假单胞菌M18株las系统负调控藤黄绿脓菌素和吩嗪-1-羧酸的合成

从假单胞菌M18株（Pseudomonassp．M18）中,克隆了las系统的双元组分lasI和lasR基因,它们的编码产物属于LuxI—LuxR调控因子．运用同源重组技术,分别构建lasI和lasR基因的染色体失活突变株,与野生型菌株相比,抗生素藤黄绿脓菌素（Pit）和吩嗪-1-羧酸（PcA）的产量均分别提高了4～5倍和2～3倍．在lasI和lasR突变株的反式互补实验中,两种抗生素的产量回复到野生型水平．pltA’-lacZ和phzA-＇lacZ翻译融合的测定结果进-步证明lasI和lasR的编码产物对Plt和PCA生物合成基因簇具有负调控作用．las系统正调控细菌的群集运动,在lasI和lasR的失活突变株中,细菌的群集运动能力消失．对lasI和lasR突变株和野生型的生长曲线的研究发现,lasI和lasR基因的编码产物对细菌的生长具有抑制作用．结果表明,las系统作为整体调控因子的编码基因,参了与细胞内多种生物活动的调控作用．     

假单胞菌gacA插入突变对藤黄绿脓菌素和吩嗪-1-羧酸合成代谢的差异性调控

假单胞菌株M18分泌藤黄绿脓菌素 (Pyoluteorin ,Plt )和吩嗪 1 羧酸 (Phenazine 1 carboxylicacid ,PCA)并抑制多种植物病菌的生长。从M18中克隆双基因调控系统gacS gacA的组成基因gacA ,并构建了该基因抗性插入突变株M18G。在KMB培养基中 ,M18G合成Plt的能力受到完全抑制 ,而PCA的积累约比野生型提高 31倍左右。Plt合成基因簇突变株M18T和在M18G基础上构建的PCA合成基因簇突变株M18GA的Plt和PCA合成的动力学变化表明 ,在M18G菌株中 ,Plt合成的抑制并不引起PCA的过量积累 ,PCA的过量积累也不引起Plt合成的抑制。由此推测 ,gacA在基因表达的水平上全局性地执行着调控功能     

RNA聚合酶σ38亚基缺失对假单胞菌抗生物质合成代谢的影响

设计引物从假单胞菌M18基因组DNA中扩增并获得rpoS基因的378bp保守区段。以此为探针,从假单胞菌基因组文库中克隆了包括rpoS基因全序列及其相邻序列的3.1kb EcoRⅠ_XhoⅠ片段。通过抗性基因（抗庆大霉素基因）的定点插入构建了σ38亚基缺失突变株M18S。HPLC检测结果显示,σ38亚基缺失引起该菌株的抗生物质合成代谢的显著变化。与野生株相比,缺失突变株的吩嗪_1_羧酸在PPM和KMB中2种培养基中合成量由58μg/mL和10.2μg/mL分别减少到20.4μg/mL和0μg/mL;而缺失突变株的藤黄绿脓菌素则相反,在PPM和KMB两种培养基中合成量由0.5μg/mL和20.5μg/mL分别提高到75.4μg/mL和185.6μg/mL。表明σ38亚基可区别性调控假单胞菌M18的抗生物质合成代谢。rpoS基因的互补实验和两种抗生素基因与β_半乳糖苷酶基因的翻译融合表达实验进一步验证了上述的结果: σ38亚基正调控吩嗪_1_羧酸的表达,而负调控藤黄绿脓菌素的表达。     

RNA聚合酶σ^38亚基缺失对假单胞菌抗生物质合成代谢的影响

设计引物从假单胞菌M18基因组DNA中扩增并获得rpoS基因的378bp保守区段。以此为探针,从假单胞菌基因组文库中克隆了包括rpoS基因全序列及其相邻序列的3·1kbEcoRⅠ-XhoⅠ片段。通过抗性基因(抗庆大霉素基因)的定点插入构建了σ38亚基缺失突变株M18S。HPLC检测结果显示,σ38亚基缺失引起该菌株的抗生物质合成代谢的显著变化。与野生株相比,缺失突变株的吩嗪-1-羧酸在PPM和KMB中2种培养基中合成量由58μg/mL和10·2μg/mL分别减少到20·4μg/mL和0μg/mL;而缺失突变株的藤黄绿脓菌素则相反,在PPM和KMB两种培养基中合成量由0·5μg/mL和20·5μg/mL分别提高到75·4μg/mL和185·6μg/mL。表明σ38亚基可区别性调控假单胞菌M18的抗生物质合成代谢。rpoS基因的互补实验和两种抗生素基因与β-半乳糖苷酶基因的翻译融合表达实验进一步验证了上述的结果:σ38亚基正调控吩嗪-1-羧酸的表达,而负调控藤黄绿脓菌素的表达。     

假单胞菌gacA插入突变对藤黄绿脓菌素和吩嗪-1-羧酸合成代谢的差异性调控

假单胞菌株M18分泌藤黄绿脓菌素(Pyoluteorin,Plt )和吩嗪1羧酸（Phenazine1carboxylic acid, PCA）并抑制多种植物病菌的生长。 从M18中克隆双基因调控系统gacS/gacA的组成基因gacA,并构建了该基因抗性插入突变株M18G。在KMB培养基中,M18G合成Plt的能力受到完全抑制,而PCA的积累约比野生型提高31倍左右。Plt合成基因簇突变株M18T和在M18G基础上构建的PCA合成基因簇突变株M18GA的Plt和PCA合成的动力学变化表明,在M18G菌株中,Plt合成的抑制并不引起PCA的过量积累,PCA的过量积累也不引起Plt合成的抑制。由此推测,gacA在基因表达的水平上全局性地执行着调控功能。     

荧光假单胞菌M18的rpoS基因克隆及其功能分析

从荧光假单胞菌 (Pseudomonasfluorescentsp .)M1 8基因组中克隆了RNA聚合酶的稳定期σs 因子编码基因rpoS ,推测其氨基酸序列与铜绿假单胞菌、荧光假单胞菌和恶臭假单胞菌的同源性分别为 99 1 %、87 35 %和87 8%。利用体外定点插入突变和同源重组技术 ,构建了M1 8的rpoS突变株M1 8R- 。对突变株M1 8R- 合成抗生素吩嗪 1 羧酸 (PCA)和藤黄绿菌素 (Plt)的动力学分析结果表明 ,在KB或PPM培养基中 ,突变株合成PCA的能力比野生型分别提高了 2 5或 5 78倍 ,但Plt的积累量不受影响。与野生型相比 ,突变株对碳源饥饿的耐性下降。同时 ,在碳源饥饿条件下对过氧化氢、乙醇和和氯化钠等环境胁迫的交叉保护性减小 ,存活率显著降低     

假单胞菌株M18 psrA突变株的构建及其对吩嗪-1-羧酸和藤黄绿菌素合成的调控

【目的】假单胞菌M18是一株能同时合成吩嗪-1-羧酸(PCA)和藤黄绿菌素(Plt)两种抗生素的植物根际促生细菌。PsrA为细菌TetR家族转录调控因子。为了研究PsrA对PCA与Plt生物合成的影响,从M18菌株基因组中扩增psrA基因。【方法】通过同源重组技术,构建庆大霉素抗性片段置换psrA的突变菌株M18psrA。利用基因互补、lacZ报告基因融合分析实验,验证PsrA对抗生素合成基因的调控作用。【结果】在PPM和KMB培养基中,分别比较野生型菌株M18和突变菌株M18psrA的PCA与Plt产量,突变菌株M18psrA的PCA产量显著下降;Plt产量显著升高,为野生型菌株的10-15倍。基因互补、lacZ报告基因融合分析,进一步证明了psrA正调控PCA的phz2合成基因簇,负调控Plt的合成基因簇。【结论】PsrA区别性调控抗生素PCA与Plt的生物合成。     

铜绿假单胞菌M18 rsmY突变株的构建及其对PCA和Plt的区别性调控

【目的】在假单胞菌中,小RNA(sRNA)参与初级和次级代谢产物、多种毒素因子以及菌群传感系统的调控,通过在植物根际促生铜绿假单胞菌M18中研究RsmY对吩嗪-1-羧酸(PCA)和藤黄绿菌素(Plt)两种抗生素的调控作用,深入了解假单胞菌中次级代谢的途径并为构建高产抗生素工程菌株提供了一定的理论基础。【方法】运用同源重组技术,构建了铜绿假单胞菌M18株的rsmY突变菌株M18RY,通过基因过表达、lacZ报告基因融合分析实验,进一步验证了RsmY对抗生素合成基因的调控作用。【结果】比较野生型M18和突变株M18RY中PCA和Plt在同一培养条件下的生物合成量,突变菌株M18RY中PCA的产量显著增加,为野生型菌株的5倍左右,而Plt的产量降为野生型的1/8。LacZ报告基因融合分析进一步证明了RsmY对PCA的负调控作用主要是通过phz2基因簇来实现的。【结论】结果表明,rsmY基因区别性调控PCA和Plt的生物合成。     

荧光假单胞菌M18rpoD克隆及其对抗生素合成的影响

荧光假单胞菌M18对多种植物病原真菌具有显著的抑制作用。荧光假单胞菌(Pseuclomones fluorescens)M18能同时合成吩嗪1羧酸（PCA）和藤黄绿菌素(Plt) 两种抗生素。从M18的基因组中克隆了rpoD基因,其相应的氨基酸序列与荧光假单胞菌CHAO中RpoD蛋白的氨基酸序列完全相同。利用基因重组技术和大肠杆菌荧光假单胞菌穿梭质粒pME6032,将rpoD置于强启动子Ptac的控制下,导入M18菌株。发现经重组质粒转化的M18,与对照相比,培养基中PCA和Plt开始累积的时间分别提前4h和8h,积累量提高1倍和6倍。     

温度对假单胞rsmA突变株M-18R合成Plt和PCA的区别性影响

次生代谢物阻遏蛋白(Repressor of secondary metabolite,Rsm)A是一种全局性调控因子,与mRNA的RBS结合,转录后水平上抑制基因翻译。运用同源重组技术,构建了假单胞茵(Pseudomonas sp．)M-18的rsmA突变菌株M-18R。在37℃、28℃恒温和短期升温(37℃、4h培养,转28℃继续培养)条件下,比较野生株M-18和突变株M-18R生物合成藤黄绿菌素(Plt)和吩嗪-1-羧酸(PCA)的量。在37℃条件下,M-18和M-18R合成这两种抗生物质的能力几乎受到完全抑制。在28℃条件下,M-18R合成P11的量约为野生型M-18的10倍,达到270μg／mL,但是合成PCA的量仅为野生型的50％。经短期升温培养,M-18的Plt合成量明显下降,PCA产量降低不显;相反,M-18R合成Plt的量达到400μg／mL,但PCA产量的变化仍不明显。推测,M-18菌株细胞内存在着某种与RsmA相关联的温度敏感因子,在RsmA缺失条件下,作为专一性激活剂促进Plt的生物合成,但是,并不参与对PCA合成的调控。     

Las-like quorum-sensing system negatively regulates both pyoluteorin and phenazine-1-carboxylic acid production in Pseudomonas sp. M18

A las-like quorum-sensing system in Pseudomonas sp. M18 was identified, which consisted of lasI and lasR genes encoding LuxI-LuxR type regulator. Several functions of the las system from strain M18 were investigated in this study. The chromosomal inactivation of either lasI or lasR by recombination increased the production of both pyoluteorin (Plt) and phenazine-1-carboxylic acid (PCA) by 4-5 fold and 2-3 fold over that of the wild type strain of M18, respectively. Production of both antibiotics was restored to wild-type levels after in trans complementation with the wild-type lasI or lasR gene. Expression of the translational fusions pltA'-'lacZ and phzA'-'lacZ further confirmed the negative effect of lasI or lasR on both biosynthetic operons, and it was also demonstrated that the las system was related to the ability of swarming motility and the inhibition of cell growth.     

Las-like quorum-sensing system negatively regulates both pyoluteorin and phenazine-1-carboxylic acid production in Pseudomonas sp. M18

A las-like quorum-sensing system in Pseudomonas sp. M18 was identified, which consisted of lasI and lasR genes encoding LuxI-LuxR type regulator. Several functions of the las system from strain M18 were investigated in this study. The chromosomal inactivation of either lasI or lasR by recombination increased the production of both pyoluteorin (Plt) and phenazine-1-carboxylic acid (PCA) by 4－5 fold and 2－3 fold over that of the wild type strain of M18, respectively. Production of both antibiotics was restored to wild-type levels after in trans complementation with the wild-type lasI or lasR gene. Ex-pression of the translational fusions pltA׳-׳lacZ and phzA׳-׳lacZ further confirmed the negative effect of lasI or lasR on both biosynthetic operons, and it was also demonstrated that the las system was related to the ability of swarming motility and the inhibition of cell growth.     

RpoS as an intermediate in RsmA-dependent regulation of secondary antifungal metabolites biosynthesis in Pseudomonas sp. M18

To investigate the regulatory mechanism governing antifungal metabolite biosynthesis, two kinds of global regulator genes in Pseudomonas sp. M18, an rpoS and an rsmA gene, were cloned and mutated by inserting with an aacC1 cassette, respectively. Two mutants showed the same regulatory mode with repression of phenazine-1-carboxylic acid and remarkable enhancement of pyoluteorin. In the rpoS-mutant, a translational rsmA'-'lacZ fusion was expressed at the same level corresponding to that in the wild-type strain. In the rsmA-mutant, however, expression of the translational rpoS'-'lacZ fusion was only about 30% of that in the wild-type strain. The results indicated that the absence of RsmA leads to repression of the rpoS gene expression, which has further been confirmed with construction of chromosomal rpoS-lacZ fusion in the rsmA-mutant and the wild-type strain, respectively. The findings showed a new regulatory cascade controlling antifungal metabolite production in Pseudomonas sp. M18, suggesting that RpoS may serve as a mediator in RsmA-dependent regulation of secondary metabolite biosynthesis.     

Phenazine-1-carboxylic acid is negatively regulated and pyoluteorin positively regulated by gacA in Pseudomonas sp. M18

The biosynthesis of antimicrobial metabolites is controlled by the GacS/GacA two-component regulatory system in Pseudomonas species. The production of phenazine-1-carboxylic acid and pyoluteorin is differentially regulated by GacA in Pseudomonas sp. M18. Pyoluteorin was reduced to nondetectable level in culture of the gacA insertional mutant strain M18G grown in King's medium B broth, whereas phenazine-1-carboxylic acid production was increased 30-fold over that of the wild-type strain. Production of both antibiotics was restored to wild-type levels after complementation in trans with the wild-type gacA gene. Expression of the translational fusions phzA'-'lacZ and pltA'-'lacZ confirmed the effect of GacA on both biosynthetic operons.     

假单胞菌M18 relA 突变株的构建及其对吩嗪-1-羧酸合成的调控

假单胞菌M18是一株能同时合成吩嗪-1-羧酸(PCA)和藤黄绿菌素两种抗生素的植物根际分离细菌。RelA催化合成的效应分子ppGpp能介导细菌因营养饥饿引起的应激反应。以M18菌株染色体DNA为模板,PCR扩增获得relA基因,通过庆大霉素抗性片段插入失活与同源重组技术,构建假单胞菌M18的relA突变菌株M18RAG。在PPM培养基中进行PCA发酵分析,发现突变菌株M18RAG的PCA产量显著升高,约为野生型菌株的1.5-2倍。relA基因反式互补实验以及phzA′-′lacZ翻译融合测定结果,均进一步证明了RelA对PCA生物合成及其基因表达具有抑制作用。     

Differential Regulation of rsmA Gene on Biosynthesis of Pyoluteorin and Phenazine-1-carboxylic Acid in Pseudomonas sp. M18

Summary Plant growth promoting rhizobacteria (PGPR) strain Pseudomonas sp. M18 can produce two different types of antibiotics, pyoluteorin (Plt) and phenazine-1-carboxylic acid (PCA). The global regulator RsmA is a translational repressor of secondary metabolism in many prokaryotes. A chromosomally rsmA inactivated mutant strain M18R was constructed to study the regulatory mechanism of Plt and PCA biosynthesis and enhancement of Plt or PCA production in Pseudomonas sp. M18. The accumulation of Plt increased six-fold over that of the wild-type strain whereas PCA production was not significantly affected in cultures of M18R. Plt production was inhibited completely but PCA biosynthesis was not altered after complementation with rsmA gene in trans in the strain of M18R. The differential activity of rsmA gene on these two operons was further confirmed by the analysis of β-galactosidase activities from translational phzA′-′lacZ and pltA′-′lacZ fusion, in which phzA is the first enzyme gene of the phenazine biosynthesis pathway and pltA is the first gene of the pyoluteorin biosynthesis pathway. The results indicate that RsmA can control Plt production negatively but not PCA production in M18, and show that the global regulator RsmA does not repress the biosynthesis of all secondary metabolites.     

假单胞菌M18rsmA-突变株的构建及其对Plt和PCA合成的区别性调控作用

假单胞菌(Pseudomonas sp．)M18是促进植物生长的根际细菌,能产生吩嗪-1-羧酸(PCA)和藤黄绿菌素(Plt)两种不同的抗生素抑制植物病原菌,保护植物免受病害。运用PCR方法,从M18基因组中,扩增出rsmA基因部分片段,并以该片段为探针,从M18的基因组柯斯文库中筛出阳性克隆,切取带有rsmA基因及两侧序列的1．5kb片段,中间插入编码Km‘的DNA片段,获得rsmA^-体外突变体。运用同源重组剔除技术,构建了M18菌株的rsmA突变株M18R^-。突变株M18R^-生物合成Plt的能力比野生型M18提高4倍,但是,PCA产量仅为野生型的20％。研究结果表明,全局性调控基因rsmA可能通过不同的机制区别性地影响Plt和PCA的生物合成。