Phage Morphogenesis Research Articles

Recognition and cleavage of the bacteriophage P1 packaging site ( pac) : I. Differential processing of the cleaved ends in vivo

The packaging of bacteriophage P1 DNA into viral capsids is initiated at a specific DNA site called pac. During packaging, that site is cleaved and at least one of the resulting ends is encapsidated into a P1 virion. We show here that pac is located on a 620 base-pair fragment of P1 DNA ( EcoRI-20). When that fragment is inserted into the chromosome of cells that are then infected with P1, packaging of host DNA into phage particles is initiated at pac and proceeds down the chromosome, unidirectionally, for about five to ten P1 “headfuls” (about 5 × 10 5 to 10 × 10 5 bases of DNA). Using an assay for pac cleavage that does not depend on DNA packaging, we have identified a set of five amber mutations that are mapped adjacent to pac, and that define a gene (gene 9) essential for pac cleavage. Amber mutations that are located in genes necessary for viral capsid formation (genes 4, 8 and 23), or in a gene necessary for “late” protein synthesis (gene 10), do not affect pac cleavage. The latter result suggests that the synthesis of the pac cleavage protein is not regulated co-ordinately with other phage morphogenesis proteins. The products of pac cleavage were analyzed using two different DNA substrates. In one case, a single copy of pac was placed in the chromosome of P1-sensitive cells. When those cells were infected with P1, we could detect the cleavage of as much as 70% of the pac-containing DNA. The pac end destined to be packaged in the virion was detected five to 20 times more efficiently than was the other end. Since this result is obtained whether or not the infecting P1 phage can encapsidate the cut pac site, the differential detection of pac ends is not simply a consequence of one end being packaged and the other not. In a second case, pac was located in cells on a small (5 × 10 3 bases) multicopy plasmid. When those cells were infected with P1, neither pac end was detected efficiently after P1 infection, unless the cells carried a recBCD − mutation. In recBCD − cells, the results with plasmid- pac substrates were similar to those obtained with chromosomally integrated pac substrates. We interpret these results to mean that, following pac cleavage, the end destined to be packaged is protected from cellular nucleases while the other end is degraded by the action of at least two nucleases, one of which is the product of the host recBCD gene. If the two ends are within several thousand base-pairs, then degradation by the RecBCD nuclease proceeds rapidly through the entire molecule and neither end is detected efficiently. If the two ends are separated by an Escherichia coli chromosome, then degradation by the RecBCD nuclease from the unprotected end does not affect recovery of the protected end. In the absence of the RecBCD nuclease, a second nuclease degrades DNA from the unprotected end. That degradation, however, rarely proceeds far enough to affect recovery of the protected pac end.

Morphogenesis of f1 filamentous bacteriophage: Increased expression of gene I inhibits bacterial growth

We have cloned the gene I sequence of the filamentous bacteriophage f1 downstream from the lambda leftward promoter on a plasmid that also contains the temperature-sensitive lambda represser, cI857. Temperature induction of gene I protein (pI) resulted in rapid cessation of growth. This inhibition appears to involve a rapid decrease in synthesis of host protein and RNA. The ability of pI to cause this inhibition is not dependent on thioredoxin, a host factor that is necessary for phage morphogenesis and has been shown by genetic data to interact with pI. The inhibition does not appear to be mediated by the amino half of the protein, as induction of an identical plasmid construction of an amber mutant positioned two-thirds along gene I, does not affect cell growth. Analysis of the transcription products from the cloned gene I confirmed previous suggestions that a transcription terminator exists in the amino-terminal portion of the gene. In addition, there is no detectable promoter activity in the 152 bases immediately upstream from the gene. These data and the inability to overproduce pI argue for down-regulation of pI production. Radioactive labeling of proteins in maxi-cells and normal Escherichia coli cells identifies pI as a protein of about 39,000 M r that partitions with the cell envelope. Pulse-chase experiments suggest that pI is not processed to any appreciable extent.

Aspects of the Growth and Regulation of the Filamentous Phages

Publisher Summary This chapter describes two series of studies of the physiology and genetics of bacteriophage fl, with implications beyond the phage itself. It discusses the regulation of filamentous phage DNA synthesis and the unexpected participation of thioredoxin in phage morphogenesis. Phages in a lytic cycle do not need to limit their overall expression— their main interest is precisely to gain maximum expression at as great an expense to the host as possible. Any control they exhibit is meant to manage their own course of development and streamline the exploitation of the hapless host. Filamentous phages (fl, M13, and fd), on the other hand, are nonlytic; instead they grow continuously as long-term parasites (8-l0) and so must consider the appropriate balance of host and phage metabolism. These phages are particularly adept at this kind of control. Thioredoxin is a heat stable, ubiquitous protein containing active-site sulfhydryl groups that readily and reversibly form a disulfide on oxidation. In addition to extensive biochemical studies, thioredoxin has also been studied by X-ray crystallographic methods. Thioredoxin is also a constituent of phage T7 DNA polymerase, an enzyme that, when isolated, contains two polypeptide chains, one is the product of T7 gene 5 and the other is thioredoxin.

The complete 30-base-pair origin region of bacteriophage phi X174 in a plasmid is both required and sufficient for in vivo rolling-circle DNA replication and packaging.

The origin of replication of the isometric single-stranded DNA bacteriophages is located in a specific sequence of 30 nucleotides, the origin region, which is highly conserved in these phage genomes. Plasmids harboring this origin region are subject to rolling-circle DNA replication and packaging of single-stranded (ss) plasmid DNA into phage coats in phi X174 or G4-phage-infected cells. This system was used to study the nucleotide sequence requirements for rolling-circle DNA replication and DNA packaging employing plasmids which contain the first 24, 25, 26, 27, 28 and the complete 30-base-pair (bp) origin region of phi X174. No difference in plasmid ss DNA packaging was observed for plasmids carrying only the 30-bp origin region and plasmids carrying the 30-bp origin region plus surrounding sequences (i.e. plasmids carrying the HaeIII restriction fragment Z6B of phi X174 replicative-form DNA). This indicates that all signals for DNA replication and phage morphogenesis are contained in the 30-bp origin region and that no contribution is made by sequences which immediately surround the origin region in the phi X174 genome. The efficiency of packaging of plasmid ssDNA for plasmids containing deletions in the right part of the origin region decreases drastically when compared with the plasmid containing the complete 30-bp origin region (for a plasmid carrying the first 28 bp of the origin region to approximately 5% and 0.5% in the phi X174 and G4 systems respectively). Previous studies [Fluit, A.C., Baas, P.D., van Boom, J.H., Veeneman, G.H. and Jansz, H.S. (1984) Nucleic Acids Res. 12, 6443--6454] have shown that the presence of the first 27 bp of the origin region is necessary as well as sufficient for cleavage of the viral strand in the origin region by phi X174 gene A protein. Moreover, Brown et al. [Brown, D.R., Schmidt-Glenewinkel, T., Reinberg, D. and Hurwitz, J. (1983) J. Biol. Chem. 258, 8402--8412] have shown that omission of the last 2 bp of the origin region does not interfere with phi X174 rolling-circle DNA replication in vitro. Our results therefore suggest that for optimal phage development in vivo, signals in the origin region are utilized which have not yet been noticed by the in vitro systems for phi X174 phage DNA replication and morphogenesis.

The J gene of bacteriophage phi X174: in vitro analysis of J protein function.

The J protein of phi X174 is a small, highly basic protein and is a component of the phage capsid. We have investigated the role of J protein during single-stranded viral DNA synthesis and phage morphogenesis by using an in vitro system composed of purified viral and host components (Aoyama et al., Proc. Natl. Acad. Sci. U.S.A. 80:4195-4199, 1983). The characterization of the products made in the presence and absence of J protein shows that J protein is not required for viral DNA synthesis, but is required for the packaging of DNA into infectious phage. The ability of J protein to bind to double-stranded DNA as well as single-stranded DNA and other interactions with DNA suggest a model in which J protein binds to double-stranded, replicative form DNA and enters the phage prohead by remaining bound to viral DNA as it is encapsidated.

Nucleotide sequence and genetic organization of the genome of the N-specific filamentous bacteriophage IKe: Comparison with the genome of the F-specific filamentous phages M13, fd and f1

The nucleotide sequence and genetic organization of the genome of the N-specific filamentous single-stranded DNA phage IKe has been established and compared with that of the F-specific filamentous phages M13, fd and f1 (Ff). The IKe DNA sequence comprises 6883 nucleotides, which is 476 (475) nucleotides more than the nucleotide sequence of the Ff genome. The data indicate that IKe and Ff have evolved from a common ancestor (overall homology approx. 55%) and that their genomes contain ten homologous genes, the order of which is identical. Similar to Ff, the major coat protein and the gene III-encoded pilot protein of IKe are synthesized via precursor molecules. The extent of homology between the genes of IKe and Ff differs significantly from one gene to another. Genes that code for viral capsid proteins are less homologous than genes whose products are involved in the processes of DNA replication and phage morphogenesis. During evolution, large nucleotide sequence rearrangements have occurred in the gene (gene III) whose product is needed for the attachment of the virion to the conjugative pili of the host cell, suggesting that these rearrangements have led to phages with different host specificities. Extensive nucleotide sequence homology was noted between the structural elements involved in DNA replication and phage morphogenesis, indicating that the mechanisms involved in DNA replication and morphogenesis are highly conserved.

The bacteriophage f1 morphogenetic signal and the gene V protein/phage single-stranded DNA complex

The region of the bacteriophage f1 genome near the gene IV/intergenic region (IG) junction previously was shown to contain a sequence necessary for efficient packaging of single-stranded (SS) DNA into phage particles (the f1 “morphogenetic signal”) [ G. P. Dotto, V. Enea, and N. D. Zinder (1981) Virology 114, 463–473]. The DNA content of f1 phage/pBR322 chimeric plasmid-transformed, phage-infected bacteria has been investigated. The chimeric plasmids, constructed by Dotto et al. (1981) , contained the f1 origins of DNA replication and, in some cases, also contained the “morphogenetic signal.” Chimeric plasmid SS DNA was detected in phage-infected bacteria harboring any one of these chimeric plasmids, and the majority of this SS DNA was found complexed to the phage gene V protein. Therefore, the “morphogenetic signal” is not required for the formation of the gene V protein/f1 SS DNA complex but instead must function at a later stage of filamentous phage morphogenesis.

Cloning vectors that yield high levels of single-stranded DNA for rapid DNA sequencing

We have constructed chimeric plasmid vectors with the origin and intergenic region from M 13 phage cloned into the PvuII (pZ145) and AhaIII (pZ150, pZ152) sites of pBR322. In the absence of M13 phage, these plasmids replicate like any other ColE1-derived plasmid and confer both ampicillin and tetracycline resistance (Amp, Tet). Upon infection with M13 phage, the viral origin present on the plasmids permits phage-directed plasmid replication and results in high yields of single-stranded (ss) plasmid DNA in M13-like particles. This ssDNA, which represents only one of the plasmid strands, is useful as a substrate for rapid DNA sequence determination by the dideoxy sequencing method described by Sanger et al. (1977). Since these plasmids contain an intact pBR322, the intergenic region can be transferred onto most pBR322 derivatives to yield ss plasmid DNA without affecting the recipient plasmid for further studies. We also constructed a deletion derivative of pZ145, plasmid pZ146, that does not exhibit interference with the growth of the M13 helper, although this plasmid is encapsidated into phage particles. This result confirms the theory that the intergenic region consists of two domains: one domain being a segment involved in phage morphogenesis and the other being a region of functional origin which interferes with M13 replication.

Involvement of the bacterial groM gene product in bacteriophage T7 reproduction. II. A reduced level of ion concentrations causes the blockage of T7 maturation in K-12-M cells.

Cellular leakage observed in Escherichia coli K-12-M shortly after T7 infection might be the cause of arrested phage morphogenesis. We observed in this strain, but not in the normal host, a drastic reduction of the intracellular concentration of potassium (60%), magnesium (40%), putrescine (90%), and spermidine (40%), whereas ATP was not significantly reduced. Leakage started about 1 min after the addition of phage and was arrested 3 to 5 min postinfection. Larger molecules such as o-nitrophenyl-beta-D-galactopyranoside could not enter the cells, showing that the permeability of the membrane was not generally affected. To prevent their leakage, we increased the outside concentrations of several small molecules and ions. The yield of progeny phage was substantially increased by the addition of 100 mM MgSO4.

Role of gene 8 product in morphogenesis of bacteriophage T3

The product of gene 8 (gp8) of T3 phage is one of the minor head proteins located at the phage head-tail junction. To determine the role of gp8, an amber (8 −) and four temperature-sensitive mutants ( ts8) were characterized by sedimentation analysis, polyacrylamide gel electrophoresis, and extract complementation. Neither DNA-containing particles nor empty particles were formed in cells infected with 8 −. In addition, prohead assembly was greatly reduced. Prohead assembly was also blocked in cells infected with all ( ts8) mutants at 42° and with some ( ts8) even at 37°. Proheads containing gp ts8 were converted to empty heads when cell lysates were treated with chloroform. The protein compositions of proheads showed that the minor head proteins, gp8, gp15, and gp16, were lost from proheads formed in cells infected with ( ts8) but these minor proteins were present in proheads formed in cells infected with double mutants of ( ts8) and 5 − or 19 −, which are defective in DNA synthesis or DNA maturation, respectively. In vitro complementation experiments suggested that a is mutation in gene 8 affected not only DNA packaging but also subsequent assembly steps. From these results, it is concluded that gp8 plays multiple roles in T3 phage morphogenesis, including prohead assembly, prohead stabilization, DNA packaging, and subsequent events.

Morphogenesis of filamentous bacteriophage f1: Orientation of extrusion and production of polyphage

Crosslinking reagents were used to interrupt the process of filamentous phage morphogenesis and investigate the orientation in which nascent virions are extruded through the host cell membrane. Infected bacteria with emerging phage particles were crosslinked with glutaraldehyde. Immunoferritin-labeling studies on these emerging phage using anti-A protein IgG suggested that extrusion begins with the C protein end. To confirm this, phage extruding from infected bacteria were frozen using the reversible crosslinker dimethyl 3,3′-dithiobis-propionimidate and fragments of emerging phage were isolated by shearing. Protein analysis of these fragments showed them to be enriched in C protein relative to A protein, as predicted if phage extrusion begins with the C protein end. The production of multiple-length phage particles (polyphage) by nonpermissive bacterial hosts infected with amber mutant phage strains was also studied. Polyphage were produced upon infection with amber mutants in genes III, VI, VII, and IX which code for proteins found at the ends of the mature phage particle. No polyphage were produced by mutants in the other genes tested. Gene III amber mutants produce noninfective polyphage, but those produced by genes VII and IX are infective. Gene VI amber mutants appear to produce unstable, noninfective polyphage particles.

Genetic studies of coliphage 186. I. Genes associated with phage morphogenesis.

In coliphage 186, 22 essential genes were defined by complementation studies with amber mutants. Eighteen genes were associated with phage morphogenesis: 11 with phage tail formation, and 7 with phage head formation. The remaining four genes are discussed in the accompanying paper (S. M. Hocking and J. B. Egan, J. Virol. 44:1068-1071, 1982).

Participation of the host protein(s) in the morphogenesis of bacteriophage P22.

Spontaneous mutants of S. typhimurium resistant to thiolutin are conditionally non-permissive for phage P22 development (Joshi and Chakravorty 1979). At 40 degree C non-infective phage particles are produced. Phage development in two nonpermissive hosts (18/MC4 and 153/MC4) has been studied in detail. The steps at which the phage morphogenesis is interfered with differ in the two mutants. The electron micrograph of the particles produced in the mutant 18/MC4 reveals the presence of normal-looking particles; these particles contain phage DNA, adsorb to the permissive host but fail to inject their DNA. The particles produced in the mutant 153/MC4 which fail to adsorb to the host are found to be tail fibre-less. These observations indicate the involvement of host protein(s) in phage P22 morphogenesis.

Evidence that the two Escherichia coli groE morphogenetic gene products interact in vivo.

The Escherichia coli groEL and groES gene products are essential for both phage morphogenesis and bacterial growth. Although the gene products have been identified, their exact roles in these processes are not known. We have isolated mutations in the groEL gene that suppress defects in the groES gene. These intergenic suppressors were shown to map in the groEL gene by a variety of genetic and biochemical analyses. These results suggest that the two morphogenetic gene products interact in vivo and help to explain why mutations in either gene exhibit the same phenotype with respect to lambda head assembly and bacterial growth.

Identification of a structural hairpin in the filamentous chimeric phage M13G ori1

M13G ori1 is a filamentous chimeric bacteriophage constructed in vitro from the replicative forms of two single-stranded DNA viruses by the insertion of a 2216-base fragment of G4 viral DNA into the intergenic region of M13 (Kaguni & Ray, 1979). The cloned insert contains a functional G4 origin of complementary strand synthesis (ori C). We have tested the model in which either the M13 ori C or the G4 ori C in M13G ori1 is located at the end of the virion. By photochemically crosslinking the DNA strands in situ with hydroxymethyl trioxsalen, we determined that the chimeric DNA is oriented with the most stable M13 hairpin, which is presumably non-essential for replication, at one end of the virus. This is the same hairpin as that found at the end of the closely related wild-type fd virion. The M13 ori C and G4 ori C are, respectively, 2333 and 1244 bases away from the end hairpin in the chimeric phage. The G4 insert is between the M13 ori C and this most stable hairpin located at the end of the virion. Thus, location of the ori C at the end of the filamentous virion is not a necessary requirement for initiation of replication. A probable role for the end hairpin in phage morphogenesis is proposed.

Identification of a second Escherichia coli groE gene whose product is necessary for bacteriophage morphogenesis.

Previous work has uncovered the existence of an Escherichia coli locus, groE, that is essential for bacterial growth, lambda phage and T4 phage head morphogenesis, and T5 phage tail assembly. Our genetic and biochemical analyses of lambda groE+ transducing phages and their deletion and point mutant derivatives show that the groE locus consists of two closely linked genes. One groE gene, groEL, has been shown to encode the synthesis of a 65,000 Mr polypeptide, whereas the second, groES, codes for the synthesis of a 15,000 Mr polypeptide. About half of the groE- bacterial isolates fall into the groES complementation group. GroE mutations in either gene cause similar phenotypes, with respect to lambda phage head morphogenesis and bacterial growth at nonpermissive temperatures.

Studies on the functional relationship between .PHI.X174 and S13 coded proteins using an in vitro DNA synthesis system.

The functional relationship between bacteriophage φX174 and SI3 gene products was investigated using an in vitro DNA synthesis system in two stages: (a), replication of the closed circular duplex form DNA (RF to RF) and (b) synthesis of single-stranded DNA from RF and formation of the intermediate 50S complex in phage morphogenesis (RF to 50S complex). Partially purified gene A products of φX174 and S13 were able to function reciprocally in the stage from RF to RF replication. In the stage of RF to 50S complex, extracts from Escherichia coli infected with φX174 catalyzed the formation of 50S complex from S13 RF DNA. Extracts from E. coli infected with SI3 also had the ability to catalyze the formation of 50S complex from φX174 RF DNA. Thus, all gene products required for DNA replication and phage morphogenesis were found to function reciprocally between φX174 and S13.

Temperature-induced synthesis of specific proteins in Escherichia coli: evidence for transcriptional control.

Synthesis of several protein chains of Escherichia coli is markedly, though transiently, induced upon shift-up of a log-phase culture to or above the critical temperature (about 34 degrees C). Such induction occurs coordinately for at least three protein chains (76K, 73K, and 64K) examined. Studies of initial kinetics of induction using a specific inhibitor of transcription (rifampin) revealed that induction occurs at the level of transcription with very little lag, though actual synthesis of messenger ribonucleic acids and proteins requires about 1 min when temperature is shifted up from 30 to 42 degrees C. Evidence suggests that E. coli cells somehow "recognize" the temperature change and activate transcription of several distinct operons, one of which contains the mop (morphogenesis of phages; groE) gene.

Genetic and physical characterization of lambda transducing phages (lambda frdA) containing the fumarate reductase gene of Escherichia coli K12.

Two types of fumarate reductase transducing phages, lambda frdA, carrying the wild-type frdA gene but differing in the orientation of a R.HindIII fragment of bacterial DNA were isolated from populations of recombinant transducing phages by their ability to complement the lesions of frdA mutants of E. coli. In lysogens, the cloned frdA gene was controlled by its own promoter and was fully responsive to normal regulatory stimuli. The lambda frdA phages would not complement the defects of succinate dehydrogenase (sdh) mutants. Genetic studies showed that the R.HindIII fragment contains ampA, the cis-acting regulatory locus for the chromosomal beta-lactamase gene ampC. No evidence for the presence of other markers was detected but the bacterial segment could be extended to produce plaque-forming phage derivatives containing the amp operon and a gene concerned with bacteriophage morphogenesis, groE(mop). A physical map of the 4.9 kb R.HindIII fragment was constructed by restriction analysis and flanking fragments were identified by DNA:DNA hybridization analysis. The frdA region contained a single asymmetric R.EcoRI target 3.33 kb from one end and the orientation of the physical map with respect to the E.coli linkage map was established.

Structure and functions of the bacteriophage P22 tail protein.

The product of gene 9 (gp9) of Salmonella typhimurium bacteriophage P22 is a multifunctional structural protein. This protein is both a specific glycosidase which imparts the adsorption characteristics of the phage for its host and a protein which participates in a specific assembly reaction during phage morphogenesis. We have begun a detailed biochemical and genetic analysis of this gene product. A relatively straightforward purification of this protein has been devised, and various physical parameters of the protein have been determined. The protein has an s(20,w) of 9.3S, a D(20,w) of 4.3 x 10(-7) cm(2)/s, and a molecular weight, as determined by sedimentation equilibrium, of 173,000. The purified protein appears as a prolate ellipsoid upon electron microscopic examination, with an axial ratio of 4:1, which is similar to the observed shape when it is attached to the phage particle. The molecular weight is consistent with the tail protein being a dimer of gp9 and each phage containing six of these dimers. An altered form of the tail protein has been purified from supF cells infected with a phage strain carrying an amber mutation in gene 9. Phage "tailed" with this altered form of gp9 adsorb to susceptible cells but form infectious centers with a severely reduced efficiency (ca. 1%). Biochemical analysis of the purified wild-type and genetically altered tail proteins suggests that loss of infectivity correlates with a loss in the glycosidase activity of the protein (2.5% residual activity). From these results we propose that the glycosidic activity of the P22 tail protein is not essential for phage assembly or adsorption of the phage to its host but is required for subsequent steps in the process of infection.