Structure and function of AvtR, a novel transcriptional regulator from a hyperthermophilic archaeal lipothrixvirus.

The structural and functional analysis of the protein AvtR encoded by Acidianus filamentous virus 6 (AFV6), which infects the archaeal genus Acidianus, revealed its unusual structure and involvement in transcriptional regulation of several viral genes. The crystal structure of AvtR (100 amino acids) at 2.6-Å resolution shows that it is constituted of a repeated ribbon-helix-helix (RHH) motif, which is found in a large family of bacterial transcriptional regulators. The known RHH proteins form dimers that interact with DNA using their ribbon to create a central ß-sheet. The repeated RHH motifs of AvtR superpose well on such dimers, but its central sheet contains an extra strand, suggesting either conformational changes or a different mode of DNA binding. Systematic evolution of ligands by exponential enrichment (SELEX) experiments combined with systematic mutational and computational analysis of the predicted site revealed 8 potential AvtR targets in the AFV6 genome. Two of these targets were studied in detail, and the complex role of AvtR in the transcriptional regulation of viral genes was established. Repressing transcription from its own gene, gp29, AvtR can also act as an activator of another gene, gp30. Its binding sites are distant from both genes' TATA boxes, and the mechanism of AvtR-dependent regulation appears to include protein oligomerization starting from the protein's initial binding sites. Many RHH transcriptional regulators of archaeal viruses could share this regulatory mechanism.

Precise excision of bacteriophage Mu DNA.

The temperate bacteriophage Mu is a transposable element that can integrate randomly into bacterial DNA, thereby creating mutations. Mutants due to an integrated Mu prophage do not give rise to revertants, as if Mu, unlike other transposable elements, were unable to excise precisely. In the present work, starting with a lacZ::Muc62(Ts) strain unable to form Lac+ colonies, we cloned a lacZ+ gene in vivo on a mini-Mu plasmid, under conditions of prophage induction. In all lac+ plasmids recovered, the wild-type sequence was restored in the region where the Mu prophage had been integrated. The recovery of lacZ+ genes shows that precise excision of Mu does indeed take place; the absence of Lac+ colonies suggests that precise excision events are systematically associated with loss of colony-forming ability.

The GemA protein of phage Mu and the GyrB gyrase subunit of Escherichia coli: the search for targets and interactions leading to the reversion of Mu-induced mutations.

The mutant bacteriophage Mugem2(Ts), known to synchronize the division of infected cells, to relax DNA supercoiling and, as prophage, to give rise to precisely excised revertants, has been thought to overexpress the gemA-mor operon, and genetic evidence suggests that the B subunit of DNA gyrase (GyrB) is the target of action of GemA. In two different double hybrid tests presented here, we find no evidence of GemA-GyrB protein-protein interaction. We do observe a GemA-GemA interaction, however, indicating that GemA can dimerize. In lacZ::Mu lysogens, overexpression of the gemA-mor operon from a plasmid, under control of the L-arabinose inducible p(araBAD) promoter, does not permit the recovery of Lac(+) revertants. These observations suggest that GyrB is not the direct target of GemA action and that the various phenotypes of Mugem2(Ts) are not caused by overexpression of the gemA-mor operon.

KorSA from the Streptomyces integrative element pSAM2 is a central transcriptional repressor: target genes and binding sites.

pSAM2, a 10.9-kb mobile integrative genetic element from Streptomyces ambofaciens, possesses, as do a majority of Streptomyces conjugative plasmids, a kil-kor system associated with its transfer. The kor function of pSAM2 was attributed to the korSA gene, but its direct role remained unclear. The present study was focused on the determination of the KorSA targets. It was shown that KorSA acts as a transcriptional repressor by binding to a conserved 17-nucleotide sequence found upstream of only two genes: its own gene, korSA, and pra, a gene positively controlling pSAM2 replication, integration, and excision. A unique feature of KorSA, compared to Kor proteins from other Streptomyces conjugative plasmids, is that it does not directly regulate pSAM2 transfer. KorSA does not bind to the pSAM2 genes coding for transfer and intramycelial spreading. Through the repression of pra, KorSA is able to negatively regulate pSAM2 functions activated by Pra and, consequently, to maintain pSAM2 integrated in the chromosome.

Replicase, excisionase, and integrase genes of the Streptomyces element pSAM2 constitute an operon positively regulated by the pra gene.

pSAM2 is a site-specific integrative element from Streptomyces ambofaciens. The pra gene described earlier as an activator of pSAM2 replication is shown here to be also involved in the activation of its integration and excision. This was evidenced with derivatives of pSAM2 mutant B3 in which the pra gene was placed under the control of the inducible tipAp promoter. Transformation of Streptomyces lividans by these derivatives was efficient only when pra expression was induced, indicating its involvement in pSAM2 integration activation. Once established, these constructions remained integrated in the chromosome under noninduced conditions. Activation of the pra expression provoked strong activation of their excision, leading to the appearance of free forms. The results of functional, transcriptional, and sequence analyses allowed to conclude that the three genes repSA, xis, and int coding for the pSAM2 replicase, excisionase, and integrase, respectively, constitute an operon directly or indirectly activated by pra.

Complete conversion of antibiotic precursor to pristinamycin IIA by overexpression of Streptomyces pristinaespiralis biosynthetic genes.

A Streptomyces pristinaespiralis strain, which produces a streptogramin antibiotic pristinamycin II (PII) as a mixture of two biologically active molecules PIIB and PIIA, was genetically engineered to produce exclusively PIIA. The snaA,B genes, which encode a PIIA synthase that performs oxidation of the precursor (PIIB) to the final product (PIIA), were integrated in the chromosome of S. pristinaespiralis using an integrative derivative of the pSAM2 genetic element from Streptomyces ambofaciens. Integration was due to site-specific recombination at the attB site of S. pristinaespiralis, and no homologous recombination at the snaA,B locus was observed. The attB site of S. pristinaespiralis was sequenced and found to overlap the 3' end of a pro-tRNA gene. The integrants were stable in industrial conditions of pristinamycin production and showed no decrease in PII biosynthesis. Western blot analysis showed a constant production of the PIIA synthase in the overall fermentation process due to expression of the cloned snaA,B genes from the constitutive ermE promoter. This allows the complete conversion of the PIIB form into PIIA.

Characterization of pra, a gene for replication control in pSAM2, the integrating element of Streptomyces ambofaciens.

pSAM2 is a genetic element found integrated in Streptomyces ambofaciens (B2) and additionally in a replicating form in two mutants B3 and B4. The presence of the pSAM2 replicating form in these mutants was the result of mutations located on pSAM2 in the pra locus, named pra3 and pra4, respectively. The pra gene is not directly involved in replication, but its inactivation led to the disappearance of the pSAM2 free form; therefore, it was considered as a replication regulator. The pra3 and pra4 mutations were located in the pra promoter and were shown to be point substitutions that increase the promoter strength. The replication regulator role of pra was demonstrated by the fact that its constitutive expression in cells harbouring pSAM2B2, which is normally only integrated, led to the appearance of the pSAM2 replicating form. Northern analysis showed that the pra gene transcript can be detected only for the replicating mutants B3 and B4 and that the three adjacent genes korSA, pra and traSA were transcribed separately. As replication of pSAM2 is not needed for its maintenance but is an indispensable stage of its transfer, the pra gene, described formally as an activator of pSAM2 replication, is patently involved in pSAM2 transfer.

Transfer functions of the conjugative integrating element pSAM2 from Streptomyces ambofaciens: characterization of a kil-kor system associated with transfer.

pSAM2 is an 11-kb integrating element from Streptomyces ambofaciens. During matings, pSAM2 can be transferred at high frequency, forming pocks, which are zones of growth inhibition of the recipient strain. The nucleotide sequences of the regions involved in pSAM2 transfer, pock formation, and maintenance have been determined. Seven putative open reading frames with the codon usage typical of Streptomyces genes have been identified: traSA (306 amino acids [aa]), orf84 (84 aa), spdA (224 aa), spdB (58 aa), spdC (51 aa), spdD (104 aa), and korSA (259 aa). traSA is essential for pSAM2 intermycelial transfer and pock formation. It could encode a protein with similarities to the major transfer protein, Tra, of pIJ101. TraSA protein contains a possible nucleotide-binding sequence and a transmembrane segment. spdA, spdB, spdC, and spdD influence pock size and transfer efficiency and may be required for intramycelial transfer. A kil-kor system similar to that of pIJ101 is associated with pSAM2 transfer: the korSA (kil-override) gene product could control the expression of the traSA gene, which has lethal effects when unregulated (Kil phenotype). The KorSA protein resembles KorA of pIJ101 and repressor proteins belonging to the GntR family. Thus, the integrating element pSAM2 possesses for transfer general features of nonintegrating Streptomyces plasmids: different genes are involved in the different steps of the intermycelial and intramycelial transfer, and a kil-kor system is associated with transfer. However, some differences in the functional properties, organization, and sizes of the transfer genes compared with those of other Streptomyces plasmids have been found.

[Chromosomal determinant of Streptomyces hygroscopicus with positive influence on resistance to bialaphos controlled by the bar gene]. / Khromosomnyi determinant Streptomyces hygroscopicus, pozitivno vliiaiushchii na uroven' ustoichivosti k bialafosu, kontroliruemyi genom bar.

Streptomyces hygroscopicus ATCC21705 is an organism producing bialaphos, a promising ecologically safe antibiotic of peptide nature. An unknown determinant was cloned within the 1.4-kb SsH fragment of chromosomal DNA of S. hygroscopicus. Its presence along with the bar gene in the hybrid plasmid PVGB21 increased expression of the phenotype resistance to bialaphos in the recipient strain of S. lividans TK 64. Plasmid PVG B22 carrying the isolated SsH fragment did not provide resistance to the antibiotic. Transcription from the cloned fragment was revealed by RNA-DNA hybridization. It was shown that the cloned determinant had a positive action on bar gene transcription correlating with a significant increase in the activity of N-acetyltransferase in the mycelium of the transformant pVG B21.

[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.

[Expression of cloned bialaphos resistance gene (bar) in Streptomyces strains]. / Ekspressiia klonirovannogo gena rezistentnosti k bialafosu (bar) v shtammakh Streptomyces.

The object of the study was a strain of Streptomyces hygroscopicus producing bialaphos, an antibiotic used as a herbicide, which is promising and ecologically safe. Molecular cloning of a bialaphos resistance gene (bar) was performed in the recipient strain, S. lividans TK64, within the 2.0-kb DNA fragment with the plasmid pIJ699. Introduction of a bar gene into another strain producing bialaphos, i.e. S. viridochromogenus Tu494, led to its higher constitutive resistance to bialaphos. The results confirmed the data on different regulation of bar (S. hygroscopicus) and pat (S. virido-chromogenus) resistance genes.

[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.

[Molecular genetic studies of the DNA promotor regions in Bacillus thuringiensis subsp. galleriae 69-6. I. Structural and functional analysis]. / Molekuliarno-geneticheskie issledovaniia promotornykh raionov DNK Bacillus thuringiensis subsp. galleriae 69-6. Soobshchenie I. Strukturno-funktsional'nyi analiz.

Three recombinant plasmids pPBT9, pPBT10 and pPBT74 carrying promoter-containing regions of DNA of Bacillus thuringiensis which are responsible for the expression of the promoterless tet gene, were studied. In the in vitro experiments, it had been shown that these promoter-active HindIII fragments of bacillar DNA contained RNA polymerase binding sites. The AluI subfragments that specifically bind to Escherichia coli RNA polymerase promote the tet gene expression, similar to the whole HindIII fragments. Sequence analysis revealed that the approximately 220 base pair AluI subfragment of the bacillar insertion of the pPBT10 plasmid contained sites typical for "-10" and "-35" homology regions of promoters specific for sigma 55-RNA polymerase from Bac. subtilis. The 1.45 kb HindII bacillar fragment of the plasmid pPBT9 had three AluI subfragments that bind to E. coli RNA polymerase. Only approximately 400 base pair AluI subfragment among these restored the tet gene expression in vivo. Bireplicon pBP plasmids were constructed that promoted the expression of the enterobacterial antibiotic resistance gene under the control of Bac. thuringiensis promoters in Bac. subtilis cells.

[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.