Watson N, Dunyak D S, Rosey E L, Slonczewski J L, Olson E R
Department of Molecular Biology, Upjohn Company, Kalamazoo, Michigan 49001.
J Bacteriol. 1992 Jan;174(2):530-40. doi: 10.1128/jb.174.2.530-540.1992.
Expression of the lysine decarboxylase gene (cadA) of Escherichia coli is induced upon external acidification. To dissect the molecular mechanisms responsible for this regulation, we analyzed a 4.2-kbp region upstream from cadA. DNA sequencing revealed two long open reading frames upstream of and on the same strand as cadA. One of these, cadB, is 444 codons long and is situated immediately upstream of cadA. Transcriptional fusions between fragments upstream of cadA and lacZ, Northern (RNA) hybridization, primer extension, and site-directed mutagenesis experiments defined a promoter, Pcad, upstream of cadB that was responsible for pH-regulated expression of cadA. Upstream of Pcad is an open reading frame, cadC, consisting of 512 codons. The predicted amino terminal region of the cadC gene product (CadC) resembles the carboxy-terminal domain of prokaryotic transcriptional activators involved in environmental sensing. Tn10 insertions within or immediately upstream of cadC abolished Pcad activity, suggesting that cadC encodes a positive transcription factor. Expression of plasmid-borne cadC in the Tn10 mutants restored Pcad activity, while introduction of a plasmid expressing truncated CadC resulted in the inability to complement. The presence of Pcad on a multicopy plasmid was found to lower expression arising from chromosomal Pcad, suggesting that a positive-acting factor is limiting. Our data suggests that cadA, cadB, and the acid-inducible Pcad comprise, at least in part, the cad operon which is under control of the cadC product.
大肠杆菌赖氨酸脱羧酶基因（cadA）的表达在外部酸化时被诱导。为了剖析负责这种调控的分子机制，我们分析了cadA上游4.2kbp的区域。DNA测序揭示了cadA上游且与cadA在同一条链上的两个长开放阅读框。其中一个是cadB，长444个密码子，紧邻cadA上游。cadA上游片段与lacZ之间的转录融合、Northern（RNA）杂交、引物延伸和定点诱变实验确定了cadB上游的一个启动子Pcad，它负责cadA的pH调控表达。Pcad上游是一个由512个密码子组成的开放阅读框cadC。cadC基因产物（CadC）预测的氨基末端区域类似于参与环境感知的原核转录激活因子的羧基末端结构域。cadC内部或紧邻cadC上游的Tn10插入消除了Pcad活性，表明cadC编码一个正转录因子。在Tn10突变体中质粒携带的cadC的表达恢复了Pcad活性，而引入表达截短的CadC的质粒导致无法互补。发现多拷贝质粒上Pcad的存在会降低染色体Pcad产生的表达，表明一个正作用因子是有限的。我们的数据表明，cadA、cadB和酸诱导的Pcad至少部分组成了受cadC产物控制的cad操纵子。
