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The expression of five genes involved in nitrogen assimilation in cyanobacteria, namely glnA, glsF, icd, ntcA, and glnB, encoding three key enzymes from that pathway (glutamine synthetase, glutamate synthase, isocitrate dehydrogenase) and two regulatory proteins (NtcA and PII), was studied in this work. Their changes under different conditions were analyzed by quantitative real-time RT-PCR. Nutrient limitation induced clear modifications on the expression of most studied genes: lack of nitrogen provoked an initial increase, followed by a marked decrease; in the cases of phosphorus and iron starvation, a general, stronger expression decrease was observed, particularly striking in the case of iron. Darkness and addition of the photosynthethic inhibitors DCMU and DBMIB also had a strong effect on gene expression. Methionine sulfoximine and azaserine, inhibitors of glutamine synthetase and glutamate synthase, respectively, provoked a sharp increase in icd expression. These results, together with previous studies, suggest that 2-oxoglutarate could be the molecule utilized by Prochlorococcus to sense the C/N balance. Besides, our results confirm the different regulation of nitrogen assimilation in Prochlorococcus with regard to other cyanobacteria.