Requirement for membrane potential in active transport of glutamine by Escherichia coli.

The effect of reducing the membrane potential on glutamine transport in cells of Escherichia coli has been investigated. Addition of valinomycin to tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid-treated E. coli cells in the presence of 20 mM exogenous potassium reduced the membrane potential, as measured by the uptake of the lipophilic cation triphenylmethylphosphonium, and caused a complete inhibition of glutamine transport. Valinomycin plus potassium also caused a rapid decrease in the intracellular levels of ATP of normal E. coli cells, but had little if any effect on the ATP levels of two mutants of E. coli carrying lesions in the energy-transducing ATP complex (unc mutants). Yet both the membrane potential and the capacity to transport glutamine were depressed in the unc mutants by valinomycin and potassium. These findings are consistent with the hypothesis that both ATP and a membrane potential are essential to the active transport of glutamine by E. coli cells.     

A membrane potential threshold for phage T4 DNA injection

DNA penetration from T4 phage adsorbed to $\text{Escherichia coli}$ was measured at different membrane potentials. There was a precipitous reduction in DNA penetration between 110 mV and 60 mV. This threshold of membrane potential for DNA penetration is independent of ΔpH and rather insensitive to external pH between 6 and 8.     

Location and unusual membrane topology of the immunity protein of the Escherichia coli phage T4.

The immunity protein (Imm) encoded by the Escherichia coli phage T4 effects exclusion of phage superinfecting cells already infected with T4. The 83-residue polypeptide possesses two long lipophilic areas (from residues 3 to 32 and 37 to 65) interrupted by a hydrophilic stretch including two positively charged residues. The charge distribution of the protein very strongly suggested that it is a plasma membrane protein with the C terminus facing the periplasm. While it could be shown that the expected location was correct, fusions of Imm to alkaline phosphatase or beta-galactosidase showed that the C terminus was at the cytosolic side of the membrane. Also, concerning function, there was almost no structural specificity to this part of the protein. Even removal of the two positively charged residues did not completely abolish function. Evidence suggesting that Imm is associated with the membrane at specific sites is presented. It is proposed that Imm is localized to the membrane with the help of a receptor and that, therefore, it does not follow the established rules for the topology of other membrane proteins. The results also suggest that Imm acts indirectly, possibly by altering the conformation of a component of a phage DNA injection site.     

Role of superinfecting phage in lysis inhibition with phage T4 in Escherichia coli

Rutberg, Blanka (Karolinska Institutet, Stockholm, Sweden), and Lars Rutberg. Role of superinfecting phage in lysis inhibition with phage T4 in Escherichia coli. J. Bacteriol. 90:891-894. 1965.-The ability of bacteriophage T4 to induce lysis inhibition upon superinfection was investigated after various treatments of the phage. This ability was found not to be a property of the external protein part of the phage, nor was it dependent on the functional and possibly structural integrity of the phage genetic material.     

Alteration of the Escherichia coli membrane by addition of bacteriophage T4 protein synthesized after infection.

Many T4-induced proteins were found associated with the Escherichia coli membrane during infection. Some of these were apparently ionically bound, but many could be identified as integral parts of the inner and outer bacterial membranes by their selective solubilities in guanidine or Sarkosyl. The synthesis and insertion of these proteins into the bacterial membrane were temporally controlled and, once in the membrane, these proteins were stably integrated. Host membrane protein synthesis continued after infection of non-UV-irradiated cells, but was not present, if the cells were irradiated. There were no major redistribution or loss of bacterial proteins from E. coli membranes as a consequence of T4 infection.     

Rifampicin resistant DNA synthesis in phage T4 infected Escherichia coli

We have found that net DNA synthesis in T4 infected cells is rifampicin resistant. This finding implies that both the initiation of each T4 genome and its elongation are rifampicin resistant processes.     

Kinetic studies on the reaction catalyzed by polynucleotide kinase from phage T4-infected Escherichia coli.

Kinetic properties of polynucleotide kinase (EC 2.7.1.78) isolated from Escherichia coli cells infected with phage T4 were investigated. The reaction depends on the concentration of MgATP, while free ATP or free Mg2+ have neither inhibitory nor accelerating effect. The initial reaction velocity was plotted against variable concentrations of ATP as the phosphate donor at various fixed concentrations of 5'-hydroxyl-DNA or -oligo(rA) as the phosphate acceptor in the presence or absence of products. The double reciprocal plot analysis of the data suggested that the reaction obeys the random sequential mechanism. Various constants were determined and the reaction mechanism was discussed.     

Non-toxic expression in Escherichia coli of a plasmid-encoded gene for phage T4 lysozyme

The phage T4 gene coding for lysozyme has been cloned into a plasmid under control of the (trp/lac) hybrid tac promoter and expressed in Escherichia coli with no significant toxic effect to actively growing cells. E. coli D1210 (lacIq) transformed with this plasmid produced active T4 lysozyme at levels up to 2% of the cellular protein after induction with isopropyl-beta-D-thiogalactoside. A strain producing active lysozyme was shown to be under a selective disadvantage when co-cultured with a similar strain producing inactive lysozyme. Purified strains, however, are reasonably stable in culture and under normal storage conditions.     

Programmed Escherichia coli cell lysis by expression of cloned T4 phage lysis genes

Self-disruptive Escherichia coli that produces foreign target protein was developed. E. coli was co-transformed with two vector plasmids, a target gene expression vector and a lysis gene expression vector. The lytic protein was produced after the expression of the target gene, resulting in simplification of the cell disruption process. In this study, the expression of cloned T4 phage gene e or t was used for the disruption of E. coli that produced beta-glucuronidase (GUS) as a model target protein. The expression of gene e did not lead to prompt cell disruption but weakened the cell wall. Resuspension with deionized water facilitated cell lysis, and GUS activity was observed in the resuspended liquid. Expression of gene e at mid logarithmic growth phase was the optimal induction period for GUS production and release. On the other hand, the expression of gene t induced immediate cell lysis, and intracellular GUS was released to the culture medium. Maximum GUS production was obtained when gene t was induced at late logarithmic growth phase.     

Effects of T4 phage infection and anaerobiosis upon nucleotide pools and mutagenesis in nucleoside diphosphokinase-defective Escherichia coli strains.

Bacteriophage T4 encodes nearly all of its own enzymes for synthesizing DNA and its precursors. An exception is nucleoside diphosphokinase (ndk gene product), which catalyzes the synthesis of ribonucleoside triphosphates and deoxyribonucleoside triphosphates (dNTPs) from the corresponding diphosphates. Surprisingly, an Escherichia coli ndk deletion strain grows normally and supports T4 infection. As shown elsewhere, these ndk mutant cells display both a mutator phenotype and deoxyribonucleotide pool abnormalities. However, after T4 infection, both dNTP pools and spontaneous mutation frequencies are near normal. An E. coli strain carrying deletions in ndk and pyrA and pyrF, the structural genes for both pyruvate kinases, also grows and supports T4 infection. We examined anaerobic E. coli cultures because of reports that in anaerobiosis, pyruvate kinase represents the major route for nucleoside triphosphate synthesis in the absence of nucleoside diphosphokinase. The dNTP pool imbalances and the mutator phenotype are less pronounced in the anaerobic than in the corresponding aerobic ndk mutant strains. Anaerobic dNTP pool data, which have not been reported before, reveal a disproportionate reduction in dGTP, relative to the other pools, when aerobic and anaerobic conditions are compared. The finding that mutagenesis and pool imbalances are mitigated in both anaerobic and T4-infected cultures provides strong, if circumstantial, evidence that the mutator phenotype of ndk mutant cells is a result of the dNTP imbalance. Also, the viability of these cells indicates the existence of a second enzyme system in addition to nucleoside diphosphokinase for nucleoside triphosphate synthesis.     

DNA injection during bacteriophage T4 infection of Escherichia coli.

The process of phage T4 DNA injection into the host cell was studied under a fluorescent microscope, using 4',6-diamidino-2-phenylindole as a DNA-specific fluorochrome. The phage DNA injection was observed when spheroplasts were infected with the artificially contracted phage particles having a protruding core. The DNA injection was mediated by the interaction of the core tip with the cytoplasmic membrane of the spheroplast. A membrane potential was not required for the process of DNA injection. On the other hand, DNA injection upon infection by intact noncontracted phage of the intact host cell was inhibited by an energy poison. Based on these observations, together with results from previous work, a model for the T4 infection process is presented, and the role of the membrane potential in the infection process is discussed.