• Microbiology and Biotechnology Letters
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• v.46 no.1
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• pp.29-39
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• 2018

Vibrio vulnificus needs various responsive mechanisms to survive and transmit successfully in alternative niches of human and marine environments, and to ensure the acquisition of steady energy supply to facilitate such unique life style. The bacterium had genetic constitution very different from that of Escherichia coli regarding metabolism of glycogen, a major energy reserve. V. vulnificus accumulated more glycogen than other bacteria and at various levels according to culture medium and carbon source supplied in excess. Glycogen was accumulated to the highest level in Luria-Bertani (3.08 mg/mg protein) and heart infusion (4.30 mg/mg protein) complex media supplemented with 1% (w/v) maltodextrin at 3 h into the stationary phase. Regarding effect of carbon source, more glycogen was accumulated when maltodextrin (2.34 mg/mg protein) was added than when glucose or maltose (0.78.1-14 mg/mg protein) was added as an excessive carbon source to M9 minimal medium, suggesting that maltodextrin metabolism might affect glycogen metabolism very closely. These results were supported by the analysis using the malP (encoding a maltodextrin phosphorylase) and malQ (encoding a 4-${\alpha}$-glucanotransferase) mutants, which accumulated much less glycogen than wild type when either glucose or maltodextrin was supplied as an excessive carbon source, but at different levels (3.1-80.3% of wild type glycogen). Therefore, multiple pathways for glycogen metabolism were likely to function in V. vulnificus and that responding to maltodextrin might be more efficient in synthesizing glycogen. All of the glycogen samples from 3 V. vulnificus strains under various conditions showed a narrow side chain length distribution with short chains (G4-G6) as major ones. Not only the comparatively large accumulation volume but also the structure of glycogen in V. vulnificus, compared to other bacteria, may explain durability of the bacterium in external environment.

• Journal of Microbiology and Biotechnology
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• v.15 no.3
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• pp.616-625
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• 2005

It is likely that maltose could provide a good substrate for the bacteria in the intestine, when the pathogenic bacteria invade and colonize in human gut. For better understanding of this organism's maltose metabolism, a mutant that was not able to grow with maltose as a sole carbon source was screened from a library of mutants constructed by a random transposon mutagenesis. By a transposon-tagging method, malPQ genes encoding a maltodextrin phosphorylase and a 4-${\alpha}$-glucanotransferase, were identified and cloned from Vibrio vulnificus. The deduced amino acid sequences of malPQ from V. vulnificus were 48 to $91\%$ similar to those of MalP and MalQ reported from other Enterobacteriaceae. Functions of malPQ genes were assessed by the construction of mutants whose malPQ genes were inactivated by allelic exchanges. When maltose was used as the sole carbon source, neither malP nor malQ mutant was able to grow to a substantial level, revealing that the MalP and MalQ are the only enzymes for metabolic utilization of maltose. The malQ mutant exhibited decreased adherence toward intestinal epithelial cells in vitro, but there was no difference in the $LD_{50}s$ of the wild-type and the malQ mutant in mice. Therefore, it appears that MalQ is less important in the pathogenesis of V. vulnificus than would have been predicted by considering maltose as a most common sugar in the intestine, but not completely dispensable for virulence in mice.

• Journal of Microbiology and Biotechnology
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• v.29 no.3
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• pp.357-366
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• 2019

We first confirmed the involvement of MalQ (4-${\alpha}$-glucanotransferase) in Escherichia coli glycogen breakdown by both in vitro and in vivo assays. In vivo tests of the knock-out mutant, ${\Delta}malQ$, showed that glycogen slowly decreased after the stationary phase compared to the wild-type strain, indicating the involvement of MalQ in glycogen degradation. In vitro assays incubated glycogen-mimic substrate, branched cyclodextrin (maltotetraosyl-${\beta}$-CD: G4-${\beta}$-CD) and glycogen phosphorylase (GlgP)-limit dextrin with a set of variable combinations of E. coli enzymes, including GlgX (debranching enzyme), MalP (maltodextrin phosphorylase), GlgP and MalQ. In the absence of GlgP, the reaction of MalP, GlgX and MalQ on substrates produced glucose-1-P (glc-1-P) 3-fold faster than without MalQ. The results revealed that MalQ led to disproportionate G4 released from GlgP-limit dextrin to another acceptor, G4, which is phosphorylated by MalP. In contrast, in the absence of MalP, the reaction of GlgX, GlgP and MalQ resulted in a 1.6-fold increased production of glc-1-P than without MalQ. The result indicated that the G4-branch chains of GlgP-limit dextrin are released by GlgX hydrolysis, and then MalQ transfers the resultant G4 either to another branch chain or another G4 that can immediately be phosphorylated into glc-1-P by GlgP. Thus, we propose a model of two possible MalQ-involved pathways in glycogen degradation. The operon structure of MalP-defecting enterobacteria strongly supports the involvement of MalQ and GlgP as alternative pathways in glycogen degradation.

• Microbiology and Biotechnology Letters
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• v.44 no.3
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• pp.363-369
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• 2016

A putative cyclomaltodextrinase gene (licd) was found from the genome of Listeria innocua ATCC 33090. The licd gene is located in the gene cluster involved in maltose/maltodextrin utilization, which consists of various genes encoding maltose phosphorylase and sugar ABC transporters. The structural gene encodes 591 amino acids with a predicted molecular mass of 68.6 kDa, which shares less than 58% of amino acid sequence identity with other known CDase family enzymes. The licd gene was cloned, and the dimeric enzyme with C-terminal six-histidines was successfully produced and purified from recombinant Escherichia coli. The enzyme showed the highest activity at pH 7.0 and 37℃. licd could hydrolyze β-cyclodextrin, starch, and maltotriose to mainly maltose, and it cleaved pullulan to panose. It could also catalyze the hydrolysis of acarbose to glucose and acarviosine-glucose. In particular, it showed significantly higher activity towards β-cyclodextrin and maltotriose than towards starch and acarbose. licd also showed transglycosylation activity, producing α-(1,6)- and/or α-(1,3)-linked transfer products from the acarbose donor and α-methyl glucopyranoside acceptor.