Reference

Lievens, S (2001) Ph.D Thesis, Dept Plantengenetica, VIB, U. Gent Acknowledgements

We thank Tomoko Kanamori for critical advice. This work was supported in part by Belgium government fellowship.

SITE DIRECTED MUTAGENESIS OF THE AZOTOBACTER VINELANDII REGULATORY FLAVOPROTEIN NifL

S. Perry1, R. Little1, F. Reyes-Ramirez2, R. Dixon1

'Dept of Molecular Microbiology, John Innes Centre, Norwich, NR4 7UH, UK 2School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK

The nitrogen fixation-specific regulatory flavoprotein, NifL, modulates the activity of the nif gene transcriptional activator NifA in response to redox, carbon and nitrogen status. Sequence analysis of Azotobacter vinelandii NifL has revealed a conserved N-terminal PAS domain and a conserved C-terminal kinase-like domain (HATPase), although NifL has no detectable kinase activity. We have performed site directed mutagenesis to determine the role of critical residues in NifL function.

Previous work has demonstrated that NifL binds FAD located in its PAS domain (Soderback et al. 1998). Mutation of three conserved residues in the PAS domain (Y60, Y83 and Y129) eliminated the redox response but did not affect the nitrogen response in vivo (Figure 1). All these mutations appeared to impair FAD binding. NifLY83A retained the response to ADP but the oxidized form of this protein was unable to inhibit NifA activity in vitro.

1 anaerobic nitrogen limited i anaerobic nitrogen excess i aerobic nitrogen limited i aerobic nitrogen excess

50000-

25000-

3000.

2000.

Y60V Y83A Y129A R272A H305N N419D G455A G480A NifA only

NifL

Figure 1. Influence of mutations on NifL activity in vivo

The central region of NifL has no significant homology to other proteins and its function is unknown. Mutation of two residues in this region (R272, and H305) eliminated the redox response but did not affect the nitrogen response in vivo (Figure 1). On purification NifL R272A was yellow in color and had FAD-specific spectral features comparable to wild type NifL. We propose that redox signaling to NifA involves the central region of NifL and that signaling of the redox and nitrogen status occurs independently.

NifL interacts with NifA by direct protein-protein interaction and this interaction is increased in the presence of ADP (Money et al. 1999), although the precise role of adenosine nucleotide binding is not understood. Three mutations in conserved residues (N419D, G455A and G480A), which have been well characterized in various histidine protein kinases, eliminated the response of NifL to both nitrogen and redox status (Figure 1). Hence, nucleotide binding is a major determinant of NifL activity.

References

Money T et al. (1999) J. Bacterid. 181, 4461-4468 Soderback E et al. (1998) Mol. Microbiol. 28, 179-192

NifA ACTIVATION OF TARGET PROMOTERS BY BINDING TO NON-CANONICAL UPSTREAM ACTIVATING SEQUENCES (UAS)

M. Martinez, M.V. Colombo, J. Palacios, J. Imperial, T. Ruiz-Argüeso Dept of Biotecnología, ETSIA, Universidad Politécnica, 28040-Madrid, Spain

1. Introduction

The prokaryotic enhancer-binding protein NifA stimulates transcription by binding to a conserved sequence (5'-TGT-N10-ACA-3') located upstream of -24/-12 promoters such as those of nitrogen fixation (nif and fix) genes. Recycling of hydrogen generated by the nitrogenase complex in legume nodules is mediated by a nickel-containing hydrogenase and contributes to increase the energy efficiency of the nitrogen-fixation process. The hydrogenase structural genes (hupSL) of Rhizobium leguminosarum bv. viciae strain UPM791 are temporally and spatially co-expressed in pea nodules with nif genes (Brito et al. 1995). The symbiotic transcription of hupSL takes place from a -24/-12 type promoter (PI), which is activated by NifA and requires IHF (Brito et al. 1997).

2. Results and Discussion

Promoter deletion assays identified an enhancer region spanning from position -173 to -140 that was essential for NifA-dependent PI activation but that contains no NifA-binding canonical UAS. Band shift experiments using purified NifA from Azotobacter vinelandii and the C-terminal half of NifA from UPM791 demonstrated that NifA binds to this region. Extensive site directed mutagenesis analysis identified three extended half-boxes (ACAA n(5) ACAA n(12) TTGT) within this region that were required for full NifA-dependent PI activation. Random mutagenesis of PI promoter identified no additional elements other than the extended half-boxes, IHF and RpoN binding sites that were required for PI promoter activity. The relative contribution of each extended half-box to activation was examined by comparing the activity associated to mutant promoters altered in one, two or the three half-boxes. The most important one was the TTGT half-box, but a strong cooperative effect among them was observed. A mechanism of activation based on a weak, cooperative binding of NifA to these half-boxes with formation of an oligomeric NifA-complex is proposed.

3. References

Brito B et al. (1995) Mol. Plant-Microbe Interact. 8, 235-240 Brito B et al. (1997) Proc. Natl. Acad. Sci USA 94, 6019-6024

4. Acknowledgements

Work was supported by grants PB95-0232 from DGICYT, BI096-0503 from CICYT, and by the Programa de Grupos Estratégicos de la Comunidad Autónoma de Madrid.

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