Plasmids and Rickettsial Evolution: Insight from Rickettsia felis

BACKGROUND: The genome sequence of Rickettsia felis revealed a number of rickettsial genetic anomalies that likely contribute not only to a large genome size relative to other rickettsiae, but also to phenotypic oddities that have confounded the categorization of R. felis as either typhus group (TG) or spotted fever group (SFG) rickettsiae. Most intriguing was the first report from rickettsiae of a conjugative plasmid (pRF) that contains 68 putative open reading frames, several of which are predicted to encode proteins with high similarity to conjugative machinery in other plasmid-containing bacteria. METHODOLOGY/PRINCIPAL FINDINGS: Using phylogeny estimation, we determined the mode of inheritance of pRF genes relative to conserved rickettsial chromosomal genes. Phylogenies of chromosomal genes were in agreement with other published rickettsial trees. However, phylogenies including pRF genes yielded different topologies and suggest a close relationship between pRF and ancestral group (AG) rickettsiae, including the recently completed genome of R. bellii str. RML369-C. This relatedness is further supported by the distribution of pRF genes across other rickettsiae, as 10 pRF genes (or inactive derivatives) also occur in AG (but not SFG) rickettsiae, with five of these genes characteristic of typical plasmids. Detailed characterization of pRF genes resulted in two novel findings: the identification of oriV and replication termination regions, and the likelihood that a second proposed plasmid, pRFδ, is an artifact of the original genome assembly. CONCLUSION/SIGNIFICANCE: Altogether, we propose a new rickettsial classification scheme with the addition of a fourth lineage, transitional group (TRG) rickettsiae, that is unique from TG and SFG rickettsiae and harbors genes from possible exchanges with AG rickettsiae via conjugation. We offer insight into the evolution of a plastic plasmid system in rickettsiae, including the role plasmids may have played in the acquirement of virulence traits in pathogenic strains, and the likely origin of plasmids within the rickettsial tree

Rickettsia Phylogenomics: Unwinding the Intricacies of Obligate Intracellular Life

BACKGROUND: Completed genome sequences are rapidly increasing for Rickettsia, obligate intracellular alpha-proteobacteria responsible for various human diseases, including epidemic typhus and Rocky Mountain spotted fever. In light of phylogeny, the establishment of orthologous groups (OGs) of open reading frames (ORFs) will distinguish the core rickettsial genes and other group specific genes (class 1 OGs or C1OGs) from those distributed indiscriminately throughout the rickettsial tree (class 2 OG or C2OGs). METHODOLOGY/PRINCIPAL FINDINGS: We present 1823 representative (no gene duplications) and 259 non-representative (at least one gene duplication) rickettsial OGs. While the highly reductive (approximately 1.2 MB) Rickettsia genomes range in predicted ORFs from 872 to 1512, a core of 752 OGs was identified, depicting the essential Rickettsia genes. Unsurprisingly, this core lacks many metabolic genes, reflecting the dependence on host resources for growth and survival. Additionally, we bolster our recent reclassification of Rickettsia by identifying OGs that define the AG (ancestral group), TG (typhus group), TRG (transitional group), and SFG (spotted fever group) rickettsiae. OGs for insect-associated species, tick-associated species and species that harbor plasmids were also predicted. Through superimposition of all OGs over robust phylogeny estimation, we discern between C1OGs and C2OGs, the latter depicting genes either decaying from the conserved C1OGs or acquired laterally. Finally, scrutiny of non-representative OGs revealed high levels of split genes versus gene duplications, with both phenomena confounding gene orthology assignment. Interestingly, non-representative OGs, as well as OGs comprised of several gene families typically involved in microbial pathogenicity and/or the acquisition of virulence factors, fall predominantly within C2OG distributions. CONCLUSION/SIGNIFICANCE: Collectively, we determined the relative conservation and distribution of 14354 predicted ORFs from 10 rickettsial genomes across robust phylogeny estimation. The data, available at PATRIC (PathoSystems Resource Integration Center), provide novel information for unwinding the intricacies associated with Rickettsia pathogenesis, expanding the range of potential diagnostic, vaccine and therapeutic targets

Phylogeny of Gammaproteobacteria▿ §

The phylogeny of the large bacterial class Gammaproteobacteria has been difficult to resolve. Here we apply a telescoping multiprotein approach to the problem for 104 diverse gammaproteobacterial genomes, based on a set of 356 protein families for the whole class and even larger sets for each of four cohesive subregions of the tree. Although the deepest divergences were resistant to full resolution, some surprising patterns were strongly supported. A representative of the Acidithiobacillales routinely appeared among the outgroup members, suggesting that in conflict with rRNA-based phylogenies this order does not belong to Gammaproteobacteria; instead, it (and, independently, “Mariprofundus”) diverged after the establishment of the Alphaproteobacteria yet before the betaproteobacteria/gammaproteobacteria split. None of the orders Alteromonadales, Pseudomonadales, or Oceanospirillales were monophyletic; we obtained strong support for clades that contain some but exclude other members of all three orders. Extreme amino acid bias in the highly A+T-rich genome of Candidatus Carsonella prevented its reliable placement within Gammaproteobacteria, and high bias caused artifacts that limited the resolution of the relationships of other insect endosymbionts, which appear to have had multiple origins, although the unbiased genome of the endosymbiont Sodalis acted as an attractor for them. Instability was observed for the root of the Enterobacteriales, with nearly equal subsets of the protein families favoring one or the other of two alternative root positions; the nematode symbiont Photorhabdus was identified as a disruptor whose omission helped stabilize the Enterobacteriales root

Annotation of <i>R. felis</i> large (pRF) and small (pRFδ) plasmid-encoded genes that are absent on the <i>R. felis</i> chromosome, and their distribution in other rickettsiae, other bacterial sequences, and other non-bacterial taxa.

a<p>ORF labels that are bolded depict putative genes that are unknown from other published rickettsiae genomes.</p>b<p>AG = ancestral group, TG = typhus group, TRG = transitional group, SFG = spotted fever group, bac = present (y) or absent (n) in a diverse array of non-rickettsial bacteria, or present in a few (f) bacteria, nb = present (y) or absent (n) in non-bacterial organisms. Presence (y) or absence (n) of putative orthologs found in other plasmids (pl) are listed. Br = <i>R. bellii</i> str. RML369-C, Bo = <i>R. bellii</i> str. OSU 85 389, Ca = <i>R. canadensis</i> str. McKiel, Pr = <i>R. prowazekii</i> str. Madrid E, Ty = <i>R. typhi</i> str. Wilmington, Ak = <i>R. akari</i> str. Hartford, Fe = <i>R. felis</i> str. URRWXCal2, Ri = <i>R. rickettsii</i>, Co = <i>R. conorii</i> str. Malish 7, and Si = <i>R. sibirica</i> str. 246. r = reduced gene relative to the plasmid gene, t = truncated gene relative to the plasmid gene, s = split gene relative to the plasmid gene.</p

Results of a BlastP search using pRF23 (parA) as a query.

<p>Only sequences with a score greater than 80 bits are shown.</p

Comparison of two hypotheses for the evolution of plasmids in rickettsiae.

<p>(A) The appearance of a plasmid system in <i>R. felis</i> (as a member of SFG rickettsiae) as recently suggested (Ogata et al., 2005b; Blanc et al. 2007). (B) Our hypothesis centered on the notion that the ancestor to all rickettsiae harbored a plasmid system with subsequent losses in the ancestors to the TG and SFG rickettsiae, and in <i>R. canadensis</i> and <i>R. bellii</i> str. RML369-C. Red = ancestral rickettsiae, Aquamarine = typhus group, light blue = transitional group, brown = true spotted fever group. Trees are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000266#pone-0000266-g002" target="_blank">Figure 2A</a>.</p

Characteristics and summary information of predicted origin of replication (<i>oriV</i>) of the pRF plasmid of <i>Rickettsia felis.</i>

<p>(A) Schematic map of the pRF with shaded regions containing the putative <i>oriV</i> (right) and replication termination region (left). The region outlined in the dark dashed line depicts the portion of the plasmid missing in pRFδ (pRF15-pRF38). Grey boxes depict genes, with gene names described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000266#pone-0000266-t001" target="_blank">Tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000266#pone-0000266-t002" target="_blank">2</a>. Red lines depict coding strands, and yellow blocks depict areas of gene overlap. (B) AT-skew of pRF, with AT-skew (blue), cumulative AT-skew (red) and minimum AT-skew (orange). (C) CG-skew of pRF, with CG-skew (blue), cumulative CG-skew (red) and maximum CG-skew (orange). Plots generated and values computed with GenSkew (<a href="http://mips.gsf.de/services/analysis/genskew" target="_blank">http://mips.gsf.de/services/analysis/genskew</a>).</p

Individual phylogeny estimations for the seven pRF proteins used in the combined analysis of pRF.

<p>(A, B) Majority rule consensus trees. (C–H) Strict consensus trees. All analyses were of amino acids from an exhaustive search under parsimony with branch support from one million bootstrap replications. Bootstrap values are placed above branches. Percentages of nodes recovered in majority rule consensus trees are shown below branches. Scores are tree lengths, with total characters and number of parsimony informative characters provided.</p

Comparison of phylogeny estimations from exclusively chromosomal proteins and proteins present on the chromosome and plasmids of <i>R. felis.</i>

<p>(A) Estimated phylogeny of 21 exclusively chromosomal proteins from 10 rickettsial strains. (B) Estimated phylogeny of 10 proteins present on the chromosome and plasmids of <i>R. felis</i>. “Ancestral” (red) refers to primitive rickettsiae with no known potential for host virulence. TG (aquamarine) = typhus group, TRG (light blue) = transitional group and SFG (brown) = spotted fever group. TG and TRG boxes depict the major differences in tree topologies. The pRF genes are boxed and shaded. Results from both analyses of amino acids are from an exhaustive search under parsimony with branch support from one million bootstrap replications.</p

Annotation of <i>R. felis</i> large (pRF) and small (pRFδ) plasmid-encoded genes that are present on the <i>R. felis</i> chromosome, and their distribution in other rickettsiae, other bacterial sequences, and other non-bacterial taxa.

a<p>ORF labels that are bolded depict putative genes that are unknown from other published rickettsiae genomes. Underlined ORFs depict sequences analyzed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000266#pone-0000266-g002" target="_blank">Figures 2B</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000266#pone-0000266-g003" target="_blank">3</a>.</p>b<p>AG = ancestral group, TG = typhus group, TRG = transitional group, SFG = spotted fever group, bac = present (y) or absent (n) in a diverse array of non-rickettsial bacteria, or present in a few (f) bacteria, nb = present (y) or absent (n) in non-bacterial organisms. Presence (y) or absence (n) of putative orthologs found in other plasmids (pl) are listed. Br = <i>R. bellii</i> str. RML369-C, Bo = <i>R. bellii</i> str. OSU 85 389, Ca = <i>R. canadensis</i> str. McKiel, Pr = <i>R. prowazekii</i> str. Madrid E, Ty = <i>R. typhi</i> str. Wilmington, Ak = <i>R. akari</i> str. Hartford, Fe = <i>R. felis</i> str. URRWXCal2, Ri = <i>R. rickettsii</i>, Co = <i>R. conorii</i> str. Malish 7, and Si = <i>R. sibirica</i> str. 246. r = reduced gene relative to the plasmid gene, t = truncated gene relative to the plasmid gene, s = split gene relative to the plasmid gene. * = similar to virD4 genes.</p