• Korean Journal of Veterinary Research
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• v.53 no.4
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• pp.211-216
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• 2013

This study was focusing on evaluating the protection of polyphosphate kinase (ppk) deleted and/or temperature-sensitive (ts) Salmonella Enteritidis (SE) as an attenuated vaccine in chickens. We constructed SEppk, SEts and SEppk::ts mutants and screened those mutants by growth capability in vitro, protection study in mice model and antibody response in chickens. Among the mutants, SEppk::ts-3 was selected because it showed higher growth capability, good protection against highly virulent SE in mice model, and good antibody response in chickens. SEppk::ts-3 also showed good protection against highly virulent SE isolate because it decreased colonization of virulent SE challenge strain in spleen, liver and cecum compared with the non-vaccinated control. The SEppk::ts-3 mutant showed cross-protection against S. Gallinarum (SG) challenge although the its cross-protection rate was a little lower than that of SG9R, a commercial vaccine against SG infection. To use for live attenuated vaccine in chickens, it should further be characterized.

• Korean Journal of Microbiology
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• v.35 no.4
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• pp.283-288
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• 1999

The effect of dissolved oxygen(DO) concentration on the growth of Pseudomonas aeruginosa and it's mutant M-10 was studied the growth kinetics and the possibility of waste treatment for reducing the amount of excess sludge. Different DO concentrations on the growth of wild type Pseudomonas aeruginosa affected to cellular yields, decreasing with increasing DO concentrations. Under these conditions, the maximum 14 folds decrease of cellular yield was achieved at 90 ppm DO levels by screened, being able to reduce their cellular yields under low DO concentration. The optimum mutation conditions were obtained by the treatment of NTG mutagen at 30${\circ}C$ for 1 hr. The growth characteristics of the selected mutant M-10 showed the same as the wild type growth kinetics. However, cellular yields are significantly decreased to 55% compared with those of wild type under DO concentrations increased. We, therefore, expect the application of the mutants to waste treatment for reducing excess sludge.

• Korean Journal of Microbiology
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• v.47 no.2
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• pp.124-129
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• 2011

The role of the ferric uptake regulator (Fur) of Helicobacter pylori in the oxidative stress was investigated in this study. A fur knockout mutant of H. pylori was constructed by replacing the fur gene with an aphA (kanamycin resistant marker) gene. Photodynamic therapy using methylene blue (MB) and 660 nm light was chosen to induce oxidative stress. The bactericidal effect of photodynamic therapy (PDT) was compared between wild type H. pylori and fur knockout mutant H. pylori. The degree of oxidative damage of DNA was confirmed using alkaline gel electrophoresis and an assay of 8-hydroxy-2-deoxyguanosine (8-OHdG). In control groups, the number of viable cells was maintained constantly during experiment. After PDT, the mutant H. pylori showed 10,000 times decreased viable cell number compared with wild type H. pylori. Depending on the exposure time of 660 nm light, the 3-fold increase in the concentration of 8-OHdG was observed in mutant H. pylori. The results of this study showed that H. pylori's Fur protein may play a role in oxidative stress induced by PDT.

• BMB Reports
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• v.34 no.1
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• pp.51-58
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• 2001

Attempts using in vitro and in vivo selection procedures have been made to search for hammerhead ribozymes that have higher activities than the wild-type ribozyme and also to determine whether other sequences might be possible in the catalytic core of the hammerhead ribozyme. Active sequences selected in the past conformed broadly to the consensus core sequence except at A9, and no sequences were associated with higher activity than that of the hammerhead with the consensus core, an indication that the consensus sequence derived from viruses and virusoids is probably the optimal sequence [Vaish et al. (1997) Biochemistry 36, 6495-6501]. Recently, during construction of ribozyme expression vectors, we isolated a mutant hammerhead ribozyme, with an insertion of G between A9 and G10.1, that appeared to show significant activity [Kawasaki et al. (1996) Nucleic Acids Res. 24, 3010-3016; Kawasaki et al. (1998) Nature 393, 284-289]. We, therefore, characterized kinetic properties of the G-inserted mutant ribozymes in terms of the NUX rule. We demonstrate that the NUX rule is basically applicable to the G-inserted ribozymes and, more importantly, one type of G-inserted ribozyme was very active with $k_{cat}$, value of $6.4\;min^{-1}$ in 50 mM Tris-HCl (pH 8.0) and 10 mM $MgCl_2$ at $37^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• v.14 no.6
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• pp.1324-1326
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• 2004

In previous studies, we isolated an isogenic LPS mutant of Bradyrhizobium japonicum 61A101C, which was completely devoid of O-polysaccharide and had altered cell surface characteristics. Subsequently, the mutated gene was identified, cloned, and used to complement the LPS mutant strain JS314 to restore the phenotype. Since it has been reported that in Escherichia coli LPS O-polysaccharide is involved in resistance to an organic acid such as acetic acid under low pH (Barna et al., Molecular Microbiology 43: 629-640, 2002), we compared the organic acid resistance of the three B. japonicum strains; wild-type 61A101C, the LPS mutant JS314, and the complemented strain to determine whether the role of O-polysaccharide in the resistance to organic acid could be generalized. Growth of all three strains was inhibited by the presence of 3 mM acetic acid under acidic condition (pH 5.5). To our surprise, however, in the presence of 2 mM acetic acid, wild-type and the complemented strains did not grow while the $LPS^-$ mutant showed a significant growth. Therefore, unlike in E. coli, the lack of O­polysaccharide of LPS appears to render B. japonicum more resistant to organic acid.

• Journal of Microbiology and Biotechnology
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• v.15 no.6
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• pp.1337-1345
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• 2005

Vibrio vulnificus is the causative agent of foodborne diseases such as gastroenteritis and life-threatening septicemia. Microbial pathogenicity is a complex phenomenon in which expression of numerous virulence factors is frequently controlled by a common regulatory system. In the present study, a mutant exhibiting decreased cytotoxic activity toward intestinal epithelial cells was screened from a library of V. vulnificus mutants constructed by a random transposon mutagenesis. By a transposon-tagging method, an open reading frame, fexA, a homologue of Escherichia coli areA, was identified and cloned. The nucleotide and deduced amino acid sequences of the fexA were analyzed, and the amino acid sequence of FexA from V. vulnificus was $84\%\;to\;97\%$ similar to those of AreA, an aerobic respiration control global regulator, from other Enterobacteriaceae. Functions of the FexA were assessed by the construction of an isogenic mutant, whose fexA gene was inactivated by allelic exchanges, and by evaluating its phenotype changes in vitro and in mice. The disruption of fexA resulted in a significant alteration in growth rate under aerobic as well as anaerobic conditions. When compared to the wild-type, the fexA mutant exhibited a substantial decrease in motility and cytotoxicity toward intestinal epithelial cell lines in vitro. Furthermore, the intraperitoneal $LD_{50}$ of the fexA mutant was approximately $10^{1}-10^{2}$ times higher than that of parental wild-type. Therefore, it appears that FexA is a novel global regulator controlling numerous genes and contributing to the pathogenesis as well as growth of V. vulnificus.

• Microbiology and Biotechnology Letters
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• v.33 no.4
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• pp.319-321
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• 2005

We have previously reported the isolation and characterization of peptides binding to $TiO_{2}$ nanoparticles from phage display peptide libraries. One of the peptides (PEP9) was selected and mutant peptide-displaying phages were produced by alanine scanning mutagenesis. The mutant phages were subjected to binding analysis to $TiO_{2}$ nanoparticles. When the proline at residue 4 was substituted by alanine, the binding activity was reduced to $10\%$ of that of wild type PEP9. Substitution of valine at residue 2, serine at residue 3, and isoleucine at residue 5 also decreased the binding to $40\%$. Based on these observations, we concluded that the three dimensional structure generated by residues 2-5 was the critical factor for the binding between PEP9 and the nanoparticle.

• Microbiology and Biotechnology Letters
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• v.28 no.3
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• pp.152-155
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• 2000

Cultural Conditions for the Improvement in Gibberellic Acid Productivity by a Mutant of Gibberellafujikuroi ATCC 12616-Gibberella fujikuroi G-36 . Dh, Young-Jun. Department of Food and Biotechnolog}'r Dongshm Umversity, Naju 520-714, Korea - A mutant Gibberella jujih/roi G- 36 was selected by metagenesis of G/bberella fitjikuroi ATCC 12616 with mutagens such as N-methy1-N'~nitro~N"nitrosoguanidine and hydroxylamine for improving productivity of gibberellic acid. The mutant strain produced gibberellic acid (70 mg/l) more than that of wilde type. A fermentation medium containing glucose, $NH_4N0_3$, $MgS0_4$, $KH_2P0_4$ and trace elements was deve]oped for the maximal production of a gibberellic acid by the mutanL The Guctuating cultural temperature that was vaded from 300e to 20DC resulted in higher GA yield than that of fixed cu1tura] temperature at $28^{\circ}C$.

• Korean Journal of Animal Reproduction
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• v.27 no.4
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• pp.299-307
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• 2003

The effects of additions/deletions in glycosylated residues of recombinant human EPO (rhEPO) produced in CHO-K1 on their secretion were examined. hEPO cDNA was amplified from human liver mRNA and cloned into the pCR2.1 TOPO. Using overlapping-extension site-directed mutagenesis method, glycosylation sites at 24th, 38th, 83rd, and 126th were respectively or accumulatively removed by substituting its asparagine (or serine) with glutamine. To add novel glycosylation sites, 69 and 105th leucine was mutated to asparagine. Mutant and wild type rhEPO constructs were cloned into the pcDNA3 expression vector with CMV promoter and transfected into CHO cell line, CHO-K1, to produce mutant rhEPO mutant rhEPO proteins. Enzyme-linked immunosorbant assay (ELISA) and Western analysis with monoclonal anti-EPO antibody were performed using supernatants of the cultures showing transient and stable expressions respectively. Addition of novel glycosylation reduced rhEPO secretion dramatically while deletion mutants had little effect except some double deletion mutants ($\Delta$24/83 and $\Delta$38/83) and triple mutant ($\Delta$24/38/83). This fact suggests that not single but combination of changes in glycosyl groups affect secretion of rhEPO in cell culture, possibly via changes in their conformations.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.249-257
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• 2002

Pseudomonas fluorescens GL20 is a plant growth-promoting rhizobacterium that produces a large amount of hydroxamate siderophore under iron-limited conditions. The strain GL20 considerably inhibited the spore germination and hyphal growth of a plant pathogenic fungus, Fusarium solani, when iron was limited, significantly suppressed the root-rot disease on beans caused by F. solani, and enhanced the plant growth. The mechanism for the beneficial effect of strain GL20 on the disease suppression was due to the siderophore production, evidenced by mutant strains derived from the strain. Analysis of the outer membrane protein profile revealed that the growth of strain GL20 induced the synthesis of specific iron-regulated outer membrane proteins with molecular masses of 85- and 90 kDa as the high-affinity receptors for the ferric siderophore. In addition, a cross-feeding assay revealed the presence of multiple inducible receptors for heterologous siderophores in the strain. In order to induce increased efficacy and potential in biological control of plant disease, a siderophore-overproducing mutant, GL20-S207, was prepared by NTG mutagenesis. The mutant GL20-S207 produced nearly 2.3 times more siderophore than the parent strain. In pot trials of beans with F. solani, the mutant increased plant growth up to 1.5 times compared with that of the parent strain. These results suggest that the plant growth-promoting P. fluorescens GL20 and the genetically bred P. fluorescens GL20-S207 can play an important role in the biological control of soil-borne plant diseases in the rhizosphere.