DGGE fingerprinting of culturable soil bacterial communities complements culture-independent analyses

Culture-dependent DGGE (CD DGGE) fingerprinting of the 16S rRNA gene was used to characterize mixed bacterial communities recovered on agar plates. Using R2A Agar as a growth medium, CD DGGE analysis resulted in clear banding patterns of sufficient complexity (16-32 major bands) and reproducibility to investigate differences in bacterial communities in a silt loam soil. Replicate CD DGGE profiles from plates inoculated with less-dilute samples (10⁻³) had a higher band count and were more similar (72-77%) than profiles from more-dilute samples (51-61%). Different culture media and incubation conditions resulted in distinct community fingerprints and increased the cumulative number of unique bands detected. When CD DGGE fingerprints were compared to profiles constructed from 16S rRNA genes obtained from culture-independent clone libraries (CB DGGE profiles) 34% of the bands were unique to the culture-dependent profiles, 32% were unique to the culture-independent profiles and 34% were found in both communities. These data demonstrate that culture-independent DGGE profiles are supplemented by the distinct bands detected in culture-dependent profiles. CD DGGE can be a useful technique to follow the dynamics of distinct culturable fractions of the soil bacterial community.     

非培养方法解析烟草根部内生细菌的群落结构

为了解烟草根部内生细菌多样性,采用改进的CTAB法提取烟草根部总DNA,利用细菌16 S rDNA基因通用引物对烟草根总DNA进行16 S rDNA扩增,构建烟草根部内生细菌16 S rDNA克隆文库;挑取具有不同酶切谱型的克隆进行测序、比对并构建16 S rDNA基因系统发育树。构建的烟草根部内生细菌16 S rDNA克隆文库中,155个克隆分属于36个不同的分类单元,Blast结果表明,大部分克隆与已知细菌的16 S rDNA序列相似性较高,分别属于变形菌门(Proteobacteria)的Alpha、Gamma、Beta亚群,放线菌门(Actinobacteria),拟杆菌门(Bacteroidetes),厚壁菌门(Fir-micutes)中的肠杆菌属(Enterobacter)、不动杆菌属(Acinetobacter)、寡养食单胞菌属(Stenotrophomonas)、假单胞菌属(Pseudomonas)、嗜甲基菌属(Methylobacillus)、食酸菌属(Acidovorax)、芽孢杆菌属(Bacillus)等16个属,其中13.5%的克隆序列与非可培养细菌(Uncultured bacteria)的相似性在93%～99%之间,假单胞菌属细菌是烟草根部内生细菌的优势种群。这些研究结果说明烟草根部存在较为丰富的内生细菌系统发育多样性,并且潜藏着未知的内生细菌资源。