Hydrogenase genes from Rhizobium leguminosarum bv viciae are controlled by the nitrogen fixation regulatory protein NifA

Rhizobium leguminosarum bv. viciae expresses an uptake hydrogenase in symbiosis with peas (Pisum sativum) but, unlike all other characterized hydrogen-oxidizing bacteria, cannot express it in free-living conditions. The hydrogenase-specific transcriptional activator gene hoxA described in other species was shown to have been inactivated in R. leguminosarum by accumulation of frameshift and deletion mutations, Symbiotic transcription of hydrogenase structural genes hupSL originates from a -24/-12 type promoter (hupS(p)). A regulatory region located in the -173 to -88 region was essential for promoter activity in R. leguminosarum. Activation of hupS(p) was observed in Klebsiella pneumoniae and Escherichia coli cells expressing the K. pneumoniae nitrogen fixation regulator NifA, and in E. coli cells expressing R. meliloti NifA. This activation required direct interaction of NifA with the essential -173 to -88 regulatory region, However, no sequences resembling known NifA-binding sites were found in or around this region, NifA-dependent activation was also observed in R. etli bean bacteroids. NifA-dependent hupS(p) activity in heterologous hosts was also absolutely dependent on the RpoN sigma-factor and on integration host factor. Proteins immunologically related to integration host factor were identified in R. leguminosarum. The data suggest that hupS(p) is structurally and functionally similar to nitrogen fixation promoters, The requirement to coordinate nitrogenase-dependent H-2 production and H-2 oxidation in nodules might be the reason for the loss of HoxA in R. leguminosarum and the concomitant NifA control of hup gene expression, This evolutionary acquired control would ensure regulated synthesis of uptake hydrogenase in the most common H-2-rich environment for rhizobia, the legume nodule.