铜绿假单胞菌重金属离子耐受性调查及相关机制的研究

Heavy Metal Resistance and Its Related Mechanisms in Pseudomonas aeruginosa

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。

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 Co 2＋,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.