Urease gene의 전이에 의한 길항세균 Bacillus sp. SH14의 길항능력 증가

최종규,김상달 한국산업미생물학회 1998 한국미생물·생명공학회지 Vol.26 No.2

최근 생물방제균으로 주목받고 있는 Enterobacter cloacae의 방제기작이 이 균에 의해 토양내에서 생산된 휘발성 ammonia이며 ammonia의 생산에는 urease가 관계한다는 보고를 근거로하여, 항생물질 생산성 균주로 선발된 우수한 길항균주에 암모니아 생성능, 즉 urease 유전자를 유전적으로 부가함으로써 항진균성 길항물질 생산과 암모니아 생산이 동시에 이루어 질 수 있는 새로운 다기능의 생물방제균을 유전적으로 육종하고자 하였다. 저병해 인삼경작지로부터 식물근부균 Fusarium solani의 생육을 강하게 억제하는 길항세균 한 균주 SH14 균주를 분리, 선발하였으며, 분리된 균주를 동정한 결과 Bacillus subtilis이거나 그 근연종으로 추정되었다. 억제기작 실험을 통해 길항균주 B. subtilis SH14에 의해 생산되는 항진균성 길항물질은 외막가수분해효소와 같은 고분자 물질이 아니라 열에 안정한 저분자의 항생물질임을 알 수 있었다. 한편 ammonia 생산을 위한 urease의 유전자는 urease 생산력이 강력한 호알칼리성 Bacillus pasteurii의 urease 생산유전자를 E. coli-Bacillus shuttle vector인 pEB203에 subcloning하였고, 이어서 pGU 366으로 명명된 이 recombinant plasmid를 선발된 항진균성 길항균주 B. subtilis SH14에 PEG-induced protoplast transformation 방법으로 도입, 발현시켰으며, 최적조건을 조사하여 90분간의 lysozyme 처리과정 후 1.5 ㎍/㎖의 DNA와 40% PEG4000의 첨가로 약 6.5×10 exp (4)의 형질전환율을 얻을 수 있었다. 아울러 암모니아 생성능이 부가된 생물방제균 B. subtilis SH14(pGU366)에 의해 식물근부균 F. solani에 대한 생육억제력이 증가되는지 여부를 억제거리 측정법과 균체중량법을 통해 확인한 결과 urease 유전자가 도입된 형질전환체 B. subtilis SH14(pGU366)의 근부균 생육억제능이 각각 36.7%, 44.0%정도로 숙주균주인 B. subtilis SH14에 비해 근부균 생육억제능을 보다 강하게 나타내었음을 알 수 있었다. 따라서 항진균성 항생물질 생산성 생물방제균 B. subtilis SH14에 외부의 urease 유전자를 도입하여 ammonia 생성능을 부가함으로써 생물방제력의 상승효과를 거둘 수 있었다. It were reported that antifungal mechanism of Enterobacter cloacae is a volatile ammonia that produced by the strain in soil, and the production of ammonia is related to the bacterial urease activity. A powerful bacterium SH14 against soilborne pathogen Fusarium solani, which cause root rot of many important crops, was selected from a ginseng pathogen supressive soil. The stain SH14 was identified as Bacillus subtilis by cultural, biochemical, morphologica method, and API test. From several in vitro tests, the antifungal substance that is produced from B. subtilis SH14 was revealed as heat-stable and low-molecular weight antibiotic substance. In order to construct the multifunctional biocontrol agent, the urease gene of Bacillus pasteurii which can produce pathogenes-supressive ammonia transfered into antifungal bacterium. First, a partial BamH I digestion fragment of plasmid pBU11 containing the alkalophilic B. pasteurii 11859 urease gene was inserted into the BamH I site of pEB203 and expressed in Escherichia coli JM109. The recombinant plasmid was designated as pGU366. The plasmid pGU366 containing urease gene was introduced into the B. subtilis SH14 with PEG-induced protoplast transformation (PIP) method. The urease gene was very stably expressed in the transformant of B. subtilis SH14. Also, the optimal conditions for tranformation were established and the highest transformation frequency was obtained by treatment of lysozyme for 90 min, and then addition of 1.5 ㎍/㎖ DNA and 40% PEG4000. From the in vitro antifungal test against F. solani, antifungal activity of B. subtilis SH14(pGU366) containing urease gene was much higher than that of the host strain. Genetical development of B. subtilis SH14 by transfer of urease gene can be responsible for enhanced biocontrol efficacy with its antibiotic action.

생물방제균 Bacillus subtilis YB-70의 외부 Urease 유전자 도입과 길항력 증강

최종규,김용수,이은탁,김상달 한국산업미생물학회 1997 한국미생물·생명공학회지 Vol.25 No.1

토양내 미생물개체군 중 우점화 능력이 강한 길항균주에 타 길항기작을 추가적으로 부가하여 다기능적 생물방제균을 육종하고자 하였다. 이를 위해 최근 생물방제균으로 주목받고 있는 Enterobacter cloacae의 길항기작이 이 균에 의해 토양내에 생산되는 휘발성 암모니아이었다는 보고를 근거로 하여, 이미 일련의 식물근부균 생물방제연구에서 선발된 식물방제균 Bacillus subtilis YB-70에 암모니아 생산에 필요한 urease 유전자를 유전적으로 부가함으로써 식물근부균 F. solani에 대한 길항력이 훨씬 강화된 생물방제균을 육종할 수 있었다. Urease 유전자는 토양세균 중 urease 생산능이 가장 큰 Bacillus pasteurii의 urease gene library (10.7 Kb)를 B. subtilis YB-70에 가장 잘 발현되는 plasmid vector pGB215-110의 HindⅢ site에 삽입하였으며, 이렇게 제작된 urease 함유 recombinant plasmid (pGU266)을 생물방제균 B. subtilis YB-70에 alkali cation competent transformaiton으로 도입시켰다. 이때 transformation의 최적 조건을 조사한 결과 PEG 40% 존재하에 2.5 ㎍/㎖의 DNA첨가로 가장 높은 형질전환율을 얻을 수 있었으며 일단 도입된 urease 유전자는 상당히 안정하게 B. subtilis YB-70 내에서 유지되었으며, 그 transformation된 recombinant plasmid의 존재도 Birnboim방법으로 agarose gel 상에서 확인할 수 있었다. 또한 urease 유전자가 도입된 형질전환체 B. subtilis (pGU266)은 urease gene이 도입되지 않은 B. subtilis YB-70에 비해 식물근부균 F. solani에 대한 길항력이 훨씬 증강됨도 확인되었다. To genetically breed powerful multifunctional antagonistic bacteria, the urease gene of alkalophilic Bacillus pasteurii was transferred into Bacillus subtilis YB-70 which had been selected as a powerful biocontrol agent against root-rotting fungus Fusarium solani. Urease gene was inserted into the HindⅢ site of pG215-110 and designated pGU266. The plasmid pGU266 containing urease gene was introduced into the B. subtilis YB-70 by alkali cation transformation system and the urease gene was very stably expressed in the transformant of B. subtilis YB-70(pGU266). The optimal conditions for the transfomation were also eveluated. From the in vitro antibiosis tests against F. solani, the antifungal activity of B. subtilis YB-70 containing urease gene was much efficient than that of the non-transformed strain. Genetic improvement of B. subtilis YB-70 by transfer of urease gene for the efficient control seemed to be responsible for enhanced plant growth and biocontrol efficacy by combinding its antibiotic action and ammonia producing ability.

Escherichia coli에서 발현된 Recominant Bacillus pasteurii Urease의 정제 및 효소학적 특성

이은탁,김상달 한국산업미생물학회 1992 한국미생물·생명공학회지 Vol.20 No.5

Bacillus pasteurii의 urease gene이 Escherichia coli HB101에서 발현된 Bacillus성 recombinant urease를 단일단백으로 정제하고 그 효소학적 특성을, 별도로 정제한 B. pasteurii urease의 그것과 비교검토하였다. B. pasteurii urease gene이 cloning된 E. coli HB101(pBU11)의 균체파쇄액으로 부터 TEAE-cellulose, DEAE-Sephadex A-50, Sephadex G-150, Sephadex G-200 등의 이온교환 크로마토그래피와 gel filtration을 이용하여 E. coli내에서 발현된 B. pasteurii성 recombinant urease를 비활성도 기준 63.1배까지 정제하였으며, 또한 B. pasteurii로부터도 비활성도 185.2배의 urease를 정제하여 disc gel electrophoresis로 단일 단백으로 정제되었음을 확인하였다. 정제된 두 urease들의 native 상태의 분자량은 공히 280,000±10,000 정도로 확인되었고, SDS-electrophoresis에 의해 subunit 유무와 분자량을 확인한 결과도 67,000 정도의 subunit 4개와 20,000의 subunit 1개로 된 α_4β구조의 동일한 효소단백으로 추정할 수 있었다. Gene donor인 B. pasteurii와 cloning된 균주 E. coli(pBU11)이 생산한 두 urease의 효소학적 특성을 비교 조사해본 결과 두 urease의 최적반응 pH는 공히 7.5로 나타났으며, pH에 대한 안정성도 두 urease가 공히 pH 5.5에서 10.5 사이에서 50% 이하로 활성이 떨어지지 않는 강한 pH 안정성을 보였다. 두 urease의 최적반응의 온도는 60℃였으며, 비교적 온도에 대한 저항이 강한 효소임을 알았다. 두 urease의 활성에 미치는 금속이온의 영향은 Ag^2+, Hg^2+ 등에서 양효소가 모두 강한 저해현상을 받는 반면, Mn^2+, Mg^2+에서는 다소 촉진되는 현상을 보였다. 효소반응 저해제들의 영향을 조사해 본 결과 ρ-CMB, acetohydroxamic acid에 두 urease가 모두 강한 저해를 받았다. 두 urease의 K_m값과 V_max값은 E. coli(pBU11)의 urease는 4.21×10^-2 mol/ℓ, 86.96 μmol/min이었고, B. pasteurii urease는 4.04×10^-2 mo1/ℓ, 160 μmol/min이었다. 따라서 B. pasteurii의 urease나 그 urease gene으로 cloning되어 E. coli내에서 발현된 recombinant urease는 분자량이나 효소학적 특성에서 거의 동일한 효소단백임을 알 수 있었다. The gene coding for urease of alkalophilic Bacillus pasteurii had been cloned in Escherichia coli previously. The urease protein was purified 63.1-fold by TEAE-cellulose, DEAE-Sephadex A-50, Sephadex G-150 and Sephadex G-200 chromatographies with a 7.3% yield from the sonicated fluid of the E. coli HB101(pBU11) encoding B. pasteurii urease gene. The ureases of E. coli (pBU11) and B. pasteurii possessed as a K_m for urea, 42.1 mM and 40.4 mM, respectively. They hydrolyzed urea with V_max of 86.9 μmol/min and 160 μmol/min, respectively. Both ureases were composed with four subunits (Mrs 67,000) and a subunit (Mr 20,000). The molecular weight of both native enzymes was Mr 280,000±10,000 determined by gel filtration chromatography and Coomassie blue staining of the subunits. The optimal reaction pH of both ureases were pH 7.5. The ureases were stabled in pH 5.5∼10.5. The optimal reaction temperature of both ureases were 60℃, and the ureases were stable for an hour at 50℃, 40min at 60℃ and 10min at 70℃ The activity of both enzymes were inhibited completely by Ag^2+, Hg^2+, Zn^2+, Cu^2+, and were inhibited 60% by Co^2+, 30% by Fe^2+ and 10% by Pb^2+. However it was increased by the addition of Sn^2+, Mn^2+, Mg^2+ at concentration of 1×10^-3 M. Both ureases were inhibited completely by ρ-CMB and atetohydroxamic acid. The urease expressed in E. coli (pBU11) was inhibited 70% by SDS. The urease of B. pasteurii was inhibited 40% by hydroxyurea, whereas the recombinant urease of E. colistrain was inhibited 17%. Both enzymes were not inhibited by cyclohexanediaminetetraacetic acid(CDTA) and ethylendiaminetetraacetic acid (EDTA).

세균성 Urease Gene에 의한 모기유충 방제균 Bacillus sphaericus 1593의 형질전환

한길환,김상달 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.4

위생곤충이나 농업해충의 방제목적으로 남용되는 화학살충제의 피해를 줄이기 위해 Bacillus sphaericus를 이용하는 생물학적 해충방제법의 연구가 진행되고 있다. 모기유충방제균인 B. sphaericus 1593의 살충단백질은 alkali 조건하에서 활성도가 증가된다. Urea의 효소적분해로 ammonia의 생산을 증강하여 독소단백질 주위의 pH를 알칼리화 할려는 시도를 하였으며, 이를 위해 세균성 urease의 생산유전자를 B. sphaericus 1593에 도입시키고자 하였다. Urease 유전자는 세균 중 urease 생산력이 가장 강한 Bacillus pasteurii의 urease 유전자를 cloning 한 재조합 plasmid pGU66을 사용하였다. B. sphaericus 1593 내에 있는 cryptic plasmid를 먼저 제거하고 원형질체 형질전환 방법으로 recombinant palsmid pGU66을 도입함으로서 80% 이상의 urease 활성 증가를 이룰 수 있었으며, 도입된 urease가 transformant 내에서 매우 안정하게 발현됨을 확인하였고, 아울러 urease 유전자로 형질전환된 B. sphaericus 1593(pGU66) 내에서도 모기유충 살충단백질이 변함없이 생산됨을 전자 현미경으로 확인할 수 있었다. Bacillus sphaericus 1593 is a larvicidal toxin-producing mosquitocidal bacterium. The toxin contains a parasporal crystalline inclusion which is composed of a protein that is activated under alkaline condition. To enhance alkaline environment around toxin protein, cryptic plasmid cured, B. sphaericus 1593 was transformed by the Bacillus pasteurii urease gene which generate ammonia from urea. Transformant produced urease at about 80% more than wild type strain. B. sphaericus 1593, and the urease gene was stably maintained. It also produced crystalline toxin protein at the same level as the wild type strain B. sphaericus 1593.

Bacillus parteurii Urease Gene의 생물방제균 Bacillus subtilis YBL-7내에서의 발현

김용수,김상달 한국산업미생물학회 1991 한국미생물·생명공학회지 Vol.19 No.4

식물근부병의 방제균으로 선발된 Bacillus subtilis YBL-7의 식물근부균 Fusarium solani에 대한 길항력을 유전공학적 조작에 의해 다목적으로 증강시킬 수 있는지를 타진하기 위해 외부유전자인 Bacillus pasteurii의 urease 유전자를 생물방제균 B. subtilis YBL-7내로 도입하고자 시도하였다. 외부 urease 유전자는 B. pasteurii의 urease gene을 shuttle vector인 pGR71의 HindIII site에 삽입하여 E. coli내에서 발현시킨 pGU66을 사용하여 형질전환시켰으며 이때의 최적 형질전환조건과 도입된 urease 유전자의 발현을 조사해 보았다. 그 결과 B. subtilis YBL-7의 원형질체를 200㎍/㎖의 lysozyme으로 42℃에서 90분 처리하였을 때 그 형질전환율이 가장 높았으며, 플라스미드 DNA 첨가농도는 1.2㎍까지는 증가하였으나 그 이상의 농도에서는 일정하였다. 또한 최종농도가 30%(v/v)의 PEG 4000를 사용한 경우에서 최적의 형질 전환율을 얻을 수 있었으며, 상기의 최적조건으로 형질전환시켜 본 결과 약 5×10 exp(-3)의 빈도를 나타냈다. 그리고 형질전환체에서 urease 생성력도 강하게 나타났으며 플라스미드 안정성도 비교적 높았다. Urease 유전자를 함유한 pGU66으로 형질전환된 형질전환체에서도 F. solani에 대한 항진균성 물질이 숙주균주와 동일하게 생성되었다. To investigate the possibility of genetic development for a multi-purpose strain of Bacillus subtilis YBL-7 against Fusarium solani causing root rot of many impotant corps, the plasmid pGU66 inserting urease gene of Bacillus pasteurii had been introduced into Bacillus subtilis YBL-7 by PEG-induced protoplast(PIP) transformation system. Protoplasts of B. subtilis YBL-7 were prepared by treating the cells with lysozyme (200㎍/㎖) in hypertonic buffer (SMMP). The highest transformation frequency was achieved when cells of the strain with lysozyme at 42℃ for 90 minutes. Optimal transformation was obtained using polyethylene glycol (MW 4000) at final concentration of 30%(V/V). The transformation frequency was increased proportionally to 1.2㎍ of plasmid DNA. At best condition, the transformation frequency (transformants/regenerants/㎍ of DNA) for pGU66 was appoximately 4×10 exp(-3). Also, the urease gene was strongly expressed in the transformants of B. subtilis YBL-7 and maintained steadily. The antifungal ability of transformant was very similar to that of B. subtilis YBL-7.

Helicobacter pylori Urease Activity is Influenced by Ferric Uptake Regulator

이종승,이지혁,이혜진,이지현,최영옥,최연호 연세대학교의과대학 2010 Yonsei medical journal Vol.51 No.1

Purpose: The role of the Ferric Uptake Regulator (FUR) in the acid resistance of Helicobacter pylori (H. pylori)has been thought to be independent of urease. However, we demonstrated in this study that Fur influences urease activity. Materials and Methods: A fur knockout mutant of H. pylori was constructed by replacing the Fur gene with a kanamycin resistant marker gene. The wild-type H. pylori and fur mutant were compared for survival. The integrity of the inner membrane of the bacteria was evaluated by confocal microscopy using membrane-permeant and -impermeant fluorescent DNA probes. Urease activity of intact H. pylori was measured between pH 3 and 8. Real time PCR of both strains was performed for urease genes including ureI, ureE, ureF, ureG, and ureH. Results: The fur deletion affected the survival of H. pylori at pH 4. The urease activity curve of the intact fur mutant showed the same shape as the wild-type but was 3-fold lower than the wild-type at a pH of less than 5. Real time PCR revealed that the expression of all genes was consistently down-regulated in the fur mutant. Conclusion:The results of this study showed that fur appears to be involved in acid resistant H. pylori urease activity.

Deletion Mutageneses of the Helicobacter pylori Urease Accessory Genes

Lee, Mann-Hyong,Sung, Jae-Young Korean Society of Life Science 1999 Journal of Life Science Vol.9 No.1

Helicobacter pylori is the etiologic agent of human gastritis and peptic ulceration and produces urease as the major protein component on its surface. H. pylori urease is known to serve as a major virulence factor and a potent immunogen. Deletion mutageneses were performed in the H. pylori urease accessory genes by using combinations of restriction enzymes and other DNA modifying enzymes in order to assess the function of these accessory gene products in the expression of the active urease. Selective disruptions in the accessory gene regions resulted in complete abolishment of the urease activity, which is consistent with other bacterial ureases. Interestingly, deletions in ureE-containing regions caused reduced expression of the structural enzyme subunits.

해수에서 urease 양성 Photobacterium sp. Strain HA-2의 분리 및 동정

김강진,노아름,박권삼 한국수산과학회 2009 한국수산과학회지 Vol.42 No.6

A urease-positive bacterium isolated from sea water was identified as Photobacterium sp. by morphological, biochemical, and 16s rRNA gene analyses and named Photobacterium sp. strain HA-2. 2.0-fold increase enzyme activity was observed in LB medium containing 3% NaCl and 0.1% urea or not and the enzyme activity was 16.0-fold lower compared to urease-positive Vibrio parahaemolyticus AQ4673 strain when grown in the LB medium containing 3% NaCl with 0.1% urea. The cloning and sequencing of Photobacterium sp. strain HA-2 urease gene cluster is currently being analyzed in our laboratory.

Effect of the Urease Accessory Genes on Activation of the Helicobacter pylori Urease Apoprotein

박정욱,Young-Cheol Kwon,정미자,전진수,박정원,박승규,Hyang-Ran Hwang,Sang-Haeng Choi,Seung-Chul Baik,강형련,윤희상,Won-Kon Lee,이광호,송재영,조명제 한국분자세포생물학회 2005 Molecules and cells Vol.20 No.3

The roles that accessory gene products play in activating the Helicobacter pylori urease apoprotein were examined. The activity of the urease apoprotein increased in the following order when it was expressed with the accessory genes: ureG < ureGH < ureFGH <ureEFGH < ureIEFGH. Moreover, stepwise additions of ureE and ureI to ureFGH significantly increased urease activity. Urease apoproteins coexpressed with ureFGH, ureEFGH, and ureIEFGH had similar low chymotrypsin susceptibilities. In vivo and in vitro activation studies showed that the cooperative effect of the accessory proteins involved processes in which the UreFGH complex, UreE, and UreI were implicated. Thus, the UreFGH complex may serve to alter the conformation of the apoprotein into one that is more competent to assemble a stable metallocenter, and that facilitates cooperative effects.

Genetic Organization of the Recombinant Bacillus pasteurii Urease Genes Expressed in Escherichia coli

KIM, SANG DAL,HAUSINGER, ROBERT P. 한국미생물 · 생명공학회 1994 Journal of microbiology and biotechnology Vol.4 No.2

The genetic organization of the urease gene cluster from an alkalophilic Bacillus pasteurii was determined by subcloning and Tn5 transposon mutagenesis of a 10.7 kilobasepair cloned fragment A region of DNA between 5.0 and 6.0 kb in length is necessary for urease activity. In vitro transcription-translation analysis of transposon insertion mutants of the cloned urease genes demonstrated that the major (M_r 67,000) and minor (M_r 20,000) structural peptides of urease are encoded at one end of the urease gene cluster and at least 3 additional polypeptides are encoded by adjacent DNA sequences.