Maeda Michihisa    Department   Undergraduate School  , School of Agriculture    Position   Professor
Language	English
Publication Date	1994/01
Type	Academic Journal
Title	Cloning,sequencing,and expression of thermophilic Bacillus sp.strain TB-90 urease gene complex in Escherichia coil
Contribution Type	Co-authored (first author)
Journal	J. Bacteriol.
Volume, Issue, Page	176(2),pp.432-442
Author and coauthor	M Maeda, M Hidaka, A Nakamura, H Masaki, and T Uozumi
Details	The urease of thermophilic Bacillus sp. strain TB-90 is composed of 3 subunits with mol. masses of 61, 12, and 11 kDa. By using synthetic oligonucleotide probes based on N-terminal amino acid sequence of each subunit, a 3.2-kb EcoRI fragment of TB-90 genomic DNA was cloned. Moreover, 2 two other DNA fragments were cloned by gene walking starting from this fragment. Finally, a 6.2-kb DNA fragment which expressed catalytically active urease in Escherichia coli was reconstructed in vitro by combining these 3 DNA fragments. Nucleotide sequencing anal. revealed that the urease gene complex consists of 9 genes, which were designated ureA, ureB, ureC, ureE, ureF, ureG, ureD, ureH, and ureI in order of arrangement. The structural genes ureA, ureB, and ureC encode the 11-, 12-, and 61-kDa subunits, resp. The deduced amino acid sequences of UreD, UreE, UreF, and UreG, the gene products of the accessory genes, are homologous to those of the corresponding Ure proteins of Klebsiella aerogenes. UreD, UreF, and UreG were essential for expression of urease activity in E. coli and are suggested to play important roles in the maturation step of the urease in a co- and/or posttranslational manner. On the other hand, UreH and UreI exhibited no significant similarity to the known accessory proteins of other bacteria. However, UreH showed 23% amino acid identity to the Alcaligenes eutrophus HoxN protein, a high-affinity nickel transporter.