Peyret J L, Bayan N, Joliff G, Gulik-Krzywicki T, Mathieu L, Schechter E, Leblon G
Laboratoire de Biologie Moléculaire des Coryné-bactéries, URA D1354 CNRS et GDR 961, Université Paris-Sud, Orsay, France.
Mol Microbiol. 1993 Jul;9(1):97-109. doi: 10.1111/j.1365-2958.1993.tb01672.x.
PS2 is one of two major proteins detected in the culture media of various Corynebacterium glutamicum strains. The coding and promoter regions of the cspB gene encoding PS2 were cloned in lambda gt11 using polyclonal antibodies raised against PS2 for screening. Expression of the cspB gene in Escherichia coli led to the production of a major anti-PS2 labelled peptide of 63,000 Da, corresponding presumably to the mature form of PS2. It was detected in the cytoplasm, periplasm and surrounding medium of E. coli. Three other slower migrating bands of 65,000 68,000 and 72,000 Da were detected. The largest one probably corresponds to the precursor form of PS2 in E. coli. Analysis of the nucleotide sequence revealed an open reading frame (ORF) of 1533 nucleotides. The deduced 510-amino-acid polypeptide had a calculated molecular mass of 55,426 Da. According to the predicted amino acid sequence, PS2 is synthesized with a N-terminal segment of 30-amino-acid residues reminiscent of eukaryotic and prokaryotic signal peptides, and a hydrophobic domain of 21 residues near the C-terminus. Although no significant homologies were found with other proteins, it appears that some characteristics and the amino acid composition of PS2 share several common features with surface-layer proteins. The cspB gene was then disrupted in C. glutamicum by gene replacement. Freeze-etching electron microscopy performed on the wild-type strain indicated that the cell wall of C. glutamicum is covered with an ordered surface of proteins (surface layer, S-layer) which is in very close contact with other cell-wall components. These structures are absent from the cspB-disrupted strain but are present after reintroduction of the cspB gene on a plasmid into this mutant. Thus we demonstrate that the S-layer protein is the product of the cspB gene.
PS2是在多种谷氨酸棒杆菌菌株的培养基中检测到的两种主要蛋白质之一。利用针对PS2产生的多克隆抗体进行筛选，将编码PS2的cspB基因的编码区和启动子区克隆到λgt11中。cspB基因在大肠杆菌中的表达导致产生了一种主要的63,000 Da抗PS2标记肽，推测它对应于PS2的成熟形式。在大肠杆菌的细胞质、周质和周围培养基中都检测到了它。还检测到另外三条迁移较慢的条带，分子量分别为65,000、68,000和72,000 Da。最大的那条条带可能对应于大肠杆菌中PS2的前体形式。对核苷酸序列的分析揭示了一个1533个核苷酸的开放阅读框（ORF）。推导的510个氨基酸的多肽计算分子量为55,426 Da。根据预测的氨基酸序列，PS2合成时带有一个30个氨基酸残基的N端片段，类似于真核和原核信号肽，以及一个靠近C端的21个残基的疏水结构域。虽然未发现与其他蛋白质有明显的同源性，但PS2的一些特征和氨基酸组成似乎与表层蛋白有几个共同特征。然后通过基因替换在谷氨酸棒杆菌中破坏了cspB基因。对野生型菌株进行的冷冻蚀刻电子显微镜观察表明，谷氨酸棒杆菌的细胞壁覆盖着一层有序的蛋白质表面（表层，S层），它与其他细胞壁成分紧密接触。这些结构在cspB基因破坏的菌株中不存在，但在将cspB基因重新导入该突变体的质粒后又出现了。因此，我们证明S层蛋白是cspB基因的产物。
