Nishibuchi M, Kaper J B
J Bacteriol. 1985 May;162(2):558-64. doi: 10.1128/jb.162.2.558-564.1985.
The gene encoding the thermostable direct hemolysin of Vibrio parahaemolyticus was characterized. This gene (designated tdh) was subcloned into pBR322 in Escherichia coli, and the functional tdh gene was localized to a 1.3-kilobase HindIII fragment. This fragment was sequenced, and the structural gene was found to encode a mature protein of 165 amino acid residues. The mature protein sequence was preceded by a putative signal peptide sequence of 24 amino acids. A putative tdh promoter, determined by its similarity to concensus sequences, was not functional in E. coli. However, a promoter that was functional in E. coli was shown to exist further upstream by use of a promoter probe plasmid. A 5.7-kilobase SalI fragment containing the structural gene and both potential promoters was cloned into a broad-host-range plasmid and mobilized into a Kanagawa phenomenon-negative V. parahaemolyticus strain. In contrast to E. coli, where the hemolysin was detected only in cell lysates, introduction of the cloned gene into V. parahaemolyticus resulted in the production of extracellular hemolysin.
对副溶血性弧菌耐热直接溶血素的编码基因进行了表征。该基因（命名为tdh）被亚克隆到大肠杆菌的pBR322中，功能性tdh基因定位于一个1.3千碱基的HindIII片段。对该片段进行了测序，发现结构基因编码一个由165个氨基酸残基组成的成熟蛋白。成熟蛋白序列之前有一个由24个氨基酸组成的推定信号肽序列。通过与共有序列的相似性确定的推定tdh启动子在大肠杆菌中无功能。然而，通过使用启动子探针质粒表明，在更上游存在一个在大肠杆菌中有功能的启动子。将一个包含结构基因和两个潜在启动子的5.7千碱基SalI片段克隆到一个广宿主范围的质粒中，并转入到一个神奈川现象阴性的副溶血性弧菌菌株中。与仅在细胞裂解物中检测到溶血素的大肠杆菌不同，将克隆基因导入副溶血性弧菌导致细胞外溶血素的产生。
