Development of a gene expression vector for Thermus thermophilus based on the promoter of the respiratory nitrate reductase.
A specific expression system for Thermus spp. is described. Plasmid pMKE1 contains replicative origins for Escherichia coli and Thermus spp., a selection gene encoding a thermostable resistance to kanamycin, and a 720 bp DNA region containing the promoter (Pnar), and the regulatory sequences of the respiratory nitrate reductase operon of Thermus thermophilus HB8. Two genes, encoding a thermophilic beta-galactosidase and an alkaline phosphatase were cloned in pMKE1 as cytoplasmic and periplasmic reporters, respectively. The expression of the reporters was specifically induced by the combined action of nitrate and anoxia in facultative anaerobic derivatives of T. thermophilus HB27 to which the gene cluster for nitrate respiration was transferred by conjugation. Overexpressions in the range of approximately 200-fold were obtained for the cytoplasmic reporter, whereas that of the periplasmic reporter was limited to approximately 20-fold, with respect to their intrinsic respective activities.
Moreno
Renata
R
Departamento de Biologi;a Molecular, Centro de Biologi;a Molecular Severo Ochoa, Universidad Autónoma de Madrid, Cantoblanco, Madrid 28049, Spain.
Zafra
Olga
O
Cava
Felipe
F
Berenguer
José
J
D000469:Alkaline Phosphatase / Q000235:genetics; D000693:Anaerobiosis; D001483:Base Sequence; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D005822:Genetic Vectors; D020071:Interspersed Repetitive Sequences; D008969:Molecular Sequence Data; D005810:Multigene Family; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics*; D009566:Nitrates / Q000502:physiology; D019897:Periplasm / Q000235:genetics; D011401:Promoter Regions, Genetic; D016963:Thermus thermophilus / Q000235:genetics* / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics
D009566:Nitrates; D009565:Nitrate Reductases; D050901:Nitrate Reductase; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase