A new component of bacteriophage Mu replicative transposition machinery: the Escherichia coli ClpX protein

We have shown previously that some particular mutations in bacteriophage Mu repressor, the frameshift vir mutations, made the protein very sensitive to the Escherichia coli ATP-dependent Clp protease. This enzyme is formed by the association between a protease subunit (ClpP) and an ATPase subunit. ClpA, the best characterized of these ATPases, is not required for the degradation of the mutant Mu repressors. Recently, a new potential ClpP associated ATPase, ClpX, has been described. We show here that this new subunit is required for Mu vir repressor degradation. Moreover, ClpX (but not ClpP) was found to be required for normal Mu replication. Thus ClpX has activities that do not require its association with ClpP. In the pathway of Mu replicative transposition, the block resides beyond the strand transfer reaction, i.e. after the transposition reaction per se is completed, suggesting that ClpX is required for the transition to the formation of the active replication complex at one Mu end. This is a new clear-cut case of the versatile activity of polypeptides that form multi-component ATP-dependent proteases.     

Structure-function relationships in the transposition protein B of bacteriophage Mu

The B-protein of phage Mu, which is required for high frequency intermolecular transposition in vivo, shows ATPase activity in vitro, binds nonspecifically to DNA, and stimulates intermolecular strand transfer. To elucidate the structural bases for B-protein function, it was subjected to limited proteolysis with two different proteases, trypsin and chymotrypsin. The resulting fragments were mapped by amino acid sequencing. These data show that the B-protein is organized in two domains: an amino-terminal domain of 25 kDa and a carboxyl-terminal domain of 8-kDa. A fragment analogous to the amino-terminal domain, produced by deleting the 3' end of a cloned B gene, proved to be insoluble and had to be renatured after elution from a sodium dodecyl sulfate gel. The renatured protein retains ATP-binding activity and to a lesser extent the DNA-binding activity of the MuB protein, but is unable to hydrolyze ATP or function in transposition. We also show in this study that efficient DNA-strand transfer by the B-protein occurs even in the absence of a detectable ATPase activity or in the presence of adenosine 5'-O-(thio)triphosphate (ATP gamma S).     

Synchronization of bacteriophage Mu DNA replicative transposition: analysis of the first round after induction.

The lytic cycle of bacteriophage Mu includes a large number of coupled DNA replication and integration events, each of which is equivalent in several respects to the process of transposition of genetic elements. To aid us in studying the process of Mu DNA replicative transposition, we developed a technique for synchronizing the first round of replication following induction of a lysogen. Synchronization was achieved by inducing a lysogen in the absence of DNA replication for a time sufficient to develop the potential for Mu DNA replication in all cells in the population; upon release of the inhibition of replication, a synchronized round of Mu DNA replication was observed. Development of the potential for Mu DNA replication in the entire population took approximately 12 min. Protein synthesis was required for development of the potential, but the requirement for protein synthesis was satisfied by approximately 9 min suggesting that other, as yet unspecified, reactions occupied the last 3 min. Replication proceeded predominantly from the left end of the prophage, though a significant amount of initiation from the right end was observed. The usefulness of the technique for studying the mechanism of replicative transposition and the end products of a single round of replication are discussed.     

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.     

Host DNA replication forks are not preferred targets for bacteriophage Mu transposition.

Bacteriophage Mu DNA integration in Escherichia coli strains infected after alignment of chromosomal replication was analyzed by a sandwich hybridization assay. The results indicated that Mu integrated into chromosomal segments at various distances from oriC with similar kinetics. In an extension of these studies, various Hfr strains were infected after alignment of chromosomal replication, and Mu transposition was shut down early after infection. The positions of integrated Mu copies were inferred from the transfer kinetics of Mu to an F- strain. Our analysis indicated that the location of Mu DNA in the host chromosome was not dependent on the positions of host replication forks at the time of infection. However, the procedure for aligning chromosomal replication affected DNA transfer by various Hfr strains differently, and this effect could account for prior results suggesting preferential integration of Mu at host replication forks.     

Electron microscopic analysis of in vitro transposition intermediates of bacteriophage Mu DNA

Bacteriophage Mu is a highly efficient transposon and the only moveable element for which an in vitro transposition system has been reported. Recently, this system has been used by Craigie and Mizuuchi [Cell 41 (1985) 867-876] to identify and biochemically characterize intermediates in the transposition process. We have utilized the in vitro transposition system to generate intermediates in the transposition process and have analyzed these intermediates by electron-microscopic methods. Partial denaturation mapping has shown the intermediates to be theta-shaped structures in which the phi X174 target DNA is joined to the mini-Mu plasmid at the ends of the Mu genome. Our results are in agreement with the previous biochemical studies and the type of intermediate we observe is exactly what is predicted by the Shapiro model of transposition [Proc. Natl. Acad. Sci. USA 76 (1979) 1933-1937].     

On the molecular length of the replicative form DNA of bacteriophage phiX174

This paper describes an electron microscopic study of the circular replicative form DNA of bacteriophage φX174. The study has been carried out using a preparative technique in which the DNA molecules are adsorbed from solution on to the cleavage surface of mica and visualized in the electron microscope as a metal-shadowed replica (Gordon &; Kleinschmidt, 1969,1970). Contour lengths of open circular molecules were measured in samples obtained from preparations in which the following experimental parameters were varied: the ionic strength of the solution from which the DNA was adsorbed on the mica and the way in which the molecules were dried before shadowing. At the 0.05 significance level, varying these parameters had no effect on the mean length and variances of samples of molecules obtained from five experiments; the samples were therefore regarded as being drawn from the same molecular population with a mean length and variance of, respectively, 1.83 μm and 0.0117 μm².It was argued that the DNA molecules adsorbed on the mica are “frozen” into the molecular conformation present in solution at the time of adsorption and that, therefore, the experimentally determined contour lengths represent authentic molecular lengths in solution. Based on current estimates of the replicative form DNA molecular weight, the mean contour length obtained was slightly but significantly larger than the length predicted for molecules in an exact B configuration. The variance was larger than could be attributed solely to experimental error, indicating that the molecular population in aqueous solution is heterogeneous in contour length. These experimental results were shown to be consistent with a model for DNA structure in aqueous solution in which individual molecules are dynamic variants of a perturbed B form structure (von Hippel &; Wong, 1971).     

Differential role of the Mu B protein in phage Mu integration vs. replication: mechanistic insights into two transposition pathways

The Mu B protein is an ATP-dependent DNA-binding protein and an allosteric activator of the Mu transposase. As a result of these activities, Mu B is instrumental in efficient transposition and target-site choice. We analysed in vivo the role of Mu B in the two different recombination reactions performed by phage Mu: non-replicative transposition, the pathway used during integration, and replicative transposition, the pathway used during lytic growth. Utilizing a sensitive PCR-based assay for Mu transposition, we found that Mu B is not required for integration, but enhances the rate and extent of the process. Furthermore, three different mutant versions of Mu B, Mu BC99Y, Mu BK106A, and Mu B1-294, stimulate integration to a similar level as the wild-type protein. In contrast, these mutant proteins fail to support Mu growth. This deficiency is attributable to a defect in formation of an essential intermediate for replicative transposition. Biochemical analysis of the Mu B mutant proteins reveals common features: the mutants retain the ability to stimulate transposase, but are defective in DNA binding and target DNA delivery. These data indicate that activation of transposase by Mu B is sufficient for robust non-replicative transposition. Efficient replicative transposition, however, demands that the Mu B protein not only activate transposase, but also bind and deliver the target DNA.     

Target immunity of Mu transposition reflects a differential distribution of Mu B protein

A DNA molecule carrying Mu end DNA sequence(s) is a poor target in the Mu DNA strand-transfer reaction, a phenomenon which is referred to as "target immunity." We find that Mu B protein stimulates intermolecular strand-transfer by binding to the target DNA. Our results show that a differential distribution of Mu B protein between "immune" and "non-immune" DNA molecules is responsible for target immunity; in the presence of Mu A protein and ATP, Mu B protein dissociates preferentially from immune DNA molecules. Hydrolysis of ATP is implicated in establishing the differential distribution of Mu B protein between immune and non-immune DNA molecules in the presence of Mu A protein; nonhydrolyzable ATP gamma S can support an efficient strand-transfer reaction even with a target DNA that is immune in a reaction with ATP.     

DNA transposition of bacteriophage Mu. A quantitative analysis of target site selection in vitro.

The Mu transpositional DNA recombination machinery selects target sites by assembling a protein-DNA complex that interacts with the target DNA and reacts whenever it locates a favorable sequence composition. Splicing of a transposon into the target generates a 5-bp duplication that reflects the original target site. Preferential usage of different target pentamers was examined with a minimal Mu in vitro system and quantitatively compiled consensus sequences for the most preferred and the least preferred sites were generated. When analyzed as base steps, preferences toward certain steps along the 5-bp target site were detected. We further show that insertion sites can be predicted on the basis of additively calculated base step values. Also surrounding sequences influence the preference of a given pentamer; a symmetrical structural component was revealed, suggesting potential hinges at and around the target site.     

Transpososomes: stable protein-DNA complexes involved in the in vitro transposition of bacteriophage Mu DNA

We report that two types of stable protein-DNA complexes, or transpososomes, are generated in vitro during the Mu DNA strand transfer reaction. The Type 1 complex is an intermediate in the reaction. Its formation requires a supercoiled mini-Mu donor plasmid, Mu A and HU protein, and Mg2+. In the Type 1 complex the two ends of Mu are held together, creating a figure eight-shaped molecule with two independent topological domains; the Mu sequences remain supercoiled while the vector DNA is relaxed because of nicking. In the presence of Mu B protein, ATP, target DNA, and Mg2+, the Type 1 complex is converted into the protein-associated product of the strand transfer reaction. In this Type 2 complex, the target DNA has been joined to the Mu DNA ends held in the synaptic complex at the center of the figure eight. Supercoils are not required for the latter reaction.     

Immunoelectron microscopic analysis of the A, B, and HU protein content of bacteriophage Mu transpososomes

Stable protein-DNA complexes or transpososomes mediate the Mu DNA strand transfer reaction in vitro (Surette, M. G., Buch, S. J., and Chaconas, G. (1987) Cell 49, 253-262; Craigie, R., and Mizuuchi, K. (1987) Cell 51, 493-501). Formation of the Type 1 complex, an intermediate in the strand transfer reaction, requires the Mu A and Escherichia coli HU proteins. Generation of the Type 2 complex, in which the Mu ends have been covalently linked to the target DNA, requires the Mu B protein, ATP, and target DNA in addition to A and HU. The protein content of these higher order synaptic complexes has been studied by immunoelectron microscopy using protein A-colloidal gold conjugates to visualize antibody-bound complexes. Under our in vitro transposition conditions, Type 1 complexes were found to contain A and HU; in addition, Type 2 complexes contained Mu B. However, both the HU and the Mu B protein were found to be loosely associated and could be quantitatively removed from the nucleoprotein core of both complexes by incubation in 0.5 M NaCl. Depletion of HU from the Type 1 complex did not affect the ability of this complex to be converted into the strand-transferred product. Hence, the indispensable role of the HU protein in the Mu DNA strand transfer reaction is limited to the formation of the Type 1 transpososome.     

A rolling circle model of the in vivo replication of bacteriophage phiX174 replicative form DNA: different fate of two types of progeny replicative form

In a preceding paper (Schröder and Kaerner, 1972) a rolling circle mechanism has been described for the replication of bacteriophage φX174 replicative form. Replication involved nicking and elongation of the viral (positive) strand component of the RF molecule resulting in the displacement of a single-strand tail of increasing length. The synthesis of the new complementary (negative) strand on the single-strand tails appears to be initiated with considerable delay and converts the tail into double-stranded DNA. Before the new negative strand is completed the replicative intermediates split into (I) a complete RF molecule containing the “old” negative and the new positive strand, and (II) a linear, partially double-stranded “tail” consisting of the complete old positive strand and a fragment of the new negative strand.The present study is concerned with the fate during RF replication of these fragments of the rolling circles. Those RFII molecules containing the old negative strands appear to go into further replication rounds repeatedly. Some of the tails were found in the infected cells in their original linear form. “Gapped” RFII molecules, which have been described earlier by Schekman and co-workers (Schekman &; Ray, 1971; Schekman et al., 1971), are supposed to originate from the tails of rolling circle intermediates by circularization of their positive strand components. Evidence is provided by our experiments that even late during RF replication these gaps are present only in the negative strands of RFII. Appropriate chase experiments indicated that the tails finally are converted to RFI molecules. Progeny RFI molecules could not be observed to start new replication rounds under our conditions although we cannot exclude that this might happen to some minor extent.The results presented suggest that the master templates for RF replication are the first negative strands to be formed, rather than the parental positive strands.     

The phiX174-type primosome promotes replisome assembly at the site of recombination in bacteriophage Mu transposition.

Initiation of Escherichia coli DNA synthesis primed by homologous recombination is believed to require the phiX174-type primosome, a mobile priming apparatus assembled without the initiator protein DnaA. We show that this primosome plays an essential role in bacteriophage Mu DNA replication by transposition. Upon promoting transfer of Mu ends to target DNA, the Mu transpososome undergoes transition to a pre-replisome that permits initiation of DNA synthesis only in the presence of primosome assembly proteins PriA, DnaT, DnaB and DnaC. These assembly proteins promote the engagement of primase and DNA polymerase III holoenzyme, initiating semi-discontinuous replication preferentially at the Mu left end. The results indicate that these proteins play a crucial role in promoting replisome assembly on a recombination intermediate.     

The solution structure of the C-terminal domain of the Mu B transposition protein

Mu B is one of four proteins required for the strand transfer step of bacteriophage Mu DNA transposition and the only one where no high resolution structural data is available. Structural work on Mu B has been hampered primarily by solubility problems and its tendency to aggregate. We have overcome this problem by determination of the three-dimensional structure of the C-terminal domain of Mu B (B(223-312)) in 1.5 M NaCl using NMR spectroscopic methods. The structure of Mu B(223-312) comprises four helices (backbone r.m.s.d. 0.46 A) arranged in a loosely packed bundle and resembles that of the N-terminal region of the replication helicase, DnaB. This structural motif is likely to be involved in the inter-domainal regulation of ATPase activity for both Mu A and DnaB. The approach described here for structural determination in high salt may be generally applicable for proteins that do not crystallize and that are plagued by solubility problems at low ionic strength.     

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.