Decarboxylase from Bacillus tequilensis and Its Application in the Biosynthesis of ß-Alanine.

L-aspartate-α-decarboxylase was extracellularly expressed to enhance its production for ß-alanine biosynthesis. L-aspartate-α-decarboxylase and cutinase were coexpressed in Escherichia coli; more than 40% of the L-aspartate-α-decarboxylase was secreted into the medium. Selection of best conditions among tested variables enhanced L-aspartate-α-decarboxylase production by the recombinant strain. The total L-aspartate-α-decarboxylase activity reached 20.3 U/mL. Analysis of the enzymatic properties showed that the optimum temperature and pH for L-aspartate-α-decarboxylase were 60 °C and 7.5, respectively. Enzyme activity was stable at pH 4.0-8.5 and displayed sufficient thermal stability at temperatures < 50 °C. In addition, enzymatic synthesis of ß-alanine was performed using extracellularly expressed L-aspartate-α-decarboxylase, and a mole conversion rate of > 99% was reached with a substrate concentration of 1.5 M. Extracellular expression of L-aspartate-α-decarboxylase resulted in increased enzyme production, indicating its possible application in the enzymatic synthesis of ß-alanine.

LasR Might Act as an Intermediate in Overproduction of Phenazines in the Absence of RpoS in Pseudomonas aeruginosa.

As an opportunistic bacterial pathogen, Pseudomonas aeruginosa PAO1 contains two phenazineproducing gene operons, phzA1B1C1D1E1F1G1 (phz1) and phzA2B2C2D2E2F2G2 (phz2), each of which is independently capable of encoding all enzymes for biosynthesizing phenazines, including phenazine-1-carboxylic acid and its derivatives. Other previous study reported that the RpoS-deficient mutant SS24 overproduced pyocyanin, a derivative of phenazine-1- carboxylic acid. However, it is not known how RpoS mediates the expression of two phz operons and regulates pyocyanin biosynthesis in detail. In this study, with deletion of the rpoS gene in the PAΔphz1 mutant and the PAΔphz2 mutant respectively, we demonstrated that RpoS exerted opposite regulatory roles on the expression of the phz1and phz2 operons. We also confirmed that the phz1 operon played a critical role and especially biosynthesized much more phenazines than the phz2 operon when the rpoS gene was knocked out in P. aeruginosa. By constructing the translational reporter fusion vector lasR'-'lacZ and the chromosomal fusion mutant PAΔlasR::lacZ, we verified that RpoS deficiency caused increased expression of lasR, a transcription regulator gene in a first quorum sensing system (las) that activates overexpression of the phz1 operon, suggesting that in the absence of RpoS, LasR might act as an intermediate in overproduction of phenazine biosynthesis mediated by the phz1 operon in P. aeruginosa.

vfr, A Global Regulatory Gene, is Required for Pyrrolnitrin but not for Phenazine-1-carboxylic Acid Biosynthesis in Pseudomonas chlororaphis G05.

In our previous study, pyrrolnitrin produced in Pseudomonas chlororaphis G05 plays more critical role in suppression of mycelial growth of some fungal pathogens that cause plant diseases in agriculture. Although some regulators for pyrrolnitrin biosynthesis were identified, the pyrrolnitrin regulation pathway was not fully constructed. During our screening novel regulator candidates, we obtained a white conjugant G05W02 while transposon mutagenesis was carried out between a fusion mutant G05ΔphzΔprn::lacZ and E. coli S17-1 (pUT/mini-Tn5Kan). By cloning and sequencing of the transposon-flanking DNA fragment, we found that a vfr gene in the conjugant G05W02 was disrupted with mini-Tn5Kan. In one other previous study on P. fluorescens, however, it was reported that the deletion of the vfr caused increased production of pyrrolnitrin and other antifungal metabolites. To confirm its regulatory function, we constructed the vfr-knockout mutant G05Δvfr and G05ΔphzΔprn::lacZΔvfr. By quantifying ß-galactosidase activities, we found that deletion of the vfr decreased the prn operon expression dramatically. Meanwhile, by quantifying pyrrolnitrin production in the mutant G05Δvfr, we found that deficiency of the Vfr caused decreased pyrrolnitrin production. However, production of phenazine-1-carboxylic acid was same to that in the wild-type strain G05. Taken together, Vfr is required for pyrrolnitrin but not for phenazine-1-carboxylic acid biosynthesis in P. chlororaphis G05.

Construction of a ß-galactosidase-gene-based fusion is convenient for screening candidate genes involved in regulation of pyrrolnitrin biosynthesis in Pseudomonas chlororaphis G05.

In our recent work, we found that pyrrolnitrin, and not phenazines, contributed to the suppression of the mycelia growth of Fusarium graminearum that causes heavy Fusarium head blight (FHB) disease in cereal crops. However, pyrrolnitrin production of Pseudomonas chlororaphis G05 in King's B medium was very low. Although a few regulatory genes mediating the prnABCD (the prn operon, pyrrolnitrin biosynthetic locus) expression have been identified, it is not enough for us to enhance pyrrolnitrin production by systematically constructing a genetically-engineered strain. To obtain new candidate genes involved in the regulation of the prn operon expression, we successfully constructed a fusion mutant G05ΔphzΔprn::lacZ, in which most of the coding regions of the prn operon and the phzABCDEFG (the phz operon, phenazine biosynthetic locus) were deleted, and the promoter region plus the first thirty condons of the prnA was in-frame fused with the truncated lacZ gene on its chromosome. The expression of the fused lacZ reporter gene driven by the promoter of the prn operon made it easy for us to detect the level of the prn expression in terms of the color variation of colonies on LB agar plates supplemented with 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal). With this fusion mutant as a recipient strain, mini-Tn5-based random insertional mutagenesis was then conducted. By picking up colonies with color change, it is possible for us to screen and identify new candidate genes involved in the regulation of the prn expression. Identification of additional regulatory genes in further work could reasonably be expected to increase pyrrolnitrin production in G05 and to improve its biological control function.

[Effects of insertional inactivation of gacS gene on two secondary metabolites in Psedomonas chlororaphis G-05].

UNLABELLED: Phenazine-1-carboxylic acid biosynthesized and secreted by Pseudomonas chlororaphis G-05 isolated from the rhizosphere of pepper in greenhouse (Huaian, China), contributes to its biological suppression of R. solani growth. OBJECTIVE: Our aim is to elucidate its biocontrol function and regulation mechanism. METHODS: We first identified the strain with biochemical method and homology comparison of 16S rDNA. A conservative DNA fragment of gacS gene was then obtained from the genomic DNA of the wild type strain G-05 by polymerase chain reaction (PCR). According to homologous recombination technology, a mutant G-05S was then created with insertional inactivation of gentamycin resistance cassette (aacC1). RESULTS: In comparison with the wild type strain G-05, the gacS-deficient mutant G-05S produced trace amount of phenazine-1-carboxylic acid in King's B ( KMB ) or Pigment Production Medium( PPM) medium, respectively. However, it produced indole-3-acetic acid (IAA) in the same way as the wild type strain. When the gacS gene was introduced with the shuttle vector pME6032, the mutant G-05S produced the same phenazine-1-carboxylic acid as the wild type strain. CONCLUSION: The regulation mediated by gacS gene on secondary metabolites is specific and differential in some biocontrol agents.

Pyrrolnitrin is more essential than phenazines for Pseudomonas chlororaphis G05 in its suppression of Fusarium graminearum.

Fusarium graminearum is the major causal agent of Fusarium head blight (FHB) disease in cereal crops worldwide. Infection with this fungal phytopathogen can regularly cause severe yield and quality losses and mycotoxin contamination in grains. In previous other studies, one research group reported that pyrrolnitrin had an ability to suppress of mycelial growth of F. graminearum. Other groups revealed that phenazine-1-carboxamide, a derivative of phenazine-1-carboxylic acid, could also inhibit the growth of F. graminearum and showed great potentials in the bioprotection of crops from FHB disease. In our recent work with Pseudomonas chlororaphis strain G05, however, we found that although the phz operon (phenazine biosynthetic gene cluster) was knocked out, the phenazine-deficient mutant G05Δphz still exhibited effective inhibition of the mycelial growth of some fungal phytopathogens in pathogen inhibition assay, especially including F. graminearum, Colletotrichum gloeosporioides, Botrytis cinerea. With our further investigations, including deletion and complementation of the prn operon (pyrrolnitrin biosynthetic gene cluster), purification and identification of fungal compounds, we first verified that not phenazines but pyrrolnitrin biosynthesized in P. chlororaphis G05 plays an essential role in growth suppression of F. graminearum and the bioprotection of cereal crops against FHB disease.

RpoS as an intermediate in RsmA-dependent regulation of secondary antifungal metabolites biosynthesis in Pseudomonas sp. M18.

To investigate the regulatory mechanism governing antifungal metabolite biosynthesis, two kinds of global regulator genes in Pseudomonas sp. M18, an rpoS and an rsmA gene, were cloned and mutated by inserting with an aacC1 cassette, respectively. Two mutants showed the same regulatory mode with repression of phenazine-1-carboxylic acid and remarkable enhancement of pyoluteorin. In the rpoS-mutant, a translational rsmA'-'lacZ fusion was expressed at the same level corresponding to that in the wild-type strain. In the rsmA-mutant, however, expression of the translational rpoS'-'lacZ fusion was only about 30% of that in the wild-type strain. The results indicated that the absence of RsmA leads to repression of the rpoS gene expression, which has further been confirmed with construction of chromosomal rpoS-lacZ fusion in the rsmA-mutant and the wild-type strain, respectively. The findings showed a new regulatory cascade controlling antifungal metabolite production in Pseudomonas sp. M18, suggesting that RpoS may serve as a mediator in RsmA-dependent regulation of secondary metabolite biosynthesis.

[Autoinduction of pyoluteorin and correlation between phenazine-1-carboxylic acid and pyoluteorin in Pseudomonas sp. M18].

A new bacterium with potential biocontrol ability, Pseudomonas sp. M18, was isolated from the soil of agricultural field in suburb of Shanghai (China). It had been demonstrated that biosynthesis and secretion of phenazine-1-carboxylic acid and pyoluteorin in Pseudomonas sp. M18 contributes to its suppression of soilborne pathogens. In order to study the correlation and regulatory mechanism of two antifungal compounds biosynthesis, the mutant M18T and M18Z1 were constructed with insertion of the gentamycin resistance gene cassette (aacC1), respectively. With introduction of the translational fusion pMEAZ (pltA'-' lacZ) into the wild type strain MI8 or the pit-mutant M18T, respectively, it was found that beta-galactosidase activities of the mutant M18T (pMEAZ) are remarkably enhanced by adding a certain amount of pyoluteorin in KMB medium. The results indicated that pyoluteorin might positively autoinduce expression of the pit gene loci. In investigating the correlation of two antifungal agents, it was showed that the pyoluteorin-negative mutant MI8T produces the same level of phenazine-1-carboxylic acid in comparison with the wild type strain M18. Overexpression of the plt gene loci does not result in decrease of phenazine-1-carboxylic acid in a pltZ-mutant of Pseudomonas sp. M18. However, the distinct decrease of phenazine-1-carboxylic acid biosynthesis does lead to enhanced biosynthesis of pyoluteorin in the mutant M18Z1. Addition of phenazine-1-carboxylic acid in KMB medium makes the mutant M18S produce less pyoluteorin. These results indicated that a special correlation of secondary antifungal agents biosynthesis seems to be existed in Pseudomonas sp. M18, i.e., production of pyoluteorin does not exert any influence on expression of the phz gene cluster, while phenazine-1-carboxylic acid makes negative impact on the biosynthesis of pyoluteorin.

[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.