벼 흰잎마름병균(Xanthomonas oryzae pv. oryzae)의 병원성 유전자의 분자유전학적 연구현황 및 비교유전체 분석

강희완,박영진,이병무,Kang, Hee-Wan,Park, Young-Jin,Lee, Byeong-Moo 한국미생물학회 2008 미생물학회지 Vol.44 No.1

본 논문은 벼 흰 잎마름병균인 Xanthomonas oryzae pv. oryzae(Xoo)의 병원성 유전자의 분자유전학적 연구현황을 기술하고자 한다. 또한 국내 고유 벼 흰 잎마름병균 KACC10331의 유전체해독 정보를 기반으로 다른 Xanthomonas의 유전체와 비교 분석함으로써, Xoo의 주요 병원성 유전자의 분자구조를 구명하고자 한다. 이를 통해 Xoo 고유 병원성 유전자 탐색 및 기능 해석을 위한 기초자료를 제공하는데 목적이 있다. Xoo 유전체에는 5개 과(family)에 속하는 9 type의 Insertion sequence(IS)가 611 copy로 존재하고 있으며, 주로 병원성 관련 유전자군 주위에 많이 분포하고 있는 것으로 나타났다. 현재까지 연구가 수행된 주요 병원성 관련 유전자인 hypersensitive response and pathogenicity (hrp) 유전자, extracellular polysaccharide (EPS) 유전자, extracellular enzyme 유전자, lipopolysaccharides (LPS) 유전자, 그리고 avilulence 유전자의 분자유전학적 연구현황을 기술하였다. Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of bacterial blight on rice. In this paper, current status on molecular genetic study of major virulence genes, hypersensitive response and pathogenicity (hrp), productions of extracellular polysaccharide (EPS), extracellular enzymes and lipopolysaccharides (LPS), avr genes were reviewed. The IS elements with 611 copies including 133 ORF IS were inserted in various regions of the Xoo genome and in expecially regions franking virulence genes. Whole genome sequence of X. oryzae pv. oryzae KACC10331 were used for defining genetic organization of the virulence genes. Futhermore, the virulence genes in Xoo genome were compared to those of other Xanthomonas species in Blast GenBank data base.

Genus-Specific Distribution and Pathovar-Specific Variation of the Glycinecin R Gene Homologs in Xanthomonas genomes

Eunjung Roh,Sunggi Heu,Eunpyo Moon 한국미생물학회 2008 The journal of microbiology Vol.46 No.6

Xanthomonas axonopodis pv. glycines produces bacteriocins called glycinecin, and two glycinecin genes, glyA and glyR, were reported previously. In this paper, we describe genomic distribution and variation of the glyR gene revealed by extensive Southern hybridization analysis. In contrast to the glyA gene present only in X. axonopodis pv. glycines, the glyR gene was found to be distributed widely in all the pathovars of Xanthomas genus. It was also found that the glyR gene is a multigene family while the glyA is a single copy gene. Moreover, the copy number and the variation of the glyR multigene are unique to each pathovar of Xanthomonas. The uniqueness can be easily detected by the patterns resulted from Southern hybridization using the genomic digests. Thus, we suggest the glyR gene can serve as a useful genus-specific and pathovarspecific DNA marker for Xanthomonas. One of the glyR homologs was further isolated from X. axonopodis pv. glycines, and analyzed to be functional with strong inhibitory activity against several members of Xanthomonas.

Biochemical Characteristics of an Alanine Racemase from Xanthomonas oryzae pv. oryzae

( Han Chul Kang ),( Sang Hong Yoon ),( Chang Muk Lee ),( Bon Sung Koo ) 한국응용생명화학회 2011 Journal of Applied Biological Chemistry (J. Appl. Vol.54 No.4

A gene encoding a putative alanine racemase in Xanthomonas. oryzae pv. oryzae was cloned, expressed and characterized. Expression of the cloned gene was performed in Escherichia coli BL21(DE3)pLys using a pET-21(a) vector harbouring 6 × histidine tag. Purification of the recombinant alanine racemase by affinity chromatography resulted in major one band by sodium dodecyl sulfate polyacryl amide gel electrophoresis analysis, showing about 45 kDa of molecular weight. The alanine racemase gene, cloned in this experiment, appears to be constitutively expressed in X. oryzae, as analyzed by reverse transcriptase polymerase chain reaction. The enzyme was the most active toward L-alanine and secondly D-alanine, showing a racemic reaction, thus the enzyme is considered as an alanine racemase. The enzyme was considerably activated by addition of pyridoxal-5-phosphate (PLP), showing that 75% increase in activity was observed at 0.3 mM, compared with control. D-Cysteine as well as L-cysteine significantly inhibited the enzyme activity. The inhibitions by cysteines were more prominent in the absence of PLP, showing 9 and 5% of control activity at 2 mM of addition, respectively. The enzyme was the most active at pH 8.0 and more stable at alkaline pHs than acidic pH condition.

Xanthan Gum Production of Xanthomonas spp. Isolated from Different Plants

Tuncay Gumus,A. Sukru Demirci,Mustafa Mirik,Muhammet Arici,Yesim Aysan 한국식품과학회 2010 Food Science and Biotechnology Vol.19 No.1

Xanthan gum were produced from the following Xanthomonas strains; standard strain Xanthomonas campestris NRRL B-1459 and isolated strains Xanthomonas arbicola pv. juglandis, Xanthomonas axonopodis pv. vesicatoria, Xanthomonas axonopodis pv. begonia,Xanthomonas axonopodis pv. dieffenbachia. The viscosity features of the xanthan gums obtained were determined at 25-80℃ with different pH values and were compared to commercial xanthan gum. Our results indicate that X. arbicola pv. juglandis showed the highest productivity (8.22±1.52 g/L gum). This was followed by X. axonopodis pv. begonia (7.74±1.30 g/L gum), and the control bacterial strain X. campestris NRRL B-1459 (7.46±0.28 g/L gum). X. axonopodis pv. vesicatoria showed the lowest productivity (6.40±0.55 g/L gum). No xanthan gum could be obtained from X. axonopodis pv. dieffenbachia. Xanthan gum produced by X. axonopodis pv. vesicatoria showed the highest viscosity value (428 mPa·sec at 1%solution) in all Xanthomonas strains isolated from plants.

암모니아 산화 세균 Xanthomonas maltophilia SU6 와 Achromobacter sp. 12A에 의한 nitrite 생성의 비교

최미경,한효영,정영희,민경희 숙명여자대학교 환경과학연구소 1995 환경과학 Vol.2 No.-

암모니아를 NO_2^-로 산화, 이용하는 미생물 균주들을 한강 주변의 토양에서 분리하였으며, 이들은 Xanthomonas maltophilia SU6와 Achromobacter sp. 12A로 동정하였다. 이 균주들을 암모니아가 첨가된 액체 최소 배지에서 2주 동안 배양한 후, 생성되는 NO_2^-의 양을 측정하여 암모니아 산화의 활성을 조사하였다. 생장 최적 pH는 Xanthomonas maltophilia SU6 와 Achromobacter sp. 12A 균주가 7.0-8.0 이었으며, 생장에 필요한 무기질소원으로서 NH_4Cl 의 최적 기질 농도는 두 균주가 모두 10mM 이었다. 암모니아 산화 균주의 활성을 높이기 위하여 Xanthomonas maltophilia SU6와 Achromobacter sp. 12A를 12시간 배양한 후에 50mM의 hydroxylamine을 배지에 첨가하였을 때 각각 467㎍/1, 641㎍/1 의 NO_2^-를 유도 생성하였다. Hydroxylamine oxidase 의 활성에 미치는 기질의 영향을 규명하기 위하여 Xanthomonas maltophilia SU6 와 Achromobacter sp. 12A를 8시간 동안 암모니아가 포함된 최소배지에서 배양한 후에, hydroxylamine 을 첨가한 결과 hydroxylamine oxidase 의 비활성은 109units/㎎ protein 와 132 units/㎎ protein 으로 증가하였다. Hydroxylamine oxidase 의 최적 pH는 7.5, 최적 온도는 30℃ 였으며, 열에 대한 안정성에서는 Xanthomonas maltophilia SU6 와 Achromobacter sp. 12A 는 50℃, 60분에서 각각 30%와 25%의 안정성을 보여주었다.

알칼리성 Protease를 생산하는 Xanthomonas sp. YL-37의 분리 및 조효소의 성질

이창호,권태종,강상모,서현효,권기석,오희목,윤병대 한국산업미생물학회 1994 한국미생물·생명공학회지 Vol.22 No.5

저온에서 높은활성을 나타내며, 계면활성제에 내성이 있는 알칼리성 protease를 생산하는 균주를 토양으로부터 분리·선발하였으며, Xanthomonas sp. YL-37로 명명하였다. 효소생산을 위한 배양 최적온도, 초발 pH, 그리고 배양시간은 각각 20℃, 11.0, 그리고 84시간이였다. Jar fermenter 배양을 했을 때 배양 후 108시간에 효소의 역가는 약 15,000 DU/㎖-broth였다. 본 알칼리성 protease의 최적온도 및 pH는 70℃ 및 10.0이였으며, 20℃ 에서도 최적활성의 약 40%를 유지하였다. PH 및 열안정성은 각각 pH 7.0∼12.0, 온도 50℃ 이하에서 비교적 안정하였다. 또한 Xanthomonas sp. YL-37이 생산하는 protease는 PMSF에 강하게 저해를 받아서 본 효소는 활성부위에 serine 잔기를 가지고 있는 일종의 serine protease로 사료된다. A bacterial strain, which showed the high protease activity at low temperature and the high tolerance for the surfactant, was isolated from soil and identified as Xanthomonas sp. YL-37. The optimal temperature, initial pH, and cultivation time for the production of the alkaline protease by Xanthomonas sp. YL-37 were 20℃, 11.0, and 84 hours, respectively. In the jar fermenter culture of Xanthomonas sp. YL-37, the alkaline protease activity was about 15,000 DU/㎖-broth after cultivating for 108 hours. The optimal pH and temperature for the protease activity were 70℃ and 11.0, respectively. The protease was relatively stable at the pH range of 7.0∼12.0 and at the temperatures below 50℃. The protease activity at 20℃ was about the level of 40% of its activity at 70℃. The enzyme was suggested as a serine protease because the enzyme activity was inhibited by phenylmethane sulfonyl fluoride, a serine modifier.

Specificity of Multiplex PCR in the Detection of Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola in Rice (Oryza sativa L.) Seeds

Da-Young Lee,Vera Cruz, C. M. 한국국제농업개발학회 2014 韓國國際農業開發學會誌 Vol.26 No.4

종자 등 유전자원의 국가간 교류는 새로운 병원균을 유입시켜 새로운 병을 일으키고 경제적인 손실을 극대화 시킬수 있는 원인을 제공하기 때문에 국가별로 검역이 더욱 강화되고 있는 실정이다. 그러한 연유로 국가별로는 자국에 존재하지 않은 유사한 병원균을 발견할 경우 종자도입을 막고 있어 불필요한 경제적 손실원인이 되기도 한다. 따라서 이러한 손실을 막기위해 각국에 검역소에서는 병원균의 형태적인 감별보다 더 정확한 검정기술이 시급한 실정이다. 본 연구에서는 다중PCR을 통하여 한번의 PCR 증폭으로 벼종자에 존재하는 벼흰잎마름병을 일으키는 Xanthomonas oryzae pv. oryzae (Xoo)과 세균성 줄무늬병을 일으키는 Xanthomonas oryzae pv.oryzicola (Xoc) 를 검출하고 이 두병원균을 판별할수 있는, 국제미작연구소가 최적화한 다중PCR 검정방법의 실질적 적용성 여부를 검증하기 위한 연구였다. 그 결과 51개의 비병원성이나 Xoo 또는 Xoc 와 유사하며 벼종자에서 분리된 균들은 다중PCR검정에서 어떤 밴드도 증폭하지 않은 반면, 대조구(positive control)에서는 알맞은 band size의 amplicon들을 증폭해냈다. 따라서 본 연구에 사용된 국제미작연구소에서 최적화된 다중 PCR방법 (IRRI-optimized multiplex PCR)은 벼종자에서 발견할수 있는 Xoo와 Xoc 의 존재 여부 뿐만 아니라 이들과 형태적으로 유사하나 비병원성균으로부터 구분해 내는데 탁월한 방법이었음을 확인할 수 있었다. One of the major hurdles faced by numerous quarantine centers is to accurately detect and distinguish non-plant pathogenic bacteria from plant pathogenic bacteria based on colony morphology. Yellow colony-forming, non-plant pathogenic bacteria found in rice seeds are often misidentified as Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), causal agents of bacterial leaf blight and bacterial leaf streak, respectively. In this study, 51 non-pathogenic, yellow-colony forming, Xoo and Xoc look-alike bacteria isolated from rice seeds, were used to test the specificity of a multiplex PCR for the detection and differentiation of Xoo and Xoc. Four primer pairs used in this multiplex PCR specific for Xanthomonas oryzae (Xo), Xoo and Xoc were used and all 51 isolates did not amplify any band, indicating that they are not Xo, Xoo or Xoc. These results imply that the multiplex PCR used in this study is robust in detecting and differentiating Xoo and Xoc from non-pathogenic, rice seed-associated, yellow colony-forming bacteria.

알칼리성 Prottease를 생산하는 Xanthomonas sp. YL-37의 분리 및 조효소의 성질

이창호,권태종,강상모,서현효,권기석,오희목,윤병대,Lee, Chang-Ho,Kwon, Tae-Jong,Kang, Sang-Mo,Suh, Hyun-Hyo,Kwon, Gi-Seok,Oh, Hee-Mock,Yoon, Byung-Dae 한국미생물 · 생명공학회 1994 한국미생물·생명공학회지 Vol.22 No.5

A bacterial strain, which showed the high protease activity at low temperature and the high tolerance for the surfactant, was isolated from soil and identified as Xanthomonas sp. YL-37. The optimal temperature, initial pH, and cultivation time for the production of the alkaline protease by Xanthomonas sp. YL-37 were 20$\circC , 11.0, and 84 hours, respectively. In the jar fermenter culture of Xanthomonas sp. YL-37, the alkaline protease activity was about 15,000 DU/ml/-broth after cultivating for 108 hours. The optimal pH and temperature for the protease activity were 70$\circC and 11.0, respectively. The protease was relatively stable at the pH range of 7.0~12.0 and at the temperatures below 50$\circC . The protease activity at 20$\circC was about the level of 40% of its activity at 70$\circC . The enzyme was suggested as a serine protease because the enzyme activity was inhibited by phenylmethane sulfonyl fluoride, a serine modifier.

Characterization of Nonhypersensitive Mutant and Nonpathogenic Mutant of Xanthomonas campestris pv. campestris

Kim, Mee Hyang,Bae, Dong Won,Lee, Jun Teak,Yun, Han Dae,Kim, Hee Kyu 경상대학교 유전공학연구소 1997 遺傳工學硏究所報 Vol.16 No.-

십자화과 식물에 검은썩음병을 일으키는 Xanthomonas campestris pv. campestris에 대하여 담배 18품종중 가장 과민반응을 강하게 나타내는 품종(Nicotiana tabacum cv. TC500)을 과민반응 비기주식물로 선발하였다. NTG 돌연변이를 실시하여 얻은 약 4,500개의 mutants들을 10^8 cells/ml의 농도로 주사 접종하여 비기주식물인 담배 잎에서 과민반응을 유도하지 않은 HR^- mutants(XHN 514-774, XHN620-831)를 선발하였고, 기주식물인 양배추 잎에서 병원성을 나타내지 않는 비병원성변이주(XPN 1001)를 선발하였다. HR^- mutants를 기주식물인 양배추에 접종한 결과 병원성을 나타내었고, nonpathogenic mutants를 비기주식물인 담배 잎에 접종한 결과 과민반응을 유도하였다. 담배 잎에서HR^- mutants 와wild type의 population을 조사한 결과 wild type 은 24시간 이후 급격히 감소하는 반면, HR^- mutants는 일정수준을 유지하였으나 72시간 이후에 감소하였다. 그리고 mutants들의protease, cellulase,amylase,polygalacturonate lyase분비여부를 조사한 결과 wild type과 비슷하게 분비하였다. We have screened hypersensitive responses of 18 cultivars of Nicotiana tabacum to Xanthomonas campestris pv. campestris. TC500 cultivar produced the most strong hypersensitive mutants (HR) to Xanthomonas campestris pv. campestris. By NTG mutagenesis, nonhypersensitive mutants (XHN 514-774, XHN620-831) were generated, which dose not induce hypersensitive response on tobacco leaves(Nicotiana tabacum cv. TC500). Also non-pathogenic mutant(XPN 1001), which does not incite any of the black rot symptoms on leaves was generated. We observed that HR^- mutants were still pathogenic on cabbage leaves. The in planta growth of wild type and HR^- mutants were examined for up to 120 hrs after inoculation: population of wild type strain increased to more than 10^8 in 24 hrs, but rapidly declined thereafter; HR^- mutants increased to more than 10^6 in 48 hrs after inoculation but subsequently stabilized and slowly decreased. We observed that wild type and these mutants produced similar amounts of degardative enzymes such as protease, pectate lyase, cellulase and amylase.

Molecular Cloning of the cel Gene from Xanthomonas campestirs pv. oryzae

YUN, HAN DAE,LIM, SUN TECH,CHUNG, MIN HWA,PARK, YONG WOO,KIM, HEE KYU,KANG, KYU YOUNG 경상대학교 유전공학연구소 1991 遺傳工學硏究所報 Vol.10 No.-

Xanthomonas campestris pv. oryzae(Ishiyama) Dye is a cacusal orginism of bacterial leaf blight of rice(Oryzae sativa). It infects the rice through hydathod or wounds. No report on infection through stomata is available. The penetration of Xanthomonas campestris pv. oryzae into the plant requires the activity of cell wall hydrolyzing enzymes. Enzymatic process of infection was evidenced by smooth surfaces without trichomes upon inoculation and many penetrating bacterial cells through stoma by scanning eletron microscopy. The presence of pathogens in the mestome sheath of rice leaf was also confirmed by transmission electron microscopy. Furthermore, Xanthomonas campestris pv. oryzae exhibited the activities of extracellular cellulase and proteases as cell wall degrading enzymes, but not polygalactronase, pectate lyase, and hemicellulase by the agar diffusion method. Thus, we constructed the genomic libary of Xanthomonas campestris pv. oryzae using the cosmid vector pSAFR₃to assess the contribution of the cell wall degrading enzymes to the pathogenicity of Xanthomonas campestris pv. oryzae. Out of one thousand transductants, one extracellular cellulase and several proteases clones were isolated. This clone, designasted pXC3-43 as cel gene plasmid, independently expresses cellulase activity in an Escherichia coli background, and was mutagenized with Tn5 to make a cel^- mutant.