KR20080097568A - Methylobacterium oryzae cbmb20 - Google Patents

Methylobacterium oryzae cbmb20 Download PDF

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KR20080097568A
KR20080097568A KR1020070042587A KR20070042587A KR20080097568A KR 20080097568 A KR20080097568 A KR 20080097568A KR 1020070042587 A KR1020070042587 A KR 1020070042587A KR 20070042587 A KR20070042587 A KR 20070042587A KR 20080097568 A KR20080097568 A KR 20080097568A
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mtb
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사동민
임우종
김경아
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충북대학교 산학협력단
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Abstract

Methylobacterium oryzae CBMB20 is provided to provide novel microorganism of Methylobacterium sp. by producing ACC deaminase promoting growth of plant. Methylobacterium oryzae sp. nov. KACC11585 is novel microorganism of Methylobacterium sp. The Methylobacterium oryzae sp. nov. KACC11585 is separated from stem tissue of the rice plant and produces ACC deaminase.

Description

에이씨씨 디아미나제를 생산하는 신규미생물{Methylobacterium oryzae CBMB20}New microorganism that produces BC deaminase {Methylobacterium oryzae CBMB20}

도1은 0.5% 메탄올(v/v) 보충 AMS 배지(글루타르알데히드/사산화 오스뮴 고착, 금/팔라듐 코팅; Hitachi S-2500C) 상에서 CBMB20T 균주 세포의 주사전자 현미경 사진 횡선, 2㎛(위), 400㎚(아래)1 is a scanning electron micrograph transverse line of CBMB20 T strain cells on 0.5% methanol (v / v) supplemented AMS medium (glutaraldehyde / osmium tetraoxide fixation, gold / palladium coating; Hitachi S-2500C), 2 μm (above) ), 400 nm (below)

도 2는 1% 메탄올(v/v) 보충 NA 배지(글루타르알데히드/사산화 오스뮴 고착, 금/팔라듐 코팅; Hitachi S-2500C) 상에서 CBMC20T 균주 세포의 주사전자 현미경 사진 횡선, 2㎛(위), 400㎚(아래)Figure 2 shows scanning electron micrograph transverse lines of CBMC20 T strain cells on 1% methanol (v / v) supplemented NA medium (glutaraldehyde / osmium tetraoxide fixation, gold / palladium coating; Hitachi S-2500C), 2 μm (upper) ), 400 nm (below)

도3은 본 발명의 신규 미생물과 Methylobacterium 속에 속한 관련 종들의 위치를 나타낸 16S rRNA 유전자 서열 비교에 기초한 계통 발생도.3 is a novel microorganism and Methylobacterium of the present invention Phylogeny based on 16S rRNA gene sequence comparison showing the location of related species in the genus.

본 발명은 신규미생물에 관한 것으로서, 보다 상세하게는 ACC(1-aminocyclopropane 1-carboxylate) 디아미나제(Deaminase)를 생산하는 신규미생물에 관한 것이다.The present invention relates to a novel microorganism, and more particularly to a novel microorganism that produces ACC (1-aminocyclopropane 1-carboxylate) deaminase (Deaminase).

ACC 디아미나제는 메틸로박테리아(Methylovacterium)에 의해 생산되며 이는 식물성장에 기여 하는 것으로 보고 되고 있다. (Madhaiyan등 2006). 이러한 ACC 디아미나제를 생산할 수 있는 메틸로박테리아(Methylovacterium) 속은 엄격한 호기성, 그램-음성, 분홍색-착색성, 임의 메탄올자화성(PPFM) 박테리아 군을 포함하며, 이들은 세린 경로에 의해 메탄올 및 포름알데히드와 같은 단일-탄소 화합물을 이용하는 것뿐만 아니라, 광범위한 다-탄소 성장 기질을 이용할 수 있는 능력을 특징으로 한다(Green, 1992). ACC deaminase is produced by Methylovacterium , which is reported to contribute to plant growth. (Madhaiyan et al. 2006). The genus Methylovacterium capable of producing these ACC deaminases includes a stringent group of aerobic, gram-negative, pink-coloring, random methanolic (PPFM) bacteria, which are produced by the serine pathway with methanol and formaldehyde. In addition to using the same single-carbon compounds, it also features the ability to use a wide range of multi-carbon growth substrates (Green, 1992).

메틸로박테리아는 프로테오박테리아의 α2 하위군으로 분류되며, 현재 유효한 공식명칭을 갖는 22종으로 구성되어 있다(Heumann, 1962; Ito & Iizuka, 1971; Kouno & Ozaki, 1975; Patt 등, 1976; Rock 등, 1976; Austin & Goodfellow, 1979; Green & Bousfield, 1983; Urakami & Komagata, 1984; Bousfield & Green, 1985; Green 등, 1988; Urakami 등, 1993; Wood 등, 1998; Doronina 등, 2000, 2002; McDonald 등, 2001; Van Aken 등, 2004; Jourand 등, 2004; Anesti 등, 2004; Gallego 등, 2005a, b, c, 2006). 또한, 포플러 나무에서 분리한 신규한 메탄-이용성 종인 Methylovacterium populi가 밝혀지기 전까지 대표 종인 Methylovacterium organophilum이 메탄을 이용해 생장하는 유일한 PPFM 박테리아였다. Methylobacterium 속의 구성원은 자연에서 어디에서나 존재하며, 토양, 민물, 호수 침전물뿐만 아니라 그 외의 다른 고체 표면에서도 발견되고(Corpe & Rheem, 1989; Lidstrom & Chistoserdova, 2002), 특히 식물과 밀접하게 관련된다고 알려져 있다(Holland & Polacco, 1994; Lidstrom & Chistoserdova, 2002; Sy 등, 2005). 메 틸로박테리아 구성원과 식물의 관계는 식물 내질에 기생하거나 공생관계까지의 범위에 이른다(Sy 등, 2001; Koenig 등, 2002; Pirttila 등, 2000; Idris 등, 2006). Methylobacterium에 의한 식물성장 촉진의 가능한 메커니즘은 인돌-3-아세트산(IAA) 같은 식물성장 호르몬, 시토키틴 또는 비타민의 생산이 포함된다(Basile 등, 1985; Koenig 등, 2002; Trotsenko 등, 2001). 게다가, Methylobacterium 구성원은 박테리아 요소에 의한 식물의 질소대사와 관련된다고 보고되고 있고,(Holland & Polacco, 1992), Methylobacterium 균주는 콩 식물 뿌리에 작은 마디를 만듦으로써 효과적인 질소 고정 공생관계를 확립할 수 있으며(Sy 등, 2001), 스코틀랜드 소나무의 싹(눈) 에 공생하며 효소적으로 질소고정을 할 수 있다. (Pirttila 등, 2000). 더욱이, 본 발명자들은 1-아미노시클로프로판-1-카르복실레이트(ACC) 디아미나제를 생산하는 Methylobacterium 균주에 의해 카놀라에서 식물성장이 증진된다는 것을 보고한 바 있다(Madhaiyan 등 2006). Methylobacteria are classified into the α2 subgroup of the proteobacteria and consist of 22 species with official names currently valid (Heumann, 1962; Ito & Iizuka, 1971; Kouno & Ozaki, 1975; Patt et al., 1976; Rock 1976; Austin & Goodfellow, 1979; Green & Bousfield, 1983; Urakami & Komagata, 1984; Bousfield & Green, 1985; Green et al., 1988; Urakami et al., 1993; Wood et al., 1998; Doronina et al., 2000, 2002; McDonald et al., 2001; Van Aken et al., 2004; Jourand et al., 2004; Anesti et al., 2004; Gallego et al., 2005a, b, c, 2006). In addition, the representative species, Methylovacterium organophilum, was the only PPFM bacterium to grow with methane until a new methane- soluble species Methylovacterium populi isolated from the poplar tree was identified. Members of the genus Methylobacterium exist everywhere in nature and are found not only in soil, fresh water, lake sediment, but also on other solid surfaces (Corpe & Rheem, 1989; Lidstrom & Chistoserdova, 2002), and are especially known to be closely related to plants. (Holland & Polacco, 1994; Lidstrom & Chistoserdova, 2002; Sy et al., 2005). Methylobacteria The relationship between members and plants ranges from parasitic or symbiotic relationships to plant internals (Sy et al., 2001; Koenig et al., 2002; Pirttila et al., 2000; Idris et al., 2006). Possible mechanisms of the plant growth promoting according to the Methylobacterium is indole-3-acetic acid (IAA) are included, such as plant growth, the production of hormones, cytokines or vitamins chitin (Basile et al., 1985; Koenig et al., 2002; Trotsenko et al., 2001). In addition, Methylobacterium members have been reported to be involved in plant nitrogen metabolism by bacterial factors (Holland & Polacco, 1992), and Methylobacterium strains can establish effective nitrogen fixation symbiosis by making small nodes in the roots of soybean plants. (Sy et al., 2001), symbiotic to the shoots (Snow) of Scottish pine and can be enzymatically nitrogen-fixed. (Pirttila et al., 2000). Furthermore, we have reported that plant growth is enhanced in canola by the Methylobacterium strain producing 1-aminocyclopropane-1-carboxylate (ACC) diminase (Madhaiyan et al. 2006).

본 발명은 따라서 식물성장을 증진시키는 ACC 디아미나제를 생산할 수 있는 Methylobacterium 속의 신규미생물을 제공함에 그 목적이 있다.It is therefore an object of the present invention to provide a novel microorganism of the genus Methylobacterium capable of producing ACC deaminase to enhance plant growth.

상기한 목적을 당설하기 위하여 연구한 결과 본 발명자들은 메틸로박테리아 (Methylobacterium)속의 신규한 균주를 벼의 줄기 조직으로부터 분리하여 이를 메틸로박테리아 오라이자 에스피. 노브(Methylobacterium oryzae sp. nov.;CBMB20T)라 명명하였으며, 이 균주는 메틸로박테리아 오라이자 씨비엠비20 (Methylobacterium oryzae CBMB 20)이라는 명칭으로 2006. 1. 6일자로 농업생명공학 연구원에 기탁번호 KACC11585로 기탁되었다. (자료 1)As a result of studying the above object, the present inventors have isolated a novel strain of the genus Methylobacterium from the stem tissue of rice, which is used as the methylobacterial orisa sp. Knob (Methylobacterium oryzae sp. Nov .; CBMB20 T ) was named, and this strain was named Methylobacterium oryzae CBMB 20 (January 6, 2006) Deposited as KACC11585. (Document 1)

이하에서는 본 발명에 따른 신규미생물을 설명하기 위해 신규미생물의 분리, 동정과 그 균학적 특성을 실시 예를 참고로 하여 보다 상세히 설명한다.Hereinafter, in order to explain the novel microorganism according to the present invention, the isolation, identification and microbial characteristics of the new microorganism will be described in more detail with reference to Examples.

[실시 예1] 균주의 분리 및 분석,Example 1 Isolation and Analysis of Strains,

본 발명의 균주는 충청북도 농업기술원(대한민국 충북 청원군(36ㅀ5′N 127ㅀ57′E)으로부터 얻은 벼(Oryza staiva L,"남평")의 줄기조직으로부터 분리 하였다.The strain of the present invention was isolated from the stem tissue of rice (Oryza staiva L, "Nampyeong") obtained from Chungcheongbuk-do Agricultural Research and Extension Services (Chungwon-gun, Chungbuk, South Korea (36 민 5'N 127 ㅀ 57'E).

이 균주는 28℃에서 필터 멸균된 시클로헥시미드(10㎍/mL) 및 메탄올을 첨가한 (0.5% v/v) 암모늄/미네랄 염(AMS) 배지(Whittenbury 등, 1970) 상에서 분리 되었다. 균주는 통상적으로 1%(v/v) 메탄올로 보충한 영양 한천(NA: Difco) 배지 또는 선택적 AMS 배지 상에서 배양, 보존시켰다. 일반적인 프로토콜에 따라서 형태학적 특성을 연구했다(Gerhardt 등, 1994). Bozzola & Russel (1998)에 의해 설명된 루틴 실험을 위해 제조한 고착 물질 상에서 주사전자현미경(SEM)으로 관찰했다(도1 및 도2 참조). 시료를 임계점 건조시키고, 시료대에 올려놓고 금/파라듐으로 스퍼터-코팅한 다음, 전기장 방출 전자원을 장착한 GEMINI 칼럼 Hitachi S-2500C SEM을 사용하여 균주를 관찰 하였다.This strain was isolated on filter sterilized cycloheximide (10 μg / mL) and methanol (0.5% v / v) ammonium / mineral salt (AMS) medium (Whittenbury et al., 1970) at 28 ° C. Strains were typically cultured and preserved on nutrient agar (NA: Difco) medium or selective AMS medium supplemented with 1% (v / v) methanol. Morphological characteristics were studied in accordance with the general protocol (Gerhardt et al., 1994). Observations were made by scanning electron microscopy (SEM) on sticking materials prepared for the routine experiments described by Bozzola & Russel (1998) (see FIGS. 1 and 2). The sample was dried at the critical point, placed on the sample stand, sputter-coated with gold / palladium, and the strains were observed using a GEMINI column Hitachi S-2500C SEM equipped with an electric field emission electron source.

Sambrook 등(1989)에 의해 설명된 대로, AMS 배지에서 배양된 세포로부터 염색체 DNA를 추출하여 정제했다. 16S rRNA 유전자를 fD1 및 rP2 (Weisberg 등, 1991) 프라이머를 사용하여 증폭했고, 이전에 설명된 대로(Brosius 등, 1978; Shima 등, 1994), 자동 DNA 염기서열 분석기 (ABI Prism 310 유전 분석기)를 사용하여 Big-Dye 프라이머법에 의해서 16S rRNA 유전자의 서열을 작성했다. CLUSTAL W(Thompson 등, 1994)에 의해 데이터를 다중정렬한 후, MECTA 버전 3.1(Kumar 등, 2004)을 이용해 계통발생적 분석을 수행하였다. Kimura 2-변수 모델(Kimura, 1980)에 따른 옵션을 사용하여 계통간 거리를 측정하였고, 이웃-결합법(Suitou & Nei, 1987)을 사용하여 클러스터링을 수행하였다. 1000회 반복의 부트스트랩 값을 사용하여 분기들의 신뢰수준을 구했다(Felsenstein, 1985).As described by Sambrook et al. (1989), chromosomal DNA was extracted and purified from cells cultured in AMS medium. The 16S rRNA gene was amplified using the fD1 and rP2 (Weisberg et al., 1991) primers, and as previously described (Brosius et al., 1978; Shima et al., 1994), an automated DNA sequencer (ABI Prism 310 genetic analyzer) was used. The sequence of 16S rRNA gene was prepared by Big-Dye primer method. After multiplexing the data by CLUSTAL W (Thompson et al., 1994), phylogenetic analysis was performed using MECTA version 3.1 (Kumar et al., 2004). The distance between lines was measured using the option according to the Kimura 2-variable model (Kimura, 1980), and clustering was performed using the neighbor-combination method (Suitou & Nei, 1987). The bootstrap value of 1000 iterations was used to determine the confidence level of branches (Felsenstein, 1985).

영양적 특징을 Biolog Microstation(MicroLog-3, 4.01B)를 사용하여 결정했다. 분석은 Biolog GN2 마이크로타이터 플레이트에서 제조자의 지침에 따라 수행했는데, 플레이트를 28℃에서 7일간 배양한 후 반응을 관찰했다. Green & Bousfield(1982)에 의해 설명된 표준 프로토콜을 사용하여 탄소원 이용시험(Biolog 제외)을 수행하였다. 다른 생리학적 및 생화학적 특성은 제조자의 지침에 따라 API ZYM 및 API ZONE 갤러리(Bio Merieux)를 사용하여 시험했다. 1% 메탄올(v/v)을 첨가한 NA에서 48시간 성장시킨 균주에서 세포 지방산을 분석했다. 세포 지방산을 메틸에스테르 유도체로 만들어서(Sasser, 1990), 미생물 확인 시스템(MIDI: Microbial ID) 소프트웨어 패키지를 이용하는 기체 크로마토그래피(Hewlett Packard 6980)에 의해 분석했다.  Nutritional characteristics were determined using Biolog Microstation (MicroLog-3, 4.01B). Assays were performed according to the manufacturer's instructions on Biolog GN2 microtiter plates, which were incubated at 28 ° C. for 7 days and observed for reaction. Carbon source utilization tests (except Biolog) were performed using the standard protocol described by Green & Bousfield (1982). Other physiological and biochemical properties were tested using API ZYM and API ZONE Gallery (Bio Merieux) according to the manufacturer's instructions. Cellular fatty acids were analyzed in strains grown for 48 hours in NA with 1% methanol (v / v). Cellular fatty acids were made into methyl ester derivatives (Sasser, 1990) and analyzed by gas chromatography (Hewlett Packard 6980) using a Microbial ID (MIDI) software package.

게놈 DNA의 G+C 함량을 역상-칼럼(Supelcosil LC18-S; Supelco)으로, DNA 가수분해 및 탈인산화(Mesbah 등, 1989)에 의해 생긴 각 뉴클레오시드를 HPLC 분석하 여 측정했다. DNA+DNA 혼성화를 Seldin & Dubnau(1985)에 의하여 설명된 필터 혼성화법에 따라 수행하였다. 프로브 표지부착 비-방사선 DIG-High Prime 시스템(Roche Diagnostics)을 사용하여 수행했고, 혼성화된 DNA를 DIG 발광 검출 키트(Roche Diagnostics)를 사용하여 가시화했다. 혼성화 온도는 60℃ 및 65℃였으며, DNA-DNA 관련성을 농도계(Bio-Rad Laboratories)를 사용하여 정량했다.G + C content of genomic DNA was determined by reverse phase-column (Supelcosil LC18-S; Supelco) and each nucleoside produced by DNA hydrolysis and dephosphorylation (Mesbah et al., 1989) by HPLC analysis. DNA + DNA hybridization was performed according to the filter hybridization method described by Seldin & Dubnau (1985). Probe labeled non-radiative DIG-High Prime system (Roche Diagnostics) was performed and hybridized DNA was visualized using DIG Luminescence Detection Kit (Roche Diagnostics). Hybridization temperatures were 60 ° C. and 65 ° C., and DNA-DNA relevance was quantified using a bio-rad laboratories.

균주 CBMB20T은 엄격한 호기성, 그램-음성, 비-포자-형성성이었고, 분홍색 내지 적색 착색된 콜로니를 형성했다. 세포는 막대 모양이었고, 대체로 분기가 있었으며, 고체 AMS 및 NA 배지 상에서 단일 또는 군집 형태로 생겼다. CMBM20T 균주의 SEM 현미경 사진은 도1 및 2로 나타내었다. mxaF 유전자의 확인은 PCR을 이용하였으며 f1003과 r1561 프라이머를 사용하였고(Mcdonald ＆ Murell, 1977), 이 mxaF 유전자는 메탄올을 이용할 때 필요한 메탄올 탈수소효소를 변화시킨다; 예상된 크기(560bp)의 PCR product가 검출되었으며, 다른 Methylobacterium종과 비슷하였다. CBMB20T종으로부터 얻은 16S rRNA 유전자 서열은 1416bp까지 계속 이어졌다. 서열 유사성 계산 및 계통발생적 분석 결과, 이 균주가 별개의 군으로서 Methylobacterium 속의 구성원임을 확인했고, Methylobacterium fujusawaense 99.1%, Methylobacterium radiotolerans 98.0% 및 Methylobacterium mesophilicum 97.4% 의 유사도로 보였다. (도3참조) 16S rRNA 유전자 서열 행렬은 표1에 나타내었다.Strain CBMB20 T was strict aerobic, gram-negative, non-spore-forming, forming pink to red colored colonies. The cells were rod-shaped, mostly branched, and appeared in single or clustered form on solid AMS and NA media. SEM micrographs of CMBM20 T strains are shown in FIGS. 1 and 2. Identification of the mxaF gene was carried out using PCR and f1003 and r1561 primers (Mcdonald & Murell, 1977), which altered the methanol dehydrogenase required when using methanol; A PCR product of the expected size (560bp) was detected, similar to other Methylobacterium species. The 16S rRNA gene sequence from CBMB20 T species continued up to 1416 bp. Sequence similarity calculations and phylogenetic analysis, he confirmed that this strain is a member of the genus Methylobacterium as a separate group, it showed a Methylobacterium fujusawaense 99.1%, Methylobacterium radiotolerans similarity of 98.0% and Methylobacterium mesophilicum 97.4%. (See FIG. 3) The 16S rRNA gene sequence matrix is shown in Table 1.

Methylobacterium 종들 사이의 표현형적 차이는 제한적이며, 대체로 탄소원 및 에너지원의 이용에 의존한다(Green & Bousfield, 1982; Green, 1992).Phenotypic differences between Methylobacterium species are limited and largely depend on the use of carbon and energy sources (Green & Bousfield, 1982; Green, 1992).

Methylobacterium 속의 다른 구성원들과 마찬가지로, 균주 CBMB20T는 메탄올 및 포르메이트와 같은 C1 기질 상에서 성장했지만, 메틸아민, 트리메틸아민 또는 포름알데히드에서는 성장하지 않았다. 균주 CBMB20T는 그것의 높은 유사도를 보이는 균주들과 몇 가지 탄소원의 이용에 있어 차이를 보였다. 이를 표2에 요약하였다. 이 균주를 다른 관련된 종들과 구별하는 탄소원 이용, 효소 활성 및 다른 생화학적 반응은 표4,5,6으로부터 알 수 있다. 균주 CBMB20T 및 CBMB110의 지방산 프로파일은 주로 시스-바센산(C18:1w7c) 및 옥타데칸산(스테아르산, C18:0)으로 구성된다. (표 3참조)Like other members of the genus Methylobacterium, strain CBMB20 T, but grown on C1 substrate, such as methanol and formate, it did not grow in the methyl amine, trimethyl amine or formaldehyde. Strain CBMB20 T differed in the use of several carbon sources from its highly similar strains. This is summarized in Table 2. The use of carbon sources, enzyme activity and other biochemical reactions to distinguish this strain from other related species can be seen from Tables 4, 5 and 6. The fatty acid profile of strains CBMB20 T and CBMB110 consists mainly of cis-basenic acid (C18: 1w7c) and octadecanoic acid (stearic acid, C18: 0). (See Table 3)

<표1> Methylobacterium 속에 대한 16S rRNA 유전자 서열 유사성 행렬(%)Table 1 16S rRNA gene sequence similarity matrix for Methylobacterium genus

균주Strain Gene Bank수탁번 호Gene Bank Accession No. 1One 22 33 44 55 66 77 88 99 1010 1111 1212 1313 1414 1515 1616 1. CBMB20T1.CBMB20T AY683045AY683045 (100)(100) 99.999.9 99.199.1 98.098.0 97.497.4 95.595.5 95.295.2 94.494.4 93.893.8 93.693.6 93.293.2 92.492.4 92.392.3 91.791.7 91.491.4 86.886.8 2. CBMB1102. CBMB110 AY683046AY683046 -- (100)(100) 99.499.4 98.098.0 97.497.4 95.795.7 95.295.2 94.494.4 93.993.9 93.693.6 93.293.2 92.592.5 92.492.4 91.991.9 91.691.6 87.187.1 3. Mtb . fujisawaense DSM 5686T3. Mtb . fujisawaense DSM 5686T AJ250801AJ250801 -- -- (100)(100) 982.982. 97.597.5 96.096.0 95.595.5 94.794.7 94.094.0 94.194.1 93.793.7 92.892.8 92.792.7 92.092.0 92.692.6 87.687.6 4. Mtb . radotolerans JCM 2831T4. Mtb . radotolerans JCM 2831T D32227D32227 -- -- -- (100)(100) 96.496.4 94.794.7 94.694.6 93.893.8 92.892.8 92.992.9 93.393.3 91.391.3 89.889.8 91.991.9 90.890.8 87.687.6 5. Mtb . mesophilicum GP34T5. Mtb . mesophilicum GP34T D32225D32225 -- -- -- -- (100)(100) 95.195.1 95.995.9 84.384.3 94.094.0 94.294.2 939.939. 93.793.7 93.893.8 92.892.8 72.572.5 87.787.7 6. Mtb . hispanicum GP34T6. Mtb . hispanicum GP34T AJ635304AJ635304 -- -- -- -- -- (100)(100) 94.994.9 92.292.2 93.993.9 92.892.8 92.492.4 93.293.2 92.592.5 92.092.0 91.591.5 85.685.6 7. Mtb . organophilum JCM 2833T7. Mtb . organophilum JCM 2833T D32226D32226 -- -- -- -- -- -- (100)(100) 95.395.3 93.593.5 95.495.4 95.095.0 93.593.5 92.992.9 92.592.5 93.593.5 88.488.4 8. Mtb . rhodinum JCM 2811T8. Mtb . rhodinum JCM 2811T D32229D32229 -- -- -- -- -- -- -- (100)(100) 93.793.7 97.597.5 97.297.2 93.493.4 94.594.5 92.392.3 93.993.9 88.388.3 9. Mtb . isbilense AR24T9. Mtb . isbilense AR24T AJ888239AJ888239 -- -- -- -- -- -- -- -- (100)(100) 93.993.9 93.693.6 95.795.7 95.695.6 97.197.1 92.892.8 87.087.0 10. Mtb . extorquens JCM 2802T10. Mtb . extorquens JCM 2802T D32224D32224 -- -- -- -- -- -- -- -- -- (100)(100) 99.499.4 94.294.2 94.294.2 92.792.7 95.595.5 88.188.1 11. Mtb . rhodesinum JCM 2810T11. Mtb . rhodesinum JCM 2810T D32228D32228 -- -- -- -- -- -- -- -- -- -- (100)(100) 93.393.3 93.993.9 92.392.3 95.295.2 87.787.7 12. Mtb . aquaticum GR16T12. Mtb . aquaticum GR16T AJ635303AJ635303 -- -- -- -- -- -- -- -- -- -- -- (100)(100) 95.795.7 94.494.4 93.393.3 86.486.4 13. Mtb . variabile GR3T13. Mtb . variabile GR3T AJ851087AJ851087 -- -- -- -- -- -- -- -- -- -- -- -- (100)(100) 93.293.2 91.891.8 86.286.2 14. Mtb . nodulans ORS 2060T14. Mtb . nodulans ORS 2060T AF220763AF220763 -- -- -- -- -- -- -- -- -- -- -- -- -- (100)(100) 90.090.0 86.886.8 15. Mtb . suomiense F20T15. Mtb . suomiense F20T AY009404AY009404 -- -- -- -- -- -- -- -- -- -- -- -- -- -- (100)(100) 85.785.7 16. Afipia felis ATCC 49715 16.Afipia felis ATCC 49715 AF338177AF338177 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- (100)(100)

<표2> Methylobacterium 종들 간 탄소-기질 활용의 차이Table 2 Methylobacterium Differences in Carbon-Substrate Utilization Between Species

탄소 및 에너지의 단독 공급원으로서 다양한 화학물들의 활용을 나타낸다.It represents the use of various chemicals as the sole source of carbon and energy.

Green(1992)으로부터 수정한 것이다.Modified from Green (1992).

종/균주: 1, CBMB20T; 2, CBMB110; 3, Mtb. radiotolerans(Ito & Iizuka, 1971의 데이터); 4, Mtb. nesophilcum(Austin & Goodellw, 1979); 5. Mtb fujisawaense(Kouno & Ozaki, 1975); 6, Mtb, suomiense(Doronina 등, 2002); 7, Mtb, extorquens(Urakami & Komagata, 1984); 8, Mtb. organophilum(Patt 등, 1976); 9, Mtb. rhodesianum(Rock 등, 1976); 10, Mtb. zatmanii(Rock 등, 1976); 11, Mtb. rhodinum(Heumann, 1962); 12, Mtb. nodulans(Jourand 등, 2004); 13, Mtb. hispanicum(Gallego 등, 2005b); 14, Mtb. populi(Van Aken 등, 2004); 15, Mtb. adhaesivum(Gallego 등, 2006).Species / strains: 1, CBMB20 T ; 2, CBMB110; 3, Mtb. radiotolerans (data of Ito & Iizuka, 1971); 4, Mtb. nesophilcum (Austin & Goodellw, 1979); 5. Mtb fujisawaense (Kouno & Ozaki, 1975); 6, Mtb, suomiense (Doronina et al., 2002); 7, Mtb, extorquens (Urakami & Komagata, 1984); 8, Mtb. organophilum (Patt et al., 1976); 9, Mtb. rhodesianum (Rock et al., 1976); 10, Mtb. zatmanii (Rock et al., 1976); 11, Mtb. rhodinum (Heumann, 1962); 12, Mtb. nodulans (Jourand et al., 2004); 13, Mtb. hispanicum (Gallego et al., 2005b); 14, Mtb. populi (Van Aken et al., 2004); 15, Mtb. adhaesivum (Gallego et al., 2006).

+, 성장; -, 비성장; V, 가변적; W, 약한성장; NA, 데이터 이용불가+, Growth; -Non-growth; V, variable; W, weak growth; NA, no data available

탄소원Carbon source 1One 22 33 44 55 66 77 88 99 1010 1111 1212 1313 1414 1515 D-글루코스D-glucose -- -- ++ ++ ++ ++ -- -- -- -- ++ -- -- -- -- 푸코스Fucos -- -- ++ ++ ++ NaNa -- -- -- -- -- -- -- -- -- D-크실로스D-Xylose -- -- ++ ++ ++ -- -- -- -- -- ++ ++ -- -- -- L-아라비노스L-Arabinose ++ ++ ++ ++ ++ -- -- -- -- -- -- ++ -- -- -- 프럭토스Fructose -- ++ -- -- -- ++ -- ++ ++ ++ ++ -- -- ++ ++ L-글루타메이트L-glutamate ++ ++ ++ ++ ++ -- ++ ++ -- -- ++ ++ ++ -- ++ 시트케이트Seat Kate -- ++ ++ ++ ++ -- -- -- -- -- ++ ++ ++ -- ++ 세바케이트Sebacate ++ -- ++ VV ++ NANA -- -- -- -- -- NANA NANA -- NANA 아세테이트acetate -- ++ ++ -- ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ 베타인Betaine -- -- ++ -- -- ++ ++ -- ++ -- ++ ++ NANA ++ NANA 타르트레이트Tartrate -- -- -- -- VV -- VV -- -- VV -- ++ NANA ++ NANA 에탄올ethanol WW ++ VV ++ ++ ++ ++ ++ ++ ++ ++ ++ NANA ++ NANA 메탄methane -- -- -- -- -- -- -- VV -- -- -- NANA NANA ++ NANA 메틸아민Methylamine -- -- -- -- -- ++ ++ ++ ++ ++ ++ -- NANA ++ NANA 디메틸아민Dimethylamine -- -- -- -- -- -- -- -- ++ -- -- NANA NANA -- NANA 트리메틸아민Trimethylamine -- -- -- -- -- -- -- ++ -- -- -- NANA NANA -- NANA 영양 한천Nutrition agar ++ ++ ++ -- ++ -- ++ ++ ++ ++ ++ NANA NANA ++ NANA

<표3><Table 3>

균주 CBMB20T 및 CBMB110 및 Methylobacterium 속에 속한 관련 종들의 세포 지방산 조성Fatty acid composition of the cells of the relevant species belonging to the genus strains CBMB20 T and CBMB110 and Methylobacterium

종/균주; 1, CBMB20T; 2, CBMB110; 3, Mtb. fujisawaense KACC 10744T; 4, Mtb. hispanicum DSM 16372T; 5, Mtb. mesophilicum DSM 1708T; 6, Mtb. orhanopgilum DSM 760T; 7, Mtb radiotolerans DSM 1819T(본 연구에서는 칼럼 1 내지 7의 데이터); 8, Mtb. populi(Van Aken 등, 2004의 데이터); 9, Mtb. suomiense(Doronina 등, 2002); 10, Mtb. Iusitanum(Doronina 등, 2002); 11, 'Mtb. goesingense'(Idris 등, 2006).Species / strains; 1, CBMB20 T ; 2, CBMB110; 3, Mtb. fujisawaense KACC 10744 T ; 4, Mtb. hispanicum DSM 16372 T ; 5, Mtb. mesophilicum DSM 1708 T ; 6, Mtb. orhanopgilum DSM 760 T ; 7, Mtb radiotolerans DSM 1819 T (data of columns 1 to 7 in this study); 8, Mtb. populi (Van Aken et al., 2004 data); 9, Mtb. suomiense (Doronina et al., 2002); 10, Mtb. Iusitanum (Doronina et al., 2002); 11, 'Mtb. goesingense '(Idris et al., 2006).

수치는 총 지방산 퍼센트이다. 모든 균주에서 0.3% 미만을 나타낸 지방산은 생략했다. ND, 층정 불가; NR, 기록 불가; ECL, 동등한 사슬 길이The figures are percent total fatty acids. Fatty acids showing less than 0.3% in all strains were omitted. ND, no stratification; NR, not recordable; ECL, equivalent chain length

지방산fatty acid 1One 22 33 44 55 66 77 88 99 1010 1111 C9:0 C 9: 0 NDND NDND NDND 1.21.2 NDND NDND NDND NRNR NRNR NRNR NRNR C12:0 C 12: 0 NDND NDND NDND 4.534.53 NDND NDND NDND NRNR NRNR NRNR NRNR C14:0 C 14: 0 NDND 0.30.3 NDND 2.352.35 NDND NDND NDND NRNR NRNR NRNR NRNR C16:0 C 16: 0 3.013.01 3.53.5 1.961.96 4.574.57 3.183.18 2.982.98 3.013.01 6.406.40 NRNR NRNR 3.33.3 C18:0 C 18: 0 4.614.61 3.663.66 5.425.42 4.574.57 4.14.1 6.276.27 5.295.29 11.911.9 NRNR NRNR 2.42.4 C18 :0 3-OHC 18 : 0 3-OH 0.770.77 0.720.72 0.690.69 NDND 1.521.52 0.490.49 0.640.64 NRNR NRNR NRNR NRNR C18 :0 ｗ7c C 18 : 0 ｗ 7 c 88.1888.18 86.7786.77 88.6588.65 68.5368.53 88.6388.63 88.1088.10 88.7688.76 86.1186.11 84.784.7 83.183.1 82.082.0 섬드 피쳐 2*Thumb feature 2 * 0.770.77 1.691.69 0.800.80 7.977.97 1.061.06 0.790.79 0.810.81 NRNR NRNR NRNR NRNR 섬드 피쳐 3*Thumb feature 3 * 1.791.79 2.12.1 1.741.74 6.276.27 0.840.84 0.690.69 0.810.81 NRNR NRNR NRNR NRNR 섬드 피쳐 4*Thumb feature 4 * NDND 0.810.81 NDND NDND NDND NDND NDND NRNR NRNR NRNR NRNR 미지 ECL 14.959Unknown ECL 14.959 0.360.36 0.460.46 0.470.47 NDND 0.450.45 0.460.46 0.500.50 NRNR NRNR NRNR NRNR

* 섬드 피쳐는 MIDI 시스템을 구비한 GLC에 의해 분리될 수 없는 2 또는 3개 지방산의 군을 나타낸다. 섬드 피쳐 2는 이소-C16 :1 I 및/또는 C14 :0 3-OH를 함유했고; 섬드 피쳐 3은 C16 :1ｗ7c 및/또는 이소-C15 :0 2-OH를 함유했고; 섬드 피쳐 4는 이소-C17 :1 I 및/또는 안테이소-C17 :0 B를 함유했다.The summ feature represents a group of two or three fatty acids that cannot be separated by GLC with a MIDI system. Sumde feature 2 contained iso-C 16 : 1 I and / or C 14 : 0 3-OH; Summ feature 3 contained C 16 : 1 ′ 7 c and / or iso-C 15 : 0 2-OH; Sumd feature 4 contained iso-C 17 : 1 I and / or anteiso-C 17 : 0 B.

<표4> CBMB20T균주와 높은 유사도를 보이는 균주들 간의 DNA-DNA 관련성<Table 4> DNA-DNA relationship among strains showing high similarity to CBMB20 T strain

StrainStrain DNA-DNA hybridization with strain CBMB20T(%)DNA-DNA hybridization with strain CBMB20 T (%) CBMB20T CBMB20 T 100.00100.00 CBMB110CBMB110 88.6388.63 Mtb. fujisawaense KACC 10744T Mtb. fujisawaense KACC 10744 T 42.0942.09 Mtb. mesophilicum DSM 1708T Mtb. mesophilicum dsm 1708 t 54.5154.51 Mtb. radiotolerans DSM 1819T Mtb. radiotolerans DSM 1819 T 63.0963.09

<표5><Table 5>

균주 CBMB20T 및 다른 Methylobacterium속들에 의한 탄소원 활용(Biolog)Carbon source utilization by the strain CBMB20 T and other Methylobacterium sokdeul (Biolog)

균주; 1, CBMB20T; 2, CBMB110; 3, Mtb. fujisawaense KACC 10744T; 4, Mtb. radiotolerans DSM 1819T; 5, Mtb. mesophilicum DSM 1708T; 6, Mtb. hispanicum DSM 16372T; 7, Mtb. organophilum DSM 760T Strains; 1, CBMB20 T ; 2, CBMB110; 3, Mtb. fujisawaense KACC 10744 T ; 4, Mtb. radiotolerans DSM 1819 T ; 5, Mtb. mesophilicum DSM 1708 T ; 6, Mtb. hispanicum DSM 16372 T ; 7, Mtb. organophilum DSM 760 T

+, 양성; -, 음성; W, 약한 활용+, Positive; -, voice; W, weak utilization

탄소원Carbon source 1One 22 33 44 55 66 77 대조군Control -- -- -- -- -- -- -- α-시클로덱스트린α-cyclodextrin -- -- -- -- -- -- -- 덱스트린dextrin -- -- -- -- -- -- -- 글리코겐Glycogen -- -- -- -- -- -- -- Tween 40Tween 40 -- -- -- -- -- ++ -- Tween 80Tween 80 -- -- -- -- ++ ++ -- N-아세틸-D-갈락토사민N-acetyl-D-galactosamine -- -- -- -- -- -- -- N-아세틸-D-클루코사민N-acetyl-D-glucosamine -- -- -- -- -- -- -- 아도니톨Adonitol -- -- -- -- -- -- -- L-아라비노스L-Arabinose ++ ++ ++ ++ ++ -- ++ D-아라비톨D-Arabitol -- -- -- -- -- -- -- D-셀룰로스D-cellulose -- -- -- -- -- -- -- i-에리트리톨i-erythritol -- -- -- -- -- -- -- D-프럭토스D-fructose -- ++ -- -- -- ++ -- L-푸코스L-fucose -- -- -- -- ++ -- -- D-갈락토스D-galactose ++ ++ ++ WW ++ -- ++ 겐티오바이오스Genthiobios -- -- -- -- -- -- -- α-D-Glucoseα-D-Glucose -- -- -- WW -- -- -- myo-Inositolmyo-Inositol -- -- -- -- -- -- -- α-D-락토스α-D-lactose -- -- -- -- -- -- -- 락툴로스Lactulose -- -- -- -- -- -- -- 말토스Maltose -- -- -- -- -- -- -- D-만니톨D-mannitol -- -- -- -- -- -- -- D-만노스D-Mannose -- -- -- -- -- -- -- D-멜리바이오스D-Melibiose -- -- -- -- -- -- -- C2-메틸 β-D-클루코시드C2-methyl β-D-glucoside -- -- -- -- -- -- -- D-프사이코스D-Psycos -- -- -- -- -- -- -- D-라피노스D-Raffinose -- -- -- -- -- -- -- L-람노스L-Rhamnose -- -- -- -- -- -- -- D-소르비톨D-sorbitol -- -- -- -- -- -- -- 수크로스Sucrose -- -- -- -- -- -- -- D-트레할로스D-trehalose -- -- -- -- -- -- -- 튜라노스Turanos -- -- -- -- -- -- -- 크실리톨Xylitol -- -- -- -- -- -- -- 피루브산 메틸 에스테르Pyruvic acid methyl ester ++ ++ ++ ++ ++ ++ ++ 숙신산 모노메틸 에스테르Succinic Monomethyl Ester ++ ++ ++ ++ ++ ++ ++ 아세트산Acetic acid -- ++ -- ++ ++ ++ -- 시스-아코니트산Cis-aconic acid -- ++ ++ ++ ++ ++ ++ 시트르산Citric acid -- ++ ++ ++ ++ WW ++ 포름산Formic acid ++ ++ ++ ++ ++ ++ ++ D-갈락톤산 락톤D-galactonic acid lactone ++ ++ ++ ++ ++ -- ++ D-갈락투론산D-galacturonic acid -- -- -- -- WW -- -- D-글루콘산D-gluconic acid ++ ++ ++ ++ ++ ++ ++ D-글루코사민산D-glucosamine acid -- -- -- -- WW -- -- D-글루쿠론산D-glucuronic acid -- -- -- -- -- -- -- α-히드록시부티르산α-hydroxybutyric acid ++ ++ ++ ++ ++ ++ ++ β-히드록시부티르산β-hydroxybutyric acid ++ ++ ++ ++ ++ ++ ++ γ-히드록시부티르산γ-hydroxybutyric acid ++ ++ ++ ++ ++ ++ ++ p-히드록시부티르산 p -hydroxybutyric acid -- -- -- -- -- -- -- 이타콘산Itaconic acid -- -- WW -- -- ++ -- α-케토부티르산α-ketobutyric acid ++ ++ ++ WW ++ ++ -- α-케토글루타르산α-ketoglutaric acid ++ ++ ++ ++ ++ ++ ++ α-케토발레르산α-ketovaleric acid ++ ++ ++ WW ++ WW -- DL-락트산DL-lactic acid ++ ++ ++ ++ ++ ++ WW 말론산 Malonic acid -- -- ++ -- WW ++ -- 프로피온산Propionic acid -- -- -- -- WW ++ -- 퀸산Quinsan -- -- -- -- -- -- -- D-사카르산D-Sakaric Acid ++ ++ ++ ++ ++ ++ ++ 세박산Sebaksan -- -- -- -- -- ++ -- 숙신산Succinic acid ++ ++ ++ ++ ++ ++ ++ 브로모숙신산Bromosuccinic acid ++ ++ ++ ++ ++ ++ ++ 숙신남산Succinnamsan ++ ++ ++ ++ ++ ++ ++ 글루쿠론아미드Glucuronamide -- -- -- -- -- -- -- L-알라닌아미드L-alanineamide ++ ++ ++ ++ ++ -- ++ D-알라닌D-alanine -- -- -- -- -- -- -- L-알라닌L-alanine -- -- -- -- -- -- -- L-알라닌 글리신L-alanine glycine -- -- ++ -- ++ -- -- L-아스파라긴L-asparagine ++ ++ ++ -- ++ ++ ++ L-아스파르트산L-aspartic acid ++ ++ ++ -- ++ ++ ++ L-글루탐산L-glutamic acid ++ ++ ++ -- ++ ++ ++ 글리실-L-아스파르트산Glysyl-L-aspartic acid -- -- -- -- -- -- -- 글리실-L-글루탐산Glysyl-L-Glutamic Acid -- -- -- -- -- -- -- L-히스티딘L-histidine -- -- -- -- -- -- -- 히드록시-L-프롤린Hydroxy-L-proline -- -- -- -- -- -- -- L-류신L-leucine -- -- -- -- -- -- -- L-오르니틴L-ornithine -- -- -- -- -- -- -- L-페닐알라닌L-phenylalanine -- -- -- -- -- -- -- L-프롤린L-proline -- -- -- -- -- -- -- L-Pyroglutamic acidL-Pyroglutamic acid ++ ++ ++ WW ++ -- ++ D-세린D-serine -- -- -- -- -- -- -- L-세린L-serine -- -- -- -- -- -- ++ L-트레오닌L-threonine -- -- -- -- -- -- -- DL-카르니틴DL-carnitine -- -- -- -- -- -- -- v-아미노부티르산v-aminobutyric acid -- -- -- -- -- -- -- 우로칸산Urokan -- -- -- -- -- -- -- 이노신Inosine -- -- -- -- -- -- -- 우리딘Urdin -- -- -- -- -- -- -- 티미딘Thymidine -- -- -- -- -- -- -- 페닐에틸아민Phenylethylamine -- -- -- -- -- -- -- 퓨트레스신Putrescine -- -- -- -- -- -- -- 2-아미노에탄올2-aminoethanol -- -- -- -- -- ++ -- 2,3-부탄디올2,3-butanediol -- -- -- -- -- -- -- 글리세롤Glycerol ++ ++ ++ ++ ++ ++ ++ DL-α-글리세롤 포스페이트DL-α-glycerol phosphate -- -- WW ++ ++ ++ ++ α-D-글루코스 1-포스페이트α-D-glucose 1-phosphate -- -- -- -- -- -- -- D-글루코스 6-포스페이트D-glucose 6-phosphate -- -- -- -- -- -- --

<표6><Table 6>

균주 CBMB20T 다른 Methylobacterium속들에 효소 활성In other strains CBMB20 T Methylobacterium sokdeul Enzyme activity

균주; 1, CBMB20T; 2, CBMB110; 3, Mtb. fujisawaense KACC 10744T; 4, Mtb. radiotolerans DSM 1819T; 5, Mtb. mesophilicum DSM 1708T; 6, Mtb. hispanicum DSM 16372T; 7, Mtb. organophilum DSM 760T Strains; 1, CBMB20 T ; 2, CBMB110; 3, Mtb. fujisawaense KACC 10744 T ; 4, Mtb. radiotolerans DSM 1819 T ; 5, Mtb. mesophilicum DSM 1708 T ; 6, Mtb. hispanicum DSM 16372 T ; 7, Mtb. organophilum DSM 760 T

데이터는 API ZYM 시스템(BioMㅹrieux)를 이용하여 얻었다. +, 양성; -, 음성; W, 약한 활용Data was obtained using the API ZYM system (BioM® rieux). +, Positive; -, voice; W, weak utilization

효소enzyme 1One 22 33 44 55 66 77 대조군Control -- -- -- -- -- -- -- 알칼리성 포스파타제Alkaline phosphatase -- -- -- -- -- -- WW 에스테라제(C4)Esterase (C 4 ) ++ -- ++ ++ ++ ++ ++ 에스테라제 리파제(C8)Esterase lipase (C 8 ) WW -- WW ++ ++ WW ++ 리파제(C14)Lipase (C 14 ) -- -- -- -- -- -- -- 류신 아릴아미다제Leucine Arylamidase ++ -- ++ ++ ++ ++ ++ 발린 아릴아미다제Valine arylamidase WW -- -- -- -- -- WW 시스틴 아릴아미다제Cystine arylamidase -- -- -- -- -- -- -- 트립신Trypsin WW -- -- -- -- -- WW α-키모트립신α-chymotrypsin WW ++ WW WW WW WW ++ 산 포스파타제Acid phosphatase -- -- -- -- -- -- -- 나프톨-AS-BI-포스포히드롤라제Naphthol-AS-BI-phosphohydrolase WW ++ WW ++ ++ WW ++ α-갈락토시다제α-galactosidase WW ++ WW WW ++ WW ++ β-갈락토시다제β-galactosidase -- -- -- -- -- -- -- β-글루쿠로니다제β-glucuronidase -- -- -- -- -- -- -- α-글루코시다제α-glucosidase -- -- -- -- -- -- -- β-글루코시다제β-glucosidase -- -- -- -- -- -- -- N-아세틸-β-글루코사미니다제N-acetyl-β-glucosaminidase -- -- -- -- -- -- -- α-만노시다제α-mannosidase -- -- -- -- -- -- -- α-푸코시다제α-fucosidase -- -- -- -- -- -- --

<표7><Table 7>

균주 CBMB20T 다른 Methylobacterium속들에 대한 생화학 시험 결과 Biochemical test results for the other strains CBMB20 T Methylobacterium sokdeul

균주; 1, CBMB20T; 2, CBMB110; 3, Mtb. fujisawaense KACC 10744T; 4, Mtb. radiotolerans DSM 1819T; 5, Mtb. mesophilicum DSM 1708T; 6, Mtb. hispanicum DSM 16372T; 7, Mtb. organophilum DSM 760T Strains; 1, CBMB20 T ; 2, CBMB110; 3, Mtb. fujisawaense KACC 10744 T ; 4, Mtb. radiotolerans DSM 1819 T ; 5, Mtb. mesophilicum DSM 1708 T ; 6, Mtb. hispanicum DSM 16372 T ; 7, Mtb. organophilum DSM 760 T

데이터는 API ZYM 시스템(BioMㅹrieux)를 이용하여 얻었다. +, 양성; -, 음성; W, 약한 활용Data was obtained using the API ZYM system (BioM® rieux). +, Positive; -, voice; W, weak utilization

생화학적 시험Biochemical test 1One 22 33 44 55 66 77 질산염의 아질산염(NO2) 환원Nitrite (NO2) Reduction of Nitrate ++ -- -- ++ -- -- -- 질산염의 질소(N2) 환원Nitrogen (N2) reduction of nitrate -- -- -- -- -- -- -- 인돌 생산(TRP)Indole Production (TRP) -- -- -- -- -- -- -- 글루코스 발효(GLU)Glucose Fermentation (GLU) -- -- -- -- -- -- -- 아르기닌 디히드롤라제(ADH)Arginine Dihydrolase (ADH) -- -- -- -- -- -- -- 유레아제(URE)Urease (URE) -- ++ -- -- -- ++ -- 에스쿨린 가수분해(ESC)Esculin hydrolysis (ESC) -- -- -- -- -- -- -- 젤라틴 가수분해(GEL)Gelatin Hydrolysis (GEL) -- -- -- -- -- -- -- β-갈락토시다제(PNG)β-galactosidase (PNG) -- -- -- -- -- -- -- D-클루코스(GLU)D-Clucos (GLU) -- -- -- ++ -- -- -- D-아라비노스(ARA)D-Arabinose (ARA) ++ ++ ++ ++ ++ -- ++ D-만노스(MNE)D-Mannose (MNE) -- -- -- -- -- -- -- D-만니톨(MAN)D-mannitol (MAN) -- -- -- -- -- -- -- N-아세틸글루코사민(NAG)N-acetylglucosamine (NAG) -- -- -- -- -- -- -- D-말토스(MAL)D-Maltose (MAL) -- -- -- -- -- -- -- 클루콘산 칼륨(GNT)Potassium Cluconate (GNT) ++ ++ ++ ++ ++ -- ++ 카프르산(CAP)Capric Acid (CAP) -- -- -- -- -- -- -- 아디프산(ADI)Adipic acid (ADI) ++ ++ ++ ++ ++ -- ++ 말산(MLT)Malic acid (MLT) ++ ++ ++ ++ ++ ++ ++ 시트르산 삼나트륨(CIT)Trisodium Citrate (CIT) -- -- -- -- ++ -- ++ 페닐아세트산(PAC)Phenylacetic acid (PAC) -- -- -- -- -- -- --

균주 CBMB20T는 질소원으로서 ACC를 이용하며, 기지균외 무감염 상태 조건 하에서 캐뉼라 묘목에서 ACC 디아미나제 활성의 존재 에틸렌 수준 저하 및 뿌리 신장 촉진이 다른 문헌에서 보고되었다(Madhaiyan 등, 2006). 이런 특수한 능력은 Methylobacterium 속의 다른 구성원들에는 시험된 적 없었다. Strain CBMB20 T uses ACC as the nitrogen source, and the presence of ACC deaminase activity in cannula seedlings under non-bacterial uninfected conditions has been reported in other literature (Madhaiyan et al., 2006). This particular ability has not been tested in other members of the genus Methylobacterium .

다른 Methylobacterium 속인 Mtb. Fujisawaens, Mtb. Radiotolerans 및 Mtb. Organophilum의 대표 균주를, 질소원으로서 30μmol ACC 를 함께 펴 바른 AMS 배지 상에서 예비 평판 분석하여 시험했으며, 그 결과 이 세 균주 모두 ACC 디아미나제 활성에 양성인 것으로 밝혀졌다. 박테리아에서 ACC 디아미나제의 존재는 식 물성장 촉진에 중요한 역할을 하며, 박테리아 세포는 에틸렌의 조기 생합성 전구체인 ACC를 흡수하여 식물 에틸렌 수준을 저하시키고, 다양한 환경 스트레스의 부정적 효과를 감소시킨다(Stearns 등, 2005). 다양한 박테리아 속에서 ACC 디아미나제의 존재가 이미 보고된 바 있다(Penrose & Glick, 2001; Belimov 등, 2001).Another Methylobacterium genus Mtb. Fujisawaens, Mtb. Radiotolerans and Mtb. Representative strains of Organophilum were tested by preliminary plate analysis on AMS medium spread with 30 μmol ACC together as a nitrogen source, and all three strains were found to be positive for ACC deaminase activity. The presence of ACC deaminase in bacteria plays an important role in promoting plant growth, and bacterial cells absorb ACC, an early biosynthetic precursor of ethylene, lowering plant ethylene levels and reducing the negative effects of various environmental stresses. Et al., 2005). The presence of ACC deaminase in various bacteria has already been reported (Penrose & Glick, 2001; Belimov et al., 2001).

[실시 예2] 균주의 동정Example 2 Identification of Strains

CBMB20T와 CBMB110의 DNA G+C 함량은 각각 70.6, 69.2mol%이다. DNA-DNA reassociation은 이 두 종의 계통학적 관계를 확인하기 위하여 실행되었고, 88.63%의 관계정도를 보여 두 종은 가깝다는 것을 확인했다. 그러나 다른 Methylobacterium 속들은 CBMB20T와 42.09-63.09% 정도의 유사성을 보여주었고, 이것은 다른 Methylobacterium 속들로부터 분리가 가능하다는 것을 보여주었다.The DNA G + C content of CBMB20 T and CBMB110 is 70.6 and 69.2 mol%, respectively. DNA-DNA reassociation was performed to confirm the phylogenetic relationship between the two species, showing a relationship of 88.63%, indicating that the two species are close. However, they showed the other Methylobacterium in CBMB20 T from 42.09 to 63.09% and the degree of similarity, which showed that the separation from the other Methylobacterium sokdeul possible.

종 정의에 있어서 DNA-DNA 관련성의 역치를 70%로 권장하고 있는 것을 고려할 때(Wayne 등, 1987), 이들 결과는 균주 CBMB20T가 이 종들 중 어떤 것에도 속하지 않는다는 것을 나타낸다 .Considering the 70% threshold for DNA-DNA relevance in species definition (Wayne et al., 1987), these results indicate that strain CBMB20 T does not belong to any of these species.

16S rRNA 유전자 서열 유사 데이터인 DNA-DNA hybridization 값과 표현형적인 특성은 CBMB20T와 CBMB110이 다른 Methlobacterium균종들과 다르다는 것을 확인 하였다. CBMB20T 종은 Methylobacterium의 새로운 종으로써 확인되었고, 이름을 Methylobacterium oryzae sp. nov. 라고 명명하였다.DNA-DNA hybridization values and phenotypic characteristics, 16S rRNA gene sequence-like data, confirmed that CBMB20 T and CBMB110 were different from other Methlobacterium species. CBMB20 T species was identified as a new species of Methylobacterium, the name Methylobacterium oryzae sp. nov. It was named.

[실시 예3] 신규미생물의 특성Example 3 Characteristics of New Microorganisms

이하에서는 메틸로박테리아 오라이자 에스피,노브,(Methylobacterium oryzae sp. nov.)라 명명 되었고 일명 CBMB20T으로 칭하며 기탁번호로 기탁된 본 발명의 신규미생물 특성을 설명한다.Hereinafter methyl bacteria come and SP, knob, (Methylobacterium oryzae sp. Nov.) LA was referred to as a Named one people CBMB20 T describes a novel microorganism characteristics of the present invention deposited as a deposit number.

세포는 엄격한 호기성, 그램-음성, 비-포자-형성성, 운동성 간상균(0.60-0.80 x 2.10-2.75㎛) 세포로서, 하나씩, 쌍으로, 또는 로제트 형태로 생긴다. 28℃ AMS 배지 상에서 96시간 후, 분홍색 내지 적색의 규칙적인 가장자리를 갖는 볼록한 모양의 반투명 콜로니를 형성하며, 이것은 서서히 성장하고, 직경은 0.2-1.2mm이다. 성장은 NA 및 Luria-Bertani, R2A, PYG, 숙시네이트, 글리세롤-펩톤 및 평판 계수 한천 상에서 발생한다. pH 5.0-10.0(최적 pH 6.8)에서 20-30℃(최적 온도 28℃)에서 성장하며, 4℃ 또는 40℃에서는 성장하지 않는다. 2.0% 이상의 NaCl의 존재 하에서 성장하지 않는다. 분홍색 착색성은 수불용성이며, 클로로포름/메탄올(1:1) 중에서 233, 358, 505 및 534nm에서 최대 흡수를 나타낸다. 케탈라제, 옥디사제, 에스테라제 및 류신 아릴아미나제 활성에 대해 양성이다. 에스테라제 리파제, 발린 아릴아미다제, 트립신, 산 포스파타제 및 나프톨-AS-BI-포스포히드라제에 대해 약한 활성이다. 펙티나제, 셀룰라제, 프로테아제 또는 리파제에 대해서는 활성이 없다. 인돌, 메틸 레드 및 Voges-Proskauer 시험은 음성이다. 젤라틴, 녹말, 지질, 카세인 및 아세쿨린은 가수분해되지 않는다. 황화수소는 생산하지 않는다. 시몬스(Simmons's) 시트레이트 시험에서는 D-아라비노스, D-크실로스 및 푸마레이트가 단독 탄소원으로서 이용된다. 수크로스, 2-프로판올, 1-부탄 올, 둘시톨, L-리신, 베타인, 옥살레이트, 타르트레이트, 살리실레이트, 포름알데히드, 메틸아민, 디메틸아민, 트리메틸아민, 디클로로메탄 또는 메탄은 사용하지 않는다. 황산암모늄, 질산칼륨, 질산나트륨, 염화암모늄, L-알라닌, L-글루타메이트, L-글루타민, 요소, ACC 및 티오시안산 칼륨이 단독 질소원으로서 이용된다. L-아스파레이트, 글리신, L-트립토판, 메틸아민 또는 시안산 칼륨은 이용하지 않는다. 탄소 흡수 시험에서 L-아라비노스, 글루콘산 칼륨, 아디프산 및 말산에 대해 양성이다. 대표 균주의 고유 항생제-내성 패턴은 암피실린, 카르베니실린, 날리딕스산, 클로람페니콜 및 스트렙토마이신에 대해서는 높은 내성을 나타내고, 가나마이신, 겐타마이신, 스펙티노마이신 및 테트라시클린에는 감수성을 나타낸다. Cells are stringent aerobic, gram-negative, non-spore-forming, motile rod (0.60-0.80 x 2.10-2.75 μm) cells, one by one, in pairs, or in rosette form. After 96 hours on 28 ° C. AMS medium, they form convex shaped translucent colonies with regular edges of pink to red, which grow slowly and are 0.2-1.2 mm in diameter. Growth occurs on NA and Luria-Bertani, R2A, PYG, succinate, glycerol-peptone and plate count agar. It grows at 20-30 ° C. (optimum temperature 28 ° C.) at pH 5.0-10.0 (optimum pH 6.8) and does not grow at 4 ° C. or 40 ° C. It does not grow in the presence of at least 2.0% NaCl. Pink coloration is water insoluble and exhibits maximum absorption at 233, 358, 505 and 534 nm in chloroform / methanol (1: 1). It is positive for ketalase, oxadiase, esterase and leucine arylaminase activity. Weak activity against esterase lipase, valine arylamidase, trypsin, acid phosphatase and naphthol-AS-BI-phosphohydrase. There is no activity against pectinase, cellulase, protease or lipase. Indole, methyl red and Voges-Proskauer tests are negative. Gelatin, starch, lipids, casein and aceculin are not hydrolyzed. Hydrogen sulfide does not produce. In Simmons's citrate test, D-arabinose, D-xylose and fumarate are used as the sole carbon source. Sucrose, 2-propanol, 1-butanol, dulsitol, L-lysine, betaine, oxalate, tartrate, salicylate, formaldehyde, methylamine, dimethylamine, trimethylamine, dichloromethane or methane are used I never do that. Ammonium sulfate, potassium nitrate, sodium nitrate, ammonium chloride, L-alanine, L-glutamate, L-glutamine, urea, ACC and potassium thiocyanate are used as the sole nitrogen source. L-aspartate, glycine, L-tryptophan, methylamine or potassium cyanate are not used. It is positive for L-arabinose, potassium gluconate, adipic acid and malic acid in the carbon uptake test. Intrinsic antibiotic-resistant patterns of representative strains show high resistance to ampicillin, carbenicillin, nalidixic acid, chloramphenicol and streptomycin, and susceptibility to kanamycin, gentamycin, spectinomycin and tetracycline.

다음의 화합물들이 단독 탄소원 및 에너지원으로서 이용된다(Biolog): L-아라비노스, D-락토스, 피루브산 메틸 에스테르, 숙신산 모노메틸 에스테르, 포름산, D-갈락톤산, 락톤, D-글루콘산, α-, β- 및 γ-히드록시부티르산, α-케토부티르산, α-케토글루타르산, α-케토발레르산, DL-락트산, 말론산, 프로피온산, D-사카르산, 세박산, 숙신산, 브로모숙신산, 숙신남산, L-알라닌아미드, L-아스파라긴, L-아스파르트산, L-글루탐산, L-피로글루탐산, 글리세롤 및 DL-α-글리세롤 포스페이트. 세포 지방산은 헥사데카노에이트(팔미트산: C16:0), 3.01%; 시스-7-옥타데카노에이트(시스-바센산; C18:1w7c), 88.18%; 옥타데카노에이트(스테아르산; C18:0), 4.61%; 및 3-히드록시옥타데카노에이트(C18:0 3-OH), 0.77%이다.The following compounds are used as sole carbon and energy sources (Biolog): L-arabinose, D-lactose, pyruvic acid methyl ester, succinic acid monomethyl ester, formic acid, D-galactonic acid, lactone, D-gluconic acid, α- , β- and γ-hydroxybutyric acid, α-ketobutyric acid, α-ketoglutaric acid, α-ketovaleric acid, DL-lactic acid, malonic acid, propionic acid, D-saconic acid, sebacic acid, succinic acid, bromo Succinic acid, succinic acid, L-alanineamide, L-asparagine, L-aspartic acid, L-glutamic acid, L-pyroglutamic acid, glycerol and DL-α-glycerol phosphate. Cellular fatty acids were hexadecanoate (palmitic acid: C16: 0), 3.01%; Cis-7-octadecanoate (cis-basenic acid; C18: 1 w 7 c ), 88.18%; Octadecanoate (stearic acid; C18: 0), 4.61%; And 3-hydroxyoctadecanoate (C18: 0 3-OH), 0.77%.

상기한 바와 같이 벼의 줄기로부터 분리한 본 발명은 신규미생물을 제공함으 로서 식물성장을 증진시키는 ACC디아미제를 신규미생물을 통해 생산가능하게 할 수 있으므로 식물성장에 기여할 것이다.As described above, the present invention isolated from the stems of rice may contribute to plant growth since it may be possible to produce ACC diamiases that promote plant growth by providing new microorganisms through new microorganisms.

Claims (1)

ACC 디아미나제를 생산할 수 있는 Methylobacterium 속의 신규미생물로서 벼의 줄기조직으로부터 분리된 Methylobacterium 오라이자 에스피, 노브,(Methylobacterium oryzae sp. nov.; KACC11585) Methylobacterium capable of producing ACC deaminase Methylobacterium Isolated from the Stem Tissue of Rice as a Novel Microorganism in the Genus Orissa sp, knob, (Methylobacterium oryzae sp.nov .; KACC11585)
KR1020070042587A 2007-05-02 2007-05-02 Methylobacterium oryzae cbmb20 KR20080097568A (en)

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