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.

SummaryAcrAB of Escherichia coli , an archetype among bacterial multidrug efflux pumps, exports an extremely wide range of substrates including solvents, dyes, detergents and antimicrobial agents. Its expression is regulated by three XylS/AraC family regulators, MarA, SoxS and Rob. Although MarA and SoxS regulation works by the alteration of their own expression levels, it was not known how Rob, which is constitutively expressed, exerts its regulatory action. We show here that the induction of the AcrAB efflux pump by decanoate and the more lipophilic unconjugated bile salts is mediated by Rob, and that the low-molecularweight inducers specifically bind to the C-terminal, non-DNA-binding domain of Rob. Induction of Rob is not needed for induction of AcrAB, and we suggest that the inducers act by producing conformational alterations in pre-existing Rob, as was suggested recently (Rosner, Dangi, Gronenborn and Martin, J Bacteriol 184: 1407-1416, 2002). Decanoate and unconjugated bile salts, which are present in the normal habitat of E. coli , were further shown to make the bacteria more resistant to lipophilic antibiotics, at least in part because of the induction of the AcrAB efflux pump. Thus, it is likely that E. coli is protecting itself by the Rob-mediated upregulation of AcrAB against the harmful effects of bile salts and fatty acids in the intestinal tract.

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Alkaline phosphatase (PhoA) fusions to TonB amino acids 32, 60, 125, 207, and 239 (the carboxy terminus) all showed high PhoA activity; a PhoA fusion to TonB amino acid 12 was inactive. The full-length TonB-PhoA fusion protein was associated with the cytoplasmic membrane and retained partial TonB function. These results support a model in which TonB is anchored in the cytoplasmic membrane by its hydrophobic amino terminus, with the remainder of the protein, including its hydrophobic carboxy terminus, extending into the periplasm.The tonB gene product is required for energy-dependent transport of large (>600 Da) nutrients across the outer membrane of Escherichia coli. TonB couples the electrochemical potential of the cytoplasmic membrane to the active transport of vitamin B12 and ferric siderophores across the outer membrane, to the energy-dependent steps of bacteriophage +80 and Ti infection, and to the entry of B-group colicins into bacteria. Thus, TonB functions to transduce energy between the cytoplasmic and outer membranes (for a review, see reference 16).To understand the mechanism of TonB-dependent energy transduction, it is important to know the disposition of TonB within the cell envelope. Hydropathy plots of the TonB amino acid sequence indicate that it is markedly hydrophilic, with the notable exceptions of short hydrophobic regions at both the amino and carboxy termini (17). One or both of these regions could serve as membrane anchors. We recently showed that TonB is membrane associated and that it extends into the periplasm sufficiently to be accessible to proteinase K (18). In addition, we showed that the amino terminus of TonB functions as an export signal but is not proteolytically cleaved (18,21). Previous studies had shown that TonB expressed during infection of UV-irradiated bacteria with XtonB transducing phage is associated with the cytoplasmic membrane based on Sarkosyl fractionation (15). These results suggested that the amino terminus of TonB serves to anchor TonB in the cytoplasmic membrane, with the bulk of the polypeptide extending into the periplasm. However, the location of the carboxy terminus remained uncertain. The 11-residue hydrophobic region near the carboxy terminus is shorter than the 21 residues thought to be required to span the lipid bilayer in an a-helix. Nevertheless, the hydropathy profile of this region is similar to that of penicillin-binding protein 5, which is known to be anchored in the cytoplasmic membrane by its carboxy terminus (19). Thus, it seemed possible that the short hydrophobic region at the carboxy terminus of TonB could be a membrane anchor.To further investigate the membrane topology of TonB, we constructed tonB-phoA gene fusions by oligonucleotidedirected deletion mutagenesis (1,2). To avoid problems with potentially toxic TonB-PhoA fusion proteins (1,3,21) (4) and CAGE/ GEM (5) software packages. Starting with pKP287, a precise deletion of the 5' tetA coding region, the tonB promoter, and sequences 5' to the tonB coding region was made by oligonucleot...