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.     

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.     

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 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.     

Ter mutation and susceptibility to phi X174 phage in E. coli K12.

The Ter-15 mutant derived from E. coli K12 W2252-11U^? RC^str (wild type I) is found to be sensitive to φx174 phage infection. Lipopolysaccharide extracted from this mutant inactivates the phage, and has core oligosaccharides identical in amounts to those in the lipopolysaccharide from wild type cells.In contrast, the Ter-21 mutant derived from E. coli K12 W2252-11U^? RC^rel (wild type II) is not sensitive to this phage infection, and its lipopolysaccharide does not inactivate the phage. Its lipopolysaccharide sugars are found to be D-glucose and D-ribose, thus differing from the lipopolysaccharide sugars of the wild type cells.     

Lysis induction of Escherichia coli by the cloned lysis protein of the phage MS2 depends on the presence of osmoregulatory membrane-derived oligosaccharides

Expression of the cloned lysis protein of phage MS2, which is sufficient to lyse wild type Escherichia coli, does not cause lysis of mutants lacking the osmoregulatory membrane-derived oligosaccharides (MDO). The lysis gene product normally found in the membrane fraction was not stably inserted into the membranes of a mdoA mutant; rather degradation and release from the membrane occurred. Gentle plasmolysis of the MDO-lacking mutant clearly showed an increased periplasmic space as compared to wild type cells. It is concluded that the MDOs play an important role in maintaining a proper arrangement of inner and outer membrane, a prerequisite for a functional insertion of the MS2 lysis protein.     

The role of bivalent ions in the inactivation of bacteriophage phi X174 by lipopolysaccharide from Escherichia coli C.

The need for Ca2+ in the inactivation of bacteriophage phi X174 by lipopolysaccharide from Escherichia coli C was confirmed. Ca2+ could be replaced almost completely by Na+, but the concentration of Na+ needed was greater by more than an order of magnitude. Other bivalent ions caused inactivation in the same way as Ca2+, and the degree of inactivation varied according to the ion. At 50% inactivation of bacteriophage, the relation between the concentrations of NaCl and of bivalent or tervalent ions (Mx+) fitted the conception that NaCl was neutralizing electrostatic repulsion between virus and lipopolysaccharide by an ionic-strength effect: that is, log[Mx+] varies inversely with square root[NaCl]. The variation in effect of bi- and ter-valent ions and the low concentration needed show that this is not an ionic-strength effect but likely to involve binding to more than one site.     

Proteolysis of bacteriophage phi X174 prohead protein gpB by a protease located in the Escherichia coli outer membrane.

The gene B protein (gpB) of bacteriophage phi X174 is required for prohead assembly and is removed from prohead during phage maturation. Protease activity was observed in isolated prohead which specifically cleaved gpB. Cleavage of gpB produced two fragments that had apparent molecular weights of 12,300 and 3,700 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal sequencing of the fragments confirmed that they resulted from the cleavage of gpB and identified the cleavage site as an Arg-Arg at amino acids 76 to 77 of the 120-amino-acid protein. gpB-specific protease activity was observed in both phi X174-infected and uninfected Escherichia coli extracts. This protease activity was localized to the outer-membrane fraction of uninfected cells. Protease activities present in the outer membrane and in isolated prohead produced identical fragments and had the same protease inhibition profile. The gpB-specific activity in uninfected cells was induced by growth at 42 degrees C and was inhibited by the protease inhibitors, 1,10-phenanthroline, EDTA, and N-ethylmaleimide.     

抗菌肽-X基因的克隆及在大肠杆菌中的表达

用PCR技术获得抗菌肽—X基因、TNFα基因,与温度诱导的表达载体pRC连接成为重组表达载体,导入大肠杆菌TG1,通过温度诱导表达重组蛋白。将重组质粒转入不同的表达菌中进行表达,经SDS—PAGE选出E.coil BL21(DE3)为最佳表达的宿主菌。培养后,离心得菌体,经超声破碎离心得包涵体,溶解后用CNBr切割并透析,最后经CM52纤维素柱分离纯化得到有活性高纯度的抗菌肽—X。     

Identification of a phi X174 coded protein involved in the inhibition of beta-galactosidase synthesis in Escherichia coli

The synthesis of β-galactosidase (EC 3.2.1.23: β-D-galactoside galactohydrolase) in $\text{Escherichia}\text{coli}$ is repressed as a result of infection with single-stranded DNA phage ØX174. An $\text{amber}$ mutant in ØX174 cistron A, which codes for two proteins, does not inhibit the enzyme synthesis while $\text{amber}$ mutants in all other genes do cause repression. A mutant near the amino-terminal end of cistron A, which produces the small 35,000 molecular weight cistron A polypeptide, also inhibits the synthesis of β-galactosidase. Inhibition is also observed in an $\text{Escherichia}\text{coli}\text{rep}$ mutant which does not support the replication of replicative-form DNA. Exogenous nucleotide bases and cyclic 3′,5′-adenosine monophosphate (cyclic AMP) do not have any effect on the degree of repression.