AUTOREGULATION OF HIP, AN OPERON THAT AFFECTS LETHALITY DUE TO INHIBITION OF PEPTIDOGLYCAN OR DNA-SYNTHESIS

The hip locus of Escherichia coli affects the frequency of persistence to the lethal consequences of selective inhibition of either DNA or p eptidoglycan synthesis. Regulation of the hip operon, which consists o f a regulatory region and two genes, hipB and hipA, was examined with strains containing a hip-lac transcriptional fusion placed in single c opy at the h 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 filtra tion and cross-linking studies, HipB is a dimer in solution. Sequence analysis revealed that HipB is a Cro-like DNA-binding protein. The int eraction of HipB with the hip regulatory region was examined by gel re tardation, DNase I protection, and methylation protection studies. Hip B binds with a K-app (K apparent) of 40 pM to four operator sites with the conserved sequence TATCCN(8)GGATA (N represents any nucleotide). Binding to the operators is nearly simultaneous acid appears to be coo perative. Analysis of the role of HipA in the regulation of the hip op eron is complicated by the toxicity of HipA in the absence of HipB. St rains disrupted in hipB but not in hipA could not be recovered. Moreov er, 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 t o the hip regulatory region directly but indirectly via HipB.