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Comparative Analysis Of Heavy-Metal Ion Sensing Mechanisms With Transcription Factors, Smtbs, From Freshwater Synechococcus Sp. PCC 7942, And Marine Synechococcus Sp. PCC 7002: Evolutionary And Structural Aspects Rahul Mahadev Shelake, Kanako Aibara, Hidenori Hayashi, Shunnosuke Abe, Eugene Hayato Morita Abstract: Metal-responsive transcription factors play a crucial role in the metal ion homeostasis with both cellular metabolism and environmental metal availability. In present work, we studied structural and evolutionary features of a transcription repressor, SmtB from freshwater Synechococcus sp. PCC 7942 and its homologs from other cyanobacteria in detail. We mined putative SmtB-like zinc sensors from all available sequenced genomes of cyanobacterial species in CyanoBase database. We found marine Synechococcus sp. PCC 7002 consists of similar homologous gene regulation system to that of PCC 7942. In both cyanobacteria, the gene expression of SmtA (metallothionein) is regulated by repressor protein SmtB and they are transcribed divergently. We compared the binding affinities of SmtBs to the operator/promoter regions of smtA and inhibitory effect of Zn2+ with electrophoretic mobility shift assay. From these analyses, we found number of complexes between recognition DNA sequences and repressor proteins depends on the structural aspects of DNA sequences of operator/promoter region. We also found that the α5 sites are completely identical in these two proteins, but differences in α3N sites and/or operator/promoter region might be responsible for dissociation of protein-DNA complexes at different zinc ion concentrations (16 µM of Zn2+ in PCC 7942 and 256 µM of Zn2+ in case of PCC 7002). Index Terms: Cyanobacteria, electrophoretic mobility shift assay, heavy metal ion sensing, phylogenetic analysis, SmtB/ArsR family. ————————————————————

1 INTRODUCTION THE metal ions are indispensible in various cellular and physiological processes. The requirement of specific metal ion (iron, manganese, zinc or cobalt) in each proteome differs between each kingdom [1]. The availability of necessary concentration of each metal is vital to survival and higher bioavailable concentrations in the environment can be lethal for physiological processes. At least 2.75 billion years ago, ability to carry out oxygenic photosynthesis was developed in cyanobacteria [2]. _______________________  Rahul M. Shelake is currently pursuing doctoral degree program in Molecular Cell Physiology Laboratory, Department of Applied Bioresources, Faculty of Agriculture, Ehime University, Matsuyama, Japan (rahultnau@gmail.com)  K. Aibara, affiliated to Department of Chemistry, Faculty of Science, Ehime University, Matsuyama, Japan (anaconda@f4.dion.ne.jp)  Dr H. Hayashi is professor at Department of Chemistry, Faculty of Science, Ehime University, Matsuyama, Japan. He is also affiliated with CSTRC and Venture Business Laboratory, Ehime University, Matsuyama, Japan (hayashi.hidenori.mj@ehime-u.ac.jp)  Dr S. Abe is professor at Molecular Cell Physiology Laboratory, Department of Applied Bioresources, Faculty of Agriculture, Ehime University, Matsuyama, Japan (abe@mcb.agr.ehime-u.ac.jp)  Dr E. H. Morita is Associate professor at Molecular Cell Physiology Laboratory, Department of Applied Bioresources, Faculty of Agriculture, Ehime University, Matsuyama, Japan; and also affiliated to CSTRC and Venture Business Laboratory, Ehime University, Matsuyama, Japan (Corresponding author: morita.hayato.mu@ehime-u.ac.jp)

The molecular oxygen (released as byproduct of the process) had large influence on biosphere and increased bio-availability of metal ions by oxidation. Numerous biochemical processes developed to deal with increased bio-available concentrations of metal ions. Hence cyanobacteria might have developed capability of acquiring specific trance metal ions from their environment [3]. Unsurprisingly, recent genome sequencing data shows many metal ions homeostatic machineries have been evolved in cyanobacteria. Metal-responsive transcription factors are the key elements contributing for the metal ion homeostasis with both cellular metabolism and environmental metal availability [1], [4], [5]. SmtB (now onwards 7942-SmtB) from freshwater cyanobacterium Synechococcus sp. PCC 7942 is a founding member of the ArsR/SmtB family of prokaryotic metalloregulatory transcriptional repressors that regulates the expression of the smt operon, which plays a role in preferably Zn2+ and also Cd2+ responses [6], [7]. It consists of two divergently transcribed genes, smtA (metallothionein, 56 a.a.) and smtB (repressor of smtA transcription, 122 a.a.) [8]. In absence of excess heavy-metal ions, SmtB binds to operator/promoter region of smtA repressing its transcription. The smtA transcription is stimulated by heavy-metal ions, and this is thought to be due to the inhibition of complex formation between recognition DNA sequences and SmtB, following heavy-metal ion binding with SmtB [7]. The mechanism of heavy metal ion sensing with SmtB is well studied in freshwater species but knowledge about zinc ion homeostasis in marine cyanobacteria is limited till date [9]. From 39 sequenced cyanobacterial genomes available in the CyanoBase, 29 of them are marine or salt-tolerant species. This provides an excellent data set for comparative genomics and provides a starting point for understanding how habitat, nutrient requirements, and the presence/absence of metal ion sensing mechanisms are correlated. In present work, we identified putative 7942-SmtB related transcription regulators among the completed cyanobacterial genomes available in 274

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CyanoBase database. The occurrence of SmtB-like zinc sensors, structural similarities/differences and evolutionary relationship among the thirty-nine cyanobacterial strains with different habitats were compared and analyzed in detail. We found SmtB from marine species Synechococcus sp. PCC 7002 (along with one more marine species) is exceptionally showing more sequence similarity with freshwater 7942-SmtB. Marine Synechococcus sp. PCC 7002 consists of similar homologous gene regulation system to that of freshwater Synechococcus sp. PCC 7942. It (formerly Agmenellum quadruplicatum) is used as ideal model systems for functional, structural and applied studies in bioengineering [10]. The gene expression of SmtA (metallothioneins) is regulated by repressor protein SmtB, in both cyanobacteria. Deletion of smt locus significantly reduces the tolerance of Synechococcus sp. PCC 7942 for Zn2+/Cd2+ [32]. Response of repressors to Zn2+ ions is specifically studied in Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803 [11] but very little is known about the physiochemical aspects of transcription repressor SmtB from PCC 7002 (now onwards 7002-SmtB). Tolerance levels to zinc ion stress are completely different in PCC 7942 and PCC 7002. In case of PCC 7942, Zn2+ of 10 µM affects the growth severely and 100 µM completely inhibits the growth. However, even 100 µM concentration of Zn2+ does not affect the growth of strain PCC 7002. These could be the interesting factors for getting more insights into the transcription repression and metal ion sensing. In order to understand in depth mechanism of metal ion sensing, we successfully amplified smtB homologs and compared encoding protein sequences and operator/promoter regions (abbreviated as o/p-7942 and o/p-7002) of smtA from these two species. We found differences in these two proteins (specifically in metal-binding sites, flexible N-terminal arm) and operator/promoter regions (presence of extra direct repeat in only o/p-7942). To elucidate the structural and functional meaning of the differences found in these systems responsible for sensing excess Zn2+, we analyzed binding affinities of recombinant SmtBs to respective operator/promoter regions with electrophoretic mobility shift assay (EMSA) technique. We found number of different sized protein-DNA complexes formed in these two repressors is not the same (in case of PCC 7942: four; and PCC 7002: five). The α5 sites are completely identical in these two proteins, but differences in α3N sites might be responsible for dissociation of protein-DNA complex at different metal ion concentrations (16 µM of Zn2+ in 7942-SmtB and 256 µM of Zn2+ in case of 7002-SmtB). This bioinformatic analysis and EMSA studies suggests that marine cyanobacterial species might have been either lost smtB gene, or whether those containing it have acquired during their evolution process.

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2.2 Sequence alignments and phylogenetic tree Multiple sequence alignment was done using the ClustalX 1.83 software [12] and adjusted manually. Phylogenetic tree was constructed with the NCBI BLASTP 2.2.28+ online program [13]. Algorithm used to create a tree from given distances (or dissimilarities) between sequences by means of Fast Minimum Evolution method [14]. Sequence alignment and further analysis was done using SmtB-7942 as reference in all habitats because it is well characterized protein with known structural features. 2.3 Chemicals and plasmids Buffers were prepared with ultrapure water (MilliQ, Millipore). Enzymes and Escherichia coli expression host (JM 109 and BL21(DE3)/pLysS) were purchased from Novagen (Japan). Plasmid purification kits were obtained from Roche Applied Sciences (Germany). All other reagents were purchased from nacalai tesque, if not stated otherwise, and were of analytical grade or better. 2.4 Cloning and protein overexpression The 7942-smtB and 7002-smtB genes were cloned in pET-21d (+) plasmid vectors at the sites of Nco1 and Xho1. Confirmed plasmids were transformed in E. coli JM 109 cells. The gene constructs of cloned plasmid were further confirmed by nucleotide sequencing (Applied Biosystems Gene Sequencer) with the use of BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). For protein expression, plasmids were further transformed and overexpressed in E. coli strain BL21(DE3)/pLysS cells harboring pET-21d (+) plasmids. The plasmids were constructed to produce recombinant proteins without any change in original amino acid sequences of native proteins. Cells were pre-cultured in 2 mL of LB (Luria-Bertani) broth with 100 mg/mL ampicillin from glycerol stock and incubated at 37 °C for 8 hrs on horizontal shaker (speed 180 rpm). Further, 1 mL from grown pre-culture was sub-cultured in 100 mL of LB broth with ampilcillin (100 μg/mL of final concentration) for 12 hrs with rotational shaking (speed 100 rpm) in conical flasks. The 12 hrs grown cells were inoculated in a baffled flask at 30 °C at a rotating speed of 160 rpm. Protein overexpression was induced by the addition of 1.0 mM isopropyl-β-D-thiogalactopyranoside (IPTG) when OD660 (Optical density) value of the culture reached to 0.4-0.6. There was less expression of recombinant proteins with regular protocol. To improve recombinant protein expression efficiency rifampicin was added to the bacterial culture as early as 30 minutes after the IPTG induction. Rifampicin concentration was standardized [15] [27] for higher level of recombinant protein expression but with lower quantity of intrinsic proteins in final crude extract. Cells were harvested after 4 hrs (of IPTG induction) by centrifugation at 6,000 g for 5 min.

2 MATERIALS AND METHODS 2.1 Identification of SmtB homologs and chromosomal localization Sequences of the putative 7942-SmtB homologs were identified employing BLAST searches from genome of various cyanobacterial species available at CyanoBase website (http://genome.microbedb.jp/cyanobase/) of Kazusa genome resources. The chromosomal localization of all of the genes encoding annotated SmtB related sensors from all cyanobacterial species was determined with genome sequences available at CyanoBase database.

2.5 Purification and quantification of recombinant proteins The entire procedure for purifying recombinant proteins from E. coli is outlined in Fig. 1. SDS-PAGE analysis was done using Atto Corporation, Japan with 15 % gels in a Tris-glycine buffer system, which allows all fragments to be resolved on the same gel. Bacterial cell pellets were resuspended in lysis buffer (4 mL/g of pellet) consisting 50 mM Tris-HCl (pH 8.0), 300 mM KCl and 1 mM EDTA. Further 7 mM βmercaptoethanol and 10 mM p-APMSF (4Amidinophenylmethanesulfonyl fluoride hydrochloride) were 275

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added to it. Recombinant proteins were obtained as soluble form in supernatant after disruption of cells by sonication (3 min x 7 times) on TOMY Ultrasonic Disruptor UD-201 (parameters used, output: 4, duty: 60). Soluble materials containing overexpressed proteins were purified with 0.5 % PEI (polyethylene glycol) treatment and then precipitated from supernatant with ammonium sulphate (65 %) treatment. Precipitated proteins were dialyzed overnight against low salt buffer (25 mM K2HPO4, 25 mM KH2PO4, 50 mM KCl, 1 mM EDTA, and 0.5 mM DTT). Majority of unwanted protein was removed by ion-exchange chromatography with a HiTrap SP HP column 1 mL (GE Healthcare). Adsorbed proteins were eluted with KCl concentration gradient, from 50 mM to 1.0 M, and then further purified with size-exclusion chromatography on Superdex 75 pg (GE Healthcare). Concentrations of purified proteins were determined by measuring the absorbance at 280 nm (A280). Extinction coefficient was estimated using Lambert-Beer equation. A Spectrophotometer (Beckman, DU® 640) was first balanced with low buffer and then used for absorbance measurements. The protein concentration was estimated with the above calculated extinction coefficients.

Fig.1. Various steps followed for purification of 2.6 EMSA analysis recombinant SmtB. For the EMSA experiments, DIG Gel Shift Kit 2nd generation (Roche Applied Science) was used. The operator/promoter region of smtA from Synechococcus sp. PCC 7942 (97 bp) and Synechococcus sp. PCC 7002 (100 bp) were used as a probe DNA in EMSA experiments and amplified by the PCR method with the genome DNA as the template. The amplified DNA fragments were labeled at the 3’-ends with digoxigenin and then used as probes. DNA binding was carried out at 25 °C for 15 min in a 20 µl reaction mixture comprising 20 mM HEPES-KOH, pH 7.6, 30 mM KCl, 10 mM (NH4)2SO4, 1.0 mM EDTA, 1.0 mM DTT, 50 ng µl–1 poly[d(I-C)], 5 ng µl–1 poly Llysine, 0.2 % Tween 20, 1.5 nM end-labeled respective probe sequences. The final concentration of SmtBs in DNA-binding experiments were adjusted in the range of 10–6 to 10–9 M. protein-DNA complexes were separated from the free DNA probe by 6 % PAGE in 0.25 X TBE running buffer. For analyses of Zn2+ inhibition, we used binding buffer without EDTA. The EDTA concentrations in protein stock solutions were 1.0 mM for the analyses in the absence of Zn2+, and 0.1

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mM for the analyses of Zn2+ inhibition, to store the proteins stably without any non-specific binding divalent cations. Protein-DNA complexes were separated from the free DNA probe by 8 % PAGE in 0.25 ×TBE running buffer (in the Zn2+ free condition) or in 0.25 ×TB running buffer (without EDTA for the Zn2+-binding conditions). Zn2+ was prepared as a 10mM Zn(CH3COO)2 stock solution (pH- 4.5). To examine the effects of the presence of metal ions on the complex formation between SmtBs and the corresponding probe DNA sequences, the stock solution of Zn2+ was added to the DNA binding solution, to give the final concentrations of 0 to 512 μM (pH 7.6).

3 RESULTS AND DISCUSSION 3.1 Comparative analysis of SmtB-like proteins from different habitats In order to determine the structural and evolutionary development of the SmtB in various cyanobacterial species, we initially mined all available sequenced genomes for homologs of 7942-SmtB protein. In previous studies, putative SmtA-like metallothioneins are discussed in detail [3]. Blindauer [3] also studied putative SmtB-like sensor proteins employing BLAST searches in NCBI but did not included some species for those genome sequencing was not completed in the year 2007-2008. There is massive data accessible in NCBI with same sequences deposited with different names and not yet classified or assigned to specific families. Thus, to identify SmtB-like proteins, we applied a selective approach and screened only Cyanobase database. CyanoBase provides an easy way of accessing the sequences and all-inclusive annotation data on the structures of the 39 cyanobacterial genomes. Mercuric acetate soak of the apo-SmtB crystallographic studies [16] shows that 7942-SmtB forms homodimeric structure with four metal-binding sites per dimer. Two symmetry-related inter-subunit sites (Fig. 2) were found to bridge the α5 helices (involving Asp104, His106, His117P, Glu120P), while two additional symmetry-related sites were observed near the α3 helices (composed of Cys14, His18, Cys61, Asp64 as α3N, and other possible site involving Cys61, Asp64, His97, H2O) [16, 17, 18]. Later studies also confirmed that the occurrence of two metal-binding sites on SmtB is not unique within the ArsR/SmtB family of metalloregulatory sensor proteins. However, site-directed mutagenesis studies showing only the α5 ligands are required for inducerresponsiveness in vivo and in vitro [18], [19], despite the α3N sites being pre-dominantly occupied in the purified protein [20], [21].

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TABLE 1 SMTB AND ITS HOMOLOGS IN SEQUENCED GENOMES OF CYANOBACTERIAL SPECIES

Fig.2. Structure of SmtB. A. Ribbon representation of the structure of homodimeric apo-SmtB determined crystallographically, created with Chimera program version 1.8, one monomer shaded blue and the other pink [16]. Each SmtB monomer contains a helix-turn-helix DNA binding domain of the winged helix family (3α-turn-αR). With the present knowledge about metal-binding, relative positions of four metal-binding sites (red) per dimer are shown on dimer structure. The N-terminal residues 1-24 were not observed crystallography studies and were added manually. B & C- Amino acid residues involved in metalbinding sites. Cys14 (circled in black) was not observed in crystal structure and it is present in N-terminal flexible arm may be playing crucial role in metal ion sensing. Previous studies suggested many marine SmtBs are expected to have different metal-binding aspects as compare to freshwater SmtB, and suggested that this divergence has influenced by the habitat of species [3] [9]. Therefore, in present work, according to habitat of the species, sequences of putative 7942-SmtB proteins (query coverage more than 60 % in BLAST analysis) were searched, aligned and compared. In some species we found more than one sequence, in such case sequences having highest similarity are used in present studies. Reference sequences of predicted transcripts, habitat, gene ID available in CyanoBase database, nucleotide size, number of amino acid residues, and chromosome location are summarized in Table 1. Following important features discovered from analysis are explained in detail.

3.1.2 N-terminal region There is noteworthy dissimilarity in N-terminal region of putative SmtBs in all studied cyanobacterial species irrespective of their habitat which might be the contributing factor for differences in sensitivity among various species. Previous studies about sensor protein CadC from ArsR/SmtB family also suggested that flexibility of the N-terminal arm is vital in forming inter-subunit α3N site and plays a regulatory role in preferential binding to larger, more thiophilic metal ions [18], [21], [22], [23].

3.1.1 SmtB occurrence Out of total 39 screened genomes available at CyanoBase, we found putative SmtB-like proteins in 7 freshwater; 22 marine (SmtB-related zinc sensors found in only 15 species); 4 hot spring; 3 sediment and rock; 3 terrestrial species (Table 1). This shows that SmtB-like proteins are found in many but not in all cyanobacteria indicating existence of other metalhandling mechanisms also suggested by Blindauer for cyanobacterial metallothioneins [3]. There may be other parallel mechanisms, either membrane-bound uptake or efflux pumps, or passive transporters such as diffusion facilitators, or proteins involved in sequestration of intracellular metal ion.

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Fig 3. Continue.

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Fig.3. Sequence alignment of putative SmtBs from (A) freshwater, (B) marine, (C) hot spring, (D) sediment and rock; (E) terrestrial species. Cyanobacterial SmtB-like proteins sequences derived from the genome project. Putative metalbinding legends corresponding to α3N and α5 sites in well characterized SmtB protein from Synechococcus sp PCC 7942 are highlighted in red and blue respectively. The variations (as compared to 7942-SmtB) in metal-binding ligands are highlighted in yellow. Green dotted line shows DNA-binding domain. In some species more than one sequence is found, in that case sequences showing higher percent identity are included. 279 IJSTR©2013 www.ijstr.org


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3.1.3 DNA-binding domain and metal-binding sites Amino acid residues (21 a.a.) occupied in helix-turn-helix motif (initially termed as metal-binding box) of SmtB are almost preserved in all considered cyanobacterial species, specifically in freshwater and terrestrial species, indicative of a common evolutionary origin for SmtB. The structural studies show that two distinct metal-binding sites are present in this family [16]. Some members of SmtB/ArsR family have only the α3/α3N site or only the α5/α5C site and some other consists of both metal-binding sites [5]. Interestingly, in present studies, we found that the metal-binding ligands of α3N or α5 or both the sites are highly conserved in specific habitat. Specifically, amino acid residues in α5 sites are completely conserved and those in α3N sites are partially conserved in SmtB compared from all freshwater species. Some putative SmtB-like proteins

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possess the α3/α3N site (most of the marine, sediment and rock growing species), others contain only the α5 site (partially conserved in hot spring species) and some others possess both the sites (freshwater and terrestrial species) (Fig. 3). Replacement of His97 with aromatic amino acid tryptophan (W) is the major change observed in marine species. Additionally, two marine cyanobacterial speciesSynechococcus sp. PCC 7002 and Acaryochloris marina MBIC11017 are the exceptions and shows conserved α5 sites. Previous studies show that only one site of metal-binding is indispensable for allosteric metalloregulation in vitro and metal sensing in vivo [17], [18], [24]. The occurrence of both metalbinding sites and its molecular mechanism of heavy metal ion sensing are not completely known.

Fig. 4. Phylogenetic tree of cyanobacterial putative SmtB-related zinc sensors, with reference to 7942-SmtB. The possible reason is the existence of both metal-binding sites in intact may improve the overall metal ion homeostasis in freshwater and terrestrial species because zinc is typically highly abundant in freshwater as compare to ocean [3].

3.2 Phylogenetic analysis The phylogenetic tree of all 32 species for putative SmtB homologs is presented in Fig. 4. Surprisingly, SmtB from studied hot spring species is showing widely distributed pattern and not showing close evolutionary origin for putative SmtBs. Freshwater species are closely related but not clustered. SmtB from Synechococcus sp. PCC 7942 and Synechococcus elongatus PCC 6301 are showing higher identity and closely related than any other species. Marine cyanobacterial species are clustered together with exception of two species- Synechococcus sp. PCC 7002 and Acaryochloris marina MBIC11017. These two species are showing similarities with freshwater and terrestrial species.

The coastal strains such as Synechococcus sp. PCC 7002 would certainly be exposed to fluctuating habitats by the addition of freshwater and nutrients from land, and tidal influences [25], [26], [27], [28]. Multiple sequence alignment shows SmtB from Synechococcus sp. PCC 7002 contain intact α5 sites as of 7942-SmtB (Fig. 3B). Additionally, phylogenetic analysis also suggests 7002-SmtB has a close evolutionary relationship with SmtB from freshwater species. To understand mechanism of Zn2+ ion sensing with SmtB in marine species, Synechococcus sp. PCC 7002, we cloned SmtB encoding gene and analyzed.

3.3 Analysis of 7942-SmtB and 7002-SmtB proteins The sequence of 7942-SmtB was compared with 7002-SmtB to understand the structural aspects of respective proteins in detail (Fig. 5). The transcription repressor 7002-SmtB shows in 64 % identity in comparison with 7942-SmtB. In particular, the amino acids of helix-turn-helix motif (15 a.a. of total 21 a.a.) 280

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highlighted by blue dotted line are highly conserved (71 %). In addition, metal-binding ligands of α5 site are identical in these species. The residues that are expected to be the ligands for Zn2+ are mostly but not completely conserved in α3N sites of 7002-SmtB and there is a major difference in N-terminal region, specifically 7942-SmtB contains extra hanging region in N-terminal flexible arm.

Fig.5. Comparison of the amino acid sequences between 7002-SmtB (108 a.a.) and 7942-SmtB (122 a.a.). Conserved amino acids are indicated by *, and helix-turnhelix motif is 2+highlighted by dotted blue line. Ligand residues for Zn are encompassed by pink boxes.

3.4 Comparative analysis of operator/promoter region All studied operons involved in metal ion sensing appear to be consists of either one or two imperfect 12-2-12 inverted repeats at the transcription initiation site of regulated gene [28]. The smt operon contains two imperfect, inverted 12-2-12 repeats of similar sequence termed ‘Bbs1’ and ‘Bbs2’ (underlined with black and green line respectively in Fig. 6) [29]. In addition, the sequence of each repeat is also conserved for other native metal ion sensors of the SmtB/ArsR family.).

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3.5 Cloning, transformation and protein expression In this study, we confirmed expression vectors, pET21d, which would overproduce a 7002-SmtB and 7942-SmtB in E. coli with no additional tags. DNA sequencing analysis of 7942smtB, 7002-smtB and its encoding proteins [7942- SmtB (122 a.a.), 7002-SmtB (108 a.a.) respectively] found to be identical with sequences available in NCBI (7942-SmtB: YP_170972.1; 7002-SmtB: YP_001735796.1). Recombinant proteins were successfully purified with the followed protocol. 3.6 Patterns of protein-DNA complex formation with EMSA As shown in Fig. 7, 1.5 nM probe DNA and SmtB (protein concentration from 10–6 to 10–9 M) forms four different sized protein-DNA complexes (C1, C2, C3, and C4) for 7942-SmtB with o/p-7942, and five different sized protein-DNA complexes (C5, C6, C7, C8, and C9) for 7002-SmtB with o/p-7002. In consideration of homology in both protein sequences and o/p regions, similar EMSA patterns are reasonable for 7002-SmtB and 7942-SmtB with o/p-7002 and o/p-7942 respectively. Furthermore, the differences in the number of complexes found in EMSA may be attributed to the presence of direct repeat sequence only in o/p-7942. In our previous studies [11], we indicated Cys14 from 7942-SmtB functions as ligand for metal ion in α3N sites and present results are in consistent and support the same. Additionally, these results also confirm that the number of different sized complexes between operator/promoter sequences and repressor proteins (SmtB homologs) depends on the structural aspects of DNA sequences of operator/promoter as suggested by Morita et al [11]. In our previous studies [29], [30], [31], we confirmed that Zn2+ specifically inhibits the protein-DNA complex formation between 7942-SmtB and operator/promoter region of smtA. The functional differences in α3N sites of 7942-SmtB, 7002SmtB (concentrations used 100 nM each) were further analyzed with EMSA technique by comparing inhibitory effect of Zn2+ (concentration from 0 to 512 µM), on protein-DNA complex formation with respective operator/promoter DNA sequences as probes. The obtained results are as shown in Fig. 8. In our experimental conditions, residual EDTA concentrations in reaction mixtures were less than 0.1 µM and its effects are considered almost negligible comparing with the Zn2+ concentrations. EMSA experiments were performed with 7942-SmtB, 7002-SmtB (100 nM each) and respective probe DNAs (1.5 nM each), in the presence of Zn2+ ranging from 0 to 512 µM. In the case of 7942-SmtB, most preferential complex of

Fig. 6. Structural comparisons of operator/promoter regions of smtA in Synechococcus sp. PCC 7002 and PCC 7942. The sequences of the operator/promoter regions of 7002-smtA (100 a.a.) and 7942-smtA (100 a.a.) are compared in Fig. 6. In o/p-7942 and o/p-7002, there are two palindromic sequences and distance between them is completely same (18 bp). However, only o/p-7942 contains extra direct repeat at downstream of second palindromic sequence.

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Fig.7. EMSA analysis of 7942-SmtB with o/p-7942, and 7002-SmtB with o/p-7002 respectively. In both the cases, probe DNA concentration is 1.5 nM and protein concentrations are 0 nM 1.0 nM, 2.2 nM, 4.6 nM, 10 nM, 22 nM, 46 nM, 100 nM, and 1.0 µM, from left to right.

Fig. 8. Inhibition of Protein-DNA complex formation between 7942-SmtB and 7002-SmtB with respective operator/promoter regions (o/p-7002 and o/p-7942) by Zinc ions. For EMSA, protein concentrations were 100 nM and respective probe DNA concentrations were 1.5 nM. The zinc ion concentration was used from left side onwards in each experiment is 0, 2, 4, 8, 16, 32, 64, 128, 256, and 512 µM.

protein-DNA dissociates at 16 µM or higher concentrations of Zn2+. But interestingly, in case of 7002-SmtB, most preferential protein-DNA complexes completely dissociates at 256 µM or higher concentrations of Zn2+. These results clearly show that zinc ion concentrations inducing inhibitory effect on proteinDNA complex formations for 7942-SmtB and 7002-SmtB are completely different. In the case of 7942-SmtB, the size of the most preferential protein-DNA complex is gradually becoming

smaller and then completely dissociated. But in the case of 7002-SmtB, most preferential protein-DNA complex is rapidly dissociated at threshold level of metal ion concentration (256µM). These results suggest that these two cyanobacterial species may have differences in the mechanism of regulation of gene expression. The ligand residues in α5 metal-binding sites (Fig. 3) in both the SmtBs are completely conserved, and those in α3N sites are homologous, but not conserved. On the 282

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basis of amino acid sequence comparisons, Cys61, Asp64, and His97 in 7942-SmtB correspond to Arg42, Glu45, and Asn58 in 7002-SmtB, respectively. With present analyses, ligand residue in 7002-SmtB corresponding to Cys14 of 7942SmtB may be the His4. The differences in the EMSA analyses between these two proteins (7942-SmtB and 7002-SmtB) with or without Zn2+, might be induced by structural differences in α3N sites, N-terminal flexible regions, and/or presence of extra direct repeat in only o/p-7942. Additionally, EMSA results for these two repressor proteins in absence/presence of Zn2+ also supports that freshwater Synechococcus sp. PCC 7942 and marine Synechococcus sp. PCC 7002 are closely related in terms of SmtB evolution. Marine cyanobacterial species might have been either lost smtB gene, or whether those containing it have acquired it during their evolution process. The marine species (Synechococcus sp. PCC 7002 and Acaryochloris marina MBIC11017) showing close evolutionary relationship with freshwater species might be served as connecting bridge in these two habitats during their evolutionary development.

REFERENCES

4 CONCLUSIONS

[5] L. S. Busenlehner, M. A. Pennella, and D. P. Giedroc, “The SmtB/ArsR family of metalloregulatory transcriptional repressors: Structural insights into prokaryotic metal resistance,” FEMS Microbiology Reviews, vol. 27, no. 2-3, pp. 131-143, 2003.

Bioinformatics analysis suggests that individual members of SmtB-related zinc sensors of cyanobacterial species from different habitat clearly derive from a common evolutionary origin and SmtB homologs are found in many but not in all cyanobacteria. Sequence alignment analysis shows that structural features of SmtB are highly conserved in specific habitat. We found, transcription regulator, SmtB from marine Synechococcus sp. PCC 7002 is closely related to homolog from freshwater Synechococcus sp. PCC 7942. EMSA analysis shows that minimum concentration to induce dissociation of protein-DNA complex in 7942-SmtB is completely different as compare to 7002-SmtB. Furthermore, patterns of most preferential protein-DNA complex dissociation for 7942-SmtB and 7002-SmtB are not similar. In case of 7942-SmtB, dissociation occurs slowly and it occurs immediately in the case of 7002-SmtB. These differences in Nterminal region of SmtB, (specifically ligand residues in α3N sites) and/or operator/promoter region of smtA among these two species are responsible for generating differences in protein-DNA complex formation and sensitivity to heavy metal ion sensing. EMSA and protein sequence analysis suggests that the ligand residue in 7002-SmtB corresponding to Cys14 of 7942-SmtB may be the His4. The knowledge of structural, functional and evolutionary aspects will contribute to our better understanding of important aspects about metal homeostasis mechanisms leading to develop new tools to enhance bioremediation technology and has been receiving more attention as an eco-friendly and efficient way of cleaning up toxic-metal contaminations.

ACKNOWLEDGMENT This research was partially supported by MEXT (Ministry of Education, Culture, Sports, Science & Technology in Japan) a Grant-in-Aid for scientific research (C), 22510234, 2010-2012 to E.H.M. and Doctoral fellowship to R.M.S. Authors are grateful to the staff of Integrated Center for Sciences, Tarumi, Ehime University for providing nucleotide sequencing facility.

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