Construction of Pseudomonas sp. M18 pqsR Mutant and Its Regulation on Plt Biosynthesis / 微生物学通报

Pseudomonas sp. M18 is one of plant growth-promoting rhizobacteria capable of producing two kinds of anti-fungal agents: phenazine-1-carboxilic acid (PCA) and pyoluteorin (Plt). The pqsR gene, which encodes a LysR family member PqsR, was amplified from chromosomal genome of strain M18. Using the homologous recombination technique, a chromosomal pqsR inactivated mutant strain M18PRG was constructed in Pseudomonas sp. M18. To study the effect of pqsR gene on Plt biosynthesis, the dynamic curves of Plt production by strains M18 and M18PRG was measured in KMB media. As a result, Plt production of the pqsR mutant was three to four folds higher than that of its parent strain M18. The Plt production was restored to the wild-type level when strain M18PRG was complemented with pqsR gene in trans. The regulation of pqsR gene on Plt production was further confirmed by the pltA′-′lacZ translational fusion analysis. These results indicate that pqsR gene negatively controls the Plt biosynthesis. Additionally, by analyzing the growth curves of wild type strain M18 and pqsR mutant, wecan readily find that PqsR has a negative influence on cell growth. It was also shown that the production of red pigments in strain M18 required the expression of pqsR gene. In conclusion, the data presented in this study clearly demonstrate that PqsR acts as a global regulator involved in many physiological activities in Pseudomonas sp. M18.

Optimal Model Design for High Production of Phenazine-1-Caboxylic Acid (PCA) by Pseudomas. spp M18G / 中国生物工程杂志

Phenazine-1-carboxylic acid(PCA)is one of the major antifungal compounds produced by plant growth-promoting rhizosphere(PGPR)pseudomonads and has been shown to contribute to the biological control of soil-borne plant pathogens.Model on secondary metabolite phenazine-1-carboxylic acid(PCA)fermentation nutrition conditions of gacA inactivate mutant Pseudomonas sp.M18G was constructed by Plackett-Burman design(PB)and Response Surface Method(RSM).In PB design four key components selected from 12 different factors have shown to play an important role for promoting PCA production.Center Composite Design(CCD)was adopted to establish a fermentation model for the four nutrition components using RSM.The optimal concentration of the four components based on analysis of regression equation were determined that soybean meal 33.4 g/L,glucose 12.7 g/L,soy peptone10.9 g/L,ethanol13.8 g/L,and the highest PCA production could reached 1.89 g/L after 60h fermentation and the yield increased to 6 fold over that before optimization.The contour graphs depicted interactions of the two nutrition components showed that soybean meal and ethanol played an even more crucial role for the highest production of PCA in fermentation.It establishes a method for high PCA production and lays a foundation for the further PCA commercial development.

Differential effect of temperature on Plt and PCA synthesis in a rsmA inactivated mutant strain of Pseudomonas sp. M-18 / 生物工程学报

Rsm (repressor of secondary metabolite) A is an mRNA binding protein which functions as a global repressor to control multiple genes at the posttranscriptional level. Using homologous recombination technique a chromosomal rsmA inactivated mutant strain M-18R 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 RsmA 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-18R were measured in KMB medium under different temperature conditions such as 37 degrees C constant, 28 degrees C constant and nonconstant (37 degrees C 4 hours at first and then 28 degrees C constant) cultivation. The synthesis of both Plt and PCA were almost inhibited in the cultures under the condition of 37 degrees C. At 28 degrees C, however, compared with the wild type strain M-18, the mutant strain produced tenfold amount of Plt, while the production of PCA decreased only about 50%. When cultivated under the nonconstant condition, the amount of Plt produced by M-18R could reach 400 microg/mL while the PCA production was not significantly affected, but in the wild type strain M-18, the amount of Plt production decreased obviously while the PCA production was not affected in comparison with the results at 28 degrees C constant. These results suggest that a temperature sensitive factor exists to function as an activator independent of RsmA to promote the synthesis of Plt in the rsmA mutant strain M-18R while it may bind with RsmA to repress the synthesis of Plt in the wild type strain M-18. But this factor did not exert any affect on the synthesis of PCA.

Construction and Characterization of a ppbR gene Mutant of Pseudomonas sp. M18 / 微生物学通报

Pseudomonas sp. M18, one of plant-growth-promoting rhizobacteria, can produce secondary metabolites including phenazine-1-carboxylic acid (PCA) and pyoluteorin (Plt). PA2572 gene coding protein is a probable two-component response regulator in Pseudomonas according to homologous speculations. In order to investigate its genetic function, PA2572 homologous gene, ppbR, was amplified from M18 genome, inactivated by inserting a Gm cassette. The resulting reconstruct was introduced into the M18 genome using homologous recombination technique, so as to obtain the null mutant M18P. The results showed that the M18P has less flagellar swimming and swarming motility, and yielded fewer PCA. The production of PCA was only 50% of the wild type. However, there was no remarkable difference between mutant and wild type in producing pyoluteorin in KMB medium.