[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.

[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.

[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.

[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.

[Differential regulation of phenazine-1-carboxylic acid and pyoluteorin production mediated by inactivated gacA in Pseudomonas sp. M18].

Pseudomonas sp. M18 is one of the plant-growth-promoting rhizobacteria, which can produce fungicides: phenazine-1-carboxylic acid (PCA) and pyoluteorin (Plt). The chromosomally gacA inactivated mutant named M18G was constructed and its PCA production was enhanced 31-fold and Plt production was almost blocked completely in KMB medium. To assess the mutual influence of two antibiotics, the plt gene cluster mutant M18T and the phz gene cluster mutant M18GA were then constructed. Non-Plt-producing M18T could synthesize the same amount of PCA as wild type strain. Plt could not be detected in M18GA while PCA production was inhibited dramatically in it. Results indicate that promotion of PCA and inhibition of Plt production in M18G do result from the inactivation of gacA gene. It suggests that the production of antibiotics in strain M18 is differentially mediated by the gacA.

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.

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.