DNA replication studies with coliphage 186. III. A single phage gene is required for phage 186 replication

We have shown that the BglII to BamHI (79.6% to 95.8%) region of the coliphage 186 chromosome can direct 186-specific replication. DNA sequencing of the region revealed five presumptive genes, CP80, CP81, CP83, CP84 and CP87. Surprisingly, alleles of the previously defined replication gene, A, were localized in both CP84 and CP87. We have successfully constructed a 186 minichromosome using the single gene CP87, and determined that CP84 was not concerned with replication, neither of a minichromosome nor of the phage. Rather, the replication defect seen with amber mutants of CP84 reflects a polarity effect on the downstream expression of CP87. We have concluded that CP87 is the only phage gene necessary for 186 replication, and have called it gene A.     

Genetic studies of coliphage 186. II. Genes associated with phage replication and host cell lysis

DNA synthesis in coliphage 186-infected cells was investigated. Phage 186 appeared to inhibit host DNA synthesis early in infection. The subsequent synthesis of phage 186 DNA was dependent on the product of 186 gene A. The product of gene B controlled both the production of late 186 proteins and the cessation of 186 DNA synthesis, and the products of genes O and P had no influence on 186 DNA synthesis. The product of gene P controlled host cell lysis, and the product of gene O may have some regulatory function.     

DNA replication studies with coliphage 186: the involvement of the Escherichia coli DnaA protein in 186 replication is indirect.

The inability of coliphage 186 to infect productively a dnaA(Ts) mutant at a restrictive temperature was confirmed. However, the requirement by 186 for DnaA is indirect, since 186 can successfully infect suppressed dnaA (null) strains. The block to 186 infection of a dnaA(Ts) strain at a restrictive temperature is at the level of replication but incompletely so, since some 20% of the phage specific replication seen with infection of a dnaA+ host does occur. A mutant screen, to isolate host mutants blocked in 186-specific replication but not in the replication of the close relative coliphage P2, which has no DnaA requirement, yielded a mutant whose locus we mapped to the rep gene. A 186 mutant able to infect this rep mutant was isolated, and the mutation was located in the phage replication initiation endonuclease gene A, suggesting direct interaction between the Rep helicase and phage endonuclease during replication. DNA sequencing indicated a glutamic acid-to-valine change at residue 155 of the 694-residue product of gene A. In the discussion, we speculate that the indirect need of DnaA function is at the level of lagging-strand synthesis in the rolling circle replication of 186.     

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).     

Packaging of coliphage lambda DNA. II. The role of the gene D protein

The gene D protein (pD) of coliphage λ is normally an essential component of the virus capsid. It acts during packaging of concatemeric λ DNA into the phage prohead and is necessary for cutting the concatemers at the cohesive end site (cos). In this report we show that cos cutting and phage production occur without pD in λ deletion mutants whose DNA content is less than 82% that of λ wild type. D-independence appears to result directly from DNA loss rather than from inactivation (or activation) of a phage gene. (1) In cells mixedly infected with undeleted λ and a deletion mutant, particles of the deletion mutant alone are efficiently produced in the absence of pD; and (2) D-independence cannot be attributed to loss of a specific segment of the phage genome. pD-deficient phage resemble pD-containing phage in head size and DNA ends; they differ in their extreme sensitivity to EDTA, greater density, and ability to accept pD.pD appears to act by stabilizing the head against disruption by overfilling with DNA rather than by changing the capacity of the head for DNA. This is shown by the observation that the amount of DNA packaged by a “headful” mechanism, normally in excess of the wild-type chromosome size, is not reduced in the absence of pD. In fact, pD is required for packaging headfuls of DNA. This implies that a mechanism exists for preventing the entry of excess DNA into the head during packaging of concatemers formed by deletion mutants, and we suggest that this is accomplished by binding of cos sites to the head.The above results show that pD is not an essential component of the nuclease that cuts λ concatemers at cos during packaging, and they imply that 82% of a wild-type chromosome length can enter the prohead in the absence of pD. Yet, pD is needed for the formation of cohesive ends after infection with undeleted phage. We propose two models to account for these observations. In the first, cos cutting is assumed to occur early during packaging. The absence of pD leads to release of packaged DNA and the loss of cohesive ends by end-joining. In the second, cos cutting is assumed to occur as a terminal event in packaging. pD promotes cos cutting indirectly through its effect on head stability. We favor the second model because it better explains the asymmetry observed in the packaging of the chromosomes of cos duplication mutants (Emmons, 1974).     

Requirement for a fluid host cell membrane in injection of coliphage T5 DNA.

Injection of T5 first-step-transfer DNA was prevented at 29 degrees C, after adsorption to an unsaturated fatty acid mutant grown on elaidate (phase transition at 35 degrees C). Local anesthetics, which increase membrane fluidity, did not inhibit injection above transition temperature and could even reverse the inhibition observed at 29 degrees C on elaidate cells.     

Adenovirus type 2 DNA replication. II. Termini of DNA replication.

Complete, mature adenovirus type 2 DNA molecules were isolated from virus-infected HeLa cells, pulse-labeled at 20 h postinfection in [3H]thymidine pulses shorter than the time necessary for one round of viral DNA replication. After digestion with the restriction endonucleases Eco RI, Hpa I, and Hind III, a temporal order of synthesis of different regions of the viral genome was established from the relative specific radioactivities in the restriction enzyme fragments. A comparison with the physical order of these fragments revealed the existence of two termini of DNA replication towards both the molecular right and left ends, respectively, of the viral chromosome.     

A new host gene (groPC) necessary for lambda DNA replication.

Summary The isolation of a bacterial mutation in a gene, designated groPC, which affects the growth of phages lambda and P2 is described. Lambda replication is severely limited in the strain, and some lambda mutations, which map in (or near) the P gene, allow growth. The gro mutation, groPC259, is recessive to wild type and maps between threonine (thr) and diaminopimelate (dapB) on the E. coli chromosome. The possibility that the groPC gene is concerned with host DNA replication is discussed.     

Stability of coliphage lambda DNA replication initiator, the lambda O protein.

The initiator of coliphage lambda DNA replication, lambda O protein, may be detected among other 35S-labeled phage and bacterial proteins by a method based on immunoprecipitation. This method makes it possible to study lambda O proteolytic degradation in lambda plasmid-harboring or lambda phage-infected cells; it avoids ultraviolet (u.v.)-irradiation of bacteria, used for depression of host protein synthesis, prior to lambda phage infection. We confirm the rapid decay of lambda O protein (half-time of 80 s), but we demonstrate the existence of a stable lambda O fraction. In the standard five minute pulse-chase experiments, 20% of synthesized lambda O is stable. The extension of the [35S]methionine pulse, possible in lambda plasmid-harboring cells, leads to a linear increase of this fraction, as if a part of the synthesized lambda O was constantly made resistant to proteolysis. Less than 5% of lambda O protein synthesized during one minute is transformed into a stable form. We presume that the stable lambda O is identical with lambda O present in the normal replication complex and thus protected from proteases. We cannot find any stable lambda O in Escherichia coli recA+ cells that were irradiated with u.v. light prior to lambda phage infection, but their recA- counterparts behave normally, suggesting that recA function interferes in the assembly of a normal replication complex in u.v.-irradiated bacteria. The stable lambda O found in lambda plasmid-harboring, amino acid-starved relA cells is responsible for the lambda O-dependent lambda plasmid replication that occurs in this system in the absence of lambda O synthesis. The existence of stable lambda O raises doubt concerning its role as the limiting initiator protein in the control of replication. Another significance of lambda O rapid degradation is proposed.     

Multiple interactions of a DNA-binding protein in vivo. II. Effects of host mutations on DNA replication of phage T4 gene 32 mutants.

A quantitative analysis has been made of the kinetics of disulphide bond formation, breakage, and rearrangement which occur during the folding and unfolding of the pancreatic trypsin inhibitor. The results have been used to infer the energetics of the protein conformational transitions which accompany each step.The folding transition is shown to be a co-operative process in which all intermediate states with one or two disulphide bonds are unstable relative to the unfolded, reduced protein and that in the fully folded conformation with three disulphide bonds. The approximate two-state nature of the transition at equilibrium was demonstrated experimentally. The folding transition of the inhibitor which has been determined kinetically is therefore analogous to that observed generally with other globular proteins.     

Genes for the establishment and maintenance of lysogeny by the temperate coliphage 186.

To identify the genes in coliphage 186 that are required for lysogeny, we isolated clear-plaque mutants. Complementation studies and DNA sequencing identified two genes, the cI gene for the immunity maintenance repressor and the cII gene, which is required only for the establishment of lysogeny. One mutant carried a change in the LexA-binding site controlling expression of the antirepression protein Tum.     

The role of DNA polymerase I-associated 5'-exonuclease in replication of coliphage M13 replicative-form DNA.

The conversion of both parental- and progeny-nascent open circular M13 RF DNA into covalently closed RF I is drastically reduced in an $\text{E. coli}$ mutant deficient in the 5′ → 3′ exonuclease associated with DNA polymerase I. The nascent progeny RF DNA also contains a significant proportion of fragments of smaller than unit length.     

Incomplete detachment of parental coliphage T4 DNA from the host cell membrane.

One-half of parental coliphage T4 DNA remains attached to the host membrane during infection and is not packaged into progeny phage. This DNA consists of equal parts of both strands.     

Effects of temperature and host cell growth phase on replication of F-specific RNA coliphage Q beta.

Human enteric viruses have been found in groundwater in the absence of fecal coliforms. Because detection of human enteric viruses is costly, time-consuming, and lacking in sensitivity, F-specific RNA (FRNA) coliphages, which infect Escherichia coli by attachment to F pili, are being examined for suitability as indicators of human enteric viruses in groundwater. Temperatures and host cell growth conditions that constrain F-pilus expression will limit FRNA coliphage replication in groundwater and wastewater, as is desirable in an indicator. Below 25 degrees C F-pilus synthesis ceases; FRNA coliphage Qbeta did not replicate below this temperature in batch cultures. One-step replication studies indicated that the replicative cycle is prolonged and that fewer progeny are released as the temperature decreases. The decreases in phage replication observed in the one-step replication studies were a consequence of fewer cells infected as the temperature was lowered or as host cells entered stationary phase. The numbers of phage particles released from infected cells did not change. The minimum temperature for replication of Qbeta, 25 degrees C, is not maintained in wastewater and does not occur in Wisconsin groundwater. On the basis of temperature and host cell growth phase, we have concluded that extensive replication of FRNA coliphages does not occur in wastewater and groundwater in Wisconsin and areas with similar cool climates.     

Genetic studies of coliphage P1. II. Relatedness to P7.

Using semiquantitative spot tests, 107 independently isolated amber mutants of P1 were shown to be rescued by a nonpermissive strain of Escherichia coli lysogenic for P7 (previously called phiamp), indicating extensive genetic relatedness between P1 and P7. The amount of rescue observed varied with mutants from different genetic linkage clusters of P1. Although these rescue tests cannot distinguish between recombination, complementation, transactivation, or combinations thereof, a major role is indicated for recombination.