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Journal of Bacteriology, May 2008, p. 3185-3191, Vol. 190, No. 9
0021-9193/08/$08.00+0     doi:10.1128/JB.00107-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Novel Arrangement of Enhancer Sequences for NifA-Dependent Activation of the Hydrogenase Gene Promoter in Rhizobium leguminosarum bv. viciae{triangledown} ,{dagger}

Marta Martínez,1,{ddagger} Maria-Victoria Colombo,1 Jose-Manuel Palacios,1 Juan Imperial,1,2 and Tomás Ruiz-Argüeso1*

Departamento de Biotecnología and Centro de Biotecnología y Genómica de Plantas (CBGP), E. T. S. Ingenieros Agrónomos, Universidad Politécnica de Madrid, Ciudad Universitaria, s/n, 28040 Madrid, Spain,1 Consejo Superior de Investigaciones Científicas, Madrid, Spain2

Received 22 January 2008/ Accepted 19 February 2008

The transcriptional activation of the NifA-dependent {sigma}54 promoter of the Rhizobium leguminosarum hydrogenase structural genes hupSL (P1) has been studied through gel retardation analysis and detailed mutagenesis. Gel retardation analysis indicated the existence of a physical interaction between NifA and the promoter. Extensive mutagenesis followed by in vivo expression analysis showed that three sequences of 4 bases each (–170 ACAA –167, –161 ACAA –158, and –145 TTGT –142) are required for maximal stimulation of in vivo transcription of the P1 promoter. The arrangement of these upstream activating sequences (ACAA N5 ACAA N12 TTGT) differs from the canonical 5'ACA N10 TGT 3' UAS structure involved in NifA-dependent activation of nif/fix genes. Mutant promoter analysis indicated that the relative contribution of each of these sequences to P1 promoter activity increases with its proximity to the transcription start site. Analysis of double mutants altered in two out of the three enhancer sequences suggests that each of these sequences functions in NifA-dependent activation of the P1 promoter in an independent but cooperative mode. The similarities and differences between cis elements of hup and nif/fix promoters suggest that the structure of the P1 promoter has adapted to activation by NifA in order to coexpress hydrogenase and nitrogenase activities in legume nodules.

* Corresponding author. Mailing address: Departamento de Biotecnología, E. T. S. Ingenieros Agrónomos, Universidad Politécnica de Madrid, Ciudad Universitaria, s/n, E-28040 Madrid, Spain. Phone: 34-913365759. Fax: 34-913365757. E-mail: t.ruizargueso{at}upm.es

{triangledown} Published ahead of print on 29 February 2008.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

{ddagger} Present address: Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus Universidad Autónoma, Cantoblanco, E-28049 Madrid, Spain.

Journal of Bacteriology, May 2008, p. 3185-3191, Vol. 190, No. 9
0021-9193/08/$08.00+0     doi:10.1128/JB.00107-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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