• Microbiology and Biotechnology Letters
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• v.49 no.1
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• pp.9-17
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• 2021

A γ-aminobutyric acid (GABA)-producing microorganism was isolated from galchi (hairtail fish, Trichiurus lepturus) jeotgal, a Korean salted and fermented seafood. The G144 isolate produced GABA excessively when incubated in MRS broth containing monosodium glutamate (MSG, 3%, w/v). G144 was identified as Lactobacillus brevis through 16S rRNA and recA gene sequencing. gadB and gadC encoding glutamate decarboxylase (GAD) and glutamate/GABA antiporter, respectively, were cloned and gadB was located downstream of gadC. The operon structure of gadCB was confirmed by reverse transcription (RT)-polymerase chain reaction. gadB was overexpressed in Escherichia coli and recombinant GAD was purified and its size was 54.4 kDa as evidenced by SDS-PAGE results. Maximum GAD activity was observed at pH 5.0 and 40℃ and the activity was dependent on pyridoxal 5'-phophate. The K_m and V_max of GAD were 8.6 mM and 0.01 mM/min, respectively.

• The Plant Pathology Journal
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• v.39 no.5
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• pp.417-429
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• 2023

Ralstonia solanacearum species complex (RSSC) is a soil borne plant pathogen causing bacterial wilt on various important crops, including Solanaceae plants. The bacterial pathogens within the RSSC produce exopolysaccharide (EPS), a highly complicated nitrogencontaining heteropolymeric polysaccharide, as a major virulence factor. However, the biosynthetic pathway of the EPS in the RSSC has not been fully characterized. To identify genes in EPS production beyond the EPS biosynthetic gene operon, we selected the EPS-defective mutants of R. pseudosolanacearum strain SL341 from Tn5-inserted mutant pool. Among several EPSdefective mutants, we identified a mutant, SL341P4, with a Tn5-insertion in a gene encoding a putative NDP-sugar epimerase, a putative membrane protein with sugar-modifying moiety, in a reverse orientation to EPS biosynthesis gene cluster. This protein showed similar to other NDP-sugar epimerases involved in EPS biosynthesis in many phytopathogens. Mutation of the NDP-sugar epimerase gene reduced EPS production and biofilm formation in R. pseudosolanacearum. Additionally, the SL341P4 mutant exhibited reduced disease severity and incidence of bacterial wilt in tomato plants compared to the wild-type SL341 without alteration of bacterial multiplication. These results indicate that the NDP-sugar epimerase gene is required for EPS production and bacterial virulence in R. pseudosolanacearum.

• Journal of Applied Biological Chemistry
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• v.66
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• pp.128-137
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• 2023

Transcriptome analysis revealed that the sinR gene encoding a transition-state regulator of Bacillus pumilus, genetically close to B. subtilis, was expressed at high levels during growth. The sinR gene is the second gene of the sinIR operon consisting of three promoters and two structural genes in B. subtilis. This study used the sinIR promoter of B. subtilis DB104 to construct a recombinant protein expression system. First, the expression ability depending on the number of sinIR promoter was investigated using enhanced green fluorescent protein (eGFP). The expression level of eGFP was slightly higher when using two promoters (Psin2) than using original promoters. The Psin2 promoter was further engineered by modifying the repressor binding site and -35 and -10 regions. Shine-Dalgarno (SD) sequence of the sinI gene was modified to the consensus sequence. Finally, combining the engineered Psin2 promoter with the modified SD sequence increased the expression level of eGFP by about 13.4-fold over the original promoter. Our results suggest that the optimized sinIR promoter could be used as a novel tool for recombinant protein expression in B. subtilis.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.758-761
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• 2001

In this study, the nar promoter of E. coli was characterized to see whether the nar promoter cloned onto pBBR122 can be used as an expression promoter of gram negative microbes. For this purpose, a plasmid with lacZ gene expressing ${\beta}-galactosidase$ instead of the structural genes of nar operon in a gram negative host strain(Agrobacterium.tumefaciens) was used to simplify an assay of induction of the nar promoter. The following effects were investigated to find optimal conditions: methods of inducing the nar promoter, optimal nitrate concentration, maximally inducing the nar promoter, the amount of expressed ${\beta}-galactosidase$ and induction ratio(specific ${\beta}-galactosidase$ activity after maximal induction/specific ${\beta}-galactosidase$ activity before induction). The following results were obtained from the experiments: the growth of Agrobacterium with E.coli nar promoter was not much affected by nitrate concentration in the shake-flask; induction of nar promoter was optimal when Agrobacterium was grown in the presence of 1% nitrate ion at the beginning of culture and when overnight culture was completely grown in the shake-flask before being transferred to other shake-flask; the amount of ${\beta}-galactosidase$ per cell and per medium volume was maximal when Agrobacterium was grown under aerobic condition to $OD_{600}$ of 1.7; then the nar promoter was induced under microaerobic and anaerobic condition made by lowering dissolved oxygen level(DO). After 2-3h of induction in the YEP medium selected as a main culture medium, the specific ${\beta}-galactosidase$ activity became about 17,000 Miller units in the fermentor cluture.

• Korean Journal of Medicinal Crop Science
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• v.13 no.3
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• pp.109-113
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• 2005

To analyze the genetic relationship 8 accessions of Ostericum koreanum Kitakawa, random amplified polymorphic DNA(RAPD) analysis was performed using 60 Operon primers. The 25 primers out of 60 random primers were amplified DNA by PCR using genomic DNA of O. koreanum. Eighty-five (49.1%) among 173 bands derived from 25 primers showed poly morphism, On the basis of similarity coefficient analysis by UPGMA, 8 accessions of O. koreanum Kitakawa could be classified into three groups at the similarity coefficient value of 0.71. Group I contained three accessions (Nam Gangwhal), Group II contained one accession (Nam Gangwhal) and Group III contained four accessions (Buk Gangwhal), The range of total genetic similarity coefficient value of 8 accessions of O. koreanum Kitakawa was $0.63{\sim}0.96$. Buk Gangwhal was flowered 18 to 26 days earlier than Nam Gangwhal, and Nam Gangwhal leaf stalk was thin and long as bolting rate high compared with Buk Gangwhal.

• KSBB Journal
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• v.11 no.2
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• pp.165-172
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• 1996

To obtain a correctly folded human proinsulin, export of proinsulin using Staphylococcal protein A signal sequence-mediated secretion pathway has been attempted in E.coli. A secretion operon for proinsulin was constructed by consecutively connecting T7 promoter, SPA ribosome binding site, SPA signal sequence gene, and human proinsulin gene. Little immunoreactive proinsulin was detected in the periplasmic space and. culture medium, and not even in cytoplasmic space. The qualitative analysis of transcribed proinsulin mRNA and the in vitro transcription/translation experiment suggests that the negligible level of proinsulin export appears to be due to intracellular degradation of proinsulin, rather than due to the blockage during translocation. However, expression of proinsulin fusion protein such as MBP-proinsulin could dramatically increase export of proinsulin in E.coli.

• BMB Reports
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• v.32 no.4
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• pp.414-418
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• 1999

A novel gene for malonyl-CoA decarboxylase was discovered in the mat operon, which encodes a set of genes involved in the malonate metabolism of Rhizobium trifolii (An and Kim, 1998). The subunit mass determined by SDS-PAGE was 53 kDa, which correspond to the deduced mass from the sequence data. The molecular mass of the native enzyme determined by field flow fractionation was 208 kDa, indicating that R. trifolii malonyl-CoA decarboxylase is homotetrameric. R. trifolii malonyl-CoA decarboxylase converted malonyl-CoA to acetyl-CoA with a specific activity of 100 unit/mg protein. Methylmalonyl-CoA was decarboxylated with a specific activity of 0.1 unit/mg protein. p-Chloromercuribenzoate inhibited this enzyme activity, suggesting that thiol group(s) is(are) essential for this enzyme catalysis. Database analysis showed that malonyl-CoA decarboxylase from R. trifolii shared 32.7% and 28.1% identity in amino acid sequence with those from goose and human, respectively, and it would be located in the cytoplasm. However, there is no sequence homology between this enzyme and that from Saccharopolyspora erythreus, suggesting that malonyl-CoA decarboxylases from human, goose, and R. trifolii are in the same class, whereas that from S. erythreus is in a different class or even a different enzyme, methylmalonyl-CoA decarboxylase. According to the homology analysis, Cys-214 among three cysteine residues in the enzyme was found in the homologous region, suggesting that the cysteine was located at or near the active site and plays a critical role in catalysis.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.48 no.4
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• pp.297-302
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• 2003

Genetic linkage maps serve the plant geneticist in a number of ways, from marker assisted selection in plant improvement to map-based cloning in molecular genetic research. Genetic map based upon DNA polymorphism is a powerful tool for the study of qualitative and quantitative traits in crops. The objective of this study was to develop genetic linkage map of soybean using the population derived from the cross of Korean soybean cultivar 'Kwangkyo, and wild accession 'IT182305'. Total 1,000 Operon random primers for RAPD marker, 49 combinations of primer for AFLP marker, and 100 Satt primers for SSR marker were used to screen parental polymorphism. Total 341 markers (242 RAPD, 83 AFLP, and 16 SSR markers) was segregated in 85 $\textrm{F}_2$ population. Forty two markers that shown significantly distorted segregation ratio (1:2:1 for codominant or 3:1 for domimant marker) were not used in mapping procedure. A linkage map was constructed by applying the computer program MAPMAKER/EXP 3.0 to the 299 marker data with LOD 4.0 and maximum distance 50 cM. 176 markers were found to be genetically linked and formed 25 linkage groups. Linkage map spanned 2,292.7 cM across all 25 linkage groups. The average linkage distance between pair of markers among all linkage groups was 13.0 cM. The number of markers per linkage group ranged from 2 to 55. The longest linkage group 3 spanned 967.4 cM with 55 makers. This map requires further saturation with more markers and agronomically important traits will be joined over it.

• Korean Journal of Food Science and Technology
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• v.50 no.4
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• pp.414-419
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• 2018

Escherichia coli O157:H7 is one of the most common foodborne pathogens responsible for outbreaks of hemorrhagic colitis, which can lead to the life-threatening hemolytic-uremic syndrome. In this study, we identified phytochemicals that specifically inhibit the expression of LEE operon in E. coli O157:H7. Among phytochemicals, diosmin decreased the adherence of E. coli O157:H7 towards Caco-2 cells in vitro (p<0.01) and its biofilm formation activity (p<0.05). Quantitative RT-PCR analysis revealed that the transcripts of Ler-regulated genes and genes related to curli production were significantly reduced in the presence of diosmin. However, diosmin does not affect bacterial viability, indicating that the resistance rate to diosmin was remarkably low. Overall, these results provide significant insights into the development of a novel anti-infective agent that is different from conventional antibiotics.

• Journal of Microbiology
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• v.40 no.4
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• pp.305-312
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• 2002

The effects of two natural polyamines (putrescine and spermidine) on the synthesis of ArcA, a response regulator of the Arc two-component signal transduction system, were studied using an E. coli mutant deficient in polyamine biosynthesis. Endogenous polyamine deficiency of the mutant resulted in marked reduction in the ArcA level determined by Western blot analysis. Putrescine supplement to the growth medium effectively increased the ArcA level of the mutant in a concentration-dependent manner. Spermidine also stimulated the ArcA level in the mutant to a greater degree than putrescine. Expression of arcA'::lacZ operon fusion in the mutant was stimulated 6-fold and 10-fold by putrescine and spermidine at a 1mM concentration, respectively, indicating that the stimulatory effect of the polyamines on ArcA synthesis is due to transcriptional induction, and that spermidine is a more potent arcA inducer than putrescine. The polyamine-dependent arcA'::lacZ induction was growth-phase-dependent and independent of either arcA or fnr which are two regulators involved in anaerobic stimulation of the Arch level. These results suggested that putrescine and spermidine polyamines may be potential intracellular signal molecules in the control of arcA expression, and thereby may play an important role in cellular metabolism.