RESUMEN
Genetic manipulation of Rhodothermus marinus has been hampered by the lack of a selection system for gene transfer. We report construction of a Rhodothermus/Escherichia coli shuttle plasmid, containing the R. marinus trpB gene, based on pUC18 and the cryptic R. marinus plasmid pRM21. A plasmid-less R. marinus recipient strain was selected on the basis of growth characteristics and absence of restriction activity. The shuttle plasmid, pRM100, was successfully introduced into a TrpB- mutant of the recipient strain using electroporation and was found to transform it to prototrophy. No loss or rearrangement of pRM100 was observed after growth for 80 generations in non-selective medium. The relative copy numbers of pRM100 and of the parental plasmid, pRM21, were determined as 7+/-1 and 42+/-4, respectively. The shuttle plasmid was used to optimize an electroporation protocol, and the maximal number of transformants obtained was 4.3+/8-0.7x10(6) cfu/microg DNA at 22.5 kV/cm, 200 Omega and 25 microF. Transformation failed, however, after chemical preparation of cells according to several protocols. This is the first report of genetic transformation in the genus Rhodothermus.
Asunto(s)
Técnicas de Transferencia de Gen , Rhodothermus/genética , Enzimas de Restricción del ADN/análisis , ADN Bacteriano/química , Electroporación , Escherichia coli , Transferencia de Gen Horizontal , Genes Bacterianos , Vectores Genéticos , Datos de Secuencia Molecular , Plásmidos , Selección Genética , Análisis de Secuencia de ADN , Transformación Bacteriana , Triptófano/biosíntesis , Triptófano/genéticaRESUMEN
A gene encoding a DNA polymerase I from the thermophilic eubacterium Rhodothermus marinus was identified. The gene was cloned, sequenced and expressed in Escherichia coli. The gene is 2772 bp long and encodes a protein of 924 amino acids with a calculated molecular mass of 104.8 kDa. Sequence analysis showed that a generally conserved Phe residue in the O-helix is substituted by a Tyr (position 756) in the R. marinus enzyme. A Tyr in this position decreases the discrimination against dideoxynucleotides which is a major advantage in DNA sequencing. The protein was purified, characterized and showed to contain specific DNA-polymerization activity of 3100 units/mg of protein, 5'-->3' exonuclease activity and a 3'-->5' proofreading activity. Its optimum activity was at 55 degrees C and it had a half-life of 2 min at 90 degrees C. A truncated form of the enzyme lacking the 5'-->3' exonuclease domain was also expressed in E. coli. It had a specific DNA-polymerization activity of 5000 units/mg of protein and lacked the 5'-->3' exonuclease activity. Its optimum activity was at 65 degrees C and it had a half-life of 11 min at 90 degrees C. It was usable for DNA sequencing. This is the first thermostable DNA polymerase described with the O-helix Phe-->Tyr substitution.
Asunto(s)
ADN Polimerasa I/química , ADN Polimerasa I/genética , ADN Polimerasa I/metabolismo , Secuencia de Aminoácidos , Bacterias/enzimología , Clonación Molecular , ADN Bacteriano/metabolismo , Escherichia coli/metabolismo , Exonucleasas/metabolismo , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Plásmidos , Reacción en Cadena de la Polimerasa , Isoformas de Proteínas , Estructura Terciaria de Proteína , ADN Polimerasa Dirigida por ARN/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factores de Tiempo , Transcripción GenéticaRESUMEN
We describe the characterisation of four thermo-stable NAD(+)-dependent DNA ligases, from Thermus thermophilus (Tth), Thermus scotoductus (Ts), Rhodothermus marinus (Rm) and Thermus aquaticus (Taq), by an assay which measures ligation rate and mismatch discrimination. Complete libraries of octa-, nona- and decanucleotides were used as substrates. The assay comprised the polymerisation of oligo-nucleotides initiated from a 17 base 'primer', using M13mp18 ssDNA as template. Polymers of ligation products were analysed by polyacrylamide gel electro-phoresis. Under optimum conditions, the enzymes produced polymers ranging from 8 to 16 additions; there was variation between enzymes and the length of the oligonucleotides had a strong effect. The optimal total oligonucleotide concentration for each library was approximately 4 nmol. We compared the rates of ligation between the four ligases using an octanucleotide library as substrate. By this criterion, the Ts and Rm ligases are far more active compared to the more commonly available thermostable ligases.
Asunto(s)
Chlorobi/enzimología , ADN Ligasas/metabolismo , Thermus/enzimología , Cartilla de ADN , ADN de Cadena Simple/metabolismo , ADN Viral/metabolismo , Biblioteca de Genes , Oligodesoxirribonucleótidos/metabolismo , Especificidad por SustratoRESUMEN
Thymidine kinase type II is an important part of the pyrimidine salvage pathway. The thymidine kinase gene from the thermophilic eubacterium Rhodothermus marinus was cloned, sequenced and overexpressed. The gene is 639 bp and encodes a protein of 213 amino acids with a calculated molecular mass of 23.6 kDa. It shows homology to other thymidine kinase proteins from eukaryotic and prokaryotic organisms. The recombinant protein is inhibited by dNTPs but not by dNDPs. It is a tetramer in its native state. Its optimum temperature of activity is 65 degrees C and it has a half life of 15 min at 90 degrees C. This is the first thymidine kinase to be described from a thermophilic bacterium.
Asunto(s)
Bacterias Gramnegativas/genética , Timidina Quinasa/genética , Secuencia de Aminoácidos , Clonación Molecular , Genes Bacterianos , Bacterias Gramnegativas/enzimología , Datos de Secuencia Molecular , Peso Molecular , Proteínas Recombinantes/biosíntesis , Timidina Quinasa/química , Timidina Quinasa/metabolismoRESUMEN
A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan, but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a nickel nitrilotriacetate column. The enzyme had a pH optimum of 6-7 and its highest measured initial activity at 100 degrees C. The heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after 8 h at 90 degrees C.
Asunto(s)
Proteínas Bacterianas/genética , Celulasa/genética , Genes Bacterianos/genética , Bacterias Aerobias Gramnegativas/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Celulasa/química , Celulasa/metabolismo , Electroforesis en Gel de Poliacrilamida , Bacterias Aerobias Gramnegativas/genética , Bacterias Aerobias Gramnegativas/crecimiento & desarrollo , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
In this paper we describe the cloning and sequence analysis of a gene encoding DNA ligase (Lig; EC 6.5.1.2) from the thermophilic bacterium Rhodothermus marinus (Rm). We also describe the overexpression of the Lig-encoding genes of Rm and the thermophile, Thermus scotoductus (Ts), in Escherichia coli, and the purification and characterization of the overproduced Lig. The Rm lig gene encodes a protein of 712 amino acids (aa) with a calculated molecular mass of 79,487 Da. Comparison with published sequences of bacterial Lig revealed significant homology between the NAD(+)-utilizing Lig, and alignment of their aa sequences revealed several blocks of conserved residues. Both of the purified Lig exhibit nick-closing activity over a wide range of temperatures. Under our assay conditions the Rm Lig was active at 5-75 degrees C with apparent optimal activity above 55 degrees C. The Ts enzyme showed activity at 15-75 degrees C with optimal activity above 65 degrees C. The half-life of the Lig at 91 degrees C was estimated to be 7 min for the Rm Lig and 26 min for the Ts Lig.
Asunto(s)
ADN Ligasas/genética , Bacterias Aerobias Gramnegativas/genética , Isoenzimas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Ligasas/aislamiento & purificación , ADN Ligasas/metabolismo , ADN Bacteriano , Escherichia coli/genética , Genes Bacterianos , Prueba de Complementación Genética , Bacterias Aerobias Gramnegativas/enzimología , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
By dideoxynucleotide sequencing of a genomic clone, we have determined the complete nucleotide sequence of the gene encoding NAD(+)-dependent DNA ligase (EC 6.5.1.2) of the thermophilic bacterium Thermus scotoductus. The gene encodes a 674-amino-acid thermostable enzyme highly similar to other bacterial DNA ligases and to parts of the deduced gene product of Escherichia coli ORF f562, 5' to the spoR gene encoding 5' guanosyl kinase.
Asunto(s)
Secuencia Conservada , ADN Ligasas/genética , Thermus/enzimología , Thermus/genética , Secuencia de Aminoácidos , Bacterias/enzimología , Bacterias/genética , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Secuencia de Consenso , ADN Ligasas/biosíntesis , Sistemas de Información , Datos de Secuencia Molecular , Ribosomas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Mutations conferring resistance to low levels of kanamycin in Escherichia coli have been mapped at 3 locations: the unc locus (min. 83), a locus we have designated kanA (MIN. 72), close to strA (rpsL), and a locus at min. 86.5 previously discovered by Plate (1976) that we have designated ecfB. The unc and ecfB mutations are associated with defects in energy metabolism, while mutations at kanA may be in the gene coding for ribosomal protein S12 (rpsL). The three types of mutations cause cross resistance to a number of different aminoglycoside antibiotics and the effects of the mutations are cumulative in combination.