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The physiological regulation of glutamine synthetase (GS; EC 6.3.1.2) in the axenic Prochlorococcus sp. strain PCC 9511 was studied. GS activity and antigen concentration were measured using the transferase and biosynthetic assays and the electroimmunoassay, respectively. GS activity decreased when cells were subjected to nitrogen starvation or cultured with oxidized nitrogen sources, which proved to be nonusable for Prochlorococcus growth. The GS activity in cultures subjected to long-term phosphorus starvation was lower than that in equivalent nitrogen-starved cultures. Azaserine, an inhibitor of glutamate synthase, provoked an increase in enzymatic activity, suggesting that glutamine is not involved in GS regulation. Darkness did not affect GS activity significantly, while the addition of diuron provoked GS inactivation. GS protein determination showed that azaserine induces an increase in the concentration of the enzyme. The unusual responses to darkness and nitrogen starvation could reflect adaptation mechanisms of Prochlorococcus for coping with a light-and nutrient-limited environment.Prochlorococcus is a marine photosynthetic prokaryote ubiquitous in most intertropical areas of the oceans and is responsible for a significant part of the global primary production (for a review, see reference 27). Its unusual photosynthetic apparatus (12,13,15,29) and wide genetic diversity have attracted increasing scientific interest in recent years. Its ability to tolerate a very wide light gradient has been linked to the cooccurrence in the field of two ecotypes with distinct irradiance optima for growth and photosynthesis (21,40). While many reports have described these features in detail, little is known about other important aspects of Prochlorococcus metabolism, such as nutrient assimilation. In particular, the nitrogen assimilatory pathways in Prochlorococcus have not yet been studied. However, it is widely accepted that nitrogen is the main limiting nutrient in the upper layer of the oceans (27), and an understanding of the mechanisms involved in nitrogen assimilation could thus provide some keys to unveiling the remarkable ability of Prochlorococcus to colonize very oligotrophic regions. Nevertheless, such studies with Prochlorococcus face two problems: first, this organism is not easy to cultivate, and second, most isolated strains or clones contain contaminant heterotrophic bacteria. Only very recently, Rippka and coworkers described the first axenic strain, PCC 9511 (31), a typical high-light-adapted Prochlorococcus ecotype, allowing proper study of nonphotosynthetic metabolic pathways.In the present work, we have studied the physiological response of glutamine synthetase (GS) in cultures of Prochlorococcus subjected to different conditions by measuring transferase and biosynthetic activities and antigen concentration. The standard nitrogen assimilatory pathway in non-nitrogenfixing cyanobacteria is composed of a complex, highly modulated system of transporters, enzymes, and regulatory proteins (6). It allows...

The dithiol forms of thioredoxin and glutaredoxin are hydrogen donors for ribonucleotide reductase. We have determined the intracellular levels of ribonucleotide reductase (RRase), thioredoxin (Trx), glutaredoxin 1 (Grx1), and glutathione (GSH) and the glutathione redox status in new Escherichia coli K12 strains lacking thioredoxin (trxA-), glutaredoxin 1 (grxA-), and/or GSH (gshA-) or overproducing Trx or Grx1 from multicopy plasmids. We propose a regulatory network in which RRase levels are balanced with those of Trx, Grx1, and GSH so that deficiency or overproduction of one component would promote the opposite effect on the others to maintain a balanced supply of deoxyribonucleotides. GSH deficiency strongly increased both Grx1 levels and RRase activity, even more than Trx deficiency. Double gshA-trxA- bacteria were viable, whereas additional deficiency in lipoate synthesis (gshA-trxA-lipA-) caused the inability to grow in minimal medium plates supplemented with acetate plus succinate instead of lipoic acid. Thus, lipoate might be the only substitute of GSH for glutaredoxin reduction in gshA-trxA- cells, although the extremely high Grx1 content (55-fold) of these bacteria suggests that electron transfer from lipoate might be an inefficient reduction mechanism of glutaredoxins. Moreover, the enhanced Grx1 level of gshA-trxA- cells could obviate the need for a large increase in RRase activity, in contrast to grxA-trxA- double mutant cells. Impairment of the sulfate assimilation pathway, leading to very low GSH concentrations, and an oxidized glutathione redox state might explain the inability of grxA-trxA- cells to grow in minimal medium. Restoration of nearly normal levels of both GSH content and redox status cure the growth defect.