Alfreider A, Vogt C, Hoffmann D, Babel W
Department of Environmental Microbiology, UFZ Centre for Environmental Research, Leipzig 04318, Germany.
Microb Ecol. 2003 May;45(4):317-28. doi: 10.1007/s00248-003-2004-9. Epub 2003 Apr 28.
To test our hypothesis that microbial autotrophic CO2 fixation plays an important role in subsurface systems of two large groundwater remediation projects, several anaerobic/microaerobic aquifer and groundwater samples were taken and used to investigate the distribution and phylogenetic diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large-subunit genes. Two primer sets were designed for amplifying partial-subunit genes of RubisCO forms I and II from the DNA, directly extracted from the samples. PCR products were used to construct five clone libraries with putative RubisCO form I sequences, and two libraries of DNA amplified by form II primers. Selected clones were screened for variation by restriction fragment length polymorphism analysis, and a total of 28 clone inserts were sequenced and further analyzed. The phylogenies constructed from amino acid sequences derived from the partial RubisCO large-subunit sequences showed a distinct pattern. Diverse sequences affiliated to the cluster of green-like type IA RubisCO sequences were found, representing various obligate and facultative chemolithoautotrophic Proteobacteria, whereas type II RubisCO sequences detected were most closely related to those of thiobacilli species. An isolate obtained from aquifer enrichment culture, which has been provisionally named Halothiobacillus sp. RA13 on the basis of its 16S rDNA sequence, was found to contain both types of RubisCO genes, i.e., forms I and II. Physiological and ecological considerations are discussed in the context of additional microbial data and physicochemical properties.
为了验证我们的假设，即微生物自养二氧化碳固定在两个大型地下水修复项目的地下系统中发挥重要作用，我们采集了几个厌氧/微需氧含水层和地下水样本，用于研究核酮糖-1,5-二磷酸羧化酶/加氧酶（RubisCO）大亚基基因的分布和系统发育多样性。设计了两组引物，用于从直接从样本中提取的DNA中扩增RubisCO I型和II型的部分亚基基因。PCR产物用于构建五个含有假定RubisCO I型序列的克隆文库，以及两个由II型引物扩增的DNA文库。通过限制性片段长度多态性分析筛选选定的克隆以检测变异，总共对28个克隆插入片段进行测序并进一步分析。由部分RubisCO大亚基序列推导的氨基酸序列构建的系统发育树显示出明显的模式。发现了与绿色样IA型RubisCO序列簇相关的多种序列，代表了各种专性和兼性化能自养变形菌，而检测到的II型RubisCO序列与硫杆菌属物种的序列关系最为密切。从含水层富集培养物中获得的一株分离物，根据其16S rDNA序列暂命名为嗜盐硫杆菌属RA13，发现其同时含有两种类型的RubisCO基因，即I型和II型。结合其他微生物数据和物理化学性质对生理和生态因素进行了讨论。
