난배양성 토양세균을 위한 신배양기술의 고찰과 향후 발전 방향

김재수,Kim, Jai-Soo 한국미생물학회 2011 미생물학회지 Vol.47 No.3

고찰을 통해 난배양성 토양세균의 특징과 배양에 성공한 사례 및 성공하기 위해 알아야 될 지식들이 무엇인지에 대해 기술하였다. 먼저 배지는 목적한 세균이 토양에서 느리게 성장하다가 실험실의 빠른 성장조건으로 전환하도록 알맞게 선택되어야 하는데 일반적으로 기질, 질소 및 인 등의 농도를 낮게 조절해야 한다. 새로운 배지를 만들기 위해서는 분자생태학적 연구도 병행되어야 한다. 세균 세포 간 음성적 상호작용을 줄이기 위해 평판배양 시 접종량도 평판 당 세포수가 50개 이하로 조절해야 한다. pH나 염농도 같은 성장조건은 실제 환경조건과 맞춰야 하며 배양온도는 낮거나 다양하게 그리고 배양기간은 길게 잡아야 한다. 새로운 배지에서 분리될 수많은 토양 미생물 콜로니들 중에서 단지 몇 개만이 난배양성이므로 이들이 기존에 배양이 되지 않았던 미생물인지를 신속 정확히 검출하는 방법이 필요하다. 또한 많은 토양세균들이 군집 내에서 서로 협력하며 살아가기 때문에 공동배양이나 상등액을 이용해서 토양 미생물을 농화증식하고 이를 순수 분리하면 배양에 성공할 수 있을 것이다. This review describes the characteristics of various unculturable soil bacteria, successfully-cultivating examples of those bacteria, and the diverse factors to be considered for successful cultivation. Most importantly, the selection of proper media is very important because unculturable bacteria demand different types of nutrients at various concentrations of substrates, nitrogens and phosphorus. To develop a new medium to successfully culture unculturable bacteria from soil, molecular ecological studies should be combined together. The inoculum size on a plate is also important: less than 50 bacterial cells are recommended to be plated on a single culture plate. The environmental factors such as pH and salt concentration of the medium need to be adjusted as similar as possible to mimic the original soil environments, and the trial of the various temperatures and extended period of cultivation are better. Since one cannot simply tell about which one was unculturable among a great number of colonies grown on a newly developed medium, some suitable detection methods and fast identification methods are required. Many soil bacteria live with cooperation one another in their communities, so that enrichment such as coculture of using other bacterial metabolites and subsequent pure cultures can also guarantee successful cultivation of the previously uncultured bacteria in soil. Here, this review will discuss for the future perspectives to culture the unculturable soil bacteria.

Fibrolytic Rumen Bacteria: Their Ecology and Functions

Koike, Satoshi,Kobayashi, Yasuo Asian Australasian Association of Animal Productio 2009 Animal Bioscience Vol.22 No.1

Among rumen microbes, bacteria play important roles in the biological degradation of plant fiber due to their large biomass and high activity. To maximize the utilization of fiber components such as cellulose and hemicellulose by ruminant animals, the ecology and functions of rumen bacteria should be understood in detail. Recent genome sequencing analyses of representative fibrolytic bacterial species revealed that the number and variety of enzymes for plant fiber digestion clearly differ between Fibrobacter succinogenes and Ruminococcus flavefaciens. Therefore, the mechanism of plant fiber digestion is also thought to differ between these two species. Ecology of individual fibrolytic bacterial species has been investigated using pure cultures and electron microscopy. Recent advances in molecular biology techniques complement the disadvantages of conventional techniques and allow accurate evaluation of the ecology of specific bacteria in mixed culture, even in situ and in vivo. Molecular monitoring of fibrolytic bacterial species in the rumen indicated the predominance of F. succinogenes. Nutritive interactions between fibrolytic and non-fibrolytic bacteria are important in maintaining and promoting fibrolytic activity, mainly in terms of crossfeeding of metabolites. Recent 16S rDNA-based analyses suggest that presently recognized fibrolytic species such as F. succinogenes and two Ruminococcus species with fibrolytic activity may represent only a small proportion of the total fibrolytic population and that uncultured bacteria may be responsible for fiber digestion in the rumen. Therefore, characterization of these unidentified bacteria is important to fully understand the physiology and ecology of fiber digestion. To achieve this, a combination of conventional and modern techniques could be useful.

신령버섯의 기형으로부터 배양불능세균의 PCR 검정

신평균 외 한국버섯학회 2010 한국버섯학회지 Vol.8 No.4

신령버섯은 항암작용과 더불어 면역강화작용 등 약용으로서 이용되고 있습니다. 최근에는 신령버섯 재배농가에서 새로운 병징 현상이 보고되어 내생세균에 대한 분자생물학적 방법으로 조사하였다. 병징을 나타내는 수집된 기형버섯을 eubacterial 16S rDNA 영역을 이용하여 PCR한 결과 기형버섯만 증폭되었다. 이 영역을 부분적으로 염기서열을 결정한 결과 CFB bacterium과 유사성이 가장 높았고, 이 염기서열을 이용하여 프라이머를 디자인한 후 nested PCR를 부위별로 확인한 결과 기형을 일으킨 갓의 주름살 부위에서 가장 강하게 증폭되었고 포자수확도 되지 않았으며 배양불능세균 group인 CFB bacterium임을 확인하였다. Agaricus blazei Murill is a important medicinal mushroom for a powerful immune system builder and tonic. Currently, it is known about a new disease phenomenon that appears to be occurring on a number of mushroom farms. We described a straightforward approach in which molecular methods was used to survey the presence of potentially endo- and epiphytic bacteria infected with the Agaricus blazei. The 16S rDNA was amplified with universal eubacterial primers directly from pure cultures of Agaricus blazei mycelium and fruit body. The 16S rDNA sequences were almost identical (96 to 97% similarity), and phylogenetic analysis placed them in a single unique rRNA branch belong to the uncultural bacterium phylogroup. PCR detection of uncultural bacterium in the malformed tissues of Agaricus blazei were carried out by using 16S rRNA sequenced specific probe. It was strongly amplified at the malformed pileus region of fruit body and also spore print was impossible.

난배양성 토양세균의 배양법 평가 및 신 분류군의 순수분리

김도형,이상훈,조재창,Kim, Do-Hyoung,Lee, Sang-Hoon,Cho, Jae-Chang 한국미생물학회 2008 미생물학회지 Vol.44 No.4

난배양성 세균의 배양효율을 증진시킬 수 있다고 보고된 배지 첨가물들이 포함된 다양한 종류의 빈영양 배지들을 대상으로 배양효율을 비교평가하고 최적의 배양조건을 모색하였으며, 평가된 배지를 사용하여 토양시료로부터 순수 분리된 난배양성 세균들의 계통분류학적 위치를 분석하였다. 배지 첨가물로는 토양의 화학적 조성을 반영하기 위한 토양추출액(soil extract), 부식질산의 유사체(humic acid analogue)인 anthraquinone disulfonate, 정족수인식 신호물질(quorum-signaling compounds)인 acyl homoserine lactones, 과산화물(exogenous peroxide)로부터 세균을 보호하기 위한 catalase가 사용되었다. $CO_2$ 과분압(5%, v/v) 조건에서 60일간 배양하였을 때, catalase가 첨가된 배지가 가장 높은 세균집락수(CFU)를 보였다. 이 배지로부터 147개의 균주를 무작위적으로 선택하여 순수분리하고 16S rRNA 유전자의 염기서열을 이용한 계통학적 분석을 실시한 결과, 순수분리된 균주의 약30%가 이전에 배양 또는 발견된 적이 없는 새로운 종(species)에 속하며, 이 중 약 25%는 새로운 과(family)에 속하는 세균일 가능성이 있는 것으로 나타났다. 또한 난배양성 토양세균으로 알려진 phylum Acidobacteria에 속하는 세균들이 성공적으로 배양되었다는 결과를 고려하면, 본 연구에서 사용된 배지 및 배양조건은 난배양성 토양세균의 배양은 물론 신 분류군의 발굴에도 큰 기여를 할 것으로 기대된다. We evaluated cultivation methods to obtain pure cultures of previously uncultivated bacteria from soil. Soil bacteria (suspensions) were inoculated onto various oligotrophic media with one of the following additives: 1) soil extract; 2) anthraquinone disulfonate (humic acid analogue); 3) acyl homoserine lactones (quorum-signaling compounds); 4) catalase (for the protection of bacteria from exogenous peroxides). After the relatively long period (60 days) of incubation with elevated concentrations of $CO_2$ (5%, v/v), the media containing catalase showed the highest colony count. We purified 147 randomly selected colonies from the media and the isolates were subjected to the phylogenetic analyses of their 16S rRNA gene sequences. Phylogenetic analysis revealed that approximately 30% of the isolates might belong to novel species or novel family, suggesting that the media and incubation conditions used could be useful for the cultivation of as-yet-uncultured bacteria. Especially, bacteria belonging to the phylum Acidobacteria, ubiquitous bacterial taxon known as an uncultured bacterial group (at least difficult to culture from environmental samples), were successfully cultured in this study.

Cultivation of Diverse Novel Marine Bacteria from Deep Ocean Sediment Using Spent Culture Supernatant of Ca. Bathyarchaeia Enrichment

Ishaq Sidra Erum,Ahmad Tariq,Liang Lewen,Xie Ruize,Yu Tiantian,Wang Yinzhao,Wang Fengping 한국미생물학회 2024 The journal of microbiology Vol.62 No.8

Most microorganisms resist pure cultivation under conventional laboratory conditions. One of the primary issues for this un-culturability is the absence of biologically produced growth-promoting factors in traditionally defined growth media. However, whether cultivating microbes by providing spent culture supernatant of pivotal microbes in the growth medium can be an effective approach to overcome this limitation is still an under-explored area of research. Here, we used the spent culture medium (SCM) method to isolate previously uncultivated marine bacteria and compared the efficiency of this method with the traditional cultivation (TC) method. In the SCM method, Ca. Bathyarchaeia-enriched supernatant (10%) was used along with recalcitrant organic substrates such as lignin, humic acid, and organic carbon mixture. Ca. Bathyarchaeia, a ubiquitous class of archaea, have the capacity to produce metabolites, making their spent culture supernatant a key source to recover new bacterial stains. Both cultivation methods resulted in the recovery of bacterial species from the phyla Pseudomonadota, Bacteroidota, Actinomycetota, and Bacillota. However, our SCM approach also led to the recovery of species from rarely cultivated groups, such as Planctomycetota, Deinococcota, and Balneolota. In terms of the isolation of new taxa, the SCM method resulted in the cultivation of 80 potential new strains, including one at the family, 16 at the genus, and 63 at the species level, with a novelty ratio of ~ 35% (80/219). In contrast, the TC method allowed the isolation of ~ 10% (19/171) novel strains at species level only. These findings suggest that the SCM approach improved the cultivation of novel and diverse bacteria.

Cloning and Characterization of Cellulase Gene (cel5C) from Cow Rumen Metagenomic Library

Min Keun Kim(김민근),Dhirendra Nath Barman(디렌 바르만),Tae Ho Kang(강태호),Jungho Kim(김정호),Hoon Kim(김훈),Han Dae Yun(윤한대) 한국생명과학회 2012 생명과학회지 Vol.22 No.4

한우의 반추위에서 게놈 DNA를 분리하여 메타게놈 은행을 구축한 다음 섬유소분해효소를 암호화하는 유전자를 클로닝 및 유전자를 선별하였다. 선별된 유전자의 DNA 염기서열 및 아미노산 서열을 분석하고 유전산물의 생화학적인 특성을 조사하였다. cel5C 유전자는 1,125 bp로 374개의 아미노산 잔기를 가진 단백질을 암호화하였으며 이 단백질 분자량은 42 kDa이었다. 이 효소의 최적 pH는 4 근방이었으며 최적 온도는 50oC 부근이었다. cel5C 유전자의 internal primer를 사용하여 인공적으로 배양할 수 있는 49종의 반추세균에서 분리한 게놈 DNA을 주형으로 PCR 분석한 결과 해당하는 밴드를 확인할 수 없었다. Cel5C는 현재로서는 배양할 수 없는 반추 미생물로 추정된다. A metagenomic library of cow rumen in the pCC1FOS phage vector was screened in E. coli EPI300 for cellulase activity on carboxymethyl cellulose agar plates. One clone was partially digested with Sau3AI, ligated into the BamHI site of the pBluescript II SK+ vector, and transformed into E. coli DH5 α. We obtained a 1.5 kb insert DNA, designated cel5C, which hydrolyzes carboxymethyl cellulose. The cel5C gene has an open reading frame (ORF) of 1,125 bp encoding 374 amino acids. It belongs to the glycosyl hydrolase family 5 with the conserved domain LIMEGFNEIN. The molecular mass of the Cel5C protein induced from E. coli DH5α, as analyzed by CMC SDS-PAGE, appeared to be approximately 42 kDa. The enzyme showed optimum cellulase activity at pH 4.0, and 50℃. We examined whether the cel5C gene comes from the 49 identified cow rumen bacteria using PCR. No PCR bands were identified, suggesting that the cel5C gene came from the unidentified cow rumen bacteria.

Cloning of α-Amylase Gene from Unculturable Bacterium Using Cow Rumen Metagenome

조수정,윤한대,Cho, Soo-Jeong,Yun-Han-Dae Korean Society of Life Science 2005 생명과학회지 Vol.15 No.6

미생물 메타게놈은 특이한 생체촉매의 다양한 원료로 제공된다. 한우의 반추위에서 게놈 DNA를 분리한 후 메타게놈 은행을 구축하고 $\alpha$-amlylase를 암호화하는 유전자를 클로닝하여 DNA 및 아미노산 서열을 밝히고 생화적 특징을 조사하였다. amyA유전자는 1,893 bp로 631개의 아미노산 잔기를 가진 단백질을 암호화였으며 효소의 분자량은 단백질 전기영동 결과 약 71,000 Da으로 확인되었다. 이 효소를 다른 아밀라제와 비교한 결과 $21-59\%$의 상동성을 보였다. AnyA는 pH $6.40\%$에서 최적 활성을 나타내었고, pl값은 5.87이었다. E. coliDH5$\alpha$에서 발현된 AmyA의 활성은 $\Mg^{2+}$(20mM), $Ca^{2+}$ (30 mM) 존재 시 그 활성이 증가하였고, $Fe^{2+}$, $Cu^{2+}$ 존재 시 저해되었다. amyA 유전자의 internal primer를 사용하여 인공적으로 배양할 수 있는 49종의 반추세균에서 분리한 게놈 DNA을 주형으로 PCR분석한 결과 해당하는 벤드를 확인할 수 없었다. AmyA는 현재로 배양할 수 없는 반추 미생물에서 온 것으로 추정된다. The metagenomes of complex microbial communities are rich sources of novel biocatalysts. The gene encoding an extracellular $\alpha$-amylase from a genomic DNA of cow rumen was cloned in Escherichia coli DH5$\alpha$ and sequenced. The $\alpha$-amylase (amyA) gene was 1,893 bp in length, encoding a protein of 631 amino acid residues with calculated molecular weight of 70,734 Da. The molecular weight of the enzyme was estimated to be about 71,000 Da by active staining of a SDS-PACE. The enzyme was 21 to $59\%$ sequence identical with other amyloyltic enzymes. The AmyA was optimally active at pH 6.0 and $40\%$. The AmyA had a calculated pI of 5.87. AmyA expressed in E. coli DH5$\alpha$ was enhanced in the presence of $Mg^{2+}$ (20 mM) and $Ca^{2+}$ (30 mM) and inhibited in the presence of $Fe^{2+}$ and $Cu^{2+}$. The origin of amyA gene could not be confirmed by PCR using internal primer of amyA gene from extracted genomic DNA of 49 species rumen culturable bacteria so far. An amyh is supposed to obtained from unculturable rumen bacterium in cow rumen environment.

신령버섯의 기형으로부터 배양불능세균의 PCR 검정

신평균,박윤정,유영복,공원식,장갑열,오세종,이금희 한국버섯학회 2010 한국버섯학회지 Vol.8 No.4

Agaricus blazei Murill is a important medicinal mushroom for a powerful immune system builder and tonic. Currently, it is known about a new disease phenomenon that appears to be occurring on a number of mushroom farms. We described a straightforward approach in which molecular methods was used to survey the presence of potentially endo- and epiphytic bacteria infected with the Agaricus blazei. The 16S rDNA was amplified with universal eubacterial primers directly from pure cultures of Agaricus blazei mycelium and fruit body. The 16S rDNA sequences were almost identical (96 to 97% similarity), and phylogenetic analysis placed them in a single unique rRNA branch belong to the uncultural bacterium phylogroup. PCR detection of uncultural bacterium in the malformed tissues of Agaricus blazei were carried out by using 16S rRNA sequenced specific probe. It was strongly amplified at the malformed pileus region of fruit body and also spore print was impossible.

High Level of Bacterial Diversity and Novel Taxa in Continental Shelf Sediment

( Jin Kyung Hong ),( Jae Chang Cho ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.6

The bacterial diversity of the continental shelf sediment in the Yellow Sea was investigated by the cloning and sequencing of PCR-amplified 16S rRNA genes. The majority of the cloned sequences were distinct phylotypes that were novel at the species level. The richness estimator indicated that the sediment sample might harbor up to 32 phylum-level taxa. A large number of low-abundance, phylum-level taxa accounted for most of the observed phylogenetic diversity at our study site, suggesting that these low-abundance taxa might play crucial roles in the shelf sediment ecosystem.

Development of a Novel Microorganism Screening System for the Isolation of Unculturable Bacteria

Sun Ja CHO,Suk Min KIM,Sun Bok LEE 한국생물공학회 2010 한국생물공학회 학술대회 Vol.2010 No.10