Brusseau G A, Bulygina E S, Hanson R S
Gray Freshwater Biological Institute, University of Minnesota, Navarre 55392.
Appl Environ Microbiol. 1994 Feb;60(2):626-36. doi: 10.1128/aem.60.2.626-636.1994.
Fifteen small-subunit rRNAs from methylotrophic bacteria have been sequenced. Comparisons of these sequences with 22 previously published sequences further defined the phylogenetic relationships among these bacteria and illustrated the agreement between phylogeny and physiological characteristics of the bacteria. Phylogenetic trees were constructed with 16S rRNA sequences from methylotrophic bacteria and representative organisms from subdivisions within the class Proteobacteria on the basis of sequence similarities by using a weighted least-mean-square difference method. The methylotrophs have been separated into coherent clusters in which bacteria shared physiological characteristics. The clusters distinguished bacteria which used either the ribulose monophosphate or serine pathway for carbon assimilation. In addition, methanotrophs and methylotrophs which do not utilize methane were found to form distinct clusters within these groups. Five new deoxyoligonucleotide probes were designed, synthesized, labelled with digoxigenin-11-ddUTP, and tested for the ability to hybridize to RNA extracted from the bacteria represented in the unique clusters and for the ability to detect RNAs purified from soils enriched for methanotrophs by exposure to a methane-air atmosphere for one month. The 16S rRNA purified from soil hybridized to the probe which was complementary to sequences present in 16S rRNA from serine pathway methanotrophs and hybridized to a lesser extent with a probe complementary to sequences in 16S rRNAs of ribulose monophosphate pathway methanotrophs. The nonradioactive detection system used performed reliably at amounts of RNA from pure cultures as small as 10 ng.
已对15种甲基营养菌的小亚基rRNA进行了测序。将这些序列与之前发表的22种序列进行比较，进一步明确了这些细菌之间的系统发育关系，并说明了细菌的系统发育与生理特征之间的一致性。基于序列相似性，采用加权最小均方差法，用甲基营养菌的16S rRNA序列和变形菌纲各亚类中的代表性生物构建了系统发育树。甲基营养菌已被分成连贯的簇，同一簇中的细菌具有共同的生理特征。这些簇区分了利用磷酸核糖或丝氨酸途径进行碳同化的细菌。此外，发现甲烷氧化菌和不利用甲烷的甲基营养菌在这些组中形成了不同的簇。设计、合成了5种新的脱氧寡核苷酸探针，用地高辛-11-ddUTP进行标记，并测试了它们与从独特簇中所代表的细菌提取的RNA杂交的能力，以及检测通过暴露于甲烷-空气气氛中一个月而富集甲烷氧化菌的土壤中纯化的RNA的能力。从土壤中纯化的16S rRNA与与丝氨酸途径甲烷氧化菌的16S rRNA中存在的序列互补的探针杂交，并与与磷酸核糖途径甲烷氧化菌的16S rRNA中的序列互补的探针在较小程度上杂交。所使用的非放射性检测系统在来自纯培养物的低至10 ng的RNA量下可靠运行。
