CLONING AND CHARACTERIZATION OF THE KATB GENE OF PSEUDOMONAS-AERUGINOSA ENCODING A HYDROGEN PEROXIDE-INDUCIBLE CATALASE - PURIFICATION OF KATB, CELLULAR-LOCALIZATION, AND DEMONSTRATION THAT IT IS ESSENTIAL FOR OPTIMAL RESISTANCE TO HYDROGEN-PEROXIDE
CLONING AND CHARACTERIZATION OF THE KATB GENE OF PSEUDOMONAS-AERUGINOSA ENCODING A HYDROGEN PEROXIDE-INDUCIBLE CATALASE - PURIFICATION OF KATB, CELLULAR-LOCALIZATION, AND DEMONSTRATION THAT IT IS ESSENTIAL FOR OPTIMAL RESISTANCE TO HYDROGEN-PEROXIDE
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DOI：
10.1128/jb.177.22.6536-6544.1995
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发表时间：
1995-11-01
影响因子：
3.2
通讯作者：
HASSETT, DJ
中科院分区：
文献类型：
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作者：
BROWN, SM;HOWELL, ML;HASSETT, DJ
铜绿假单胞菌是一种专性需氧菌，在环境中几乎无处不在。在有氧呼吸过程中，分子氧的代谢可导致活性氧中间体的产生，其中之一包括过氧化氢。为了抵消这种化合物的潜在毒性作用，铜绿假单胞菌拥有两种含血红素的过氧化氢酶，可以解毒过氧化氢。本研究克隆了铜绿假单胞菌过氧化氢酶基因katB。该基因被克隆在5.4-kb的EcoRI片段上，由1，539 bp组成，编码513个氨基酸。铜绿假单胞菌katB的氨基酸序列与来自相关物种假单胞菌的过氧化氢酶的氨基酸序列相似，具有65%的同一性。katB基因被定位于铜绿假单胞菌染色体的71- 75-min区域，具有编码锰和铁超氧化物歧化酶的sodA和sodB基因的相同区域。将其克隆到大肠杆菌过氧化氢酶缺陷型突变株（UM 255）中，表达了重组铜绿假单胞菌KatB（229 U/mg），并使该菌株对过氧化氢的抗性几乎等同于野生型大肠杆菌菌株（HB 101）。KatB蛋白被纯化至均一性，并被确定为类似于228 kDa的四聚体，有趣的是，KatB在正常铜绿假单胞菌生长周期中不产生，并且过氧化氢酶活性在非粘液样中比在粘液样、产生藻酸盐的生物体中更大。当暴露于过氧化氢和更大程度上的百草枯时，总过氧化氢酶活性分别升高7至16倍。此外，KatB活性的增加导致对过氧化氢的抗性显著增加，KatB定位于细胞质，而KatA，“管家"酶，在细胞质和周质提取物中均被检测到，铜绿假单胞菌katB突变体对过氧化氢的敏感性比野生型细菌高50%，表明KatB对于铜绿假单胞菌对外源性过氧化氢的最佳抗性是必需的。
Pseudomonas aeruginosa is an obligate aerobe that is virtually ubiquitous in the environment. During aerobic respiration, the metabolism of dioxygen can lead to the production of reactive oxygen intermediates, one of which includes hydrogen peroxide. To counteract the potentially toxic effects of this compound, P. aeruginosa possesses two heme-containing catalases which detoxify hydrogen peroxide. In this study, we have cloned katB, encoding one catalase gene of P. aeruginosa. The gene was cloned on a 5.4-kb EcoRI fragment and is composed of 1,539 bp, encoding 513 amino acids, The amino acid sequence of the P, aeruginosa katB was similar to 65% identical to that of a catalase from a related species, Pseudomonas syringae, The katB gene was mapped to the 71- to 75-min region of the P. aeruginosa chromosome, the identical region which harbors both sodA and sodB genes encoding both manganese and iron superoxide dismutases. When cloned into a catalase deficient mutant of Escherichia coli (UM255), the recombinant P. aeruginosa KatB was expressed (229 U/mg) and afforded this strain resistance to hydrogen peroxide nearly equivalent to that of the wild-type E, coli strain (HB101), The KatB protein was purified to homogeneity and determined to be a tetramer of similar to 228 kDa, which was in good agreement with the predicted protein size derived from the translated katB gene, Interestingly, KatB was not produced during the normal P, aeruginosa growth cycle, and catalase activity was greater in nonmucoid than in mucoid, alginate-producing organisms. When exposed to hydrogen peroxide and, to a greater extent, paraquat, total catalase activity was elevated 7- to 16-fold, respectively, In addition, an increase in KatB activity caused a marked increase in resistance to hydrogen peroxide, KatB was localized to the cytoplasm, while KatA, the ''housekeeping'' enzyme, was detected in both cytoplasmic and periplasmic extracts, A P, aeruginosa katB mutant demonstrated 50% greater sensitivity to hydrogen peroxide than wild-type bacteria, suggesting that KatB is essential for optimal resistance of P. aeruginosa to exogenous hydrogen peroxide.