Abstract
The control of nitrogen metabolism in the model cyanobacterium Anabaena sp. strain PCC7120 is a complex process orchestrated by master regulators such as NtcA, HetR, and FurC (PerR). These proteins establish complex networks with secondary regulators, finely tuning cellular metabolism in response to diverse, often undefined environmental signals. The XRE-like Alr1976 and the MerR-like All0345 proteins are two predicted transcriptional regulators regulated by FurC. While All0345 is widespread, both proteins have homologs conserved across several bacterial phyla, with alr1976 often followed by a gene encoding a Zn-metalloprotease. Previous transcriptomic analyses showed that furC overexpression affected alr1976 expression slightly more under nitrogen-deficient conditions, while changes in all0345 expression were exclusively observed under N deficiency, pointing them as critical candidates for understanding the finer details of nitrogen control in Anabaena. This work shows that both Alr1976 (NsrX) and All0345 (NsrM) are potentially modulated by NtcA and work as nitrogen secondary regulators. Electrophoretic mobility shift assays and transcriptomic analyses of ∆nsrX and ∆nsrM deletion strains indicate that both regulators act as repressors of key genes involved in nitrogen metabolism and heterocyst development. Notably, the ∆nsrM strain showed earlier heterocyst formation at 24 h of nitrogen step-down. NsrX and NsrM display distinct requirements for optimal DNA-binding activity to nitrogen metabolism genes (presence of Mn and reducing environment, respectively), suggesting they respond to different environmental stimuli. This differential signal integration likely enables master regulators FurC and NtcA to exert more precise control over shared targets, thereby refining the intricate network of nitrogen metabolic regulation in Anabaena. IMPORTANCE: Filamentous, nitrogen-fixing cyanobacteria are valuable organisms for biotechnology applications and as models for the study of multicellularity in prokaryotes. Understanding the regulation of nitrogen fixation and heterocyst development is essential for optimizing their use in synthetic biology and as biofertilizers. This study identifies two novel nitrogen secondary regulators, Alr1976 (NsrX) and All0345 (NsrM), as part of the intricate regulatory circuit governing nitrogen metabolism in the model cyanobacterium Anabaena sp. strain PCC7120. Genes encoding NsrX and NsrM are regulated by both FurC (PerR) and NtcA, therefore taking part in the NtcA-PerR network that modulates nitrogen metabolism and heterocyst differentiation genes.