Characterization of an oxygen-dependent inducible promoter, the Escherichia coli nar promoter, in gram-negative host strains.
The Escherichia coli nar promoter is maximally induced under anaerobic conditions in the presence of nitrate ion or under anaerobic only conditions, depending on the genotype of the E. coli nar promoter. Previously, we found that the E. coli nar promoter has some desirable characteristics as an inducible promoter in the E. coli host strains. In this study, the E. coli nar promoter with lacZ gene at the downstream was cloned onto a broad-host-range Gram-negative vector, pBBR122. It was then induced in some other Gram-negative host strains, such as Agrobacterium, Pseudomonas, and Rhizobium, to determine whether the E. coli nar promoter could be used as an inducible promoter in these strains. From shake-flask experiments it was found that the wild-type E. coli nar promoter cloned onto pBBR122, pNW61, was suppressed under aerobic conditions in an Agrobacterium host strain, was partially induced under microaerobic only conditions, and was maximally induced under microaerobic conditions in the presence of nitrate ion. Whereas the mutant-type E. coli nar promoter cloned onto pBBR122, pNW618, was suppressed under aerobic conditions and was maximally induced under microaerobic conditions, regardless of the presence of nitrate ion. This kind of induction pattern observed for the E. coli nar promoters in the Agrobacterium host strain was similar to that observed for the E. coli nar promoters in the E. coli host strain. On the other hand, it was found that both of the E. coli nar promoters, pNW61 and pNW618, in a Pseudomonas host strain were partially induced under aerobic conditions and were maximally induced under microaerobic conditions, regardless of the presence of nitrate. Finally, it was found that both of the E. coli nar promoters in a Rhizobium host strain were minimally induced, regardless of the presence of oxygen or nitrate ion. Similar induction patterns for the three strains were also observed from fermentor experiments in which the dissolved oxygen (DO) level was tightly controlled. From an evolutionary point of view, the results from the three Gram-negative host strains indicate that the E. coli nar promoter system, including the promoter and regulatory proteins, was best conserved in the Agrobacterium host strain and the least conserved in the Rhizobium host strain. From an industrial point of view, the results indicate that the E. coli nar promoter system can be used as an oxygen-dependent inducible promoter in both Agrobacterium and Pseudomonas host strains.
Lee
Kil Ho
KH
School of Chemical Engineering and Technology, Yeungnam University, Kyungbuk, Korea.
Cho
Moo Hwan
MH
Chung
Taeowan
T
Chang
Ho Nam
HN
Lim
Sun Ha
SH
Lee
Jongwon
J
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006090:Gram-Negative Bacteria / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics* / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity
D009566:Nitrates; D011994:Recombinant Proteins; D009565:Nitrate Reductases; D050901:Nitrate Reductase; D010100:Oxygen