Cloning and Molecular Analysis of the 2-Keto-3-Deoxy Gluconate Kinase (kdgK) Gene from Serrtia marcescens

유주순,김혜선,문종환,정수열,최용락 한국생명과학회 1998 한국생명과학회 학술발표회 Vol.20 No.-

We have get several clones from Serratia which stimulate the cells to use maltose as a carbon source in E. coli TP2139(Δ lac, Δcrp). One of the cloned genes, pCKB17, was further analyzed. In order to find whether the increased expression of the gene under the direction of maltose metabolism, we constructed several subclones. The nucleotide sequence of the 2.9 kb BamHI fragment was determined. Sequence analysis revealed the presence of two open reading frames, one is truncated ORF and the other was identified as the kdgK gene. The deduced KdgK amino acid sequence corresponds to a protein of 308 amino acid with a molecular mass of 34,000 Da. The deduced amino acid sequence of Serratia kdgK gene is highly homologous to the KDG kinase gene of Erwinia chrysanthemi, fructokinase gene of Klebsiella pneumoniae, dmpI gene of Pseudomonas putida. At the 3´end of the kdgK gene and surrounding the kdgK translational stop, there is a GC-rich inverted repeat sequence, a putative transcription terminator site. In the 5´-untranslated end of kdgK, we found a sequence showing good homology with the consensus established for the KdgR-binding site.

Serratia marcescens에서 cAMP receptor protein(CRP) 유전자의 클로닝 해석

유주순,김혜선,문종환,정수열,최용락,Yoo, Ju-soon,Kim, Hae-Sun,Moon, Jong-Hwan,Chung, Soo-Yeol,Choi, Yong-Lark 한국생명과학회 1998 생명과학회지 Vol.8 No.3

전사조절인자로서 잘 알려져 있는 cAMP receptor protein(CRP)은 cAMP와 DNA에 결합하는 특별한 활성을 가지고 있으며, cAMP-CRP complex를 형성하여 수많은 유전자의 발현조절에 관여한다. 이러한 측면에서 cAMP-CRP의 조절은 어떤 면에서 총체적 조절체계라고까지 한다.본 연구는 Serratia 균주에서 crp 유전자의 분자적 특성 및 cAMP에 의한 발현조절을 받는 분자기구를 해석하고자 유전자를 클로닝하고 발현을 확인하였다. MacConkey 배지에서 maltose를 탄소원으로 충분히 이용하지 못하는 대장균 TP2139(${\Delta}crp$,${\Delta}lac$를 숙주로 이용하고, 염색체 DNA를 library로 작성하여 얻은 형질전환체 약 일만개의 콜로니에서 red colony를 나타내는 5종류의 양성 클론을 얻었다. 이들 클론을 Southern 방법으로 확인한 결과 3kh의 단편을 가진 pCKB12클론이 crp유전자를 coding하고 있음을 확인하였다. glpD-lacZ 융합 plasmid인 pLDC6의 BamHI부위에 pCKB12의 3kb 단편을 삽입시킨 재조합 plasmid pLDC6-Scrp를 작성하여, 클로닝된 Serratia의 crp유전자가 대장균에서 유전자 전사조절에 미치는 영향을 확인한 결과 cAMP-CRP 복합체 형성에 의한 전사조절 기능이 확인되어졌다. One of the better-characterized transcription factor of E. coli is the cAMP receptor protein(CRP) and the CRP binds cAMP and DNA. The cAMP-CRP complex is involved in regulation of many genes at bacteria. The cAMP-CRP regulatory element represents, in some respects, a global regulatory network. The aim of this work was to study the structure and the mechanisms controlling the expression of CRP in Serratia marcescens. We have been get 5 different clones from Serratia which stimulated the cells to use maltose as a sole carbon source in E. coli TP2139. The crp gene clone, pCKB12, was confirmed by Southern hybridization with E. coli crp gene. The location of the crp gene was determined by construction subclones carrying various portions of pCKB12. To investigate the potential role of CRP in E. coli, lacZ fused plasmids were constructed and investigated the ${\beta}$-galactosidase activity of the fused plasmid. The Serratiamarcescens cAMP receptor protein can substitute the E. coli CRP in transcriptional activation at the lacZ gene. These results suggest that Serratia marcescens cAMP receptor protein complex functions to regulate several promoters in E. coli.

Poster 발표 논문 초록 - 생화학 / 분자생물학 / 생리학 분야 PP-11 : Serratia marcescens 의 aconitase ( acn ) 유전자의 구조해석과 발현

유주순,김혜선,이승진,정수열,최용락 한국응용생명화학회(구 한국농화학회) 1999 한국응용생명화학회 학술발표회 Vol.1999 No.1

포스터 초록 ( 미생물학 ) : Cellulose 생합성 균주의 분리 동정 및 특성

유주순,이승진,이희영,정수열,최용락 한국응용생명화학회(구 한국농화학회) 1995 한국응용생명화학회 학술발표회 Vol.1995 No.2

Molecular Cloning and Characterization of CMCase gene (celC) from Salmonella typhimurium UR

유주순,Youn-Ju Jung,Soo-Yeol Chung,이영춘,최용락 한국미생물학회 2004 The journal of microbiology Vol.42 No.3

The sequence coding for carboxymethylcellulase (CMCase, CelC) was isolated from the DNA of Salmonella typhimurium UR1. Comparison between the deduced amino acid sequence of CelC (368 amino acid residues, Molecular mass 41 kDa) and that of the previously published CMCase revealed that this enzyme belongs to the cellulase family 8 and D. The protein was overproduced in Escherichia coli using T7 expression system, and its activity was confirmed by CMC-SDS-PAGE. When the overexpressed CelC protein was tested on cellulose-type substrates, the recombinant protein is able to degrade cellulose- type substrates, such as CM-cellulose, xylan, avicel, lichenan, and laminarin. Optimal temperature and pH for enzyme activity were found to be 50oC and pH 6.5, respectively.

Analysis of cAMP regulation-related genes in Serratia

유주순 한국생명과학회 2001 한국생명과학회 학술발표회 Vol.33 No.-

Five different clones obtained from Serratia marcescens relate cAMP regulation in E. coli TP2139 (Δlac, Δcrp). pCKB12, 13, and 15. The crp gene clone, pCKBl2, was confirmed by Southern hybridization and the stimulation of the β-galactosidase activity. The nucleotide sequence of the crp region consisting of 1979 bp was determined. The sequencing of the fragment led to the identification of two open reading frames and one of these, the crp gene, encoded 210 amino acids, and the other encoded a truncated protein. The S. marcescens and E. coli crp genes presented a higher degree of divergence in their nucleotide sequence with 120 changes, however, the corresponding amino acid sequences presented only two amino acid differences. Not yet, an analysis of the amino acid divergence, revealed that the catabolite gene activator protein, the crp gene product, is the most conserved protein observed so far. Using a crp-lac protein fusion, it was demonstrated that S. marcescens CRP can repress its own expression, probably via a mechanism similar to that previously described for the E. coli crp gene. A DNA fragment pCKB13 containing two genes encoding CoA transferase was isolated from a genomic DNA library of S. marcescens KCTC2172. The complete nucleotide sequence of pCKB13 consisting of 2081 bp was determined. Sequencing of the fragment led to the identification of two open reading frames showing high homology with Coenzyme A (CoA) transferases, Acetoacetyl CoA transferase (Acot) and Succinyl CoA transferase (Scot), enzymes catalyzing the reversible transfer of CoA from one carboxylic acid to another. Therefore, we have confirmed that the clone, pCKB13 codes for Coenzyme A transferase gene by partial nucleotide sequencing in the terminal region. So pSC0123 and pSC0989 were designed to get upstream of scotB, scotA and putatived promotor region. The truncated protein of ScotA gene is located directly upstream of scotB, with a same direction of transcription. Active site and CoA binding site motif of ScotA and B was highly conversed. The enzyme activity of Coenzyme A transferase increased after introduction of the multicopy of the cloned gene in E. coli. The recombinant protein overexpressed by multicopy and induction with IPTG, the polypeptide of 42-kDa, was confirmed by SDS-PAGE. The protein was purified to homogeneity through two sequential chromatographic techniques including DEAF-sepharose ion exchange and CM-sepharose. The nucleotide sequence of the 2.9 kb BamHI fragment of pCKB15 was determined. Sequence analysis revealed the presence of two open reading frames, one of the truncated ORF was identified as the aconitase gene.

sfs1 유전자의 cAMP-cAMP receptor protein에 의한 발현 조절

유주순,이승진,이희영,정수열,최용락,Yoo, Ju-Soon,Lee, Seung-Jin,Lee, Hee-Young,Chung, Soo-Yeol,Choi, Yong-Lark 한국응용생명화학회 1996 Applied Biological Chemistry (Appl Biol Chem) Vol.44 No.1

We have cloned several E. coli sfs genes which stimulate mal gene expression with $crp^{{\ast}1}$). One the genes (pPVC2) was sequenced and potential CRP binding site is located in the upstream of the putative promoter in the regulatory region. In order to investigate the regulation of the sfs1 gene by the cAMP-CRP complex, we have constructed the sfs-lacZ fusion gene in this research. The overall transcriptional stimulations of sfs1 gene in the presence cAMP were confirmed by ${\beta}-galactosidase$ activity and Western blot analysis of sfs1-lacZ fusion gene. Transcriptional regulation by cAMP-CRP was also confirmed by Northern blot analysis. End-labelled DNA of the DNA fragment in sfs1 regulation region were used for gel retardation assay to examine the CRP-DNA complex in the presence of cAMP. Results here indicate that CRP binding site in the regulatory region of sfs1 gene is positive regulator for the expression of sfs1 gene. $crp^{\ast}$ 유전자가 도입된 대장균 MK2001($crp^{{\ast}1}$}, cya::km)을 숙주로 사용하여 mal 유전자 발현을 촉진시키는 유전자의 하나인 sfs1(sugar fermentation stimulation)의 구조해석 결과에 의하면, 잠정적인 sfs1의 promoter 영역에는 CRP 단백질과의 결합영역으로 보이는 염기배열이 존재하였다. 본 실험에서는 sfs1 유전자의 cAMP-CRP에 의한 발현 조절을 확인하고자, lacZ 와의 융합 유전자를 작성하였다. 작성된 융합 유전자는 cya 결손주인 Tp2010에서 cAMP의 첨가에 의해 ${\beta}-galactosidase$ 활성이 크게 증가하였으며, Western blotting의 실험에서도 같은 결과를 나타냈다. in vivo에서 발현이 확인된 전사산물은 cAMP에 의해 전사 촉진이 일어났으며, CRP의 결합부위로 예상되는 DNA 영역은 cAMP가 존재하면 CRP 단백질과 결합하는 특성을 나타내었다. 이상의 결과로 보아, sfs1 유전자의 발현은 UMP-CRP에 의한 전사촉진 현상을 받는 것으로 나타났다.

Purification and Characterization of Thermostable β-1,3-1,4 Glucanase from Bacillus sp. A8-8

유주순,정수열,이상철,정연주,최용락,이용석,박인혜,김선희,Masaaki Yasuda 한국생물공학회 2007 Biotechnology and Bioprocess Engineering Vol.12 No.3

In this study, the extracellular enzyme activity of Bacillus sp. A8-8 was detected on LB agar plates containing 0.5% of the following substrates: carboxymethylcellulose (CMC), xylan, cellulose, and casein, respectively. The β-1,3-1,4 glucanase produced from Bacillus sp. A8-8 was purified by ammonium sulfate and hydrophobic chromatography. The molecular size of the protein was estimated by SDS-PAGE as approximately 33 kDa. The optimum pH and temperature for the enzyme activity were 6.0 and 60℃, respectiveley. However, enzyme activity was shown over a broad range of pH values and temperatures. The purified β-1,3-1,4 glucanase retained over 70% of its original activity after incubation at 80℃ for 2 h, and showed over 40% of its original activity within the pH range of 9 to 12. This suggests that β-1,3-1,4 glucanase from Bacillus sp. A8-8 is thermostable and alkalistable. In addition, β-1,3-1,4 glucanase had higher substrate specificity to lichenan than to CMC. Finally, the activity of the endoglucanase was inhibited by Fe3+, Mg2+, and Mn2+ ions. However Co2+ and Ca2+ ions were increased its activity.

포스터 발표논문 초록 ( 생화학 / 분자생물학 분야 Poster 초록 ) : Salmonella typhimurium에서 cellulose 생합성 operon의 클로닝 및 해석

유주순,이승진,정수열,최용락 한국응용생명화학회(구 한국농화학회) 1997 한국응용생명화학회 학술발표회 Vol.1997 No.1

Cloning and Molecular Analysis of the Acetoacetate and Acetyl CoA Acetyltrasferase Gene from S. marcescens

유주순,김혜선,이승진,정수열,최용락 한국생명과학회 1998 한국생명과학회 학술발표회 Vol.20 No.-

We have get several clones from Serratia which stimulate the cells to use maltose as a carbon source in E. coli TP2139(Δlac, Δcrp). One of the cloned genes, pCKB13, was further analyzed. In order to find whether the increased expression of the gene under the direction of maltose metabolism, we constructed several recombinant subclones. The nucleotide sequence of the 2.0 kb BamHI fragment was determined. Sequence analysis revealed the presence of two open reading frames, one is identified as acetoactate CoA (acoA) and the other was as the acetoacetyl CoA transferase (acoB) gene. The deduced amino acid sequence ActA and ActB corresponds to protein with 208 and 393 amino acid. The deduced amino acid sequence of Serratia acoA gene is highly homologous to the lpsJ gene of Xanthomonas campestris, scoB gene of Mycobacterium tuberculosis, yxjE gene of Bacillus subtilis and Helicobacter pylori, acoA gene of E. coli and Haemophilus and succinyl CoA transferase gene of Homo sapiens. The acoB gene is highly homologous to the acetyl CoA acetyltransferase gene of E. coli, Thiocysis sp,. Acinetobacter sp., Thermoanaerobacterium sp., Alcaligenes sp., and Clostridium sp. At the 3´end of the actB gene and surrounding the actB translational stop, there is a GC-rich inverted repeat sequence, a putative transcription terminator site.