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J Plant Mol Biol Biotechnol 2010 1 (1): 21-23

Journal of Plant Molecular Biology & Biotechnology


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Short Communication

Molecular Cloning and Sequencing of GlucosylGlycerol Biosynthesis Genes in Marine Cyanobacteria, Synechocystis spp.
Lawrance ANBU RAJAN*, Gopinathan SREEJA, Yeragudipati RAJALAKSHMI, Vetrivel UMASHANKAR
School of Biosciences, Department of Bioinformatics, SRM University, Ramapuram, Chennai, Tamil Nadu, INDIA. Received: 11.09.2010 Accepted: 23.09.2010 Published: 23.09.2010

Abstract The cyanobacterium Synechocystis spp. was known to adjust various levels of salinity ranging from freshwater to twice the seawater salt concentrations by accumulating the compatible solute glucosylglycerol. In this study, glucosyl glycerol phosphate synthase (GgpS) and salt tolerant proteinA (stpA) genes encoding for glucosylglycerol biosynthesis were PCR amplified from genomic DNA of Synechocystis spp. The amplified genes were cloned and nucleotide sequences were determined. The sequencing results showed that GgpS and stpA genes contain ORF of 1500 bp and 1269 bp long, encoding 499 and 422 amino acids respectively (GenBank accession nos. FJ823262 and FJ895835).
Keywords: glucosylglycerol phosphate, glucosylglycerol phosphate synthase, sequencing, vector. *Corresponding Author: L. Anbu Rajan, E. mail: anburajanl@yahoo.co.in, Phone: +91-96795 50065

INTRODUCTION

Cyanobacteria are phototrophic prokaryotes which has successfully evolved mechanisms for salt adaptation. The best characterized cyanobacteria in relation to salt and osmotic stress response is the moderately halotolerant cyanobacterium Synechocystis sp. strain PCC 6803 (Kay et al. 1998). It is able to acclimate to high concentrations of NaCl up to 1.2 M by the de novo synthesis of the compatible solute glucosylglycerol (GG) (Fulda et al. 1989). The synthesis or uptake of compatible solute by Synechocystis spp. is to stabilize the osmotic potential of the cytoplasm and to maintain the structure of complex proteins at enhanced concentrations of ions (Reed et al. 1985). Glucosylglycerol is synthesized from ADP-glucose and glycerol-3-phosphate via the intermediate glucosylglycerol phosphate (GGP) with the cooperation of glucosylglycerol phosphate synthase (GgpS) and Glucosylglycerol phosphate phosphatase (stpA). In this study, the (GgpS) and (stpA) genes, responsible for the biosynthesis of glucosylglycerol was cloned and sequenced from marine cyanobacteria, Synechocystis spp.

MATERIALS AND METHODS Cultivation and Isolation of Genomic DNA Cyanobacteria (Synechocystis spp.) isolated from marine source was grown aerobically in BG-11 medium by maintaining the culture at 25C with a photosynthetic photon flux density of approximately 5 mol m-2 sec-1 and a light regime of 12 h light/12 h dark. The genomic DNA was isolated from the culture as described by (Xiaoqiang et al. 2000). Polymerase Chain Reaction The Glucosylglycerol biosynthesis genes, GgpS and stpA were amplified by using gene specific primers. The PCR reaction was done with the final volume of 50 L that contained: 0.5 M each of forward and reverse primers, (GgpSF: 5-GGAAGATCTATGAATTCATCCCTTGTGATC-3;
GgpSR: 5-CGGGGTACCCCTAAACTCTAACTTTGG-3; stpAF:5-GGCCGCGGATCCATGGTATTACACCAACAACGT-3; stpAR: CCGGAATTCCTACTGGGAAAAATGGACTCTTCGGCG-3)

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J Plant Mol Biol Biotechnol 2010 1 (1): 21-23


1.0 L of genomic DNA, 200 M of dNTPs, 1X Taq buffer, 2.5 mM MgCl2, 1U Taq DNA polymerase (MBI Fermentas, Hanover, Maryland, USA) and autoclaved Millipore water. The PCR was performed using a Master cycler (Eppendorf, Germany) with the following conditions; initial denaturation at 94C for 3 min, followed by 30 repeated cycles of 94C for 30 sec, 50C for 1 min and 72C for 2 min and final extension at 72C for 5 min. The PCR amplified product was analyzed on 1% agarose gel along with DNA ladder (MBI Fermentas) and documented using a gel documentation system (Alpha Imager 1220, Alpha Innotech Corporation, San Leandro, CA, USA). Cloning and Sequencing of PCR Amplicons The amplified DNA fragments of GgpS and stpA genes were gel eluted by Perfectprep Gel Cleanup Kit (Eppendorf). The purified gene amplicons were ligated into the cloning vector pTZ57R/T (MBI Fermentas). The ligated gene constructs were transformed into E. coli JM109 strain and plated on LB agar containing ampicillin (100 g/mL), IPTG (50 M) and X-gal (80 g/mL). The plates were incubated at 37C and the transformants were selected and inoculated in 5 mL LB broth containing ampicillin. The recombinant plasmids were isolated from the overnight culture by alkaline lysis method (Sambrook and Russell 2001). The recombinant plasmids were characterized for the presence of inserts by double digestion with BglII & KpnI for GgpS and BamHI & EcoRI for stpA respectively. The characterized plasmids were later on sequenced using M13 forward primer in ABI PRISM 377 genetic analyzer (Applied Biosystems, Perkin Elmer Co., Foster City, CA, USA). RESULTS The GgpS and stpA genes encode the glucosylglycerol phosphate synthase and glucosylglycerol phosphate phosphatase respectively. Together these proteins constitute the glucosylglycerol biosynthesis pathway. The GgpS and stpA genes were PCR amplified and is encoded by polynucleotides of 1500 bp and 1269 bp respectively (Figure 1).

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masses of 56 kDa and 46 kDa, based on the in silico estimates (Figure 3 & 4). The amino acid analysis also revealed that the GgpS and stpA genes encoded protein belongs to the family of glycosyl transferase and glucosyl glycerol phosphate phosphatase respectively.

Figure 3 Translated sequence of GgpS gene

Figure 1 Agarose gel electrophoresis of PCR amplicons of stpA and GgpS of Synechocystis: Lane a, 1kb DNA ladder; Lane b, stpA amplicon (1269 bp); Lane c, GgpS amplicon (1500 bp).

The GgpS and stpA encodes proteins of 499 and 422 amino acids respectively with the calculated molecular

Figure 4 Translated sequence of stpA gene

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J Plant Mol Biol Biotechnol 2010 1 (1): 21-23


After PCR amplification, the amplicons were purified from the agarose gel and ligated into pTZ57R/T. The recombinant transformants with GgpS and stpA genes were also confirmed by double digestion with restriction enzymes (Figure 2). The nucleotide sequence of inserts GgpS and stpA genes were submitted to GenBank and have been given accession nos. FJ823262 and FJ895835. ACKNOWLEDGEMENTS

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Authors are grateful to the Pro-Vice Chancellor, SRM University, Ramapuram, Chennai for providing the necessary facilities to carry out this research work. REFERENCES
Fulda S, Hagemann M, Libbert E, 1989. Release of glucosylglycerol from the cyanobacterium Synechocystis sp. SAG 92.79 by hypo-osmotic shock Archives of Microbiology 153: 405408 Kay M, Ellen Z, Thomas K, Anja K, Martin H, 1998. The GgpS gene from Synechocystis sp. strain PCC 6803 encoding glucosylglycerol phosphate synthase is involved in osmolyte synthesis. Journal of Bacteriology 180: 48434849 Mikkat S, Effmert U, Martin H, 1997. Uptake and use of the osmoprotective compounds trehalose, glucosylglycerol, and sucrose by the cyanobacterium Synechocystis sp. PCC6803. Archives of Microbiology 167: 112118

Figure 2 Agarose gel electrophoresis of digested clones in plasmid pTZ57R/T (2886 bp): Lane a, GgpS gene (1500 bp); Lane b, 1kb DNA ladder; Lane c, stpA gene (1269 bp).

Pfenning N, 1978. General physiology and ecology of photosynthetic bacteria. In Clayton RK, Sistrom WR, Eds, The Photosynthetic bacteria. Plenum Press, New York. 318 Reed RH, Stewart WDP, 1985. Osmotic adjustment and organic solute accumulation in unicellular cyanobacteria from freshwater and marine habitats. Marine Biology 88: 19 Sambrook J, Russell DW, 2001. Molecular cloning: A laboratory manual, 3rd ed. New York: Cold spring harbor laboratory press. Xiaoqiang W, Aliza Z, Sammy B, 2000. A simplified protocol for preparing DNA from filamentous cyanobacteria. Plant Molecular Biology Reporter 18: 385392

DISCUSSION Cyanobacteria are prokaryotic microorganisms that perform oxygenic photosynthesis (Pfenning 1978). In cyanobacteria, glucosylglycerol and trehalose were accumulated in high levels to protect the cells against the deleterious effects of salt stress (Mikkat et al. 1997). The biosynthetic pathway of glucosylglycerol initiates from the precursor ADP-glucose and glycerol-3-phosphate, utilizing glucosylglycerol phosphate synthase and glucosylglycerol phosphate phosphatase as intermediate. As of now, only minimum reports on the characterization of GgpS and stpA genes have been reported. As the first step towards the molecular characterization of glucosylglycerol phosphate synthase and glucosylglycerol phosphate phosphatase, in this study we cloned and analyzed the GgpS and stpA genes from marine cyanobacteria, Synechocystis spp. In conclusion, based on molecular analysis, the enzyme encoded by GgpS and stpA in Synechocystis belongs to glycosyl transferase and glucosylglycerol phosphate phosphatase family respectively. To our knowledge, this study represents the first instance in which GgpS and stpA genes from marine cyanobacteria has been cloned and characterized in detail. The heterologous expression of glucosylglycerol biosynthesis genes will certainly pave the way to overcome the demand of this osmolyte in modern biotechnological as well as pharmacological applications.

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