Hendrixson D R, Akerley B J, DiRita V J
Unit for Laboratory Animal Medicine, and Department of Microbiology and Immunology, 5641 Medical Science II, Box 0620, University of Michigan Medical School, Ann Arbor, MI 48109-0620, USA.
Mol Microbiol. 2001 Apr;40(1):214-24. doi: 10.1046/j.1365-2958.2001.02376.x.
Campylobacter jejuni constitutes the leading cause of bacterial gastroenteritis in the United States and a major cause of diarrhoea worldwide. Little is known about virulence mechanisms in this organism because of the scarcity of suitable genetic tools. We have developed an efficient system of in vitro transposon mutagenesis using a mariner-based transposon and purified mariner transposase. Through in vitro transposition of C. jejuni chromosomal DNA followed by natural transformation of the transposed DNA, large random transposon mutant libraries consisting of approximately 16 000 individual mutants were generated. The first genetic screen of C. jejuni using a transposon-generated mutant library identified 28 mutants defective for flagellar motility, one of the few known virulence determinants of this pathogen. We developed a second genetic system, which allows for the construction of defined chromosomal deletions in C. jejuni, and demonstrated the requirement of sigma28 and sigma54 for motility. In addition, we show that sigma28 is involved in the transcription of flaA and that sigma54 is required for transcription of three other flagellar genes, flaB and flgDE. We also identified two previously uncharacterized genes required for motility encoding proteins that we call CetA and CetB, which mediate energy taxis responses. Through our analysis of the Cet proteins, we propose a unique mechanism for sensing energy levels and mediating energy taxis in C. jejuni.
空肠弯曲菌是美国细菌性肠胃炎的主要病因，也是全球腹泻的主要病因。由于缺乏合适的遗传工具，人们对这种生物体的致病机制知之甚少。我们利用基于水手座的转座子和纯化的水手座转座酶，开发了一种高效的体外转座子诱变系统。通过对空肠弯曲菌染色体DNA进行体外转座，然后对转座后的DNA进行自然转化，产生了由大约16000个单个突变体组成的大型随机转座子突变体文库。利用转座子产生的突变体文库对空肠弯曲菌进行的首次遗传筛选，鉴定出28个鞭毛运动缺陷的突变体，鞭毛运动是这种病原体为数不多的已知致病决定因素之一。我们开发了第二个遗传系统，该系统允许在空肠弯曲菌中构建特定的染色体缺失，并证明了sigma28和sigma54对运动的必要性。此外，我们表明sigma28参与flaA的转录，而sigma54是其他三个鞭毛基因flaB和flgDE转录所必需的。我们还鉴定出两个以前未被表征的运动所需基因，它们编码我们称为CetA和CetB的蛋白质，这些蛋白质介导能量趋化反应。通过对Cet蛋白的分析，我们提出了一种在空肠弯曲菌中感知能量水平和介导能量趋化的独特机制。
