Characterization of a new internal promoter (P3) for Rhizobium leguminosarum hydrogenase accessory genes hupGHIJ.

Synthesis of the Rhizobium leguminosarum [NiFe] hydrogenase requires the participation of 16 accessory genes (hupCDEFGHIJKhypABFCDEX) besides the genes encoding the structural proteins (hupSL). Transcription of hupSL is controlled by a -24/-12-type promoter (P(1)), located upstream of hupS and regulated by NifA. In this work, a second -24/-12-type promoter (P(3)), located upstream of the hupG gene and transcribing hupGHIJ genes in R. leguminosarum pea (Pisum sativum L.) bacteroids, has been identified in the hup gene cluster. Promoter P(3) was also active in R. leguminosarum free-living cells, as evidenced by genetic complementation of hydrogenase mutants. Both NifA and NtrC activated P(3) expression in the heterologous host Klebsiella pneumoniae. Also, P(3) activity was highly stimulated by K. pneumoniae NifA in Escherichia coli. This NifA activation of P(3) expression only required the sigma(54)-binding site, and it was independent of any cis-acting element upstream of the sigma(54) box, which suggests a direct interaction of free NifA with the RNA polymerase holoenzyme. P(3)-dependent hupGHIJ expression in pea nodules started in interzone II/III, spanned through nitrogen-fixing zone III, and was coincident with the NifA-dependent nifH expression pattern. However, P(3) was dispensable for hupGHIJ transcription and hydrogenase activity in pea bacteroids due to transcription initiated at P(1). This fact and the lack of an activator recruitment system suggest that P(3) plays a secondary role in symbiotic hupGHIJ expression.