ABSTRACT
The cyanobacterial protein NtcA is a global transcriptional regulator of genes involved in nitrogen assimilation that are subjected to ammonium-promoted repression and is itself controlled by positive autoregulation. Strain CSI70 derived from Synechococcus sp. strain PCC 7942 was constructed to overexpress an additional ntcA gene copy from a constitutive promoter. This strain exhibited high levels of the NtcA protein both in the presence and in the absence of ammonium. However, expression of the NtcA-dependent nir operon and glnA gene (tested by RNA/DNA hybridization and enzyme activity) was still subjected to nitrogen regulation. These results indicate in vivo regulation of the activity of NtcA at activation of transcription of nitrogen-regulated genes as a function of the nitrogen status of the cell.
Subject(s)
Bacterial Proteins , Cyanobacteria/genetics , Cyanobacteria/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Activation/genetics , Gene Expression Regulation, Bacterial , Glutamate-Ammonia Ligase/metabolism , Nitrate Reductase , Nitrate Reductases/metabolism , Nitrite Reductases/metabolism , Nitrogen/metabolism , Quaternary Ammonium Compounds/metabolismABSTRACT
Synthesis of nitrate reductase in the unicellular cyanobacterium Synechococcus sp. strain PCC 7942 took place at a slow rate when the cells were incubated without a supply of inorganic carbon, but addition to these cells of CO(2)/bicarbonate or, in a Synechococcus strain transformed with a gene encoding a 2-oxoglutarate permease, 2-oxoglutarate stimulated expression of the enzyme. Induction by 2-oxoglutarate was also observed at the mRNA level for two nitrogen-regulated genes, nir and amt1, but not for the photosystem II D1 protein-encoding gene psbA1. Our results are consistent with a role of 2-oxoglutarate in nitrogen control in cyanobacteria.
Subject(s)
Bacterial Proteins , Carbon/pharmacology , Cyanobacteria/drug effects , Gene Expression Regulation, Bacterial/drug effects , Ketoglutaric Acids/pharmacology , Nitrate Reductases/metabolism , Nitrogen/metabolism , Carbon/metabolism , Cyanobacteria/enzymology , Cyanobacteria/genetics , Cyanobacteria/metabolism , DNA-Binding Proteins/metabolism , Dicarboxylic Acid Transporters/metabolism , Escherichia coli Proteins/metabolism , Genes, Regulator , Nitrate Reductase , Transcription Factors/metabolismABSTRACT
Dissociation constant (K(d)) was determined for the binding of the transcriptional regulator NtcA to a number of NtcA-activated promoters of the cyanobacterium Synechococcus sp. PCC 7942, as well as to mutant versions of those promoters altered in their NtcA-binding sites. K(d) values obtained ranged from 27 nM for the NtcA-binding site in the glnA promoter to ca. 1.4 microM for the site in the glnB promoter. Changes in the sequence signature of NtcA-binding sites (i.e. GTAN(8)TAC) either completely prevented binding of NtcA or drastically decreased its affinity for the promoter revealing that the GTN(10)AC subset is essential for binding. The importance of the flanking A/T-rich sequences and of a conserved CA dimer found in between the GTA/TAC triplets was also stressed. Thus, this structure seems to be optimal for the binding of NtcA. Comparison of K(d) values for NtcA binding to native promoters showed that differences in the sequence of their NtcA binding sites determine extensive differences in the affinity of the regulator for those sites.
Subject(s)
Bacterial Proteins/genetics , Cyanobacteria/genetics , DNA-Binding Proteins/genetics , Membrane Transport Proteins/genetics , Nitrate Reductases/genetics , Nitrite Reductases/genetics , Promoter Regions, Genetic , Transcription Factors/genetics , Amino Acid Sequence , Binding Sites , Consensus Sequence , DNA-Binding Proteins/chemistry , Molecular Sequence Data , Mutation , Nitrate Reductase , Transcription Factors/chemistryABSTRACT
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.
