In vivo mutagenesis of bacteriophage Mu transposase.

We devised a method for isolating mutations in the bacteriophage Mu A gene which encodes the phage transposase. Nine new conditional defective A mutations were isolated. These, as well as eight previously isolated mutations, were mapped with a set of defined deletions which divided the gene into 13 100- to 200-base-pair segments. Phages carrying these mutations were analyzed for their ability to lysogenize and to transpose in nonpermissive hosts. One Aam mutation, Aam7110, known to retain the capacity to support lysogenization of a sup0 host (M. M. Howe, K. J. O'Day, and D. W. Shultz, Virology 93:303-319, 1979) and to map 91 base pairs from the 3' end of the gene (R. M. Harshey and S. D. Cuneo, J. Genet. 65:159-174, 1987) was shown to be able to complement other A mutations for lysogenization, although it was incapable of catalyzing either the replication of Mu DNA or the massive conservative integration required for phage growth. Four Ats mutations which map at different positions in the gene were able to catalyze lysogenization but not phage growth at the nonpermissive temperature. Phages carrying mutations located at different positions in the Mu B gene (which encodes a product necessary for efficient integration and lytic replication) were all able to lysogenize at the same frequency. These results suggest that the ability of Mu to lysogenize is not strictly correlated with its ability to perform massive conservative and replicative transposition.     

Stoichiometric use of the transposase of bacteriophage Mu

The transposase of bacteriophage Mu (gene A protein) mediates the coupled replication and integration processes that constitute transposition during the lytic cycle. Our previous results showed that the activity of the A protein is unstable, as its continued synthesis is required to maintain Mu DNA replication throughout the lytic cycle. We present here the results of experiments in which the A protein is used stoichiometrically and must be synthesized de novo for each round of Mu DNA replication. Induction of a Mu lysogen in the absence of DNA replication allows accumulation of potential for a single round of Mu DNA replication. Once achieved, this potential is stable even in the absence of further protein synthesis. Release of inhibition of DNA replication leads to a single semi-conservative replicative transposition event, followed by later rounds only if additional synthesis of the A protein is allowed.     

Instability of transposase activity: Evidence from bacteriophage Mu DNA replication

Transposition of genetic elements involves coupled replication and integration events catalyzed in part by a class of proteins called transposases. We have asked whether the transposase activity of bacteriophage Mu (the Mu A protein) is stable and capable of catalyzing multiple rounds of coupled replication/integration, or whether its continued synthesis is required to maintain Mu DNA replication. Inhibition of protein synthesis during the lytic cycle with chloramphenicol inhibited Mu DNA synthesis with a half-life of approximately 3 min, demonstrating a need for continued protein synthesis to maintain Mu DNA replication. Synthesis of specific Mu-encoded proteins was inhibited by infecting a host carrying a temperature-sensitive suppressor, at permissive temperature, with Mu amber phages, then shifting to nonpermissive temperature. When Aam phages were used, Mu DNA replication was inhibited with kinetics essentially identical to those with chloramphenicol addition; hence, it is likely that continued synthesis of the Mu A protein is required to maintain Mu DNA replication. The data suggest that the activity of the Mu A protein is unstable, and raise the possibility that the Mu A protein and other transposases may be used stoichiometrically rather than catalytically.     

Characterization of functionally important sites in the bacteriophage Mu transposase protein

The 663 amino acid Mu transposase protein is absolutely required for Mu DNA transposition. Mutant proteins were constructed in vitro in order to locate regions of transposase that may be important for the catalysis of DNA transposition. Deletions in the A gene, which encodes the transposase, yielded two stable mutant proteins that aid in defining the end-specific DNA-binding domain. Linker insertion mutagenesis at eight sites in the Mu A gene generated two proteins, FF6 and FF14 (resulting from two and four amino acid insertions, respectively, at position 408), which were thermolabile for DNA binding in vitro at 43°C. However, transposition activity in vivo was severely reduced for all mutant proteins at 37°C, except those with insertions at positions 328 and 624. In addition, site-specific mutagenesis was performed to alter tyrosine 414, which is situated in a region that displays amino acid homology to the active sites of a number of nicking/closing enzymes. Tyrosine 414 may reside within an important, yet non-essential, site of transposase, as an aspartate-substituted protein had a drastically reduced frequency of transposition, while the remaining mutants yielded reduced, but substantial, frequencies of Mu transposition in vivo.     

Instability of bacteriophage Mu transposase and the role of host Hfl protein

The activity of the transposase of bacteriophage Mu is unstable, requiring the protein to be synthesized throughout the lytic cycle (Pato and Reich, 1982). Using Western blot analysis, we analysed the stability of the transposase protein during the lytic cycle and found that it, too, is unstable. The instability of the protein is observed both in the presence and the absence of Mu DNA replication, and is independent of other Mu-encoded proteins and the transposase binding sites at the Mu genome ends. Stability of the protein is enhanced in host strains mutated at the hfl locus; however, stability of the transposase activity is not enhanced in these strains, suggesting that functional inactivation of the protein is not simply a result of its proteolysis.     

Cloning of the A gene of bacteriophage Mu and purification of its product, the Mu transposase

The bacteriophage Mu transposase (the Mu A gene product), which is absolutely required for both integration of Mu and replicative transposition during the lytic cycle, has been overproduced by cloning the gene on a plasmid under the control of the phage lambda PL promoter. The protein has been purified to near homogeneity from the lysate of heat-induced cells of a strain carrying the plasmid. The purified protein is active as judged by its ability to complement Mu A- cell extracts for supporting Mu transposition in a cell-free reaction.     

Characterization of amber mutations in bacteriophage Mu transposase: a functional analysis of the protein

We have characterized a series of amber mutations in the A gene of bacteriophage Mu encoding the phage transposase. We tested different activities of these mutant proteins either in a sup0 strain or in different sup bacteria. In conjunction with the results described in the accompanying paper by Bétermier et al. (1989) we find that the C-terminus of the protein is not absolutely essential for global transposase function, but is essential for phage growth. Specific binding to Mu ends is defined by a more central domain. Our results also reinforce the previous findings (Bétermier et al., 1987) that more than one protein may be specified by the A gene.     

Characterisation of an IS1 induced mutation in the carboxy-terminal end of bacteriophage Mu transposase which affects several functional domains of the protein

We show that a mutation in bacteriophage Mu transposase (pA) which was isolated as a deletion of the C-terminal end of the protein actually consists of the replacement of the last 16 amino acids (which are mostly hydrophilic) by 26 mostly hydrophobic amino acids. This change almost completely inactivates the in vivo enzyme activity as well as its capacity to bind Mu ends in vitro, although the end-binding domain of the protein resides at least 150 amino acids from the C-terminus. This sharply contrasts with the properties of a series of missense mutations and short C-terminal deletions in pA described earlier which only slightly decrease the overall transposase activity.     

Effect of mutations in the C-terminal domain of Mu B on DNA binding and interactions with Mu A transposase

Bacteriophage Mu transposition requires two phage-encoded proteins, the transposase, Mu A, and an accessory protein, Mu B. Mu B is an ATP-dependent DNA-binding protein that is required for target capture and target immunity and is an allosteric activator of transpososome function. The recent NMR structure of the C-terminal domain of Mu B (Mu B223-312) revealed that there is a patch of positively charged residues on the solvent-exposed surface. This patch may be responsible for the nonspecific DNA binding activity displayed by the purified Mu B223-312 peptide. We show that mutations of three lysine residues within this patch completely abolish nonspecific DNA binding of the C-terminal peptide (Mu B223- 312). To determine how this DNA binding activity affects transposition we mutated these lysine residues in the full-length protein. The full-length protein carrying all three mutations was deficient in both strand transfer and allosteric activation of transpososome function but retained ATPase activity. Peptide binding studies also revealed that this patch of basic residues within the C-terminal domain of Mu B is within a region of the protein that interacts directly with Mu A. Thus, we conclude that this protein segment contributes to both DNA binding and protein-protein contacts with the Mu transposase.     

Solution structure of the Mu end DNA-binding ibeta subdomain of phage Mu transposase: modular DNA recognition by two tethered domains.

The phage Mu transposase (MuA) binds to the ends of the Mu genome during the assembly of higher order nucleoprotein complexes. We investigate the structure and function of the MuA end-binding domain (Ibetagamma). The three-dimensional solution structure of the Ibeta subdomain (residues 77-174) has been determined using multidimensional NMR spectroscopy. It comprises five alpha-helices, including a helix-turn-helix (HTH) DNA-binding motif formed by helices 3 and 4, and can be subdivided into two interacting structural elements. The structure has an elongated disc-like appearance from which protrudes the recognition helix of the HTH motif. The topology of helices 2-4 is very similar to that of helices 1-3 of the previously determined solution structure of the MuA Igamma subdomain and to that of the homeodomain family of HTH DNA-binding proteins. We show that each of the two subdomains binds to one half of the 22 bp recognition sequence, Ibeta to the more conserved Mu end distal half (beta subsite) and Igamma to the Mu end proximal half (gamma subsite) of the consensus Mu end-binding site. The complete Ibetagamma domain binds the recognition sequence with a 100- to 1000-fold higher affinity than the two subdomains independently, indicating a cooperative effect. Our results show that the Mu end DNA-binding domain of MuA has a modular organization, with each module acting on a specific part of the 22 bp binding site. Based on the present binding data and the structures of the Ibeta and Igamma subdomains, a model for the interaction of the complete Ibetagamma domain with DNA is proposed.     

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.     

Functional domains of the IS1 transposase: analysis in vivo and in vitro

The IS1 bacterial insertion sequence family, considered to be restricted to Enterobacteria, has now been extended to other Eubacteria and to Archaebacteria, reviving interest in its study. To analyse the functional domains of the InsAB' transposase of IS1A, a representative of this family, we used an in vivo system which measures IS1-promoted rescue of a temperature-sensitive pSC101 plasmid by fusion with a pBR322::IS1 derivative. We also describe the partial purification of the IS1 transposase and the development of several in vitro assays for transposase activity. These included a DNA band shift assay, a transposase-mediated cleavage assay and an integration assay. Alignments of IS family members (http://www-is.biotoul.fr) not only confirmed the presence of an N-terminal helix-turn-helix and a C-terminal DDE motif in InsAB', but also revealed a putative N-terminal zinc finger. We have combined the in vitro and in vivo tests to carry out a functional analysis of InsAB' using a series of site-directed InsAB' mutants based on these alignments. The results demonstrate that appropriate mutations in the zinc finger and helix-turn-helix motifs result in loss of binding activity to the ends of IS1 whereas mutations in the DDE domain are affected in subsequent transposition steps but not in end binding.     

Carboxyl-terminal mutants of phage Mu transposase

A study of the properties of deletion mutants at the 3’ end ofA, the gene encoding the transposase protein of phage Mu, shows that the mutants are defective in the high-frequency non-replicative transposition observed early after Mu infection as well as the high-frequency replicative transposition observed during Mu lytic growth. They show near-normal levels of lysogenization, low frequency transposition and precise excision. The mutants behave as if they are “blind” to the presence of Mu B, a protein whose function is essential for the high frequency of both replicative and non-replicative Mudna transposition. We have sequenced these deletion mutants as well as the amber mutant A 7110 which is known to be defective in replicative transposition.A 7110 maps at the 3’ end of geneA. We suggest that the carboxyl-terminal region of the A-protein is involved in protein-protein interactions, especially with the B-protein. We also show in this study that mutations upstream of the Shine-Dalgarno sequence of geneA and within the preceding genener, perturb the synthesis of A-protein and that higher levels of A-protein cause an inhibition ofA activity.     

A subsequence-specific DNA-binding domain resides in the 13 kDa amino terminus of the bacteriophage Mu transposase protein

We have previously reported that the 13 kDa amino terminus of the 70 kDa bacteriophage D108 transposase protein (A gene product) contains a two-component, sequence-specific DNA-binding domain which specifically binds to the related bacteriophage Mu's right end (attR) in vitro. To extend these studies, we examined the ability of the 13 kDa amino terminus of the Mu transposase protein to bind specifically to Mu attR in crude extracts. Here we report that the Mu transposase protein also contains a Mu attR specific DNA-binding domain, located in a putative alpha-helix-turn-alpha-helix region, in the amino terminal 13 kDa portion of the 70 kDa transposase protein as part of a 23 kDa fusion protein with beta-lactamase. We purified for this attR-specific DNA-binding activity and ultimately obtained a single polypeptide of the predicted molecular weight for the A'--'bla fusion protein. We found that the pure protein bound to the Mu attR site in a different manner compared with the entire Mu transposase protein as determined by DNase I-footprinting. Our results may suggest the presence of a potential primordial DNA-binding site (5'-PuCGAAA-3') located several times within attR, at the ends of Mu and D108 DNA, and at the extremities of other prokaryotic class II elements that catalyze 5 base pair duplications at the site of element insertion. The dissection of the functional domains of the related phage Mu and D108 transposase proteins will provide clues to the mechanisms and evolution of DNA transposition as a mode of mobile genetic element propagation.     

Simultaneous expression of a bacteriophage Mu transposase and repressor: a way of preventing killing due to mini-Mu replication

In vitro studies of bacteriophage Mu transposition have shown that the phage-encoded transposase and repressor bind the same sequences on the phage genome. We attempted to test that prediction in vivo and found that Mu repressor directly inhibits transposition. We also found that, in the absence of repressor, constitutive expression of Mu transposition functions pA and pB is lethal in Escherichia coli strains lysogenic for a mini-Mu and that this is the result of intensive replication of the mini-Mu. These findings have important consequences where such mini-Mus are used as genetic tools. We also tested whether in Erwinia chrysanthemi the effect of transposition functions on a resident mini-Mu was the same as in E. coli. We observed that expression of pA alone was lethal in E. chrysanthemi and that a large fraction of the survivors underwent precise excision of the mini-Mu.     

Use of bacteriophage Mu to isolate deletions in the his-nif region of Klebsiella pneumoniae.

Klebsiella pneumoniae M5a1 is naturally resistant to infection by bacteriophage Mu. Mutants of K. pneumoniae sensitive to Mu infection were isolated and found to support both lytic and lysogenic development of Mu. K. pneumoniae lysogens containing a heat-inducible Mu prophage integrated in his were isolated. Strains carrying deletions extending from his into nif were obtained after heat treatment of these lysogens. Such deletions should be useful for determining the map order and cistronic organization of the nif genes.     

Defective prophages of bacteriophage Mu

Summary A method is described for the isolation of thermoinducible defective Mu lysogens. Four of these defective lysogens were studied more extensively. By marker-rescue experiments it was shown that the strain harbouring the smallest defective prophage contains the immunity gene cts and the genes A and B; the strain with the largest defective prophage still contains all the known essential genes of Mu, A to S (see Fig. 1).After induction at 43° C all the defective lysogens are killed, whereas no lysis occurs.Although in all the thermoinducible defective lysogens the A and B gene products could be demonstrated by complementation, these gene products are not responsible for the killing of the host, suggesting the presence of another unknown early gene product of Mu. The level of complementation of a mutation in gene A is reduced by the presence in the cell of another defective Mu prophage containing the G part of Mu. This effect on A gene complementation is markedly enhanced when the defective prophage, containing the G part, is located on an episome instead of on the chromosome. Complementation of late genes by a defective prophage located on the chromosome, is extremely low or undetectable. A stimulation of complementation by a factor of 10 to 40 was found when the same defective prophage was situated on a F factor. A possible explanation for this episome effect will be discussed.     

Regulation of bacteriophage Mu transposition

Bacteriophage Mu is a transposon and a temperate phage which has become a paradigm for the study of the molecular mechanism of transposition. As a prophage, Mu has also been used to study some aspects of the influence of the host cell growth phase on the regulation of transposition. Through the years several host proteins have been identified which play a key role in the replication of the Mu genome by successive rounds of replicative transposition as well as in the maintenance of the repressed prophage state. In this review we have attempted to summarize all these findings with the purpose of emphasizing the benefit the virus and the host cell can gain from those phage-host interactions.