Role of premature translational termination in the regulation of expression of the phi X174 lysis gene

Expression of the phi X174 lysis (E) gene, a member of an overlapping gene pair, appears to depend on a frameshift-induced chain termination by ribosomes translating the upstream D gene. A -1 reading frameshift, possibly induced by misreading of an alanine codon as a doublet, causes ribosomes to terminate translation at two different sites, suggesting two modes of regulating expression of the E gene. One frameshift can cause translational termination at a stop codon(s) near the E gene ribosome binding site (RBS), resulting in reinitiation by ribosomes at the E gene RBS. Termination at a second site some 70 bases upstream from the E gene RBS, while too far away to allow ribosomal re-initiation at the E gene RBS, probably results in an unmasking of the message, allowing entry of a new ribosome at the E gene RBS.     

Expression of bacteriophage PhiX174 lysis gene E in Staphylococcus carnosus TM300

Abstract Expression of the cloned PhiX174 gene E causes lysis of the Gram-negative bacterium Escherichia coli , which led to the proposal that a two-membrane system is necessary for the protein E lysis function. Gene E was cloned in an E. coli/Bacillus subtilis shuttle vector and expressed in the Gram-positive bacterium Staphylococcus carnosus TM300. Regulated gene E expression had a lethal effect on S. carnosus ; however, no lysis was detected, lending support to the hypothesis.     

Altered temperature induction sensitivity of the lambda pR/cI857 system for controlled gene E expression in Escherichia coli

Cell lysis of Gram-negative bacteria can be efficiently achieved by expression of the cloned lysis gene E of bacteriophage PhiX174. Gene E expression is tightly controlled by the rightward lambda pR promoter and the temperature-sensitive repressor cI857 on lysis plasmid pAW12. The resulting empty bacterial cell envelopes, called bacterial ghosts, are currently under investigation as candidate vaccines. Expression of gene E is stringently repressed at temperatures up to 30 degrees C, whereas gene E expression, and thus cell lysis, is induced at temperatures higher than 30 degrees C due to thermal inactivation of the cI857 repressor. As a consequence, the production of ghosts requires that bacteria have to be grown at 28 degrees C before the lysis process is induced. In order to reflect the growth temperature of pathogenic bacteria in vivo, it seemed favorable to extend the heat stability of the lambda pR promoter/cI857 repressor system, allowing pathogens to grow at 37 degrees C before induction of lysis. In this study we describe a mutation in the lambda pR promoter, which allows stringent repression of gene E expression at temperatures up to 36 degrees C, but still permits induction of cell lysis at 42 degrees C.     

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.     

Mutations in the new gene stIII of bacteriophage T4B suppressing the lysis defect of gene stII and a gene e mutant.

Mutations of bacteriophage T4B were found which suppress the lysis defect of both gene stII mutants and gene e mutants. The suppressor mutations belong to a new gene, stIII, of phage T4B. Gene stIII is located on the genetic map of T4B between genes stI and e. stIII mutants sometimes form star plaques on Escherichia coli B. The latent period on E. coli 594, but not E. coli B, is shorter with stIII mutants than that with wild-type phage. The premature lysis of E. coli 594 infected with stIII phage does not depend on the expression of both stII+ and e+ function. StIII allele is dominant over the stIII+ with respect to both the ability to suppress the stII defect and the early lysis of infected E. coli 594 cultures.     

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.     

Escherichia coli ghost production by expression of lysis gene E and Staphylococcal nuclease

The production of bacterial ghosts from Escherichia coli is accomplished by the controlled expression of phage phiX174 lysis gene E and, in contrast to other gram-negative bacterial species, is accompanied by the rare detection of nonlysed, reproductive cells within the ghost preparation. To overcome this problem, the expression of a secondary killing gene was suggested to give rise to the complete genetic inactivation of the bacterial samples. The expression of staphylococcal nuclease A in E. coli resulted in intracellular accumulation of the protein and degradation of the host DNA into fragments shorter than 100 bp. Two expression systems for the nuclease are presented and were combined with the protein E-mediated lysis system. Under optimized conditions for the coexpression of gene E and the staphylococcal nuclease, the concentration of viable cells fell below the lower limit of detection, whereas the rates of ghost formation were not affected. With regard to the absence of reproductive cells from the ghost fractions, the reduction of viability could be determined as being at least 7 to 8 orders of magnitude. The lysis process was characterized by electrophoretic analysis and absolute quantification of the genetic material within the cells and the culture supernatant via real-time PCR. The ongoing degradation of the bacterial nucleic acids resulted in a continuous quantitative clearance of the genetic material associated with the lysing cells until the concentrations fell below the detection limits of either assay. No functional, released genetic units (genes) were detected within the supernatant during the lysis process, including nuclease expression.     

Changes in host cell phospholipid composition of φX174 gene E product

Abstract Expression of bacteriophage φX174 gene E from plasmid pUH51 induced lysis of Escherichia coli . Before onset of bacterial lysis, cellular phospholipase activity was induced due to the presence of gene E product within the cells. By comparison of the lytic behaviour of phospholipase-negative E. coli strains with the corresponding wild-type strain it was found that neither the action of detergent-resistant phospholipase A nor of detergent-sensitive phospholipase were essential for the lysis-inducing properties of the gene E product. It was concluded that induction of phospholipases after expression of the φX174 gene E was a consequence of membrane perturbation caused by the integration of the gene E product into the cytoplasmic membrane of E. coli .     

Binding of mammalian ribosomes to MS2 phage RNA reveals an overlapping gene encoding a lysis function.

The main binding site for mammalian ribosomes on the single-stranded RNA of bacteriophage MS2 is located nine tenths of the way through the coat protein gene. Translation initiated at an AUG triplet in the +1 frame yields a 75 amino acid polypeptide which terminates within the synthetase gene at a UAA codon, also in the +1 frame. Partial amino acid sequence analysis of the product synthesized in relatively large amounts by mammalian ribosomes confirms this assignment of the overlapping cistron. The same protein is made in an E. coli cell-free system, but only in very small amounts. Analysis of the translation products directed by RNA from op3, a UGA nonsense mutant of phage f2, identifies the overlapping cistron as a lysis gene. In this paper we show that the op3 mutation is a C yield U transition occurring in the second codon of the synthetase cistron, which explains the lowered production of phage replicase (as well as lack of lysis) upon op3 infection of nonpermissive cells. We discuss the properties of the overlapping gene in relation to its lysis function, recognition of the lysis initiator region by E. coli versus eucaryotic ribosomes and op3 as a ribosome binding site mutant for the f2 synthetase cistron.     

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.     

Role of ribosome recycling factor (RRF) in translational coupling

RNA phage GA coat and lysis protein expression are translationally coupled through an overlapping termination and initiation codon UAAUG. Essential for this coupling are the proximity of the termination codon of the upstream coat gene to the initiation codon of the lysis gene (either a <3 nucleotide separation or physical closeness through a possible hairpin structure) but not the Shine-Dalgarno sequence. This suggests that the ribosomes completing the coat gene translation are exclusively responsible for translation of the lysis gene. Inactivation of ribosome recycling factor (RRF), which normally releases ribosomes at the termination codon, did not influence the expression of the reporter gene fused to the lysis gene. This suggests the possibility that RRF may not release ribosomes from the junction UAAUG. However, RRF is essential for correct ribosomal recognition of the AUG codon as the initiation site for the lysis gene.