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Journal of Bacteriology, October 1999, p. 6535-6539, Vol. 181, No. 20
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Genetic Analysis of nif Regulatory Genes by Utilizing the Yeast Two-Hybrid System Detected Formation of a NifL-NifA Complex That Is Implicated in Regulated Expression of nif Genes

Shi Lei, Lakshmidevi Pulakat, and Narasaiah Gavini*

Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 43403

Received 18 May 1999/Accepted 23 July 1999

In diazotrophic organisms, nitrogenase synthesis and activity are tightly regulated. Two genes, nifL and nifA, are implicated as playing a major role in this regulation. NifA is a transcriptional activator, and its activity is inhibited by NifL in response to availability of excess fixed nitrogen and high O2 tension. It was postulated that NifL binds to NifA to inhibit NifA-mediated transcriptional activation of nif genes. Mutational analysis combined with transcriptional activation studies clearly is in agreement with the proposal that NifL interacts with NifA. However, several attempts to identify NifA-NifL interactions by using methods such as coimmunoprecipitations and chemical cross-linking experiments failed to detect direct interactions between these proteins. Here we have taken a genetic approach, the use of a yeast two-hybrid protein-protein interaction assay system, to investigate NifL interaction with NifA. A DNA fragment corresponding to the kinase-like domain of nifL was PCR amplified and was used to generate translation fusions with the DNA binding domain and the DNA activation domain of the yeast transcriptional activator GAL4 in yeast two-hybrid vectors. Similarly, a DNA fragment corresponding to the catalytic domain of nifA was PCR amplified and used to generate translation fusions with the DNA-binding domain and the DNA-activation domain of GAL4 in yeast two-hybrid vectors. After introducing appropriate plasmid combinations in yeast cells, the existance of direct interaction between NifA and NifL was analyzed with the MATCHMAKER yeast two-hybrid system by testing for the expression of lacZ and his3 genes. These analyses showed that the kinase-like domain of NifL directly interacts with the catalytic domain of NifA.

* Corresponding author. Mailing address: Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403. Phone: (419) 372-2279. Fax: (419) 372-2024. E-mail: ngavini{at}bgnet.bgsu.edu.

Journal of Bacteriology, October 1999, p. 6535-6539, Vol. 181, No. 20
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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