Characterization of a linear extrachromosomal DNA element (pBL1) isolated after interspecific mating between Streptomyces bambergiensis and S. lividans.

Streptomyces bambergiensis S712 harbours a giant linear plasmid PSB1 of 640 kb. After mating with the plasmidless S. lividans strain TK64, conjugants carrying a smaller extrachromosomal DNA element, pBL1, were identified. pBL1 is a 43-kb linear DNA molecule bound to a protein which protects it from attack by both 3'- and 5'-exonucleases. The absence of this protein drastically reduces the transforming efficiency of pBL1. pBL1 shares homology with linear plasmids and chromosomal DNA from S. bambergiensis strains.

[Restriction mapping of plasmid pSa1, isolated from Streptomyces albus G strain]. / Restriktsionnoe kartirovanie plazmidy pSa1, vydelennoi iz shtamma Streptomyces albus G.

A novel plasmid designated pSa1 has been isolated from Streptomyces albus G strain producing SalGI restriction endonuclease. Molecular weight of the plasmid is 3.4 +/- 0.2 mD. The action of 12 restriction endonucleases on the plasmid DNA was studied. Restriction map of pSa1 DNA was established for SmaI, HindII, XbaI and KpnI endonucleases.

[Molecular cloning and study of the expression of a region of a diphtheria toxin gene encoding fragment A of the Streptomyces lividans 66 strain]. / Molekuliarnoe klonirovanie i izuchenie ékspressii chasti gena difteriinogo toksina, kodiruiushchego fragment A, v shtamme Streptomyces lividans 66.

The shuttle plasmid pVG202 containing a part of diphtheria toxin gene coding for fragment A has been constructed. S. lividans strain 66 has been transformed by the plasmid pVG202 DNA. The presence of the hybrid plasmid in S. lividans 66 cells determines the production of catalytically active toxoid secreted into the cultural liquid medium. The deleted plasmid pVG205 which determines for the increased catalytic activity has been selected and shown to be stably inherited by the bacterial cells.

[Actinomycetes--the test-organisms of genetic engineering]. / Aktinomitsety--ob''ekty genno-inzhenernykh issledovanii.

The paper contains a short review of the data on using the methods of genetic engineering in studies of genetics and molecular biology in Streptomyces. The techniques of DNA introduction into actinomycetes and wide-spread vectors are briefly described. The origin of the actinomycete plasmids as chromosomal segments capable of autonomous replication is discussed. In this view, it is suggested that genetic instability in actinomycetes is connected with excision of specific DNA sequences from the chromosome at frequencies characteristic of recombination events. Also, amplification of short DNA segments within the chromosome resulting in tandem repeats is a consequence of unequal crossing over between direct repeats flanking the amplifying DNA and, possibly, of induction of replication of this DNA. The data on molecular cloning of actinomycete genes for primary metabolism and those for resistance to and biosynthesis of antibiotics, on using actinomycetes as the hosts for foreign genes to be expressed, as well as on analysis of nucleotide sequences of actinomycete DNA, are presented.

[Restriction of two-replicon shuttle Escherichia coli-Streptomyces plasmids in Streptomyces lividans strain 66]. / Restriktsiia dvureplikonnykh chelnochnykh plasmid Escherichia coli-Streptomyces v shtamme Streptomyces lividans 66.

The shuttle Escherichia coli - Streptomyces plasmids were used to transform S. lividans 66. Plasmid DNAs isolated from this strain transform it 10-1000-fold more efficiently than DNAs from E. coli. Rare transformant cured from most restricted plasmid is more efficient recipient of plasmid DNA from E. coli and has the property of R +/- M+ mutant. Restriction in S. lividans 66 correlates with the appearance in DNA from E. coli of the sites susceptible to Scg2I restriction endonuclease. The latter was isolated earlier from recombinant strain Rcg2, a hybrid between S. griseus Kr. 15 and S. coelicolor A3(2). Scg2I possesses the recognition sequence CCTAGG, like EcoRII, MvaI and Eco dcm methylase. The DNA resistant to Scg2I cleavage retained this ability after in vitro modification by EcoRII methylase. So, the resistance of DNA to Scg2I cleavage is not connected with methylation at 4th and 5th position of second cytosine in the recognition sequence. Neither restriction of plasmid DNA in S. lividans 66 is dependent on dcm modification in E. coli, though its dependence on dam modification is not excluded. It is assumed that the restriction in S. lividans 66 is specified by endonuclease analogous to Scg2I.

[Identification of the restriction and modification systems in Streptomyces strains]. / Identifikatsiia sistem restriktsii i modifikatsii u shtammov Streptomyces.

Host range of actinophage phi C31, VP5 and Pg81 in respect to 109 strains of Streptomyces genus and hybrid strain Rcg2 from the cross S. coelicolor A3(2)XS. griseus Kr was studied. The existence of RM-systems in strains S. griseus Kr15, S. griseus Kr20, Rcg2, S. griseofovillus 43 was shown using phage Pg81. Mutants of Pg81 were observed which to some extent lost snesitivity to RM-system in the strain Rcg2. The presence of RM-system in S. lividans 67 was demonstrated by the phage VP5.

[Actinomycete plasmid and integrative vectors based on DNA of the temperate Phi C31 actinophage, determining limitation of lytic development of phage Phi C31, not dependent on repressor]. / Aktinomitsetnye plazmidnye i integrativnye vektory na osnove DNK umerennogo aktinofaga Phi C31, opredeliaiushchie organichenie liticheskogo razvitiia faga Phi C 31, ne zavisimoe ot repressora.

Bireplicon plasmids were constructed. The plasmids consist of DNAs of the streptomycete plasmid pIJ702, the Escherichia coli plasmid pUC19 and the phi C31 actinophage genome fragment encoding the function of the site-specific integration into the chromosome. Part of the plasmids transformed Streptomyces lividans TK64 to thiostreptone resistance. The DNA transforming activity depended on the mutual orientations of the blocks used for the construction and probably depended on the structural stability of the plasmids in S. lividans. The integrative vectors consisting of the pUC19 plasmid DNA and the phage genome fragment with the integrative function efficiently transformed S. lividans. No phage plagues were detected with the standard procedure of integrants' infection by phi C31 phage, despite the absence of the phi C31 phage repressor gene in the integrated DNA. The phi C31 phage mutants including clear and virulent ones were not capable of lytic growth on the integrants. The region determining the limitation of the phi C31 phage lytic development was localized by the deletion analysis of the bireplicon plasmids. As a result actinomycete monoreplicon plasmids were formed. The region is the 1.3 kb phage fragment whose right end maps at 0.2 kb preceding the right end of the phi C31 phage genome linear map.

[The methods of protoplast formation and transformation of Streptomyces strains]. / Metody protoplastirovaniia i transformatsii shtammov Streptomyces.

Factors influencing formation, regeneration and transformation of protoplasts in streptomyces are described. Conditions for formation and regeneration of protoplasts in 4 industrial strains producing the macrolide antibiotic tylosin were studied. It was demonstrated possible to apply the method for transformation of the S. lividans type culture to 3 industrial strains of S. griseus producing grisin, an antibiotic used as a feed additive. Potential increasing of the efficiency of protoplast transformation and transfection in various actinomycetous strains including industrial ones is discussed. The stimulating effect of lyposomes on transformation of protoplasts in S. lividans 66 with DNA of plasmids pVG101 and pIJ350 as well as transfection with DNA of phages SH10 and KS404 was shown. The tylosin resistance genes in S. fradiae strain B45 were cloned which enabled isolating the cluster of the genes participating in tylosin biosynthesis.

[Molecular cloning of tylosin-producing Streptomyces fradiae B-45 genes determining increased inducible resistance to tylosin in Streptomyces lividans TK64]. / Molekuliarnoe klonirovanie iz shtamma-produtsenta tilozona Streptomyces fradiae B-45 genov, opredeliaiushchikh v shtamme Streptomyces lividans TK64 povyshennuiu indutsibel'nuiu ustoichivost' k tilozinu.

DNA of S. fradiae B-45 partially cleaved by Sau3A restrictase was cloned in S. lividans TK64 in the plasmid vector pIJ702. Three recombinant plasmids pVG251, pVG262, and pVG253 with tlr1, tlr2 and tlr3 genes were isolated from the transformed clones of S. lividans TK64 with higher inducible resistance to tylosin as compared to the plasmid-free strain. DNA-DNA blot hybridization was performed between the total DNA cleaved by several restrictases from S. fradiae B-45 and some other strains and the DNA probes containing the tlr genes. It was shown that tlr1 and tlr3 genes were unique in S. fradiae B-45. Sequences homologous to tlr2 gene were present both in DNA of S. fradiae B-45 in 7 copies and in strains of S. antibiotics and S. hygroscopicus producing respectively oleandomycin and turimycin.

[Functioning of integrative vectors, based on phage phi C 31, in the strain Streptomyces bambergiensis 712]. / Funktsionirovanie integrativnykh vektorov, sozdannykh na osnove faga phiC31, v shtamme Streptomyces bambergiensis 712.

Actinomycete integrative vectors were constructed. The vectors contain the Escherichia coli plasmid ColE1 replicon, the thiostrepton resistance gene used for selection in Streptomyces and a fragment of the phi C31 actinophage genome with integrative functions. The pS133 and PS135 vector DNAs transformed Streptomyces bambergiensis 712, a strain producing the phosphoglycolipid antibiotic moenomycin. Two types of the transformants were detected. The first type was not affected in the ability to produce moenomycin and the vector pS135 DNA was shown to integrate into the S. bambergiensis 712 genome by the site-specific pattern with the psi C31 phage DNA fragment. The second type of the transformants lost the ability to produce moenomycin. The Southern analysis and cloning of the inserted DNA indicated that in this case the vector pS135 and pS133 DNAs also integrated specifically into the genome but the integration took place not within the phage DNA fragment. Its realization was suggested to proceed via homologous recombination.

[Comparative study of Streptomyces--producer of aminoglycoside antibiotics, monomycin and kanamycin, and the strain 344 obtained by fusion of their protoplasts by the method of restrictive total DNA fingerprinting]. / Sravnitel'noe izuchenie streptomitsetov--produtsentov monomitsina a kanamitsina i shtamma 344, poluchennogo pri zliianii ikh proto- plastov metodom restriktsionnykh "fingerprintov" total'noi DNK.

The method of total DNA restriction finger prints was applied to the study of Streptomyces monomycini INA 1465 producing monomycin, Streptomyces kanamyceticus INA K-13 producing kanamycin and strain 344 isolated after fusion of the protoplasts of strain 1465 and K-13, which produced albofungin and chloralbofungin, aminoglycoside antibiotics. For preparing the finger prints of the strains splitting by endonucleases BamHI, PstI, PvuII, and BgIII was used. The finger prints showed that strain 344 was related to the strain of S. monomycini and markedly differed from the strain of S. kanamyceticus. Strain 344 was likely to result from reconstruction (probably 20-kb deletion) of the genome of S. monomycini INA 1465 induced by the preparation and regeneration of its protoplasts. The reconstruction could affect the genome area with localization of the genes involved in monomycin biosynthesis and monomycin resistance genes.

[Effect of plasmid pIJ350 on genetic stability of antibiotic production by Streptomyces bambergiensis]. / Vliianie plazmidy pIJ350 na geneticheskuiu stabil'nost' priznaka antibiotikoobrazovaniia u Streptomyces bambergiensis.

It was shown that S. bambergiensis S800 was genetically instable with respect to the property of the antibiotic production (Ant) while in strain S712 of S. bambergiensis this property was stable. Transformation of S. bambergiensis protoplasts with pIJ350 plasmid DNA and analysis of the transformants screening revealed induction of the Ant instability in both the strains. In case of plasmids pVG101 and pIJ943 this effect was not shown. Analysis of the S800 (pIJ350) transformant screening revealed five groups of mutants differing in the antibiotic production level and the presence of pIJ350 plasmid. Restriction analysis of the total DNA of the mutants showed that there were large deletions in the genome of two of them. Retransformation of the mutants with pIJ350 plasmid DNA showed that in all the cases induction of the instability was lacking. The behaviour of the spontaneous mutants Ant- of strain S800 with respect to pIJ350 plasmid was analogous to that of the mutants Ant- from the transformant S800 (pIJ350) screening. A hypothetic model for the determinant incompatibility with pIJ350 plasmid genetically linked to the Ant property in the genome of S. bambergiensis and unstable in strain S800 was proposed.