Integration of phage Mu into the RP4::D3112A-plasmid in Escherichia coli cells

Hybrid plasmids obtained as a result of Mu phage insertions into the RP4::D3112 plasmid in Escherichia coli cells were studied. Stable maintenance of RP4::D3112 plasmid in E. coli cells was provided by using the D3112 phage genome with a point polar mutation in the A gene which prevented early genes' expression. The presence of D3112A- in the RP4 plasmid has been shown to have no effect on efficiency of phage Mu transposition into this plasmid. Moreover, RP4 and D3112 genomes were equivalent targets for Mu integration. The integration of transposable phage into genome of nonrelated phage can be used as one of the approaches to construct recombinant phage genomes in vivo in the absence of DNA homology.     

The infection of bacilli by Mu cts phage integrated into the plasmid

The infection of Bacillus thuringiensis, B. cereus, B. mesentericus and B. polymyxa strains with temperate E. coli bacteriophage Mu cts62 integrated into plasmid RP4 under conditions of conjugative transfer is shown possible. The investigated strains of bacilli are not able to produce intact phage particles but they acquire the thermosensitive property determined by the phage genome. Gel electrophoresis and blot hybridization of DNA have confirmed the transfer of Mu cts62 genome or a part of it in the investigated strains of bacilli.     

The Tra2 core of the IncP(alpha) plasmid RP4 is required for intergeneric mating between Escherichia coli and Streptomyces lividans.

Escherichia coli cells and Streptomyces mycelia are able to form close contacts in the absence of a conjugative system which might facilitate intergeneric plasmid transfer without the genes required for mating pair formation (Tra2) of the RP4 plasmid. The same Tra2 genes found to be essential for RP4 plasmid transfer, RSF1010 mobilization, and donor-specific phage propagation in E. coli were also required for intergeneric transfer between E. coli and Streptomyces lividans.     

Compatibility of transposable phages of Escherichia coli and Pseudomonas aeruginosa. I. Co-development of phages Mu and D3112 and integration of phage D3112 into RP4::Mu plasmid in Pseudomonas aeruginosa cells

The possibility of using a model system (which included RP4::Mu plasmid and D3112 phage in Pseudomonas aeruginosa cells) for analysis of compatibility of transposable Escherichia coli phage Mu and P. aeruginosa phage D3112, as phages and transposons, was studied. No interaction was observed during the vegetative growth of phages. The majority of the hybrid RP4::Mu plasmids lost the Mu DNA after insertion of D3112 into RP4::Mu. The phenomenon was not a result of transposition immunity. We consider the loss of the Mu DNA as a consequence either of plasmid RP4::Mu instability in P. aeruginosa cells, because of the lack of functional Mu repressor, or of some D3112-encoded activity involved in its transposition. For the inambiguous conclusion on compatibility of two phages as transposons, it is necessary to modify the model system, eliminating the possibility of Mu phage replication--transposition.     

The genetic transfer, heredity and phenotypic expression of plasmid RP4::Mu cts genes in Bacillus cereus

The transcipients were obtained in intrageneric matings of E.coli donor harbouring the plasmid PR4::Mu cts 62 with Bac. cereus GP7 recipient cells with the frequency 10(-9). The transcipient clone Bac. cereus 682 was selected having stably inherited and expressed the hybrid plasmid PR4::Mu cts 62 genes for antibiotic resistance and temperature sensitivity. Production of the bacteriophage Mu cts 62 particles was not registered in the bacillary transcipient cells. The plasmid RP4::Mu cts 62 genes were localized in the chromosome of Bac. cereus 682 transcipient by the blot-hybridization technique with 32P-labelled DNA of the bacteriophage Mu cts 62 and the plasmid PR4. The transcipient of Bac. cereus 682 has the donor properties and transfers the RP4::Mu cts 62 genes to recipient cells of Bac. cereus DSM 318 with the frequency of 10(-6)-10(-7). The expression and transfer of the gram-negative plasmid genes in gram-positive bacterial cells are discussed.     

Genetic mapping of tyramine oxidase and arylsulfatase genes and their regulation in intergeneric hybrids of enteric bacteria.

The genes for arylsulfatase (atsA) and tyramine oxidase (tynA) have been mapped in Klebsiella aerogenes by P1 transduction. They are linked to gdhD and trp in the order atsA-tynA-gdhD-trp-pyrF. Complementation analysis using F' episomes from Escherichia coli suggested an analogous location of these genes in E. coli, although arylsulfatase activity was not detected in E. coli. P1 phage and F' episomes were used to create intergeneric hybrid strains of enteric bacteria by transfer of the ats and tyn genes between K. aerogenes, E. coli, and Salmonella typhimurium. Intergeneric transduction of the tynK gene from K. aerogenes to an E. coli restrictionless strain was one to two orders less frequent than that of the leuK gene. The tyramine oxidase of E. coli and S. typhimurium in regulatory activity resemble very closely the enzyme of K. aerogenes. The atsE gene from E. coli was expressed, and latent arylsulfatase protein was formed in K. aerogenes and S typhimurium. The results of tyramine oxidase and arylsulfatase synthesis in intergeneric hybrids of enteric bacteria suggest that the system for regulation of enzyme synthesis is conserved more than the structure or function of enzyme protein during evolution.     

In Vivo Cloning of Arylsulfatase-Tyramine Oxidase Genes in rec Strains of Klebsiella aerogenes

A general, genetic technique for in vivo cloning of bacterial genes is presented. We previously introduced the Mu phage into various genera of bacteria including Klebsiella aerogenes with RP4 : : Mu. Using these strains carrying RP4 : : Mu cts and thermo-inducible Mu prophage in the chromosome, we cloned in vivo the arylsulfatase (ats) and tyramine oxidase (tyn) genes by partial thermo-induction. The donor strains carrying the recombinant plasmids were conjugated with K. aerogenes rec strains, which were isolated as UV-sensitive mutants. The resultant recombinant plasmids, pAT1 and pAT2, were purified and used for the transformation of mutant strains deficient in the ats and tyn genes. The ats-tyn genes seemed to be transposed into the RP4::Mu cts plasmid together with other chromosomal DNA fragments. This in vivo cloning method is applicable to a wide variety of gram-negative bacteria.     

Adsorptive capacity of bacteriophage Mu on Escherichia coli K-12 strains with deletions in the attlambda region]

Escherichia coli strains with deletions in att lambda region were obtained. The comparison of the extent of deletions with the sensitivity of the corresponding mutant clones to phage Mu showed that the gene controlling the sensitivity of E. coli K-12 to the phage Mu is located in nad A-gal region of the bacterial chromosome. It is shown that the resistance of E. coli strains which had lost the region of bacterial chromosome between nad A gene and genes of gal-operon have adsorption character. Deletion of the nad A-gal region does not affect the adsorption of other phages (lambda, P1 and T4). Thus, the gene, located in this region, is responsible for the specific adsorption of the phage Mu.     

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.     

Internal EcoRI-generated deletion in prophage Mu DNA

The hybrid plasmid consisting of the plasmid pRP1.2 (derivative of RP4) genome and deleted prophage Mucts 62 genome which lost the central EcoRI fragment of DNA was constructed. The ability of deleted Mu phage to carry out E. coli chromosomal genes transposition was still retained.     

Transduction of chromosomal genes between enteric bacteria by bacteriophage P1.

We have used P1 transduction to create intergeneric hybrid strains of enteric bacteria by moving the genA and hut genes between Klebsiella aerogenes, Escherichia coli and Salmonella typhimurium. The use of E. coli as the recipient in such transductions permits the construction of episomes and specialized transducing phage containing non-E. coli material. The effect of host restriction modification and deoxyribonucleic acid homology on the frequency of intergeneric transduction of these loci has been examined.     

Infection of Salmonella typhimurium with coliphage Mu d1 (Apr lac): construction of pyr::lac gene fusions.

A procedure was developed for introducing the coliphage Mu d1 (Apr lac) into Salmonella typhimurium in order to construct gene fusions that place the structural genes of the lac operon under the control of the promoter-regulatory region of other genes. To introduce Mu d1 from Escherichia coli K-12 into S. typhimurium, which is normally not a host for Mu, we first constructed an E. coli double lysogen carrying the defective Mu d1 phage and a Mu-P1 hybrid helper phage (MuhP1) that confers the P1 host range. A lysate prepared from this strain was used to infect a P1-sensitive (i.e., galE), restriction-deficient, modification-proficient strain of S. typhimurium, and a double lysogen carrying Mu d1 and MuhP1 was isolated. Induction of the latter strain produced lysates capable of infecting and generating gene fusions in P1-sensitive strains of S. typhimurium. In this paper we describe the construction of pyr::lac fusions by this technique.     

Erwinia carotovora as a recipient system for the natural hybrid plasmid RP4::Mu cts62

The plasmid RP4::Mu cts62 is transferred from Escherichia coli cells into a recipient strain Erwinia carotovora 268 by conjugation with the frequency 1.5-5 x 10(-7) per donor cell. The maximal frequencies of transfer are obtained by cultivation of donor and recipient cells for 3-5 h on the filters. Structural and functional validity of the plasmid in transconjugants is expressed in preservation of all antibiotic-resistant markers of RP4, thermosensitivity to growth at 42 degrees C as well as spontaneous and thermally-induced production and zygotic induction of bacteriophage determined by the genome of Mu cts62, total length of the plasmid restricts. Location and orientation of Mu cts62 genome in the plasmid restricts. Location and orientation of Mu cts62 genome in the plasmid RP4::Mu cts62 in Erwinia carotovora transconjugant cells has been determined. A single bacteriophage genome has been shown to transpose into the chromosome of the cell with the elimination of RP4 fragment under the conditions of thermal induction.     

Conjugation in Rhodopseudomonas sphaeroides mediated by R plasmids

Plasmids R68.45, RP4, RP4::Mu cts62, RP1ts::Tn10, RP1ts::Tn9, Rts1 and RP41 were transferred into cells of photosynthetic nitrogen-fixation bacterium Rhodopseudomonas sphaeroides from Escherichia coli and Pseudomonas aeruginosa. The transfer of plasmids occurred with high frequency of 10(-1) to 10(-2) per donor cell in all cases. Mobilization of R. sphaeroides 2R chromosome was obtained by RP4 and Rts1 plasmids at a frequency of 10(-7) to 10(-8) per donor cell in all cases. Mobilization of R. sphaeroides 2R chromosome was obtained by RP4 and Rts1 plasmids at a frequency of 10(-7) to 10(-8) per donor cell. Bacteriophage Mu cts62 could be induced from the plasmid DNA in R. sphaeroides 2R cells and was capable of the lytic growth and producing phage progeny. It was demonstrated that an increase in the efficiency of donor chromosomal genes transfer into recipient cells could be achieved in crosses with the donor carrying RP4::Mcts62 plasmid.     

Behaviour of temperate phage Mu in Salmonella typhi

We have developed a convenient system for genetic analysis of Salmonella typhi exploiting the properties of the mutator phage Mu. In spite of the fact that wild-type Salmonella typhi strains do not allow Mu to form plaques on them, we have shown that these strains are actually sensitive to the phage. It proved possible to use Mu to induce mutations and to promote intra- and interspecific genetic transfer, without having to introduce the phage into the bacteria by means other than infection. Furthermore, we isolated Salmonella typhi derivatives on which Mu formed plaques, and studied the behaviour of Mu in these and wild-type strains.     

Genetic study of the effect of lambda phage on Mu phage production in Escherichia coli K-12 strains with Mu--lambda--Mu structures

The interaction of temperate bacteriophages Mu and lambda is studied during their simultaneous induction in specially constructed heterolysogenic strains of Escherichia coli bearing trimeric Mu--lambda--Mu structures. These strains were obtained by the MU-mediated integration of phage lambda circular genomes. Heterolysogenic strains of E. coli were used for studying phage lambda eliminating effect on Mu development with a simultaneous induction of prophages in the same cell. The results of the study allow the localization of the region of phage lambda genome incorporating gene (genes) lambda, which produces an eliminating effect on Mu development.     

The cell wall receptor for bacteriophage Mu G(−) in Erwinia and Escherichia coli C

Abstract Adsorption of bacteriophage Mu with its invertible DNA segment in the G(−) orientation requires a terminal glucose residue for binding to the core lipopolysaccharide (LPS) of Gram-negative bacteria. Analysis of a Mu-resistant mutant shows that the receptor for Mu G(−) in Erwinia B374 is a Glc-β1,6-Glc disaccharide. A spontaneously occurring host-range mutant, Mu G(−)h101, grows on Escherichia coli C. The loss of the terminal β1,3-linked glucose from the LPS of E. coli C leads to resistance to the phage Mu. These mutants are also resistant to phage P1 and D108 which have largely homologous G segments. This shows that Mu G(+) and G(−) phage particles differ with respect to their cell-wall receptors in the type of glycosidic linkage of a terminal glucose residue: α1, 2 for G(+) and β1,6 for G(−).     

Use of deletion mutants of plasmid RP4::D3112 for the genetic analysis of Pseudomonas aeruginosa bacteriophage D3112

The hybrid plasmid RP4::D3112 becomes unstable in Escherichia coli K-12 cells under certain growth conditions. The deletion mutants of this plasmid are formed at a high frequency. All the deletions selected have a specific feature: they start in the left end, at the point of joining of plasmid and phage DNA, and remove different portions of the phage genome. The deletion mutants have been used for genetic mapping of D3112. We have localized the repressor gene cI (0-1.3 kb), 3 early genes (1.3-14.2 kb) and two groups of late genes (14.2-29.9 and 29.9-38 kb). Electron microscope studies of RP4::D3112 DNA and its deletion derivatives have shown that integration of D3112 genome in RP4 occurs through the ends of the genome, without permutations. It appears that bacterial nucleotide sequences joined to DNA from mature D3112 particles, to the right end of D3112 genome, are lost. Thus, transposable phages D3112 of Pseudomonas aeruginosa and E. coli Mu phage have some similarities in the genome organization and in the way of their integration into the host DNA.     

Bacteriophage Mu genome sequence: analysis and comparison with Mu-like prophages in Haemophilus, Neisseria and Deinococcus

We report the complete 36,717 bp genome sequence of bacteriophage Mu and provide an analysis of the sequence, both with regard to the new genes and other genetic features revealed by the sequence itself and by a comparison to eight complete or nearly complete Mu-like prophage genomes found in the genomes of a diverse group of bacteria. The comparative studies confirm that members of the Mu-related family of phage genomes are genetically mosaic with respect to each other, as seen in other groups of phages such as the phage lambda-related group of phages of enteric hosts and the phage L5-related group of mycobacteriophages. Mu also possesses segments of similarity, typically gene-sized, to genomes of otherwise non-Mu-like phages. The comparisons show that some well-known features of the Mu genome, including the invertible segment encoding tail fiber sequences, are not present in most members of the Mu genome sequence family examined here, suggesting that their presence may be relatively volatile over evolutionary time.The head and tail-encoding structural genes of Mu have only very weak similarity to the corresponding genes of other well-studied phage types. However, these weak similarities, and in some cases biochemical data, can be used to establish tentative functional assignments for 12 of the head and tail genes. These assignments are strongly supported by the fact that the order of gene functions assigned in this way conforms to the strongly conserved order of head and tail genes established in a wide variety of other phages. We show that the Mu head assembly scaffolding protein is encoded by a gene nested in-frame within the C-terminal half of another gene that encodes the putative head maturation protease. This is reminiscent of the arrangement established for phage lambda.     

Intergeneric mobilization of Rhizobium nif genes to Agrobacterium and Klebsiella.

Summary The P-1 incompatibility group plasmid RP1 transfers itself from Escherichia coli J53 to the clover endosymbiont bacterium Rhizobium trifolii strain T1 at low frequency in agar surface matings. R. trifolii T1 R-plasmid recipients display a phenotype identical to the wild-type parent strain in all respects except RP1 antibiotic resistances, allowing straightforward donor counterselection and differentiation of excojugants in further intergeneric plasmid transfer experiments. Hence RP1 can readily transfer itself intergenerically from R. trifolii T1 to the related plant pathogenic organism Agrobacterium tumefaciens and to a strain of the free-living diazotroph Klebsiella pneumoniae.Using R. trifolii T1 (RP1) as donor and as recipient LBA 4006, an avirulent strain of A. tumefaciens lacking the tumour-inducing (Ti) plasmid, selection was made for intergeneric transfer of the R-plasmid and its potential as vector of nitrogen-fixation genes evaluated by subsequent indirect screening. Exconjugant Agrobacteria were obtained which carried RP1 resistance markers and, given specific physiological conditions, would reduce acetylene under air. This is the first report of expression of nif genes in a hybrid strain of A. tumefaciens and is of interest since the Ti plasmid of this organism has been suggested as a natural vector for the introduction of these genes into plants. Plasmid RP1 also cotransferred Rhizobium nif genes to KP52, a strain of K. pneumoniae M5al, with deletion by phage eduction of the chromosomal genes for histidine biosynthesis, one of the nif regulatory genes (nif A), the gene for molybdenum cofactor (nif B) and for an electron transport protein of the nitrogen-fixation pathway (nif F). KP52 exconjugants carried RP1 drug resistances and reduced acetylene under anaerobic conditions.