Bacteriophage Mu DNA replication in vitro

An in vitro system for bacteriophage Mu DNA replication using lysates on cellophane discs is described. Mu replication was monitored by DNA hybridization. Using a thermoinducible Mu lysogen, 30-50% of all DNA synthesis in vitro was Mu-specific. Mu DNA synthesis is semidiscontinuous. In the presence of the DNA ligase inhibitor NMN, about one-half of the DNA was in Okazaki pieces and one-half in large DNA. The Mu Okazaki pieces hybridized mainly to the Mu light strand; the large DNA hybridized mainly to the Mu heavy strand. Okazaki pieces isolated from uninfected cells also hybridized to 2000-3000 bases of host DNA present in Mu-separated strands. However, the host Okazaki pieces hybridize to both Mu strands symmetrically. Most, if not all, host sequences were represented in mature Mu viral DNA. The in vitro data are most consistent with models in which Mu sequences, oriented randomly in both directions in the host chromosome, have recruited a bacterial replisome which traverses the Mu genome from left to right.     

Bacteriophage Mu DNA circularizes following infection of Escherichia coli

Mu DNA, isolated from infected cells or minicells, has been shown to be held by proteins in twisted and open circular forms. Circularization does not require protein synthesis in the infected cells. A 64,000-dalton polypeptide is injected into the infected cell with Mu DNA and co-sediments with Mu DNA through sucrose gradients. Circularization of the infecting Mu DNA does not require removal of the Escherichia coli DNA sequences which are attached to both ends of the Mu genome in the viral particle.     

Replacement of the Bacteriophage Mu Strong Gyrase Site and Effect on Mu DNA Replication

The bacteriophage Mu strong gyrase site (SGS) is required for efficient replicative transposition and functions by promoting the synapsis of prophage termini. To look for other sites which could substitute for the SGS in promoting Mu replication, we have replaced the SGS in the middle of the Mu genome with fragments of DNA from various sources. A central fragment from the transposing virus D108 allowed efficient Mu replication and was shown to contain a strong gyrase site. However, neither the strong gyrase site from the plasmid pSC101 nor the major gyrase site from pBR322 could promote efficient Mu replication, even though the pSC101 site is a stronger gyrase site than the Mu SGS as assayed by cleavage in the presence of gyrase and the quinolone enoxacin. To look for SGS-like sites in the Escherichia coli chromosome which might be involved in organizing nucleoid structure, fragments of E. coli chromosomal DNA were substituted for the SGS: first, repeat sequences associated with gyrase binding (bacterial interspersed mosaic elements), and, second, random fragments of the entire chromosome. No fragments were found that could replace the SGS in promoting efficient Mu replication. These results demonstrate that the gyrase sites from the transposing phages possess unusual properties and emphasize the need to determine the basis of these properties.     

Bacteriophage Mu DNA replication is stimulated by non-essential early functions

Summary The replication of a spontaneous Kil^– mutant of bacteriophage Mu has been investigated. The Kil^– mutation (Mucts62-13/4), which was introduced into a defective prophage, is pleiotrophic, leading to the loss of also the Gam, Cim and Sot functions. The mutation is caused by an insertion with the characteristics of IS1, located just outside the B gene.Mucts62-13/4 phages form extremely small plaques on wildtype indicator strains. The replication of the insertion mutant as compared to Mucts62 is strongly reduced. Normal replication could be restored by relieving the polarity of the insertion or by complementation with defective prophages which express all early functions. Apparently, early genes other than A and B are involved in normal Mu DNA replication.     

Temperature-Sensitive Mutants of Bacteriophage Mu

Temperature-sensitive mutants of bacteriophage Mu, which grow at 32 C but not at 42 C, have been isolated. These mutants fall into two groups. Group 1 mutants fail to lyse host cells at nonpermissive temperatures, whereas lysis occurs normally with the group 2 mutants. All of the group 1 mutants apparently belong to the cistrons mapping to the left of gene C, whereas the group 2 mutants have lesions in various genes between D and S.     

Synthesis of the Unusual DNA of Bacillus subtilis Bacteriophage SP-15

Cultures of Bacillus subtilis infected with phage SP-15 were examined to investigate the metabolic origin of two of the unique components of the phage DNA: the component responsible for the unusually high buoyant density in CsCl and the unusual pyrimidine, 5-(4', 5'-dihydroxypentyl) uracil (DHPU). Newly synthesized pulse-labeled DNA was light in buoyant density and shifted to the high density of mature phage DNA upon further incubation. Parental DNA was converted to a light-density intermediate form prior to replication. When labeled uracil, thymidine, or DHPU were added to infected cells, it was found that only uracil served as the precursor to DHPU and thymine in phage DNA. Analysis of the bases from hydrolyzed DNA of labeled phage or infected cells indicated that the uracil was incorporated into the DNA as such (presumably via deoxyuridine triphosphate) and later converted to DHPU and thymine at the macromolecular level. The sequence of events after phage infection appeared to be: (i) injection of parental DNA; (ii) conversion of parental DNA to a light form; (iii) DNA replication, yielding light DNA containing uracil; (iv) conversion of uracil to DHPU and thymine; and (v) addition of the heavy component.     

Bacteriophage Mu: a transposing replicon

Mu DNA replication has been carried out in vitro on cellophane discs in the presence of dBUTP. If the DNA is sheared to 80 kb pieces, the Mu replicas band anomalously in CsCl gradients between hybrid and light DNA density positions. The intermediate density DNA comprises semiconservatively replicated Mu sequences, flanked by unreplicated light DNA. This and previous data are consistent with replication occurring within Mu boundaries. Both the synthesis of Mu sequences and the intermediate density DNA are abolished by protein synthesis inhibition in vivo just prior to lysis on cellophane discs. These observations indicate that at least some steps in bona fide Mu transposition-replication are being observed in vitro.     

Bacteriophage Mu integration in yeast and mammalian genomes

Genomic parasites have evolved distinctive lifestyles to optimize replication in the context of the genomes they inhabit. Here, we introduced new DNA into eukaryotic cells using bacteriophage Mu DNA transposition complexes, termed ‘transpososomes’. Following electroporation of transpososomes and selection for marker gene expression, efficient integration was verified in yeast, mouse and human genomes. Although Mu has evolved in prokaryotes, strong biases were seen in the target site distributions in eukaryotic genomes, and these biases differed between yeast and mammals. In Saccharomyces cerevisiae transposons accumulated outside of genes, consistent with selection against gene disruption. In mouse and human cells, transposons accumulated within genes, which previous work suggests is a favorable location for efficient expression of selectable markers. Naturally occurring transposons and viruses in yeast and mammals show related, but more extreme, targeting biases, suggesting that they are responding to the same pressures. These data help clarify the constraints exerted by genome structure on genomic parasites, and illustrate the wide utility of the Mu transpososome technology for gene transfer in eukaryotic cells.     

Efficient Insertion Mutagenesis Strategy for Bacterial Genomes Involving Electroporation of In Vitro-Assembled DNA Transposition Complexes of Bacteriophage Mu

An efficient insertion mutagenesis strategy for bacterial genomes based on the phage Mu DNA transposition reaction was developed. Incubation of MuA transposase protein with artificial mini-Mu transposon DNA in the absence of divalent cations in vitro resulted in stable but inactive Mu DNA transposition complexes, or transpososomes. Following delivery into bacterial cells by electroporation, the complexes were activated for DNA transposition chemistry after encountering divalent metal ions within the cells. Mini-Mu transposons were integrated into bacterial chromosomes with efficiencies ranging from 10⁴ to 10⁶ CFU/μg of input transposon DNA in the four species tested, i.e., Escherichia coli, Salmonella enterica serovar Typhimurium, Erwinia carotovora, and Yersinia enterocolitica. Efficiency of integration was influenced mostly by the competence status of a given strain or batch of bacteria. An accurate 5-bp target site duplication flanking the transposon, a hallmark of Mu transposition, was generated upon mini-Mu integration into the genome, indicating that a genuine DNA transposition reaction was reproduced within the cells of the bacteria studied. This insertion mutagenesis strategy for microbial genomes may be applicable to a variety of organisms provided that a means to introduce DNA into their cells is available.     

Dissection of the DNA Mimicry of the Bacteriophage T7 Ocr Protein using Chemical Modification

The homodimeric Ocr (overcome classical restriction) protein of bacteriophage T7 is a molecular mimic of double-stranded DNA and a highly effective competitive inhibitor of the bacterial type I restriction/modification system. The surface of Ocr is replete with acidic residues that mimic the phosphate backbone of DNA. In addition, Ocr also mimics the overall dimensions of a bent 24-bp DNA molecule. In this study, we attempted to delineate these two mechanisms of DNA mimicry by chemically modifying the negative charges on the Ocr surface. Our analysis reveals that removal of about 46% of the carboxylate groups per Ocr monomer results in an ∼ 50-fold reduction in binding affinity for a methyltransferase from a model type I restriction/modification system. The reduced affinity between Ocr with this degree of modification and the methyltransferase is comparable with the affinity of DNA for the methyltransferase. Additional modification to remove ∼ 86% of the carboxylate groups further reduces its binding affinity, although the modified Ocr still binds to the methyltransferase via a mechanism attributable to the shape mimicry of a bent DNA molecule. Our results show that the electrostatic mimicry of Ocr increases the binding affinity for its target enzyme by up to ∼ 800-fold.     

Bacteriophage DNA reptation]

The kinetics of reptation process of dsDNA leaving the phage head is analysed theoretically. It is assumed that the process is caused by DNA free energy decrease when it is leaving the head (DNA has to be in a globular state) for its surroundings where it is transformed into a coil state. For the analysis we have used the results of previous paper on equilibrium theory of DNA intraphage globule. Three possible cases for the ejection process friction are considered: friction in the tail-part channel, that of DNA segments with each other in the whole globule volume (it is essential for the collective way of the globule decondensation with simultaneous movement of all the loops--the first type way), the globule friction with internal capsid surface (it is most essential for the decondensation by the way of the globule rotation as a whole "spool"--the second type way). The first way would correspond to the greatest ejection time. The known experimental data on distinguishing ejection kinetics for phages with short and long tail-parts allow us to formulate arguments in favor of realization of the second way in nature.     

Kinetics of Mu DNA synthesis

Summary Mu specific DNA synthesis starts at 10 min after infection. All essential amber mutants of Mu were tested for the ability to replicate in a non permissive host. Except for the amber mutants A and B, which were already known to be blocked in Mu DNA synthesis (Wijffelman et al., 1974), all the other mutants showed normal Mu DNA replication.Using mitomycin C-treated cells Mu DNA synthesis was found to start at about 20 min after induction. However using the much more sensitive method of DNA-RNA hybridization, it was found that the DNA synthesis starts already at 10 min after induction, and that at 20 min after induction about 7 copies of the Mu DNA are present per cell.     

Restriction and Modification of Bacteriophage in Bacillus stearothermophilus

Host-controlled restriction and modification of TP-1C phage and infectious phage DNA occurs in Bacillus stearothermophilus and is subject to control by TP-8 or TP-12 prophage.