荧光假单胞菌2P24中RstA蛋白的功能鉴定
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摘要
探究荧光假单胞菌2P24中OmpR家族转录因子RstA的功能,明确其对EmhABC外排泵的调控作用及机制。利用共适应分析预测RstA的潜在功能;采用同源重组技术构建rstA、emhABC基因缺失菌株ΔrstA和ΔemhABC,检测野生型、ΔrstA、ΔemhABC对多种抗生素的敏感性;通过qRT-PCR和β-半乳糖苷酶实验检测emhABC在野生型和ΔrstA菌株中的转录、表达水平;表达纯化His-RstA蛋白并经凝胶阻滞实验检测RstA蛋白与emhABC基因启动子区域的结合活性。结果显示,RstA同EmhABC存在共适应性,并预测RstA与多种抗生素胁迫环境的适应相关;ΔrstA和ΔemhABC对多种抗生素耐受性下降;与野生株相比,突变株ΔrstA中emhABC的转录、表达水平均下降超过3倍;成功表达纯化His-RstA蛋白,经凝胶阻滞实验显示重组His-RstA蛋白可与emhABC基因启动子区域特异性结合。OmpR家族转录因子RstA通过结合在emhABC上游启动子区域正向调控EmhABC的表达,并影响荧光假单胞菌2P24的多重耐药性。
This work is to identify the function of transcriptional regulator RstA from OmpR subfamily and clarify the role of RstA in the regulation of efflux pump EmhABC in Pseudomonas fluorescens strain 2P24. Cofitness data was obtained from Cofitness Browser to explore potential functions of RstA. The rstA and emhABC-deficient mutant strain ΔrstA and ΔemhABC were constructed by homologous recombination and minimum inhibitory concentration(MIC)values of wild-type strain,ΔrstA and ΔemhABC to several antibiotics were detected. Quantitative real-time PCR assay and β-galactosidase assay were performed to detect the transcriptional levels of emhABC in the wild-type strain and ΔrstA.Electrophoretic mobility shift assay(EMSA)of the purified His-RstA protein were employed to access the interaction between RstA and the emhABC promotor. As results,RstA presented similar fitness pattern with EmhABC,which was predicted to be responsible for adapting several antibiotics stress conditions,and the tolerances of ΔrstA and ΔemhABC to multiple antibiotics decreased. Comparing to the wild-type strain,ΔrstA showed 3-fold down-regulation to the transcription and expression of emhABC. Results of EMSA indicated that the recombinant purified protein His-RstA was able to bind to the promoter of emhABC specifically. Transcriptional regulator RstA directly activates the expression of efflux pump EmhABC by binding to the emhABC promotor and contributes to multi-antibiotic resistance in P. fluorescens strain 2P24.
This work is to identify the function of transcriptional regulator RstA from OmpR subfamily and clarify the role of RstA in the regulation of efflux pump EmhABC in Pseudomonas fluorescens strain 2P24. Cofitness data was obtained from Cofitness Browser to explore potential functions of RstA. The rstA and emhABC-deficient mutant strain ΔrstA and ΔemhABC were constructed by homologous recombination and minimum inhibitory concentration(MIC)values of wild-type strain,ΔrstA and ΔemhABC to several antibiotics were detected. Quantitative real-time PCR assay and β-galactosidase assay were performed to detect the transcriptional levels of emhABC in the wild-type strain and ΔrstA.Electrophoretic mobility shift assay(EMSA)of the purified His-RstA protein were employed to access the interaction between RstA and the emhABC promotor. As results,RstA presented similar fitness pattern with EmhABC,which was predicted to be responsible for adapting several antibiotics stress conditions,and the tolerances of ΔrstA and ΔemhABC to multiple antibiotics decreased. Comparing to the wild-type strain,ΔrstA showed 3-fold down-regulation to the transcription and expression of emhABC. Results of EMSA indicated that the recombinant purified protein His-RstA was able to bind to the promoter of emhABC specifically. Transcriptional regulator RstA directly activates the expression of efflux pump EmhABC by binding to the emhABC promotor and contributes to multi-antibiotic resistance in P. fluorescens strain 2P24.
引文
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[16]Yan X, Yang R, Zhao RX, et al. Transcriptional regulator PhlH modulates 2, 4-diacetylphloroglucinol biosynthesis in response to the biosynthetic intermediate and end product[J]. Applied and Environmental Microbiology, 2017, 83(21):e01419-17.
[17] Hellman LM, Fried MG. Electrophoretic mobility shift assay(EMSA)for detecting protein-nucleic acid interactions[J].Nature Protocols, 2007, 2(8):1849-1861.
[18]魏海雷,张力群.荧光假单胞杆菌2p24中生防相关调控基因gacS的克隆和功能分析[J].微生物学报, 2005, 45(3):368-272.
[19]Zhang W, Zhao Z, Zhang B, et al. Posttranscriptional regulation of 2, 4-diacetylphloroglucinol production by GidA and TrmE in Pseudomonas fluorescens 2P24[J]. Applied and Environ Microbiology, 2014, 80(13):3972-3981.
[20]Wei HL, Zhang LQ. Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24[J]. Antonie Van Leeuwenhoek, 2006, 89(2):267-280.
[21]Li X, Gu GQ, Chen W, et al. The outer membrane protein OprF and the sigma factor SigX regulate antibiotic production in Pseudomonas fluorescens 2P24[J]. Microbiological Research,2018, 206:159-167.
[22]Liu P, Zhang W, Zhang LQ, et al. Supramolecular structure and functional analysis of the type III secretion system in Pseudomonas fluorescens 2P24[J]. Frontiers in Plant Science, 2016, 6:1190.
[23]Martinez-Hackert E, Stock AM. The DNA-binding domain of OmpR:crystal structures of a winged helix transcription factor[J]. Structure, 1997, 5(1):109-124.
[24]Stock AM, Robinson VL, Goudreau PN. Two-component signal transduction[J]. Annual Review of Biochemistry, 2000, 69(1):183-215.
[25]Groisman EA. Feedback control of two-component regulatory systems[J]. Annual Review of Microbiology, 2016, 70(1):103-124.
[26]Li YC, Chang CK, Chang CF, et al. Structural dynamics of the twocomponent response regulator RstA in recognition of promoter DNA element[J]. Nucleic Acids Research, 2014, 42(13):8777-8788.
[2]Scales BS, Dickson RP, LiPuma JJ et al. Microbiology, genomics,and clinical significance of the Pseudomonas fluorescens species complex, an unappreciated colonizer of humans[J]. Clinical Microbiology Reviews, 2014, 27(4):927-948.
[3]Goldberg JB, Hancock RE W, Parales RE, et al. Pseudomonas2007[J]. Journal of Bacteriology, 2008, 190(8):2649-2662.
[4]Haas D, Défago G. Biological control of soil-borne pathogens by fluorescent pseudomonads[J]. Nature Reviews Microbiology,2005, 3(4):307-319.
[5] Weller DM, Landa BB, Mavrodi OV, et al. Role of 2, 4-diacetylphloroglucinol-producing fluorescent pseudomonas spp. in the defense of plant roots[J]. Plant Biology, 2007, 9(1):4-20.
[6]Kittinger C, Lipp M, Baumert R, et al. Antibiotic resistance patterns of Pseudomonas spp. isolated from the River Danube[J]. Frontiers in Microbiology, 2016, 7:586.
[7]Zhou Y, Xu YB, Xu JX, et al. Combined toxic effects of heavy metals and antibiotics on a Pseudomonas fluorescens strain Zy2 isolated from Swine wastewater[J]. International Journal of Molecular Sciences, 2015, 16(2):2839-2850.
[8]Martinez JL, Sánchez MB, Martínez-Solano L, et al. Functional role of bacterial multidrug efflux pumps in microbial natural ecosystems[J]. FEMS Microbiology Reviews, 2009, 33(2):430-449.
[9]Tian T, Wu XG, Duan HM, et al. The resistance-nodulationdivision efflux pump EmhABC influences the production of 2,4-diacetylphloroglucinol in Pseudomonas fluorescens 2P24[J].Microbiology, 2010, 156(1):39-48.
[10]Wetmore KM, Price MN, Waters RJ, et al. Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly barcoded transposons[J]. MBio, 2015, 6(3):e00306-15.
[11]Price MN, Wetmore KM, Waters RJ, et al. Mutant phenotypes for thousands of bacterial genes of unknown function[J]. Nature,2018, 557(7706):503-509.
[12]Zhao C, Zhao J, Wang W, et al. Expression of MLAA34-HSP70fusion gene constructed by SOE-PCR[J]. Pakistan Journal of Pharmaceutical Sciences, 2017, 30:1125-1127.
[13]King EO, Ward MK, Raney DE. Two simple media for the demonstration of pyocyanin and fluorescin[J]. The Journal of Laboratory and Clinical Medicine, 1954, 44(2):301-307.
[14]Schwalbe R, Steele-Moore L, Goodwin AC, Antimicrobial susceptibility testing protocols[M]. Boca Raton:CRC Press,2007.
[15]Yan Q, Wu XG, Wei HL, et al. Differential control of the PcoI/PcoR quorum-sensing system in Pseudomonas fluorescens 2P24 by sigma factor RpoS and the GacS/GacA two-component regulatory system[J]. Microbiological Research, 2009, 164(1):18-26.
[16]Yan X, Yang R, Zhao RX, et al. Transcriptional regulator PhlH modulates 2, 4-diacetylphloroglucinol biosynthesis in response to the biosynthetic intermediate and end product[J]. Applied and Environmental Microbiology, 2017, 83(21):e01419-17.
[17] Hellman LM, Fried MG. Electrophoretic mobility shift assay(EMSA)for detecting protein-nucleic acid interactions[J].Nature Protocols, 2007, 2(8):1849-1861.
[18]魏海雷,张力群.荧光假单胞杆菌2p24中生防相关调控基因gacS的克隆和功能分析[J].微生物学报, 2005, 45(3):368-272.
[19]Zhang W, Zhao Z, Zhang B, et al. Posttranscriptional regulation of 2, 4-diacetylphloroglucinol production by GidA and TrmE in Pseudomonas fluorescens 2P24[J]. Applied and Environ Microbiology, 2014, 80(13):3972-3981.
[20]Wei HL, Zhang LQ. Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24[J]. Antonie Van Leeuwenhoek, 2006, 89(2):267-280.
[21]Li X, Gu GQ, Chen W, et al. The outer membrane protein OprF and the sigma factor SigX regulate antibiotic production in Pseudomonas fluorescens 2P24[J]. Microbiological Research,2018, 206:159-167.
[22]Liu P, Zhang W, Zhang LQ, et al. Supramolecular structure and functional analysis of the type III secretion system in Pseudomonas fluorescens 2P24[J]. Frontiers in Plant Science, 2016, 6:1190.
[23]Martinez-Hackert E, Stock AM. The DNA-binding domain of OmpR:crystal structures of a winged helix transcription factor[J]. Structure, 1997, 5(1):109-124.
[24]Stock AM, Robinson VL, Goudreau PN. Two-component signal transduction[J]. Annual Review of Biochemistry, 2000, 69(1):183-215.
[25]Groisman EA. Feedback control of two-component regulatory systems[J]. Annual Review of Microbiology, 2016, 70(1):103-124.
[26]Li YC, Chang CK, Chang CF, et al. Structural dynamics of the twocomponent response regulator RstA in recognition of promoter DNA element[J]. Nucleic Acids Research, 2014, 42(13):8777-8788.