Previous studies have shown that the regulation of nitrogenase synthesis and activity in Rhodobacter capsulatus is complex (Masepohl, Klipp 1996). Transcription of Fe-protein requires active NifA protein and R. capsulatus contains two copies of NifA, NifAl and NifA2, which can function in this regard. Transcription of NifA requires the R. capsulatus NtrC and NtrB homologs whose activity is modulated in response to the nitrogen status of the cell. Once synthesized, the activity of NifA is controlled by fixed nitrogen in a manner that has been unclear. After its synthesis, nitrogenase is subject to several controls. Reversible ADP-ribosylation of Fe-protein, carried out by DraT and DraG, has the potential for modulating nitrogenase activity in response to the addition of NH3 or sudden changes in light intensity. As well, there appears to be an additional mechanism that is capable of regulating nitrogenase activity in R. capsulatus, a control over nitrogenase activity that is independent of the ADP-ribosylation system since it can be observed in strains that lack DraT, DraG. These systems for the regulation of nitrogenase activity are in turn sensitive to the cellular nitrogen status. Not only are these responses seen in response to sudden changes in environmental conditions, e.g. addition of NH3, light deprivation, but they are also observed with cells that are under steady state conditions. Thus cells that are actively fixing N2, cells growing on glutamate, or cells grown on moderate amounts of limiting NH3 all show some degree of ADP-ribosylation of Fe-protein (Yakunin et al. 1999). The degree of modification observed has been shown to be correlated with the intracellular pool of fixed nitrogen.
In terms of fast, short-term responses, highly nitrogen-limited cultures show a classical nitrogenase switch-off effect, with complete inhibition of nitrogenase activity following ammonia addition, a period of no nitrogenase activity that is proportional to the amount of NH3 added, and then complete recovery of nitrogenase activity upon exhaustion of the added NH3. Moderately nitrogen-limited (MNL) cultures show a different response, a "magnitude" response, with a decrease in in vivo nitrogenase activity that is proportional to the amount of added ammonia (Yakunin, Hallenbeck 1998). Since we found that MNL cultures appeared to have a decreased level of high-affinity ammonium transport compared with HNL cultures, we hypothesized that the switch-off process responded to a signal generated by transport of ammonium by the high-affinity system. Here we describe the presence of two putative ammonium transporters in R. capsulatus, AmtB and ArntY and describe the effects of mutations in the corresponding genes on these processes. As well, R. capsulatus possesses two Pu homologs, GlnB and GlnK, and we have carried out mutagenesis of the corresponding genes. GlnK and GlnB appear to be involved at all levels of nitrogenase regulation.
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