Characterization of cyanobacterial glycogen isolated from the wild type and from a mutant lacking of branching enzyme.
Cyanobacteria produce glycogen as their primary form of carbohydrate storage. The genomic DNA sequence of Synechocystis sp. PCC6803 indicates that this strain encodes one glycogen-branching enzyme (GBE) and two isoforms of glycogen synthase (GS). To confirm the putative GBE and to demonstrate the presence of only one GBE gene, we generated a mutant lacking the putative GBE gene, sll0158, by replacing it with a kanamycin resistance gene through homologous recombination. GBE in sll0158(-) mutant was eliminated; the mutant strain produced less glucan, equivalent to 48% of that produced by the wild type. In contrast to the wild-type strain that had 74% of the glucan being water-soluble, the mutant had only 14% of the glucan water-soluble. Molecular structures of glucans produced by the mutant and the wild type were characterized by using high-performance size-exclusion and anion-exchange chromatography. The glycogen produced by the wild type displayed a molecular mass of 6.6 x 10(7) daltons (degree of polymerization (DP) 40700) and 10% branch linkages, and the alpha-D-glucan produced by the mutant displayed a molecular mass of 4.7-5.6 x 10(3) daltons (DP 29-35) with slight branch linkages. The results indicated that sll0158 was the major functional GBE gene in Synechocystis sp. PCC6803.
Yoo
Sang-Ho
SH
Department of Food Science and Human Nutrition, 2312 Food Science Building, Iowa State University, Ames, IA 50011, USA.
Spalding
Martin H
MH
Jane
Jay-lin
JL
D015061:1,4-alpha-Glucan Branching Enzyme / Q000235:genetics*; D002845:Chromatography; D000458:Cyanobacteria / Q000201:enzymology*; D006003:Glycogen / Q000096:biosynthesis / Q000737:chemistry*; D006006:Glycogen Synthase / Q000235:genetics; D008970:Molecular Weight; D009154:Mutation; D015202:Protein Engineering; D012995:Solubility
D006003:Glycogen; D006006:Glycogen Synthase; D015061:1,4-alpha-Glucan Branching Enzyme