Uchiyama Hirofumi, Hashidoko Yasuyuki, Kuriyama Yuhki, Tahara Satoshi
Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan.
Biosci Biotechnol Biochem. 2008 Jan;72(1):116-23. doi: 10.1271/bbb.70496. Epub 2008 Jan 7.
A 7.1-kbp DNA fragment isolated from a wild strain of Klebsiella oxytoca was sequenced, leading to the identification of 10 open-reading frames (ORFs), including a 504-bp Pad gene. The Pad gene of the Gram-negative bacterium was subsequently expressed in Escherichia coli as a chimeric Pad. The deduced amino acid (AA) sequence of the Pad gene from wild-type K. oxytoca showed approximately 50% homology to those of other bacterial PADs from Gram-positive bacilli plus a coccus. These data and a genomic library search of some gamma-proteobacteria, including E. coli and Vibrio sp., indicated that PAD of K. oxytoca is a member of the bacterial PAD family characteristic of Gram-negative bacteria. Using Pad-specific PCR primers designed from the Gram-negative bacterial Pad of K. oxytoca, Pad genes of two further strains of K. oxytoca, another wild isolate and JCM 1665 and two PAD-positive Enterobacter spp. were successfully amplified for specific Pad detection.
从产酸克雷伯菌野生菌株中分离出一个7.1千碱基对的DNA片段并进行测序，从而鉴定出10个开放阅读框（ORF），其中包括一个504碱基对的Pad基因。随后，该革兰氏阴性菌的Pad基因在大肠杆菌中作为嵌合Pad表达。野生型产酸克雷伯菌Pad基因推导的氨基酸（AA）序列与来自革兰氏阳性杆菌和球菌的其他细菌PAD的序列显示出约50%的同源性。这些数据以及对包括大肠杆菌和弧菌属在内的一些γ-变形菌的基因组文库搜索表明，产酸克雷伯菌的PAD是革兰氏阴性细菌特有的细菌PAD家族的一员。使用根据产酸克雷伯菌革兰氏阴性细菌Pad设计的Pad特异性PCR引物，成功扩增出另外两株产酸克雷伯菌、另一野生分离株和JCM 1665以及两株PAD阳性肠杆菌属细菌的Pad基因，用于特异性Pad检测。
