Identification of PamA as a PII-binding Membrane Protein Important in Nitrogen-related and Sugar-catabolic Gene Expression in Synechocystis sp. PCC 6803

Abstract

The PII signaling protein plays a pivotal role in the coordination of carbon and nitrogen metabolism in a wide variety of bacteria, Archaea, and plant chloroplasts. By using a yeast two-hybrid screening system, we identified a transmembrane protein, designated PamA (encoded by sll0985), as a PII-binding protein in Synechocystis sp. PCC 6803. The interaction between PII and PamA was confirmed in vitro, and the interaction was inhibited in the presence of ATP and 2-oxoglutarate, whereas the interaction was not influenced by the phosphorylation status of PII. Northern blot analyses revealed that the transcripts of a set of nitrogen-related genes, including nblA, nrtABCD, and ureG, were decreased in a pamA deletion mutant. The mRNA and protein levels of a group 2 factor SigE were also reduced by the pamA mutation, and transcripts for sugar catabolic genes, such as gap1, zwf, and gnd that are under the control of SigE, were consequently decreased in the pamA mutant. In addition, the pamA mutant was found to be unable to grow in glucose-containing media. These results indicate that PamA has a role in the transcript control of genes for nitrogen and sugar metabolism in Synechocystis sp. PCC 6803.Cyanobacteria are prokaryotes that perform oxygenic photosynthesis similar to that in higher plants and algae. These bacteria coordinately regulate various aspects of cellular metabolism in response to changes in their environment. Carbon and nitrogen are important for cell growth, and the complex metabolism of each of these elements is regulated in synchrony under all conditions. The internal balance of carbon and nitrogen in unicellular cyanobacteria is monitored by the PII sensor protein, which is highly conserved among bacteria, Archaea, and plant chloroplasts (1-3).It has been shown that PII protein regulates nitrogen metabolism in unicellular cyanobacteria (4). As in Escherichia coli and related bacteria, the PII protein of Synechococcus sp. PCC 7942 binds to ATP and 2-OG 2 in a synergistic manner (5). The intracellular 2-OG level is assumed to reflect not only the carbon status but also the nitrogen status in cyanobacteria because of the lack of canonical 2-OG dehydrogenase (6). Therefore, PII is considered to be able to integrate energy, carbon, and nitrogen signals by monitoring ATP and 2-OG levels (4). In addition to ATP and 2-OG binding, Synechococcus and Synechocystis PII proteins are phosphorylated at a serine residue under nitrogen starvation (7,8). This phosphorylation level is also affected by carbon status (9), and recent analyses revealed that PII dephosphorylation specifically responded to intracellular 2-OG concentrations (10).A PII-deficient mutant (MP2) of Synechococcus sp. PCC 7942 does not inhibit the nitrate/nitrite transporter in the presence of ammonium, which is the preferred nitrogen source for unicellular cyanobacteria; however, it inhibits the nitrate/nitrite transporter in the wild-type strain (11). High affinity bicarbonate transporters are constitutively activated regardless of the ambient carb...

Physiological Roles of the cyAbrB Transcriptional Regulator Pair Sll0822 and Sll0359 in Synechocystis sp. strain PCC 6803

“…Aliquots of cells corresponding to 1 ml of culture at an OD 730 of 1.5 were harvested. The total sugar content of each sample was determined by the method described previously (18). In short, cells were suspended in sulfuric acid solution and boiled for 40 min.…”

Role of the Synechococcus PCC 7942 nitrogen regulator protein PipX in NtcA-controlled processes

“…Using yeast twohybrid approaches, three cyanobacterial P II receptors could recently be identified: the enzyme N-acetylglutamate kinase (NAGK) (Burillo et al, 2004;Heinrich et al, 2004) and the regulatory factor PipX (Burillo et al, 2004;Espinosa et al, 2006) in Synechococcus, and the non-conserved membrane protein PamA in Synechocystis sp. PCC 6803 (Osanai et al, 2005). The physiological role of the P II interaction with these protein receptors is only understood in the case of NAGK.…”

Effects of spontaneous mutations in PipX functions and regulatory complexes on the cyanobacterium Synechococcus elongatus strain PCC 7942

“…The non-conserved membrane protein PamA has been identified as a P II receptor in Synechocystis sp. PCC 6803 (Osanai et al, 2005). Structural and functional details are only known for the P II -NAGK complex (Llacer et al, 2007).…”