Except for a small fraction of persisters, 10-6 to 10-5, Escherichia coli K-12 is killed by prolonged inhibition of murein synthesis. The progeny of persisters are neither more resistant to inhibition of murein synthesis nor more likely to persist than normal cells. Mutants have been isolated in which a larger fraction, 10-2 persists. The persistent response of the mutants, Hip (high persistence), is to inhibition of murein synthesis at early or late steps by antibiotics (phosphomycin, cycloserine, and ampicillin) or by metabolic block (starvation for diaminopimelic acid). Killing of the parent strain by each of the four inhibitors has two phases: The first is rapid and lasts about 30 min; the second is slower, but still substantial, and lasts 3 to 4 h. The first phase also occurs in the Hip mutants, but then viability of the mutants remains constant after about 30 min. Neither tolerance, resistance, impaired growth, nor reversion of spheroplasts accounts for high-frequency persistence. Two of the mutations map at 33.8 min in a region containing few other recognized functions. This position and the phenotypes define hipA as a newly recognized gene. Transposons TnS and TnlO have been inserted close to hipA making it possible to explore the molecular genetics of persistence, a long recognized but poorly understood phenomenon.

The hipA gene at 33.8 min on the Escherichia coli chromosome controls the frequency of persistence upon inhibition of murein synthesis; for strains bearing hipA+ the frequency is 10-6, and for hipA-strains the frequency is 10-2. hip' has been cloned by selection for a kanamycin resistance determinant at 33.9 min. hipA+ is dominant over hipA-in both recA+ and recA backgrounds. The smallest DNA insert which contains hipA+, as determined by the ability of the plasmids to complement hipA-strains, is 1,885 base pairs. Both orientations of hipA+ are obtained when the cloning site of vector is remote from strong promoters; both orientations complement hipA-, and both encode a unique peptide of 50,000 Mr. The probable direction of transcription has been deduced from the pattern of peptides encoded by plasmids from which either end of the insert and adjacent vector sequences have been deleted. This information and the recovery of only one orientation of hipA+ when the cloning site is close to a strong promoter suggest that a high level of expression of the gene is not tolerated by E. col.

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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 aft 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.

Mutations in hipA, a gene of Escherichia coli K-12, greatly reduce the lethality of selective inhibition of peptidoglycan synthesis. These mutations have also been found to reduce the lethality that accompanies either selective inhibition of DNA synthesis or heat shock of strains defective in htpR. In addition, the mutant alleles of hipA are responsible for a reversible cold-sensitive block in cell division and synthesis of macromolecules, particularly peptidoglycan. Recombination between the chromosome of hipA mutants and plasmids containing noncomplementing fragments of hipA revealed that-the mutations responsible for both cold sensitivity and reduced lethality were probably identical and, in any case, lay within the first 360 base pairs of the coding region of hipA, probably within the first 50 base pairs. We suggest that the pleiotropic effects of mutations in hipA reflect the involvement of this gene in cell division.Mutations in hipA, a gene of Escherichia coli, suppress the lethal effects of inhibition of peptidoglycan synthesis, wallless' death, caused by an antibiotic'or genetic block in the early, middle, or terminal steps (8). A 1,883-base-pair (bp) fragment from the chromosome, the minimum length of fragment capable of recA-independent restoration of wallless death in hipA mutants, encodes a weakly expressed peptide of 50,000 Mr which may be toxic when overexpressed (9). Because of the strong temporal linkage of wall-less death to an event of the division cycle, termination of chromosome replication (5), it was suggested that hipA might be involved in cell division (8).We now report that hipA, in addition to governing the frequency of wall-less death, also controls death that ordinarily accompanies selective inhibition of DNA synthesis or heat shock of strains incapable of a normal response to heat shock because of mutation in htpR. The mutations in hipA that uncouple lethality from these inhibitions also cause a reversible cold-sensitive block in cell division. In view of this and earlier indications that hipA has a role in cell division, it may be of special interest that the position of hipA on the E. coli chromosome, min 33.8 (9), places it in or near the terminus of clockwise replication (4). MATERIALS AND METHODSBacterial strains, plasmids, and media. The strains used in this work are listed in Table 1. Plasmids containing the two orientations of a 1,883-bp'fragment that includes the 1,321-bp coding region of hipA, pHM519 and pHM520, and plasmids lacking either 360 bp from the 5' end of the coding region, pHM519d, or 225 bp from the 3' end, pHM520d, were derived from pACYC177 (9). pGK610 was constructed by inserting a 610-bp BglII-HindIII fragment from the hipA region into a promoter detection vector (11), pKO11. pGK610 contains 250 bp from the 3' end of a preceding gene, hipB, and the first 360 bp of the coding region of hipA. pGK300 was constructed by inserting a 300-bp BglII-HpaI * Corresponding author. fragment of the hipA region into pKO11. It contains 250 bp from the 3' end of hipB and th...