비배양에 근거한 16S rDNA-DGGE fingerprinting 방법을 적용하여 공생세균 군집구조를 조사하였다. Zobell 배지와 천일염 배지를 사용하여 총 164균주를 선별하였다. 이들 균주로 부터 증폭한 16S rDNA를 제한효소 HaeIII와 MspI을 사용하여 절단한 후 각각의 다른 RFLP 패턴으로 구분하였다. RFLP패턴으로부터 유래한 16S rDNA의 염기서열 분석 결과, 알려진 염기서열들과 95% 이상의 유사도를 나타내었으며 Proteobacteria (Alphaproteobacteria, Gammaproteobacteria), Actinobacteria, Firmicutes, Bacteroidetes의 4개의 문이 나타났으며 우점 군집은 Alphaproteobacteria였다. 해면에서 분리한 total gDNA로 부터 증폭한 16S rDNA의 DGGE 분석 결과 5개의 DGGE band가 확인 되었고, 각각의 band의 염기서열 분석 결과 알려진 염기서열들과 96% 이상의 유사도를 나타내었으며 band로부터 밝혀진 모든 서열들은 배양되지 않은 세균 클론들과 높은 상동성을 나타내었다. DGGE에 의한 공생세균 군집은 Proteobacteria (Alphaproteobacteria, Gammaproteobacteria), Actinobacteria, Spirochetes, Chloroflexi로 4개의 문(phylum)으로 나타났다. Spirastrella abata의 공생세균 군집에 대한 RFLP와 DGGE 적용 결과, Alphaproteobacteria, Gammaproteobacteria, Actinobacteria의 공통 세균 그룹이 발견되었으나 전체적인 공생세균 군집구조는 분석 방법에 따른 차이를 나타내었다.
Culture-dependent RFLP and culture-independent DGGE were employed to investigate the bacterial community associated with the marine sponge Spirastrella abata. A total of 164 bacterial strains associated with the sponge were cultivated using Zobell and Natural sea salt media. PCR amplicons of the 16S rDNA from the bacterial strains were digested with the restriction enzymes HaeIII and MspI, and then assigned into different groups according to their restriction patterns. The 16S rDNA sequences derived from RFLP patterns showed more than 95% similarities compared with known bacterial species, and the isolates belonged to four phyla, Proteobacteria (Alphaproteobacteria, Gammaproteobacteria), Actinobacteria, Firmicutes, and Bacteriodetes, of which Alphaproteobacteria was dominant. DGGE fingerprinting of 16S rDNAs amplified from the sponge- derived total gDNA showed five major DGGE bands, and their sequences showed more than 96% similarities compared with available sequences. The sequences derived from DGGE bands revealed high similarity with the uncultured bacterial clones. DGGE revealed that bacterial community consisted of four phyla, including Proteobacteria (Alphaproteobacteria, Gammaproteobacteria), Actinobacteria, Spirochetes, and Chloroflexi. Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria were commonly found in bacteria associated with S. abata by both RFLP and DGGE methods; however, overall bacterial community in the sponge differed depending on the analysis methods.
