Intermediates in bacteriophage Mu lysogenization of Escherichia coli him hosts.

Characterization of a putative intermediate in the Mu lysogenization pathway is possible in a variant Escherichia coli himD strain which exhibits greatly diminished lysogen formation. In this strain, most infecting Mu genomes form stable, transcribable, nonreplicating structures. Many of these genomes can be mobilized to form lysogens by a second Mu infection, which can be delayed by at least 100 min. This intermediate structure can be formed in the absence of Mu A or B function. We suggest that the inferred intermediate could be the previously reported protein-linked circular form of the Mu genome. Providing Mu B function from a plasmid enhances Mu lysogenization in this him strain, and the enhancement is much greater when both Mu A and B functions are provided.     

Growth of bacteriophage Mu in Escherichia coli dnaA mutants.

In one-step growth experiments we found that bacteriophage Mu grew less efficiently in nonreplicating dnaA mutants than in dnaA+ strains of Escherichia coli. Phage development in dnaA hosts was characterized by latent periods that were 15 to 30 min longer and an average burst size that was reduced by 1.5- to 4-fold. The differences in phage Mu development in dnaA and dnaA+ strains were most pronounced in cells infected at a low multiplicity and became less pronounced in cells infected at a high multiplicity. Many of these differences could be eliminated by allowing the arrested dnaA cells to restart chromosome replication just before infection. In continuous labeling experiments we found that infected dnaA strains incorporated 5 to 40 times more [methyl-3H]thymidine than did uninfected cells, depending on the multiplicity of infection. DNA-DNA hybridization assays showed that greater than 90% of this label was contained in phage Mu DNA sequences and that only small amounts of the label appeared in E. coli sequences. In contrast, substantial amounts of label were incorporated into both host and viral DNA sequences in infected dnaA+ cells. Although our results indicated that phage Mu development is not absolutely dependent on concurrent host chromosomal DNA replication, they did strongly suggest that host replication is necessary for optimal growth of this phage.     

Lysogenization by bacteriophage lambda

Summary An easy and sensitive way of measuring the proportion of E. coli cells which are lysogenized by lambda phage or lambda mutants has been devised. With this assay it was possible to analyse the lysogenic response as a function of the average phage input per cell. The results indicate that lysogenization of exponentially growing cells requires that the cells are infected by at least two phages able to replicate, or three or four phages unable to replicate.     

Behavior of bacteriophage Mu DNA upon infecton of Escherichia coli cells.

The question whether the ends of bacteriophage Mu DNA are fused to form a ring in host cells is critical to the understanding of the mechanism of integrative recombination between Mu DNA and host DNA. We have examined the fate of ³²P-labeled Mu DNA, after infection of sensitive and immune (lysogenic) cells, by sedimentation in sucrose gradients, ethidium bromide/CsCl density centrifugation and by electrophoresis of parental Mu DNA and its fragments in agarose gels. We find that the parental Mu DNA cannot be detected as covalently closed circles at any stage during the Mu life cycle. An interesting form of Mu DNA can be seen after superinfection of immune cells. This form sediments about twice as fast as the mature phage DNA marker in neutral sucrose gradients but yields linear molecules upon phenol extraction. Upon infection of sensitive cells, most of the parental DNA associates with a large complex, presumably containing the host chromosome. When Mu-sensitive cells are infected with unlabeled Mu particles and Mu DNA examined at different times after infection by fractionation in 0.3% agarose gels and hybridization with ³²P-labeled Mu DNA, Mu sequences are found to appear with the bulk host DNA as the phage lytic cycle progresses. However, no distinct replicative or integrative intermediate of Mu, that behaves differently from linear Mu DNA and is separate from the host DNA, can be detected.     

Mutants of Escherichia coli defective for replicative transposition of bacteriophage Mu.

We isolated 142 Hir- (host inhibition of replication) mutants of an Escherichia coli K-12 Mu cts Kil- lysogen that survived heat induction and the killing effect of Mu replicative transposition. All the 86 mutations induced by insertion of Tn5 or a kanamycin-resistant derivative of Tn10 and approximately one-third of the spontaneous mutations were found by P1 transduction to be linked to either zdh-201::Tn10 or Tn10-1230, indicating their location in or near himA or hip, respectively. For a representative group of these mutations, complementation by a plasmid carrying the himA+ gene or by a lambda hip+ transducing phage confirmed their identification as himA or hip mutations, respectively. Some of the remaining spontaneously occurring mutations were located in gyrA or gyrB, the genes encoding DNA gyrase. Mutations in gyrA were identified by P1 linkage to zei::Tn10 and a Nalr gyrA allele; those in gyrB were defined by linkage to tna::Tn10 and to a gyrB(Ts) allele. In strains carrying these gyrA or gyrB mutations, pBR322 plasmid DNA exhibited altered levels of supercoiling. The extent of growth of Mu cts differed in the various gyrase mutants tested. Phage production in one gyrA mutant was severely reduced, but it was only delayed and slightly reduced in other gyrA and gyrB mutants. In contrast, growth of a Kil- Mu was greatly reduced in all gyrase mutant hosts tested.     

Lysogenization of Escherichia coli by bacteriophage Lambda: complementary activity of the host's DNA polymerase I and ligase and bacteriophage replication proteins Q and P.

When bacteriophage lambda DNA replication is blocked by mutation in phage genes O or P, the efficiency of lysogenization drops to a very low value unless high multiplicities of infecting phage are used. Our results show that even at high multiplicity, lambda O or P mutants cannot efficiently lysogenize some hosts that are defective in either DNA polymerase I or DNA ligase. Covalent closure of infecting DNA molecules, a preliminary step for insertion according to Campbell's model and an obvious candidate for this lysogenization defect, appears to occur normally under our conditions. In addition, prophage excision as measured by the frequency of curing O- and P- lysogens seemed normal when tested in the poll- strain. These results suggest that the Escherichia coli enzymes DNA polymerase I and ligase, and phage proteins O and P, are able to provide some complementary activity whose function is required specifically for prophage integration.     

Stabilization of bacteriophage Mu repressor-operator complexes by the Escherichia coli integration host factor protein

All of the previously described effects of integration host factor (IHF) on bacteriophage Mu development have supported the view that IHF favours transposition-replication over the alternative state of lysogenic phage growth. In this report we show that, consistent with a model in which Mu repressor binding to its operators requires a particular topology of the operator DNA, IHF stimulates repressor binding to the O1 and O2 operators and enhances Mu repression. IHF would thus be one of the keys, besides supercoiling and the H-NS protein, that lock the operator region into the appropriate topological conformation for high-affinity binding not only of the phage transposase but also of the phage repressor.     

Polypeptides encoded by the early region of bacteriophage Mu synthesized in minicells of Escherichia coli

The preferential interaction of calf brain tubulin with glycerol in an aqueous buffer (0.01 m-NaP_i, 0.02 m-NaCl, 10^?4m-GTP, pH 7.0) has been investigated by densimetry. The apparent specific volumes of tubulin at constant chemical potential of the diffusible components were determined at 0, 10, 20 and 30% ($\text{v}\text{v}$) glycerol. Application of multicomponent solution thermodynamics shows that tubulin is preferentially hydrated in aqueous glycerol solvent and that such interaction results in thermodynamic destabilization of the system by raising the chemical potentials of both glycerol and tubulin. Interpreted in terms of the Wyman linkage function, the unfavorable free energy change brought about by the preferential protein-glycerol interaction can account for the glycerol enhancement of tubulin self-assembly in vitro into microtubules as well as offer a rationale for glycerol stabilization of the native tubulin conformation.     

Virulence in bacteriophage Mu: a case of trans-dominant proteolysis by the Escherichia coli Clp serine protease.

The importance of proteases in gene regulation is well documented in both prokaryotic and eukaryotic systems. Here we describe the first example of genetic regulation controlled by the Escherichia coli Clp ATP-dependent serine protease. Virulent mutants of bacteriophage Mu, which carry a particular mutation in their repressor gene (vir mutation), successfully infect Mu lysogens and induce the resident Mu prophage. We show that the mutated repressors have an abnormally short half-life due to an increased susceptibility to Clp-dependent degradation. This susceptibility is communicated to the wild type repressor present in the same cell, which provides the Muvir phages with their trans-dominant phenotype. To our knowledge this is the first case where the instability of a mutant protein is shown to trigger the degradation of its wild type parent.     

Involvement of Escherichia coli K-12 DNA polymerase I in the growth of bacteriophage Mu.

We examined several aspects of bacteriophage Mu development in Escherichia coli strains that carry mutations in the polA structural gene for DNA polymerase I (PolI). We found that polA mutants were markedly less efficient than PolI wild-type (PolI+) strains in their capacity to form stable Mu lysogens and to support normal lytic growth of phage Mu. The frequency of lysogenization was determined for polA mutants and their isogenic PolI+ derivatives, with the result that mutants were lysogenized 3 to 8 times less frequently than were PolI+ cells. In one-step growth experiments, we found that phage Mu grew less efficiently in polA cells than in PolI+ cells, as evidenced by a 50 to 100% increase in the latent period and a 20 to 40% decrease in mean burst size in mutant cells. A further difference noted in infected polA strains was a 10-fold reduction in the frequency of Mu-mediated transposition of chromosomal genes to an F plasmid. Pulse labeling and DNA-DNA hybridization assays to measure the rate of phage Mu DNA synthesis after the induction of thermosensitive prophages indicated that phage Mu replication began at about the same time in both polA and PolI+ strains, but proceeded at a slower rate in polA cells. We conclude that PolI is normally involved in the replication and integration of phage Mu. However, since phage Mu does not exhibit an absolute requirement for normal levels of PolI, it appears that residual PolI activity in the mutant strains, other cellular enzymes, or both can partially compensate for the absence of normal PolI activity.     

ClpX protein of Escherichia coli activates bacteriophage Mu transposase in the strand transfer complex for initiation of Mu DNA synthesis.

During transposition bacteriophage Mu transposase (MuA) catalyzes the transfer of a DNA strand at each Mu end to target DNA and then remains tightly bound to the Mu ends. Initiation of Mu DNA replication on the resulting strand transfer complex (STC1) requires specific host replication proteins and host factors from two partially purified enzyme fractions designated Mu replication factors alpha and beta (MRFalpha and beta). Escherichia coli ClpX protein, a molecular chaperone, is a component required for MRFalpha activity, which removes MuA from DNA for the establishment of a Mu replication fork. ClpX protein alters the conformation of DNA-bound MuA and converts STC1 to a less stable form (STC2). One or more additional components of MRFalpha (MRFalpha2) displace MuA from STC2 to form a nucleoprotein complex (STC3), that requires the specific replication proteins and MRFbeta for Mu DNA synthesis. MuA present in STC2 is essential for its conversion to STC3. If MuA is removed from STC2, Mu DNA synthesis no longer requires MRFalpha2, MRFbeta and the specific replication proteins. These results indicate that ClpX protein activates MuA in STC1 so that it can recruit crucial host factors needed to initiate Mu DNA synthesis by specific replication enzymes.     

Transduction of bacteriophage Mu by bacteriophage T1.

Phage T1 transduces phage Mu PFU from Mu-lysogenic donor cells to sensitive recipient cells. The efficiency of transduction depends on the chromosomal location of the Mu prophage. T1, therefore, appears to package different regions of the bacterial chromosome with different efficiencies. Although T1 transduces bacterial markers with different efficiencies, there is no direct correlation between the efficiency of transduction of a bacterial marker and the efficiency of transduction of Mu PFU from donor cells with the Mu prophage located in that marker.     

Genetic analysis of Escherichia coli min81 mutation blocking the development of bacteriophage Mu

Data characterizing mim81 mutation obtained by the method for direct selection of transposition mutations are presented. The development of Mu is shown to be dramatically suppressed in the mutant strain both upon infection and after induction from the lysogenic state. Frequencies of lysogenization and mini-Mu-dependent formation of cointegrates in the mutant strain are comparable with those in the wild-type strain. Mu development prohibition is removed if expression of early Mu gene is provided from the modified Pe promoter. The results obtained make us believe that the mechanism of mim81 mutation action involves reduction of early gene expression to the level that is sufficient for Mu DNA integration into the chromosome during infection and for single replicative events, but insufficient for vegetative development of bacteriophage Mu.