Lin J T, Stewart V
Section of Microbiology, Cornell Unversity, Ithaca, NY 14853, USA.
J Mol Biol. 1996 Mar 1;256(3):423-35. doi: 10.1006/jmbi.1996.0098.
Klebsiella pneumoniae can use nitrate and nitrite as sole nitrogen sources during aerobic growth. Nitrate is converted through nitrite to ammonium by assimilatory nitrate and nitrite reductase, respectively. Enzymes required for nitrate assimilation are encoded by the nasFEDCBA operon of K. pneumoniae; nasF operon expression is subject to both general nitrogen control and pathway-specific nitrate/nitrite induction, mediated by the NtrC and NasR proteins, respectively. Sequence inspection revealed a presumptive sigmaN (sigma54)-dependent promoter as well as two presumptive upstream NtrC protein binding sites. Site-specific mutational and primer extension analyses confirmed the identity of the sigmaN-dependent promoter. Deletions removing the apparent NtrC protein binding sites greatly reduced NtrC-dependent regulation, indicating that these sites are involved in general nitrogen control. However, deletions removing most of the sequence upstream of the promoter had little effect on nitrate/nitrite regulation, suggesting that the nasF leader region is involved in nitrate/nitrite regulation. The 119 nucleotide long transcribed leader region contains an apparent factor-independent transcription terminator. Promoter replacement experiments demonstrated that the leader region is involved in nitrate/nitrite regulation of nasF operon expression. Deletions removing the transcription terminator structure resulted in a nitrate-blind constitutive phenotype, indicating that the transcription terminator structure serves a negative function. Other deletions, removing proximal portions of the leader region, resulted in an uninducible phenotype, indicating that this region serves a positive function. These results indicate that nitrate/nitrite regulation of nasF operon expression is determined by a transcription attenuation mechanism. We hypothesize that in the absence of nitrate or nitrite, the terminator structure abrogates transcription readthrough into the nasF operon. In the presence of nitrate or nitrite, the NasR protein mediates transcription antitermination, thereby allowing transcription to proceed into the nasF operon.
肺炎克雷伯菌在有氧生长过程中可利用硝酸盐和亚硝酸盐作为唯一氮源。硝酸盐分别通过同化硝酸盐还原酶和亚硝酸盐还原酶转化为亚硝酸盐，再转化为铵。硝酸盐同化所需的酶由肺炎克雷伯菌的nasFEDCBA操纵子编码；nasF操纵子的表达受一般氮调控以及由NtrC和NasR蛋白分别介导的途径特异性硝酸盐/亚硝酸盐诱导。序列检查揭示了一个推测的依赖sigmaN（sigma54）的启动子以及两个推测的上游NtrC蛋白结合位点。位点特异性突变和引物延伸分析证实了依赖sigmaN的启动子的身份。去除明显的NtrC蛋白结合位点的缺失大大降低了NtrC依赖的调控，表明这些位点参与一般氮调控。然而，去除启动子上游大部分序列的缺失对硝酸盐/亚硝酸盐调控影响很小，表明nasF前导区参与硝酸盐/亚硝酸盐调控。119个核苷酸长的转录前导区包含一个明显的不依赖因子的转录终止子。启动子替换实验表明前导区参与nasF操纵子表达的硝酸盐/亚硝酸盐调控。去除转录终止子结构的缺失导致硝酸盐不敏感的组成型表型，表明转录终止子结构起负向作用。其他去除前导区近端部分的缺失导致不可诱导的表型，表明该区域起正向作用。这些结果表明，nasF操纵子表达的硝酸盐/亚硝酸盐调控由转录衰减机制决定。我们假设在没有硝酸盐或亚硝酸盐的情况下，终止子结构消除了转录通读进入nasF操纵子。在有硝酸盐或亚硝酸盐的情况下，NasR蛋白介导转录抗终止，从而允许转录进入nasF操纵子。
