Four types of protease negative mutants of Bacillus amyloliquefaciens A50 were selected after four stages of step-by-step UV light mutagenesis. EDTA, and PMSF were used as inhibitors of protease activity to characterize the protease negative mutants with regard to the protease type. The electrophoretic patterns of proteases from culture medium of B. amyloliquefaciens protease deficient mutants were studied. The proinsulin stability in the culture medium of different mutants was analysed. Protease deficient B. amyloliquefaciens strain A50-32 may be used as a recipient for cloning and expression of a foreign proteins genes.

The processes of replication and transposition of Pseudomonas aeruginosa transposable phage D3112 in cells of Escherichia coli (D3112) and E. coli (RP4::D3112) were studied. D3112 genome is a "silent cassette" ("conex-phage"--conditionally expressible) in E. coli cells incubated at 42 degrees C. Two compulsory conditions for D3112 genome expression are incubation at 30 degrees C and the presence in cells of RP4 plasmid. Processes of replication and transposition in E. coli are coupled. RP4 plasmid stimulates D3112 DNA synthesis in E. coli at least by two order of magnitude. In correspondence with this observation is the fact that when Mg2+ is present in high concentration (0.1 M) in a cultural medium, the production of mature phage is enhanced by two order of magnitude in E. coli (RP4::D3112) or in E. coli (D3112, RP4) cells, and is approx. 10(-1)-10(-2) phage per cell. No influence of Mg on phage production is observed in E. coli (D3112) cells.

Electron microscopical examination of the new virulent bacteriophage phi KZ, specific for Pseudomonas aeruginosa, has revealed an unusual structure in its capsid. In the center of the phage head is a cylinder of low electron density ("inner body"), surrounded by fibrous material which is packed around the inner body in a spoollike manner. The inner body itself has a springlike appearance. These structures disappear after adsorption of phage particles to bacteria. Various morphological forms, which can be interpreted as intermediate steps in phi KZ DNA condensation, have been seen in ultrathin sections of phi KZ-infected cells.

The study of the ultrathin sections of cells infected with virulent phage phi KZ has confirmed the presence of a specific cylindrical formation, an inner body, in the head of this phage and revealed the spiral structure of this inner body. The formation of DNA condensates whose structure resembles a spring wound around the core (the inner body) has been shown to occur in the cells in the process of the ultracellular development of phage phi KZ. This development leads to characteristic changes in the cellular structure, and in particular in the cell walls and the nucleoid.

It has been demonstrated that the genome of phage D3112 of Preudomonas aeruginosa can be transposed into Escherichia coli chromosome as a component of the hybrid plasmid RP4 TcrKms::D3112. Also, transposition of D3112 from E. coli (D3112) chromosome into RP4 plasmid occurs. The phage stimulates the chromosome mobilizing activity of RP4 plasmid, similar to other transposons. E. coli (RP4::D3112) cells were previously shown to form no colonies at 30 degrees C. Auxotrophic mutants and mutants incapable of utilizing different carbohydrates were found among E. coli clones survived after a long incubation at 30 degrees C (at frequencies approximately 10(-3) - 10(-4). These mutants inherited stably the capability to produce D3112 phage. E. coli auxotrophic mutants have arisen indeed as a consequence of phage integration into the E. coli chromosome, since prototrophic transductants derived from these mutants after their treatment with generalized transducing P1 phage have lost the ability to produce D3112 phage. Clones with mutations in Km or Tc genes of RP4 plasmid, occurring at high frequencies (about 3%) were found after introduction of RP4 into E. coli (D3112). These mutant RP4 plasmids carry insertions of D3112 genomes. Clones of E. coli which lost mutant plasmids still produce D3112 and retain their initial auxotrophic mutations.

It has been shown that D3112 prophage can be integrated into different chromosomal sites of Pseudomonas aeruginosa. The other Mu-like phages (B3, B39, PM69) are capable to insert their genomes during infection process into the plasmids RPL11, RMS148, RMS163. Their integration is occasionally accompanied by formation of mutations in plasmid genes. The certain types of auxotrophic and morphological mutants (thi, met, pigmented, met - pigmented) can be found at a frequency about 10% among survivors after a long (48 h) incubation at 42 degrees C of PAO (D3112cts15) or PAO (B39cts1) lysogens. The spectrum of mutants might depend on the time of heat induction. After a short exposure (10-20 min), arg and pigmented mutants can be found. Accumulation of certain kinds of mutants after heat induction is quite a specific phenomenon for Mu-like phages; heat induction of PAO (F116ts245) does not lead to selection of these specific bacterial mutants (F116 is unrelated to Mu-like phages and has extrachromosomal location).

The behavior of Escherichia coli cells carrying RP4 plasmid which contains the genome of a Mu-like D3112 phage specific for Pseudomonas aeruginosa was studied. Two different types of D3112 genome expression were revealed in E. coli. The first is BP4-dependent expression. In this case, expression of certain D3112 genes designated as "kil" only takes place when RP4 is present. As a result, cell division stops at 30 degrees C and cells form filaments. Cell division is not blocked at 42 degrees C. The second type of D3112 genome expression is RP4-independent. A small number of phage is produced independently of RP4 plasmid but this does not take place at 42 degrees C. No detectable quantity of the functionally active repressor of the phage was determined in E. coli (D3112). It is possible that the only cause for cell stability of E. coli (D3112) or E. coli (RP4::D3112) at 42 degrees C in the absence of the repressor is the fact of an extremely poor expression of D3112. In another heterologous system, P. putida both ways of phage development (lytic and lysogenic) are observed. This special state of D3112 genome in E. coli cells is proposed to be named "conditionally expressible prophage" or, in short, "conex-phage", to distinguish it from a classical lysogenic state when stability is determined by repressor activity. Specific blockade of cell division, due to D3112 expression, was also found in P. putida cells. It is evident that the kil function of D3112 is not specific to recognize the difference between division machinery of bacteria belonging to distinct species or genera. Protein synthesis is needed to stop cell division and during a short time period this process could be reversible. Isolation of E. coli (D3112) which lost RP4 plasmid may be regarded as an evidence for D3112 transposition in E. coli. Some possibilities for using the system to look for E. coli mutants with modified expression of foreign genes are considered.

The genome of a Mu-like bacteriophage D3112 specific for Pseudomonas aeruginosa was integrated in vivo into the RP4 plasmid. The fact of integration has been proved by two experiments: 1. The loss of RP4 plasmid is accompanied by loss of D3112 prophage; 2. Transfer of the plasmid by conjugation from Pseudomonas aeruginosa into bacteria of other species - P. putida PgG1 or Escherichia coli C600 leads to the occurrence of clones of these species which liberate phage capable of growing on the lawn of P. aeruginosa bacteria. The integrated state of D3112 inserted into RP4 is stable in P. aeruginosa, E. coli, P. putida. The transfer frequency of RP4 with integrated D3112 prophage into different bacteria which do not contain homoimmune prophage is essentially lower than that of the RP4 having no D3112 prophage. Specific manifestation of D3112 genome activity in E. coli cells is the sensitivity of cell growth to lower temperature (30 degrees C) of incubation.

A group of ts mutants of phi KZ phage specific for Pseudomonas aeruginosa was isolated and studied. A phi KZ phage particle has a unique feature: a specific structure as an internal body concerned with DNA packaging which is present in the phage capsid. Ts mutants were distributed into complementation groups. The time of the development block when grown at elevated temperatures was determined for mutants from several groups, the exact stage of the block having been defined for some late ts mutants. As was found for ts759, the stage of connection of heads containing DNA and the inner body with tails was blocked. The results obtained with ts759 allowed to prove the circular location of fibrous material (DNA?) around the inner body. The conditions for phage crosses were revealed. The genetic map of phi KZ is circular. It may be concluded, considering the linear phi KZ DNA structure in capsids, that circular permutations of phage DNA take place during phage maturation or DNA packaging.

A virulent phage of Pseudomonas aeruginosa, phi KZ which is able to package DNA of 210 MD is a general transducer. The transducing activity of phi KZ can be revealed when using recipient bacteria containing RMS148 plasmid (the P7 group of incompatibility). This plasmid blocks the development of phi KZ and prevents the phage to kill bacteria. General transduction mediated by phi KZ may be useful for the primary localization of genetic markers in P. aeruginosa.

Several different hybrids have been isolated in a cross between heteroimmune Mu-like Pseudomonas aeruginosa bacteriophages D3112 and B39. Analysis of hybrid phage DNAs treated with specific endonucleases and heteroduplex studies have permitted to map the immunity region of phages in the left part of their genomes. Also, a ts-mutation in the wild-type B39 and some genetic factors influencing the growth of D3112 in P. aeruginosa strain PA0 (B39) and in bacteria harbouring RPL11 plasmid, have been localized. All recombinants studied have arisen by double crossingover and can be considered as substitutions of different continuous parts of the D3112 genome by fragments of the B39 genome. A comparison of distribution of non-homologous regions in B39 and D3112 genomes as well as locations of endonuclease-sensitive sites have given some unexpected results. In contrast to the B39-D3112 pair, the other pair of related Mu-like phages, B3-D3112, have no visible homology within the most part of their genomes.

Using UV-spectroscopy and circular dichroism (CD) phi KZ bacteriophage and its DNA were investigated. From the value of optical densities in the 320--400 nm range the size of the phi KZ bacteriophage's head was determined; the diameter of phi KZ bacteriophage head was found to be equal to 1300 +/- 100 A. phi KZ bacteriophage DNA has block structure and the GC-pair content is equal to 43.8 +/- 0.3%. phi KZ bacteriophage CD spectrum has an unusual profile (in comparison with known bacteriophage CD spectra); this spectrum is similar to the CD spectra of DNA in polyethylene glycole solution. To our knowledge CD spectra of such type were not obtained for other bacteriophages.

A study of physico-chemical properties of the nucleic acid of phage Brevibacterium flavum was made. It was found that the phage nucleic acid is a double-stranded DNA. The content of GC pairs was determined by the temperature of DNA melting and by the chromatographic method. In both cases it was (52 +/- 2) mole %. It has been shown that endonuclease Eco RI treatment of phiB DNA forms 12 fragments. The molecular weight of phiB DNA has been determined by two independent methods: by measuring the contous length of DNA and by the sum of molecular weights of DNA restricts obtained after hydrolysis by endonuclease Eco RI and was found to be (30 +/- 1) . 10(6) dalton. Electron microscopy investigations detected rod and circular DNA in either monomeric or dimeric forms. The polarity of DNA arrangement in the phiB phage particle was determined by using the partial denaturation maps.