McDevitt D, Francois P, Vaudaux P, Foster T J
Microbiology Department, Moyne Institute, Trinity College, Dublin, Ireland.
Mol Microbiol. 1994 Jan;11(2):237-48. doi: 10.1111/j.1365-2958.1994.tb00304.x.
Four mutants of Staphylococcus aureus strain Newman that were defective in the fibrinogen receptor (clumping factor) were isolated by transposon Tn917 mutagenesis. Southern hybridization analysis of the mutants identified transposon-host DNA junction fragments, one of which was cloned and used to generate a probe to identify and clone the wild-type clumping factor locus (clfA). The mutants failed to form clumps in soluble fibrinogen and adhered poorly to polymethylmethacrylate (PMMA) coverslips coated with fibrinogen. A single copy of the clfA gene, when introduced into the chromosome of the mutant strains, fully complemented the clumping deficiency of these strains and restored the ability of these mutants to adhere to fibrinogen-coated PMMA. In addition, the cloned clfA gene on a shuttle plasmid allowed the weakly clumping strain 8325-4 to form clumps with the same avidity as the wild-type strain Newman and also significantly enhanced the adherence of 8325-4 strains. Thus the formation of clumps in soluble fibrinogen correlated with adherence of bacteria to solid-phase fibrinogen. The clfA gene encodes a fibrinogen-binding protein with an apparent molecular mass of c. 130 kDa. The amino acid sequence of the protein was deduced from the DNA sequence; it was predicted that a 896 residue protein (molecular mass 92 kDa) would be expressed. The putative ClfA protein has features that suggest that it is associated with the cell surface. Furthermore it contains a novel 308 residue region comprising dipeptide repeats predominantly of Asp and Ser ending 28 residues upstream from the LPXTG motif common to wall-associated proteins. Significant homology was found between the ClfA protein and the fibronectin-binding proteins of S. aureus, particularly in the N- and C-termini.
通过转座子Tn917诱变，分离出了金黄色葡萄球菌纽曼菌株的4个纤维蛋白原受体（聚集因子）缺陷型突变体。对这些突变体进行的Southern杂交分析鉴定出转座子 - 宿主DNA连接片段，其中一个片段被克隆，并用于制备探针以鉴定和克隆野生型聚集因子基因座（clfA）。这些突变体在可溶性纤维蛋白原中无法形成菌团，并且在涂有纤维蛋白原的聚甲基丙烯酸甲酯（PMMA）盖玻片上的黏附能力很差。当将单个拷贝的clfA基因导入突变菌株的染色体时，可完全弥补这些菌株的聚集缺陷，并恢复这些突变体黏附于纤维蛋白原包被的PMMA的能力。此外，穿梭质粒上克隆的clfA基因使弱聚集菌株8325 - 4能够以与野生型菌株纽曼相同的亲和力形成菌团，并且还显著增强了8325 - 4菌株的黏附能力。因此，在可溶性纤维蛋白原中形成菌团与细菌对固相纤维蛋白原的黏附相关。clfA基因编码一种表观分子量约为130 kDa的纤维蛋白原结合蛋白。该蛋白的氨基酸序列由DNA序列推导得出；预计将表达一种896个残基的蛋白（分子量92 kDa）。推测的ClfA蛋白具有表明其与细胞表面相关的特征。此外，它包含一个新的308个残基的区域，该区域由主要为天冬氨酸和丝氨酸的二肽重复序列组成，在与细胞壁相关蛋白共有的LPXTG基序上游28个残基处结束。ClfA蛋白与金黄色葡萄球菌的纤连蛋白结合蛋白之间存在显著同源性，特别是在N端和C端。
