Ma J F, Ochsner U A, Klotz M G, Nanayakkara V K, Howell M L, Johnson Z, Posey J E, Vasil M L, Monaco J J, Hassett D J
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524, USA.
J Bacteriol. 1999 Jun;181(12):3730-42. doi: 10.1128/JB.181.12.3730-3742.1999.
We have cloned a 3.6-kb genomic DNA fragment from Pseudomonas aeruginosa harboring the rpoA, rplQ, katA, and bfrA genes. These loci are predicted to encode, respectively, (i) the alpha subunit of RNA polymerase; (ii) the L17 ribosomal protein; (iii) the major catalase, KatA; and (iv) one of two iron storage proteins called bacterioferritin A (BfrA; cytochrome b1 or b557). Our goal was to determine the contributions of KatA and BfrA to the resistance of P. aeruginosa to hydrogen peroxide (H2O2). When provided on a multicopy plasmid, the P. aeruginosa katA gene complemented a catalase-deficient strain of Escherichia coli. The katA gene was found to contain two translational start codons encoding a heteromultimer of approximately 160 to 170 kDa and having an apparent Km for H2O2 of 44.7 mM. Isogenic katA and bfrA mutants were hypersusceptible to H2O2, while a katA bfrA double mutant demonstrated the greatest sensitivity. The katA and katA bfrA mutants possessed no detectable catalase activity. Interestingly, a bfrA mutant expressed only approximately 47% the KatA activity of wild-type organisms, despite possessing wild-type katA transcription and translation. Plasmids harboring bfrA genes encoding BfrA altered at critical amino acids essential for ferroxidase activity could not restore wild-type catalase activity in the bfrA mutant. RNase protection assays revealed that katA and bfrA are on different transcripts, the levels of which are increased by both iron and H2O2. Mass spectrometry analysis of whole cells revealed no significant difference in total cellular iron levels in the bfrA, katA, and katA bfrA mutants relative to wild-type bacteria. Our results suggest that P. aeruginosa BfrA may be required as one source of iron for the heme prosthetic group of KatA and thus for protection against H2O2.
我们从铜绿假单胞菌中克隆了一个3.6 kb的基因组DNA片段，该片段含有rpoA、rplQ、katA和bfrA基因。预计这些基因座分别编码：（i）RNA聚合酶的α亚基；（ii）L17核糖体蛋白；（iii）主要过氧化氢酶KatA；（iv）两种铁储存蛋白之一，称为细菌铁蛋白A（BfrA；细胞色素b1或b557）。我们的目标是确定KatA和BfrA对铜绿假单胞菌对过氧化氢（H2O2）抗性的贡献。当在多拷贝质粒上提供时，铜绿假单胞菌katA基因可互补大肠杆菌的过氧化氢酶缺陷菌株。发现katA基因包含两个翻译起始密码子，编码一个约160至170 kDa的异源多聚体，其对H2O2的表观Km为44.7 mM。同基因的katA和bfrA突变体对H2O2高度敏感，而katA bfrA双突变体表现出最大的敏感性。katA和katA bfrA突变体没有可检测到的过氧化氢酶活性。有趣的是，尽管bfrA突变体具有野生型katA转录和翻译，但它表达的KatA活性仅约为野生型生物体的47%。携带编码在铁氧化酶活性必需的关键氨基酸处发生改变的BfrA的bfrA基因的质粒不能恢复bfrA突变体中的野生型过氧化氢酶活性。核糖核酸酶保护分析表明，katA和bfrA位于不同的转录本上，其水平在铁和H2O2的作用下均会增加。全细胞的质谱分析显示，相对于野生型细菌，bfrA、katA和katA bfrA突变体的总细胞铁水平没有显著差异。我们的结果表明，铜绿假单胞菌BfrA可能是KatA血红素辅基的铁来源之一，因此对于抵抗H2O2是必需的。
