假单胞菌Tw224抗砷基因簇的功能研究

长期在砷污染环境生存的细菌逐渐演化出抗砷机制,旨在评价分离自湖南铅锌矿区污染土壤的Pseudomonas sp.Tw224抗砷能力和抗重金属谱;揭示该菌的抗砷基因/基因簇的功能.在砷或重金属存在条件下测量细菌的生长情况,对该菌的抗砷能力和抗重金属谱进行评价;通过基因组框架图分析该菌的抗砷基因/基因簇;采用代谢工程法构建...

Functions of Arsenic-resistant Gene Cluster in Pseudomonas sp.Tw224

The mechanisms of arsenic resistance have been gradually evolved in bacteria living in arsenic-contaminated environment.The purpose of this paper is to evaluate the resistance of Pseudomonas sp.Tw224 isolated from contaminated soil of lead-zinc mine in Hunan province to arsenic and its heavy metal-resistant spectrum,and to reveal its function of arsenic-resistant gene/gene clusters.The growth of the bacteria was measured in the presence of arsenic or heavy metals,and their resistances to arsenic and heavy metal-resistant spectrum were evaluated.The arsenic-resistant gene/gene clusters of the bacteria were analyzed by a draft sequence of genome.And finally the gene cluster-deleted mutants were constructed by metabolic engineering method to reveal the functions of the gene clusters.The delayed period prolonged,and the maximum bacterial concentration reduced in the presence of arsenic.The maximum As⁵⁺-resistant and As³⁺-resistant concentrations of Tw224 were 600 and 20 mmol/L,respectively.Moreover,Tw224 was also resistant to Cu,Ni,Zn,Cd,Cr,Ag and Hg at varying level.A total of 20 genes(1 arsH,4 arsC,2 arsB,2 ACR(3)and 11 arsR)related to arsenic resistance were found in the genome of the strain,and arsH-arsC2-arsB-arsR(ars1)and arsH-arsB-arsC2(ars2)existed in the form of gene clusters with the length of 2.8 kb and 2.2 kb,respectively.The ars1-inactivated Pseudomonas sp.QSA1,ars2-inactivated Pseudomonas sp.QSA2,and ars1-ars2-inactivated Pseudomonas sp.QSARS were successfully constructed by homologous recombinant double exchange method.In the absence of arsenic,the growths of the three mutants were not affected,and the minimum inhibitory concentrations of arsenic to QSA1 and QSA2 reduced to 4% and 80% of that of wild one,respectively.The arsenic reduction capacity of the two mutants was about 9.8% and 53.8% of that of wild one at 48 h,respectively.Pseudomonas sp.QSARS,a double gene clusters-deleted mutant,almost completely lost its resistance to arsenic and arsenic reduction ability.In conclusion,Tw224 had strong resistance to arsenic and a broad spectrum of resistance to heavy metals.There are a large number of heavy metal-resistant related genes in its genome,among which there are two arsenic-resistant related gene clusters.The ars1 gene cluster played a major role and the ars2 played an auxiliary role in arsenic-resistant process.