Chitinase Determinants of 'Vibrio vulnificus': Gene Cloning and Applications of a Chitinase Probe

摘要

To initiate study of the genetic control of chitinolytic activity in vibrios, the chitobiase gene was isolated by cloning chromosomal DNA prepared from Vibrio vulnificus. Chimeric plasmids were constructed from Sau3A I partial digests of chromosomal DNA by ligating 5 to 15-Kilobase fragments into the BamHI site, i.e., in the Tc(sub r) gene, of pBR322 Am(sup r)Tc(sub r). The resulting plasmids were transformed into Escherichia coli DH1. Chitobiase activity of the insert-bearing clones was detected by using a chromogenic substrate, p-nitrophenyl-N-acetyl beta, D-glucosaminide, and confirmed by the appearance of a fluorescent end product from the hydrolysis of 4-methylumbelliferyl-beta, D-N-N'-diacetylchitiobiose. Physical mapping of plasmids containing the chitinase determinants indicate that transcription of these genes in E. coli may be initiated at a V. vulnificus promoter.