Analysis of seven putative Na+/H+ antiporters of Arthrospira platensis NIES-39 using transcription profiling and In-silico studies: an indication towards alkaline pH acclimation

Na+/H+ antiporters mediated pH regulation is one of the known mechanism(s), which advocates a possible role of the antiporters in the alkaline pH tolerance of Arthrospira platensis NIES-39 too. Seven putative Na+/H+ antiporters have been reported in A. platensis. We have characterized these seven antiporters, where A1, Q2, L2 and L6 belong to the CPA1 family whereas C5, D5 and O6 belong to CPA2 family through various In-silico analysis. Conserved domain analysis of these seven putative antiporters indicate the presence of nine different kinds of domains. Out of these nine domains, six domains function as monovalent cation-proton antiporters and two as the universal stress protein (Usp) category. Transcription profile of these seven antiporters was also generated at three different pH and time frames which showed a significant difference in the m-RNA levels at pH 7, 9 and at 11 along with a temporal pattern of the expression profile. The Insilico and the real time PCR analysis, put together, suggest the active participation of these seven putative Na+/H+ antiporters in alkaline pH homeostasis of this cyanobacterial strain where CPA1 subfamily antiporters play a major role.


Introduction
Ion and pH homeostasis are fundamental regulators of cellular processes. It is reported that among prokaryotic and eukaryotic organisms, pH is regulated by antiporters specially Na + /H + antiporters and Mrp-like antiporters [4]. Antiporters maintain a stable pH inside the cell under alkaline conditions [27] and help in the cell volume regulation [24].
All the Na + /H + antiporters ranging from prokaryotes to human belong to CPA super family [25].
CPA family is further subdivided in to CPA1 and CPA2 subfamily. The structure and function of 3 Na + /H + antiporters was first studied in E.coli by Padan et al. [15,11]. Padan et al. in 1989 studied the role of nhaA gene (Na + /H + antiporter) in pH regulation and salt stress by chromosome deletion. Later crystallographic structures of the antiporters along with the functionally important residues became available for other prokaryotes, belonging to CPA1 and CPA2 family.
Further, gene knockout studies of Na + /H + antiporters in Synechocystis sp. PCC 6803 strongly supported their active participation in pH regulation along with salinity stress [40]. Deletion of plant genes which encodes the vacuolar and cytoplasmic membrane Na + /H + antiporters decreases the plant's salt tolerance [2,30] whereas over expression is used to produce salt-resistant plants [1].
Many reports thus, suggest the role of Na + /H + antiporters in pH regulation. A. platensis NIES-39 was isolated from a soda lake, which can withstand the alkaline pH (up to pH 11). Its genome is composed of a single, circular double stranded chromosome of 6.8 Mb, without any plasmids [13]. The mechanism behind pH homeostasis has not been elucidated so far in this organism; however, seven Na + /H + antiporters are reported but still putative. Through In-silico analysis these seven putative antiporters are compared with the CPA1 and CPA2 family proteins along with their functionally conserved domains. The differential and temporal transcript expression profile of the Na + /H + antiporters under different pH regimes was also studied, to appraise their possible role during pH stress.

Sequence analysis and domain search
A. platensis NIES-39 antiporter's protein sequences were retrieved from NCBI database [10].
Pairwise and Multiple sequence alignments for Na + /H + antiporters were conducted using 4 ClustalW servers [37]. The domains and motifs of these antiporters were analyzed using the conserved domain (CD) search tools, NCBI [22]. The conservation patterns of the amino acid positions, in the CPA1 and CPA2 family, were analysed using sequence logos [9]. Protein sequences of the A. platensis antiporters were retrieved from NCBI and abbreviated as

Maintaining Arthrospira platensis NIES-39
A. platensis was procured from National Institute for Environmental Studies, Japan, and maintained aseptically in SOT medium (pH 9-9.5) at 30º C temperatures with 4 klux light intensity and a photoperiod of 16h light/8h dark with continues shaking.

Sample collection at different pH
A. platensis was grown at different pH i.e. pH 7, 9 and 11. pH 9 being the optimum pH for the growth whereas 7 and 11 pH were the points used to study pH dependent response of the antiporters. The cells were collected at 0 th , 1 st , 4 th and 6 th hour. Buffered medium was used to maintain a stable pH and the fluctuations were timely monitored.

RNA Isolation, c-DNA synthesis and real time PCR
The pH treated cells were collected in RNA protect Bacteria Reagent (QIAGEN). Total RNA was extracted using RNeasy mini kit (QIAGEN) and quantified using GE Healthcare Nanodrop. c-DNA was synthesized using Quantitect Reverse Transcription Kit (QIAGEN). iQ SYBR Green Supermix (BIO-RAD) to a final concentration of 1x was used to study the expression profile by semi-quantitative real time PCR. iQ5 multicolour real time PCR detection system from BIO-5 RAD was used for the PCR. 16s RNA was used as internal control for normalizing the calculations. Primers were designed using Primer3 [38] and the stability parameters were analyzed using IDT Oligoanalyzer 3.1. Primers were synthesized from SIGMA-ALDRICH. The PCR conditions used for the real time PCR starts with an initial denaturation of 3 minutes at 95º C followed by 45 cycles of 95º C-10 seconds, 56º C 30 seconds, 72º C 50 seconds each after which the fluorescence of the product was recorded at 72º C at the end of every cycle. Final extension was given for 10 minutes at 72º C. The calculation of the fold values was done using ∆∆ Ct method [21].

In-silico characterization of putative antiporters of Arthrospira platensis NIES-39
The amino acid sequence length of the seven antiporters from A. platensis varies from 409 to 691 (table 1). Pairwise global sequences alignment shows that on an average 17.54% (Standard deviation of 3.58) sequence identity exists among all seven antiporters with maximum 29% sequence identity between D5 and O6 antiporters. Lowest sequence identity of 13.3% is observed between Q2 and D5. Conserved domain database (CDD) search tool identifies nine (among them six are unique) different domains among seven antiporters of A. platensis (table 1).
Coverage span analysis of each domain reveals that each antiporter of A. platensis contains at least one domain belonging to NhaP, Na + /H + exchanger, KefB and Asp-A1a exchanger superfamily which exchanges alkali cations such as Na + , Li + , K + , Rb + , Ca 2+ and NH 4+ against protons [31] across the plasma membrane. Universal stress protein (USP) like domain along with USP A domain, Na + /H + antiporter C and TrkA domain along with TrkA-N are other three domains present among the antiporters of A. platensis. It has been shown that the expression of USP is enhanced when the bacterial cell is exposed to stress agents [35]. TrkA is a constituent of 6 K + uptake systems and is peripherally bound to the inner side of the cytoplasmic membrane [32].
The presence of all stress related domains within the seven antiporters indicates the possible role of these antiporters in the alkaline stress tolerance mechanism.    antiporters, Asn158 was replaced with non-polar residues. Interestingly, even the residues (Ile151, Phe355 and Gly359) whose structural rearrangement is responsible for blocking of ion transportation [41]  and O6 is replaced with polar Thr side chain. In case of O6 antiporter the functionally important Asp157 is replaced with polar Asn. The sequence analysis also shows occurrence of a considerable amount of acidic and basic sidechains (13% to 16%) which is also observed as a characteristic feature of CPA2 family protein.

Transcription profile of seven putative Na + /H + antiporters of A. platensis NIES-39
Many studies have reported the positive correlation of the Na + /H + antiporter involvement during alkaline stress tolerance mechanism. A similar role of the antiporters could be related in our cyanobacteria. Transcription profile based upon the Real-Time PCR for these seven putative Na + /H + antiporters showed a differential temporal expression pattern with a significant enhancement at pH 11 which was recorded as-A1 showed 40-fold; C5, 11-fold; D5, 7-fold; L2, 10-fold; L6, 49-fold; O6, 11-fold; Q2, 14-fold increase (figure 1a-g). After 1 hour of incubation D5 showed a significant expression while antiporters A1, C5, L6, O6, and Q2 were expressed at the 4 th hour of incubation. Antiporters L2 and L6 showed their significant expression on the 6 th 11 hour of incubation. Exceptionally, L2 showed 14-fold higher expression at pH 7 as compared with the other antiporters. A differential expression was found for different antiporters at pH 7, 9 and 11. We have also observed that the expression of these antiporters under pH stress is temporally regulated which throw light on the actual importance of the seven antiporters in alkaline stress tolerance mechanism.
As seen from our results antiporters, A1 and L6 showed higher level of expression upon subjecting the cyanobacteria to pH 11 as compared to pH 9. Our In-silico analysis shows that A1 houses NhaP, Na + /H + Exchanger, USP like, Usp A domains and antiporters L6 houses NhaP P, TrkA-N, TrkA domains. Interestingly, only the antiporter, A1 shows the unique UspA domain among the 177 Na + /H + antiporters studied from 37 sequenced and annotated cyanobacteria (data not given and to be communicated soon) using various Bio-Informatics tools and databases.
Many reports [35,21] suggest categorically role of UspA in stresses like metabolic, oxidative and temperature. UspA is also found to be associated with pH stress, where its expression is upregulated under alkaline condition in E. coli K-12 [42]. Our experiments show that subjecting the cyanobacteria to pH 7 lead to 14-fold higher expressions of the antiporter L2, housing NhaP and Na + /H + Exchanger domains ( Figure 1d). However, this result could suggest a possible dual role of the antiporter in pH regulation (acidification and alkalization). Also these three antiporters belong to CPA1 superfamily, which suggest an active role played by CPA1 in A.
platensis as compared to CPA2 superfamily.