RESUMEN
Mutations in the rpoH gene, encoding sigma 32, an alternative factor required for transcription of the heat shock genes, result in the extensive aggregation of virtually all cellular proteins and formation of inclusion bodies both under stress and non-stress conditions. Inhibitors of protein synthesis suppress this aggregation, suggesting that newly synthesized proteins preferentially aggregate in rpoH mutants. These data suggest that the heat shock proteins are involved in acquisition of the soluble state (i.e. correct conformation) of the bulk of intracellular proteins after their translation.
Asunto(s)
Escherichia coli/genética , Proteínas de Choque Térmico/genética , Mutación , Escherichia coli/fisiología , Escherichia coli/ultraestructura , Genes Bacterianos , Proteínas de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/química , Conformación ProteicaRESUMEN
The plasmid composition of S. sonnei standard strains has been studied by the method of electron microscopy of the preparations of plasmid DNA. In S. sonnei cells I-941-HP, phase I, plasmids of 2,500; 5,000; 5,600; 6,100 and 6,800 base pairs, as well as plasmids of 85,000-117,000 and 170,000-235,000 base pairs have been detected. In S. sonnei cells, phase II, plasmids of 2,500; 4,900 and 6,100 base pairs, as well as plasmids of 85,000-109,000 base pairs, have been found. Thus, virulent S. sonnei in phase I contain additional plasmids of 5,600; 6,800; 110,000-117,000 and 170,000-237,000 base pairs. The range of plasmid lengths between 85,000-117,000 and 170,000-237,000 base pairs exceeds the usual background of electron-microscopic studies, which makes it possible to come to the conclusion on the intrastrain heterogeneity of these classes of plasmids. The suggestion has been made that the transition of S. sonnei from phase I to phase II is linked with the loss of fragments of the genetic material, limited by inverted DNA repetitions.
Asunto(s)
Plásmidos/genética , Shigella sonnei/genética , Composición de Base/genética , ADN Bacteriano/genética , ADN Bacteriano/ultraestructura , Electroforesis , Microscopía Electrónica , Peso Molecular , Replicón/genética , Shigella sonnei/ultraestructura , Virulencia/genéticaRESUMEN
Genetic and restriction (for enzymes EcoRI, BamHI and HindIII) maps of the relatively broad host range plasmid R906 are constructed. There are two non-essential regions on the R906 DNA which can be deleted and cloned. Non-essential regions confer a resistance to different agents and restriction sites are clustered in these regions. Essential and conjugativity genes are located in two other DNA regions approximately at 0-23 and 29-44 kb of the R906 map. These large regions share a high level of homology with Inc-1 group plasmids R751 and RP4 according to Southern-blot hybridization and heteroduplex analyses. A transposon-like structure is found on the R751 DNA among R751/R906 heteroduplex molecules. This transposon of total length 5.1 kb has 1.4 kb inverted repeats at the ends. Bla genes of R906 and RP4 plasmids do not have homologous sequences. Data evidence that IncP-1 group plasmids irrespective to their original bacterial source and range of coded antibiotic resistance have very similar molecular organization. The role of possible factors which are responsible for the broad host range property of the IncP-1 group plasmids is discussed.
Asunto(s)
ADN Bacteriano/genética , Genes Bacterianos , Factores R , Bordetella/genética , Inversión Cromosómica , Mapeo Cromosómico , Clonación Molecular , Enzimas de Restricción del ADN , Elementos Transponibles de ADN , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
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.
Asunto(s)
Bacillus thuringiensis/genética , ADN Bacteriano/genética , Regiones Promotoras Genéticas , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Regulación de la Expresión Génica , Microscopía Electrónica , Plásmidos , Recombinación Genética , ReplicónRESUMEN
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.
Asunto(s)
Plásmidos , Pseudomonas aeruginosa/genética , Autorradiografía , Secuencia de Bases , Mapeo Cromosómico , ADN Bacteriano/genética , Electroforesis en Gel de Agar , Genes Bacterianos , Hibridación Genética , Ácidos Nucleicos Heterodúplex/genética , Hibridación de Ácido NucleicoRESUMEN
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.
Asunto(s)
Genética Microbiana , Plásmidos , Bacterias/efectos de los fármacos , Bacterias/genética , Bacterias/efectos de la radiación , Conjugación Genética , Cruzamientos Genéticos , Enzimas de Restricción del ADN/farmacología , ADN Bacteriano/genética , ADN Bacteriano/efectos de la radiación , Mutación , Hibridación de Ácido Nucleico/efectos de los fármacos , Plásmidos/efectos de los fármacos , Plásmidos/efectos de la radiación , Rayos UltravioletaRESUMEN
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.
Asunto(s)
Bacillus thuringiensis/genética , Clonación Molecular , Plásmidos , Toxinas Bacterianas/genética , ADN Bacteriano/genética , Escherichia coli/genética , Genes Bacterianos , Ácidos Nucleicos Heterodúplex/genética , Hibridación de Ácido NucleicoRESUMEN
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.
