Kinsella N, Guerry P, Cooney J, Trust T J
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
J Bacteriol. 1997 Aug;179(15):4647-53. doi: 10.1128/jb.179.15.4647-4653.1997.
The flgE gene encoding the flagellar hook protein of Campylobacter coli VC167-T1 was cloned by immunoscreening of a genomic library constructed in lambdaZAP Express. The flgE DNA sequence was 2,553 bp in length and encoded a protein with a deduced molecular mass of 90,639 Da. The sequence had significant homology to the 5' and 3' sequences of the flgE genes of Helicobacter pylori, Treponema phagedenis, and Salmonella typhimurium. Primer extension analysis indicated that the VC167 flgE gene is controlled by a sigma54 promoter. PCR analysis showed that the flgE gene size and the 5' and 3' DNA sequences were conserved among C. coli and C. jejuni strains. Southern hybridization analyses confirmed that there is considerable sequence identity among the hook genes of C. coli and C. jejuni but that there are also regions within the genes which differ. Mutants of C. coli defective in hook production were generated by allele replacement. These mutants were nonmotile and lacked flagellar filaments. Analyses of flgE mutants indicated that the carboxy terminus of FlgE is necessary for assembly of the hook structure but not for secretion of FlgE and that, unlike salmonellae, the lack of flgE expression does not result in repression of flagellin expression.
通过对构建于λZAP Express载体中的空肠弯曲菌VC167-T1基因组文库进行免疫筛选,克隆出了编码空肠弯曲菌鞭毛钩蛋白的flgE基因。flgE DNA序列长度为2553 bp,编码一种推导分子量为90639 Da的蛋白质。该序列与幽门螺杆菌、蚀疮假单胞菌和鼠伤寒沙门氏菌的flgE基因的5'和3'序列具有显著同源性。引物延伸分析表明,VC167 flgE基因受σ54启动子调控。PCR分析显示,flgE基因大小以及5'和3' DNA序列在空肠弯曲菌和空肠结肠弯曲菌菌株中是保守的。Southern杂交分析证实,空肠弯曲菌和空肠结肠弯曲菌的钩基因之间存在相当程度的序列同一性,但基因内也存在不同的区域。通过等位基因置换产生了钩产生缺陷的空肠弯曲菌突变体。这些突变体无运动性且缺乏鞭毛丝。对flgE突变体的分析表明,FlgE的羧基末端对于钩结构的组装是必需的,但对于FlgE的分泌不是必需的,并且与沙门氏菌不同,flgE表达的缺乏不会导致鞭毛蛋白表达的抑制。