• 한국축산식품학회지
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• 제30권6호
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• pp.946-950
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• 2010

This study evaluated the antimicrobial effects of meat processing-related organic acids on Burkholderia thailandensis (Burkholderia pseudomallei surrogate) with different water activities. B. thailandensis KACC12027 (4 log CFU/mL) was inoculated in microwell plates containing tryptic soy broth pH-adjusted to 4, 5, 6, and 7 with ascorbic acid, citric acid, and lactic acid and with water activities adjusted to 0.94, 0.96, 0.98, and 1.0 with NaCl, followed by incubation at $35^{\circ}C$ for 30 h. The optical density (OD) of the samples was measured at 0, 3, 6, 12, 24, and 30 h at 595 nm to estimate the growth of B. thailandensis. Growth of B. thailandensis was observed only at water activity of 1.0. In general, more bacterial growth (p<0.05) was observed at pH 6 than at pH 7, and the antimicrobial effects of the organic acids on B. thailandensis were in the following order: Ascorbic acid > lactic acid > citric acid after incubation at $35^{\circ}C$ for 30 h. These results indicate that organic acids in meat processing-related formulations should be useful in decreasing the risk related to an emerging high risk agent (B. pseudomallei).

• BMB Reports
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• 제37권5호
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• pp.556-564
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• 2004

A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2% glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.

• Molecules and Cells
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• 제27권2호
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• pp.237-241
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• 2009

Pathogens Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) contain a large number (> 12,000) of Simple Sequence Repeats (SSRs). To study the extent to which these features have contributed to the diversification of genes, we have conducted comparative studies with nineteen genomes of these bacteria. We found 210 genes with characteristic types of SSR variations. SSRs with nonamer repeat units were the most abundant, followed by hexamers and trimers. Amino acids with smaller and nonpolar R-groups are preferred to be encoded by the variant SSRs, perhaps due to their minimal impacts to protein functionality. A majority of these genes appears to code for surface or secreted proteins that may directly interact with the host factors during pathogenesis or other environmental factors. There also are others that encode diverse functions in the cytoplasm, and this protein variability may reflect an extensive involvement of phase variation in survival and adaptation of these pathogens.

• Journal of Microbiology and Biotechnology
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• 제28권3호
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• pp.498-502
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• 2018

Molecular imaging is a powerful method for tracking various infectious disease-causing pathogens in host organisms. Currently, a dual molecular imaging method that can provide temporal and spatial information on infected hosts at the organism, organ, tissue, and cellular levels simultaneously has not been reported for Burkholderia pseudomallei, a high-risk pathogen that causes melioidosis. In this study, we have established an experimental method that provides spatiotemporal information on infected hosts using luminescent and fluorescent dual-labeled B. pseudomallei. Using this method, we visualized B. pseudomallei infection at the organism, organ, and tissue levels in a BALB/c mouse model by detecting its luminescence and fluorescence. The infection of B. pseudomallei at the cellular level was also visualized by its emitted fluorescence in infected macrophage cells. This method could be an extremely useful and applicable tool to study the pathogenesis of B. pseudomallei-related infectious diseases.

• Journal of Microbiology and Biotechnology
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• 제33권1호
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• pp.15-27
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• 2023

The incidence of melioidosis cases caused by the gram-negative pathogen Burkholderia pseudomallei (BP) is seeing an increasing trend that has spread beyond its previously known endemic regions. Biofilms produced by BP have been associated with antimicrobial therapy limitation and relapse melioidosis, thus making it urgently necessary to understand the mechanisms of biofilm formation and their role in BP biology. Microbial cells aggregate and enclose within a self-produced matrix of extracellular polymeric substances (EPSs) to form biofilm. The transition mechanism of bacterial cells from planktonic state to initiate biofilm formation, which involves the formation of surface attachment microcolonies and the maturation of the biofilm matrix, is a dynamic and complex process. Despite the emerging findings on the biofilm formation process, systemic knowledge on the molecular mechanisms of biofilm formation in BP remains fractured. This review provides insights into the signaling systems, matrix composition, and the biosynthesis regulation of EPSs (exopolysaccharide, eDNA and proteins) that facilitate the formation of biofilms in order to present an overview of our current knowledge and the questions that remain regarding BP biofilms.

• Journal of Microbiology and Biotechnology
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• 제22권10호
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• pp.1336-1342
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• 2012

In Burkholderia pseudomallei, the pathogen that causes melioidosis, the gene cluster encoding the capsular polysaccharide, is located on chromosome 1. Among the 19 capsular genes in this cluster, wzm has not been thoroughly studied. To study the function of wzm, we generated a deletion mutant and compared it with the wild-type strain. The mutant produced less biofilm in minimal media and was more sensitive to desiccation and oxidative stress compared with the wild-type strain, indicating that wzm is involved in biofilm formation and membrane integrity. Scanning electron microscopy showed that the bacterial cells of the mutant strain have more defined surfaces with indentations, whereas cells of the wild-type strain do not.

• BMB Reports
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• 제36권5호
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• pp.493-498
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• 2003

The scFv antibody towards the Burkholderia pseudomallei exotoxin was previously constructed by phage display and exhibited good specificity towards the exotoxin. We report here the optimization of the scFv expression in an E. coli expression system. Four different E. coli strains (ER2537, TG1, HB2151, and XL1-Blue) were examined for optimal expression of the scFv protein. Two types of carbon source (i.e. 0.2% glucose and 0.2% glycerol) were also tested for their ability to induce the scFv expression. Cells that carried the scFv construct were grown at $30^{\circ}C$ and induced with 0.05 mM IPTG. The expression was then monitored by SDS-PAGE, Western blotting, and indirect ELISA. The Western blot profile showed different levels of the scFv expression among the host strains; XL1-Blue exhibited the highest level of the scFv protein expression. Glycerol at a concentration of 0.2% (v/v) significantly increased the scFv protein expression level when compared to 0.2% (w/v) glucose. Further optimization demonstrated that the scFv protein expression in XL1-Blue was the most optimal with a glycerol concentration as low as 0.05%. However, by indirect ELISA, only the scFv protein that was expressed in 0.2% (v/v) glycerol exhibited high specificity towards the Burkholderia pseudomallei exotoxin.

• BMB Reports
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• 제34권6호
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• pp.509-516
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• 2001

A genomic DNA fragment that contains the gene, which codes for a novel extracellular serine protease in Burkholderia pseudomallei, was cloned by using pQE40 as a vector. It was maintained in Escherichia coli JM109. The expression of the gene(s) resulted in the production of a 52 kDa protease. The recombinant protease was purified from the culture filtrate via ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography. The purified protease had an optimum pH and temperature of pH 8.9 and $38^{\circ}C$, respectively. The protease activity was inhibited by EGTA, EDTA, and PMSF, but not 1,10-phenanthroline. The first 11 amino acid residues from the N-terminus of the purified protease were identified as LAPNDPYYYGY. PNDPYY was found to show homology to the Bacillus cereus microbial serine protease and B. subtilis PD498 serine protease. These results indicate that the protease that was purified in this study is an extracellular calcium-dependent serine protease. The purified protease was able to digest the human serum 19A, IgG, albumin, and transferrin, as well as bovine muscle actin and myosin. Furthermore, it was able to promote or cause dermonecrosis in experimental rabbits. These results propose the possible role of a novel B. pseudomallei extracellular calcium-dependent serine protease in the virulence of the pathogen.

• Journal of Microbiology and Biotechnology
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• 제32권5호
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• pp.551-563
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• 2022

L-asparaginase (E.C. 3.5.1.1) purified from bacterial cells is widely used in the food industry, as well as in the treatment of childhood acute lymphoblastic leukemia. In the present study, the Burkholderia pseudomallei L-asparaginase gene was cloned into the pGEX-2T DNA plasmid, expressed in E. coli BL21 (DE3) pLysS, and purified to homogeneity using Glutathione Sepharose chromatography with 7.26 purification fold and 16.01% recovery. The purified enzyme exhibited a molecular weight of ~33.6 kDa with SDS-PAGE and showed maximal activity at 50℃ and pH 8.0. It retained 95.1, 89.6%, and 70.2% initial activity after 60 min at 30℃, 40℃, and 50℃, respectively. The enzyme reserved its activity at 30℃ and 37℃ up to 24 h. The enzyme had optimum pH of 8 and reserved 50% activity up to 24 h. The recombinant enzyme showed the highest substrate specificity towards L-asparaginase substrate, while no detectable specificity was observed for L-glutamine, urea, and acrylamide at 10 mM concentration. THP-1, a human leukemia cell line, displayed significant morphological alterations after being treated with recombinant L-asparaginase and the IC₅₀ of the purified enzyme was recorded as 0.8 IU. Furthermore, the purified recombinant Lasparaginase improved cytotoxicity in liver cancer HepG2 and breast cancer MCF-7 cell lines, with IC₅₀ values of 1.53 and 18 IU, respectively.

• The Plant Pathology Journal
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• 제33권2호
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• pp.148-162
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• 2017

As a step towards discovering novel pathogenesis-related proteins, we performed a genome scale computational identification and characterization of secreted and transmembrane (TM) proteins, which are mainly responsible for bacteria-host interactions and interactions with other bacteria, in the genomes of six representative Burkholderia species. The species comprised plant pathogens (B. glumae BGR1, B. gladioli BSR3), human pathogens (B. pseudomallei K96243, B. cepacia LO6), and plant-growth promoting endophytes (Burkholderia sp. KJ006, B. phytofirmans PsJN). The proportions of putative classically secreted proteins (CSPs) and TM proteins among the species were relatively high, up to approximately 20%. Lower proportions of putative type 3 non-classically secreted proteins (T3NCSPs) (~10%) and unclassified non-classically secreted proteins (NCSPs) (~5%) were observed. The numbers of TM proteins among the three clusters (plant pathogens, human pathogens, and endophytes) were different, while the distribution of these proteins according to the number of TM domains was conserved in which TM proteins possessing 1, 2, 4, or 12 TM domains were the dominant groups in all species. In addition, we observed conservation in the protein size distribution of the secreted protein groups among the species. There were species-specific differences in the functional characteristics of these proteins in the various groups of CSPs, T3NCSPs, and unclassified NCSPs. Furthermore, we assigned the complete sets of the conserved and unique NCSP candidates of the collected Burkholderia species using sequence similarity searching. This study could provide new insights into the relationship among plant-pathogenic, humanpathogenic, and endophytic bacteria.