해양 미세조류인 파블로바 비리디스는 빨리 자라며, DHA, EPA와 같은 해양생물을 키우는데 필수적인 영양요소를 축적하는 능력을 가지고 있어 어류와 새우류 치어 사육에 이용되어 왔다. 본 연구에서는 파블로바 비리디스와이 미세조류의 표면에 붙어서는 세균과의 공생적 상호작용을 연구하였다. 무균의 파블로바 비리디스 균주는 항생제 혼합액을 포함하는 액체배지에 반복 배양함으로써 얻어졌다. 무균상태는 항생제를 포함하지 않는 배지에 3번 계대배양한 후 확인하였다. 무균상태의 조류는 이 조류로부터 분리되었으며, 임의로 I1–I5로 명명한 세균과 혼합배양하면서 조류의 성장 촉진 효과를 조사하였다. 모든 세균이 파블로바 비리디스의 생장을 촉진하였으며, 그 중 I3로 명명한 세균이 5가지 세균 중 가장 효과가 좋았다. 혼합배양 상태에서 파블로바 비리디스의 세포 수는 대조구에 비하여 유의하게 많았다. I3의 16S rRNA 유전자에 대한 염기서열 분석 결과 시트로박터 종의 그것과 97%의 염기서열 상동성을 보였다. I3을 파블로바 비리디스와 혼합배양할 경우 I3의 성장도 유의하게 증가하였으며, 이것은 조류와 그 표면에 부착하여 살아가는 세균들 사이에 공생관계가 존재한다는 것을 제시한다. 미세조류와 세균과의 공생관계는 전자현미경적 관찰을 이용하여 확인하였다.
해양 미세조류인 파블로바 비리디스는 빨리 자라며, DHA, EPA와 같은 해양생물을 키우는데 필수적인 영양요소를 축적하는 능력을 가지고 있어 어류와 새우류 치어 사육에 이용되어 왔다. 본 연구에서는 파블로바 비리디스와이 미세조류의 표면에 붙어서는 세균과의 공생적 상호작용을 연구하였다. 무균의 파블로바 비리디스 균주는 항생제 혼합액을 포함하는 액체배지에 반복 배양함으로써 얻어졌다. 무균상태는 항생제를 포함하지 않는 배지에 3번 계대배양한 후 확인하였다. 무균상태의 조류는 이 조류로부터 분리되었으며, 임의로 I1–I5로 명명한 세균과 혼합배양하면서 조류의 성장 촉진 효과를 조사하였다. 모든 세균이 파블로바 비리디스의 생장을 촉진하였으며, 그 중 I3로 명명한 세균이 5가지 세균 중 가장 효과가 좋았다. 혼합배양 상태에서 파블로바 비리디스의 세포 수는 대조구에 비하여 유의하게 많았다. I3의 16S rRNA 유전자에 대한 염기서열 분석 결과 시트로박터 종의 그것과 97%의 염기서열 상동성을 보였다. I3을 파블로바 비리디스와 혼합배양할 경우 I3의 성장도 유의하게 증가하였으며, 이것은 조류와 그 표면에 부착하여 살아가는 세균들 사이에 공생관계가 존재한다는 것을 제시한다. 미세조류와 세균과의 공생관계는 전자현미경적 관찰을 이용하여 확인하였다.
The marine microalga Pavlova viridis can grow fast and has the ability to accumulate essential nutrients for culturing marine animals, such as EPA and DHA, and it has been used as food for raring larval fish and prawn. The symbiotic relationship between the flagellate microalga Pavlova viridis and i...
The marine microalga Pavlova viridis can grow fast and has the ability to accumulate essential nutrients for culturing marine animals, such as EPA and DHA, and it has been used as food for raring larval fish and prawn. The symbiotic relationship between the flagellate microalga Pavlova viridis and its associated bacteria was investigated. An axenic culture of P. viridis was obtained by repeated treatment of the microalga with an antibiotic cocktail. The axenic status was confirmed after sub-culturing three times in a sterile f/2 medium without an antibiotic. The axenic alga was then co-inoculated with five bacteria, arbitrarily designated as I1–I5, isolated from the alga to test the growth promotion of the algae. All bacterial strains promoted the growth of P. viridis, and bacterial isolate I3 was the most effective among the five bacteria tested. The cell number of P. viridis in the co-culture with I3 was significantly higher than that of the control culture. A sequence analysis of the 16S rRNA gene isolated from I3 revealed a 97% nucleotide sequence similarity to that of Citrobacter sp. The growth of strain I3 was also significantly enhanced by co-culturing with P. viridis, indicating a symbiotic relationship between the microalga and its associated bacterium. The association between the microalga and bacterium was confirmed by scanning electron microscopy.
The marine microalga Pavlova viridis can grow fast and has the ability to accumulate essential nutrients for culturing marine animals, such as EPA and DHA, and it has been used as food for raring larval fish and prawn. The symbiotic relationship between the flagellate microalga Pavlova viridis and its associated bacteria was investigated. An axenic culture of P. viridis was obtained by repeated treatment of the microalga with an antibiotic cocktail. The axenic status was confirmed after sub-culturing three times in a sterile f/2 medium without an antibiotic. The axenic alga was then co-inoculated with five bacteria, arbitrarily designated as I1–I5, isolated from the alga to test the growth promotion of the algae. All bacterial strains promoted the growth of P. viridis, and bacterial isolate I3 was the most effective among the five bacteria tested. The cell number of P. viridis in the co-culture with I3 was significantly higher than that of the control culture. A sequence analysis of the 16S rRNA gene isolated from I3 revealed a 97% nucleotide sequence similarity to that of Citrobacter sp. The growth of strain I3 was also significantly enhanced by co-culturing with P. viridis, indicating a symbiotic relationship between the microalga and its associated bacterium. The association between the microalga and bacterium was confirmed by scanning electron microscopy.
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제안 방법
8-ml microcentrifuge tube. The cells were sequentially dehydrated with 1.5 ml of 20%, 30%, 50%, 70%, 80%, 85%, 90%, 95%, and 100% ethanol for 30 min each. After two more dehydration steps at 100% ethanol for 30 min, the cells were stored overnight in 100% ethanol.
The opti- mum temperature was determined by inoculating a 40-µl ali- quot of bacterial isolate I3 culture to 4 ml of LB broth and incubating the culture at 4℃, 20℃, 25℃, 30℃, 37℃, or 45℃ with shaking at 150 rpm for 24 hr. The optimum NaCl con- centration for bacterial growth was examined by preparing LB broth containing 0%, 1%, 2%, 3%, 4%, 5%, 7%, and 10% NaCl and inoculating 4 ml of broth with a 40-μl aliquot of bacterial isolate I3 culture, followed by incubation at 37℃ with shaking at 150 rpm for 24 hr. The optimum pH was examined by preparing LB broth containing 1% NaCl with the pH adjusted to 3, 4, 5, 6, 7, 8, 9, or 10; then 4 ml of broth was inoculated with a 40-μl aliquot of bacterial isolate I3 culture and incubated at 37℃ with shaking at 150 rpm for 24 hr.
대상 데이터
The PCR products were visualized in a 1% agarose gel. The amplified DNA fragments were purified from agarose gels using a Gel SV kit (Gene All, Seoul, Korea) and were sequenced using 18S rRNA gene primers (512F/978R; G&C Bio, Daejon, Korea). Homology analysis was carried out using BLAST (http:// blast.
The marine microalga P. viridis was obtained from the Korea Marine Microalgae Culture Center, Pukyong National University, Busan, Korea. The alga was cultured in f/2 me- dium [14, 15] at 20℃ under a 16:8 hr light:dark cycle with a light intensity of 30 μmol m–2 s–1 [1].
이론/모형
The PCR product was sequenced using 16S rRNA gene primers (27F and 1492R). Homology analysis was carried out using EzTaxon (http://www.ezbiocloud.net/).
성능/효과
2. An UPGMA phylogenetic tree constructed based on the 16S rRNA gene of the isolate 3. Citrobacter species showing the high sequence similarity in the analysis using Eztaxon were included and Escherichia coli was included as an outgroup. The result is from 1,000 bootstraps.
The sequences were determined and compared with sequences in the database using EzTaxon. The 16S rRNA sequence of bacterial strains I1–I5 showed 99.950%, 99.034%, 99.69%, 99.217%, and 98.825% sequence identity to that of Kocuria marina (AY 211385), Staphylococcus cohnii (D83361), Citrobacter freundii (ANAV01000046), Micrococcus yunnanensis (Fj214355), and Staphylococcus haemolyticus (L37600), respectively.
Homology analysis was carried out from the GenBank database using BLAST. The results indicated 99% nucleotide sequence iden- tity to that of the 18S rRNA sequence of P. viridis (GenBank accession number HQ877913).
후속연구
The bac- teria-free filtrate of bacterial strain I3 also promoted the growth of the microalga. These results suggest that the alga produces some organic compounds that increase the growth of the bacterium, and that the bacterial strain produces some metabolites that promote the growth of the microalga, in- dicating that these two organisms have developed a sym- biotic relationship and future study for the identification of the substances involved in the interaction will clarify the relationships. Because the interactions between algae and bacteria occur in an aquatic environment where growth-pro- moting metabolites can diffuse quickly, close contact is re- quired between the two organisms.
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