Abstract
Rickettsia bellii is an obligate intracellular bacterium that is one of the few rickettsiae that encode a complete set of conjugative transfer (tra) genes involved in bacterial conjugation and has been shown to exhibit pili-like structures. The reductive genomes of rickettsiae beg the question whether the tra genes are nonfunctional or functioning to enhance the genetic plasticity and biology of rickettsiae. We characterized the transcriptional dynamics of R. bellii tra genes in comparison to genes transcribed stably and above the background level to understand when and at what levels the tra genes are active or whether the tra genes are degenerative. We 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. To validate the use of metG with other rickettsial genes exhibiting variable transcriptional patterns we examined its use with sca2 and rickA, genes involved in actin based motility. Both were shown to be up-regulated at different times of replication in Vero cells, showing variable and stable transcription levels of rickA and sca2, respectively. traA Ti was up-regulated at 72 hours post inoculation in the tick cell line ISE6, but showed no apparent changes in the monkey cell line Vero and mouse cell line L929. 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). Thus, the up-regulation of the tra genes indicated that the integrity and activity of each gene were intact and may facilitate the search for the optimal conditions necessary to demonstrate conjugation in rickettsiae.
Highlights
Rickettsiae are obligate-intracellular, coccobacillary, Gram-negative bacteria that include invertebrate symbionts
The overall domains of R. bellii TraATi are similar to the well characterized conjugal relaxases of other bacteria; we suspect that R. bellii TraATi expression may be a good indicator of the time of induction for potential bacterial conjugation
To verify that there were no differences in rickettsial numbers determined from rickettsiae prepared (RP) vs. whole cell (WC), R. bellii DNA was prepared and the gltA genomic copy numbers in each preparation were compared (Table 1)
Summary
Rickettsiae are obligate-intracellular, coccobacillary, Gram-negative bacteria that include invertebrate symbionts. Genetic analysis of rickettsiae has revealed that plasmids are widely distributed within the genus [1,2] and tra genes are common [3], suggesting that rickettsiae can transfer mobile genetic elements, i.e. plasmids, to receptive bacteria via conjugation. Several other rickettsiae, e.g. Rickettsia felis strain California 2, only encode a few of the tra genes, suggestive of an incomplete conjugation system [2]. The tra clusters of R. bellii RML 369-C, R. felis LSU-lb, and R. massiliae MTU are similar at the nucleotide sequence level, gene order, and gene orientation to at least one of the tra clusters of R. buchneri, suggesting multiple lateral gene transfer events between rickettsiae [7,10]. Bacterial conjugation has not been documented in any rickettsiae
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