Genome sequence of the endosymbiont Rickettsia peacockii and comparison with virulent Rickettsia rickettsii: identification of virulence factors

Abstract

Rickettsia peacockii, also known as the East Side Agent, is a non-pathogenic obligate intracellular bacterium found as an endosymbiont in Dermacentor andersoni ticks in the western USA and Canada. Its presence in ticks is correlated with reduced prevalence of Rickettsia rickettsii, the agent of Rocky Mountain Spotted Fever. It has been proposed that a virulent SFG rickettsia underwent changes to become the East Side Agent. We determined the genome sequence of R. peacockii and provide a comparison to a closely related virulent R. rickettsii. The presence of 42 chromosomal copies of the ISRpe1 transposon in the genome of R. peacockii is associated with a lack of synteny with the genome of R. rickettsii and numerous deletions via recombination between transposon copies. The plasmid contains a number of genes from distantly related organisms, such as part of the glycosylation island of Pseudomonas aeruginosa. Genes deleted or mutated in R. peacockii which may relate to loss of virulence include those coding for an ankyrin repeat containing protein, DsbA, RickA, protease II, OmpA, ScaI, and a putative phosphoethanolamine transferase. The gene coding for the ankyrin repeat containing protein is especially implicated as it is mutated in R. rickettsii strain Iowa, which has attenuated virulence. Presence of numerous copies of the ISRpe1 transposon, likely acquired by lateral transfer from a Cardinium species, are associated with extensive genomic reorganization and deletions. The deletion and mutation of genes possibly involved in loss of virulence have been identified by this genomic comparison. It also illustrates that the introduction of a transposon into the genome can have varied effects; either correlating with an increase in pathogenicity as in Francisella tularensis or a loss of pathogenicity as in R. peacockii and the recombination enabled by multiple transposon copies can cause significant deletions in some genomes while not in others.

Figure 1. Alignment of Rickettsia rickettsii and Rickettsia peacockii genomes.

The alignment of the Rickettsia rickettsii SS and Rickettsia peacockii genomes using progressive Mauve with default parameters shows the lack of synteny between the genomes of these closely related organisms. The breakpoints of the syntenic blocks in R. peacockii are largely associated (31 of 37) with the ISRpe1 transposon, indicated with black arrows. The genome on top is that of R. rickettsii SS (reference genome) and that below is R. peacockii.

Figure 2. Phylogenetic analysis of ISRpe1 transposon.

Neighbor joining (NJ) and maximum parsimony (MP) analyses included 14 taxa. Exclusion of gaps left 309 amino acids for the analyses; 105 amino acids were constant, 36 of the variable amino acids were parsimony uninformative and 168 of the variable amino acids were parsimony informative. Bootstrap analysis involved 2,000 replicates: top number is NJ bootstrap value and bottom number the MP bootstrap value. Genbank references for the proteins found in Text S7.

Figure 3. Maximum parsimony analysis of 27 parA proteins.

Exclusion of gaps left 166 amino acids for the analysis; 8 amino acids were constant, 2 of the variable amino acids were parsimony uninformative and 156 of the variable amino acids were parsimony informative. Bootstrap analysis involved 1,000 replicates: numbers at selected branches are the NJ (MP/NJ) bootstrap values that were ≥50%. The top two or three blastP hits to rickettsial plasmid parA's with E = >1e−30 were chosen for the analysis. The parA from the Trichoplax adhaerens genome project is likely from a bacterium associated with this simplest of eukaryotes as several contigs have homology to Rickettsiales. The parA proteins from the rickettsial endosymbiont of Ixodes scapularis (REIS) were added to the analysis following PCR and sequencing to confirm the presence of the genes in our REIS isolate (Baldridge et. al., in preparation). Maximum parsimony and neighbor joining (not shown) phylograms were congruent. Genbank references for the proteins found in Text S7.