Comparative Genomics and Transcriptomics of Trait-gene Association

Background: The Order Rickettsiales includes important tick-borne pathogens, from Rickettsia rickettsii, which causes Rocky Mountain spotted fever, to Anaplasma marginale, the most prevalent vector-borne pathogen of cattle. Although most pathogens in this Order are transmitted by arthropod vectors, little is known about the microbial determinants of transmission. A. marginale provides unique tools for studying the determinants of transmission, with multiple strain sequences available that display distinct and reproducible transmission phenotypes. The closed core A. marginale genome suggests that any phenotypic differences are due to single nucleotide polymorphisms (SNPs). We combined DNA/RNA comparative genomic approaches using strains with different tick transmission phenotypes and identified genes that segregate with transmissibility.

Results: Comparison of seven strains with different transmission phenotypes generated a list of SNPs affecting 18 genes and nine promoters. Transcriptional analysis found two candidate genes downstream from promoter SNPs that were differentially transcribed. To corroborate the comparative genomics approach we used three RNA-seq platforms to analyze the transcriptomes from two A. marginale strains with different transmission phenotypes. RNA-seq analysis confirmed the comparative genomics data and found 10 additional genes whose transcription between strains with distinct transmission efficiencies was significantly different. Six regions of the genome that contained no annotation were found to be transcriptionally active, and two of these newly identified transcripts were differentially transcribed.

Conclusions: This approach identified 30 genes and two novel transcripts potentially involved in tick transmission. We describe the transcriptome of an obligate intracellular bacterium in depth, while employing massive parallel sequencing to dissect an important trait in bacterial pathogenesis.