新疆冷泉沉积物葡萄糖利用细菌群落多样性的稳定同位素标记分析

The glucose-utilizing bacterial diversity in the cold spring sediment of Shawan, Xinjiang,based on stable isotope probing

新疆沙湾冷泉低盐且不含硫化物,为了解其沉积物细菌群落的碳源利用,以13C稳定同位素标记的葡萄糖作为底物培养沉积物中的细菌,通过提取和分离带有13C标记的总DNA,结合16S rDNA-PCR克隆文库法以及限制性片段长度多态性方法,对冷泉沉积物中葡萄糖利用细菌群落多样性进行分析。随机挑取417个阳性克隆,HaeIII酶切分型,共获得27个OTUs。经测序、序列同源性对比和系统发育学分析,归为细菌域中的9个门,即厚壁菌门(Firmicutes)、绿弯菌门(Chloroflexi)、硝化螺旋菌门(Nitrospirae)、放线菌门(Actinobacteria)、变形杆菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、浮霉菌门(Planctomycetes)、蓝细菌门(Cyanobacteria)和酸杆菌门(Acidobacteria),其中,厚壁菌门和变形杆菌门为优势类群,分别占克隆文库的28.3%和38.6%。与前期研究比较,以葡萄糖为碳源的细菌OTUs仅占总菌群的31%,表明该环境中可能存在其它碳源利用方式的细菌群落。

Most methods for investigating bacterial diversity of soil microorganisms was lack of the ability to establish a causal relationship between function and community compositions.Recently,using stable isotopes to label phylogenetically informative biomolecules（phospholipid fatty acids,DNA,or RNA）,has the advantage of providing more definitive evidence that a detected population is active in a specific process,if that process leads to assimilation of C or N into cellular constituents.In practice,Nucleic acid SIP is considerably more sensitive and more informative for taxonomic purposes,particularly among of those related organisms,and thus is more generally useful. Shawan cold spring provides a typical non-sulfide cold spring environment in the arid and semi-arid areas of Xinjiang.In the previous research,the first overview of microbial diversity in the Shawan cold spring had provided,and the results showed that bacterial communities in the Shawan cold spring were diverse which comprised of 11 phyla and maybe exist a diversity of functional groups.In this paper,we surveyed the carbon sources utilization functional bacteria and their diversity in the sediment of the cold spring by stable isotope probing（SIP） and 16S rDNA clone library.The functional bacteria were enriched using 13C-labeled glucose as substrate,and then 13C-labeled genomic DNA was separated directly from the sediment samples and then it used as template for the PCR amplification of bacterial 16S rRNA gene and construction of the gene clone library.Based on restriction fragment length polymorphism（RFLP）,417 positive clones were picked up and grouped into 27 operational taxonomic units（OTUs） by Hae III digestion.Subsequently,unique clones were sequenced.The blast search in Genbank and phyogenetic sequence analysis showed that 27 OTUs were divided into 9 phyla of bacterial domain i.e.,Firmicutes,Chloroflexi,Nitrospirae,Actinobacteria,Proteobacteria,Bacteroidetes,Planctomycetes,Cyanobacteria and Acidobacteria.Of those,Firmicutes and Proteobacteria were the dominant groups,and accounted for 28.3% and 38.6% of the total clone library respectively. Compared with 86 OTUs in the previous publication,Gemmatimonadetes,Chlorobi and Verrucomicrobia were not captured in glucose-utilizing bacteria,and the number of OTUs in other phyla decreased evidently,especially in Acidobacteria,only 6.45% of OTUs was observed in this research（48% in the previous research）.In sum,that only 31% OTUs utilized glucose as carbon source in the system,suggesting there maybe exist other carbon sources utilization bacteria in the environment.In addition,we found that the incubating time and substrate concentration are key factors for the detection of some species that in low lever in the environment.As the result of enriching in this paper,a lots of OTUs were detected for the first time,such as the phylum Chloroflexi and genus Desulfuromonas.Particularly,genus Desulfuromonas was an important supplement to previous research,because strains belonged to the genus could reduce the sulfur to H2S,and the reason why the genus exited in a non-sulfide cold spring need further study.Additionally,compared with previous research,we found that the proportion of OTUs in Firmicutes was increased obviously from 1 to 7,which were divided into order Clostridiales and order Lactobacillales,and replaced Acidobacteria as the second dominant groups.Hence,the results indicated that an appropriate and reasonable pre-culture could be a useful supplement to study microbial composition and diversity in environmental samples by using culture-independent methods.