假单胞菌M18pqsR突变株的构建及其对藤黄绿菌素合成的调控

Construction of Pseudomonas sp. M18 pqsR Mutant and Its Regulation on Plt Biosynthesis

假单胞菌M18是一株能同时合成藤黄绿脓菌素（Plt）和吩嗪-1-羧酸（PCA）两种抗生物质的植物根际促生细菌。运用PCR方法, 从M18基因组中扩增得到pqsR基因, 该基因编码LysR家族调控蛋白PqsR。通过同源重组技术, 构建假单胞菌M18的pqsR突变菌株M18PRG。比较野生型菌株M18和突变菌株M18PRG在KMB培养基的Plt产量, 发现M18PRG 菌株合成Plt的量约为野生型M18菌株的3-4倍。在pqsR突变株的反式互补实验中, Plt的产量回复到野生型水平。pltA′-′lacZ翻译融合的测定结果进一步证明PqsR对Plt生物合成基因簇具有负调控作用。分析M18野生型及其pqsR突变株的生长曲线, 发现PqsR对细菌的生长具有抑制作用。另外, 我们还发现pqsR基因调控红色色素的产生。上述结果表明, 在假单胞菌M18中, PqsR作为全局性调控因子参与了细胞内多种生理活动的调控。

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, we can 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.