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

嗜热芽孢杆菌。TB-90尿素酶由三个亚基组成，分子量分别为61、12和11 kDa。根据Tb-90各亚基N端氨基酸序列，利用人工合成的寡核苷酸探针，克隆了一段3.2kb的TB-90基因组DNA的EcoRI酶切片段，并以此为起点，用基因步行法克隆了另外两个DNA片段。最后，我们通过将这三个DNA片段组合在一起，在体外构建了一个在大肠杆菌中表达催化活性尿素酶的6.2kb的DNA片段。核苷酸序列分析表明，该尿素酶基因复合体由9个基因组成，按排列顺序依次命名为ureB、ureE、ureE、ureF、ureG、ureH和ureI。结构基因UREA、UREA和UREC分别编码11-12和61-kDa亚基。4个辅助基因的基因产物OreE、UreE、UreF和UreG的推导氨基酸序列与产气克氏杆菌相应的Ure蛋白同源。URE、UreF和UreG是在大肠杆菌中表达尿素酶活性所必需的，并被认为以共翻译和/或翻译后的方式在尿素酶的成熟过程中发挥重要作用。另一方面，UreH和UreI与其他细菌已知的辅助蛋白没有明显的相似性。然而，UreH与富营养性产碱菌HoxN蛋白(一种高亲和力镍转运蛋白)的氨基酸同源性为23%。

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.