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- Characterization of a cold-adapted debranching enzyme and its role in glycogen metabolism and virulence of Vibrio vulnificus MO6-24/O
- Ah-Reum Han , Haeyoung Kim , Jong-Tae Park , Jung-Wan Kim
- J. Microbiol. 2022;60(4):375-386. Published online February 14, 2022
- DOI: https://doi.org/10.1007/s12275-022-1507-3
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Abstract
- Vibrio vulnificus MO6-24/O has three genes annotated as debranching enzymes or pullulanase genes. Among them, the gene encoded by VVMO6_03032 (vvde1) shares a higher similarity at the amino acid sequence level to the glycogen debranching enzymes, AmyX of Bacillus subtilis (40.5%) and GlgX of Escherichia coli (55.5%), than those encoded by the other two genes. The vvde1 gene encoded a protein with a molecular mass of 75.56 kDa and purified Vvde1 efficiently hydrolyzed glycogen and pullulan to shorter chains of maltodextrin and maltotriose (G3), respectively. However, it hydrolyzed amylopectin and soluble starch far less efficiently, and β-cyclodextrin (β-CD) only rarely. The optimal pH and temperature of Vvde1 was 6.5 and 25°C, respectively. Vvde1 was a cold-adapted debranching enzyme with more than 60% residual activity at 5°C. It could maintain stability for 2 days at 25°C and 1 day at 35°C, but it destabilized drastically at 40°C. The Vvde1 activity was inhibited considerably by Cu2+, Hg2+, and Zn2+, while it was slightly enhanced by Co2+, Ca2+, Ni2+, and Fe2+. The vvde1 knock-out mutant accumulated more glycogen than the wild-type in media supplemented with 1.0% maltodextrin; however, the side chain length distribution of glycogen was similar to that of the wild-type except G3, which was much more abundant in the mutant. Therefore, Vvde1 seemed to debranch glycogen with the degree of polymerization 3 (DP3) as the specific target branch length. Virulence of the pathogen against Caenorhabditis elegans was attenuated significantly by the vvde1 mutation. These results suggest that Vvde1 might be a unique glycogen debranching enzyme that is involved in both glycogen utilization and shaping of glycogen molecules, and contributes toward virulence of the pathogen.
- Molecular Bases of High-Level Streptomycin Resistance in Pseudomonas marginalis and Pseudomonas syringae pv. actinidiae
- Hyo Shim Han^ , Hye Young Nam^ , Young Jin Koh^ , Jae-Seoun Hur^ , Jae Sung Jung^
- J. Microbiol. 2003;41(1):16-21.
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Abstract
- We have collected eight high-level streptomycin-resistant strains of Pseudomonas marginalis and P. syringae pv. actinidiae which were isolated from kiwifruit orchards in Korea and Japan. The molecular mechanisms of resistance were investigated by the PCR, susceptibility tests, and nucleotide sequence analysis. Of the eight high-level streptomycin-resistant strains, four harbored strA-strB genes, which encode streptomycin-inactivating enzymes. While the three Korean strains of P. marginalis did not have plasmid and carried the resistant genes in the chromosomes, the Japanese strain of P. syringae pv. actinidiae had a plasmid containing strA-strB genes. The myomycin susceptibility test demonstrated that the high-level resistance to streptomycin of the remaining four strains is associated with mutations in the rpsL gene. Nucleotide sequence analyses revealed that they contain a single base-pair mutation in codon 43 of their rpsL gene.
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