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J. Bacteriol. doi:10.1128/JB.00107-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Novel arrangement of enhancer sequences for NifA-dependent activation of the hydrogenase gene promoter in Rhizobium leguminosarum bv viciae

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; Consejo Superior de Investigaciones Científicas

^* To whom correspondence should be addressed. Email: t.ruizargueso{at}upm.es.

	Abstract

The transcriptional activation of the NifA-dependent ⁵⁴ promoter of 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 four 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 on 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.