PII protein is essential for transcriptional regulation of anf gene cluster for iron-only nitrogenase in Rhodopseudomonas palustris.

In addition to catalyzing the biological nitrogen fixation, iron-only (Fe-only) nitrogenase is also able to reduce carbon dioxide (CO2) to formate (HCOO-) and methane (CH4). AnfA is responsible for the transcriptional activation of the anf gene cluster for Fe-only nitrogenase, whose expression is repressed by fixed nitrogen. However, it remains unclear how AnfA is regulated to control the expression of Fe-only nitrogenase. Here, we found that in Rhodopseudomonas palustris, PII proteins play a critical role in regulating the expression of Fe-only nitrogenase genes via AnfA. We hypothesize that the deuridylylated PII protein GlnK1, which was upregulated in the presence of ammonium (NH4+), could inhibit AnfA activity by forming a potential AnfA-GlnK1 complex. This likely serves as a fail-safe mechanism to prevent R. palustris from expressing Fe-only nitrogenase when AnfA is accidentally expressed under nitrogen-excess conditions. The uridylylated PII protein GlnK2UMP, which was upregulated in response to nitrogen starvation, stimulated the expression of an active AnfA hexamer that further activated the expression of Fe-only nitrogenase under nitrogen-fixing and Mo-free conditions. This study provides new insights into the regulation of Fe-only nitrogenase in R. palustris.IMPORTANCEThe expression and maturation of nitrogenase are tightly regulated by ambient nitrogen levels, which limits the persistence and efficiency of biological nitrogen fixation. This study offers new insights into the regulatory mechanism of AnfA by PII proteins in Rhodopseudomonas palustris. Understanding the regulation of AnfA, the transcriptional activator of the Fe-only nitrogenase gene cluster, could provide strategies to better control the expression of iron-only nitrogenase. Nitrogen-fixing bacteria that constitutively express iron-only nitrogenase have the potential to be developed into promising biofertilizers, as their nitrogen-fixing activity is enhanced and independent of molybdenum availability in the soil.