Biocontrol strain Pseudomonas PA1201 produces pyoluteorin(Plt),which is an antimicrobial secondary metabolite.Plt represents a promising candidate pesticide due to its broad-spectrum antifungal and antibacterial activ...Biocontrol strain Pseudomonas PA1201 produces pyoluteorin(Plt),which is an antimicrobial secondary metabolite.Plt represents a promising candidate pesticide due to its broad-spectrum antifungal and antibacterial activity.Although PA1201 contains a complete genetic cluster for Plt biosynthesis,it fails to produce detectable level of Plt when grown in media typically used for Pseudomonas strains.In this study,minimum medium(MM)was found to favor Plt biosynthesis.Using the medium M,which contains all the salts of MM medium except for mannitol,as a basal medium,we compared 10 carbon sources for their ability to promote Plt biosynthesis.Fructose,mannitol,and glycerol promoted Plt biosynthesis,with fructose being the most effective carbon source.Glucose or succinic acid had no significant effect on Plt biosynthesis,but effectively antagonized fructose-dependent synthesis of Plt.Promoter-lacZ fusion reporter strains demonstrated that fructose acted through activation of the pltLABCDEFG(pltL)operon but had no effect on other genes of plt gene cluster;glucose or succinic acid antagonized fructose-dependent pltL induction.Mechanistically,fructose-mediated Plt synthesis involved carbon catabolism repression.The two-component system CbrA/CbrB and small RNA catabolite repression control Z(crcZ)were essential for fructose-induced Plt synthesis.The small RNA binding protein Hfq and Crc negatively regulated fructose-induced Plt.Taken together,this study provides a new model of fructose-dependent Plt production in PA1201 that can help improve Plt yield by biosynthetic approaches.展开更多
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 i...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.展开更多
The pltR gene, coding a putative LysR-type regulator, was identified upstream Plt biosynthetic gene cluster in Pseudomonas sp. M18 using bioinformatics technology. The null mutation of pltR resulted in mutant M18TRG (...The pltR gene, coding a putative LysR-type regulator, was identified upstream Plt biosynthetic gene cluster in Pseudomonas sp. M18 using bioinformatics technology. The null mutation of pltR resulted in mutant M18TRG (pltR::Gm) by recombination and its Plt (Pyoluteorin) production declined to 30% while PCA (Phenazine-1-carboxylic acid) production remained unchanged as compared with the wild-type M18 grown in King’s Medium B. After complementation, Plt production of mutant M18TRG was restored to the level in wild-type M18. Overexpression of pltR in M18 led to 13-fold enhancement of Plt produc-tion over the wild-type M18 strain. However, PCA production was unchanged under this condition. These data suggested that PltR was a positive regulator on Plt production. Plt itself, however, could not regulate expression of pltR. Expression of the plt-lacZ transcriptional fusion in mutant M18TRG de-clined obviously as compared with the wild-type M18, which further proved that PltR could regulate expression of Plt biosynthetic genes at the transcriptional level. In addition, the investigation on the pltR expression in gacA mutant M18G and rsmA mutant M18R disclosed that PltR was involved in the positive regulation of gacA on Plt production while being excluded from the negative control caused by rsmA.展开更多
文摘Biocontrol strain Pseudomonas PA1201 produces pyoluteorin(Plt),which is an antimicrobial secondary metabolite.Plt represents a promising candidate pesticide due to its broad-spectrum antifungal and antibacterial activity.Although PA1201 contains a complete genetic cluster for Plt biosynthesis,it fails to produce detectable level of Plt when grown in media typically used for Pseudomonas strains.In this study,minimum medium(MM)was found to favor Plt biosynthesis.Using the medium M,which contains all the salts of MM medium except for mannitol,as a basal medium,we compared 10 carbon sources for their ability to promote Plt biosynthesis.Fructose,mannitol,and glycerol promoted Plt biosynthesis,with fructose being the most effective carbon source.Glucose or succinic acid had no significant effect on Plt biosynthesis,but effectively antagonized fructose-dependent synthesis of Plt.Promoter-lacZ fusion reporter strains demonstrated that fructose acted through activation of the pltLABCDEFG(pltL)operon but had no effect on other genes of plt gene cluster;glucose or succinic acid antagonized fructose-dependent pltL induction.Mechanistically,fructose-mediated Plt synthesis involved carbon catabolism repression.The two-component system CbrA/CbrB and small RNA catabolite repression control Z(crcZ)were essential for fructose-induced Plt synthesis.The small RNA binding protein Hfq and Crc negatively regulated fructose-induced Plt.Taken together,this study provides a new model of fructose-dependent Plt production in PA1201 that can help improve Plt yield by biosynthetic approaches.
基金the Science Program of the China National 10th "Five-year Plan" (Grant No. 2004BAAA308A21-6)the National Natural Science Foundation of China (Grant No. 3037004)+1 种基金the National High Technology Research and Development Program (863 Program) of China (Grant No. 2007AA02Z215)New Century Excellent Talent Program (NCET) in China and Shanghai Leading Academic Discipline (Project Number: B203)
文摘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.
基金Supported by the Science Program of the China National 10th "Five-year Plan" (Grant No. 2004BA308A21-6)the National Natural Science Foundation of China (Grant No. 30370041) and NCET in China
文摘The pltR gene, coding a putative LysR-type regulator, was identified upstream Plt biosynthetic gene cluster in Pseudomonas sp. M18 using bioinformatics technology. The null mutation of pltR resulted in mutant M18TRG (pltR::Gm) by recombination and its Plt (Pyoluteorin) production declined to 30% while PCA (Phenazine-1-carboxylic acid) production remained unchanged as compared with the wild-type M18 grown in King’s Medium B. After complementation, Plt production of mutant M18TRG was restored to the level in wild-type M18. Overexpression of pltR in M18 led to 13-fold enhancement of Plt produc-tion over the wild-type M18 strain. However, PCA production was unchanged under this condition. These data suggested that PltR was a positive regulator on Plt production. Plt itself, however, could not regulate expression of pltR. Expression of the plt-lacZ transcriptional fusion in mutant M18TRG de-clined obviously as compared with the wild-type M18, which further proved that PltR could regulate expression of Plt biosynthetic genes at the transcriptional level. In addition, the investigation on the pltR expression in gacA mutant M18G and rsmA mutant M18R disclosed that PltR was involved in the positive regulation of gacA on Plt production while being excluded from the negative control caused by rsmA.
