Caballero Armando, Thibodeaux Brett, Marquart Mary, Traidej Mullika, O'Callaghan Richard
Department of Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.
Invest Ophthalmol Vis Sci. 2004 Feb;45(2):522-30. doi: 10.1167/iovs.03-1050.
To determine the distribution of the protease IV gene, the production of this and other proteases by multiple strains of Pseudomonas, and the virulence of a mutant specifically deficient in protease IV.
The protease IV gene was cloned, its sequence analyzed, and its chromosomal location determined by pulse-field gel electrophoresis. Three PCR reactions were used to detect the protease IV gene in 30 Pseudomonas isolates and protease production was determined by Western blot analysis, colorimetric assay, and zymography. An allelic replacement mutant deficient in the protease IV gene was analyzed for enzyme production, corneal growth, and corneal virulence.
The protease IV gene was identified in all P. aeruginosa, but none of the non-aeruginosa strains tested. The protease IV genes of strains PA103-29 and PAO1 were in a common chromosomal site and had 98.5% sequence identity with variations occurring mainly in the promoter region. The protease IV activity of the 23 wild-type P. aeruginosa strains tested varied from 2.3 to 221.5 x 10(-3) U/mg protein in the culture supernatant. Protease IV was produced by all P. aeruginosa wild-type strains. A protease IV-deficient mutant derived from strain PA103-29 had reduced virulence compared with its parent strain and unexpectedly produced alkaline protease.
The protease IV gene and its product are common to P. aeruginosa, but not to other Pseudomonas species. Protease IV activity varies among P. aeruginosa strains, and a mutant specifically deficient in this activity produced alkaline protease and had reduced corneal virulence.
确定蛋白酶IV基因的分布、多种假单胞菌菌株产生该蛋白酶及其他蛋白酶的情况，以及蛋白酶IV特异性缺陷突变体的毒力。
克隆蛋白酶IV基因，分析其序列，并通过脉冲场凝胶电泳确定其染色体定位。使用三种聚合酶链反应（PCR）检测30株假单胞菌分离株中的蛋白酶IV基因，并通过蛋白质印迹分析、比色测定法和酶谱法测定蛋白酶的产生情况。分析蛋白酶IV基因缺陷的等位基因替换突变体的酶产生、角膜生长和角膜毒力。
在所有铜绿假单胞菌中均鉴定出蛋白酶IV基因，但在所测试的非铜绿假单胞菌菌株中均未发现。菌株PA103 - 29和PAO1的蛋白酶IV基因位于共同的染色体位点，序列同一性为98.5%，变异主要发生在启动子区域。所测试的23株野生型铜绿假单胞菌菌株在培养上清液中的蛋白酶IV活性为2.3至221.5×10⁻³U/mg蛋白质。所有铜绿假单胞菌野生型菌株均产生蛋白酶IV。源自菌株PA103 - 29的蛋白酶IV缺陷突变体与其亲本菌株相比毒力降低，且意外地产生碱性蛋白酶。
蛋白酶IV基因及其产物是铜绿假单胞菌所特有的，而非其他假单胞菌属物种。蛋白酶IV活性在铜绿假单胞菌菌株之间存在差异，特异性缺乏该活性的突变体产生碱性蛋白酶且角膜毒力降低。
