Mutagenesis of Burkholderia pseudomallei with Tn5-OT182: Isolation of motility mutants and molecular characterization of the flagellin structural gene
Mutagenesis of Burkholderia pseudomallei with Tn5-OT182: Isolation of motility mutants and molecular characterization of the flagellin structural gene
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DOI：
10.1128/jb.179.7.2116-2125.1997
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发表时间：
1997-04-01
影响因子：
3.2
通讯作者：
Woods, DE
中科院分区：
文献类型：
--
作者：
DeShazer, D;Brett, PJ;Woods, DE
鼻疽伯克霍尔德氏菌是热带地区，特别是东南亚和澳大利亚北部的人类和动物病原体，目前对该生物的遗传学和分子生物学知之甚少，在本报告中，我们描述了类鼻疽伯克霍尔德氏菌与转座子Tn5-OT182的诱变。每个初始供体细胞以4.6 x 10(-4)的频率获得B.pseudomallei 1026b转座子突变体，并将转座子随机插入染色体中，我们使用Tn5-OT182鉴定鞭毛蛋白结构基因fliC。我们筛选了 3,500 个转座子突变体并鉴定了 28 个运动突变体。 Tn5-OT182 整合到 19 个独特的基因位点，编码与大肠杆菌和鼠伤寒沙门氏菌鞭毛和趋化蛋白同源的蛋白质。两个突变体 MM35 和 MM36 在 fliC 中包含 Tn5-OT182 整合。我们对fliC进行了克隆和测序，并用其反式互补MM35和MM36，通过引物延伸分析鉴定了fliC转录起始位点和sigma(F)样启动子，我们观察到细菌生长过程中两个不同的fliC-lacZ转录融合体的表达存在显着差异，表明fliC中存在潜在的基因内转录终止子。在糖尿病大鼠或叙利亚仓鼠中，1026b 的毒力与 MM36 的毒力没有显着差异，这表明鞭毛和/或运动性可能不是这些拟鼻疽伯克氏菌感染动物模型中的毒力决定因素。基于多种细菌物种的鞭毛蛋白的系统发育分析支持最近拟鼻疽伯克氏菌从假单胞菌属转移到 伯克霍尔德氏菌。
Burkholderia pseudomallei is a human and animal pathogen in tropical regions, especially Southeast Asia and northern Australia, Currently little is known about the genetics and molecular biology of this organism, In this report, we describe the mutagenesis of B. pseudomallei with the transposon Tn5-OT182. B. pseudomallei 1026b transposon mutants were obtained at a frequency of 4.6 x 10(-4) per initial donor cell, and the transposon inserted randomly into the chromosome, We used Tn5-OT182 to identify the flagellin structural gene, fliC. We screened 3,500 transposon mutants and identified 28 motility mutants. Tn5-OT182 integrated into 19 unique genetic loci encoding proteins with homology to Escherichia coli and Salmonella typhimurium flagellar and chemotaxis proteins, Two mutants, MM35 and MM36, contained Tn5-OT182 integrations in fliC. We cloned and sequenced fliC and used it to complement MM35 and MM36 in trans, The fliC transcriptional start site and a sigma(F)-like promoter were identified by primer extension analysis, We observed a significant difference in the expression of two distinct fliC-lacZ transcriptional fusions during bacterial growth, suggesting the presence of a latent intragenic transcriptional terminator in fliC. There was no significant difference in the virulence of 1026b compared to that of MM36 in diabetic rats or Syrian hamsters, suggesting that flagella and/or motility are probably not virulence determinants in these animal models of B. pseudomallei infection, A phylogenetic analysis based on the flagellins from a variety of bacterial species supported the recent transfer of B. pseudomallei from the genus Pseudomonas to Burkholderia.