rpoB sequence analysis of cultured Tropheryma whippelii
rpoB sequence analysis of cultured Tropheryma whippelii
复制标题
DOI：
10.1128/jcm.39.7.2425-2430.2001
复制
发表时间：
2001-07-01
影响因子：
9.4
通讯作者：
Raoult, D
中科院分区：
文献类型：
--
作者：
Drancourt, M;Carlioz, A;Raoult, D
直到最近，还没有分离出Tropheryma whippelii，因此遗传学研究仅限于基于保守基因的PCR扩增。在这项研究中，我们确定了核苷酸序列的rpoB（编码RNA聚合酶的β亚基）从培养的T。whippelii的基因组步移。霸王whippelii rpoB基因全长3,657 bp，GC含量为50.4%，编码1,218个氨基酸，分子量为138 kDa。比较T. whippelii RpoB与其他真细菌RpoB蛋白的序列相似性从57.19（肺炎支原体）到74.63%（结核分枝杆菌）。基于其RpoB序列与其他细菌的序列比较的系统发育分析的T，whippelii是一致的，以前来自165核糖体DNA（rDNA）序列，表明它属于放线菌分支。序列比较允许设计特异于T.白藓当将该引物组合到PCR中时，可以检测T。whippelii rpoB在三个16 S rDNA PCR阳性活检标本中的三个和七个阴性对照中的零个中表达。因此，rpoB可以在PCR介导的检测和鉴定这种新出现的细菌物种中被靶向。这种方法以前已被证明是有用的相关分枝杆菌的鉴定。这项研究强调了一种涉及分离然后繁殖新兴细菌的方法是快速获得这些病原体的广泛分子知识的有用方法。
Until recently no isolate of Tropheryma whippelii was available, and therefore genetic studies were limited to those based on PCR amplification of conserved genes. In this study we determined the nucleotide sequence of rpoB (encoding the Beta-subunit of RNA polymerase) from a cultured strain of T. whippelii using degenerate consensus PCR and genome walking. The T. whippelii rpoB consists of 3,657 bp with a 50.4% GC content and encodes 1,218 amino acids with a calculated molecular mass of 138 kDa. Comparison of T. whippelii RpoB with other eubacterial RpoB proteins indicated sequence similarity ranging from 57.19 (Mycoplasma pneumoniae) to 74.63% (Mycobacterium tuberculosis). Phylogenetic analysis of T, whippelii based on comparison of its RpoB sequence with sequences available for other bacteria was consistent with that previously derived from the 165 ribosomal DNA (rDNA) sequence, indicating that it belongs to the actinomyces clade. The sequence comparison allowed the design of a primer pair, TwrpoB.F and TwrpoB.R, specific for T. whippelii rpoB. When incorporated into a PCR, this primer pair allowed the detection of T. whippelii rpoB in three of three 16S rDNA PCR-positive biopsy specimens and zero of seven negative controls. rpoB could therefore be targeted in PCR-mediated detection and identification of this emerging bacterial species. This approach has previously been shown useful for the identification of related mycobacteria. This study underscores that a method involving isolation and then propagation of emerging bacteria is a useful way to quickly achieve extensive molecular knowledge of these pathogens.