铜绿假单胞菌重金属离子耐受性调查及相关机制的研究

生物技术通报 ›› 2013, Vol. 0 ›› Issue (6): 160-166.

铜绿假单胞菌重金属离子耐受性调查及相关机制的研究

李群¹, 杨洪江¹, 林书祥², 王维², 叶羽洁¹

(1. 工业发酵微生物教育部重点实验室天津市工业微生物重点实验室天津科技大学生物工程学院, 天津 300457;2.天津市儿童医院, 天津 300074)

收稿日期:2013-06-20 修回日期:2013-06-20 出版日期:2013-06-20 发布日期:2013-06-20
作者简介:李群, 女, 硕士研究生, 研究方向: 工业微生物育种;E-mail: lxflq1986@163.com
基金资助:
国家自然科学基金面上项目(30970114)

Heavy Metal Resistance and Its Related Mechanisms in Pseudomonas aeruginosa

Li Qun¹ Yang Hongjiang¹ Lin Shuxiang² Wang Wei² Ye Yujie¹

(1. Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457;2. Tianjin Children’s Hospital, Tianjin 300074)

Received:2013-06-20 Revised:2013-06-20 Published:2013-06-20 Online:2013-06-20

摘要/Abstract

摘要： 旨在调查铜绿假单胞菌临床菌株对多种重金属离子的耐受性, 并对相关机制进行初步研究。测定临床菌株在含有重金属离子培养基中的生长状况;构建转录融合报告基因, 分析铜绿假单胞菌中RND外排泵CzcCBA的表达水平;PCR方法结合DNA测序分析调节基因czcR和czcS的序列差异。结果显示, 158株铜绿假单胞菌临床菌株中, 大多数耐受0.002 mol/L Co²⁺, 1株不能在0.001 mol/L Co²⁺条件下生长, 对照菌株ATCC27853在0.003 mol/L Co²⁺条件下能够生长。分析其中2株临床菌株和对照菌株ATCC27853对其它重金属离子的耐受性, 结果显示其耐受趋势与Co²⁺一致。进一步分析了转录融合报告基因czcC-lacZ在临床菌株中的表达水平发现, β-半乳糖苷酶活力与重金属离子耐受性一致, 重金属离子能够诱导β-半乳糖苷酶活力的提高。对操纵子czcCBA的调节基因czcR和czcS序列分析显示, 3株菌株中调节蛋白CzcR的氨基酸序列相同, 而蛋白CzcS的氨基酸序列则存在多处差异。得出结论, 铜绿假单胞菌临床菌株对重金属离子存在耐受性, 耐受程度与外排泵CzcCBA的表达水平相关, 调节蛋白CzcS某些氨基酸的突变, 可能改变了操纵子czcCBA的表达水平。

关键词: 铜绿假单胞菌, 重金属离子耐受性, RND外排泵CzcCBA, 双因子调控系统czcRS

Abstract: It was to investigate the prevalence of heavy metals resistance in Pseudomonas aeruginosa clinical strains and study the related mechanisms. Measurement of bacterial growths in the medium with various heavy metals;construction of transcription fusion reporter gene for analyzing the expression level of the RND efflux pump CzcCBA;analysis of the regulatory gene czcRS with PCR method and sequence alignment. Results showed that among 158 clinical strains, the majority was able to thrive in the presence of 0.002 mol/L Co²⁺, 1 strains didn’t grow in the presence of 0.001 mol/L Co²⁺, and control strain ATCC27853 could tolerate 0.003 mol/L Co²⁺ during the incubation. Two clinical strains and strain ATCC 27853 were selected for other heavy metals tolerance analysis and the resistance profiles were similar to that of Co²⁺. To investigate the mechanisms of heavy metal resistance, the expression level of gene czcC was determined via detecting β-galactosidase activity encoded by the transcription fusion reporter gene czcC-lacZ. The results showed that β-galactosidase activity corresponded with their respective resistance to heavy metals and heavy metals could induce β-galactosidase activity increase in each strain. Two-component regulatory genes czcR and czcS of the czcCBA were amplified and their sequences were analyzed. Comparing with the genome of strain PAO1, regulator CzcR had the same sequence in all the three strains, while each regulator CzcS had multiple amino acids substitutions in each strain. Clinical strains of P. aeruginosa were resistant to heavy metals. The expression level of RND efflux pump czcCBA contributed to heavy metals resistance. Mutations in regulator CzcS might play key role in the expression variation of operon czcCBA.

Key words: Pseudomonas aeruginosa, Heavy metals resistence, RND efflux pump, CzcCBA, Two-component regulatory system czcRS

李群, 杨洪江, 林书祥, 王维, 叶羽洁. 铜绿假单胞菌重金属离子耐受性调查及相关机制的研究[J]. 生物技术通报, 2013, 0(6): 160-166.

Li Qun, Yang Hongjiang, Lin Shuxiang, Wang Wei, Ye Yujie. Heavy Metal Resistance and Its Related Mechanisms in Pseudomonas aeruginosa[J]. Biotechnology Bulletin, 2013, 0(6): 160-166.

参考文献

[1] Choy MH, Stapleton F, Willcox MD, Zhu H. Comparison of virulence factors in Pseudomonas aeruginosa strains isolated from contact lens- and non-contact lens-related keratitis [J]. J Med Microbiol, 2008, 57(Pt12):1539-46.
[2] Matsui H, Wagner VE, Hill DB, et al. A physical linkage between cystic fibrosis airway surface dehydration and Pseudomonas aeruginosa biofilms [J]. Proc Natl Acad Sci USA, 2006, 103(48):18131-18136.
[3] Lyczak JB, Cannon CL, Pier GB. Establishment of Pseudomonas aeruginosa infection:lessons from a versatile opportunist [J]. Microbes Infect, 2000, 2(9):1051-1060.
[4] Stover CK, Pham XQ, Erwin AL, et al. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen [J]. Nature, 2000, 406(6799):959-964.
[5] Pirnay JP, Bilocq F, Pot B, et al. Pseudomonas aeruginosa population structure revisited [J]. PLoS One, 2009, 4(11):e7740.
[6] Mima T, Kohira N, Li Y, et al. Gene cloning and characteristics of the RND-type multidrug efflux pump MuxABC-OpmB possessing two RND components in Pseudomonas aeruginosa [J]. Microbiology, 2009, 155(Pt11):3509-3517.
[7] Lister PD, Wolter DJ, Hanson ND. Antibacterial-resistant Pseudomo-nas aeruginosa:clinical impact and complex regulation of chromos-omally encoded resistance mechanisms [J]. Clin Microbiol Rev, 2009, 22(4):582-610.
[8] Hassan MT, van der Lelie D, Springael D, et al. Identification of a gene cluster, czr, involved in cadmium and zinc resistance in Pseudomonas aeruginosa [J]. Gene, 1999, 238(2):417-425.
[9] Vander Lelie D, Schwuchow T, Schwidetzky U, et al. Two-component regulatory system involved in transcriptional control of heavy-metal homoeostasis in Alcaligenes eutrophus [J]. Mol Microbiol, 1997, 23(3):493-503.
[10] Munkelt D, Grass G, Nies DH. The chromosomally encoded cation diffusion facilitator proteins DmeF and FieF from Wautersia metallidurans CH34 are transporters of broad metal specificity [J]. J Bacteriol, 2004, 186(23):8036-8043.
[11] Leedjarv A, Ivask A, Virta M. Interplay of different transporters in the mediation of divalent heavy metal resistance in Pseudomonas putida KT2440 [J]. J Bacteriol, 2008, 190(8):2680-2689.
[12] Grosse C, Grass G, Anton A, et al. Transcriptional organization of the czc heavy-metal homeostasis determinant from Alcaligenes eutrophus [J]. J Bacteriol, 1999, 181(8):2385-2393.
[13] Perron K, Caille O, Rossier C, et al. CzcR-CzcS, a two-component system involved in heavy metal and carbapenem resistance in Pseudomonas aeruginosa [J]. J Biol Chem, 2004, 279(10):8761-8768.
[14] 巴兆粉, 杨洪江, 林书祥, 等. 一株铜绿假单胞菌临床菌株耐药的分子机制研究[J]. 临床儿科杂志, 2012, 30(6):515-518.
[15] Dieppois G, Ducret V, Caille O, Perron K. The transcriptional regulator CzcR modulates antibiotic resistance and quorum sensing in Pseudomonas aeruginosa [J]. PLoS One, 2012, 7(5):e38148.
[16] Schaber JA, Carty NL, McDonald NA, et al. Analysis of quorum sensing-deficient clinical isolates of Pseudomonas aeruginosa [J]. J Med Microbiol, 2004, 53(Pt9):841-853.
[17] 吴国营, 杨洪江, 巴兆粉, 等. 铜绿假单胞菌临床菌株TTSS表达水平及相关因素分析[J]. 中国生物化学与分子生物学报, 2012, 28(2):158-163.
[18] Gimmestad M, Sletta H, Ertesvag H, et al. The Pseudomonas fluorescens AlgG protein, but not its mannuronan C-5-epimerase activity, is needed for alginate polymer formation [J]. J Bacteriol, 2003, 185(12):3515-3523.
[19] 杨洪江, 魏东盛, 李明春, 邢来君. 铜绿假单胞菌popN^-突变子Ⅲ型分泌水平及与蛋白酶关系的研究[J].生物工程学报, 2007, 23(5):846-851.
[20] Caille O, Rossier C, Perron K. A copper-activated two-component system interacts with zinc and imipenem resistance in Pseudomonas aeruginosa [J]. J Bacteriol, 2007, 189(13):4561-4568.
[21] Totten PA, Lory S. Characterization of the type a flagellin gene from Pseudomonas aeruginosa PAK [J]. J Bacteriol, 1990, 172(12):7188-7199.
[22] Choi KH, Schweizer HP. mini-Tn7 insertion in bacteria with single attTn7 sites:example Pseudomonas aeruginosa [J]. Nat Protoc, 2006, 1(1):153-161.
[23] 杨洪江, 李明春, 魏东盛, 邢来军.铜绿假单胞菌pcr2基因功能的研究[J].微生物学报, 2007, 47(5):779-784.
[24] Miller JH. Experiments in molecular genetics [M]. New York:Cold Spring Harbor Laboratory Press, 1972.
[25] Wilson K. Preparation of genomic DNA from bacteria [J]. Curr Protoc Mol Biol, 2001, 00:241-245.
[26] Gooderham WJ, Hancock RE. Regulation of virulence and antibiotic resistance by two-component regulatory systems in Pseudomonas aeruginosa [J]. Fems Microbiol Rev, 2009, 33(2):279-294.
[27] Ballesta S, Conejo MC, Garcia I, et al. Survival and resistance to imipenem of Pseudomonas aeruginosa on latex gloves [J]. J Antimicrob Chemother, 2006, 57(5):1010-1012.
[28] Holder IA. Pseudomonas immunotherapy:a historical overview [J]. Vaccine, 2004, 22(7):831-839.
[29] Deredjian A, Colinon C, Brothier E, et al. Antibiotic and metal resistance among hospital and outdoor strains of Pseudomonas aeruginosa [J]. Res Microbiol, 2011, 162(7):689-700.