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Expression of the nifA gene of Herbaspirillum seropedicae: role of the NtrC and NifA binding sites and of the -24/-12 promoter element

BBSRC, IPSR – Nitrogen Fixation Laboratory, University of Norwich, Norwich, UK¹
Departamento de Bioquímica – UFPR, C. Postal 19046, 81531-970, Curitiba, PR, Brazil²
Departamento de Farmacologia, UFPR, 81531-990, Curitiba, PR, Brazil³

Author for correspondence: E. M. Souza. Tel: +55 41 266 2042. Fax: +55 41 366 4398. e-mail: souzaem{at}bio.ufpr.br

The nifA promoter of Herbaspirillum seropedicae contains potential NtrC, NifA and IHF binding sites together with a -12/-24 ^N-dependent promoter. This region has now been investigated by deletion mutagenesis for the effect of NtrC and NifA on the expression of a nifA::lacZ fusion. A 5’ end to the RNA was identified at position 641, 12 bp downstream from the -12/-24 promoter. Footprinting experiments showed that the G residues at positions -26 and -9 are hypermethylated, and that the region from -10 to +10 is partially melted under nitrogen-fixing conditions, confirming that this is the active nifA promoter. In H. seropedicae nifA expression from the ^N-dependent promoter is repressed by fixed nitrogen but not by oxygen and is probably activated by the NtrC protein. NifA protein is apparently not essential for nifA expression but it can still bind the NifA upstream activating sequence.

Keywords: Herbaspirillum seropedicae, nitrogen fixation, nifA gene

Abbreviations: DMS, dimethysulphate; UAS, upstream activating sequence

^a Present address: Departamento de Bioquímica – UFPR, C. Postal 19046, 81531-970, Curitiba, PR, Brazil.

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