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.     

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.     

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.     

Genes and regulatory sequences of bacteriophage phi X174

Fragments of the DNA of bacteriophage phi X174 were inserted in the plasmids pACYC177 and pBR322, in order to test the in vivo effects of separate phage genes and regulatory sequences. The phi X174 inserts were identified by recombination and complementation with phage mutants, followed by restriction enzyme analysis. The genes B, C, F and G can be maintained stably in the cell even when there is efficient expression of these viral genes. Recombinant plasmids with the complete genes D and E can only be maintained when the expression of these genes is completely blocked. Expression of complete H and J genes could not yet be demonstrated. The intact gene A was apparently lethal for the host cell, as it was never found in the recombinants. The genes F and G are expressed, even when they are not preceded by one of the well characterized viral or plasmid promoter sequences. Screening of the nucleotide sequence of phi X174 gives two promoter-like sequences just in front of the two genes. Viral sequences with replication signals (the phi X174 (+) origin of replication, the initiation site for complementary strand synthesis and the incompatibility sequence) appeared to be functional also when inserted in recombinant plasmids. A plasmid with the phi X (+) origin can be forced to a rolling circle mode of replication. The A protein produced by infecting phages works in trans on the cloned viral origin. The (-) origin can function as initiation signal for complementary strand synthesis during transduction of single-stranded plasmid DNA. The intracellular presence of the incompatibility sequence on a plasmid prevents propagation of infecting phages.     

Inducible cell lysis system for the study of natural transformation and environmental fate of DNA released by cell death.

Two novel conditional broad-host-range cell lysis systems have been developed for the study of natural transformation in bacteria and the environmental fate of DNA released by cell death. Plasmid pDKL02 consists of lysis genes S, R, and Rz from bacteriophage lambda under the control of the Ptac promoter. The addition of inducer to Escherichia coli, Acinetobacter calcoaceticus, or Pseudomonas stutzeri containing plasmid pDKL02 resulted in cell lysis coincident with the release of high amounts of nucleic acids into the surrounding medium. The utility of this lysis system for the study of natural transformation with DNA released from lysed cells was assessed with differentially marked but otherwise isogenic donor-recipient pairs of P. stutzeri JM300 and A. calcoaceticus BD4. Transformation frequencies obtained with lysis-released DNA and DNA purified by conventional methods and assessed by the use of antibiotic resistance (P. stutzeri) or amino acid prototrophy (A. calcoaceticus) for markers were comparable. A second cell lysis plasmid, pDKL01, contains the lysis gene E from bacteriophage phi X174 and causes lysis of E. coli and P. stutzeri bacteria by activating cellular autolysins. Whereas DNA released from pDKL02-containing bacteria persists in the culture broth for days, that from induced pDKL01-containing bacteria is degraded immediately after release. The lysis system involving pDKL02 is thus useful for the study of both the fate of DNA released naturally into the environment by dead cells and gene transfer by natural transformation in the environment in that biochemically unmanipulated DNA containing defined sequences and coding for selective phenotypes can be released into a selected environment at a specific time point. This will allow kinetic measurements that will answer some of the current ecological questions about the fate and biological potential of environmental DNA to be made.     

Repressor titration: a novel system for selection and stable maintenance of recombinant plasmids.

The propagation of recombinant plasmids in bacterial hosts, particularly in Escherichia coli, is essential for the amplification and manipulation of cloned DNA and the production of recombinant proteins. The isolation of bacterial transformants and subsequent stable plasmid maintenance have traditionally been accomplished using plasmid-borne selectable marker genes. Here we describe a novel system that employs plasmid-mediated repressor titration to activate a chromosomal selectable marker, removing the requirement for a plasmid-borne marker gene. A modified E.coli host strain containing a conditionally essential chromosomal gene (kan) under the control of the lac operator/promoter, lac O/P, has been constructed. In the absence of an inducer (allolactose or IPTG) this strain, DH1 lackan , cannot grow on kanamycin-containing media due to the repression of kan expression by LacI protein binding to lac O/P. Transformation with a high copy-number plasmid containing the lac operator, lac O, effectively induces kan expression by titrating LacI from the operator. This strain thus allows the selection of plasmids without antibiotic resistance genes (they need only contain lac O and an origin of replication) which have clear advantages for use as gene therapy vectors. Regulation in the same way of an essential, endogenous bacterial gene will allow the production of recombinant therapeutics devoid of residual antibiotic contamination.     

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.     

Mutational analysis of the bacteriophage phi X174 replication origin

Bacteriophage phi X174 mutants within the 30 base-pair replication origin were constructed using oligodeoxynucleotide-directed mutagenesis. A total of 18 viable base substitution mutants at 13 different positions within the origin region were obtained. The majority of these ori mutants have a plaque morphology and burst size comparable to that of wild-type phi X174. Two phi X174 ori mutants with a reduced growth ability spontaneously acquired additional mutations that enhanced the growth rate. The additional mutation was located at the same site as the original mutation or was located in the N-terminal part of the gene A protein. This latter secondary mutation is responsible for a better binding and/or recognition of the gene A protein to the mutated origin. In a Darwinian experiment wild-type phi X174 outgrows all phi X174 ori mutants, indicating the superiority of the wild-type ori sequence for the reproduction of bacteriophage phi 174. Insertions and deletions were constructed at different positions within the phi X174 replication origin cloned in a plasmid. Small insertions and deletions in the A + T-rich spacer region do not inhibit phi X174 gene A protein cleavage in vitro, but severely impair packaging of single-stranded plasmid DNA in viral coats.     

Regions of incompatibility in single-stranded DNA bacteriophages phi X174 and G4.

The intracellular presence of a recombinant plasmid containing the intercistronic region between the genes H and A of bacteriophage phi X174 strongly inhibits the conversion of infecting single-stranded phi X DNA to parental replicative-form DNA. Also, transfection with single-stranded or double-stranded phi X174 DNA of spheroplasts from a strain containing such a "reduction" plasmid shows a strong decrease in phage yield. This phenomenon, the phi X reduction effect, was studied in more detail by using the phi X174 packaging system, by which plasmid DNA strands that contain the phi X(+) origin of replication were packaged as single-stranded DNA into phi X phage coats. These "plasmid particles" can transduce phi X-sensitive host cells to the antibiotic resistance coded for by the vector part of the plasmid. The phi X reduction sequence in the resident plasmid strongly affected the efficiency of the transduction process, but only when the transducing plasmid depended on primosome-mediated initiation of DNA synthesis for its conversion to double-stranded DNA. The combination of these results led to a model for the reduction effect in which the phi X reduction sequence interacted with an intracellular component that was present in limiting amounts and that specified the site at which phi X174 replicative-form DNA replication takes place. The phi X reduction sequence functioned as a viral incompatibility element in a way similar to the membrane attachment site model for plasmid incompatibility. In the DNA of bacteriophage G4, a sequence with a similar biological effect on infecting phages was identified. This reduction sequence not only inhibited phage G4 propagation, but also phi X174 infection.     

Expression of a gene in a 400-base-pair fragment of colicin plasmid ColE2-P9 is sufficient to cause host cell lysis.

The colicin E2 immunity (ceiB) and lysis (celB) genes of colicin plasmid ColE2-P9 were cloned as a 900-base-pair insert under the control of the lac promoter in high-copy-number plasmid pUR222. Hosts carrying this plasmid were immune to colicin E2, produced increased amounts of immunity protein (molecular weight, 9,000) and two smaller proteins (molecular weights, 5,000 and 3,000), and lysed when incubated in medium containing isopropyl-beta-D-thiogalactopyranoside (IPTG). A 400-base-pair lacp-distal fragment derived from the insert in this plasmid was recloned in the same orientation into pUR222. Although hosts carrying this plasmid also lysed when grown in the presence of IPTG, they were sensitive to colicin E2 and produced increased amounts of the 5,000- and 3,000-molecular-weight proteins (but not the full-length immunity protein) when treated with IPTG. The results were consistent with the idea that expression of celB (production of the 5,000- and 3,000-molecular-weight proteins) is sufficient to cause host cell lysis in the absence of colicin production and derepression of the host cell SOS system.     

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.     

Assembly of bacteriophage phi X174: identification of a virion capsid precursor and proposal of a model for the functions of bacteriophage gene products during morphogenesis.

A capsomeric structure sedimenting with an S value of 108 in sucrose gradients was isolated from Escherichia coli infected with bacteriophage phi X174. The 108S material contained viral proteins F, G, H, and D, and the relative amounts of these proteins in the 108S material were similar to those in the infectious 132S particle, which has previously been described as a possible intermediate in the assembly of 114S phage particles. Electron micrographs indicated that the size and shape of the 108S material resemble those of the 132S particle. The 108S material contained no DNA, and its formation occurred independently of DNA synthesis. The 108S material accumulated in infected cells when viral DNA replication was prevented either by mutation in phage genes A or C or by removal of thymidine from a culture infected with wild-type phage or with a lysis gene E mutant. Upon restoration of thymidine to cells infected with the lysis gene E mutant and then starved of thymidine, the accumulated 108S material was converted to 132S particles and to 114S phage particles, implying that the 108S material is a precursor of phage particles. A model that proposes possible functions for the products of phi X174 genes A, B, C, D, F, and G during viral replication and phage maturation is described.     

Construction and characterization of recombinant plasmid DNAs containing sequences of the origin of bacteriophage phi X174 DNA replication

The synthetic DNA fragment (formula, see text) (corresponding to nucleotides 4299-4314 of the phi X DNA sequence) was cloned into either the AmpR gene or the KmR gene of plasmid pACYC 177. The DNA sequence of the KmR gene around the insertion site was determined by nucleotide sequence analysis of the pACYC 177 FnudII restriction DNA fragment N6 (345 b.p.). Of five selected plasmid DNAs, which contained inserted DNA sequences in the antibiotic resistance genes, the nucleotide sequences at and around these insertions were determined. Two recombinant plasmids (pFH 704 and pFH 614) contain the hexadecamer sequence in tandem (tail-to-tail and tail-to-head). In the recombinant plasmids pFH 812, pFH 903 and pFH 807 the DNA sequence homology with the phi X origin region was 14 (No. 4300-4313), 16 (No. 4299-4314) and 20 nucleotides (No. 4299-4318), respectively. None of the supercoiled recombinant plasmid DNAs is nicked upon incubation with phi X gene A protein. Moreover, the recombinant plasmid RFI DNAs cannot act as substitutes for phi X RFI DNA in the in vitro (+) strand synthesizing system. It has been shown earlier that single-stranded DNA, which contains the decamer sequence CAACTTGATA is efficiently nicked by the phi X gene A protein. The present results indicate that for nicking of double-stranded supercoiled DNA nucleotide sequence homology with the phi X origin region of more than 20 nucleotides is required. These results suggest a model for initiation of phi X RF DNA replication, which involves the presence of the recognition sequence CAACTTGATA of the phi X gene A protein as well as a second specific nucleotide sequence which is required for the binding of the phi X gene A protein. This binding causes local unwinding of the DNA double helix and exposure of the recognition sequence in a single-stranded form, which then can be nicked by phi X gene A protein.     

Gene X of bacteriophage f1 is required for phage DNA synthesis. Mutagenesis of in-frame overlapping genes

The gene II protein of bacteriophage f1 is a site-specific endonuclease required for initiation of phage viral strand DNA synthesis. Within gene II is another gene, X, encoding a protein of unknown function identical to the C-terminal 27% of the gene II protein, and separately translated from codon 300 (AUG) of gene II. By oligonucleotide mutagenesis, we constructed phage mutants in which this codon has been changed to UAG (amber) or UUG (leucine), and propagated them on cells carrying a cloned copy of gene X on a plasmid. The amber mutant makes no gene X protein, and cannot grow in the absence of the complementing plasmid; the leucine-inserting mutant can make gene X protein, and grows normally without the plasmid. Without gene X protein, phage DNA synthesis (particularly viral strand synthesis) is impaired. We discuss this finding in the context of other known in-frame overlapping genes (particularly genes A and A* of phage phi X174), many of which are also involved in the specific initiation of DNA synthesis, and suggest applications for the mutagenic strategy we employed.     

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.