Norwood D A, Sands J A
Department of Biological Sciences, Lehigh University, Bethlehem, PA 18015, USA.
Gene. 1997 Nov 12;201(1-2):159-68. doi: 10.1016/s0378-1119(97)00443-5.
Clostridium difficile is a causative agent in antibiotically induced diarrhea and pseudomembraneous colitis. The ability of strains of C. difficile to cause disease depends upon the presence of two toxin genes and their corresponding proteins, designated toxin A and toxin B. Previous studies conducted in this laboratory indicated that toxigenic strains of C. difficile possess both toxin genes, whereas non-toxigenic strains do not. Likewise, the studies showed that toxigenic and non-toxigenic strains of C. difficile differ significantly in chromosomal organization by ribotype analysis. Therefore, the chromosomal organization of a reference strain was investigated. Pulsed field gel electrophoresis was utilized to generate a physical map of the chromosome of the toxigenic Clostridium difficile strain ATCC 43594. Restriction digestions of whole chromosomes with the enzymes NruI and SacII generated consistent macrofragment profiles. NruI digestion resulted in 14 discernible bands containing 16 fragments of DNA. SacII digestions resulted in 14 discernible bands containing 15 fragments of DNA. Restriction digestions with both SacII and NruI resulted in 21 discernible bands containing 31 fragments of DNA. Probing of single and double digests with an extensive set of NruI and SacII single- and double-digest bands clarified the location of individual fragments in relation to one another, resulting in a restriction map of the chromosome. PCR-generated probes of five loci of C. difficile were used to map the location of seven genes on the chromosome. Finally, the addition of all fragments from NruI, SacII and NruI/SacII digestions resulted in an approximate chromosome size of 4.4 Mb.
艰难梭菌是抗生素诱导性腹泻和伪膜性结肠炎的病原体。艰难梭菌菌株致病的能力取决于两个毒素基因及其相应蛋白质的存在，分别称为毒素A和毒素B。本实验室先前进行的研究表明，产毒素的艰难梭菌菌株同时拥有这两个毒素基因，而非产毒素菌株则没有。同样，这些研究表明，通过核糖体分型分析，产毒素和不产毒素的艰难梭菌菌株在染色体组织上有显著差异。因此，对一株参考菌株的染色体组织进行了研究。利用脉冲场凝胶电泳生成了产毒素艰难梭菌菌株ATCC 43594染色体的物理图谱。用NruI和SacII酶对完整染色体进行限制性消化产生了一致的大片段图谱。NruI消化产生了14条可分辨的条带，包含16个DNA片段。SacII消化产生了14条可分辨的条带，包含15个DNA片段。用SacII和NruI进行限制性消化产生了21条可分辨的条带，包含31个DNA片段。用大量NruI和SacII单酶切及双酶切条带对单酶切和双酶切产物进行探针杂交，阐明了各个片段相互之间的位置，从而得到了染色体的限制性图谱。用PCR产生的艰难梭菌五个位点的探针来确定七个基因在染色体上的位置。最后，将NruI、SacII和NruI/SacII消化产生的所有片段相加，得出染色体大小约为4.4 Mb。
