rpoB Sequence Analysis of Cultured Tropheryma whippelii

Abstract

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 ␤-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 16S 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.

Quantitative Detection of Tropheryma whipplei DNA by Real-Time PCR

“…We used primers tws3f and tws4r to target a 489-bp fragment of the 16S-23S ribosomal DNA intergenic spacer (ITS) (5) and primers TWRPOB.F and TWRPOB.R to target a 650-bp fragment of the ␤-subunit of the RNA polymerase gene (rpoB) (1). Either 25 mg of tissue or 1 ml of aspirate from each sample was used for DNA extraction, as previously described (3).…”

Microbial degradation of disodium terephthalate by Rhodococcus sp. 9-003

“…The DNA sequencing indicated strain 9-003 was close matched with Rhodococcus globerulus, the percent difference between strain 9-003 and Rhodococcus globerulus was 0.20%. The phylogenetic relationships were analyzed by the neighbor-joining method, and the results also supported that strain 9-003 was near-perfect match of the Rhodococcus genus and globerulus species [18][19][20]. Analysis of gene sequence and phylogenetic relationships suggested strain 9-003 is a member of Rhodococcus sp.…”

Identification of Bacillus anthracis by rpoB Sequence Analysis and Multiplex PCR

“…The rpoB gene, encoding the RNA polymerase ␤-subunit, has been used as a marker for bacterial identification and for phylogenetic study (5,6,14,16,17,20,25). Recently, the rpoB gene was used for the real-time PCR detection of B. anthracis (23); however, false-positive results were observed.…”