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At least 19 recordsLinked to original sources

PubMed2004

Crystallization and preliminary X-ray analysis of gene product 44 from bacteriophage Mu.

Bacteriophage Mu baseplate protein gene product 44 (gp44) is an essential protein required for the assembly of viable phages. To investigate the roles of gp44 in baseplate assembly and infection, gp44 was crystallized at pH 6.0 in the presence of 20% 2-methyl-2,4-pentanediol. The crystals belong to space group R3, with unit-cell parameters a = b = 127.47, c = 63.97 A. The crystals diffract X-rays to at least 2.1 A resolution and are stable in the X-ray beam and are therefore appropriate for structure determination. Native data have been collected to 2.1 A resolution using a DIP6040 image-plate system at beamline BL44XU at the SPring-8 facility in Japan.

Bacteriophage mu↗

PubMed1984

The nucleotide sequence of the B gene of bacteriophage Mu.

Bacteriophage Mu is a highly efficient transposon which requires the products of the Mu A and B genes in order to transpose at a normal frequency. We have determined the nucleotide sequence of the B gene as well as that of the A-B intergenic region upstream of B. The protein product of the gene contains 312 amino acids and has a predicted molecular weight of 35,061. As expected, there do not appear to be any potential promoter sequences in the intergenic region prior to the gene, but it is preceded by a strong Shine-Dalgarno sequence. The intergenic region does not contain any obvious transcription termination sequences. The frequency of optimal codon usage is similar to that for other transposon and phage genes, and the amino acid composition is comparable to that of an "average" E. coli protein. A region near the amino terminus of the protein resembles the highly conserved bihelical fold which is involved in DNA contact and sequence specific recognition in a number of DNA binding proteins.

Amino Acid Sequence↗

PubMed1977

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.

Coliphages↗

PubMed1991

Specificity of mini-Mu bacteriophage insertions in a small plasmid.

Target site selection for bacteriophage Mu transposition was studied in pools of over 10(7) independent mini-Mu insertions in pUC9, selected by transduction of the plasmid. Insertions in both orientations were clustered in three regions and, within these, at preferred sites.

Bacteriophage mu↗

PubMed1984

Plasmid insertion mutagenesis and lac gene fusion with mini-mu bacteriophage transposons.

Small bacteriophage Mu transposable elements containing the lac operon structural genes were constructed to facilitate the isolation and use of Mu insertions and lac gene fusions. These mini-Mu elements have selectable genes for either ampicillin or kanamycin resistance and can be used to form both transcriptional and translational lac gene fusions. Some of the mini-Mu-lac elements constructed are deleted for the Mu A and B transposition genes and form stable insertions that cannot undergo transposition unless complemented for these functions. A procedure was developed for selecting mini-Mu insertions specifically into plasmids, including commonly used high-copy-number cloning vectors such as pBR322. Mu insertions in pBR322 were found to be distributed around the plasmid, but insertions in certain regions occurred more frequently than in others.

Ampicillin↗

PubMed2003

The Gam protein of bacteriophage Mu is an orthologue of eukaryotic Ku.

Mu bacteriophage inserts its DNA into the genome of host bacteria and is used as a model for DNA transposition events in other systems. The eukaryotic Ku protein has key roles in DNA repair and in certain transposition events. Here we show that the Gam protein of phage Mu is conserved in bacteria, has sequence homology with both subunits of Ku, and has the potential to adopt a similar architecture to the core DNA-binding region of Ku. Through biochemical studies, we demonstrate that Gam and the related protein of Haemophilus influenzae display DNA binding characteristics remarkably similar to those of human Ku. In addition, we show that Gam can interfere with Ty1 retrotransposition in Saccharomyces cerevisiae. These data reveal structural and functional parallels between bacteriophage Gam and eukaryotic Ku and suggest that their functions have been evolutionarily conserved.

Amino Acid Sequence↗

PubMed1985

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.

Amino Acids↗

PubMed1986

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].

Bacteriophage mu↗

PubMed1979

Unusual modification of bacteriophage Mu DNA.

Bacteriophage Mu DNA was labeled after induction in the presence of [2-(3)H]adenine or [8-(3)H]adenine. Both Mu mom(+).dam(+) DNA and Mu mom(-).dam(+) DNA have similar N(6)-methyladenine (MeAde) contents, as well as similar frequencies of MeAde nearest neighbors. Both DNAs are sensitive to in vitro cleavage by R.DpnI but resistant to cleavage by R.DpnII. These results indicate that the mom(+) protein does not alter the sequence specificity of the host dam(+) methylase to produce MeAde at new sites. However, we have discovered a new modified base, denoted A(x), in Mu mom(+).dam(+) DNA; approximately 15% of the adenine residues are modified to A(x). Although the precise nature of the modification is not yet defined, analysis by electrophoresis and chromatography indicates that the N(6)-amino group is not the site of modification, and that the added moiety contains a free carboxyl group. A(x) is not present in Mu mom(+).dam(+) or Mu mom(-).dam(+) phage DNA or in cellular DNA from uninduced Mu mom(+).dam(+) lysogens. These results suggest that expression of the dam(+) and mom(+) genes are required for the A(x) modification and that this modification is responsible for protecting Mu DNA against certain restriction nucleases. Mu mom(+).dam(-) DNA and Mu mom(-).dam(-) DNA contain a very low level of MeAde (ca. 1 MeAde per 5,000 adenine residues). Since the only nearest neighbor to MeAde appears to be cytosine, we suggest that the methylated sequence is 5'... C-A(*)-C... 3' and that this methylation is mediated by the EcoK modification enzyme.

Adenine↗

PubMed1994

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.

Bacteriophage mu↗

PubMed1991

Analysis of the IHF binding site in the regulatory region of bacteriophage Mu.

In bacteriophage Mu the converging early and repressor transcriptions are both stimulated by binding of IHF to the same region, which is located just upstream of the early promoter (Pe) and 100 base pairs downstream of the repressor promoter (Pc). Within this region two sequences are present (ihfa and ihfb) that match the consensus sequence for IHF binding. These sequences are partially overlapping and in inverted orientation. In this paper we describe the effect of mutations in the non-overlapping part of ihfa and ihfb on the binding of IHF. We show that IHF has a very strong preference to bind to ihfb even when a mutated ihfa has a better match with the consensus. A stretch of A residues located nine base pairs from the ihfb sequence appears to play an important role in the stability of the DNA-IHF complex, but not in the discrimination between the two putative binding sites. In addition we describe the effect of the mutations on the stimulation of early and repressor transcription. We show that for activation of the Pc promoter a stable complex between IHF and the DNA is required, whereas for normal Pe stimulation a much weaker DNA-IHF interaction is sufficient.

Bacterial Proteins↗

PubMed1974

Isolation and characterization of mutants of colicin plasmids E1 and E2 after Mu bacteriophage infection.

Cells colicinogenic for the colicin plasmids E1 or E2 (Col E1 and Col E2, respectively) were selected for a loss of colicin production after infection with bacteriophage Mu. Extrachromosomal deoxyribonucleic acid that was larger than the original colicin plasmids was found in such cells. A small insertion mutant in Col E1 deoxyribonucleic acid affecting active colicin production without affecting either expression of colicin immunity or Col E1 deoxyribonucleic acid replication was found. Cells carrying this Col E1 plasmid mutant do not exhibit the lethal event associated with colicin E1 induction, suggesting that synthesis of active colicin is required for killing during induction. The altered Col E2 plasmid, containing an insertion at least as large as phage Mu, was maintained unstably in the mutants examined.

Carbon Radioisotopes↗

PubMed1987

Cloning of genes from members of the family Enterobacteriaceae with mini-Mu bacteriophage containing plasmid replicons.

An in vivo cloning system that uses derivatives of the Escherichia coli bacteriophage Mu with plasmid replicons has been extended to five different species of the family Enterobacteriaceae. Mu and these mini-Mu replicon elements were introduced into strains of E. coli, Shigella flexneri, Salmonella typhimurium, Citrobacter freundii, and Proteus mirabilis by infection, by transformation, or by conjugation with newly constructed broad-host-range plasmids containing insertions of these elements. Lysates from these cells, lysogenic for Mu and mini-Mu elements, were used to infect sensitive recipient strains of E. coli, S. typhimurium, and C. freundii. Drug-resistant transductants had mini-Mu replicon elements with inserts of different DNA sequences. All of the lysogens made could be induced to yield high phage titers, including those coming from strains that were resistant to Mu and Mu derivatives. Clones of 10 particular genes were isolated by their ability to complement specific mutations in the recipient strains, even in the presence of the E. coli K-12 restriction system. Some of the mini-Mu replicon elements used contained lac gene fusing segments and resulted in fusions of the lac operon to control regions in the cloned sequences.

Citrobacter↗

PubMed1993

Cloning of toxic genes with mini-mu derivative of bacteriophage mu.

We describe an in vivo cloning method using mini-Mu phage for genes, which cannot be cloned on multicopy vectors, mainly for their toxicity. We have successfully cloned succinate dehydrogenase (sdh) gene E. coli which was inactivated with defined insertion of fragment Kmr by this method. The most of obtained Kmr clones of mini-Mu transductants have contained the sequence of whole sdh gene. The intact gene of sdh can be reconstructed by site-directed mutagenesis.

Bacteriophage mu↗

PubMed1984

[Effect of the polymerase activity of DNA polymerase I on the development of moderate bacteriophages Mu, lambda red- and lambda red-gam-. II. The effect of the polymerase activity of DNA polymerase I on the development of bacteriophage Mu].

The paper reports on the influence of polymerizing activity of DNA-polymerase I on different developmental stages of temperate bacteriophage Mu in Escherichia coli K-12 cells. This activity is shown to be necessary for optimization of phage Mu primary integration into cell chromosomes. The relative frequency of Mu integration into bacterial chromosomes is 5-6 times lower in polA cells than in isogenic polA+ control strains, the phage yield from cells being delayed during the phage infectious development, but not in the course of induction from the prophage state. Data have been obtained that show the process of phage Mu DNA integration into the plasmid pRP1 .2 and the process of Mu transposition from the cell chromosome into the plasmid to be independent of the polymerizing activity of DNA-polymerase I.

Adsorption↗

PubMed1986

Transposition and replication of maxi-Mu derivatives of bacteriophage Mu.

The insertion of DNA fragments within the lac sequence of a MudI(Ap,lac) prophage resulted in the formation of a set of maxi-Mu genomes which were 39.8, 59, 85.6, and 88.2 kb long, respectively. The comparison of these maxi-Mu's with the 37.2-kb-long parental MudI(Ap,lac) indicated that the transposition frequency decreased as the length of the prophage increased. No replication of the two longest maxi-Mu's could be detected. The 59- and the 39.8-kb-long chimeric genomes were noted to replicate at approximately 1-2 and 30%, respectively, of the rate found with the MudI(Ap,lac) prophage. The length dependence of the transposition and replication could be explained by the impairment of an early step of the transposition/replication mechanism.

Coliphages↗

PubMed1985

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.

Bacteriophage lambda↗

PubMed1995

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.

Bacterial Proteins↗