Molecular analysis of the transfer (tra) genes of Rickettsia bellii RML 369-C and their function in bacterial conjugation

Abstract

Rickettsia bellii is an obligate intracellular bacterium. Among the Rickettsiales, R. bellii is unusual because it encodes a complete set of transfer (tra) genes and pili-like structures associated with bacterial conjugation. To investigate the relationship of the tra genes with bacterial conjugation, I first characterized the tra genes into multiple operons and predicted the cellular localization of each of the Tra proteins using predicted transmembrane-spanning domains and signal peptides in the context of current knowledge of the bacterial conjugation system used by E. coli. I also characterized the transcriptional dynamics of R. bellii tra genes in comparison to stably transcribed genes to understand when the tra genes are actively transcribed during the rickettsial life cycle. I determined that the best reference genes, out of 10 tested, were methionyl tRNA ligase (metG) or a combination of metG and ribonucleoside diphosphate reductase 2 subunit beta (nrdF), using statistical algorithms from two different programs: Normfinder and BestKeeper. The transcription of tra genes was positively correlated with one another and up-regulated from 12 to 72 hours post inoculation (HPI) when compared to RBE_0422 (an inactivated transposase-derivative found within the tra cluster) suggesting that bacterial conjugation may occur at later exponential growth or early stationary growth. Furthermore, a complementation assay was designed using traDF of R. bellii to rescue a traD mutant E. coli in transferring DNA. However, TraDF expression was not detected in two different E. coli expression strains. The data suggest that future experiments to express rickettsial Tra proteins in E. coli should be undertaken to exploit the R. bellii bacterial conjugation system and connect the rickettsial Tra proteins into functional genetic transfer systems.