ABSTRACT
The recombinant bacteriophages with the genomes containing the DNA fragments of bacteria Erwinia chrysanthemi, including the pectatelyase gene, were constructed on the base of Pseudomonas aeruginosa temperate bacteriophage SM. The gene transferred into Pseudomonas aeruginosa PAO1 cells by transfection is expressed in the new bacterial host. The restriction maps of the recombinant bacteriophages are constructed and the position of an insert is defined. Bacteriophage SM was found to be capable of reproducing in Pseudomonas aeruginosa PAO1 cells when its DNA was shortened to 88% or increased to 111% of the normal genome length. Except for bacteriophage SM, the recombinant bacteriophage SM-2 with an unique restriction endonuclease site for XbaI can also be used as a vector for cloning. Bacteriophage SM capacity in cloning of heterological DNA at HindIII sites is not less than 8 Md, capacity of bacteriophage SM-2 is not less than 5 and 8 Md at XbaI and HindIII sites respectively.
Subject(s)
Bacteriophages/genetics , Pseudomonas aeruginosa/genetics , Cloning, Molecular , DNA, Bacterial/genetics , Dickeya chrysanthemi/genetics , Gene Expression , Genes, Bacterial , Genes, Viral , Genetic Vectors , Restriction Mapping , TransfectionABSTRACT
Pseudomonas aeruginosa PAO SM-prophage was localized on the chromosome between thr-9001 and pur-66 locuses on 42-43 min of chromosomal genetic map. The location of prophage was identified on the basis of prophage linkage with the above-mentioned markers and confirmed by the purine, hypoxanthine and threonine deletions in course of thermoinduction of SM cts6 prophage from lysogens. The decrease for two orders in lysogenization frequency of thr mutants by SM bacteriophage suggests the integration of SM prophage in these cells into some other region of chromosome.
Subject(s)
Bacteriophages/genetics , Chromosomes, Bacterial , Lysogeny , Genetic Markers , Mutation , Pseudomonas aeruginosa/geneticsABSTRACT
Factors affecting the efficiency of transfection of Ps. aeruginosa PAO1 cells by the temperate SM bacteriophage DNA have been determined. The efficiency of transfection by DNA preparations isolated from the wild type bacteriophage SMc+ or its thermoinducible mutant SM cts6 is practically the same. The frequency of transfection is (7-9) X 10(4) of infectious centers per mkg of transfecting DNA. Variability in the frequencies of transfection has been registered depending of the infection conditions or on the transfer of the Ps. aeruginosa PAO1 recipient strain population into the competence phase. The efficiency of transfection is increased by the addition of Ca2+ or Mg2+ ions affecting the adsorption and absorbtion of phage DNA by the recipient cells. Optimal concentrations of the bivalent metal ions are 0.15M CaCl2 and 0.2M MgCl2. The results obtained have been used for optimizing the conditions of Ps. aeruginosa PAO1 transfection by SM bacteriophage DNA.
Subject(s)
Bacteriophages/genetics , Pseudomonas aeruginosa/genetics , Transfection , DNA, Viral/genetics , MutationABSTRACT
The DNA of temperate phage SM P. aeruginosa has one PvuII site, two BamHI sites, three HindIII sites and five EcoRI sites. Using these restrictases the physical map of the phage genome has been constructed. The DNA of phage SM has in their structure cohesive ends similar to cos-sites of phage lambda DNA. EcoRI-fragments with cohesive ends have molecular masses 2.9 and 4.9 MDa.
Subject(s)
Bacteriophages/genetics , DNA, Viral/genetics , Bacteriophages/analysis , Chromosome Mapping , DNA Restriction Enzymes , DNA, Viral/analysis , Genetic Markers , Molecular Weight , Pseudomonas aeruginosaABSTRACT
76 mutants with impaired ability to lysogenize host cells were isolated in SM phage after mutagenesis using several chemical mutagens. By means of complementation test, these mutants were distributed into two groups, cI and cII. The mutants of the cI group were similar phenotypically to the cI mutants of phage lambda defective in synthesis of repressor. The mutants of the cII group establish and support the lysogenic state in infected cells with very low frequency. Temperature-sensitive mutants belonging to 13 complementation groups and nonlysogenizing mutants of the cI and cII groups were used in genetic mapping of SM phage. Mutual positions of markers and relative distances between them were determined by the method of two-factorial crosses. The greatest distance equal to 20 units of recombination was determined between ts 88 marker and one of early genes marked with ts 105 mutation. The genes cI and cII are closely linked to each other and also to ts 105 marker and are situated at one end of the genetic map.
Subject(s)
Bacteriophages/genetics , Genes, Viral , Lysogeny , Chromosome Mapping , Mutation , Pseudomonas aeruginosa , Recombination, GeneticABSTRACT
The inheritance of plasmids Rms163 and R74 by Pseudomonas aeruginosa strain PAO hs been shown to effect the reproduction of a temperature bacteriophage SM. The decrease in plating efficiency of bacteriophage on Pseudomonas aeruginosa PAO (rms163) lawn is explained by the high degree of cell lysogenization by bacteriophage. Plasmid R74 inhibits bacteriophage SM propagation ultimately, evidently due to interruption of definite stages in vegetative development of bacteriophage by the products of plasmid specific genes.
Subject(s)
Bacteriophages/genetics , R Factors , Virus Replication , Bacteriophages/physiology , Lysogeny , Pseudomonas aeruginosa/geneticsABSTRACT
Seventy three temperature-sensitive mutants of Pseudomonas aeruginosa SM phage have been obtained using different mutagens and assigned to thirteen complementation groups. Representative mutants of each group have been studied with the aim of characterizing tentatively the time of genes expression in infected bacteria. Two genes appear to function during the first minutes after infection, whereas the remaining genes are needed for late functions. Most of the temperature-sensitive functions in the different mutants are reversible, i.e. they become active when the infected cells are shifted-down to the permissive temperature.
Subject(s)
Bacteriophages/genetics , Genetic Complementation Test , Mutation , Bacteriolysis , Bacteriophages/physiology , Gene Expression Regulation , Genes, Viral , Lysogeny , Pseudomonas aeruginosa , Temperature , Time Factors , Virus ActivationABSTRACT
The temperate bacteriophage SM is not serologically related to the known transducing phages F116, G101, B3 of Pseudomonas aeruginosa. The strains with auxotrophic mutations within the wide ranges of the genetic map of P. aeruginosa strain PAO1 were used for studying the transducing activity of the SM phage. All of the 7 bacterial markers tested are transduced with SM phage grown on a prototrophic donor strain. The frequency of transduction of separate bacterial markers using the wild type SM phage is 2.3 to 4.6 X 10(-8). Linked ilv202+ - met28+ markers are cotransduced with SM phage at a frequency of about 1.5%.
Subject(s)
Bacteriophages/genetics , Transduction, Genetic , Genetic Markers , Pseudomonas aeruginosaABSTRACT
Base ratio of DNA from 21 bacteriophage of Pseudomonas was determined by chemical hydrolysis and paper chromatography. Obtained values of the GC pair content were compared with melting temperature of DNA in 0,1 X SSC. The content of GC pairs correlates with melting temperature by equation %GC = 2,53 (Tm - 53,4). The content of GC pair for DNA from 30 bacteriophages of Pseudomonas was calculated. Some speculations concerning the distribution in DNA base ratio of bacteriophages of Pseudomonas are discussed.
Subject(s)
Bacteriophages/genetics , DNA, Viral/analysis , Pseudomonas/genetics , Base Composition , Nucleic Acid Denaturation , Osmolar Concentration , Solvents , Species SpecificityABSTRACT
Fifty-two bacteriophages active against Pseudomonas bacteria were isolated from nautral sources and their following properties were studied: the morphology of negative colonies, the spectrum of lytic action, and the susceptibility to certain chemical and physical factors. One phage was shown to contain lipids. The content of GC pairs in the DNA of the phages determined from the melting temperature varied within the range of 46 to 67%.
