Study of Vitreoscilla globin (vgb) gene expression and promoter activity in E. coli through transcriptional fusion.
Bacterial hemoglobin (VtHb) is produced by the gram-negative bacterium, Vitreoscilla, in large quantity in response to hypoxic environmental conditions. The vgb gene coding for VtHb has been cloned in E. coli where it is expressed strongly by its natural promoter. The expression of the vgb gene in Vitreoscilla is transcriptionally regulated by oxygen. When E. coli cells were shifted from 20% to 5% oxygen, vgb specific transcript increased. In E. coli cells with plasmids carrying transcriptional fusions of the vgb gene promoter to either CAT (chloramphenicol acetyl transferase) or xylE (catechol-2,3-dioxygenase) genes, the promoter activity depended on the oxygen level. The concentration of CAT and xylE gene products in cells grown under 5% oxygen was 5-7 times that of aerobically (20% oxygen) grown cells. When the vgb gene promoter was deleted, VtHb was not produced under any conditions. When the promoter was replaced by the E. coli tac promoter, hypoxic oxygen did not affect the level of expression of vgb, but adding IPTG did increase the expression of this gene. These results indicate that the vgb gene promoter is transcriptionally regulated by oxygen even in E. coli, and that microaerobiosis is sufficient to induce vgb expression. The size of S1 nuclease-resistant hybrids, prepared using RNA transcripts protected with restriction enzyme fragments containing the promoter proximal region of vgb, was the same for both Vitreoscilla and E. coli, further evidence that the same promoter is used in both organisms. Transcriptional fusion of the vgb gene promoter to the xylE reporter gene on the broad host range plasmid, pKD-49, was used to demonstrate that the vgb promoter can be expressed in other gram-negative organisms, including Pseudomonas, Azotobacter, and Rhizobium.
Dikshit
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Department of Biology, Illinois Institute of Technology, Chicago 60616.
Dikshit
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Webster
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D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006090:Gram-Negative Bacteria / Q000235:genetics*; D006454:Hemoglobins / Q000235:genetics*; D010101:Oxygen Consumption / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
D006454:Hemoglobins