Bernard Labedan

Probes of membrane potential in Escherichia coli cells

Membrane potential (A

The energetics of the injection process of bacteriophage lambda DNA and the role of the ptsMpel-encoded protein

) of cells which are too small to allow the use of microelectrodes must be indirectly determined. In bacterial cells, labelled permeant cations which distribute across the cell membrane in response to a potential difference (negative inside) are currently used [l]. At thermodynamic equilibrium, the membrane potential is related to the concentration of labelled monovalent ion C’ inside and outside the cell by the Nernst equation:

Location of the first step transfer fragment and single-strand interruptions in T5st0 bacteriophage DNA☆

We have examined the nature of the role played in the process of phage lambda DNA injection by the bacterial protein coded by the ptsM/pel gene. Neither the specific inhibition of the activity of the PtsM protein, nor the addition of inhibitors of phosphotransferase system modified the efficiency of lambda DNA penetration. Thus, the PtsM/Pel protein does not seem to play a role through its transport function, although we have confirmed that it must be present for a successful lambda DNA injection. Moreover, the presence of various metabolic inhibitors (uncouplers, cyanide, arsenate) separately or together, or even harsher methods of energy depletion did not prevent lambda DNA penetration, suggesting that DNA is entering the cell cytoplasm by diffusion.

A very early step in the T5 DNA injection process.

Abstract When Escherichia coli F is infected with T5st0 phage and arrested at the first step of transfer (Lanni, 1968) then centrifuged, the phage capsids are liberated into the medium and the naked phage chromosome, linked to the bacterial membrane by its first step transfer end, is uncoiled without breakage. These statements are based on the study of the distribution of labelled phage DNA between the bacteria and the surrounding medium and have been confirmed by direct observation in the electron microscope. The attached and naked T5st0 chromosome may easily be broken by moderate shearing forces into a small number of precisely sized fragments. By sedimentation analysis on neutral sucrose gradients, it was possible to locate the preferential breaking points at distances of 8, 19, 35 and 60% of the total length of the molecule, measured from the first step transfer end. It is suggested that these weak points coincide with the single-strand breaks present on one of the two chains of the T5st0 chromosome. Evidence is also presented that the first step transfer fragment is (i) immediately attached after injection to a fast sedimenting bacterial component, possibly membrane, (ii) localized at only one end of the chromosome and (iii) possibly separated by a single-strand interruption from the rest of the DNA molecule. It is concluded that the precise location of the preferential breaking points implies that the viral chromosome must be injected into the host following a unique direction.

In vitro study of the phage T5 DNA injection process: Use of columns of Escherichia coli membranes immobilized on kieselguhr☆

Abstract After phage T5 irreversibly adsorbs onto its specific host cell receptor, its DNA is released from the head of the capsid and appears to be immediately attached to the cell envelope. The DNA-attachment step takes place before the sensitization step to deprivation of calcium and before the transfer of the first-step-DNA fragment. Phages having an aberrant injection are deficient for this DNA-attachment step. The DNA attachment itself seems relatively weak, in contrast to the firm link mediated by the injected FST-DNA fragment. In the attached position, the T5 DNA is protected from host periplasmic nucleases even when it has been freed from its capsid by low-speed centrifugation of the infected bacteria. This naked, attached DNA may inject its FST segment after transfer of the centrifuged bacteria from 0 to 37°. Therefore, this DNA-attachment step appears as a transient state before the penetration of the FST-DNA fragment and the subsequent transient block of the chromosome injection.

Fractionation of high molecular-weight duplex DNA molecules on dilute agarose gels

Abstract A new technique using columns of Escherichia coli membranes immobilized on kieselguhr is described. On these columns the phage T5 can adsorb, and different known steps of its DNA injection process can be reproduced. By using different membranous materials to build the columns and different treatments to elute the phage DNA, it is concluded that (1) the presence of the outer membrane is required to allow both the phage adsorption and the DNA attachment step, (2) arrest of the injection after first-step transfer of DNA requires the presence of the cytoplasmic membrane, and (3) the phage DNA must use some nuclease-free way to attain the host cell cytoplasm.

PROTEIN EVOLUTION VIEWED THROUGH ESCHERICHIA COLI PROTEIN SEQUENCES : INTRODUCING THE NOTION OF A STRUCTURAL SEGMENT OF HOMOLOGY, THE MODULE

Abstract Fractionation of high molecular weight duplex DNAs was studied by electrophoresis on 0.05 to 1% agarose gels. Only the most diluted gels allowed the separation of T4 and T7 phages DNAs. The method makes use of progressive melting of the gel columns and may be applied to composite acrylamide agarose gels.