Analysis of thermophilic Bacillus sp.Strain TB-90 urease gene and its product

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The thermophilic Bacillus sp. TB-90 urease is composed of three subunits with molecular masses of 61, 12 and 11 kDa. By using synthetic oligonucleotide probes based on N-terminal amino acid sequences of each subunit, we cloned a 3.2-kb EcoRI-digested fragment of TB-90 genomic DNA.Moreover, we cloned other two DNA fragments by gene walking starting from this fragment. Finally, we in vitro reconstructed a 6.2-kb DNA fragment which expressed catalytically active urease in E.coli by combining these three DNA fragments. The nucleotide sequencing analysis revealed that the urease gene complex consists of nine 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, respectively. The deduced amino acid sequences of UreD, UreE, UreF and UreG, the gene products of four accessory genes, and homologous to those of the corresponding Ure proteins of Kkebsiella aerogenes. The UreD, UreF and UreG were essential for expression of urease activity in E coli and were suggested to play important roles in the maturation step of the urease in co- and/or post-translational manners. On the other hand, UreH and UreI exhibited no significant similarity to the known accessory proteins of other bacteria. However, UreH showed a similarity of 23% amino acid identity to Alcaligenes eutrophus HoxN protein, a high-affinity nickel transporter.