藤黄绿脓菌素的自诱导及假单胞菌M18抗生物质代谢相关性初步分析

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

假单胞菌M18的生防功能归功于其分泌吩嗪-1-羧酸和藤黄绿脓菌素。为了研究抗生物质合成代谢相关性及调控机制,分别构建了两种抗生物质合成基因簇插入突变株M18T和M18Z1。用翻译融合表达载体pMEAZ(pltA′-′lacZ)分别转化野生株和突变株M18T、发酵培养并测定β-半乳糖苷酶活性,结果显示,添加藤黄绿脓菌素使突变株M18T(pMEAZ)的β-半乳糖苷酶活性比野生株M18(pMEAZ)增加约6倍,表明藤黄绿脓菌素对自身基因簇具正向自诱导作用。抗生物质的测定结果显示,突变株M18T无藤黄绿脓菌素合成,而吩嗪-1-羧酸的合成量与野生株相同;突变株M18Z1与野生株相比,吩嗪-1-羧酸明显减少,藤黄绿脓菌素却显著提高。过量的吩嗪-1-羧酸又抑制藤黄绿脓菌素的合成。表明,假单胞菌M18中独有的代谢相关方式为:藤黄绿脓菌素不影响吩嗪-1-羧酸,但吩嗪-1-羧酸负调控藤黄绿脓菌素。

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 M18 or the pit-mutant M18T, respectively, it was found that β-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 M18T produces the same level of phenazine-1-carboxylic acid in comparison with the wild type strain M18. Overexpression of the pit 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-l-carboxylic acid makes negative impact on the biosynthesis of pyoluteorin.