The isolation and characterisation of a plaque-forming derivative of bacteriophage Mu carrying a fragment of Tn3 conferring ampicillin resistance.

Summary We have isolated a plaque-forming derivative of phage Mu which carries a determinant for Ap^R. The biological properties of this MuAp phage are similar to those of normal Mu. Its genome contains a 1.1 kb substitution where Mu DNA from the right end of the G region has been replaced by a similar length of DNA from the transposon Tn3. This fragment of Tn3 DNA carries the Ap^R gene, but is no longer capable of independent transposition.     

A novel illegitimate recombination event: precise excision and reintegration with the Mu gem mutant prophage

The bacteriophage Mu is known to insert its DNA more or less randomly within the Escherichia coli chromosome, as do transposable elements, but unlike the latter, precise excision of the prophage, thereby restoring the original sequence, is not observed with wild-type Mu, although it has been reported with certain defective mutants. We show here that the mutant prophage Mu gem2ts can excise precisely from at least three separate loci —malT, Iac and thyA (selected as Mal⁺, Lac⁺ and Thy⁺, respectively). This excision occurs under permissive conditions for phage development, is observed in fully immune (c⁺) lysogens, and is independent of RecA and of Mu transposase. Mu gemts2 excision is invariably accompanied by reintegration of a Mu gem2ts prophage elsewhere in the chromosome, in the case of Mal⁺ revertants, this prophage is systematically located at 94min on the E. coli chromosome. Mu gem2ts excision therefore sheds some light on the long-standing paradox of the lack of precise Mu excisio.     

Cloning of a restriction fragment of phage Mu DNA coding for early functions

Summary The DNA of an E. coli K12 strain harboring ten wildtype Mu prophages was restricted with endonuclease EcoRI, and the fragments ligated into the plasmid vector pMB9. Upon transformation of a strain carrying a heat inducible (Mu cts62) prophage, one temperature-resistant transformant was isolated. This transformant strain harbors the hybrid plasmid pKN001, containing the EcoRI.C fragment of Mu DNA as shown by restriction and heteroduplex analysis. Stable transformants of pKN001 are immune to superinfection with phage Mu. Transformation of superinfection with phage Mu. Transformation of Mu sensitive bacteria with pKN001 results in killing of the recipients (10^-4 surviving bacteria). The killing function is not expressed upon transformation of Mu-immune (lysogenic) bacteria.This paper is dedicated by EGB to Dr. Luis F. Leloir, on the occasion of his 70th birthday     

Transfer of RP4::Mu plasmids to Agrobacterium tumefaciens

Transfers of RP4::Mu plasmids from Escherichia coli to Agrobacterium tumefaciens are very inefficient in contrast to the very efficient transfer of RP4. Apparently, one or more Mu functions prevent RPR::Mu plasmids from establishing in some Gram-negatives other than E. coli. This problem was eliminated by the use of a mutant Mu prophage, Mu cts62r23, in RP4. Moreover, the transfer of RP4::Mu cts62r23 to the Agrobacterium strain C58 was found to be affected by a restriction modification system. The target for this restriction was located on Mu DNA and not on RP4 DNA. The plaque-forming phage production of Mu cts62r23 in Agrobacterium was found to be 10⁶ times lower than in E. coli.     

Nif-hybrids of Enterobacter cloacae: Selection for nif-gene integration with nif-plasmids containing the Mu transposon

Summary The nitrogen fixation (nif)-gene group of Klebsiella can be transferred onto Enterobacter cloacae by conjugation, using Escherichia coli donor cells carrying the composite self-transmissible nif-plasmid pRD1. To enforce integration and stabilisation, in the present study a derivative of pRD1, viz plasmid pCE1, containing the Mu transposon was used. pCE1:: Mu cts makes Enterobacter cloacae cells nif ⁺, and sensitive to temperature induction of Mu. Few cells survive treatment at 42°C. Seventy-two isolates thus obtained were screened for location of their nif-genes. At least four were found to contain the nif-genes integrated into the chromosome. This was documented by gel electrophoresis of their DNA, and by Southern hybridisation of their DNA with Klebsiella nif-KDH DNA as radioactive probe. The Mu transposon had also become part of their chromosome.     

Synchronous division induced in Escherichia coli K12 by gemts mutants of phage Mu

Summary Infection with the bacteriophage mutant Mu c ⁺ gemts2 at 42° C induces synchrony in cell division in cultures of Escherichia coli K12. This synchrony may last for several cycles and is not only due to selection since synchronization is observed even when bacterial survival to the infection is over 80% as in lysogens for Mu c ⁺ gemts2. The mechanism by which sycnhrony is induced is not known, but since the product of Mu gene gem (previously called lig) has been shown to interact with the enzymatic system in the bacteria controlling the degree of DNA supercoiling, the phenomenon could be a consequence of this interaction.     

Host controlled restriction and modification of bacteriophage Mu and Mu-promoted chromosome mobilization in Citrobacter freundii

Summary Bacteriophage Mu grown on Escherichia coli K12 (Mu. K) is restricted by wild type Citrobacter freundii. In two C. freundii mutants, where the restriction of foreign F factors is absent (de Graaff and Stouthamer, 1971), the restriction for Mu. K, although at a lower level, still exists. Consequently two host specificity systems exist in C. freundii, one affecting mainly the acceptance of foreign plasmidal and chromosomal DNA and one affecting foreign DNA of bacteriophage Mu. Mu is able to lysogenize C. freundii and to induce mutations at random in its chromosome. Furthermore Mu is able to promote the mobilization of the C. freundii chromosome in strains carrying F factors. Mu promoted integration of F ts 114 lac ⁺ into the C. freundii chromosome was observed, resulting in the formation of stable Hfr strains. In this way it is possible to devise a method for chromosome transfer in other genera than E. coli to which plasmids of E. coli can be transferred, but in which no chromosome mobilization is possible because of poor DNA homology between the foreign plasmid and the host chromosome.     

Mini-muduction: A new mode of gene transfer mediated by mini-Mu

Summary We compared the transducing properties of Mucts62 and Mucts62/mini-Mu lysates, using Mu immune and non immune Rec⁺ and recA recipient strains. The Mu/mini-Mu lysates transduced all bacterial markers tested 10 times more efficiently than the Mucts62 lysates in Rec⁺ recipients. Most of the transductants obtained after infection with the Mu/mini-Mu lysates result from the substitution of the mutated gene of the recipient by the wild type allele from the donor, most probably carried on the gigantic variable end linked to the mini-Mu genome.Moreover the Mu/mini-Mu lysates gave a new type of Rec-independent transduction that we called mini-muduction. Mini-muduction requires the activity of Mu gene A and provides transductants which carry the transduced marker surrounded by two mini-Mu genomes similarly oriented, and inserted at random location in the recipient chromosome. The mini-Mu/transduced DNA/mini-Mu structures are able to transpose spontaneously, for instance into a transmissible plasmid, in the presence of Mu gene A product.     

Specificity of bacteriophage Mu excision

Summary To study the excision of bacteriophage Mu at the DNA sequence level, the Mu-derived phage placMu3 was transposed to the transcribed but non-translated leader region of a plasmid-borne tetracycline (tet) resistance gene. Revertants (excision products) were then selected by Tet⁺ restoration of Tet⁺ and characterized. Of 21 independent Tet⁺ revertants, 17 contained simple deletions of most or all of placMu3, while the other four contained more complex rearrangements in which one end of placMu3 had been transposed, and most of the prophage had been deleted. The deletion endpoints were found in short direct repeats in each of the complex rearrangements and in 11 of the 17 simple deletion excisants. The results suggest models of slipped mispairing of template and nascent DNA strands facilitated by proteins of the Mu transposition machinery.     

Evolution of the long range structure of satellite DNAs in the genus Apodemus.

The temperate bacteriophage Mu causes mutations by inserting its DNA randomly into the genes of its host bacterium Escherichia coli. It is shown here that Mu DNA can be precisely excised from the different integration sites and that as a result wild-type function of the gene into which Mu was inserted is restored. The excision of Mu DNA is observable only if the Mu prophage carries mutations at the X locus. Thus, lac⁺ revertants from six strains, containing heat-inducible prophage Mu cts62 at different locations in the Z gene of the lac operon, were readily obtained by first introducing the X mutation into Mu cts62. The lac⁺ revertants produced wild-type β-galactosidase, and no trace of Mu DNA could be detected in them; this indicates that the junction of Mu DNA and host DNA can be specifically recognized. However, the excision of Mu DNA is generally not perfect, because in most cases it does not lead to the wild-type genotype. The function of gene A of Mu appears to be required for excision. Since the lethal functions of Mu are completely blocked in the Mu cts62 X prophage, the X locus probably has a regulatory function. At least one X mutation is caused by an insertion of about 900 base-pairs in Mu DNA. The discovery of the X mutants opens the way for studying the reversible interaction of the host and Mu chromosomes, and for using Mu to manipulate the host genome in various ways.     

Behavior of bacteriophage Mu DNA upon infecton of Escherichia coli cells.

The question whether the ends of bacteriophage Mu DNA are fused to form a ring in host cells is critical to the understanding of the mechanism of integrative recombination between Mu DNA and host DNA. We have examined the fate of ³²P-labeled Mu DNA, after infection of sensitive and immune (lysogenic) cells, by sedimentation in sucrose gradients, ethidium bromide/CsCl density centrifugation and by electrophoresis of parental Mu DNA and its fragments in agarose gels. We find that the parental Mu DNA cannot be detected as covalently closed circles at any stage during the Mu life cycle. An interesting form of Mu DNA can be seen after superinfection of immune cells. This form sediments about twice as fast as the mature phage DNA marker in neutral sucrose gradients but yields linear molecules upon phenol extraction. Upon infection of sensitive cells, most of the parental DNA associates with a large complex, presumably containing the host chromosome. When Mu-sensitive cells are infected with unlabeled Mu particles and Mu DNA examined at different times after infection by fractionation in 0.3% agarose gels and hybridization with ³²P-labeled Mu DNA, Mu sequences are found to appear with the bulk host DNA as the phage lytic cycle progresses. However, no distinct replicative or integrative intermediate of Mu, that behaves differently from linear Mu DNA and is separate from the host DNA, can be detected.     

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.     

Preferential generalized transduction by bacteriophage Mu

Summary The generalized transduction by bacteriophage Mu was found to be preferential for the 0–1 min segment of the E. coli K12 chromosome. This transduction pattern is obtained with phage lysates grown on all F^-, F⁺ and Hfr tested, and is not marker-specific.Phages grown by both lytic infection and by heat induction of prophages at different locations of the host's chromosome show the same transduction pattern, indicating that generation of transducing DNA does not directly depend on excision events. Conjugation of independently obtained Muc ⁺-lysogenic strains of HfrC with a multiauxotrophic F^- recipient strain lysogenic for a Mucts62 prophage, shows that transfer of the temperature-resistance character (Muc ⁺) is not preferentially linked to the 0–1 min segment. The lysogenizing integrations do therefore not take place within the segment preferentially transduced by the phage.A model¹ for the generation of the transducing DNA is proposed, which assumes that for its replication, Mu DNA is integrated close to the 0–1 min segment of the host chromosome, which is then preferentially replicated and packaged into the phage heads.     

In vitro constructed plasmids containing both ends of bacteriophage Mu DNA express phage functions

Summary The construction of a plasmid carrying the right end PstI·B fragment of bacteriophage Mu DNA and of plasmids containing in addition the left end EcoRI·C fragment of Mu DNA into the vector pBR322 is described. Inversion of the G segment still occurs in all these plasmids. By marker rescue and complementation experiments the right PstI cleavage site was located to the left of gene Q. The composite plasmids inheriting also the left end EcoRI fragment of Mu DNA express both the immunity and killing functions of Mu and direct the in vitro synthesis of presumably Mu-specific polypeptides. These results demonstrate that Mu-specific functions can be analyzed from cloned fragments.     

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.     

Mu DNA replication in vitro: Criteria for initiation

Summary An in vitro system for investigating Mu replication and transposition using film lysates has recently been described (Higgins et al. 1983). Under most conditions examined, little or no replication initiation takes place in vitro. The data are consistent with Mu specific replication forks being initiated in vivo, and completing but not reinitiating a round of replication in vitro. Since Mu DNA replication is from left to right, an excess of right end sequences compared to left end sequences are replicated on the film lysates.Two conditions reported to specifically decrease Mu DNA replication in vivo (Pato and Reich 1982) were assessed for their effects on in vitro replication. Protein synthesis inhibition in vivo drastically decreased Mu specific DNA synthesis both in vivo and in the film lysates. However, temperature-sensitive (ts) A cells (A ts) incubated at the non-permissive temperature gave increased Mu synthesis at the permissive temperature in vitro. These conditions result in preferential mobilization of Mu specific forks, equal replication of the left and right end sequences of Mu, and meet minimal criteria for Mu replication initiation in the Ats lysates. The results are consistent with the Mu A protein limiting the initiation of Mu replication in vitro.     

Bacteriophage Mu-1: A tool to transpose and to localize bacterial genes

In a previous publication (Faelen et al., 1975), it was predicted that the temperate phage Mu-1 would mediate transposition of bacterial genes. Here we show that this is indeed the case. By mating either induced F′ strains (which carry a thermoinducible Mu prophage in the bacterial chromosome), or sensitive F′ infected with Mu, with appropriate recipients, we were able to isolate new F′ episomes which carry various lengths of bacterial DNA. The frequency of transposition of a given marker can be as high as 10^?4. The episomes which carry the transposed DNA always carry Mu as well. When this is coupled with the fact that induction or infection with Mu is necessary for transposition to occur, it is probable that both Mu enzymes and Mu DNA are required by the transposition process. Episomes selected for the presence of a given marker were analyzed for the presence of unselected markers. It was found that: (1) only markers linked to the selected marker can be cotransposed with it; (2) when two markers are simultaneously transposed, all markers lying between them on the chromosome are also transposed; (3) the frequency at which an unselected marker is cotransposed is in some way related to the distance between that marker and the selected marker; (4) the transposition process occurs in both Rec⁺ and Rec^? strains. Mu-mediated transposition offers a new way to isolate F′ episomes and to localize and order bacterial genes as far apart as three minutes.     

A general method for isolation of Mu d1-8(Aprlac) operon fusions in Salmonella typhimurium LT2 from Tn10 insertion strains: chlC::Mu d1-8

Summary A general method was developed for the isolation of Salmonella thyphimurium LT2 Mu d1–8 (Ap^r lac) operon fusions in a gene displacing a Tn10 insertion. Random Mu d1–8 fusion pools were prepared to grow phage P22 lysates which transduced chlC::Tn10 to Ap^rTet^s on fusaric acidampicillin plates. Among these Ap^rTet^s potential chlC::Mu d1–8 fusions, a simple spot test identified the fusions that were closely linked to the Tn10 insertion in chlC. Out of 68 Ap^rTet^s isolates 7 chlC::Mu d1–8 fusions with a nitrate-induced Lac⁺ phenotype were obtained. When oxrA::Tn10 was transduced into these chlC::Mu d1–8 fusions, they became Lac^- even in the presence of nitrate, confirming that they were chlC::Mu d1–8 fusions.