Glycogen Synthase Isoforms in Synechocystis sp. PCC6803: Identification of Different Roles to Produce Glycogen by Targeted Mutagenesis.

Synechocystis sp. PCC6803 belongs to cyanobacteria which carry out photosynthesis and has recently become of interest due to the evolutionary link between bacteria and plant species. Similar to other bacteria, the primary carbohydrate storage source of Synechocystis sp. PCC6803 is glycogen. While most bacteria are not known to have any isoforms of glycogen synthase, analysis of the genomic DNA sequence of Synechocystis sp. PCC6803 predicts that this strain encodes two isoforms of glycogen synthase (GS) for synthesizing glycogen structure. To examine the functions of the putative GS genes, each gene (sll1393 or sll0945) was disrupted by double cross-over homologous recombination. Zymogram analysis of the two GS disruption mutants allowed the identification of a protein band corresponding to each GS isoform. Results showed that two GS isoforms (GSI and GSII) are present in Synechocystis sp. PCC6803, and both are involved in glycogen biosynthesis with different elongation properties: GSI is processive and GSII is distributive. Total GS activities in the mutant strains were not affected and were compensated by the remaining isoform. Analysis of the branch-structure of glycogen revealed that the sll1393− mutant (GSI−) produced glycogen containing more intermediate-length chains (DP 8–18) at the expense of shorter and longer chains compared with the wild-type strain. The sll0945− mutant (GSII−) produced glycogen similar to the wild-type, with only a slightly higher proportion of short chains (DP 4–11). The current study suggests that GS isoforms in Synechocystis sp. PCC6803 have different elongation specificities in the biosynthesis of glycogen, combined with ADP-glucose pyrophosphorylase and glycogen branching enzyme

Glycogen Synthase Isoforms in Synechocystis sp. PCC6803: Identification of Different Roles to Produce Glycogen by Targeted Mutagenesis

Synechocystis sp. PCC6803 belongs to cyanobacteria which carry out photosynthesis and has recently become of interest due to the evolutionary link between bacteria and plant species. Similar to other bacteria, the primary carbohydrate storage source of Synechocystis sp. PCC6803 is glycogen. While most bacteria are not known to have any isoforms of glycogen synthase, analysis of the genomic DNA sequence of Synechocystis sp. PCC6803 predicts that this strain encodes two isoforms of glycogen synthase (GS) for synthesizing glycogen structure. To examine the functions of the putative GS genes, each gene (sll1393 or sll0945) was disrupted by double cross-over homologous recombination. Zymogram analysis of the two GS disruption mutants allowed the identification of a protein band corresponding to each GS isoform. Results showed that two GS isoforms (GSI and GSII) are present in Synechocystis sp. PCC6803, and both are involved in glycogen biosynthesis with different elongation properties: GSI is processive and GSII is distributive. Total GS activities in the mutant strains were not affected and were compensated by the remaining isoform. Analysis of the branch-structure of glycogen revealed that the sll1393− mutant (GSI−) produced glycogen containing more intermediate-length chains (DP 8–18) at the expense of shorter and longer chains compared with the wild-type strain. The sll0945− mutant (GSII−) produced glycogen similar to the wild-type, with only a slightly higher proportion of short chains (DP 4–11). The current study suggests that GS isoforms in Synechocystis sp. PCC6803 have different elongation specificities in the biosynthesis of glycogen, combined with ADP-glucose pyrophosphorylase and glycogen branching enzyme.This is an article published as Yoo S-H, Lee B-H, Moon Y, Spalding MH, Jane J-l (2014) Glycogen Synthase Isoforms in Synechocystis sp. PCC6803: Identification of Different Roles to Produce Glycogen by Targeted Mutagenesis. PLoS ONE 9(3): e91524. doi:10.1371/journal.pone.0091524. Posted with permission.</p

Gene structure of <i>sll</i>1393 (A) and <i>sll</i>0945 (B) on <i>Synechocystis</i> sp. PCC 6803 genomic DNA.

<p>The targeted replacement of each GS gene with the Km^R gene was achieved by homologous recombination. (C) PCR analysis of <i>sll</i>1393 and <i>sll</i>0945 genes using genomic DNA from wild-type, <i>sll</i>1393⁻, and <i>sll</i>0945⁻ strains as templates. For comparison, PCR products of intact genes in wild-type were loaded next (left lane) to the corresponding mutated genes. Lane1 is the size marker (1 kb plus DNA ladder, Life Technologies). DNA bands on lane 2 (WT) and 3 (M1) were amplified using prA1 and prA4; Lane 4 (WT) and 5 (M2) were amplified using prB1 and prB4.</p

Figure 4

<p>A. Branch chain-length distribution of glycogens from WT (), M1 (▪), and M2 (□). Glycogen samples were treated with a debranching enzyme, isoamylase, and the resulting debranched-samples were separated on an HPAEC system. The peak area was calculated and normalized from the chain-profile chromatogram. B. The comparison of difference in the normalized peak area calculated from M1-WT (□), M2-WT (○), or M1-M2 (⋄).</p

Effects of <i>sll</i>1393 and <i>sll</i>0945 deletions on glycogen accumulation and structure, using commercial rabbit-liver glycogen and waxy maize starch for comparison.

a<p>Values given are means ± standard deviation obtained from at least three independent experiments.</p>b<p>Calculated based on peak area of each chain on HPAEC chromatograms.</p><p><i>Number</i>-average degree of polymerization = Σ peak area/Σ (peak area/number of glucose of each chain). Values given are means ± standard deviation obtained from at least three independent experiments.</p>c<p>Not Applicable.</p

Branch structure of glycogen and starch.

a<p>Values given were determined from at least two duplications.</p>b<p>ECL (exterior chain length) = CL (which is equal to DP_n in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091524#pone-0091524-t003" target="_blank">Table 3</a>)×β-amylolysis (%)+2.0.</p>c<p>The ratio was calculated based on the mole fraction of (G2+G3) stubs released, by isoamylase and pullulanase hydrolysis, from β-limit dextrin.</p

Photomixotrophic growth of WT (▵), M1 (□), M2 (○), and M12 (⋄) strains of <i>Synechocystis</i> sp. PCC6803.

<p>Cells were grown in a BG-11 medium containing 5 mM glucose and the number of cells were counted in the exponential phase. The same amount of the cells were inoculated in the new media. Each culture was inoculated with an equal number of exponential-phase cells, and growth of the cultures was monitored by measuring absorbance at 730 nm at various time intervals.</p

Oligonucleotide sequences used to construct 3 pSHK1393 and pSHK0945 plasmids.

a<p>Final constructs were used to replace endogenous <i>sll</i>1393 and <i>sll</i>0945 genes, respectively, by homologous recombination.</p><p>The position of the oligonucleotides in the nucleotide sequences of the <i>sll</i>1393 and <i>sll</i>0945 structural DNA are indicated. The restriction sites introduced by PCR amplification are underlined.</p