key: cord-0023191-celko0yd
authors: Swapnil, Prashant; Meena, Mukesh; Rai, Ashwani K.
title: Molecular interaction of nitrate transporter proteins with recombinant glycinebetaine results in efficient nitrate uptake in the cyanobacterium Anabaena PCC 7120
date: 2021-11-18
journal: PLoS One
DOI: 10.1371/journal.pone.0257870
sha: 9f94a35ba026a066505eb179fb7269323faca39e
doc_id: 23191
cord_uid: celko0yd

Nitrate transport in cyanobacteria is mediated by ABC-transporter, which consists of a highly conserved ATP binding cassette (ABC) and a less conserved transmembrane domain (TMD). Under salt stress, recombinant glycinebetaine (GB) not only protected the rate of nitrate transport in transgenic Anabaena PCC 7120, rather stimulated the rate by interacting with the ABC-transporter proteins. In silico analyses revealed that nrtA protein consisted of 427 amino acids, the majority of which were hydrophobic and contained a Tat (twin-arginine translocation) signal profile of 34 amino acids (1–34). The nrtC subunit of 657 amino acids contained two hydrophobic distinct domains; the N-terminal (5–228 amino acids), which was 59% identical to nrtD (the ATP-binding subunit) and the C-terminal (268–591), 28.2% identical to nrtA, suggesting C-terminal as a solute binding domain and N-terminal as ATP binding domain. Subunit nrtD consisted of 277 amino acids and its N-terminal (21–254) was an ATP binding motif. Phylogenetic analysis revealed that nitrate-ABC-transporter proteins are highly conserved among the cyanobacterial species, though variation existed in sequences resulting in several subclades. Nostoc PCC 7120 was very close to Anabaena variabilis ATCC 29413, Anabaena sp. 4–3 and Anabaena sp. CA = ATCC 33047. On the other, Nostoc spp. NIES-3756 and PCC 7524 were often found in the same subclade suggesting more work before referring it to Anabaena PCC 7120 or Nostoc PCC 7120. The molecular interaction of nitrate with nrtA was hydrophilic, while hydrophobic with nrtC and nrtD. GB interaction with nrtACD was hydrophobic and showed higher affinity compared to nitrate.

Nitrogen is a fundamental nutrient required by plants and microorganisms in high amounts and is often a limiting factor for their growth and yield. Except for N 2 -fixing prokaryotes and those living in association with them, the rest of the life forms depend upon combined forms of nitrogen viz., nitrate, nitrite, ammonium, and organic nitrogen. Nitrate is the most oxidized and stable form of combined-N, and is predominantly available as an immediate source of N a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 for most of the plants and microorganisms in terrestrial and aquatic ecosystems. In the presence of nitrate, N 2 -fixing cyanobacteria cease the energy effective process of N 2 -fixation and switch over to utilizing available nitrate. However, nitrate concentration in natural environments is relatively low (< 0.01-50 μM) [1] . Hence, the initial step in nitrate assimilation, uptake of nitrate into the cell may become limiting. Cyanobacteria possess an active nitrate transport system to meet their N requirement [2] . The high-affinity ABC-nitrate transporter in cyanobacteria belongs to the ATP-binding cassette (ABC) super-family and comprises four domains (Fig 1) ; two ABC proteins: (1) a cytoplasmic ATPase (nrtD) and (2) ATPase/ nitrate binding fusion protein (nrtC), and two TMDs (transmembrane domains) proteins, (3) the periplasmic nitrate binding lipoprotein (nrtA) and (4) an integral membrane permease (nrtB) [3, 4] . The TMDs possess additional short extracytoplasmic binding proteins that provide critical contact to ABCs to function [5] . After entry into the cells, nitrate is reduced to nitrite and ammonium in two sequential enzymic reductions mediated by nitrate reductase and nitrite reductase, respectively, and then incorporated into carbon skeletons through GS/GOGAT (glutamine synthetase/ glutamine 2-oxoglutarate aminotransferase) pathway yielding amino acids.

In cyanobacteria, nitrate assimilation is directly linked to photosynthesis and is well documented [6] . In addition to the consumption of ATP during the uptake of nitrate, 8 ederived from photolysis of water are donated by ferredoxin to convert a molecule of nitrate into one ammonium molecule. However, most of the studies on nitrate assimilation are centered on the latter stages of nitrate assimilation, the first step being the transport of nitrate into the cell is the least studied [7] . Until recently, the mechanism of the ABC-nitrate transporter proteins in cyanobacteria has been overlooked. Salinity impairs nitrate uptake/ assimilation in cyanobacteria [6, 8] . Based on the kinetics and depressed transport of nitrate by an ionophore (monensin), sodium/nitrate symport has been proposed in Anacystis nidulans R2 [9, 10] . A mutant of Anabaena PCC 7120 defective in nitrate uptake could not take up Na + , indicated for NO 3 / Na + symport [11] . Conversely, no ABC-transporter driven by the electrochemical gradient of an ion has so far been reported. Furthermore, nitrate is noted to increase the adaptability of cyanobacterial cells to salinity by interacting with Na + carrier and limiting Na + influx [12] [13] [14] . To The TMD proteins of the ABC-nitrate transporter, periplasmic subunit nrtA scavenges nitrate of the solution and transports across the lipid bilayer of the membrane through nrtB pore. When nitrate level rises in the cell, ABC protein, NrtC binds to nitrate and acts as negative regulator by inhibiting nitrate uptake, while nrtD binds and hydrolyzes ATP to induce conformational changes in TMDs (nrtAB). Extra cytoplasmic helices running parallel to the cytoplasmic membrane provide contact to TMDs proteins. (B) Interaction of GB with nrtACD proteins.

https://doi.org/10.1371/journal.pone.0257870.g001 overcome the salt toxicity, halophilic and halotolerant forms synthesize/ accumulate lowmolecular-weight organic compatible solutes. Glycinebetaine (N,N,N-trimethyl glycine), hereafter referred to as GB is a universal compatible solute synthesized by both prokaryotes and eukaryotes. In addition to maintaining cell osmolarity, GB is suggested to act as molecular chaperones protecting the native structure of proteins and membrane integrity [15] .

In the present investigation, we used a freshwater diazotrophic cyanobacterium Anabaena PCC 7120 wild type (WT) and its transformant possessing N-methyltransferase genes (ApGSMT-DMT; glycine sarcosine methyltransferase and sarcosine dimethylglycine methyltransferase) from a halophilic cyanobacterium Aphanothece halophytica [6, 16] . The transformed cyanobacterium de novo synthesized and accumulated GB. We noted that salinity reduced the rate of nitrate transport in WT cells, while the rate increased in the ApGSMT-DMT transformed cells that synthesized recombinant GB. An attempt is made to unveil the mechanism(s) by which recombinant GB increases the efficiency of nitrate transporter system, and consequently its assimilation.

Cyanobacterium Anabaena sp. PCC 7120 (hereafter referred to as Anabaena 7120; also known as Nostoc sp. PCC 7120) WT cells were grown and maintained in BG11 0 medium [17] . The transformed Anabaena 7120 containing ApGSMT-DMT genes from Aphanothece halophytica (on the expression cassette pRL488 + ApGSMT-DMT) was grown in BG11 0 + 0.1 M NaCl + Km 10 (kanamycin 10 μg ml -1 ), since it was unable to grow without NaCl [6, 16] . The cultures were grown at 28 ± 1˚C with an average illumination of 70 μE m 2 s 1 provided with daylight fluorescent lamps for 14 h a day.

Exponential phase Anabaena 7120 WT and transgenic cells growing on N 2 in the presence and absence of 0.1 M NaCl were harvested and suspended in their respective fresh media to get a cell density of 70-80 μg chl ml -1 . To study the kinetics of nitrate uptake, the concentration of nitrate in the uptake mixture (nutrient solution with and without 0.1 M NaCl) was varied between 40-500 μM. The uptake experiments were performed under standard growth conditions (temperature 28 ± 1˚C and light intensity 70 μE m 2 s 1 ), and samples (0.5 ml) were withdrawn at 10 min of incubation. Uptake of nitrate was determined by measuring the depletion of nitrate in the uptake mixture [18] . Since the uptake was linear up to 10 min, the initial uptake rate was determined as to nitrate taken up by the cells during the first 10 min. The assay was performed in triplicate with independent cultures.

The amino acid sequences for the nitrate transporter proteins NrtA (accession no. BAB75032.1), NrtC (accession no. WP_010994786.1) and NrtD (accession no. WP_010994787) of Nostoc (Anabaena) 7120 were retrieved from National Centre for Biotechnology Information (NCBI) protein database, PDB (www.rcsb.org; http://www.ncbi.nlm.nih. gov/protein). The most suitable template for each protein was identified using BLASTP (protein BLAST, http://blast.ncbi.nlm.nih.gov/Blast.cgi) and selected for further bioinformatics approach. Protein domains, families and functional sites, patterns, and profile search were performed using PROSITE server [http://prosite.expasy.org/] [19] [20] [21] . The retrieved protein sequences were used for multiple sequence alignment using ClustalW (http://www.ebi.ac.uk/ Tools/msa/clustalw2/). Sequences were aligned across the entire length or only in certain regions [22] . The top 100 sequences of each nrtA, nrtC and nrtD were taken for the phylogenetic analysis using PHYLIP (Phylogeny Inference Package) programs Seqboot, Protdist, Neighbour, and Consense to generate a phylogenetic tree [23] [24] [25] . Mega 5.0 and Tree view were used to view the phylograms [26].

The nrtACD proteins of Nostoc PCC 7120 were used for template search on the NCBI PDB database for structure prediction [27, 28] . The selected templates were used for homology modeling employing Easy Modeller 4.0 [29] . It is a standalone tool with an intuitive interface that clearly defines the different steps of homology modeling for comparable protein structures based on MODELLER [30, 31] . Since no models were available for Anabaena 7120 nrtACD proteins in the PDB database, an approach was made for constructing their 3D structures using homology modeling. The validation of a generated model of nrtACD proteins was evaluated for stereochemical quality using assessment of Ramachandran Plot (RAMPAGE) server (http://mordred.boc.cam.ac.uk/~rapper/rampage.php), PROCHEK [32] and PDBSum [33] [34] [35] . The quality of the predicted model was calculated by the SAVES server (http://services. mbi.ucla.edu/SAVES/). Statistical analysis of non-bonded interactions among different atom types of the generated model was estimated using ERRAT server [36, 37] , while compatibility of an atomic model (3D) with its own amino acid sequence (1D) was tested by Verify3D [37] . Potential errors in the 3D model and Z-score values were calculated and compared with a template by the ProSA-web server, which ensured overall quality as well as determined that the input structure is within the range of scores typically found for the native proteins of similar size [38] . After complete evaluation and visualization (Discovery Studio 3.0) sequence of the model is then deposited to PMDB (Protein Model Data Base) [39] .

The chemical compounds GB (CID: 247) and nitrate (CID: 943) were retrieved using Pubchem compound database as SDF file [40] and converted to PDB using Discovery Studio Visualizer. The active sites and their residues for selected structures were identified using the MetaPocket server. The server identifies pockets on protein surfaces to predict ligand-binding sites [41, 42] . The retrieved PDB files for GB and nitrate were imported to the Patchdock server program (http://www.molegro.com/products.php) for docking with transporter proteins nrtACD models [43] [44] [45] .

Salinity (0.1 M NaCl) reduced the rate of nitrate transport into the wild-type cells of Anabaena 7120. In contrast, under identical salinity, transport rate increased into the transgenic cells (transformed with ApGSMT-DMT) that synthesized and accumulated GB (Fig 2A) . Determination of kinetics of the nitrate transport ( Fig 2B) showed that salinity increased the K s value (32.3μM) in WT, while the value was lowered (Ks 17.8 μM) in ApGSMT-DMT transformed cells compared to control (K s = 19.5 μM), i.e., transport in absence of NaCl. This indicated the increased affinity of GB synthesizing cells towards nitrate [6] . The V max value (26.8 μmol mg protein -1 min -1 ) was the highest for GB synthesizing cells, followed by control (22.6 μmol mg protein -1 min -1 ), and was the lowest (21.9 μmol mg protein -1 min -1 ) for NaCl-exposed cells. This revealed that salinization of the nutrient solution reduced the efficiency of the nitrate transport system, while GB stimulated it. Possibly, recombinant GB interacted positively with nitrate transporter proteins resulting in efficient nitrate transport. To unveil the mechanism of their interactions, we performed in silico analyses. 

Prosite analysis (S1 Fig) revealed that nrtA protein was 427 amino acids long and contained a Tat (twin-arginine translocation) signal profile (PS51318; score 8.195) of 34 amino acids necessary to initiate the transport of folded proteins [46] . The Tat signal peptide consisted of three domains, the positively charged N-terminal domain, hydrophobic domain, and Cterminal domain. The majority of the amino acids were hydrophobic in nature.

Prosite results for nrtC protein revealed that it contains 657 amino acids (S2 Fig) and two functional motifs, ABC transporter 2 (5-239 amino acids) and ABC transporter 1 (139-153 amino acids) with Walker A (GHSGCGKS, [42] [43] [44] [45] [46] [47] [48] [49] and Walker B (LLLD, 160-163), which is responsible for ATP binding and hydrolysis. Latter, it was preceded by an alpha-helical subdomain and a signature motif (LSGGMKQR, 139-146). Sequence alignment results revealed that the nrtC subunit contained two distinct domains; one was an N-terminal domain (5-239 amino acids) mostly composed of hydrophobic residues, and the other C-terminal domain (268-591 amino acids) possessing mostly hydrophobic amino acids. N-terminal domain (5-228 amino acids) of nrtC showed 59% identity and 77% similarity with nrtD (the ATP-binding subunit), whereas the C-terminal domain (268-591 amino acids) was 28.2% identical and 50% similar to nrtA, suggesting that the C-terminal domain of nrtC was a solute binding domain and N-terminal as ATP binding.

NrtD protein was found 277 amino acids long and contained ATP binding motif (PS50893; score 26.484) located at N-terminal (21-254 amino acids) (S3 Fig). The majority of the amino acids were hydrophobic.

The nrtACD protein sequences of Nostoc 7120 (target organism) were taken as input to find out the similar protein sequences in different cyanobacterial species using BLASTP. The searche against non-redundant data base and protein data bank resulted in 103, 86 and 106 homologs for nrtA, nrtC and nrtD, respectively (S1-S3 Tables), and were used for multiple sequence alignment (S4-S6 Figs). The phylogenetic analysis of nrtA, nrtC and nrtD revealed different clustering with respect to cyanobacterial nrt proteins. The nrtA cladogram (Fig 3) shows two clades in which clade 1 comprises two subclades and clade 2 comprises seven small subclades. The two subclades of clade 1 are differentiated with one having Anabaena and Nostoc species and the other containing the Microcystis and Gemnocyctis species. The subclades one, two, three and six of clad 2 showed Agrobacterium and Rhizobium, Bradyrhizobium species, Thioalkalivibrio and Psuedomonas, Thermosynechococcus and Leptolyngbya as dominating species in the phylogenetic tree, respectively. The nrtC phyllogram (Fig 4) also has two clades each having two subclades comprising of Anabaena, Nostoc, Fischerella, Planktothrix, Leptolyngbya and Microcystis species. Phylogeny of nrtD protein showed eight clades (Fig 5) , where clade seven had six subclades. The phylogenetic tree was comprised of Nostoc, Anabaena, Fischerella and Calothrix species. Clade one was dominated by the presence of Nostoc and Anabaena species.

Species like Hapalosiphon sp., Mastigocladus laminosus and Calothrix sp. had close homologues of nrtA protein, Tolipothrix sp. PCC of both the nrtC and nrtD proteins, while Fortiacontorta species appeared to have a close homologue of all three nrtACD proteins of Anabaena. Overall phylogenetic results thus indicated that the nitrate-ABCtransporter proteins (nrtACD) homology is highly conserved among the cyanobacterial species. 

The target proteins (nrtACD) of Nostoc sp. PCC 7120 were used for 3D structure prediction using templates (Fig 6A-6C) . Details of similarity for each selected template are presented ( Table 1 ). The results obtained by RAMPAGE for stereochemical quality of nrtA showed that 368 residues (96.1%) lies in the favored region, 15 residues (3.9%) lies in the allowed and none in the outliner region (Fig 7A) . For nrtC protein, 335 residues (93.8%) lies in the favored region, 18 residues (5.0%) lies in the allowed region and 4 residues (1.1%) lies in the outliner region (Fig 7B) , and for nrtD, 126 residues (94.7%) lies in the favored region, 6 residues (4.5%) lies in the allowed region and 1 residue (0.8%) lies in the outliner region (Fig 7C) . PROCHEK output in the form of Ramachandran plot for nrtA showed 304 residues (91.8%) lies in the most favored region, 24 residues (7.3%) lies in the additional allowed region, 3 residues (0.9%) lies in generously allowed regions and none in the disallowed region (Fig 8A) . For nrtC protein, 281 residues (90.6%) lies in the most favored region, 23 residues (7.4%) lies in the additional allowed region, 5 residues (1.6%) lies in generously allowed regions and 1 residue (0.3%) lies in the disallowed region (Fig 8B) , whereas for nrtD, the 102 residues (90.3%) lies in the most favored region, 10 residues (8.8%) lies in the additional allowed region, 1 residues (0.9%) lies in generously allowed regions and none in the disallowed region ( Fig 8C) . ERRAT web server was used to check the overall quality factor of nrtACD proteins which was found 73.21 in nrtA (Fig 9) , 68.58 in nrtC (Fig 10) and 66.94% in nrtD (Fig 11) protein models, while the Verify 3D was used to determine the compatibility of atomic model (3D) with its own amino acid sequence (1D) by assigning a structural class which is based on its position and environment (alpha, beta, loop, polar and nonpolar), and the compatibility was found more than or equal to 0.2 averaged 3D-1D score for the 95.84, 95.26 and 89.92% amino acid residue of NrtA, NrtC and NrtD, respectively. Hence, more than 80% amino acid score with the template protein demonstrated the good quality of the model proteins. The overall model quality was checked by the ProSA-web tool [35] by determining their Z-score values. Z-score value for nrtA, NrtC and NrtD was found -9.68 (Fig 12A) , -8.57 (Fig 12B) and -5.2 (Fig 12C) , respectively. The results taken together suggested a close similarity between the template and the predicted structures and reinforced a good quality of the modeled protein. 

Metapocket server predicted three possible active sites in nrtACD proteins ( Table 2 ). Active site one in the nrtA protein ( Fig 13A) and active site two in nrtC ( Fig 14A) and nrtD proteins (Fig 15A) appeared as the prominent binding sites ( Table 2 ). The polar hydrophilic and positively charged amino acids Lys 251 , Lys 295 and Arg 298 , neutral Ser 290 , and negatively charged Asp 246 , and hydrophobic neutral amino acid Pro 48 of nrtA protein interacted in docking with nitrate ( Fig 13B) . The majority of the molecular interactions were hydrophilic with H-bonding between Arg298:NH1-O2, and Arg298:NH1-O2. On the other, the majority of the molecular interactions of nrtA with GB were hydrophobic. Five aliphatic hydrophobic neutral amino The results of nrtD/nitrate docking showed that the major portion of the amino acid residues that interacted with nitrate was hydrophobic and contains aliphatic hydrophobic neutral Leu 17 , Leu 5 and Val 3 , polar hydrophobic neutral Cys 4 and aromatic hydrophobic neutral Phe 2 with hydrogen bond in between Cys 4 : SG-O 2 (Fig 15B) . Similarly, the majority of nrtD amino acids interacting with GB were hydrophobic involved aliphatic hydrophobic neutral (Leu 127 , Ala 13 (Fig 15B) . PatchDock server docking calculation for the ligands GB and nitrate with the receptor proteins nrtACD presented a higher score solution, larger acquired docking area and lesser atomic contact energy (ACE) for GB compared to nitrate (Table 3) , revealing a higher affinity of GB towards receptor proteins compared to nitrate. 

Nitrate (NO 3 − ) is the main nitrogen source for plants and their mobilization is regulated by various transporters in the plant body [47] . To transport nitrate into the cell, nrtA protein of ABC-transporter binds to nitrate and delivers it to the nrtB (Fig 1) . NrtA has a high-affinity with the nitrate ABC transporter. The passage of nitrate transportation through the pore is linked to ATP binding and its consequent hydrolysis on the P-loop of the ABC domain. This brings conformational changes in the transmembrane domains and allows nitrate to cross the lipid bilayer of the membrane. Short cytoplasmic coupling helices provide critical contacts to TMD with ATP domain [4, 5, 48] . When nitrate concentration becomes high in the cell, nrtC binds to nitrate allosterically and inhibits nitrate transport [49, 50] . 

Salinity reduced the nitrate transport into WT cells (Fig 2) , while the rate of transport was increased into the transgenic cells (synthesizing recombinant GB). This means that in addition to protecting nitrate transport from salt toxicity, recombinant GB increased the efficiency of nitrate transport system. This increased efficiency might be due to the interaction of GB with the nitrate transporter proteins. NaCl stress increases Na + concentration in the cells, which disrupts the water structure and reduces hydrophobic interactions and hydrostatic forces within proteins [51] . Hence, NaCl-induced inhibition of nitrate transport is due to the disturbed hydrophobic-electrostatic balance necessary to maintain the protein structure [52, 53] . Compatible solute GB can interact with both the hydrophilic and hydrophobic regions of macromolecules [54] and can replace water at the surface of proteins [55] enabling the Nrt proteins to stabilize their native structure.

In silico analyses revealed that the deduced periplasmic protein nrtA in Anabaena 7120 that traps nitrate of the ambient solution was 427 amino acids long, the majority of which were hydrophobic as reported for Synechococcus sp. PCC7942 [56] . The molecular interaction of nrtA with nitrate ( Fig 13B) was hydrophilic (involving Lys 251 , Lys 295 and Arg 298 , Ser 290 , Asp 246 , and Pro 48 with H-bonding at Arg 298 ). The protein contained a Tat signal profile of 34 amino acids (1-34) necessary to initiate the transport [46] . Tat protein transport system has been reported in most bacteria and archaea. In cyanobacteria, Tat consensus motifs have been reported in deduced protein PhoD of Aphanothece halophytica exhibiting alkaline phosphatase (assimilate phosphorous under salt stress) and phosphodiesterase activities [57] . The presence of Tat motifs also supports the capturing of nitrate by pushing nrtA off the membrane and passing to the nrtB [4] . Since one nrtA protein binds to one molecule of nitrate [58] , nrtA becomes the most abundant protein in the nitrate-grown cells [59] .

The N-terminal (5-239 amino acids) and C-terminal (268-591 amino acids) of nrtC protein of Anabaena 7120 contained 657 amino acids that are mostly hydrophobic. Therefore, the interaction of nrtC with nitrate was also found hydrophobic. The hydrophilic negatively charged amino acid residues (Asp 158 , Phe 81 , Ser 82 and Leu 85 ) were buried in the center with Fig 14B) . The nrtC protein in Synechococcus sp. PCC 7942, is reported to be 659 amino acids long with the deduced amino acid sequences 1-254 for the N-terminal domain, and 279-659 for the C-terminal domain [56] . In Phormidium laminosum, the N-terminal (1-262) contained mostly hydrophobic residues, while the C-terminal (281-626) was highly hydrophilic [60] . In Anabaena 7120, the N-terminal of nrtC contained two functional motifs, ABC transporter 2 (5-239 amino acids) and ABC transporter 1 (139-153 amino acids) consisting of Walker A (GHSGCGKS, [42] [43] [44] [45] [46] [47] [48] [49] and Walker B (LLLD, 160-163) similar to that reported in Phormidium laminosum [58] , and was responsible for ATP binding and hydrolysis.

The C-terminal domain (268-591 amino acids) of nrtC in Anabaena 7120 was 28.2% identical and 50% similar to nrtA. In Synechocystis sp. PCC 6803, C-terminal domain was >30% identical and 50% similar to nrtA [4] , while in Synechococcus sp. PCC 7942, the identity of Cterminal domain of nrtC (amino acid residues 279-659) to nrtA was 30% [3, 56] . This further affirmed that the C-terminal domain of nrtC protein is a solute-binding domain. The deduced protein NrtD of Anabaena 7120 was of 277 amino acids, the majority of which were hydrophobic. The interaction of nrtD with nitrate was found hydrophobic and included Leu 17 , Leu 5 Val 3 , Cys 4 , and Phe 2 with hydrogen bond between Cys 4 : SG-O 2 (Fig 15B) . The N-terminal domain (5-228 amino acids) showed 59% identity and 77% similarity with nrtD (the ATPbinding subunit). At N-terminal 21-254 amino acid sequences were ATP binding motif. In Synechococcus sp. PCC7942, the nrtD protein consisted of 274 amino acids and showed 58% identity to the N-terminal domain of nrtC [3, 56] . This protein domain of the ABC transporter is responsible to bind and hydrolyze ATP to energize the nitrate transport system. 

Ammonium is preferred over nitrate when available in the solution and inhibits nitrate uptake [49, 61] . When intracellular nitrate level goes up, the C-terminal domain of nrtC, which is structurally similar to nrtA, may bind to nitrate allosterically as an effector-binding domain. Hence, nrtC role is suggested as a negative regulator involved in the ammoniumpromoted inhibition of nitrate transport activity [49, 62] . The interactions of GB with deduced nrtACD proteins of Anabaena PCC 7120 were hydrophobic and showed higher affinity towards GB compared to nitrate. Further, docking of GB was more efficient (with maximum docking score) with nrtC than that of nrtA and nrtD. Among the osmoprotectants, GB is by far the most effective and the most characterized osmolyte to date [63] , allowing the cells to grow luxuriantly under salt stress. The level of GB in transgenic Anabaena 7120 was low (7.5 μmol g −1 dry weight) and insufficient to contribute significantly to cellular osmotic potential, yet GB conferred considerable tolerance to salt-stress [6] . Other than the osmoprotective role, the mechanisms put forth to explain the GB enhanced stress tolerance include: (1) stabilization of proteins native structure and maintenance of membrane integrity, and (2) a regulatory function in the expression of genes i.e., induction of specific genes that led to increased stress tolerance [64, 65] , including photosynthetic and respiratory electron transport [6] . It suggested that both the roles of GB, stabilization of protein native structure and regulatory function in gene expression are responsible for the efficient nitrate transport in GB synthesizing and accumulating transgenic Anabaena PCC 7120. Salinity reduces the thermodynamic activity of water, thereby displacing the water of hydration from the protein surfaces [66] causing protein unfolding. GB induces the folding of unfolded (inactive) proteins in vitro and in vivo in a chaperone-like manner by decreasing the exposure of protein surface to solvent by drastically decreasing the water activity [66] . Experimentation regarding in situ evidence of the GB interaction with the proteins is difficult, hence scanty [67] . However, the periplasmic protein ProX of the ATP-transport system and ProU of Escherichia coli is reported to bind GB with high affinity [68, 69] . The receptor protein ProX undergoes a conformational change to form a site/cavity to accommodate GB and has an evenly negative surface potential [70] . The two conserved cis-proline align the Trp65 and Trp188 residues in a way to form an almost rectangular box in the polypeptide backbone of tryptophan residues [70, 71] . Possibly, nitrate transporter proteins nrtACD bind with GB in a similar fashion resulting in efficient nitrate transport. To our knowledge, this is the first report of GB interaction with ABC-nitrate transporter proteins and may open the way for further experimentation.

Salinity is one of the major factors limiting plant metabolic activities, growth, biomass and yield. GB is a universal osmoprotectant synthesized by prokaryotes and eukaryotes, including some crop plants to counter various stresses including salinity, and is also beneficial to humans. Nitrogen is the most limiting nutrient for plant growth and is often available as nitrate-N in the natural ecosystems, though at μM concentrations. The cyanobacterium Anabaena 7120 transported nitrate into the cells through an active process mediated by ABCtransporter proteins nrtABCD. The ApGSMT-DMT transformed cells possessed Ń -methyltransferase activities and de novo synthesized and accumulated intracellular recombinant GB. Salinity reduced the transport of nitrate into the cells. The recombinant GB not only protected the nitrate transport into the cells but increased the rate of transport under salinity. The low amount of intracellular GB (7.5 μmol g −1 dry weight) was insufficient to act as a cellular osmoticum. It is noted that the increased rate of nitrate transport in GB accumulating cells was due to the interaction of GB with ABC-nitrate transporter proteins and also by the participation of GB in the regulation of other intracellular metabolic processes. Thus, the results provided important bearing on the mechanism of GB interaction with ABC-nitrate transporter proteins. 

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Enzyme activities in concentrated solutions of glycinebetaine and other solutes

Proteins, enzymes and inorganic ions

The role of glycine betaine in the protection of plants from stress: clues from transgenic plants

Plant cellular and molecular responses to high salinity

Identification and characterization of a gene cluster involved in nitrate transport in the cyanobacterium Synechococcus sp. PCC7942

An alkaline phosphatase/ phosphodiesterase, PhoD, induced by salt stress and secreted out of the cells of Aphanothece halophytica, a halotolerant cyanobacterium

Substrate-binding lipoprotein of the cyanobacterium Synechococcus sp. strain PCC 7942 involved in the transport of nitrate and nitrite

Genetically engineered mutant of the cyanobacterium Synechococcus PCC 7942 defective in nitrate transport

Characterization of the N-terminal domain of NrtC, the ATPbinding subunit of ABC-type nitrate transporter of the cyanobacterium Phormidium laminosum

Regulation of nitrate uptake in Nostoc muscorum by glutamine synthetase

Characterization of the nitrate-nitrite transporter of the major facilitator superfamily (the nrtP gene product) from the cyanobacterium Nostoc punctiforme strain ATCC 29133

Roles of cytoplasmic osmolytes, water, and crowding in the response of Escherichia coli to osmotic stress: biophysical basis of osmoprotection by glycine betaine

Membrane-trafficking RabA4c involved in the effect of glycine betaine on recovery from chilling stress in Arabidopsis

The FRO 2 ferric reductase is required for glycine betaine's effect on chilling tolerance in Arabidopsis roots

Responses of E. coli to osmotic stress: large changes in amounts of cytoplasmic solutes and water

Glycine betaine-assisted protein folding in a lysA mutant of Escherichia coli

Purification and characterization of a glycine betaine binding protein from Escherichia coli

The osmoprotectant proline betaine is a major substrate for the binding-protein-dependent transport system ProU of Escherichia coli K-12

Cation-π interactions as determinants for binding of the compatible solutes glycine betaine and proline betaine by the periplasmic ligand-binding protein ProX from Escherichia coli

Regulation of WRKY genes in plant defense with beneficial fungus Trichoderma: Current perspectives and future prospects

Authors acknowledge the groups whose literatures have been cited to write this manuscript.