The cyanobacterial NtcA global nitrogen regulator belongs to the catabolite activator protein (CAP) family and activates transcription of nitrogen assimilation genes in response to nitrogen step-down. The binding affinity of NtcA towards a DNA fragment carrying the promoter of the glnA gene from Synechococcus sp. PCC 7942, analyzed in vitro by band-shift assay, was increased five-fold by 2-oxoglutarate in the presence of Mg 2+ ions. The 2-oxoglutarate effect peaked at about 0.6 mM, a rather physiological concentration for this compound under nitrogen-limiting conditions, and could be partially reproduced by 3-oxoglutarate but not by oxaloacetate or glutamate. These results suggest 2-oxoglutarate as a signal of the C to N balance of the cells to regulate NtcA activity and provide a new example of regulation in the versatile CAP family of proteins. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
A number of cyanobacteria from different taxonomic groups exhibited very low levels of uptake of 2-[U-14 C] oxoglutarate. Synechococcus sp. strain PCC 7942 was transformed with DNA constructs carrying the Escherichia coli kgtP gene encoding a 2-oxoglutarate permease and a kanamycin resistance gene cassette. The Synechococcus sp. strains bearing the kgtP gene incorporated 2-oxoglutarate into the cells through an active transport process. About 75% of the radioactivity from the 2-[U-14 C]oxoglutarate taken up that was recovered in soluble metabolites was found as glutamate and glutamine. 2-Oxoglutarate was, however, detrimental to the growth of a Synechococcus sp. strain bearing the kgtP gene.The dominant mode of growth of cyanobacteria is photoautotrophy. These organisms show very simple nutritional requirements and are able to grow in water supplemented with a few mineral salts. An analysis of the putative transport proteins encoded in the chromosome of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 (11) has shown a relatively high proportion of transport systems whose predicted substrates would be ions (metals plus anions), i.e., 47% of all putative transporters, compared to 16 and 21% in Escherichia coli and Bacillus subtilis, respectively (22). On the other hand, only 6% of the putative transporters of strain PCC 6803 are predicted to have carbon compounds (carboxylates plus carbohydrates) as substrates, while carbon compound transporters represent 28 and 20% in E. coli and B. subtilis, respectively (22). One of the few carbon compound transporters experimentally identified to date in Synechocystis sp. strain PCC 6803 is the glucose permease encoded by the glcP (or gtr) gene (27,34). This is a monocomponent permease consisting of a polypeptide of 468 amino acids with 12 putative membrane-spanning segments which is homologous to sugar transporters from several biological sources.In cyanobacteria, the carbon compound that provides the skeleton for the assimilation of inorganic nitrogen, via the glutamine synthetase-glutamate synthase pathway, is 2-oxoglutarate (5, 18). In the unicellular cyanobacterium Synechococcus sp. strain PCC 7942, 2-oxoglutarate also appears to have a key regulatory role in the integration of carbon and nitrogen metabolisms, e.g., it determines the phosphorylation level of the P II protein (glnB gene product) (6, 10), which is required for the inhibition by ammonium of nitrate uptake to take place (12). We were interested in studying the fate and roles of 2-oxoglutarate in cyanobacteria, but found that these organisms take up 2-oxoglutarate very poorly when it is supplied to the cells at a low concentration (see below). We therefore sought the construction of a strain of Synechococcus sp. able to transport 2-oxoglutarate.The kgtP gene of E. coli encodes a 432-amino-acid polypeptide which bears 12 putative membrane-spanning regions and mediates 2-oxoglutarate transport (28). Because the KgtP protein is similar (28% overall amino acid sequence identity) to Synechocystis s...
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