Identification of glycine betaine as compatible solute in Synechococcus sp. WH8102 and characterization of its N-methyltransferase genes involved in betaine synthesis.
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 identified 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 purified by Ni-NTA His x bind resins. Their substrate specificities 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 confirmed the pathway of de novo synthesizing betaine from glycine existing in these marine cyanobacteria.
Lu
Wei-Dong
WD
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. luweidong401@hotmail.com
Chi
Zhen-Ming
ZM
Su
Chuan-Dong
CD
D000595:Amino Acid Sequence; D001622:Betaine / Q000378:metabolism*; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology; D050938:Glycine N-Methyltransferase / Q000235:genetics* / Q000378:metabolism; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009994:Osmolar Concentration; D010802:Phylogeny; D011994:Recombinant Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D013379:Substrate Specificity; D046940:Synechococcus / Q000378:metabolism*
D003470:Culture Media; D011994:Recombinant Proteins; D001622:Betaine; D008780:Methyltransferases; D050938:Glycine N-Methyltransferase