Lytic action of cloned phi X174 gene E.

The phi X174 lysis gene E was placed under control of the lac promoter by cloning into the multicopy plasmid pBH20. Other phi X174 gene sequences were removed by nuclease digestion. Expression of gene E was shown to be necessary and sufficient to produce lysis phenomena exhibited by infection with intact phage. Lysis, its inhibition by MgSO4 and spermine, its progression through a spheroplasting stage, and its dependence on an early chloramphenicol-sensitive step were reproduced in clones induced for expression of the E gene product. Escherichia coli clones carrying the E gene not under lac control, and clones under lac control but only minimally induced for gene E expression, exhibited morphological aberrations consistent with the view that the mechanism by which gene E mediates cell lysis is related to host cell division processes.     

Lysis of Escherichia coli by the bacteriophage phi X174 E protein: inhibition of lysis by heat shock proteins.

Lysis of Escherichia coli by the cloned E protein of bacteriophage phi X174 was more rapid than expected when bacteria were shifted from 30 to 42 degrees C at the time of E induction. Since such treatment also induces the heat shock response, we investigated the effect of heat shock proteins on lysis. An rpoH mutant was more sensitive to lysis by E, but a secondary suppressor mutation restored lysis resistance to parental levels, which suggests that the sigma 32 subunit itself did not directly increase lysis resistance. At 30 degrees C, mutants in five heat shock genes (dnaK, dnaJ, groEL, groES, and grpE) were more sensitive to lysis than were their wild-type parents. The magnitude of lysis sensitivity varied with mutation and strain background, with dnaK, dnaJ, and groES mutants consistently exhibiting the greatest sensitivities. Extended protection against lysis occurred when overproduction of heat shock proteins was induced artificially in cells that contained a plasmid with the rpoH+ gene under control of the tac promoter. This protective effect was completely abolished by mutations in dnaK, dnaJ, or groES but not by grpE or groEL mutations. Altered membrane behavior probably explains the contradiction whereby an actual temperature shift sensitized cells to lysis, but production of heat shock proteins exhibited protective effects. The results demonstrate that E-induced lysis can be divided into two distinct operations which may now be studied separately. They also emphasize a role for heat shock proteins under non-heat-shock conditions and suggest cautious interpretation of lysis phenomena in systems where E protein production is under control of a temperature-sensitive repressor.     

Differential induction of Escherichia coli autolysis by penicillin and the bacteriophage phi X174 gene E product.

The behavior of the temperature-sensitive, penicillin-tolerant Escherichia coli mutant VC44 to endogenously induced autolysis by the bacteriophage phi X174 gene E product (gpE) was investigated. Expression of the cloned phi X174 lysis gene showed that cultures of strain VC44 grown at the restricted temperature were fully sensitive to endogenously induced autolysis. The results revealed that the modes of E. coli lysis induction by gpE and by penicillin differ and that the trigger mechanisms for autolysis depend greatly on the specific inducer used.     

Proton-motive-force-dependent step in the pathway to lysis of Escherichia coli induced by bacteriophage phi X174 gene E product.

We examined the cellular effects after the expression of the cloned lysis gene E of bacteriophage phi X174. Chloramphenicol prevented lysis only when added within the first minute of derepression of E synthesis, indicating that a time lag of several minutes exists between the synthesis of the E protein and the onset of cell lysis. Experiments with protonophores showed the existence of a subsequent step dependent on proton motive force at about 3 to 5 min before lysis.     

Biochemical characterization of phi X174-protein-E-mediated lysis of Escherichia coli

Energetic and permeability properties of Escherichia coli cells were determined prior to and during lysis caused by expression of the cloned gene E of bacteriophage phi X174. Before onset of cell lysis the transmembrane gradients for K+, Na+ or Mg2+/ions, the level of ATP and the membrane potential, were unaffected. All these parameters changed simultaneously at the time of lysis onset, as monitored by measurements of culture turbidity as well as by determining the various specifications over a period of 1 min. During cell lysis chromosomal DNA was fragmented whereas plasmid DNA was liberated in its intact supercoiled form. Cytoplasmic constituents were released almost entirely, as indicated by the activity of beta-galactosidase in the supernatant fraction of protein-E-lysed cells. Periplasmic enzymes were only found in limited amounts in the cell supernatant and most remained associated with the cell ghosts. Such ghosts exhibited no gross cell damage or morphological alterations when compared with intact E. coli by light microscopy. All parameters investigated indicated that protein-E-mediated lysis of E. coli is caused by the formation of a transmembrane tunnel structure through the envelope complex of the bacterium.     

Evaluation of the interaction of phi X174 gene products E and K in E-mediated lysis of Escherichia coli.

Gene K of bacteriophage phi X174 was cloned, and its gene product was localized in the cell envelope of Escherichia coli. Compared with the sole expression of the phi X174 lysis gene E, the simultaneous expression of the K and E genes had no effect on scheduling of cell lysis. Therefore, a direct interaction of proteins E and K could be excluded. In contrast, phi X174 infection of a host carrying a plasmid expressing gene K resulted in a delayed lysis and an apparent increase in phage titer.     

DNA synthesis in Escherichia coli cells infected with gene H mutants of bacteriophage phi X174.

Escherichia coli cells infected with gene H mutants of bacteriophage phi X174 produce two types of particles. The 110S particles contain single-stranded circular DNA; the 110S particles are not infectious, although their DNA is infectious for E. coli spheroplasts. The second type of particles, 70S particles, contain a fragment of single-stranded DNA ranging from 0.2 to 0.5 genome in length. This fragment DNA anneals only to restriction enzyme fragments of replicative-form DNA from the portion of the molecule corresponding to the origin and early region of phi X174 single-stranded synthesis, although full-round single-stranded DNA synthesis is occurring in the H mutant-infected cells. Different H mutant phages produce different proportions of 70S to 110S particles; those mutants producing the most 70S also exhibit the largest amount of degradation of intracellularly labeled DNA during infection. These results suggest that in H mutant-infected cells, full-length single-stranded DNA is synthesized; varying amounts of degradation of the single-stranded material occur, and the resulting fragment DNA is subsequently incorporated into 70S particles.     

Reduced synthesis of beta-galactosidase in Escherichia coli infected with phage phi X 174.

The synthesis of beta-galactosidase (EC 3.2.1.23;beta-D-galactoside galactohydrolase) in E. coli was repressed as a result of infection with single-stranded DNA phage phi chi 174. Evidence is presented to show that this repression was not due to the restricted entry of the inducer molecules into the infected cells but to some phage-specified product(s). It was further shown that either the infected cells synthesized a fewer number of enzyme-specific mRNA or all such molecules were translated with a reduced efficiency; the half-lives of the mRNA's remained more or less unaffected.     

Dependence of the activity of phi X 174 B-promoter in the expression of Escherichia coli gal-operon on the number of its copies and their orientation

Promoter activities of different restriction fragments of the R8 DNA region of phage phi X 174 were compared. The studied DNA fragments included HindII fragment R8 (B-promoter), its left portion 49 nucleotide long, and the central segment containing 113 nucleotides generated by AluI. The promoter activity of these fragments was quantitated by the appearance of uridyltransferase and galactokinase activities in Escherichia coli clones carrying plasmids pHD68-17. The gal-promoters of these plasmids was substituted for the three aforementioned restriction fragments. The R8 region and its central part (BII-promoter) had comparable promoter activities while the left part containing the putative BI-promoter, did not induce clones with the expressed gal-operon. Clones containing 1, 2, 3 copies of the promoter fragment R8 were selected. No clones were revealed with more copies. All selected di- and tri-promoter clusters in plasmids had the same correct orientation of all inserted promoters with respect to the gal-operon. The expression of the gal-operon in E. coli was nearly directly proportional to the number of the phi X 174 B-promoters inserted before the operon.     

The Escherichia coli FKBP-type PPIase SlyD is required for the stabilization of the E lysis protein of bacteriophage phi X174

Most bacteriophages abruptly terminate their vegetative cycle by causing lysis of the host cell. The ssDNA phage phi X174 uses a single lysis gene, E, encoding a 91-amino-acid membrane protein that causes lysis of Escherichia coli by inhibiting MraY, a conserved enzyme of murein biosynthesis. Recessive mutations in the host gene slyD (sensitivity to lysis) absolutely block E-mediated lysis and phi X174 plaque formation. The slyD gene encodes a FKBP-type peptidyl-prolyl cis-trans isomerase (PPIase). To investigate the molecular basis of this unique FKBP-dependence, spontaneous plaque-forming mutants of phi X174 were isolated on a slyD lawn. All of these Epos ('plates on slyD') suppressors encode proteins with either a R3H or L19F change. The double mutant was also isolated and generated the largest plaques on the slyD lawn. A c-myc epitope tag sequence was incorporated into the parental E and Epos genes without effect on lytic function. Western blots and pulse-chase labelling experiments showed that both Epos and E are highly unstable in a slyD background; however, Epos is synthesized at a higher rate, allowing a lysis-sufficient level of Epos to accumulate. Our results indicate that SlyD is required for stabilizing the E protein and allowing it to accumulate to the levels required to exert its lytic effect. These data are discussed in terms of a model for the specific role of the SlyD PPIase in E folding, and of the use of the very strict SlyD- dependence phenotype for identifying elements of PPIase selectivity.     

The effect of multivalent ions on the inactivation of bacteriophage phi X174 by lipopolysaccharide from Escherichia coli C.

The inactivation of bacteriophage phi X174 by lipopolysaccharide from Escherichia coli C is promoted by multivalent metal ions and by polyamines. The effect of the two types of cation is similar, and the concentration causing 50% inactivation varies inversely with the charge on the cation, although quadrivalent amines are less active than expected. The increase in activity as the charge rises suggests that electrostatic binding is overwhelmingly important.     

Endogenous transmembrane tunnel formation mediated by phi X174 lysis protein E.

Biochemical and genetic studies have suggested that a transmembrane tunnel structure penetrating the inner and outer membranes is formed during the lytic action of bacteriophage phi X174 protein E. In this study we directly visualized the lysis tunnel by using high-magnification scanning and transmission electron microscopy.     

Requirement for a functional host cell autolytic enzyme system for lysis of Escherichia coli by bacteriophage phi X174

Escherichia coli VC30 is a temperature-sensitive mutant which is defective in autolysis. Strain VC30 lyses at 30 degrees C when treated with beta-lactam antibiotics or D-cycloserine or when deprived of diaminiopimelic acid. The same treatments inhibit growth of the mutant at 42 degrees C but do not cause lysis. Strain VC30 was used here to investigate the mechanism of host cell lysis induced by bacteriophage phi X 174. Strain VC30 was transformed with plasmid pUH12, which carries the cloned lysis gene (gene E) of phage phi X174 under the control of the lac operator-promoter, and with plasmid pMC7, which encodes the lac repressor to keep the E gene silent. Infection of strain VC30(pUH12)(pMC7) with phage phi X174 culminated in lysis at 30 degrees C. At 42 degrees C, intracellular phage development was normal, but lysis did not occur unless a temperature downshift to 30 degrees C was imposed. Similarly, induction of the cloned phi X174 gene E with isopropyl-beta-D-thiogalactoside resulted in lysis at 30 degrees C but not at 42 degrees C. Temperature downshift of the induced culture to 30 degrees C resulted in lysis even in the presence of chloramphenicol. These results indicate that host cell lysis by phage phi X174 is dependent on a functional cellular autolytic enzyme system.