Silverman P M, Wickersham E, Harris R
Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City 73104.
J Mol Biol. 1991 Mar 5;218(1):119-28. doi: 10.1016/0022-2836(91)90878-a.
F plasmid DNA transfer (tra) gene expression in Escherichia coli is regulated by chromosome- and F-encoded gene products. To study the relationship among these regulatory factors, we constructed low-copy plasmids containing a phi(traY'-'lacZ)hyb gene that couples beta-galactosidase and Lac permease synthesis to the F plasmid traY promoter. Wild-type transformants maintained high levels of beta-galactosidase over a broad range of culture densities. Primer extension analysis of tra mRNA from F'lac and phi(traY'-'lacZ)hyb strains indicated very similar, though not identical, transcription initiation sites. Moreover, phi(traY'-'lacZ)hyb gene expression required both TraJ and SfrA, as does tra gene expression in F+ strains. beta-Galactosidase activity was reduced approximately 30-fold in the absence of TraJ, which could be supplied in cis or in trans. In a two-plasmid system in which TraJ was supplied in trans by a lac-traJ operon fusion, phi(traY'-'lacZ)hyb expression was a linear, saturable function of traJ expression. Enzyme activity was reduced approximately tenfold in sfrA mutants. That reduction could not be attributed to an effect on the TraJ level. Several other cellular or environmental variables had only a modest effect on phi(traY'-'lacZ)hyb expression. Hyperexpression was observed at high cell density (twofold) and in anaerobic cultures (1.2- to 1.5-fold). In contrast, expression was reduced twofold in integration host factor mutants.
大肠杆菌中F质粒DNA转移（tra）基因的表达受染色体和F编码的基因产物调控。为了研究这些调控因子之间的关系，我们构建了含有phi（traY'-'lacZ）杂交基因的低拷贝质粒，该基因将β-半乳糖苷酶和乳糖通透酶的合成与F质粒traY启动子偶联。野生型转化体在广泛的培养密度范围内维持高水平的β-半乳糖苷酶。对F'lac和phi（traY'-'lacZ）杂交菌株的tra mRNA进行引物延伸分析表明，转录起始位点非常相似，但不完全相同。此外，phi（traY'-'lacZ）杂交基因的表达需要TraJ和SfrA，F+菌株中的tra基因表达也是如此。在没有TraJ的情况下，β-半乳糖苷酶活性降低约30倍，TraJ可以顺式或反式提供。在一个双质粒系统中，通过lac-traJ操纵子融合反式提供TraJ，phi（traY'-'lacZ）杂交基因的表达是traJ表达的线性、可饱和函数。在sfrA突变体中，酶活性降低约10倍。这种降低不能归因于对TraJ水平的影响。其他几个细胞或环境变量对phi（traY'-'lacZ）杂交基因的表达只有适度的影响。在高细胞密度（两倍）和厌氧培养（1.2至1.5倍）中观察到超表达。相反，在整合宿主因子突变体中，表达降低两倍。
