Nitrite assimilation enzyme NirD regulates (p)ppGpp homeostasis under nitrogen limitation in Xanthomonas campestris...

Guanosine tetraphosphate and pentaphosphate, collectively known as (p)ppGpp, are central alarmones that regulate the bacterial stringent response to nutrient limitation and environmental stress. In the plant pathogen Xanthomonas campestris pv. campestris (Xcc) strain 8004, (p)ppGpp metabolism is primarily governed by RelA/SpoT homologue (RSH) enzymes, including the monofunctional synthetase RelA, the bifunctional synthetase-hydrolase SpoT, and a small alarmone hydrolase SAH. However, the regulatory mechanisms governing RelA activity in Xcc 8004 remain to be fully elucidated. Here, we leverage the utilization of nitrate-induced nitrogen limitation, stemming from the inherently limited nitrate assimilation capacity exhibited by Xcc 8004, to investigate the intricacies of physiological and molecular regulatory processes within the stringent response. The results show that under nitrogen-limited conditions, phosphorylated NtrC transcriptionally activates relA, resulting in increased (p)ppGpp accumulation. This activation induces the NasT antitermination system, and promoting the expression of the nitrite reductase subunit nirD. The NirD directly interacts with the TGS and hydrolase domains of RelA, attenuating its synthetase activity and thereby preventing excessive (p)ppGpp production. Collectively, these findings delineate an NtrC-dependent regulatory circuit that couples nitrogen sensing to post-translational control of RelA via NirD, thereby ensuring balanced (p)ppGpp homeostasis during nitrogen stress. This work reveals a hitherto unrecognized mechanism for fine-tuning the stringent response in an aerobic plant pathogen and highlights the integration of nitrogen metabolism with stress signaling to optimize bacterial adaptation.