Craig-Mylius K A, Weiss A A
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, 231 Bethesda Ave., Cincinnati, OH 45267-0524, USA.
FEMS Microbiol Lett. 1999 Oct 15;179(2):479-84. doi: 10.1111/j.1574-6968.1999.tb08766.x.
The nine ptl genes (A-I) are required for efficient secretion of pertussis toxin past the outer membrane. Mutations were made in ptlA-H by filling in unique restriction sites, generating in-frame deletions, or inserting a FLAG epitope tag. The mutations were cloned into a suicide shuttle plasmid containing the ptxptl operon and introduced into the adenylate cyclase locus of the chromosome of a Bordetella pertussis strain deleted for ptx. The wild-type ptxptl operon restored pertussis toxin expression and secretion. The ptl mutant constructs also restored expression of periplasmic pertussis toxin to the ptx deletion strain but the mutants had a statistically significant decrease in secretion of pertussis toxin of between 5- to 35-fold, suggesting all of the ptl genes must be intact for efficient pertussis toxin secretion. The mutations were also introduced into the adenylate cyclase locus of a wild-type ptxptl strain, resulting in a ptl diploid strain. The PtlC, PtlD, PtlE, PtlF, PtlG and PtlH mutants exerted dominance over the wild-type allele.
九个ptl基因（A - I）是百日咳毒素有效分泌穿过外膜所必需的。通过填补独特的限制性酶切位点、产生框内缺失或插入FLAG表位标签，对ptlA - H进行了突变。这些突变被克隆到一个含有ptxptl操纵子的自杀穿梭质粒中，并导入缺失ptx的百日咳博德特氏菌菌株染色体的腺苷酸环化酶基因座。野生型ptxptl操纵子恢复了百日咳毒素的表达和分泌。ptl突变体构建体也使周质百日咳毒素在ptx缺失菌株中恢复表达，但突变体的百日咳毒素分泌在统计学上显著降低了5至35倍，这表明所有ptl基因必须完整才能实现有效的百日咳毒素分泌。这些突变也被导入野生型ptxptl菌株的腺苷酸环化酶基因座，产生了一个ptl二倍体菌株。PtlC、PtlD、PtlE、PtlF、PtlG和PtlH突变体对野生型等位基因表现出显性作用。
