Stress responses in Prochlorococcus MIT9313 vs. SS120 involve differential expression of genes encoding proteases ClpP, FtsH and Lon.

Prochlorococcus is a marine cyanobacterium responsible for a significant part of global primary production as well as being one of the most abundant organisms on Earth. Protein turnover is an essential and poorly understood aspect of the cyanobacterial response to environmental stresses. In the present work, cultures of the SS120 and MIT9313 strains were subjected to several conditions, and quantitative real time RT-PCR was used to measure changes in the expression of genes encoding three representative ATP-dependent proteases. We found common responses to conditions such as aging. However, the expression pattern under nutrient starvation was strikingly different in the two strains, probably reflecting the different regulatory backgrounds of the two ecotypes here studied.

Expression of genes involved in nitrogen assimilation and the C/N balance sensing in Prochlorococcus sp. strain SS120.

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.

Physiological role and regulation of glutamate dehydrogenase in Prochlorococcus sp. strain MIT9313.

Glutamate dehydrogenase is an enzyme catalysing a reaction for ammonium assimilation, alternative to those performed by glutamine synthetase and glutamate synthase. In the genus Prochlorococcus, genomic studies have shown the presence of the gdhA gene (encoding glutamate dehydrogenase) in only four of the sequenced strains, including MIT9313. We studied the physiological regulation of glutamate dehydrogenase in this strain, by measuring the expression of gdhA, the intracellular concentration of the enzyme and its activity. Our goal was to clarify the physiological role of glutamate dehydrogenase, in order to understand why it has been selectively conserved in certain strains. Studies performed in cultures under nitrogen starvation, or with inhibitors of the nitrogen assimilation, suggest that the main role of glutamate dehydrogenase is not the assimilation of ammonium. Glutamate dehydrogenase activity and gdhA expression increased along the growth of cultures. Besides, we found a significant upregulation in gene expression when cultures were grown on glutamate as nitrogen source. We suggest that the main physiological role of glutamate dehydrogenase in Prochlorococcus MIT9313 is the utilization of glutamate to produce ammonium and 2-oxoglutarate, and amino acid recycling, thus enabling to use amino acids as nitrogen source. Therefore we propose that glutamate dehydrogenase is present in the genome of strains for whom the utilization of amino acids is most important.