[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.

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.