RESUMEN
Se describe un método simple y altamente eficiente para transformar directamente Agrobacterium tumefaciens con ADN plasmídico. El protocolo se basa en la electropermeabilización de la pared celular bajo la acción de un campo electrico de alto voltaje y se obtienen resultados reproducibles transformantes por microgramo de ADN plasmídico. Usualmente, la electroporación se realiza en una solución de polietilén-glicol al 15 % y una magnitud del campo eléctrico de 12.5 kV/cm. La utilidad de este método de transformación es demostrada mediante el establecimiento de una genoteca de Arabidopsis thaliana directamente en Agrobacterium tumefaciens. Nuestros resultados ofrecen interesantes perspectivas para la transferencia de bancos de genes y la complementación génica en plantas prescindiendo del uso de Escherichia coli como hospedero intermediario (AU)
Asunto(s)
Agrobacterium , ADN , Biblioteca de Genes , Pared CelularRESUMEN
Se describe un método simple y altamente eficiente para transformar directamente Agrobacterium tumefaciens con ADN plasmídico. El protocolo se basa en la electropermeabilización de la pared celular bajo la acción de un campo electrico de alto voltaje y se obtienen resultados reproducibles transformantes por microgramo de ADN plasmídico. Usualmente, la electroporación se realiza en una solución de polietilén-glicol al 15 % y una magnitud del campo eléctrico de 12.5 kV/cm. La utilidad de este método de transformación es demostrada mediante el establecimiento de una genoteca de Arabidopsis thaliana directamente en Agrobacterium tumefaciens. Nuestros resultados ofrecen interesantes perspectivas para la transferencia de bancos de genes y la complementación génica en plantas prescindiendo del uso de Escherichia coli como hospedero intermediario
Asunto(s)
Agrobacterium , Pared Celular , ADN , Biblioteca de GenesRESUMEN
The analysis of the virulence determinants of phytopathogenic Rhodococcus fascians has been hampered by the lack of a system for introducing exogenous DNA. We investigated the possibility of genetic transformation of R. fascians by high-voltage electroporation of intact bacterial cells in the presence of plasmid DNA. Electrotransformation in R. fascians D188 resulted in transformation frequencies ranging from 10/mug of DNA to 10/mug of DNA, depending on the DNA concentration. The effects of different electrical parameters and composition of electroporation medium on transformation efficiency are presented. By this transformation method, a cloning vector (pRF28) for R. fascians based on an indigenous 160-kilobase (chloramphenicol and cadmium resistance-encoding) plasmid pRF2 from strain NCPPB 1675 was developed. The origin of replication and the chloramphenicol resistance gene on pRF28 were used to construct cloning vectors that are capable of replication in R. fascians and Escherichia coli. The electroporation method presented was efficient enough to allow detection of the rare integration of replication-deficient pRF28 derivatives in the R. fascians D188 genome via either homologous or illegitimate recombination.
RESUMEN
Recombinant clones containing the manganese superoxide dismutase (MnSOD) gene of Bacillus stearothermophilus were isolated with an oligonucleotide probe designed to match a part of the previously determined amino acid sequence. Complementation analyses, performed by introducing each plasmid into a superoxide dismutase-deficient mutant of Escherichia coli, allowed us to define the region of DNA which encodes the MnSOD structural gene and to identify a promoter region immediately upstream from the gene. These data were subsequently confirmed by DNA sequencing. Since MnSOD is normally restricted to the mitochondria in eucaryotes, we were interested (i) in determining whether B. stearothermophilus MnSOD could function in eucaryotic cytosol and (ii) in determining whether MnSOD could replace the structurally unrelated copper/zinc superoxide dismutase (Cu/ZnSOD) which is normally found there. To test this, the sequence encoding bacterial MnSOD was cloned into a yeast expression vector and subsequently introduced into a Cu/ZnSOD-deficient mutant of the yeast Saccharomyces cerevisiae. Functional expression of the protein was demonstrated, and complementation tests revealed that the protein was able to provide tolerance at wild-type levels to conditions which are normally restrictive for this mutant. Thus, in spite of the evolutionary unrelatedness of these two enzymes, Cu/ZnSOD can be functionally replaced by MnSOD in yeast cytosol.
Asunto(s)
Genes Bacterianos , Prueba de Complementación Genética , Geobacillus stearothermophilus/genética , Isoenzimas/genética , Saccharomyces cerevisiae/genética , Superóxido Dismutasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Biblioteca de Genes , Geobacillus stearothermophilus/enzimología , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Saccharomyces cerevisiae/enzimologíaRESUMEN
The Bacillus subtilis phage phi 105 repressor, a lambda repressor-like transcriptional regulatory protein, was overproduced in Escherichia coli and purified to near homogeneity in order to examine its in vitro DNA-binding properties. The active form of repressor appears to be a tetramer. DNase I protection experiments demonstrate that repressor can specifically bind to six distinct sites, all located within the phi 105 EcoRI-F immunity region (immF). Three of these sites had been identified earlier as functional operators by genetic analysis. They share a common 14-base pair, asymmetric "core" sequence, 5'-GACGGAAATACAAG-3', termed OR. The three additional sites show significant homology with OR. For an individual binding site, hydroxyl-radical footprinting reveals symmetrical repressor-DNA interactions established via one side of the helix. Dimethyl sulfate protection indicates that guanines at the conserved OR base pair positions 1, 3, and 4 may participate in sequence-specific interactions with repressor in agreement with a previously proposed recognition model. However, since the OR sequence is not symmetrical with respect to this GNCG motif, at present it remains difficult to completely understand the molecular basis of this interaction.
Asunto(s)
Bacillus subtilis/genética , Bacteriófagos/genética , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas Represoras/aislamiento & purificación , Factores de Transcripción/aislamiento & purificación , Bacillus subtilis/metabolismo , Bacteriófagos/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasa I , Escherichia coli/genética , Hidróxidos , Radical Hidroxilo , Metilación , Peso Molecular , Mutación , Mapeo Nucleótido , Regiones Operadoras Genéticas , Proteínas Represoras/biosíntesis , Proteínas Represoras/metabolismo , Ésteres del Ácido SulfúricoRESUMEN
A commercial grass silage starter strain of Lactobacillus plantarum was transformed by high-frequency electroporation with plasmids containing an alpha-amylase gene from Bacillus stearothermophilus and an endoglucanase gene from Clostridium thermocellum. Both genes were expressed from their native regulatory signals, and active enzymes were found in the supernatant. However, the segregational stability of the transforming plasmids was rather low. Therefore, the transforming genes were inserted in the L. plantarum chromosome by means of single homologous recombination. In the majority of the transformants, this led to extremely stable segregation and expression of the transforming genes, without generating secondary mutations in the host. Increased selective pressure led to tandem amplification of the transforming DNA. The transformed strains demonstrated the ability of L. plantarum to express heterologous gene products; they can be used to detect the inoculum in silage ecology studies; and they demonstrate the feasibility of engineering truly cellulolytic silage starter bacteria.
Asunto(s)
Celulasa/genética , Cromosomas Bacterianos/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Lactobacillus/enzimología , alfa-Amilasas/genética , Celulasa/biosíntesis , Celulasa/metabolismo , Clonación Molecular , ADN Bacteriano/genética , Escherichia coli/genética , Amplificación de Genes , Lactobacillus/genética , Fenotipo , Plásmidos , Recombinación Genética , Mapeo Restrictivo , Transformación Bacteriana , alfa-Amilasas/biosíntesisAsunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/genética , Genes Bacterianos , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Secuencia de Aminoácidos , Bacillus subtilis/genética , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Datos de Secuencia MolecularRESUMEN
Two plasmids, pLAB1000 and pLAB2000 (3.3 and 9.1 kb, respectively), have been isolated from a grass silage strain of Lactobacillus hilgardii. Both plasmids were cloned in Escherichia coli and characterized through restriction mapping. A 1.6-kb XbaI-SacI fragment of pLAB1000 appeared to be sufficient for autonomous replication in Lactobacillus plantarum and in Bacillus subtilis. Different shuttle vectors for E. coli and gram-positive bacteria were developed using the pLAB1000 plasmid. These could stably be maintained in Lactobacillus, Enterococcus, and Bacillus under selective conditions. Plasmids sharing DNA homologies with pLAB1000 have been observed in different strains of the related species L. plantarum.
Asunto(s)
Bacterias Grampositivas/genética , Lactobacillus/genética , Plásmidos , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Replicón , Mapeo Restrictivo , Especificidad de la Especie , Transformación BacterianaRESUMEN
The presence of a 138-kilobase plasmid (pD188) correlated with increased resistance to cadmium in Rhodococcus fascians D188. This plasmid could be transferred by a conjugation-like system in matings between R. fascians strains. Transconjugants expressed the cadmium resistance and could be used as donors in subsequent matings. Four other R. fascians strains (NCPPB 1488, NCPPB 1675, NCPPB 2551, and ATCC 12974) could also be used as donors for cadmium resistance in matings. Strain NCPPB 1675 showed a 100% cotransfer of cadmium and chloramphenicol resistance markers.
Asunto(s)
Cadmio/farmacología , Conjugación Genética , Factores R , Rhodococcus/genética , ADN Bacteriano/genética , Farmacorresistencia Microbiana , Marcadores Genéticos , Cinética , Mutación , Hibridación de Ácido Nucleico , Rhodococcus/efectos de los fármacosRESUMEN
The Bacillus subtilis phage phi 105 repressor specifically recognizes a 14-bp operator sequence which does not exhibit 2-fold rotational symmetry. To facilitate a genetic analysis of this sequence-dependent DNA binding a B. subtilis strain was constructed in which mutations affecting the phi 105 repressor-operator interaction cause a selectable phenotype, chloramphenicol resistance. After in vivo mutagenesis, we isolated and mapped 22 different mutations in the repressor coding sequence, 15 of which are missense substitutions. These are exclusively located in the N-terminal part (positions 1-43) of the 144 residue long polypeptide. Two nonsense mutants, at positions 70 and 89, respectively, still show partial repressor activity. These data suggest that the phi 105 repressor consists of at least two independently folding structural domains, of which the N-terminal is involved in operator binding. Twelve missense mutations are clustered in a region extending from Gln-18 to Arg-37, which we propose to be the DNA-binding alpha-helix--beta-turn--alpha-helix motif, common to all lambda Cro-like repressors. The second ('recognition') helix shows significant homology with the corresponding sequence in Tn3 resolvase, and there is also a striking similarity between the phi 105 operator and the consensus sequence for a Tn3 res half-site. Based on these observations, and on the previously isolated phi 105 0c mutants, we tentatively assign some specific contacts between base pairs from the first half of a phi 105 operator site and amino acids from the repressor's 'recognition helix'.
Asunto(s)
Bacillus subtilis , Bacteriófagos/genética , ADN Bacteriano/genética , Regiones Operadoras Genéticas , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Bacteriófagos/metabolismo , Mapeo Cromosómico , ADN Bacteriano/metabolismo , Mutación , Proteínas Represoras/genéticaRESUMEN
We present the first evidence, in Bacillus subtilis, for gene regulation through the classical mechanism of repressor-operator interaction. The EcoRI-F immunity region (immF) of lysogenic phage phi 105 contains two promoters, PM and PR, in divergent orientations. PM initiates transcription of the phi 105 repressor (c phi 105) gene, whereas PR most probably signals the onset of the lytic pathway. Fusions between each of these promoters and the cat-86 gene were constructed, and in-vivo promoter activities were determined, in both the presence and absence of the functional c phi 105 product, using S1 nuclease analysis and chloramphenicol acetyl transferase assays. The results showed that transcription from PM is stimulated, whereas PR activity is negatively controlled by the repressor. This differential regulation appears to be mediated by recognition of a 14 base-pair (bp) sequence, 5' GACGGAAATACAAG 3', three identical copies of which are present as direct repeats. Two copies, OR1 and OR2, are located closely together in the non-transcribed region between PM and PR, but do not overlap with the -35 and -10 regions of these promoters. The third copy, OR3, is located some 250 bp downstream from PR, within the coding region (ORF3) of the proximal gene of the PR transcription unit. When a 231 bp restriction fragment containing only OR3 was inserted between a strong constitutive promoter (P138) and the cat-86 gene, the in-vivo expression of chloramphenicol resistance was considerably reduced in the presence, but not in the absence, of phi 105 repressor. This hybrid P138-OR-cat-86 construct was subsequently used to select in vivo for operator-constitutive (Oc) mutations. Of 25 Oc mutants analyzed, all showed base alterations or deletions affecting the 14 bp sequence. We show further that insertion of a chemically synthesized oligonucleotide, containing the 14 bp OR sequence, at a site more than 100 bp downstream from the constitutive P138 is sufficient for transcription to become negatively controlled by phi 105 repressor. In comparison with previously identified Gram-negative bacterial and phage operators, the most unusual aspect of the phi 105 OR sequence appears to be its complete lack of 2-fold rotational symmetry.
Asunto(s)
Bacteriófagos/genética , Regiones Operadoras Genéticas , Transcripción Genética , Bacillus subtilis/genética , Secuencia de Bases , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas , ARN Viral , Secuencias Repetitivas de Ácidos Nucleicos , Proteínas RepresorasRESUMEN
We have identified and sequenced a bacteriophage phi 105 gene encoding an immunity repressor, the first to be characterized from a temperate phage infecting a Gram-positive host. Using superinfection immunity as an assay for repressor function, the phi 105 repressor gene was located within a 740-bp PvuII-HindIII subfragment near the left end of the phi 105 EcoRI-F fragment. We show that the repressor is specified by the 5'-proximal coding sequence of a translationally overlapping gene pair, transcribed from right to left on the conventional phi 105 map. Comparison of its amino acid sequence (146 residues) with that of a large number of Gram-negative bacterial and phage repressors revealed a putative DNA-binding region between positions 20 and 39. The coding region is preceded by a strong Shine-Dalgarno sequence 5' AAAGGAG 3'. Deletion analysis of the 5'-flanking DNA allowed to identify transcriptional control elements. Their structure, 5' TTGTAT 3' at -35 and 5' TATAAT 3' at -10, strongly suggests that the phi 105 repressor gene is transcribed by the major vegetative form of B. subtilis RNA polymerase, as would be expected for an early phage gene.
Asunto(s)
Bacteriófagos/inmunología , Genes Virales , Proteínas Represoras/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Bacillus subtilis/inmunología , Bacteriófagos/genética , Secuencia de Bases , Codón , Enzimas de Restricción del ADN , ADN Recombinante , ADN Viral/genética , ADN Viral/metabolismo , Plásmidos , Conformación Proteica , Transcripción GenéticaRESUMEN
Two different PstI fragments of temperate phage phi 105 DNA are shown to confer superinfection immunity upon Bacillus subtilis when inserted into the multicopy cloning vector pE194 cop-6. The 2.3 kb PstI fragment I is located almost entirely within EcoRI fragment F and encompasses a region previously known to encode a repressor. The other fragment, PstI-E (4.3 kb) maps inside the EcoRI-B fragment, and allows an explanation of the clear-plaque phenotype of the deletion mutant phi 105DII:6c. The two regions can be distinguished functionally, since only the PstI fragment I product interacts with a specific phi 105 promoter-operator site.
Asunto(s)
Bacillus subtilis/genética , Bacteriófagos/genética , Genes Virales , Bacteriófagos/inmunología , Deleción Cromosómica , Enzimas de Restricción del ADN , Inmunidad , Fenotipo , Plásmidos , TemperaturaRESUMEN
A Bacillus subtilis/Escherichia coli shuttle plasmid vector containing a transcriptionally silent chloramphenicol acetyl transferase gene (cat-86) was constructed by ligation of pPL603 (Williams et al., 1981a) and pUC8 (Vieira and Messing, 1982) at their unique EcoRI sites. Using this "promoter probe" vector we have obtained, by direct Cm resistance selection, a collection of cloned Sau3A fragments from the temperate phage phi 105 genome exhibiting promoter activity in B. subtilis. 18 promoter plasmids were subsequently transferred to an acceptor cell containing a functional repressor gene of phage phi 105 inserted into the temperature-sensitive replicon pE194. A repressor-controlled promoter was identified on the basis of its ability to confer thermo-inducible Cm resistance. The promoter is located on a 650-bp Sau3A fragment, mapping within the 3.2-kb EcoRI-F fragment, which also contains the phi 105 repressor gene. By assaying cloned subfragments of EcoRI-F for expression of immunity against phi 105 infection, the repressor gene could be assigned to a 1.1-kb EcoRI-HindIII fragment, which partially overlaps the promoter fragment. Taken together, these results suggest that, like the cI-coded repressor in coliphage lambda, the phi 105 repressor interacts with an operator sequence mapping very close to its own gene.
Asunto(s)
Bacillus subtilis/genética , Bacteriófagos/genética , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Acetiltransferasas/biosíntesis , Acetiltransferasas/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Cloranfenicol O-Acetiltransferasa , Escherichia coli/genética , Genes Bacterianos , Genes Virales , Vectores Genéticos , Calor , Proteínas Represoras/genética , Proteínas Virales/genéticaRESUMEN
Up to thirteen T-DNA-encoded, polyadenylated transcripts of different relative abundance were detected by Northern blot hybridization in the tobacco nopaline BT37 crown gall teratoma tissue. Their sizes range from 900 to 2,700 bases. The polarity of eight of the thirteen transcripts was assigned by hybridization of labeled RNA to single-stranded DNA fragments of the T-region obtained by cloning in an M13 vector. Both strands of the T-DNA are transcribed. Our data indicate that most, if not all, transcripts are generated via independent promoter and poly(A)-addition sites on the T-DNA. Comparison of T-DNA-encoded transcripts present in crown gall tumors showing teratoma-like growth (BT37) with those from an unorganized tumor line (W38C58) reveals that this difference in phenotype is accompanied by a difference in the expression of the T-DNA. T-DNA sequences common to both octopine and nopaline tumors encode at least five, and probably six, cross-hybridizing transcripts of the same size, location, polarity and function. These transcripts are involved in the process of plant tumor formation and maintenance.
Asunto(s)
Expresión Génica , Tumores de Planta , Plásmidos , Biosíntesis de Proteínas , Rhizobium/genética , Transcripción Genética , Arginina/análogos & derivados , Arginina/biosíntesis , Secuencia de Bases , Enzimas de Restricción del ADN , Desoxirribonucleasa HindIII , Hibridación de Ácido Nucleico , Plantas/genética , Plantas Tóxicas , NicotianaRESUMEN
Sequences in the 3'-untranslated region of two different octopine T-DNA genes were analyzed with regard to their significance in polyadenylation. Poly(A) addition sites were localized precisely by S1 nuclease mapping with T-DNA-derived mRNAs isolated from tobacco. The gene encoding transcript 7' contains two AATAAA hexanucleotides, respectively 119 bp and 170 bp downstream of the TAA stop codon. A single poly(A) site was mapped 24-25 bp downstream of the first AATAAA. Further, we show that a mutant octopine synthase gene, which has lost part of its 3'-untranslated region by deletion, is still active. This mutant gene terminates 19 bp upstream from the major wild-type polyadenylation site. The deletion also removes the AATAAT signal preceding this site. The mutant octopine synthase gene contains a minimum of four different poly(A) sites. The most prominent of these sites is identical to the minor poly(A) site of the wild-type gene, and is preceded by a sequence AATGAATATA. Three other sites are located within the adjacent plant DNA, giving rise to hybrid T-DNA/plant DNA transcripts. The two most distal sites are probably dependent on a motif AATAAATAAA, found 29 bp away from the T-DNA/plant DNA junction.
Asunto(s)
Agrobacterium tumefaciens/genética , Arginina/análogos & derivados , ADN de Plantas/análisis , Poli A , Aminoácido Oxidorreductasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , ADN Bacteriano , Genes Bacterianos , Modelos Genéticos , Datos de Secuencia Molecular , ARN Mensajero , Análisis de Secuencia de ADN , Nicotiana/genéticaRESUMEN
We have determined the complete nucleotide sequence of the gene for the crown gall enzyme, octopine synthase. The sequence was derived from cloned fragments of the Agrobacterium tumefaciens Ti plasmid Ach5. It displayed a continuous open reading frame encoding a polypeptide chain of 358 amino acids. The nucleotide positions corresponding to the 5' end and poly(A) addition site of the mature octopine synthase mRNA from a tobacco tumor cell line were determined by S1 nuclease mapping. Two sequences closely resembling transcriptional control regions found in eukaryotic genes transcribed by RNA polymerase II were identified in the flanking genomic DNA: a sequence 5'-TATTTAAA-3' was located 32 base pairs upstream from the initiation site of transcription, and a hexanucleotide 5'-AATAAT-3' occurred 17 base pairs in front of the poly(A) addition site. No Shine-Dalgarno sequence was present in the untranslated 5' leader sequence. The observations indicate that this DNA sequence, although naturally carried by a bacterial plasmid, is programmed as a functional plant gene.
Asunto(s)
Aminoácido Oxidorreductasas/genética , Arginina/análogos & derivados , Plásmidos , Rhizobium/genética , Secuencia de Aminoácidos , Arginina/genética , Secuencia de Bases , Mapeo Cromosómico , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
The DNA sequence of the nopaline synthase gene (nos) from Agrobacterium tumefaciens Ti plasmid pTiT37 and adjacent regions up to the right border of the T-DNA was determined. The 5' and 3' termini of the polyadenylated nos mRNA, isolated from a T37 tobacco teratoma tumor line, were localized by S1 mapping. The final mRNA is unspliced, encoded by a region of about 1450 bp, and specifies an open reading frame of 413 amino acids. Potential transcriptional signals in the 5' flanking DNA, such as CATAAA ("TATA box") and GGTCACTAT ("CAT box"), bear close resemblance to other eukaryotic promoters. Two putative polyadenylation signals, AATAAA and AATAAT, are found about 135 and 50 bp from the 3' end, respectively. This study may provide information for the development of expression vectors for genes in plant cells; moreover, the structural gene can be used as an easy screenable marker.