Beta-1,4-glucanase-like protein from the cyanobacterium Synechocystis PCC6803 is a beta-1,3-1,4-glucanase and functions in salt stress tolerance.
In the present study, we characterized the gene (Cyanobase accession number slr0897) designated Ssglc encoding a beta-1,4-glucanase-like protein (SsGlc) from Synechocystis PCC6803. The deduced amino acid sequence for Ssglc showed a high degree of similarity to sequences of GH (glycoside hydrolase) family 9 beta-1,4-glucanases (cellulases) from various sources. Surprisingly, the recombinant protein obtained from the Escherichia coli expression system was able to hydrolyse barley beta-glucan and lichenan (beta-1,3-1,4-glucan), but not cellulose (beta-1,4-glucan), curdlan (beta-1,3-glucan), or laminarin (beta-1,3-1,6-glucan). A 1H-NMR analysis of the enzymatic products revealed that the enzyme hydrolyses the beta-1,4-glycosidic linkage of barley beta-glucan through an inverting mechanism. The data indicated that SsGlc was a novel type of GH9 glucanase which could specifically hydrolyse the beta-1,3-1,4-linkage of glucan. The growth of mutant Synechocystis cells in which the Ssglc gene was disrupted by a kanamycin-resistance cartridge gene was almost the same as that of the wild-type cells under continuous light (40 micromol of photons/m2 per s), a 12 h light (40 micromol of photons/m2 per s)/12 h dark cycle, cold stress (4 degrees C), and high light stress (200 micromol of photons/m2 per s). However, under salt stress (300-450 mM NaCl), growth of the Ssglc-disrupted mutant cells was significantly inhibited as compared with that of the wild-type cells. The Ssglc-disrupted mutant cells showed a decreased rate of O2 consumption and NaHCO3-dependent O2 evolution as compared with the wild-type cells under salt stress. Under osmotic stress (100-400 mM sorbitol), there was no difference in growth between the wild-type and the Ssglc-disrupted mutant cells. These results suggest that SsGlc functions in salt stress tolerance in Synechocystis PCC6803.
Tamoi
Masahiro
M
Department of Advanced Bioscience, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan.
Kurotaki
Hideki
H
Fukamizo
Tamo
T
D001426:Bacterial Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018390:Gene Targeting; D005936:Glucans / Q000737:chemistry / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D001467:Hordeum / Q000737:chemistry; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009997:Osmotic Pressure; D010100:Oxygen / Q000378:metabolism; D010788:Photosynthesis / Q000502:physiology; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012492:Salts / Q000737:chemistry / Q000378:metabolism*; D017693:Sodium Bicarbonate / Q000378:metabolism; D013379:Substrate Specificity; D046939:Synechocystis / Q000201:enzymology* / Q000502:physiology; D047071:beta-Glucans / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D005936:Glucans; D011994:Recombinant Proteins; D012492:Salts; D047071:beta-Glucans; D017693:Sodium Bicarbonate; D006026:Glycoside Hydrolases; C015968:lichenin; D010100:Oxygen