The Role of Glutamine Synthetase in Regulating Ammonium Assimilation and Iron-Only Nitrogenase Expression in a Photosynthetic Diazotroph

The photosynthetic diazotrophs, such as Rhodopseudomonas palustris, can utilize light energy to drive the conversion of carbon dioxide (CO2) to a much more powerful greenhouse gas methane (CH4) by Fe-only nitrogenase, which is strictly regulated in response to the ammonium, a substrate of glutamine synthetase for the biosynthesis of glutamine. However, the primary glutamine synthetase for ammonium assimilation and its role in nitrogenase regulation remain unclear in R. palustris. Glutamine synthetase (GS) is responsible for the ammonium assimilation into glutamine, which serves as an important nitrogen donor for the synthesis of biomolecules and also plays a key role in regulating the nitrogen fixation catalyzed by nitrogenase. Rhodopseudomonas palustris, whose genome encodes 4 putative GSs and 3 nitrogenases, is an attractive photosynthetic diazotroph for studies of nitrogenase regulation, as it can produce the powerful greenhouse gas (methane) by iron-only (Fe-only) nitrogenase using light energy. However, the primary GS enzyme for ammonium assimilation and its role in nitrogenase regulation remain elusive in R. palustris. Here, we show that GlnA1, whose activity is finely regulated by reversible adenylylation/deadenylylation of Tyr(398) residue, is primarily responsible for ammonium assimilation as the preferred GS in R. palustris. The inactivation of GlnA1 makes R. palustris shift to use the alternative GlnA2 for ammonium assimilation, resulting in the expression of Fe-only nitrogenase even in the presence of ammonium. We present a model, showing how R. palustris responds to ammonium availability and further regulates the expression of Fe-only nitrogenase. These data may contribute to the design of promising strategies for a better control of greenhouse gas emissions.IMPORTANCE The photosynthetic diazotrophs, such as Rhodopseudomonas palustris, can utilize light energy to drive the conversion of carbon dioxide (CO2) to a much more powerful greenhouse gas methane (CH4) by Fe-only nitrogenase, which is strictly regulated in response to the ammonium, a substrate of glutamine synthetase for the biosynthesis of glutamine. However, the primary glutamine synthetase for ammonium assimilation and its role in nitrogenase regulation remain unclear in R. palustris. This study shows that GlnA1 is the primary glutamine synthetase for ammonium assimilation, and also plays a key role in Fe-only nitrogenase regulation in R. palustris. For the first time, a R. palustris mutant capable of expressing Fe-only nitrogenase even in the presence of ammonium is obtained by inactivation of GlnA1. A better understanding of the Fe-only nitrogenase regulation achieved in this study provide us with new insights into the efficient control of CH4 emissions.