No abstract

The nrtA gene, which has been proposed to be involved in nitrate transport of Synechococcus sp. PCC7942 (Anacystis nidulans R2), was mapped at 3.9 kb upstream of the nitrate reductase gene, narB. Three closely linked genes (designated nrtB, nrtC, and nrtD), which encode proteins of 279, 659, and 274 amino acids, respectively, were found between the nrtA and narB genes. NrtB is a hydrophobic protein having structural similarity to the integral membrane components of bacterial transport systems that are dependent on periplasmic substrate-binding proteins. The N-terminal portion of NrtC (amino acid residues 1-254) and NrtD are 58% identical to each other in their amino acid sequences, and resemble the ATP-binding components of binding protein-dependent transport systems. The C-terminal portion of NrtC is 30% identical to NrtA. Mutants constructed by interrupting each of nrtB and nrtC were unable to grow on nitrate, and the nrtD mutant required high concentration of nitrate for growth. The rate of nitrate-dependent O2 evolution (photosynthetic O2 evolution coupled to nitrate reduction) in wild-type cells measured in the presence of L-methionine D,L-sulfoximine and glycolaldehyde showed a dual-phase relationship with nitrate concentration. It followed saturation kinetics up to 10 mM nitrate (the concentration required for half-saturation = 1 microM), and the reaction rate then increased above the saturation level of the first phase as the nitrate concentration increased. The high-affinity phase of nitrate-dependent O2 evolution was absent in the nrtD mutant. The results suggest that there are two independent mechanisms of nitrate uptake and that the nrtB-nrtC-nrtD cluster encodes a high-affinity nitrate transport system.

The transcription factor NtcA is a global regulator of nitrogen homeostasis in cyanobacteria. It thus positively regulates the expression of genes related to nitrogen assimilation such as glnA (which encodes glutamine synthetase) and ntcA itself in response to nitrogen shortage or depletion. The binding of NtcA to the glnA and ntcA promoters of Synechococcus sp. PCC 7942 in vitro now has been shown to be enhanced by 2-oxoglutarate. In vitro analysis of gene transcription also revealed that the interaction of NtcA with its promoter element was not sufficient for activation of transcription, and 2-oxoglutarate was required for transcriptional initiation by NtcA. Given that the intracellular concentration of 2-oxoglutarate is inversely related to nitrogen availability, it is proposed that this metabolite functions as a signaling molecule that transmits information on cellular nitrogen status to NtcA and thereby regulates the transcription of genes related to nitrogen assimilation in cyanobacteria.regulatory factor ͉ cyanobacteria ͉ RNA polymerase ͉ nitrogen assimilation

The cmpABCD operon of Synechococcus sp. strain PCC 7942, encoding a high-affinity bicarbonate transporter, is transcribed only under CO 2 -limited conditions. In Synechocystis sp. strain PCC 6803, the slr0040, slr0041, slr0043, and slr0044 genes, forming an operon with a putative porin gene (slr0042), were identified as the cmpA, cmpB, cmpC, and cmpD genes, respectively, on the basis of their strong similarities to the corresponding Synechococcus cmp genes and their induction under low CO 2 conditions. Immediately upstream of and transcribed divergently from the Synechocystis cmp operon is a gene (sll0030) encoding a homolog of CbbR, a LysR family transcriptional regulator of the CO 2 fixation operons of chemoautotrophic and purple photosynthetic bacteria. Inactivation of sll0030, but not of another closely related cbbR homolog (sll1594), abolished low CO 2 induction of cmp operon expression. Gel retardation assays showed specific binding of the Sll0030 protein to the sll0030-cmpA intergenic region, suggesting that the protein activates transcription of the cmp operon by interacting with its regulatory region. A cbbR homolog similar to sll0030 and sll1594 was cloned from Synechococcus sp. strain PCC 7942 and shown to be involved in the low CO 2 -induced activation of the cmp operon. We hence designated the Synechocystis sll0030 gene and the Synechococcus cbbR homolog cmpR. In the mutants of the cbbR homologs, upregulation of ribulose-1,5-bisphosphate carboxylase/oxygenase operon expression by CO 2 limitation was either unaffected (strain PCC 6803) or enhanced (strain PCC 7942), suggesting existence of other low CO 2 -responsive transcriptional regulator(s) in cyanobacteria.Cyanobacteria fix CO 2 efficiently despite the low affinity and selectivity of their ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) for CO 2 , because they possess a CO 2 -concentrating mechanism (CCM) to elevate the CO 2 concentration around the active site of Rubisco (11,21). The CCM involves the abilities to actively transport HCO 3 Ϫ into the cell, to convert CO 2 to HCO 3 Ϫ intracellularly, and to effectively convert HCO 3 Ϫ into CO 2 in carboxysomes, the polyhedral inclusion bodies to which Rubisco is localized. It is supposed that the conversion of CO 2 to HCO 3 Ϫ in the cytoplasm not only helps to maintain high intracellular HCO 3 Ϫ concentrations but also allows diffusion of CO 2 from external medium into the cytoplasm (10). Biosynthesis of the components of CCM, including Rubisco, is supposed to be controlled by CO 2 availability (11, 21); however, detailed studies on the transcriptional regulation of the CCM-related genes are yet to be performed, and the underlying molecular mechanism is unknown.Among the CCM-related genes, the cmp operon of Synechococcus sp. strain PCC 7942, encoding a high-affinity bicarbonate transporter (12,18), is known to be a typical low CO 2 -inducible transcription unit; the cmp operon mRNA and the CmpA protein, which is by far the most abundant protein among the proteins encoded by the operon, a...

Nitrate, the major source of nitrogen for photosynthetic organisms, is actively transported into the cell prior to its reduction to ammonium by the sequential action of nitrate reductase (NR) 1 and nitrite reductase (NiR) (1). The transport of nitrate has been the least understood step of nitrate assimilation. Recently, nitrate transporter genes have been identified in various organisms (2-7), offering the opportunity to investigate the structure-function relationship and the regulation of the nitrate transporters.The nitrate transporter genes of the cyanobacterium Synechococcus sp. strain PCC 7942 (nrtABCD, Refs. 2-4) are clustered with the NR gene (narB) and the NiR gene (nirA) to form the nirA-nrtABCD-narB operon (8). nrtB encodes a hydrophobic protein with structural similarities to the integral membrane components of ABC (ATP-binding cassette) transporters, and nrtC and nrtD encode proteins that resemble the ATPbinding proteins of ABC transporters, indicating that the nitrate transporter belongs to the ABC superfamily of transporters (9). The product of nrtA is the 45-kDa cytoplasmic membrane protein that is abundant in nitrate-grown cells (2). The hydrophilicity of the deduced amino acid sequence, the occurrence of a putative signal peptide, and its abundance seem to suggest that the 45-kDa protein is the substratebinding protein of the nitrate transporter (4). However, the membrane-bound nature of the protein, which is not typical of the substrate-binding proteins of Gram-negative bacteria, has made biochemical studies difficult, leaving its function unclear.On the basis of competitive interaction between nitrate and nitrite utilization (10) and the competitive inhibition by nitrite of nitrate transport (11), the cyanobacterial nitrate transporter has been assumed to transport nitrite as well. Luque et al. (12) verified this assumption by showing the inability of nrtD mutants to utilize low concentrations of nitrite. It is deduced that nrtB and nrtC, encoding the other components of the membrane transporter complex, are also involved in nitrite transport. It is yet to be examined whether the nrtA-encoded 45-kDa protein is involved in the transport of nitrite.In this work, we clarified the function of the nrtA gene product by molecular biological and biochemical analyses. Characterization of the mutants of Synechococcus sp. strain PCC 7942 with defined deletions in the nirA-nrtABCD-narB operon showed that nrtA is essential for the activity of the nitrate/nitrite transporter. Recombinant NrtA bound both nitrate and nitrite with high affinity. Studies using an inhibitor of lipoprotein processing indicated that the nrtA-encoded 45-kDa protein is a lipoprotein. We concluded that the nrtA gene product is a nitrate/nitrite-binding lipoprotein. On the basis of reported amino acid sequences of the putative substrate-binding proteins of ABC transporters from cyanobacteria, we propose that lipoprotein modification of substrate-binding proteins, which is unusual in other Gram-negative bacteria, is common in cyanoba...