假单胞菌M18中藤黄绿菌素合成限速酶基因的鉴定及表达优化

【目的】假单胞菌株M18中负责抗真菌剂藤黄绿菌素(Plt)合成的结构基因包括pltLABCDEFG、pltM。为了鉴定Plt合成限速酶的基因,分别将9个结构基因过表达。【方法】以M18菌株染色体DNA为模板,PCR扩增这9个Plt合成基因的编码区,分别克隆到穿梭载体pME6032的tac启动子下游,构建9个结构基因的过表达载体,并转入假单胞菌株M18中。在KMB培养基中进行Plt发酵分析。【结果】分别携带pltC、pltD、pltF过表达载体的菌株与携带空质粒的菌株相比,Plt产量分别提高了96%、78%、75%。对重组菌株进行IPTG诱导浓度和诱导时间的优化,确定IPTG最佳诱导浓度为1.0 mmol/L,最佳诱导时间为培养6 h。【结论】pltC、pltD、pltF分别编码的Ⅰ型聚酮合成酶、卤化酶、乙酰CoA合成酶可能为Plt生物合成的限速酶。按照优化条件发酵,携带pltD、pltF过表达质粒的菌株产量分别上升77.5%、159.1%。     

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.     

Genetic diversity of phenazine- and pyoluteorin-producing pseudomonads isolated from green pepper rhizosphere

The genetic diversity among indigenous phenazine-1-carboxylic acid (PCA)-producing and pyoluteorin (Plt)-producing isolates of pseudomonads screened from green pepper rhizosphere was exploited in this study. A total of 48 bacterium isolates producing one or both of these antibiotics were screened from green pepper rhizosphere in diverse regions in China. Among these isolates, 45 could produce PCA, 3 could produce both PCA and Plt, and none could produce Plt only. Based on the restriction patterns of partial 16S and 16S-23S internal transcribed spacer (ITS) PCR fragments generated by enzyme HaeIII or HinfI, these isolates fell into 19 or 17 distinct groups respectively, indicating that there was a significant diversity among them. Polygenetic analysis of the partial 16S rDNA and 16S-23S ITS sequence from the representative in each group in the context of similar sequence from previously described bacterial species indicated that most isolates were closely related to the species of Pseudomonas fluorescens, P. putida, and Stenotrophomonas maltophilia. Some of these representatives of these isolates, then, are likely to be novel strains or species in these two genera. The GenBank accession numbers for DNA sequences of the partial 16S rDNA with ITS region in each isolate determined in this study were: GP30 DQ003219; GP127 DQ003220; GP83 DQ003221; GP42, DQ003222; GP59 DQ003223; GP50 DQ003224; GP36 DQ003225; GP110 DQ003226; GP26 DQ003227; GP37 DQ003228; GP60 DQ003229; GP31 DQ003230; GP57 DQ003231; GP75 DQ003232; GP115 DQ003233; GP65 DQ003234; GP32 DQ003235; GP76 DQ003236; GP78 DQ003237.     

假单胞菌H78中PhoR/B系统对磷元素吸收及其藤黄绿菌素合成的调控

【目的】研究根际荧光假单胞菌(Pseudomonas protegens)H78中双组分系统PhoR/B对Pst磷转运系统以及Plt生物合成的调控作用。【方法】通过同源重组的方法敲除pho R和pho B基因;使用lac Z报告基因融合质粒研究PhoR/B系统对Pst磷转运系统表达的调控;在不同磷浓度下测定H78野生型及H78pho BR突变株的生长,并在KMB培养基中测定其Plt产量。【结果】H78野生型中pst S′-′lac Z融合质粒表达的Lac Z酶活是H78pho BR突变株的15倍;在磷饥饿条件下H78的生长是H78pho BR菌株的3倍;在KMB培养基中,H78的Plt产量为H78phoBR菌株的2倍。【结论】PhoR/B系统正调控Pst转运系统的表达;在磷饥饿条件下,PhoR/B促进磷元素的吸收和利用;PhoR/B同时对Plt生物合成存在一定程度的正调控作用。     

假单胞菌M18株pltZ基因转录阻抑藤黄绿菌素ABC转运系统

假单胞菌(Pseudomonassp .)M18株的藤黄绿菌素(Pyoluteorin ,Plt )生物合成基因簇下游存在一个Plt生物合成负调控基因pltZ和一个负责Plt分泌及自身抗性的ABC(ATP_bindingcassette)转运系统基因簇。利用启动子探针载体pME6 0 15和pME6 5 2 2分别构建ABC转运基因pltH与lacZ的翻译和转录融合表达质粒pHZLF和pHZCF ,分别引入野生型假单胞菌M18株和pltZ突变菌株M18Z。半乳糖苷酶活性的测定结果表明:在pltZ突变株M18Z中,pltH’-‘lacZ翻译融合表达水平约比野生型提高3 7～8 4倍,pltH’‘lacZ转录融合表达水平显著提高了2 8～7 4倍,表明pltZ能在转录水平上阻抑PltABC转运系统的表达,pltZ很可能通过阻抑PltABC转运系统的表达,间接地负调控Plt的生物合成     

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     

荧光假单胞菌M18 rpoD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(P1t)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.