Chen Yi-Ywan M, Burne Robert A
Department of Oral Biology, University of Florida, Gainesville, Florida 32610, USA.
J Bacteriol. 2003 Dec;185(23):6773-9. doi: 10.1128/JB.185.23.6773-6779.2003.
Ureases are multisubunit enzymes requiring Ni(2+) for activity. The low pH-inducible urease gene cluster in Streptococcus salivarius 57.I is organized as an operon, beginning with ureI, followed by ureABC (structural genes), and ureEFGD (accessory genes). Urease biogenesis also requires a high-affinity Ni(2+) uptake system. By searching the partial genome sequence of a closely related organism, Streptococcus thermophilus LMG18311, three open reading frame (ORFs) homologous to those encoding proteins involved in cobalamin biosynthesis and cobalt transport (cbiMQO) were identified immediately 3' to the ure operon. To determine whether these genes were involved in urease biogenesis by catalyzing Ni(2+) uptake in S. salivarius, regions 3' to ureD were amplified by PCRs from S. salivarius by using primers identical to the S. thermophilus sequences. Sequence analysis of the products revealed three ORFs. Reverse transcriptase PCR was used to demonstrate that the ORFs are transcribed as part of the ure operon. Insertional inactivation of ORF1 with a polar kanamycin marker completely abolished urease activity and the ability to accumulate (63)Ni(2+) during growth. Supplementation of the growth medium with NiCl(2) at concentrations as low as 2.5 micro M partially restored urease activity in the mutant. Both wild-type and mutant strains showed enhanced urease activity when exogenous Ni(2+) was provided at neutral pH. Enhancement of urease activity by adding nickel was regulated at the posttranslational level. Thus, ORF1, ORF2, and ORF3 are part of the ure operon, and these genes, designated ureM, ureQ, and ureO, respectively, likely encode a Ni(2+)-specific ATP-binding cassette transporter.
脲酶是一种多亚基酶，其活性需要Ni(2+)。唾液链球菌57.I中低pH诱导型脲酶基因簇被组织成一个操纵子，从ureI开始，接着是ureABC（结构基因）和ureEFGD（辅助基因）。脲酶的生物合成还需要一个高亲和力的Ni(2+)摄取系统。通过搜索密切相关的嗜热链球菌LMG18311的部分基因组序列，在脲操纵子的3'端立即鉴定出三个与参与钴胺素生物合成和钴转运的蛋白质编码基因（cbiMQO）同源的开放阅读框（ORF）。为了确定这些基因是否通过催化唾液链球菌中Ni(2+)的摄取参与脲酶的生物合成，使用与嗜热链球菌序列相同的引物，通过PCR从唾液链球菌中扩增ureD 3'端区域。对产物的序列分析揭示了三个ORF。逆转录酶PCR用于证明这些ORF作为脲操纵子的一部分被转录。用极性卡那霉素标记对ORF1进行插入失活完全消除了脲酶活性以及生长过程中积累(63)Ni(2+)的能力。在生长培养基中添加低至2.5 μM的NiCl(2)可部分恢复突变体中的脲酶活性。当在中性pH下提供外源Ni(2+)时，野生型和突变株均显示出增强的脲酶活性。添加镍对脲酶活性的增强是在翻译后水平上调控的。因此，ORF1、ORF2和ORF3是脲操纵子的一部分，这些基因分别命名为ureM、ureQ和ureO，可能编码一种Ni(2+)特异性ATP结合盒转运蛋白。
