Phage Mu Gam protein promotes NHEJ in concert with Escherichia coli ligase.

The Gam protein of transposable phage Mu is an ortholog of eukaryotic and bacterial Ku proteins, which carry out nonhomologous DNA end joining (NHEJ) with the help of dedicated ATP-dependent ligases. Many bacteria carry Gam homologs associated with either complete or defective Mu-like prophages, but the role of Gam in the life cycle of Mu or in bacteria is unknown. Here, we show that MuGam is part of a two-component bacterial NHEJ DNA repair system. Ensemble and single-molecule experiments reveal that MuGam binds to DNA ends, slows the progress of RecBCD exonuclease, promotes binding of NAD+-dependent Escherichia coli ligase A, and stimulates ligation. In vivo, Gam equally promotes both precise and imprecise joining of restriction enzyme-digested linear plasmid DNA, as well as of a double-strand break (DSB) at an engineered I-SceI site in the chromosome. Cell survival after the induced DSB is specific to the stationary phase. In long-term growth competition experiments, particularly upon treatment with a clastogen, the presence of gam in a Mu lysogen confers a distinct fitness advantage. We also show that the role of Gam in the life of phage Mu is related not to transposition but to protection of genomic Mu copies from RecBCD when viral DNA packaging begins. Taken together, our data show that MuGam provides bacteria with an NHEJ system and suggest that the resulting fitness advantage is a reason that bacteria continue to retain the gam gene in the absence of an intact prophage.

Mu insertions are repaired by the double-strand break repair pathway of Escherichia coli.

Mu is both a transposable element and a temperate bacteriophage. During lytic growth, it amplifies its genome by replicative transposition. During infection, it integrates into the Escherichia coli chromosome through a mechanism not requiring extensive DNA replication. In the latter pathway, the transposition intermediate is repaired by transposase-mediated resecting of the 5' flaps attached to the ends of the incoming Mu genome, followed by filling the remaining 5 bp gaps at each end of the Mu insertion. It is widely assumed that the gaps are repaired by a gap-filling host polymerase. Using the E. coli Keio Collection to screen for mutants defective in recovery of stable Mu insertions, we show in this study that the gaps are repaired by the machinery responsible for the repair of double-strand breaks in E. coli-the replication restart proteins PriA-DnaT and homologous recombination proteins RecABC. We discuss alternate models for recombinational repair of the Mu gaps.

The C-terminal domain is sufficient for host-binding activity of the Mu phage tail-spike protein.

The Mu phage virion contains tail-spike proteins beneath the baseplate, which it uses to adsorb to the outer membrane of Escherichia coli during the infection process. The tail spikes are composed of gene product 45 (gp45), which contains 197 amino acid residues. In this study, we purified and characterized both the full-length and the C-terminal domains of recombinant gp45 to identify the functional and structural domains. Limited proteolysis resulted in a Ser64-Gln197 sequence, which was composed of a stable C-terminal domain. Analytical ultracentrifugation of the recombinant C-terminal domain (gp45-C) indicated that the molecular weight of gp45-C was about 58 kDa and formed a trimeric protomer in solution. Coprecipitation experiments and a quartz crystal microbalance (QCM) demonstrated that gp45-C irreversibly binds to the E. coli membrane. These results indicate that gp45 shows behaviors similar to tail-spike proteins of other phages; however, gp45 did not show significant sequence homology with the other phage tail-spike structures that have been identified.

Mu-like prophage strong gyrase site sequences: analysis of properties required for promoting efficient mu DNA replication.

The bacteriophage Mu genome contains a centrally located strong gyrase site (SGS) that is required for efficient prophage replication. To aid in studying the unusual properties of the SGS, we sought other gyrase sites that might be able to substitute for the SGS in Mu replication. Five candidate sites were obtained by PCR from Mu-like prophage sequences present in Escherichia coli O157:H7 Sakai, Haemophilus influenzae Rd, Salmonella enterica serovar Typhi CT18, and two strains of Neisseria meningitidis. Each of the sites was used to replace the natural Mu SGS to form recombinant prophages, and the effects on Mu replication and host lysis were determined. The site from the E. coli prophage supported markedly enhanced replication and host lysis over that observed with a Mu derivative lacking the SGS, those from the N. meningitidis prophages allowed a small enhancement, and the sites from the Haemophilus and Salmonella prophages gave none. Each of the candidate sites was cleaved specifically by E. coli DNA gyrase both in vitro and in vivo. Supercoiling assays performed in vitro, with the five sites or the Mu SGS individually cloned into a pUC19 reporter plasmid, showed that the Mu SGS and the E. coli or N. meningitidis sequences allowed an enhancement of processive, gyrase-dependent supercoiling, whereas the H. influenzae or Salmonella serovar Typhi sequences did not. While consistent with a requirement for enhanced processivity of supercoiling for a site to function in Mu replication, these data suggest that other factors are also important. The relevance of these observations to an understanding of the function of the SGS is discussed.

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.

Precise excision of bacteriophage Mu DNA.

The temperate bacteriophage Mu is a transposable element that can integrate randomly into bacterial DNA, thereby creating mutations. Mutants due to an integrated Mu prophage do not give rise to revertants, as if Mu, unlike other transposable elements, were unable to excise precisely. In the present work, starting with a lacZ::Muc62(Ts) strain unable to form Lac+ colonies, we cloned a lacZ+ gene in vivo on a mini-Mu plasmid, under conditions of prophage induction. In all lac+ plasmids recovered, the wild-type sequence was restored in the region where the Mu prophage had been integrated. The recovery of lacZ+ genes shows that precise excision of Mu does indeed take place; the absence of Lac+ colonies suggests that precise excision events are systematically associated with loss of colony-forming ability.

SmpB, a unique RNA-binding protein essential for the peptide-tagging activity of SsrA (tmRNA).

In bacteria, SsrA RNA recognizes ribosomes stalled on defective messages and acts as a tRNA and mRNA to mediate the addition of a short peptide tag to the C-terminus of the partially synthesized nascent polypeptide chain. The SsrA-tagged protein is then degraded by C-terminal-specific proteases. SmpB, a unique RNA-binding protein that is conserved throughout the bacterial kingdom, is shown here to be an essential component of the SsrA quality-control system. Deletion of the smpB gene in Escherichia coli results in the same phenotypes observed in ssrA-defective cells, including a variety of phage development defects and the failure to tag proteins translated from defective mRNAs. Purified SmpB binds specifically and with high affinity to SsrA RNA and is required for stable association of SsrA with ribosomes in vivo. Formation of an SmpB-SsrA complex appears to be critical in mediating SsrA activity after aminoacylation with alanine but prior to the transpeptidation reaction that couples this alanine to the nascent chain. SsrA RNA is present at wild-type levels in the smpB mutant arguing against a model of SsrA action that involves direct competition for transcription factors.

A spontaneous Escherichia coli K12 mutant which inhibits the excision-reintegration process of Mu gem2ts.

Escherichia coli K12 strains lysogenic for Mu gem2ts with the prophage inserted in a target gene (i.e., lacZ::Mu gem2ts lysogenic strains) revert to Lac+ by prophage precise excision with a relatively high frequency (about 1 x 10(-6)). The revertants obtained are still lysogens with the prophage inserted elsewhere in the bacterial chromosome. We have observed that, with the time of storage in stabs, bacterial cultures lysogenic for Mu gem2ts lose the ability to excise the prophage. The mutation responsible for this effect was co-transducible with the gyrB gene. After the removal of the prophage by P1 vir transduction from these strains, one randomly chosen clone, R3538, was further analyzed. It shows an increment of DNA supercoiling of plasmid pAT153, used as a reporter, and a reduced beta-galactosidase activity. On the other hand, R3538 is totally permissive to both lytic and lysogenic cycles of bacteriophage Mu.

Regulatory factors acting at the bacteriophage Mu middle promoter.

Lytic development of bacteriophage Mu proceeds through three phases of transcription: early, middle, and late. Initiation of middle transcription from Pm requires the phage-encoded activator, Mor. An examination of the sequences surrounding the promoter revealed possible binding sites for Mu proteins A and c, as well as for Escherichia coli integration host factor. Promoter fragments containing 5' and 3' deletions were fused to the lacZ reporter gene and assayed for activity after induction of a Mu prophage or a plasmid-borne mor gene. Sequences upstream of position -62 and downstream of +10 were dispensable for promoter activity. In DNase I footprinting with both crude extract and purified protein, Mor protected Pm sequences from position -56 to -33. Mutations disrupting the dyad symmetry of the terminator of early transcription overlapping the Mor binding site did not reduce promoter activity, suggesting that the symmetry per se is not required for Mor binding or Pm activation. Purified Mu lysogenic repressor (c) also bound to Pm, overlapping the Mor binding site. Production of large amounts of repressor in vivo reduced Mor-dependent promoter activity nearly 10-fold. Promoters with mutations in the repressor binding site showed a reduction in this repressor-mediated inhibition of Pm activity.

Characterization of Mu prophage lacking the central strong gyrase binding site: localization of the block in replication.

Bacteriophage Mu contains an unusually strong DNA gyrase binding site (SGS), located near the center of its genome, that is required for efficient Mu DNA replication (M. L. Pato, Proc. Natl. Acad. Sci. USA 91:7056-7060, 1994; M. L. Pato, M. M. Howe, and N. P. Higgins, Proc. Natl. Acad. Sci. USA 87:8716-8720, 1990). Replication of wild-type Mu initiates about 10 min after induction of a lysogen, while replication in the absence of the SGS is delayed about an hour. To determine which step in the replication pathway is blocked in the absence of the SGS, we inactivated the SGS by deletion and by insertion and studied the effects of these alterations on various stages of Mu DNA replication. Following induction in the absence of a functional SGS, early transcription and synthesis of the Mu-encoded replication proteins occurred normally. However, neither strand transfer nor cleavage at the Mu genome termini could be detected 40 min after induction. The data are most consistent with a requirement for the SGS in the efficient synapsis of the Mu prophage termini to form a separate chromosomal domain.

The Escherichia coli DnaK chaperone machine and bacteriophage Mu late transcription.

Bacteriophage Mu does not grow on temperature-sensitive E. coli dnaK mutants at elevated temperatures because of a defect in late transcription. As the Mu-encoded C protein is required for activation of transcription from the phage late promoters, we attempted to determine if DnaK and its accessory proteins DnaJ and GrpE are required for synthesis of C protein or at a later step. We found that the chaperones act in Mu late transcription beyond C-protein synthesis, and that C-protein stability is decreased in the mutant hosts. This suggests that the DnaK chaperone machine may be required for the proper folding and/or multimerization of C protein.

Participation of the bacteriophage Mu A protein and host factors in the initiation of Mu DNA synthesis in vitro.

During bacteriophage Mu transposition, strand transfer is catalyzed in the presence of phage-encoded A and B proteins and Escherichia coli HU protein, attaching Mu ends to target DNA and creating an intermediate in transposition. Bacteriophage Mu A protein, which remains tightly bound to the Mu ends in the native strand-transfer intermediate, blocked initiation of Mu DNA replication by a system of 8 host proteins (DnaB helicase, DnaC protein, DnaG primase, DNA polymerase III holoenzyme, DNA polymerase I, DNA gyrase, DNA ligase, and single-strand binding protein). This 8-protein system had all enzymatic activities to convert the deproteinized intermediate to a cointegrate; however, additional host factor(s) were required to replicate the native intermediate. While replication of the native intermediate absolutely required DnaB helicase, DnaC protein, and DNA polymerase III holoenzyme, the specific requirements were relaxed for the deproteinized intermediate. Other host factors were able to replace these specific factors. These results indicate that Mu A protein, in conjunction with additional host factor(s), acts to promote assembly of specific host replication proteins at the Mu replication fork. This process may alter the stable interaction of Mu A protein with the ends to allow initiation of Mu DNA synthesis.

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.

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.

[Some developmental features of Escherichia coli Mu phage in Pseudomonas aeruginosa cells]. / Nekotorye osobennosti razvitiia faga Mu Escherichia coli v kletkakh Pseudomonas aeruginosa.

In order to determine the replication-transposition (RT) efficiency of Escherichia coli phage Mu in Pseudomonas aeruginosa cells, the change of Mu DNA copy number after transfer of P. aeruginosa (RP4::Mu) from 42 (the condition of RP4::Mu plasmid stability and low phage production level in P. aeruginosa) to 30 degrees C (the condition of RP4::Mu plasmid instability and higher phage production level in P. aeruginosa) was analysed. It was shown that the temperature shift causes no increase in Mu DNA copy number, although free phage DNA is revealed after transfer of the cells at 30 degrees C. Considering that the studied cells contained also a linear RP4 DNA and the free Mu DNA hybridized with the RP4 DNA, we proposed that the mature Mu DNA arises as a result of Mu genome packaging from the original plasmid. So, the Mu genome RT is uneffective in P. aeruginosa and all of the phage particles released from P. aeruginosa (RP4::Mu) cells contain Mu DNA apparently originated from the DNA of hybrid plasmid RP4::Mu. Moreover, these results suggest that the Mu DNA packaging is not effective in P. aeruginosa (taking into account that the P. aeruginosa (RP4::Mu) cells release about 10(-2) p.f.u./cell and that originally the copy number of RP4::Mu > or = 1).

Amino acid substitution in the C-terminal arm domain of HU-2 results in an enhanced affinity for DNA.

Three mutants of the Escherichia coli hupA gene, encoding the HU-2 protein, were constructed by synthetic oligodeoxyribonucleotide-directed, site-specific mutagenesis on M13mp18 vectors. The resulting HupAN10, HupAN11 and HupAN12 proteins contained Thr59-->Lys, Gln64-->Lys and Asn53-->Arg substitutions, respectively. These amino acid (aa) changes increased the positive charge of the N-terminal half of the two-strand, antiparallel beta-ribbon of the arm structure, which is believed to be a domain for DNA binding. The three mutant proteins bound to DNA more tightly than wild-type HU-2, and their affinities for DNA increased in the order of HupAN10, HupAN11, HupAN12. The mutant proteins showed a slightly increased HU activity for supporting Mu phage development. A mutant HU-2 protein with increased basicity, but with an altered aa sequence in the arm region due to a frameshift mutation, was also constructed. This mutant protein showed a reduced affinity to DNA and was unable to support Mu growth, suggesting that a unique aa sequence of the arm domain, rather than mere basicity of this domain, is required for efficient binding to DNA.

Chimeric HU-IHF proteins that alter DNA-binding ability.

Chimeric proteins between Escherichia coli histone-like HU and IHF were constructed by genetic engineering, in which part of the arm region was replaced by the corresponding region of IHF alpha (designated as HupANhimA) or IHF beta (HupANhimD); alternatively, an alpha-helix 2-beta 1 region was replaced by the corresponding region of IHF alpha (HupAXhimA) or IHF beta (HupAXhimD) (symbols N and X indicate NotI and XhoI junctions). These proteins were synthesized in a hupA-hupB double-deletion mutant. HupANhimA exhibited marked reduction in nonspecific DNA binding in vitro, and a drastic loss of HU activity in replicative transposition of Mu phage in vivo. HupANhimD also showed a significant reduction in the ability for DNA binding, though this protein supported Mu phage development. In contrast, the other two chimeric HU proteins showed only slight changes in nonspecific DNA-binding ability: they retained activities for transposition of Mu phage in vivo. These observations confirm that the flexible arm of HU-2, a domain proposed for DNA binding [Tanaka et al., Nature 310 (1984) 376-381; Goshima et al., Gene 96 (1990) 141-145], plays an important role in the physiological function of this protein. The results indicate that a unique conformation of the arm structure of HU protein, particularly the N-terminal half of a two-strand antiparallel beta-ribbon of the structure, is important for the DNA-binding ability of this protein.

Global changes in gene expression in Escherichia coli K12 induced by bacteriophage Mu Gem protein.

We have studied the growth properties of some Mu lysogens with respect to the non-lysogenic strain and have observed that the division time in minimal medium was increased over 4-fold when the bacteria carried the prophage mutated in the gem gene (Mu gem3). Since this phage gene has previously been shown to be involved in modulation of expression of host genes, we have analysed the proteins extracted from lysogens and non-lysogens as a rapid assay of global gene expression. The pattern of proteins extracted showed marked quantitative variations between non-lysogens, lysogens for wild-type Mu and lysogens for phage Mu gem3. These effects were no longer as evident when the strains were grown in rich medium. This dramatic change in the physiological state of the lysogenic strain versus the non-lysogenic in particular growth conditions extends the concept of lysogeny. For many years, the prophage has been considered only as a potentially lethal factor, while here it also appears as a genetic element capable of profoundly modifying host biology.

Identification of a positive regulator of the Mu middle operon.

Transcription of bacteriophage Mu occurs in a regulatory cascade consisting of three phases: early, middle, and late. The 1.2-kb middle transcript is initiated at Pm and encodes the C protein, the activator of late transcription. A plasmid containing a Pm-lacZ operon fusion was constructed. beta-Galactosidase expression from the plasmid increased 23-fold after Mu prophage induction. Infection of plasmid-containing cells with lambda phages carrying different segment of the Mu early region localized the Pm-lacZ transactivation function to the region containing open reading frames E16 and E17. Deletion and linker insertion analyses of plasmids containing this region identified E17 as the transactivator; therefore we call this gene mor, for middle operon regulator. Expression of mor under the control of a T7 promoter and T7 RNA polymerase resulted in the production of a single polypeptide of 17 kDa as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Insertion of a linker into mor substantially reduced the ability of Mu to form plaques. When growth of the mor mutant was assayed in liquid, lysis was delayed by about 50 min and the burst size was approximately one-fifth that of wild-type Mu. The mor requirement for plaque formation and normal growth kinetics was abolished when C protein was provided in trans, indicating that the primary function of Mor is to provide sufficient C for late gene expression. Comparison of the predicted amino acid sequence of Mor with other proteins revealed that Mor and C share substantial amino acid sequence homology.