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Characterization of the oxygen-dependent promoter of the Vitreoscilla hemoglobin gene in Escherichia coli

Khosla, Chaitan and Bailey, James E. (1989) Characterization of the oxygen-dependent promoter of the Vitreoscilla hemoglobin gene in Escherichia coli. Journal of Bacteriology, 171 (11). pp. 5995-6004. ISSN 0021-9193. http://resolver.caltech.edu/CaltechAUTHORS:20120524-104235138

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Abstract

The gene coding for the Vitreoscilla hemoglobin (VHb) molecule has been cloned and functionally expressed in Escherichia coli. By using a plasmid-encoded gene as well as single-copy integrants, the oxygen-dependent VHb gene (VHb) promoter was shown to be functional in E. coli. The promoter was maximally induced under microaerobic conditions (dissolved oxygen levels of less than 2% air saturation). Direct analysis of mRNA levels as well as the use of gene fusions with lacZ showed that oxygen-dependent regulation occurred at the level of transcription. Transcriptional activity decreased substantially under anaerobic conditions, suggesting the presence of a regulatory mechanism that is maximally induced under hypoxic but not completely anaerobic conditions in E. coli. Primer extension analysis was used to identify the existence of two overlapping promoters within a 150-base-pair region upstream of the structural VHb gene. The oxygen-dependent activity of both promoters was qualitatively similar, suggesting the existence of a common mechanism by which available oxygen concentrations influence expression from the two promoters. Analysis of promoter activity in crp and cya mutants showed that both cyclic AMP and catabolite activator protein were required for full activity of the promoter. The VHb promoter contained a region of significant homology to the catabolite activator protein-binding site near the E. coli lac promoter.


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http://jb.asm.org/content/171/11/5995PublisherUNSPECIFIED
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC210464/PubMed Central IDUNSPECIFIED
Additional Information:© 1989 American Society for Microbiology. Received 7 November 1988; Accepted 19 July 1989. We acknowledge George Weinstock for providing several strains and plasmids used during the course of this work. We also thank one of the reviewers of the manuscript for suggesting the possible existence of two distinct transcripts in the experiment described in the legend to Fig. 2. This work has been supported by the Energy Conversion and Utilization Technologies Program of the U.S. Department of Energy.
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Department of Energy (DOE) Energy Conversion and Utilization Technologies Program UNSPECIFIED
Record Number:CaltechAUTHORS:20120524-104235138
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20120524-104235138
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:31631
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:06 Jun 2012 17:56
Last Modified:06 Jun 2012 17:56

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