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Applied and Environmental Microbiology, July 2005, p. 3716-3724, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3716-3724.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification of a Positive Transcription Regulatory Element within the Coding Region of the nifLA Operon in Azotobacter vinelandii

Ranjana Mitra,* Hirendra K. Das, and Aparna Dixit

Gene Regulation Laboratory, Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India

Received 14 October 2004/ Accepted 9 February 2005

Nitrogen fixation in Azotobacter vinelandii is regulated by the nifLA operon. NifA activates the transcription of nif genes, while NifL antagonizes the transcriptional activator NifA in response to fixed nitrogen and molecular oxygen levels. However, transcriptional regulation of the nifLA operon of A. vinelandii itself is not fully understood. Using the S1 nuclease assay, we mapped the transcription start site of the nifLA operon, showing it to be similar to the {sigma}54-dependent promoters. We also identified a positive cis-acting regulatory element (+134 to +790) of the nifLA operon within the coding region of the nifL gene of A. vinelandii. Deletion of this element results in complete loss of promoter activity. Several protein factors bind to this region, and the specific binding sites have been mapped by DNase I foot printing. Two of these sites, namely dR1 (+134 to +204) and dR2 (+745 to +765), are involved in regulating the nifLA promoter activity. The absence of NtrC-like binding sites in the upstream region of the nifLA operon in A. vinelandii makes the identification of these downstream elements a highly significant finding. The interaction of the promoter with the proteins binding to the dR2 region spanning +745 to +765 appears to be dependent on the face of the helix as introduction of 4 bases just before this region completely disrupts promoter activity. Thus, the positive regulatory element present within the BglII-BglII fragment may play, in part; an important role in nifLA regulation in A. vinelandii.

* Corresponding author. Present address: Division of Haematology and Oncology, Indiana University, Department of Medicine, 1044 West Walnut Street, R4-202, Indianapolis, IN 46202-5121. Phone: (317) 274-8498. Fax: (317) 274-0396. E-mail: ranmitra{at}iupui.edu or rini98{at}hotmail.com.

Applied and Environmental Microbiology, July 2005, p. 3716-3724, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3716-3724.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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