Insertional inactivation of an Escherichia coli urease gene by IS3411.
Insertional inactivation of an Escherichia coli urease gene by IS3411.
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IS3411 插入使大肠杆菌脲酶基因失活。
DOI：
10.1128/jb.174.3.883-888.1992
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发表时间：
1992
影响因子：
3.2
通讯作者：
Gutman,DM
中科院分区：
文献类型：
--
作者：
Collins,CM;Gutman,DM
溶脲大肠埃希菌是一种罕见的临床分离株，可在各种肠外感染部位发现，主要是尿路。在这些分离株中，大约25%的菌株尿素酶阳性表型不稳定，并且尿素酶阴性分离物的产生频率很高。我们研究了其中一个分离株的尿素酶阳性到阴性转化的性质，命名为E.Coli1021。用从7个独立的E.Coli1021尿素酶阴性分离物中提取的基因组DNA进行Southern杂交实验，发现在尿素酶基因簇中存在1.3kb的DNA插入。从一个尿素酶阴性分离物中克隆出含有DNA插入片段的DNA片段。该克隆的DNA片段能够与接合质粒pOX38形成共整合，表明DNA插入是一个转座元件。DNA序列分析表明，该插入片段为ureG中的IS3411元件。在插入元件两侧发现了3个碱基对的靶复制(CTG)。通过聚合酶链式反应从其他6个尿素酶阴性分离物中扩增出跨越插入位点的DNA。扩增片段的DNA序列分析表明，在所检测的所有尿素酶阴性分离物中，都在同一位置发现了IS3411元件。这些数据表明，在E.Coli1021中，IS3411在CTG位点高频转位到ureG，扰乱了该基因，消除了尿素酶的活性。
Ureolytic Escherichia coli are unusual clinical isolates that are found at various extraintestinal sites of infection, predominantly the urinary tract. The urease-positive phenotype is unstable in approximately 25% of these isolates, and urease-negative segregants are produced at a high frequency. We have studied the nature of the urease-positive-to-negative transition in one of these isolates, designated E. coli 1021. Southern hybridization experiments with genomic DNA extracted from seven independent E. coli 1021 urease-negative segregants revealed the presence of a 1.3-kb DNA insertion in the urease gene cluster. A DNA fragment containing the DNA insertion was cloned from one of the urease-negative segregants. This cloned DNA fragment was capable of mediating cointegrate formation with the conjugative plasmid pOX38, suggesting that the DNA insertion was a transposable element. The insert was identified as an IS3411 element in ureG by DNA sequence analysis. A 3-bp target duplication (CTG) flanking the insertion element was found. DNA spanning the insertion site was amplified from the other six urease-negative segregants by using the polymerase chain reaction. The DNA sequence of the amplified fragments indicated that an IS3411 element was found in an identical site in all urease-negative segregants examined. These data suggest that in E. coli 1021, IS3411 transposes at a high frequency into ureG at a CTG site, disrupting this gene and eliminating urease activity.