Fauconnier A, Veithen A, Gueirard P, Antoine R, Wacheul L, Locht C, Bollen A, Godfroid E
Service de Génétique Appliquée, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, Belgium.
Int J Med Microbiol. 2001 Mar;290(8):693-705. doi: 10.1016/S1438-4221(01)80009-6.
Multiple sequence comparisons of proteins of the LcrD/FlbF family allowed the design of primers that specifically amplify sequences coding for type III secretion components. Amplification of Bordetella pertussis DNA with these primers yielded a fragment that was further used as a probe for screening a genomic library. The nucleotide sequence of a positive clone revealed a 2100-bp gene, called bcrD, which specifies a 75-kDa polypeptide homologous to the Yersinia LcrD protein. Chromosome walking allowed the characterization of a 35-kb DNA segment that contains the entire locus and flanking housekeeping genes. The B. pertussis type III secretion locus consists of more than 30 open reading frames (ORFs), most of which are identical to annotated genes of Bordetella spp and share similarities with known type III secretion genes of related bacteria. In order to assess the function of this locus, we engineered a bcrD null mutant. However, none of the tested phenotypes, such as protein secretion, cellular invasion, cytotoxicity or mouse lung colonization, differentiated the mutant from its parental strain. Studies of bcrD and bscN expressions indicated that, under our experimental conditions, these genes are not expressed in vitro. Restriction analyses on pulsed-field gel electrophoresis allowed the type III locus mapping at coordinate position 1,590 kb on the Tohama I strain chromosome.
对LcrD/FlbF家族蛋白质进行多序列比较后，设计出了能特异性扩增编码III型分泌成分序列的引物。用这些引物扩增百日咳博德特氏菌DNA，得到一个片段，该片段进一步用作筛选基因组文库的探针。一个阳性克隆的核苷酸序列揭示了一个2100 bp的基因，称为bcrD，它编码一种与耶尔森氏菌LcrD蛋白同源的75 kDa多肽。染色体步移法对一个35 kb的DNA片段进行了表征，该片段包含整个基因座和侧翼管家基因。百日咳博德特氏菌III型分泌基因座由30多个开放阅读框（ORF）组成，其中大多数与博德特氏菌属的注释基因相同，并与相关细菌已知的III型分泌基因具有相似性。为了评估该基因座的功能，我们构建了一个bcrD缺失突变体。然而，在蛋白质分泌、细胞侵袭、细胞毒性或小鼠肺部定殖等所有测试表型中，突变体与其亲本菌株均无差异。对bcrD和bscN表达的研究表明，在我们的实验条件下，这些基因在体外不表达。通过脉冲场凝胶电泳进行的限制性分析，将III型基因座定位在Tohama I菌株染色体上的1590 kb坐标位置。
