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（Pseudomonas sp.）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_binding cassette）转运系统基因簇。利用启动子探针载体pME6015和pME6522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’-‘lacZ翻译融合表达水平约比野生型提高3.7～8.4倍,pltH’‘lacZ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Plt ABC转运系统的表达,pltZ很可能通过阻抑Plt ABC转运系统的表达,间接地负调控Plt的生物合成。     

假单胞菌M18 pqsR突变株的构建及其对藤黄绿菌素合成的调控

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。     

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。     

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。     

假单胞菌株M18中pqsA突变株的构建及其对plt的调控

【目的】根际铜绿假单胞菌M18能产生藤黄绿菌素(Plt)和吩嗪-1-羧酸(PCA)两种主要的抗生素。其PqsR/PQS群体感应系统由应答调控蛋白PqsR与信号分子PQS组成。前期研究已经表明pqsR负调控Plt生物合成及基因簇表达。本论文旨在研究PQS分子对Plt合成及基因表达的调控作用。【方法】从M18基因组中扩增PQS合成基因pqsA,通过同源重组技术构建假单胞菌M18的pqsA突变菌株M18pqsA。利用lacZ报告基因分析、信号分子添加实验等,研究PQS对Plt合成及基因表达的调控作用。【结果】在KMB培养基中,分别比较野生型菌株M18和突变菌株M18pqsA的Plt产量,突变菌株的Plt产量存在较小幅度的升高,约为野生型菌株的1.53倍。添加PQS对plt表达存在一定程度但不是很显著的负调控作用。【结论】PQS分子对Plt生物合成及基因表达存在部分负调控作用。     

假单胞菌M-18qscR突变株的构建及其对抗生素合成的调控

在革兰氏阴性菌中,全局性调控因子QscR参与菌群传感调节系统,调节多种毒素因子、次生代谢产物、稳定期基因以及参与生物膜形成的基因的表达,它通过与靶基因DNA启动子的调节元件结合,调节基因转录。假单胞菌株(Pseudomonas sp.)M-18是促进植物生长的根际细菌,能同时分泌藤黄绿菌素(pyoluterion,Plt)和吩嗪-1-羧酸(phenazine-1-carboxylicacid,PCA)。运用同源重组技术,构建了假单胞菌(Pseudomonas sp.)M-18株的qscR突变菌株M-18Q。比较野生株M-18和突变株M-18Q生物合成PCA和Plt的产量,在28℃恒温条件下,在PPM和KMB培养基中M-18Q菌株合成PCA的量分别约为野生型M-18菌株的4～6倍和3～5倍,分别达到480μg/mL和140μg/mL。在PPM培养基中,野生株M-18和突变株M-18Q几乎都没有Plt的合成,而在KMB培养基中,突变菌株和野生型M-18合成Plt的量基本一致。反式互补实验表明,在qscR突变株M-18Q中,PCA生物合成受到抑制而Plt的生物合成却不受影响。phzA基因是吩嗪合成基因簇中第一个基因,phzA‘-’lacZ翻译融合实验表明,qscR基因产物通过抑制PCA合成基因簇的表达,实施负调控作用。结果表明qscR基因是作为一个全局调控基因区别性地调控PCA和Plt的生物合成。     

rpoS基因插入突变对铜绿假单胞菌两个吩嗪合成基因簇的调控

【目的】为了研究铜绿假单胞菌rpoS基因对吩嗪(Phenazine)合成基因簇phz1和phz2的调控方式与机制。【方法】采用抗庆大霉素基因(gentamycin resistance cassette,aacC1)插入失活的策略构建了rpoS基因突变株PA-SG;同时利用lacZ的翻译融合表达载体pME6015,构建了phz1′-′lacZ和phz2′-′lacZ翻译融合表达载体pMEZ1和pMEZ2。采用电转化法分别将pMEZ1、pMEZ2和pME6015导入铜绿假单胞菌突变株PA-SG和野生株PAO1,用Miller法检测融合β-半乳糖苷酶活性。【结果】在KMB或PPM培养基中,pMEZ1在突变株PA-SG中的表达均增强,为野生株的4-5倍;而pMEZ2在突变株PA-SG中的表达均降低,野生株是突变株的2-3倍。【结论】由此推测,铜绿假单胞菌rpoS基因对两个不同吩嗪合成基因簇的调控作用具有特异性,在一定程度上,rpoS负调控phz1,正调控phz2。     

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M18调控因子GacA与RsmA的相关性及对抗生物质合成代谢调控机制的分析

假单胞菌M18是一株可同时合成并分泌吩嗪-1-羧酸(Phenazine-1-carboxylic acid,PCA)和藤黄绿脓菌素(Pyoluteorin,Plt)两种抗生物质的生防菌株。为了进一步研究假单胞菌M18抗生物质合成代谢的调控方式与机制,在分别构建gacAr、smA等单基因突变株基础上,又构建了gacArsmA双基因突变株M18GR以及gacA′-l′acZ和rsmA′-′lacZ等翻译融合表达载体(pMEGA和pMERA)。通过在PPM和KMB两种培养基中发酵培养和两种抗生物质PCA和Plt的HPLC定量测定显示,双突变株M18GR的PCA和Plt的合成量不论在PPM还是在KMB培养基中都介于单突变株M18G和M18R之间。由实验结果分析推测,两种调控因子对抗生物质合成的调控作用不是发生在转录水平,很可能发生在转录后水平。由β-半乳糖苷酶的定量分析表明,在假单胞菌M18中,两种调控因子不存在自诱导机制;虽然GacA未调控RsmA的合成,但RsmA可能部分正向调控GacA的表达。     

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

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。     

Diversity of chromosomal AmpC beta-lactamases from Enterobacter cloacae isolates in a Portuguese hospital

A new regulator gene named pltZ, which is located downstream of the plt gene cluster in the genome of Pseudomonas sp. M18, was identified, sequenced and characterized in this report. The deduced amino acid sequence of PltZ shares significant homology with other bacterial regulators in the TetR family. The chromosomal pltZ disruption mutant gave rise to 4.4-fold enhancement of pyoluteorin biosynthesis but did not exert significant influence on the accumulation of phenazine-1-carboxylic acid compared with the wild-type M18. The negative regulation of pltZ on pyoluteorin biosynthesis was further confirmed by multiplied pltZ gene dosage experiments and pltA'-'lacZ translational fusion analyses.     

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.     

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.     

Positive regulation of pyoluteorin biosynthesis in Pseudomonas sp. M18 by quorum-sensing regulator VqsR

The biocontrol rhizobacterium Pseudomonas sp. M18 can produce two kinds of antibiotics, namely pyoluteorin (Plt) and phenazine-1-carboxylic acid (PCA), and is antagonistic against a number of soilborne phytopathogens. In this study, a luxR-type quorum-sensing regulatory gene, vqsR, was identified and characterized immediately downstream of the Plt gene cluster in strain M18. A vqsR-inactivated mutant led to a significant decrease in the production of Plt and its biosynthetic gene expression. However, this was restored when introducing the vqsR gene by cloning into the plasmid pME6032 in trans. The vqsR mutation did not exert any obvious influence on the production of PCA and its biosynthetic gene expression and the production of Nacylhomoserine lactones (C4 and C8-HSLs) and their biosynthetic gene rhlI expression. Accordingly, these results introduce VqsR as a regulator of Plt production in Pseudomonas spp., and suggest that the regulatory mechanism of vqsR in strain M18 is distinct from that in P. aeruginosa. In addition, it was demonstrated that vqsR mutation did not have any obvious impact on the expression of Plt-specific ABC transporters and other secondary metabolic global regulators, including GacA, RpoS, and RsmA.