Identification of glycine betaine as compatible solute in Synechococcus sp. WH8102 and characterization of its N -methyl
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O RI G I NAL PAPE R
IdentiWcation of glycine betaine as compatible solute in Synechococcus sp. WH8102 and characterization of its N-methyltransferase genes involved in betaine synthesis Wei-Dong Lu · Zhen-Ming Chi · Chuan-Dong Su
Received: 13 March 2006 / Revised: 30 July 2006 / Accepted: 21 August 2006 / Published online: 22 September 2006 © Springer-Verlag 2006
Abstract Biosynthesis of glycine betaine from simple carbon sources as compatible solute is rare among aerobic heterotrophic eubacteria, and appears to be almost exclusive to the non-halophilic and slightly halophilic phototrophic cyanobacteria. Although Synechococcus sp. WH8102 (CCMP2370), a unicellular marine cyanobacterium, could grow up to additional 2.5% (w/v) NaCl in SN medium, natural abundance 13C nuclear magnetic resonance spectroscopy identiWed glycine betaine as its major compatible solute. Intracellular glycine betaine concentrations were dependent on the osmolarity of the growth medium over the range up to additional 2% NaCl in SN medium, increasing from 6.8 § 1.5 to 62.3 § 5.5 mg/g dw. The ORFs SYNW1914 and SYNW1913 from Synechococcus sp. WH8102 were found as the homologous genes coding for glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase, heterologously over-expressed respectively as soluble fraction in Escherichia coli BL21(DE3)pLysS and puriWed by Ni-NTA His•bind resins. Their substrate speciWcities and the values of the kinetic parameters were determined by TLC and 1H NMR spectroscopy. RT-PCR analysis revealed that the two ORFs were both transcribed in cells of Synechococcus sp. WH8102 growing in SN medium without additional NaCl, which
conWrmed the pathway of de novo synthesizing betaine from glycine existing in these marine cyanobacteria. Keywords Synechococcus sp. · WH8102 · Glycine betaine · Glycine sarcosine N-methyltransferase · Dimethylglycine N-methyltransferase Abbreviations GSDMT Glycine–sarcosine–dimethylglycine methyltransferase GSMT Glycine sarcosine methyltransferase SDMT Sarcosine–dimethylglycine methyltransferase DMT N,N-dimethylglycine methyltransferase GNMT Glycine N-methyltransferase PEAMT Phosphoethanolamine N-methyltransferase AhGSMT Aphanothece halophytica GSMT AhDMT Aphanothece halophytica DMT AdoMet S-Adenosylmethionine dw Dry weight ORF Open reading frame CV Column volumes
Introduction W.-D. Lu · Z.-M. Chi (&) · C.-D. Su UNESCO, the Chinese Center of Marine Biotechnology, College of Marine Life Sciences, Ocean University of China, QingDao, 266003, Shandong Province, People’s Republic of China e-mail: [email protected] W.-D. Lu e-mail: [email protected]
Cyanobacteria (oxygenic photosynthetic prokaryotes) are capable of growth and multiplication in widespread environments including those of widely diVering salinity. They cope with osmotic stress in the environment by accumulation of certain small organic compounds termed ‘compatible solutes’ intracellularly in order to maintain a positive cellular turgor (Ladas and
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Papageorgiou 2000). In earlier studies
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