Latent Nitrate Transport Activity of a Novel Sulfate Permease-like Protein of the CyanobacteriumSynechococcus elongatus

Shin-ichi Maeda, Chieko Sugita, Mamoru Sugita, Tatsuo Omata
2006 Journal of Biological Chemistry  
The Synechococcus elongatus mutant lacking the nrtABCD gene cluster (NA3) is defective in active nitrate transport and requires high nitrate concentrations (>30 mM) for sustained growth. Prolonged incubation of NA3 in medium containing 2 mM nitrate led to isolation of a pseudorevertant (NA3R) capable of transport of millimolar concentrations of nitrate, from which three mutants with improved affinity for nitrate were obtained. We identified three genes responsible for the latent transport
more » ... ent transport activity for nitrate: ltnA, which encodes a response regulator with no effector domain; ltnB, which encodes a hybrid histidine kinase with two receiver domains; and ltnT, which encodes a sulfate permease-like protein with a putative cyclic nucleoside monophosphate (cNMP)-binding domain. Missense mutations of the high affinity derivatives of NA3R were found in ltnT, verifying that LtnT acts as the transporter. Overexpression of truncated LtnT lacking the cNMP-binding domain (but not full-length LtnT) conferred nitrate transport activity on NA3, suggesting that the cNMP-binding domain inhibits transport under normal conditions. A nonsense mutation in ltnB that resulted in elimination of the receiver domains of the encoded protein was responsible for expression of nitrate transport activity in NA3R. Expression of LtnB derivatives lacking the receiver domains also conferred low affinity nitrate transport activity on NA3. The phosphoryl group of the histidine kinase domain of LtnB was transferred to Asp 52 of LtnA in vitro. Overexpression of LtnA (but not LtnA(D52E)) led to manifestation of the latent nitrate transport activity in NA3, indicating involvement of phosphorylated LtnA in activation of the novel transporter. Nitrate is a major source of nitrogen for plants, algae, fungi, and many species of bacteria (1, 2). It is transported into the cells by an active nitrate transport system (NRT) 2 and reduced to nitrite by nitrate reductase. Nitrite is further reduced to ammonium by nitrite reductase, and the resulting ammonium is fixed as the amide group of Gln by glutamine synthetase. Three distinct types of nitrate transport systems have been identified among the large variety of organisms that assimilate nitrate, viz. NRT1, NRT2, and ATP-binding cassette (ABC)-type NRT. NRT1 and NRT2 possess 12 membrane-spanning regions and belong to the major facilitator superfamily of secondary active transport systems (3). ABC-type NRT is a primary active transport system consisting of a substrate-binding protein and a membrane transporter complex (4). Although NRT2 is widespread and found in plants, algae, fungi, the marine strains of cyanobacteria, and some species of heterotrophic bacteria, NRT1 has been found only in vascular plants. ABC-type NRT is present only in prokaryotic organisms, including the freshwater strains of cyanobacteria and some heterotrophic bacteria. The cyanobacterium Synechococcus elongatus has an ABC-type NRT encoded by four genes: nrtA encodes the substrate-binding protein; nrtB encodes the integral membrane component of the transporter; and nrtC and nrtD encode the ABC proteins (5-7). The genes encoding NRT, nitrate reductase (narB) (8, 9) , and nitrite reductase (nirA) (10, 11) form an operon, nirA-nrtABCD-narB (nirA operon) (11). The NA3 mutant, which was constructed by deleting the nrtABCD genes from the nirA operon, lacks NRT activity and requires high concentrations of nitrate (Ͼ30 mM) for sustained growth (12). In this study, we isolated and characterized a pseudorevertant of NA3 (NA3R) that is capable of uptake of low concentrations of nitrate. Genetic analysis of NA3R and its derivatives revealed that a novel sulfate permease-like protein of unknown substrate specificity shows low affinity nitrate transport activity. We also shown that a two-component regulatory system involving a hybrid histidine kinase and a response regulator with no effector domain is required for activation of the transporter. Possible roles of the phosphotransfer reaction via the two-component system in the regulation of the activity of the novel nitrate transport system are discussed. EXPERIMENTAL PROCEDURES Strains and Growth Conditions-A derivative of S. elongatus that was cured of the resident small plasmid pUH24 (R2-SPc (13); hereafter designated as the wild-type strain) and the mutant strains derived from it were grown photoautotrophically at 30°C under continuous illumination provided by fluorescent lamps (70 microeinsteins m Ϫ2 s Ϫ1 ). The basal medium used was a nitrogen-free medium obtained by modification of the BG11 medium (14) as described previously (15) . Ammonium-containing medium and nitrite-containing medium were prepared by the addition of 3.75 mM (NH 4 ) 2 SO 4 and 5 mM NaNO 2 , respectively to the basal medium. Nitrate-containing medium was prepared by the addition of KNO 3 at the indicated concentrations to the basal medium. Solid medium was prepared by adding 1.5% Bacto agar (Difco) to the liquid medium. All media were buffered with 20 mM HEPES-KOH (pH 8.2). When appropriate, kanamycin and/or chloramphenicol was added to the media at 25 and 10 g/ml, respectively. The Synechococcus strains and plasmids used in this study are listed in Table 1 . Isolation of a Pseudorevertant of NA3 and Its Derivatives-Cells of the NA3 mutant of S. elongatus strain PCC 7942 (lacking the nitrate/nitrite transporter genes nrtABCD) were inoculated into liquid medium containing 2 mM nitrate and cultivated with occasional dilution with fresh *
doi:10.1074/jbc.m513196200 pmid:16407232 fatcat:cublldicjjeulmk2xypfopdki4