Molecular interaction of nitrate transporter proteins with recombinant glycinebetaine results in efficient nitrate uptake in the cyanobacterium Anabaena PCC 7120.

Prashant Swapnil,Mukesh Meena,Ashwani K Rai,Anjani Kumar Tiwari

doi:10.1371/journal.pone.0257870

Prashant Swapnil, Mukesh Meena + Show 2 more

Open Access

https://doi.org/10.1371/journal.pone.0257870

Copy DOI

Abstract

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.

Highlights

Nitrogen is a fundamental nutrient required by plants and microorganisms in high amounts and is often a limiting factor for their growth and yield
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 and (2) ATPase/ nitrate binding fusion protein, and two Transmembrane domains (TMDs) proteins, (3) the periplasmic nitrate binding lipoprotein and (4) an integral membrane permease [3, 4]
Recombinant GB interacted positively with nitrate transporter proteins resulting in efficient nitrate transport

Summary

Introduction

Nitrogen is a fundamental nutrient required by plants and microorganisms in high amounts and is often a limiting factor for their growth and yield. In the presence of nitrate, N2-fixing cyanobacteria cease the energy effective process of N2-fixation and switch over to utilizing available nitrate. 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]. 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 incorporated into carbon skeletons through GS/GOGAT (glutamine synthetase/ glutamine 2-oxoglutarate aminotransferase) pathway yielding amino acids

Methods

Results

Discussion

Conclusion