Muse Wilson B, Rosario Christopher J, Bender Robert A
Department of Molecular Cellular and Developmental Biology, The University of Michigan, Ann Arbor, Michigan 48109-1048, USA.
J Bacteriol. 2003 May;185(9):2920-6. doi: 10.1128/JB.185.9.2920-2926.2003.
Transcription of the cytosine deaminase (codBA) operon of Escherichia coli is regulated by nitrogen, with about three times more codBA expression in cells grown in nitrogen-limiting medium than in nitrogen-excess medium. Beta-galactosidase expression from codBp-lacZ operon fusions showed that the nitrogen assimilation control protein NAC was necessary for this regulation. In vitro transcription from the codBA promoter with purified RNA polymerase was stimulated by the addition of purified NAC, confirming that no other factors are required. Gel mobility shifts and DNase I footprints showed that NAC binds to a site centered at position -59 relative to the start site of transcription and that mutants that cannot bind NAC there cannot activate transcription. When a longer promoter region (positions -120 to +67) was used, a double footprint was seen with a second 26-bp footprint separated from the first by a hypersensitive site. When a shorter fragment was used (positions -83 to +67), only the primary footprint was seen. Nevertheless, both the shorter and longer fragments showed NAC-mediated regulation in vivo. Cytosine deaminase expression in Klebsiella pneumoniae was also regulated by nitrogen in a NAC-dependent manner. K. pneumoniae differs from E. coli in having two cytosine deaminase genes, an intervening open reading frame between the codB and codA orthologs, and a different response to hypoxanthine which increased cod expression in K. pneumoniae but decreased it in E. coli.
大肠杆菌胞嘧啶脱氨酶（codBA）操纵子的转录受氮调控，在氮限制培养基中生长的细胞中，codBA的表达量比在氮过量培养基中高约三倍。来自codBp-lacZ操纵子融合体的β-半乳糖苷酶表达表明，氮同化控制蛋白NAC是这种调控所必需的。用纯化的RNA聚合酶从codBA启动子进行体外转录时，添加纯化的NAC可刺激转录，这证实不需要其他因子。凝胶迁移率变动分析和DNase I足迹分析表明，NAC结合在相对于转录起始位点位于-59位置的中心位点，在该位点不能结合NAC的突变体不能激活转录。当使用更长的启动子区域（-120至+67位置）时，会出现双重足迹，第二个26bp的足迹与第一个足迹被一个超敏位点隔开。当使用较短的片段（-83至+67位置）时，只看到主要足迹。然而，较短和较长的片段在体内均显示出NAC介导的调控。肺炎克雷伯菌中的胞嘧啶脱氨酶表达也以NAC依赖的方式受氮调控。肺炎克雷伯菌与大肠杆菌的不同之处在于，它有两个胞嘧啶脱氨酶基因，在codB和codA直系同源基因之间有一个间隔的开放阅读框，并且对次黄嘌呤的反应不同，次黄嘌呤在肺炎克雷伯菌中增加cod表达，但在大肠杆菌中降低cod表达。
