[Molecular cloning and functional analysis of DNA regions of plasmid RP4 determining the incompatibility properties]. / Molekuliarnoe klonirovanie i funktsional'nyi analiz raionov DNK plazmidy RP4, determiniruiushchei svoistva nesovmestimosti.

Sau3A-generated DNA fragments determining incompatibility functions of the plasmid RP4 were cloned on the vectors pTK16 and pBR322. Inc+ recombinant plasmids were divided into two types: 1) expressing incompatibility only towards the homologous RP4 replicon, 2) expressing incompatibility - both towards the homologous RP4 replicon and towards the heterologous replicons of plasmids R906 and R751. For one member of the first type plasmids it was shown that the cloned Inc+-specific insertion derived from the region of location of the EcoRI restriction site. The majority of the Inc+ recombinant plasmids showed asymmetric expression of incompatibility, predominantly eliminating the resident IncP plasmid.

[Properties of potential vectors--derivatives of the broad-host--range plasmid RP4]. / Svoistva potentsial'nykh vektorov--proizvodnykh plazmidy s shirokim spektrom khoziaev RP4.

Nonconjugative deletion and recombinant derivatives of the RP4 plasmid are constructed. The plasmids can be used as vectors because they have relatively small molecular weights, unique cleavage sites for enzymes EcoRI, XhoI, BamHI, PstI, KpnI, BglII, SalGI and HindIII (the plasmids pRP401 and pRP417 having six of these sites), and easily tested phenotypes (Tcr, Apr and Gal+). In addition, all of them retain the broad host range property. Also, the plasmid pRP420 is a multicopy derivative capable of amplification. The plasmids are mobilized by conjugative plasmids pRK2013 and Flac from Escherichia coli cells into Rhizobium meliloti and Pseudomonas aeruginosa strains. Flac-mediated mobilization of the pRP417 plasmid which has an internal deletion of the transposon Tn1, is decreased, in comparison with the nondeleted plasmid. ColE1 replication machinery is inhibited for RP4--ColE1 recombinant derivatives, if both components are joined via EcoRI restriction site. This inhibition does not depend on the orientation of joined molecules. ColE1 replication machinery is functional, if delta RP4 and ColE1-like plasmids are joined via PstI cleavage site.

[Molecular genetic studies of the DNA promotor regions in Bacillus thuringiensis subsp. galleriae 69-6. II. Increased level of gene expression resulting from IS1 element integration]. / Molekuliarno-geneticheskie issledovaniia promotornykh raionov DNK Bacillus thuringiensis subsp. galleriae 69-6. Soobshchenie II. Povyshenie urovnia ékspressii gena v rezul'tate integratsii IS1-élementa.

The 1.45 kb promoter containing HindIII fragment of Bacillus thuringiensis DNA promotes the expression of the tet gene of recombinant pPBT9 plasmid in Escherichia coli cells. Spontaneous mutants of this plasmid were isolated and analysed. They are responsible for an increase in the level of tetracycline resistance. This 3-fold increase resulted from integration of IS1 element into the bacillar promoter containing HindIII fragment, which led to formation of a mutant pPBT9::IS1 plasmid. The IS sequence integrated was defined as an IS1 element of the E. coli HB101 chromosomal DNA. The integration site of IS1 was localized.

[Bacteriocinogenic plasmids of Bacillus thuringiensis]. / Bakteriotsinogennye plazmidy Bacillus thuringiensis.

When strains of Bacillus thuringiensis v. morrisoni or v. darmstadiensis were plated on solid medium, the appearance of oligosporogenic (Ospo) mutants and bacteriocin non-producing clones (Thc-) was observed. The comparative analysis of the plasmid content of original and mutant strains revealed that the appearance of Ospo and Thc- phenotypes correlated with the loss of certain plasmids.

[Homology of plasmids with a wide range of hosts]. / Gomologiia plazmid s shirokim spektrom khoziaev.

According to blotting hybridization and heteroduplex analysis, plasmids R751, R906 and RP4 of Inc Pi group have continuous regions of homology. These homologous regions were mapped on the R751 and RP4-derived pRP401 deletion mutant DNAs. The plasmid pRP401 (m.w. 21.9 kg) retains the broad host range property and has two regions of intensive homology with other Inc P-1 plasmid DNAs. These regions are localized at 8.2-12.0 kb and 13.9-21.9 kb of the physical map of pRP401 plasmid. Homologous regions of pRP401 DNA include at least the replication genes (oriV, trfA, trfB) as well as genes kilB, korA, korB and probably kilC. The data strongly point out that the broad host range plasmids have the same principle of structural and functional organization.

[Enterobacterial gene expression of antibiotic resistance under the control of Bacillus thuringiensis regulatory signals in gram-negative and gram-positive bacteria]. / Ekspressiia énterobakterial'nogo gena antibiotikoustoichivosti pod kontrolem reguliatornykh signalov Bacillus thuringiensis v gramotritsatel'noi i grampolozhitel'noi bakteriiakh.

Two recombinant plasmids pPBT9 and pPBT74 carrying HindIII promoter-active DNA fragments of Bacillus thuringiensis onto the vector pGA24 were studied. The cloned fragment of pPBT9 plasmid has many homologous regions in Bac. thuringiensis genome. This fragment contains three Escherichia coli RNA polymerase binding sites, one of which is responsible for promoting tetracycline resistance of the promoter-deficient enterobacterial tet gene in E. coli cells. The bacillar fragment of pPBT74 plasmid has a single RNA polymerase binding site. Plasmids pPBT9 and pPBT74 were joined to the bacillar vectors pBD8 and pBD12 to obtain bireplicon plasmids which replicate in E. coli and Bac. subtilis cells. The novel bireplicon plasmids pSTS98, pSTS912 and pSTS748 promote the expression of the enterobacterial tet gene under control of regulatory signals of cloned DNA fragments of Bac. thuringiensis in Bac. subtilis. Bireplicon pSTS228 plasmid containing its own enterobacterial promoter of tet gene was not able to express tetracycline resistance in Bac. subtilis cells. Most of the bireplicon plasmids studied were unstable in Bac. subtilis but not in E. coli cells.

[Comparative analysis of the P-1-group plasmids of incompatibility]. / Sravnitel'nyi analiz plazmid P-1-gruppy nesovmestimosti.

Wide host range plasmids (IncP-1) R906, R751 and R702 have several cleavage sites for BamHI, HindIII and EcoRI enzymes, in contrast to RP4 plasmid. Using these enzymes, deletion mutants of R906 plasmid have been obtained in vitro which only lost short DNA fragments (1 to 14 kb). A narrow host range pAV1 plasmid of the same incompatibility group has been transformed into the cells of Escherichia coli. pAV1 is stably maintained in the new host and retains its narrow host range in the course of conjugation. Different restriction fragments of R702, R751, R906 and R906-derived deletion mutants hybridize with the nick-translated probe of RP4 DNA. It is suggested that the wide host range plasmids have a similarity in structural and functional organization.

[Distribution, homology and cloning of cryptic Bacillus thuringiensis plasmids]. / Rasprostranennost', gomologiia i klonirovanie kripticheskikh plazmid Bacillus thuringiensis.

The 69-6 strain of Bacillus thuringiensis subsp. galleriae harbours at least 7 cryptic plasmids (pBTG1 - pBTG7) with molecular lengths 8,4 to 15,7 kb. According to hybridization analysis, the plasmid pBTG2 (8,7 kb) and other plasmids of the same host strain as well as cryptic plasmids of the strains belonging to 10 other serotypes of Bac. thuringiensis share detectable homology. As shown by the data of heteroduplex analysis, about 60% of pBTG1 and pBTG2 genomes have homologous DNA sequences. These data point out tht some plasmid genes are conserved in Bac. thuringiensis. BasmHI-, EcoRI- and HindIII-generated fragments of Bac. thuringiensis subsp. galleriae strain 69-6 are cloned on the pBR325 vehicle in the cells of Escherichia coli.

Transformation of Bacillus thuringiensis subsp. galleria protoplasts by plasmid pBC16.

Protoplasts of the entomopathogenic bacterium Bacillus thuringiensis subsp. galleria were transformed by plasmid pBC16. The frequency of transformation was much lower than that of Bacillus subtilis. All isolated B. thuringiensis transformants were characterized by increased sensitivity to lysozyme as compared with the original strain.

[Cloning and the expression of the DNA promotor fragments of Bacillus thuringiensis in Escherichia coli cells]. / Klonirovanie i ékspressiia promotornykh uchastkov DNK Bacillus thuringiensis v kletkakh Escherichia coli.

The promoter-containing fragments of Bacillus thuringiensis subsp. galleria 69-6 DNA have been cloned on the pGA24 vector in Escherichia coli cells. The recombinant plasmids make cells resistant to tetracycline in a wide range. The level of tetracycline-resistance does not depend on the length of a foreign insertion. New polypeptides are synthesized on the template of the recombinant plasmids in vitro. The data point out the presence in Bac. thuringiensis DNA of many genes which are able to express in E. coli cells.

Expression of E. coli genes carried by hybrids of plasmid RP4.

Hybrids of the RP4 plasmid, containing bacteriophage Mu and chromosomal genes of Escherichia coli, were transferred into Salmonella typhimurium, Pseudomonas putida, Pseudomonas aeruginosa and Proteus mirabilis. The individual genes of the arginine, histidine, leucine and threonine operons were expressed in these microorganisms.

[Phage pf16 interrelationships with Pseudomonas putida bacteria. I. Unstable transductants and mutants of Pseudomonas putida PfG1 resistant to phage pf16]. / Izuchenie vzaimootnoshenii faga pf16 s bakteriiami Pseudomonas putida. Soobshchenie I. Nestabil'nye transduktanty i mutanty Pseudomonas putida PpG1, ustoichivye k fagu pf16.

The frequencies of transduction of the chromosomal genes by pf16 in Pseudomonas putida PgG1 are dependent on the marker transduced and unpredictable. Histidine and isoleucine-valine positive transductants, which are resistant to pf16, have been selected in the crosses with low (about 10(-8) for phage units) frequencies of transduction. Some of these transductants carry new mutations. The transductants are unstable for phage resistance and histidine or isoleucine-valine positive characters. There is no correlation between segregations of the auxotrophic and phage sensitive clones. Among 200 tested mutants of P. putida line PpG1, which were obtained by the direct selection for the resistance of pf16, 3 mutants are found to be auxotrophic for histidine and one mutant unstable for the phage resistance character. The culture medium of such histidine negative and phage resistant mutants as much as phage resistant transductants lyse the lawn of the sensitive to pf16 strains, but viable phages are produced only by those unstable for the phage resistance character. These phages are very similar to pf16 for such characters as host range, size and morphology of plaques, latent period, burst size, transducing ability and the degree of inactivation by pf16 antiserum. The pf16 resistance of mutants and transductants is caused by the disturbing of phage adsorption. Possible mechanisms for the lisogenization of P. putida by pf16 are discussed.

[Control of plasmid incompatibility: characteristics of the bireplicon hybrid pAS8 and its deletion mutants]. / Kontrol' plazmidnoi nesovmestimosti: kharakteristika dvureplikonnogo gibrida pAS8 i ego deletsionnykh mutantov.

The phenomenon of incompatibility has been investigated using deletion mutants of hybrid bireplicon plasmid pAS8. The hybrid pAS8 displays incompatibility specific for both components of its structure. In contrast to P-specificity of pAS8, functions of ColE1-specificity are not effectively expressed. Expression of ColE1-specificity in pAS8 plasmid and its derivatives is characterized by different directions and this is due to the presence or absence of genes of RP4 replication machinery in the plasmid DNA. Mutant plasmids show different efficiency of P-specificity depending on the extension of deletion in the region of essential genes of the RP4 component. Some of the mutants, in spite of the loss of replication genes, including origin of vegetative replication, are incompatible with the representatives of the Inc P group in both directions of testing. Different character and the level of expression of ColE1- and P-specificity in the pAS8 hybrid and its deletion derivatives are not associated with change in the number of plasmid DNA copies, for all of them are subjects to stringent control of replication. The data suggest the existence of incompatibility functions control mechanism which does not seem to include replication genes. Possible ways of realization of the inc genes functions are discussed.

Mapping of RP4 plasmid using deletion mutants of pAS8 hybrid (RP4--ColE1).

We used the hybrid plasmid pAS8 in order to conduct the genetic analysis of RP4 plasmid. The presence of two replicons in the hybrid plasmid permitted to expand the spectrum of deletion mutants of RP4 isolated, which are capable to autonomous replication. The shortening of the hybrid plasmid was achieved by P22 transduction, by induction of deletion mutants using mitomycin C, as well as by seletion of Tra- mutants on the basis of resistance of cells to P-specific phages. These techniques have lead to isolation of clones possessing different combinations of plasmid resistance determinants. Comparison of phenotypic characteristics of deletion plasmids pAS9, pAS10, pAS11, pAS12 and pAS10-2 permitted to propose the map for pAS8 plasmid with the following sequence of markers: tra--kam--ColE1--amp--tet... Heteroduplex analysis of deletion mutants of pAS8 permitted to construct a physical map and to elaborate in greater detail the functional map of RP4 plasmid. The correlation between the ability of mutants to replicate in polA(TS) strain at nonpermissive conditions and the length of the deleted segment permitted to map rep genes of RP4 on a region with coordinates 9.8--17.3 kb. A relationship between the manifestation of incompatibility of mutants with Inc P-1 plasmids and the length of deletions points out that inc genes are located on DNA region with coordinates 2.1--9.8 kb. The analysis of replication of deletion mutants and the manifestation of incompatibility just as of the data about the size of appropriate deletions permitted to make the conclusion about the functional and genetic independence of the replication control and incompatibility control in RP4 plasmid.