转录调节因子σ~(38)介导铜绿假单胞菌绿脓菌素合成代谢调控
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摘要
【目的】为了进一步鉴定铜绿假单胞菌转录调控因子σ~(38)对2个拷贝吩嗪合成基因簇(phz A1-G1和phz A2-G2)的具体调控方式并推定介导绿脓菌素合成代谢的可能调控机制。【方法】根据铜绿假单胞菌基因组信息,利用同源重组原理构建rpo S基因缺失突变株Δrpo S以及克隆全长rpo S基因作互补分析;再以单一吩嗪基因簇缺失突变株Δphz1和Δphz2为出发菌株,分别构建rpo S缺失突变株Δrpo Sphz1和rpo S插入突变株Δrpo Sphz2,测定并比较野生株及相关突变株的绿脓菌素合成量,初步推定σ~(38)因子对2个不同吩嗪基因簇表达的调控方式。【结果】在GA培养基中,突变株Δrpo S的绿脓菌素合成量比野生株显著增加;互补分析证实,σ~(38)可使突变株Δrpo S的绿脓菌素降低并接近野生株PAO1水平;与对照株Δphz1相比,突变株Δrpo Sphz1的绿脓菌素合成量因σ~(38)因子缺失而显著减少;而与对照株Δphz2相比,突变株Δrpo Sphz2的绿脓菌素合成量因σ~(38)因子缺失显著增加。【结论】转录调控因子σ~(38)对铜绿假单胞菌绿脓菌素的合成代谢的确具一定的负调控作用;结合已报道的研究结果,初步推定:σ~(38)因子通过负调控吩嗪基因簇phz1,正调控吩嗪基因簇phz2的表达实现对绿脓菌素合成代谢的调控。
Pyocyanin,an important virulence factor,is synthesized and secreted by Pseudomonas aeruginosa PAO1 and plays a critical role in pathogen-host interaction during infection.Sigma~(38)(σ~(38),σ~S) is a central regulator for many virulence production in pathogens.[Objective] Our aim is to identify expression and regulation of two phenazine-producing operons mediated by the sigma~(38) factor in Pseudomonas aeruginosa PAO1.[Methods] We first cloned the flanking fragments of rpo S from the chromosomal DNA of P.aeruginosa PAO1 and constructed the deletion mutant ?rpo S with the insertion of gentamycin resistance cassette(aac C1).Complementation of rpo S was then carried out after construction and introduction of p ME10S(containing the whole rpo S region).Finally,we created the mutant ?rpo Sphz1 and ?rpo Sphz2,and measured pyocyanin production by these mutants in GA medium,using the parental strain ?phz1 and ?phz2 as controls.[Results] In GA medium,pyocyanin production by mutant ?rpo S increased dramatically in comparison with the wild-type strain PAO1.Production of pyocyanin,however,was decreased to the level of the wild-type strain with complementation of the derivative ?rpo S harboring p ME10 S.Mutant ?rpo Sphz2 produced much more pyocyanin than mutant ?phz2.Mutant ?rpo Sphz1,however,produced much less pyocyanin than mutant ?phz1.[Conclusion] By positively regulating the expression of phz2 and negatively regulating the phz1,sigma~(38 )factor exerts negative modulation on pyocyanin biosynthesis in P.aeruginosa PAO1.
Pyocyanin,an important virulence factor,is synthesized and secreted by Pseudomonas aeruginosa PAO1 and plays a critical role in pathogen-host interaction during infection.Sigma~(38)(σ~(38),σ~S) is a central regulator for many virulence production in pathogens.[Objective] Our aim is to identify expression and regulation of two phenazine-producing operons mediated by the sigma~(38) factor in Pseudomonas aeruginosa PAO1.[Methods] We first cloned the flanking fragments of rpo S from the chromosomal DNA of P.aeruginosa PAO1 and constructed the deletion mutant ?rpo S with the insertion of gentamycin resistance cassette(aac C1).Complementation of rpo S was then carried out after construction and introduction of p ME10S(containing the whole rpo S region).Finally,we created the mutant ?rpo Sphz1 and ?rpo Sphz2,and measured pyocyanin production by these mutants in GA medium,using the parental strain ?phz1 and ?phz2 as controls.[Results] In GA medium,pyocyanin production by mutant ?rpo S increased dramatically in comparison with the wild-type strain PAO1.Production of pyocyanin,however,was decreased to the level of the wild-type strain with complementation of the derivative ?rpo S harboring p ME10 S.Mutant ?rpo Sphz2 produced much more pyocyanin than mutant ?phz2.Mutant ?rpo Sphz1,however,produced much less pyocyanin than mutant ?phz1.[Conclusion] By positively regulating the expression of phz2 and negatively regulating the phz1,sigma~(38 )factor exerts negative modulation on pyocyanin biosynthesis in P.aeruginosa PAO1.
引文
[1]Kung VL,Khare S,Stehlik C,Bacon EM,Hughes AJ,Hauser AR.An rhs gene of Pseudomonas aeruginosa encodes a virulence protein that activates the inflammasome.Proceedings of the National Academy of Sciences of the Unite States of America,2012,109(4):1275–1280.
[2]Heurlier K,Williams F,Heeb S,Dormond C,Pessi G,Singer D,Cámara M,Williams P,Haas D.Positive control of swarming,rhamnolipid synthesis,and lipase production by the posttranscriptional Rsm A/Rsm Z system in Pseudomonas aeruginosa PAO1.Journal of Bacteriology,2004,186(10):2936–2945.
[3]Hunter RC,Klepac-Ceraj V,Lorenzi MM,Grotzinger H,Martin TR,Newman DK.Phenazine content in the cystic fibrosis respiratory tract negatively correlates with lung function and microbial complexity.American Journal of Respiratory Cell and Molecular Biology,2012,47(6):738–745.
[4]Mavrodi DV,Bonsall RF,Delaney SM,Soule MJ,Phillips G,Thomashow LS.Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1.Journal of Bacteriology,2001,183(21):6454–6465.
[5]Potvin E,Sanschagrin F,Levesque RC.Sigma factors in Pseudomonas aeruginosa.FEMS Microbiology Review,2008,32(1):38–55.
[6]Dong T,Schellhorn HE.Role of Rpo S in virulence of pathogens.Infection and Immunity,2010,78(3):887–897.
[7]Suh SJ,Silo-Suh L,Woods DE,Hassett DJ,West SE,Ohman DE.Effect of rpo S mutation on the stress response and expression of virulence factors in Pseudomonas aeruginosa.Journal of Bacteriology,1999,181(13):3890–3897.
[8]Sambrook J,Frisch EF,Maniatis T.Molecular cloning:a laboratory manual.2nd ed.New York:Cold Spring Harbor Laboratory Press,1989.
[9]Chieda Y,Iiyama K,Yasunaga-Aoki C,Lee JM,Kusakabe T,Shimizu S.Pathogenicity of gac A mutant of Pseudomonas aeruginosa PA01 in the silkworm,Bombyx mori.FEMS Microbiology Letters,2005,244(1):181–186.
[10]Hoang TT,Karkhoff-Schweizer RR,Kutchma AJ,Schweizer HP.A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences:application for isolation of unmarked Pseudomonas aeruginosa mutants.Gene,1998,212(1):77–86.
[11]Cui QN,Lv HN,Qi ZZ,Jiang B,Xiao B,Liu LD,Ge YH,Hu XM.Cross-regulation between the phz1 and phz2 operons maintain a balanced level of phenazine biosynthesis in Pseudomonas aeruginosa PAO1.PLo S One,2016,11(1):e0144447.
[12]Schweizer HD.Small broad-host-range gentamycin resistance gene cassettes for site-specific insertion and deletion mutagenesis.Bio Techniques,1993,15(5):831–834.
[13]Heeb S,Itoh Y,Nishijyo T,Schnider U,Keel C,Wade J,Walsh U,O'Gara F,Haas D.Small,stable shuttle vectors based on the minimal p VS1 replicon for use in gram-negative,plant-associated bacteria.Molecular Plant-Microbe Interaction,2000,13(2):232–237.
[14]Liang HH,Li LL,Dong ZL,Surette MG,Duan KM.The Yeb C family protein PA0964 negatively regulates the Pseudomonas aeruginosa quinolone signal system and pyocyanin production.Journal of Bacteriology,2008,190(18):6217–6227.
[15]Zhou JF,Ge YH,Liu T,Cheng XH,Wang L,Gao XX.Effect of rpo S mutation on two gene clusters of phenazine in Psedomonas aeruginosa PAO1.Acta Microbiolgica Sinica,2010,50(3):411–417.(in Chinese)周金凤,葛宜和,刘婷,程显好,王磊,高兴喜.rpo S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.微生物学报,2010,50(3):411–417.
[16]Jimenez PN,Koch G,Thompson JA,Xavier KB,Cool RH,Quax WJ.The multiple signaling systems regulating virulence in Pseudomonas aeruginosa.Microbiology and Molecular Biology Reviews,2012,76(1):46–65.
[17]Whiteley M,Lee KM,Greenberg EP.Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa.Proceedings of the National Academy of Sciences of the Unite States of America,1999,96(24):13904–13909.
[18]Lee JH,Lequette Y,Greenberg EP.Activity of purified Qsc R,a Pseudomonas aeruginosa orphan quorum-sensing transcription factor.Molecular Microbiology,2006,59(2):602–609.
[19]Zhang Y,Cui QN,Zhao Z,Ming YF,Chi XY,Feng ZB,Cheng SW,Xie WH,Ge YH.Positive regulation in expression of the phenazine-producing operon phz2 mediated by Pip in Pseudomonas aeruginosa PAO1.Acta Microbiologica Sinica,2013,53(2):127–135.(in Chinese)张圆,崔钦娜,赵哲,明永飞,迟晓艳,冯志彬,程仕伟,解卫海,葛宜和.Pip介导铜绿假单胞菌吩嗪基因簇phz2的表达.微生物学报,2013,53(2):127–135.
[2]Heurlier K,Williams F,Heeb S,Dormond C,Pessi G,Singer D,Cámara M,Williams P,Haas D.Positive control of swarming,rhamnolipid synthesis,and lipase production by the posttranscriptional Rsm A/Rsm Z system in Pseudomonas aeruginosa PAO1.Journal of Bacteriology,2004,186(10):2936–2945.
[3]Hunter RC,Klepac-Ceraj V,Lorenzi MM,Grotzinger H,Martin TR,Newman DK.Phenazine content in the cystic fibrosis respiratory tract negatively correlates with lung function and microbial complexity.American Journal of Respiratory Cell and Molecular Biology,2012,47(6):738–745.
[4]Mavrodi DV,Bonsall RF,Delaney SM,Soule MJ,Phillips G,Thomashow LS.Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1.Journal of Bacteriology,2001,183(21):6454–6465.
[5]Potvin E,Sanschagrin F,Levesque RC.Sigma factors in Pseudomonas aeruginosa.FEMS Microbiology Review,2008,32(1):38–55.
[6]Dong T,Schellhorn HE.Role of Rpo S in virulence of pathogens.Infection and Immunity,2010,78(3):887–897.
[7]Suh SJ,Silo-Suh L,Woods DE,Hassett DJ,West SE,Ohman DE.Effect of rpo S mutation on the stress response and expression of virulence factors in Pseudomonas aeruginosa.Journal of Bacteriology,1999,181(13):3890–3897.
[8]Sambrook J,Frisch EF,Maniatis T.Molecular cloning:a laboratory manual.2nd ed.New York:Cold Spring Harbor Laboratory Press,1989.
[9]Chieda Y,Iiyama K,Yasunaga-Aoki C,Lee JM,Kusakabe T,Shimizu S.Pathogenicity of gac A mutant of Pseudomonas aeruginosa PA01 in the silkworm,Bombyx mori.FEMS Microbiology Letters,2005,244(1):181–186.
[10]Hoang TT,Karkhoff-Schweizer RR,Kutchma AJ,Schweizer HP.A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences:application for isolation of unmarked Pseudomonas aeruginosa mutants.Gene,1998,212(1):77–86.
[11]Cui QN,Lv HN,Qi ZZ,Jiang B,Xiao B,Liu LD,Ge YH,Hu XM.Cross-regulation between the phz1 and phz2 operons maintain a balanced level of phenazine biosynthesis in Pseudomonas aeruginosa PAO1.PLo S One,2016,11(1):e0144447.
[12]Schweizer HD.Small broad-host-range gentamycin resistance gene cassettes for site-specific insertion and deletion mutagenesis.Bio Techniques,1993,15(5):831–834.
[13]Heeb S,Itoh Y,Nishijyo T,Schnider U,Keel C,Wade J,Walsh U,O'Gara F,Haas D.Small,stable shuttle vectors based on the minimal p VS1 replicon for use in gram-negative,plant-associated bacteria.Molecular Plant-Microbe Interaction,2000,13(2):232–237.
[14]Liang HH,Li LL,Dong ZL,Surette MG,Duan KM.The Yeb C family protein PA0964 negatively regulates the Pseudomonas aeruginosa quinolone signal system and pyocyanin production.Journal of Bacteriology,2008,190(18):6217–6227.
[15]Zhou JF,Ge YH,Liu T,Cheng XH,Wang L,Gao XX.Effect of rpo S mutation on two gene clusters of phenazine in Psedomonas aeruginosa PAO1.Acta Microbiolgica Sinica,2010,50(3):411–417.(in Chinese)周金凤,葛宜和,刘婷,程显好,王磊,高兴喜.rpo S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.微生物学报,2010,50(3):411–417.
[16]Jimenez PN,Koch G,Thompson JA,Xavier KB,Cool RH,Quax WJ.The multiple signaling systems regulating virulence in Pseudomonas aeruginosa.Microbiology and Molecular Biology Reviews,2012,76(1):46–65.
[17]Whiteley M,Lee KM,Greenberg EP.Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa.Proceedings of the National Academy of Sciences of the Unite States of America,1999,96(24):13904–13909.
[18]Lee JH,Lequette Y,Greenberg EP.Activity of purified Qsc R,a Pseudomonas aeruginosa orphan quorum-sensing transcription factor.Molecular Microbiology,2006,59(2):602–609.
[19]Zhang Y,Cui QN,Zhao Z,Ming YF,Chi XY,Feng ZB,Cheng SW,Xie WH,Ge YH.Positive regulation in expression of the phenazine-producing operon phz2 mediated by Pip in Pseudomonas aeruginosa PAO1.Acta Microbiologica Sinica,2013,53(2):127–135.(in Chinese)张圆,崔钦娜,赵哲,明永飞,迟晓艳,冯志彬,程仕伟,解卫海,葛宜和.Pip介导铜绿假单胞菌吩嗪基因簇phz2的表达.微生物学报,2013,53(2):127–135.