Molecular characterization of hip, an operon of Escherichia coli that affects persistence to inhibition of peptidoglycan or DNA synthesis

The vast majority of a population of Escherichia coli is killed after prolonged inhibition of either peptidoglycan or DNA synthesis. However, a small but clinically significant fraction, 10{dollar}sp{lcub}-6{rcub}{dollar} to 10{dollar}sp{lcub}-5{rcub}{dollar}, survives, a phenomenon termed persistence. Analysis was initiated by isolating mutants with high frequency persistence, 10{dollar}sp{lcub}-2{rcub}{dollar} to 10{dollar}sp{lcub}-1{rcub}{dollar}. These Hip mutations mapped to minute 33.8, hipA. The molecular analysis of the hip locus is the focus of this dissertation. An additional Hip mutant was isolated. Genetic characterization showed that this mutation is in hipB, one of the two genes of the hip operon.; Analysis of the consequences of altered expression of hip suggested that elevated amounts of HipA relative to HipB produce the Hip{dollar}sp-{dollar} phenotype. Overexpression of hipA causes hip{dollar}sp+{dollar} strains to appear Hip{dollar}sp-{dollar}, an anticomplementary effect. But overexpression of hipBA is not anticomplementary nor is overexpression of hipB in strains lacking hipA. Further increases in hipA expression are toxic; this toxicity is exacerbated in strains defective in or lacking hipB. Thus, chromosomal disruptions specific for hipB could not be recovered. Deletions spanning hipB and hipA and insertions in hipA result in the Hip{dollar}sp+{dollar} phenotype.; Overexpression of hipBA causes an intriguing but as yet unexplained increase in the intragenic reversion of dapE6 strains to DAP{dollar}sp+{dollar}.; The hip promoter is autoregulated. HipB represses transcription from the hip promoter. HipB is a Cro-like protein that forms dimers in solution and binds with a K{dollar}sb{lcub}rm app{rcub}{dollar} of 40 pM to four operator sites in the hip regulatory region. Binding is nearly simultaneous and appears to be cooperative. HipA modestly enhances repression by HipB. However, HipA does not bind to the hip promoter directly but indirectly via HipB.; Regulation of hip may have additional complexities. For example, Integration Host Factor binds specifically to the regulatory region; however, there is as yet no evidence that this site is physiologically relevant. In addition, it is known that the hip operon is growth rate and temperature regulated.