The sequenced members of a novel family of small, hydrophobic, bacterial multidrug-resistance efflux proteins, which we have designated the small multidrug resistance (SMR) protein family, are identified and analysed. Two distinct clusters of proteins were identified within this family: (i) small multidrug efflux systems; and (ii) Sug proteins, potentially involved in the suppression of groEL mutations. Hydropathy and residue distribution analyses of this family suggest a structural model in which the polypeptide chain spans the membrane four times as mildly amphipathic alpha-helices. The roles of specific residues, a possible mechanistic model of drug efflux, and the primary physiological role(s) of the SMR proteins are discussed.
The first stages of infection by phage T4 may be divided into energy-dependent and energy-independent processes. Irreversible adsorption, unplugging, and initial exposure of the DNA terminus may occur at 4VC, or at 370C in bacteria whose energy-yielding metabolism has been poisoned. DNA injection into the cytoplasm needs higher temperatures and energy from the host cell. The nature of this energy requirement was deduced from the use of metabolic inhibitors. Our results show that T4 DNA injection specifically requires the presence of a protonmotive force across the cytoplasmic membrane of the host. Moreover, the chemical gradient (ApH) does not appear to be essential, but the membrane potential (A+/) is required. The specific transfer of nucleic acid from one cell to another of the same species must require energy. Though there have been occasional studies on the energy requirements for DNA transformation (1) and conjugation (2), most studies on the mechanism of phage T4 injection have dealt with the requirements and mechanisms of irreversible attachment (3), restriction (4), or expression (5). The actual process of DNA injection has not yet been studied in detail. This is due in part to the inability to isolate experimentally the stages directly preceding and following the exit of the DNA from the phage particle, and its entry into and traversal across the cytoplasmic membrane. There have been various theoretical reports on the energy requirement for T4 phage DNA ejection (6, 7) and transport (8), but in the absence of a clear experimental definition of the stages of DNA injection it has been difficult to study this phenomenon. In this paper we directly measure the penetration of DNA into the cell cytoplasm in the absence of DNA expression and describe a simple way to separate the DNA injection process itself from T4 irreversible adsorption and initiation of the release of DNA from the capsid (DNA exposure).We demonstrate below that injection requires the protonmotive force established across the host cell cytoplasmic membrane. Kalasauskaite and Grinius (9) have independently suggested the same requirement. Furthermore, we show that the membrane potential, and not the pH gradient, is a major requirement for T4 DNA injection.MATERIALS AND METHODS Strains. Escherichia coli B and K-12 (exonuclease V+, unc+) and E. coli JC7729 (exonuclease V-, unc+), previously obtained from A. J. Clark, were from our laboratory stocks. E. coli G6 (exonuclease V+, ATPase-, uncA-) was obtained from S. E. Luria's laboratory (10).T4D and T4 2-(amN51) were from our laboratory stocks and were grown respectively on E. coli B and E. coli JC7729.32P-Labeled phages were prepared and purified as described (11). After purification, T4 2-phage stocks contained no more than 3% radioactive material not precipitable by trichloroacetic acid.Colicin K was a generous gift of M. Weiss. Infection Conditions. Bacteria were grown in P broth (12) to 1-2 X 108 per ml, washed, and concentrated 10-fold in fresh broth buffered at pH 7 by 0.1 M Tris-...
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