Black D S, Irwin B, Moyed H S
Department of Microbiology and Molecular Genetics, California College of Medicine, University of California, Irvine 92717.
J Bacteriol. 1994 Jul;176(13):4081-91. doi: 10.1128/jb.176.13.4081-4091.1994.
The hip locus of Escherichia coli affects the frequency of persistence to the lethal consequences of selective inhibition of either DNA or peptidoglycan synthesis. Regulation of the hip operon, which consists of a regulatory region and two genes, hipB and hipA, was examined with strains containing a hip-lac transcriptional fusion placed in single copy at the lambda att site. Disruption of the hip locus increased activity from the fusion 16-fold. Repression was restored by supplying HipB in trans. HipB was overexpressed and purified. On the basis of gel filtration and cross-linking studies, HipB is a dimer in solution. Sequence analysis revealed that HipB is a Cro-like DNA-binding protein. The interaction of HipB with the hip regulatory region was examined by gel retardation, DNase I protection, and methylation protection studies. HipB binds with a Kapp (K apparent) of 40 pM to four operator sites with the conserved sequence TATCCN8GGATA (N represents any nucleotide). Binding to the operators is nearly simultaneous and appears to be cooperative. Analysis of the role of HipA in the regulation of the hip operon is complicated by the toxicity of HipA in the absence of HipB. Strains disrupted in hipB but not in hipA could not be recovered. Moreover, hipA-containing plasmids cannot be replicated in strains defective in or lacking hipB. HipA is found exclusively in a tight complex with HipB. Although disruption of hipA slightly increased expression from the hip-lac fusion, in vitro studies suggest that HipA does not bind to the hip regulatory region directly but indirectly via HipB.
大肠杆菌的hip位点影响对DNA或肽聚糖合成选择性抑制的致死后果的持续频率。由一个调控区域以及两个基因hipB和hipA组成的hip操纵子的调控，通过将单拷贝的hip-lac转录融合体置于λ附着位点的菌株进行了检测。hip位点的破坏使融合体的活性增加了16倍。通过反式提供HipB恢复了抑制作用。HipB被过量表达并纯化。基于凝胶过滤和交联研究，HipB在溶液中是二聚体。序列分析表明HipB是一种Cro样DNA结合蛋白。通过凝胶阻滞、DNase I保护和甲基化保护研究检测了HipB与hip调控区域的相互作用。HipB以40 pM的表观解离常数（Kapp）与四个具有保守序列TATCCN8GGATA（N代表任何核苷酸）的操纵子位点结合。与操纵子的结合几乎是同时发生的，并且似乎是协同的。由于在没有HipB的情况下HipA具有毒性，因此对HipA在hip操纵子调控中的作用分析变得复杂。在hipB但不在hipA中被破坏的菌株无法恢复。此外，含有hipA的质粒不能在有缺陷或缺乏hipB的菌株中复制。HipA仅存在于与HipB的紧密复合物中。尽管hipA的破坏略微增加了hip-lac融合体的表达，但体外研究表明HipA不直接与hip调控区域结合，而是通过HipB间接结合。
