Calabi E, Ward S, Wren B, Paxton T, Panico M, Morris H, Dell A, Dougan G, Fairweather N
Department of Biology and Biochemistry, Imperial College, South Kensington, London SW7 2AY, UK.
Mol Microbiol. 2001 Jun;40(5):1187-99. doi: 10.1046/j.1365-2958.2001.02461.x.
Many bacteria express a surface-exposed proteinaceous layer, termed the S-layer, which forms a regular two-dimensional array visible by electron microscopy. Clostridium difficile is unusual in expressing two S-layer proteins (SLPs), which are of varying size in a number of strains. In an approach combining molecular biology with mass spectrometric sequencing strategies, we have identified the structural gene (slpA) for the S-layer from three strains of C. difficile. Both proteins are derived from a common precursor, and processing involves the removal of a signal peptide and a second cleavage to release the two mature SLPs. To our knowledge, this is the first example in which two SLPs have been shown to derive from a single gene product through post-translational processing, rather than from the expression of separate genes. The higher molecular weight (MW) SLP is highly conserved among the three strains, whereas the lower MW SLP shows considerable sequence diversity, reflecting the results from Western blotting. The high-MW SLP shows weak homology to N-acetyl muramoyl-L-alanine amidase from Bacillus subtilis, and both the native SLP from C. difficile and a recombinant protein expressed in Escherichia coli were found to display amidase activity by zymography. The high-MW SLPs showed evidence of glycosylation, whereas the lower MW proteins did not. A family of genes with sequence homology to the amidase domain of the high-MW SLP was identified in the C. difficile strain 630 genome, some of which are located in the same region of the genome as slpA and were shown by reverse transcription-polymerase chain reaction (RT-PCR) analysis to be transcribed.
许多细菌表达一种表面暴露的蛋白质层，称为S层，通过电子显微镜可见其形成规则的二维阵列。艰难梭菌不同寻常之处在于它表达两种S层蛋白（SLP），在许多菌株中这两种蛋白大小各异。通过将分子生物学与质谱测序策略相结合的方法，我们从三株艰难梭菌中鉴定出了S层的结构基因（slpA）。这两种蛋白均来源于一个共同的前体，加工过程包括去除一个信号肽和第二次切割以释放两种成熟的SLP。据我们所知，这是首次证明两种SLP是通过翻译后加工从单一基因产物衍生而来，而非来自单独基因的表达。高分子量（MW）的SLP在这三株菌株中高度保守，而低分子量的SLP则表现出相当大的序列多样性，这与蛋白质印迹法的结果相符。高分子量的SLP与枯草芽孢杆菌的N - 乙酰胞壁酰 - L - 丙氨酸酰胺酶有弱同源性，并且通过酶谱分析发现，艰难梭菌的天然SLP和在大肠杆菌中表达的重组蛋白均具有酰胺酶活性。高分子量的SLP有糖基化的证据，而低分子量的蛋白质则没有。在艰难梭菌菌株630基因组中鉴定出了一个与高分子量SLP的酰胺酶结构域具有序列同源性的基因家族，其中一些基因位于与slpA相同的基因组区域，并且通过逆转录 - 聚合酶链反应（RT - PCR）分析表明它们是可转录的。
