rpoB sequence analysis of cultured Tropheryma whippelii

Until recently no isolate of Tropheryma whippelii was available, and theref ore genetic studies were limited to those based on PCR amplification of con served genes. In this study we determined the nucleotide sequence of rpoB ( encoding the Beta-subunit of RNA polymerase) from a cultured strain of T. w hippelii using degenerate consensus PCR and genome walking. The T. whippeli i 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. whippe lii RpoB with other eubacterial RpoB proteins indicated sequence similarity ranging from 57.19 (Mycoplasma pneumoniae) to 74.63% (Mycobacterium tuberc ulosis). Phylogenetic analysis of T, whippelii based on comparison of its R poB sequence with sequences available for other bacteria was consistent wit h that previously derived from the 165 ribosomal DNA (rDNA) sequence, indic ating that it belongs to the actinomyces clade. The sequence comparison all owed the design of a primer pair, TwrpoB.F and TwrpoB.R, specific for T. wh ippelii rpoB. When incorporated into a PCR, this primer pair allowed the de tection of T. whippelii rpoB in three of three 16S rDNA PCR-positive biopsy specimens and zero of seven negative controls. rpoB could therefore be tar geted in PCR-mediated detection and identification of this emerging bacteri al species. This approach has previously been shown useful for the identifi cation of related mycobacteria. This study underscores that a method involv ing isolation and then propagation of emerging bacteria is a useful way to quickly achieve extensive molecular knowledge of these pathogens.