[Use of a plasmid with integrative function of phage phiC31 for transfer of cloned genes into Streptomyces strains]. / Ispol'zovanie plazmid s integrativnoi funktsiei faga phiC31 dlia perenosa klonirovannykh genov v shtammy Streptomyces.

Opportunities for application of integrative vectors carrying the attP site and the int gene of the temperate actinophage phi C31 for cloning genes in Streptomyces strains were demonstrated. The behavior of the integrative vectors pZAT22 and pTO1 in the model strain S. lividans TK64 and in the bialaphos-producing strain S. hygroscopicus, respectively, was characterized. Restriction maps of the S. lividans and S. hygroscopicus chromosomal regions containing attB sites were constructed. The bar gene of resistance to bialaphos was incorporated into the chromosome of the model strain S. lividans via the integrative pZAT22 vector, the level of expression of this gene within the chromosome of a heterologous host was determined. The possibility of amplification of the bar gene in the chromosome of the strain S. lividans was shown. The conjugative integrative vector pTO1, which carried the cloned bar gene, was introduced into the bialaphos-producing strain by intergeneric matings of Escherichia coli and S. hygroscopicus. The effect of an additional copy of the gene in the chromosome of S. hygroscopicus on strain resistance and productivity was studied.

[Intergeneric Escherichia coli-Streptomyces conjugation as a means for the transfer of conjugative plasmids into producers of antibiotics chlortetracycline and bialaphos]. / Mezhrodovaia kon"iugatsiia Escherichia coli-Streptomyces kak sposob peredachi kon"iugativnykh plazmid v produtsenty antibiotikov khlortetratsiklina i bialafosa.

A system for introduction of plasmids into industrial producers of antibiotics chlortetracycline and bialaphos using intergeneric conjugation of Escherichia coli and Streptomyces was developed. Low level stability of inheritance of autonomously replicating DNA in recipient strains was shown. Site-specific integration of the conjugative-integrative vector pTO1 provided stable plasmid maintenance within the chromosomes of Streptomyces aureofaciens and S. hygroscopicus. Phenomenon of disturbance in differentiation and antibiotic production, resulting from pTO1 integration into S. hygroscopicus chromosome, was discovered.

[Cloning of grisin resistance gene gsr and the study of its functioning in Streptomyces griseus Kr. strain]. / Klonirovanie gena ustoichivosti k grizinu gsr i izuchenie ego funktsionirovaniia v shtamme-produtsente Streptomyces Kr.

S. griseus Kr. is a commercial strain producing grisin, an antibiotic of the streptothricin group used as a feed additive. It was shown earlier that genetic instability of the strain was very high which was evident from a high frequency of nonreverting Grn- Grns mutants. With densitographic analysis of chromosomal DNA electrophoregrams and DNA-DNA hybridization it was revealed that the molecular basis of the genetic instability of the S. griseus strain was deletion of a DNA fragment about 20 kb in size containing a grisin resistance gene. The resistance gene designated as gsr was cloned to S. lividans TK 64 within the plasmid vector pIJ699. The restriction map of a cloned DNA fragment with a gsr gene was constructed and its similarity to that of a nat gene resistant to norseothricin, another streptothricin was observed. Introduction of a gsr gene within the multicopy plasmid pIJ699 into S. griseus 212, a highly productive strain synthesiing the antibiotic, led to an increase in its resistance and productivity. Proceeding from the preliminary data on possible linkage of a gsr gene and grisin biosynthesis genes, it appeared possible to use the cloned gene as a molecular probe in cloning the biosynthesis genes.

[Molecular cloning of chlortetracycline resistance gene from chlortetracycline producer Streptomyces aureofaciens]. / Molekuliarnoe klonirovanie gena rezistentnosti k khlortetratsiklinu iz shtamma-producenta Streptomyces aureofaciens.

The chlortetracycline (CT) resistance gene ctr was cloned from S. aureofaciens 633, a strain producing the antibiotic. The 6.6-kb DNA Bam HI fragment containing the resistance gene was cloned with the plasmid vector pIJ699. Comparison of the restriction maps of the cloned gene and the oxytetracycline (OT) resistance gene otrA from S. rimosus revealed their similarity which enabled identification of the cloned resistance gene as otrA. Investigation of the resistance determinants in S. aureofaciens 633 made it possible to identify a mtr gene(s). It was demonstrated that introduction of a ctrA gene into S. lividance provided a simultaneous increase in the resistance of the recipient strain to CT and a number of macrolide antibiotics. The CT resistance determinants in S. lividans TK64 showed properties of exogenous induction by CT and the macrolide antibiotics similar to the properties of the mtr gene(s) of S. aureofaciens. Possible adaptation properties of mtr genes are discussed.

[Determinants of resistance to chlortetracycline and other antibiotics in chlortetracycline-producing strain of Streptomyces aureofaciens]. / Determinanty ustoichivosti k khlortetratsiklinu i drugim antibiotikam u shtamma Streptomyces aureofaciens--produtsenta khlortetratsiklina.

Data are presented on resistance of Streptomyces aureofaciens strain TB-633 FU--the producer of chlortetracycline (CTC) to autogenous antibiotics and a number of other antibiotics. It is demonstrated that resistance to CTC is specified by ctr genes of constitutive expression as well as by inducible genes. CTC and ethidium bromide may serve as efficient inductors of inducible ctr genes. The induction process is accompanied by increase in antibiotic biosynthesis level. Genes responsible for strain resistance to a number of macrolide antibiotics and thiostrepton are inducible and only function in the presence of appropriate antibiotics in the medium. The action of inducible mtr gene(s) is described in detail. The gene(s) simultaneously ensure increase in resistance to CTC and a number of macrolide antibiotics in the presence of exogenous inductors in media, such as both CTC and macrolide antibiotics. Mutants have been isolated which provide constitutive level of resistance to these antibiotics. A series of ctr and mtr mutants have increased CTC biosynthesis as compared to the initial level. Data on comparative analysis of the results obtained from hybridization of fragments of S. aureofaciens and S. rimosus DNAs to actI and actIII genes, responsible for polyketide synthases' synthesis, demonstrate that genes for CTC and OTC biosynthesis are situated on DNA fragments of similar size. This determines the strategy for cloning ctr and mtr genes as well as genes for CTC biosynthesis from S. aureofaciens.

[Characterization of multiple changes of antibiotic resistance characters in Streptomyces coelicolor A3(2)]. / Kharakteristika mnozhestvennogo izmeneniia priznakov ustoichivnosti k antibiotikam u Streptomyces coelicolor A3(2).

Among mutants of Streptomyces coelicolor A3(2) studied which were sensitive to chloramphenicol (Cmls), strains sensitive to a number of antibiotics (ristomycin, tetracycline, polymyxin, lincomycin) amount of 46%. Antibiotic-sensitive mutants are capable to form different classes of resistant revertants with frequency varying from 10(-2) to 10(-6) in independent strains. Ristomycin-sensitive clones (Rims) have been found to occur with high frequency in Cmls strains and Cmlr revertants. Mutations mediating the Rims phenotype are mapped in a locus linked to the gene for resistance to chloramphenicol. The results obtained are discussed, in accordance with the notion about possible role of cml mutation in induction of secondary mutational changes in the genome of S. coelicolor A3(2).

[Genetic mapping of unstable chloramphenicol resistance determinant in Streptomyces coelicolor A3(2)]. / Geneticheskoe kartirovanie nestabil'nogo determinanta ustoichivosti k khloramfenikolu u Streptomyces coelicolor A3(2).

The results indicative of chromosomal localization of the unstable chloramphenicol resistance determinant in Streptomyces coelicolor A3(2) have been obtained. Independent mutations specifying chloramphenicol sensitivity in different strains of S. coelicolor A3(2), S18 and A617M are localized in the same region flanked by markers argA1 and cysD18 on the genetic map. Mutations restoring chloramphenicol resistance are also localized in this region. Different locations of the genetically unstable determinant of chloramphenicol resistance detected in various laboratories are discussed, in relation to the results showing that transfer to chloramphenicol sensitivity is due to a set of various rearrangements (deletions, amplifications, deamplifications, etc.), differing in separate variants.

[Selection of strains of Streptomyces griseus Kr., the producer of a streptothricin antibiotic grisin, using the method of protoplast fusion]. / Selektsiia shtammov Streptomyces griseus Kr., produtsenta streptotritsinovogo antibiotika grizina, s ispol'zovaniem metoda sliianiia protoplastov.

The effect of protoplasting on antibiotic activity of the grisin-producing organism was shown. High frequency of Grn- mutants after strain VG307f protoplasting and no capacity in these mutants for reversion to the initial Grn+ phenotype were shown. The reversion frequency was less than 10(-8). Moreover, it was shown that all the Grn- mutants lost their stability (GrnR) to the effect of their own antibiotic. With respect to strain VG212 there was noted a significant increase in the number of both the minus and the plus variants after the protoplast formation and regeneration. Fusing of protoplasts of strains VG307f and VG212 belonging to the divergent lines in selection of S. griseus Kr. yielded the phage stable strain VG7849 with high levels of the antibiotic production and improved technological properties.

[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.

[Genetic control of Actinomycetes resistance to antibiotics]. / Geneticheskii kontrol' ustoichivosti aktinomitsetov k antibiotikam.

The results of studies on genetic control of resistance to antibiotics in Streptomyces strains are discussed. Cloning and sequence analysis of resistance genes yield information concerning their expression in homo- and heterologous systems, allow analysis of signal sequences responsible for initiation of transcription and translation. Cloning of genes coding for resistance to neomycin,viomycin, thiostrepton in Streptomyces and Bac. licheniformis ermD gene made them convenient selective markers for constructing vector molecules, useful for identification of homology regions in S. fradiae aph gene and TnS of E. coli; the site homologous to ermD gene has been thus revealed in S. erythreus chromosome. Possibilities of the studies aimed at elucidation of instability of many actinomycete characters using determinants of natural multiple resistance to antibiotics as a model are demonstrated. It has been shown that genetic instability is not related to the loss of plasmids and is associated with genes having chromosomal location. Simultaneous high frequency loss of a number of resistance characters determined by non-linked genes suggests the participation in gene activity regulation of actinomycete genome rearrangements. This is confirmed by evidence for such rearrangements found in strains with mutant phenotypes, including deletions in tyrosinase and streptomycin phosphotransferase genes in Mel- and StrS strains of S. reticuli and S. glaucescens.

[Physical mapping of Streptomyces coelicolor A3(2) actinophages. VII. Formation of deletions in the region of phi C43 insertion sequence]. / Fizicheskoe kartirovanie akinofagov Streptomyces coelicolor A3(2). VII. Obrazovanie deletsii v oblasti vstavochnoi posledovatel'nosti faga phi C43.

The correlation between the plaque morphology and the presence of the insertion sequence at the phage genomes have been found for different variants of phi C43, obtained by spontaneous induction of strain S. lividans 803. Phages carrying complete insertion have Tum phenotype and they are represented in 4% of induced lysate. Phages carrying the part of insertion have Tu phenotype, their portion in induced lysate is about 26%. Phages without insertion have Stu phenotype and they are the majority of the lysate (70%). All variants phi C43, except wild-type phage, have lost the fragment of phage genome. Phage phi C43ins1, carrying the complete insertion give rise spontaneously at the frequency 10(-2) to deletion derivatives. Heteroduplex experiments show that in the deletion derivatives phi C43ins1 also in the Stu and Tu variants phi C43 one of the termini of the deletions is located within the insertion or at its end. Considerable portion of deletion variants in induced lysate phi C43 also the formation of deletion derivatives phi C43ins1 with high frequency strongly suggest that insertion sequence generates deletions similar to IS of Tn elements.

[Genetic characteristics of a new phage resistance trait in Streptomyces coelicolor A3(2)]. / Geneticheskaia kharakteristika novogo priznaka fagoustoichivosti u Streptomyces coelicolor A3(2).

Phage resistance was investigated in the system of Streptomyces coelicolor A3(2) and phi C31 actinophage. Resistance of A3(2) strain to phi C31 was shown to involve a novel mechanism responsible for the arrest of phage intracellular growth in the whole cell population. The phage resistance character designated Pgl+ (for "phage growth limiting") is determined by a gene located on the A3(2) chromosome. The gene (pgl) controls phage "modification" which results in an inability of phage to lyse lysogenize Pgl+ host. Instability of the Pgl+ character was revealed. Pgl+ strains segregate Pgl variants at a high frequency, the majority of Pgl strains reverting to the initial Pgl+ phenotype with the same high frequency. Reversible Pgl+ in equilibrium Pgl transitions are a common feature of A3(2) cell population.

[Physical mapping of actinophage Streptomyces coelicolor A3(2). VI. The use of deletion mutants of actinophage phi C31 for construction of phage vectors]. / Fizicheskoe kartirovanie aktinofagov Streptomyces coelicolor A3(2). VI. Ispol'zovanie deletsionnykh mutantov aktinofaga phi C31 dlia konstruirovaniia fagovogo vektora.

Nonessential region responsible for G function has been identified in theta C31 phage genome by means of deletion mutants. The mutant phenotype is expressed upon theta C31 phage propagation in Streptomyces albus G strains differing in functioning of restriction and modification systems. Based on their increased resistance to EDTA, deletions were located in theta C31 delta 10 and theta C31 delta 65 phage mutants. Data are presented on physical mapping of nonessention region of theta C31 phage. The total length of this region is 24.1% of the overall length of DNA molecules. The DNA segment of 19.1% of the whole genome contains overlapped deletions. Theta C31 actinophage is proposed to be used as a cloning vector for Streptomyces. Various deletion mutants obtained, with the capacity of about 3 thousands base pairs may serve as "insertion vectors". The presence of the stretched nonessential genome region allows to use theta C31 phage as a "replacement vector". Then, insertion of foreign DNA to replace the EcoRI--C fragment of theta C31 DNA of 6.4 x 10(3) base pairs is possible. The phages comprising hybrid molecules may be selected for G and Lyg phenotypes.

[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.

[Physical mapping of Streptomyces coelicolor A3(2). V. Structural modifications of actinophage phiC43 DNA molecules]. / Fizicheskoe kartirovanie aktinofagov Streptomyces coelicolor A3(2). V. Strukturnye modifikatsii molekul DNK aktinofaga phiC43.

As shown by genetical and physical methods, the total preparation of phiC43 phage obtained after spontaneous induction of the prophage from S. lividans 803 strain is a heterogenous population. The wild-type phage (phi C43 wt) is only represented in 5--10% of the population. The majority of phage variants are not able to establish the lysogenic state. The structure of DNA molecules of some phages from the total preparations was characterized by electron microscopy of DNA heteroduplexes. Molecules of phiC43 wt DNA appeared to be completely homologous to those of recently studied phiC62 phage, except for two small regions of approximately 0.3 kb in the central part. Phage variants defective in establishment of the lysogenic state were distributed to two groups. One of them consists of deletion variants, the other--deletion/insertion variants. Deletions in DNA molecule of all nonlysogenizing phage overlap. The region of overlapping seems to be responsible for establishment of the lysogenic state. In the same region, deletion of DNA molecules of mutant phiC311 yg2 has been located. Three deletion/insertion variants contain homologous foreign sequences of various length. It is likely that these insertions are fragments of the host chromosomal DNA.

[Plasmid SCP2 transfer by Streptomyces coelicolor A3(2) actinophages]. / Perenos plazmidy SCP2 aktinofagami Streptomyces coelicolor A3(2).

The ability of VP5 and phi c31 actinophages of Streptomyces coelicolor A3(2) to transfer plasmid SCP2 genes was tested. Infection with both phages led to formation of variants having the traits characteristics of plasmid bearing strains. The fertility properties of the variants suggested the presence of SCP2 in the autonomous state. Analysis of extrachromosomal DNA isolated from these variants demonstrated its identity with the plasmid DNA from the donor strain. Based on characteristics of the variants obtained, it was suggested that the whole SCP2 plasmid had been transferred by actinophages via general transduction.

[Actinophage phi C31 restriction and modification]. / Restriktsiia i modifikatsiia aktinofaga Fi C31.

Actinophage phi C31 was shown to be insensitive to a number of restriction and modification systems (RM). Phage sensitivity to RM systems of those strains to which phage cannot absorb, may be tested using protoplast transfection. For instance, the absence of phi C13 transfection in Streptomyces griseus Kr15 protoplasts, as compared to efficient transfection in protoplasts of R- M+ mutant of this strain seems to imply the sensitivity of phi C31 to the RM system of S. griseus KR15. Restriction of mutant phi C31 phage modified by S. albus G Rm system has been detected in S. coelicolor A3(2). This effect being dependent on a previous host may indicate that the mutant phage was rendered sensitive to an Rm system of S. coelicolor A3(2).