Department of Molecular Evolution, University of Uppsala, Sweden
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Abstract |
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Introduction |
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In humans, repeated sequences represent about 50% of the entire genome (International Human Genome Sequencing Consortium, 2001
). These consist of transposon-derived repeats, processed pseudogenes, short sequence repeats, and large segmental duplications. Mobile elements, such as insertion sequences and transposons normally encode one or several enzymes required for transposition between the 10- to 40-bp inverted repeats flanking the element (Mahillon and Chandler 1998
; Mahillon, Leonard, and Chandler 1999
). Short repetitive elements previously described in the
-proteobacteria include the 125-bp intergenic repeat unit and the 152-bp-long RSA repeat (Bachellier, Clement, and Hofnung 1999
; Rudd 1999
).
A novel type of repetitive element was recently identified in Rickettsia conorii, called the Rickettsia Palindromic Element (RPE) (Ogata et al. 2000
). This element represents the first example of a repetitive element that is inserted into protein-coding genes in bacteria (Ogata et al. 2000
). However, the timing of acquisition is unclear, and it is not known whether the 45 RPEs in the R. conorii genome are related by species divergence or by recent intragenomic duplications. One extreme interpretation is that the RPEs are modern-day remnants of a highly mobile RNA world (Dwyer 2001
). On the other extreme, it has been suggested that RPE elements have spread by intragenomic proliferation subsequent to speciation within the genus Rickettsia (Ogata, Audic, and Claverie 2001
).
Here, we have studied the evolution of RPEs positioned downstream of the elongation factor genes in 10 species of Rickettsia. The genes encoding elongation factor Tu (tufA) and elongation factor G (fus) are normally colocated in the streptomycin (str) operon (Sicheritz-Ponten and Andersson 1997
). Most proteobacteria have one additional gene coding for elongation factor Tu (tufB), which is part of the tufB gene operon (Sicheritz-Ponten and Andersson 1997
). It has been suggested that the elongation factor genes were part of a large cluster of ribosomal protein genes (the so-called superribosomal protein operon) in the common ancestor of bacteria and archaea (Keeling, Charlebois, and Doolittle 1994
). The presumed ancestral gene order is conserved in bacteria such as Escherichia coli and Bacillus subtilis (Wächtershäuser 1998
).
It has been very informative to compare these conserved genomic structures with those found in R. prowazekii. This species is unique among the proteobacteria in that it has only one copy of the tuf gene that is not contained within the conventional str operon or within the typical tufB gene neighborhood (Syvänen et al. 1996
). Upstream of the single tuf gene, we have identified two of the tRNA genes (tRNATyr and tRNAGly) that are located upstream of the tufB gene in E. coli (Syvänen et al. 1996
). Downstream of this gene, we have found the S10 ribosomal protein gene operon that is located downstream of the tufA gene in E. coli (Syvänen et al. 1996
). The chimeric disposition of the single tuf gene is thought to be the result of an intrachromosomal recombination event that caused an inversion of the segment flanked by the two ancestral tuf genes, followed by a deletion of one tuf gene (Andersson and Kurland 1995
; Syvänen et al. 1996
).
In this article, we have examined the arrangement of the elongation factor genes in several -proteobacterial species and studied the phylogeny of RPEs located in the spacer region downstream of the elongation factor genes in 10 Rickettsia species. The Typhus Group Rickettsia (TG) includes species such as R. prowazekii and R. typhi that are transmitted by insects. The Spotted Fever Group Rickettsia (SFG) are transmitted by ticks and include species such as R. rickettsii, R. parkeri, R. sibirica, R. montana, R. rhipicephali, R. felis and R. helvetica. We show that the acquisition of RPEs as well as the rearrangement of the elongation factor genes occurred before the divergence of the TG and the SFG, but subsequent to divergence of genera within the
-proteobacteria.
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Materials and Methods |
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Polymerase Chain Reaction Amplification
We designed a set of degenerate primers (table 1
) based on highly conserved parts of the tuf, rpsJ, fus, secE, trpT, and nusG genes. The primers were constructed so that the codon bias of Rickettsia (Andersson et al. 1998
) was taken into account. An additional set of species-specific primers was designed in order to cover missing parts and to deal with the difficulties of cross-species polymerase chain reaction (PCR) amplification.
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Sequencing
Plasmid clones and overlapping PCR products were sequenced on both strands using the PCR primers as well as internal oligomers. Sequence reactions were carried out using terminator cycle sequencing based on the Sangers dideoxy chain termination method. Reactions included 3060 ng PCR product and 3.2 pmol primer in addition to dNTPs, fluorescent ddNTPs, and thermostable DNA polymerase provided in ABI PRISMTM Big Dye Terminator Cycle Sequencing Kit (Perkin Elmer). Thermal cycling was performed using a Peltier Thermal Cycler 225 (MS Research) in 25 cycles repeating the following steps: 96°C for 10 s, annealing temperature for 5 s, and 60°C for 4 min. Amplifications were separated on a 5% long ranger denaturing polyacrylamide gel on an ABI sequenator (Perkin Elmer).
Sequence Analysis
The sequences were assembled and edited using STADEN software (Staden 1996
). Processed consensus sequences were aligned with the aid of ClustalW software (Thompson, Higgins, and Gibson 1994
). We calculated the overall G+C content and the G+C content at third codon positions with the help of CodonW software (Lloyd and Sharp 1992
). Pairwise distances for synonymous and nonsynonymous substitutions, Ks and Ka values, were calculated for the fus and the tuf genes separately using Li's method with the aid of MATDISLI software (Li 1993
). The phylogenetic reconstructions were accomplished using maximum parsimony (MP) and neighbor-joining (NJ) (Saitou and Nei 1987
) methods in Phylo_win (Galtier, Gouy, and Gautier 1996
) and PAUP (Swofford 1998
) software.
Nucleotide Sequence Accession Numbers
The nucleotide sequences obtained in this study have been given the following GenBank accession numbers: The tuf gene and downstream sequences in R. parkeri, AF502180; R. sibirica, AF502181; R. rickettsii, AF502179; R. montana, AF502183; R. rhipicephali, AF502182; R. felis, AF502185; R. helvetica, AF502184; R. typhi, AF502186; and R. bellii, AF502187. The fus gene and downstream sequences in R. parkeri, AF502171; R. sibirica, AF502172; R. rickettsii, AF502170; R. montana, AF502174; R. rhipicephali, AF502173; R. felis, AF502178; R. helvetica, AF502175; R. typhi, AF502176; and R. bellii, AF502177. The B. henselae tufA gene, AY099295; tufB gene, AY099292; and fus gene, AY099293. The R. capsulatus fus and tufA genes, AY099291; and tufB gene, AY099294.
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Results |
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Members of the TG have GC3S values that are about 3%5% lower those of members of the SFG, as also observed for several other genes (Andersson and Andersson 1997
, 1999a
, 1999b
, 2001
). In contrast to the strong effect on silent sites, the biased mutation pressure has only altered the amino acid composition features of the elongation factors to a minor extent. For EF-G, the average ratio of amino acids encoded by A+Trich codons over those encoded by G+Crich codons (Andersson and Sharp 1996
) is 1.35 in Rickettsia compared with 0.99 in R. capsulatus, and no variation in the overall amino acid composition is observed for EF-Tu. This suggests that the elongation factors are suitable as phylogenetic markers despite the strong mutation pressures acting on the
-proteobacterial genomes.
Phylogenetic Relationships Inferred from the Elongation Factors
To establish the phylogenetic context of the -proteobacterial species selected for analysis, we performed a phylogenetic reconstruction based on the combined amino acid sequences of the two elongation factors (fig. 1
). The EF-Tu and EF-G sequences from Rickettsia, B. henselae, R. capsulatus, and Agrobacterium tumefaciens were first aligned with the corresponding sequences from representative bacterial species. The
-proteobacterial elongation factors are of similar sizes, with a few exceptions. For example, B. henselae and A. tumefaciens share an insertion of five amino acids in EF-G with the consensus sequence GRDG(K/R), and all Rickettsia species are characterized by a unique insertion of nine amino acids with the consensus sequence VKDLKDEDK.
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We utilized the same methods to establish the phylogenetic relationships among the rickettsial species selected for analysis (fig. 1B
). Because of their close relationships, this analysis was based on the nucleotide sequences of the fus and tuf genes. The inferred tree separates the TG (R. prowazekii and R. typhi) from the SFG (R. rickettii, R. sibirica, R. parkeri, R. montana, R. rhipicephali, R. felis, and R. helvetica), whereas R. bellii represents an earlier diverging species. The results further suggest that the pathogenic species R. parkeri, R. sibirica, and R. rickettsii are phylogenetically distinct from the nonpathogenic species R. rhipicephali and R. montana (97% bootstrap support). R. felis and R. helvetica are placed as early diverging species within the SFG in this tree. The general features of this phylogeny match the tree topology previously obtained with small and large ribosomal RNA as well as with the citrate synthase gene sequences (Roux and Raoult 1995
; Stothard and Fuerst 1995
; Roux et al. 1997
; Andersson et al. 1999
).
Likewise, the frequencies of nonsynonymous (Ka) and synonymous (Ks) substitutions distinguish the TG from the SFG (table 2
). The mean Ka and Ks values for comparisons across the two groups are 0.046 and 0.15 substitutions per position for the tuf gene, respectively. These values are comparable to the mean Ka and Ks values of 0.035 and 0.33 substitutions per site for the fus gene. R. bellii is equally distant from both of these two groups, as inferred from Ka and Ks values in the range of 0.15 and 0.35 for the tuf gene and 0.04 and 0.66 for the fus gene. These synonymous substitution frequencies are consistent with previous estimates for these species based on other gene sequences (Andersson et al. 1998
; Andersson and Andersson 1999a
, 1999b
). Taken together, our analysis confirms that the elongation factor genes sequenced here accurately reflect the evolution of the
-proteobacterial genomes in which they reside.
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RPEs Downstream of the Elongation Factor Genes
The variation in sizes and G+C content values of the tuf-rpsJ and the fus-secE intergenic regions in Rickettsia correlates with the presence of RPEs (compare table 3
and fig. 2B
). Thus, the RPE-containing tuf-rpsJ intergenic regions of R. rickettsii, R. sibirica, R. parkeri, R. rhipicephali, and R. helvetica are 200 bp in size and have mean G+C content values of 32%37%, as expected for coding sequences in Rickettsia (Andersson et al. 1998
). In contrast, the RPE-lacking spacers of R. prowazekii, R. typhi, R. montana, R. felis, and R. bellii are approximately 100 bp shorter and have G+C content values of only 17%20%, as expected for noncoding sequences (Andersson et al. 1998
). Likewise, the fus-secE spacer region varies in size from 205 nucleotides in R. bellii that lacks an RPE to about 400 bp in the RPE-containing spacers. The high G+C content of the fus-secE spacer regions is explained by the presence of a gene coding for tRNATrp in this region.
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Any pairs of RPEs related by proliferation within the R. conorii genome would easily be recognized as a cluster in this analysis, as inferred from the short branches and high bootstrap support values for the RPE-tuf and RPE-fus clusters (fig. 3 ). No such strong clustering is observed. On the contrary, the 19 RPEs located inside coding regions were found to be more divergent from each other than either of the RPE-tuf and RPE-fus sequences were among the pathogenic SFG (R. rickettsii, R. sibirica, R. conorii, and R. parkeri) (fig. 3 ). Only two significant clusters could be identified among the coding RPEs: ubiH and RP167 (65%87% bootstrap support) and orf3 and rlpA (71%77% bootstrap support), but even for these, branch lengths were much longer than for RPE-fus and RPE-tuf in the pathogenic SFG clusters. No additional clusters with strong bootstrap support values were observed for the 25 RPEs located in noncoding regions (fig. 3 ) and for the complete set of RPEs (data not shown). This suggests that the RPEs identified in the R. conorii genome are the result of proliferation before speciation within the SFG. However, because of the short sizes of the RPEs, it is difficult to elucidate the exact order of duplication and divergence.
Loss and Deterioration of RPEs
The placement of R. montana and R. felis in phylogenetic trees based on the elongation factor genes (fig. 1B
) and the RPE-fus sequences (fig. 3A
) suggests that the absence of an RPE-tuf sequence in these two species is best explained by two independent losses. A 7-bp repeated sequence (AAGATGT) flanking the RPE-tuf element may have served as the site of excision (fig. 4A
). RPE-tuf sequences could also not be identified in R. prowazekii, R. typhi, and R. bellii. These were most likely lost by a similar mechanism. R. prowazekii and R. typhi, members of the typhus group, were found to contain highly fragmented sequence remnants of the RPE-fus sequences identified in the SFG (fig. 4B
). In these species, at least six deletion events have to be inferred, ranging in size from one to 18 nucleotides, some of which seem to have been acquired before the separation of the two species. An extreme example of RPE-deterioration is observed in the early diverging species R. bellii, where only nine nucleotides align perfectly with the RPEs in the other species, indicating that more than 95% of the RPE has been eliminated from this species.
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Discussion |
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Genomic streamlining and rearrangements may result from a variety of processes, for example by intrachromosomal recombination at duplicated genes and repeated sequences. Indeed, we have previously suggested that a recombination event between two ancestral tuf genes in Rickettsia caused an inversion of the intervening segment (Andersson and Kurland 1995
; Syvänen et al. 1996
). The rate of recombination at such sites will eventually decrease as more and more of the repeated sequences are consumed in the recombination process, unless the rate of repeat regeneration is very fast. It is therefore not surprising that highly reduced genomes often have very low frequencies of repeated sequences (Andersson et al. 1998
; Stephens et al. 1998
; Frank, Amiri, and Andersson 2002). In contrast to the scarcity of repeated sequences in the R. prowazekii genome, however, as many as 45 copies of a repeated palindromic element were found in the genome of its close relative, R. conorii (Ogata et al. 2000
). Even more astonishing was that as many as 19 of these are located inside open reading frames (ORFs; Ogata et al. 2000
). When the R. conorii RPEs were used to search for similar sequences in the R. prowazekii genome, 10 highly divergent RPEs were identified in protein-coding genes also in this species (Ogata et al. 2000
).
When were the RPEs acquired in Rickettsia? Dwyer has suggested that RPEs are modern-day vestiges of the early RNA world that have survived through evolution more or less intact (Dwyer 2001
). If so, they would have arisen thousands of millions of years ago. Ogata and his colleagues, on the other hand, believe that RPE insertion occurred after the divergence of speciation in Rickettsia (Ogata, Audic, and Claverie 2001
); if so, they would only be a few million years old. In order to place the acquisition of the RPEs in the evolutionary history of Rickettsia, we have superimposed the distribution of the RPEs located in the downstream regions of the elongation factor genes onto a phylogeny of Rickettsia inferred from the elongation factor genes. The analyses strongly suggest that the proliferation of RPEs predate speciation within the genus Rickettsia.
First, the high degree of sequence conservation and the finding of identical insertion sites for RPEs in a variety of Rickettsia species, including members from both the TG and the SFG, argues that the acquisition of RPEs occurred early in the lineage leading to Rickettsia. Some of the ancestral RPEs may already have been lost or they may no longer be recognizable by sequence similarity searches. Indeed, the highly fragmented RPE-fus sequences in R. prowazekii and R. typhi were identified only because of their location at an RPE-insertion site in the other species. Thus, the most parsimonious explanation for species-specific differences in the host proteins targeted by RPEs is recent RPE-loss in some lineages, rather than recent RPE-gain in the other lineages.
The ancestry of each RPE can be studied in more detail by exploiting the fact that any recent proliferation would become visible in the phylogenetic reconstructions as clusters of similar repeats. However, there is no support for a recent amplification, or any evidence that RPEs can proliferate in a selfish manner. On the contrary, the phylogenetic relationship of the RPE-tuf and RPE-fus sequences showed that these are related by vertical descent rather than by a recent intragenomic expansion. This suggests that the RPEs flourished before speciation within the genus Rickettsia and that there are no or only few recently born RPEs. These results are incompatible with the idea that the RPEs correspond to recently proliferating sequences (Ogata et al. 2000
).
Is there any functional role associated with the spread of RPEs? Dwyer has noted that many proteins in multigene families have evolved from smaller duplication units encoded by short inverted repeat segments that resemble transposable genetic elements (Dwyer 1998
). These transposable exons, "trexons," are similar to the RPEs in that they encode a helix and a short turn or loop. It has therefore been suggested that a putative function of the RPEs and the trexons was to support protein modularity by encoding protein segments that are specialized for participating in protein-protein and protein-DNA interactions (Dwyer 1998
). Although it cannot be ruled out that the RPEs at some point or another contributed important functional characteristics to the genes in which they were inserted, our analyses suggest that modern RPEs are nothing but silent passengers in the genomes in which they reside.
The survival rate of selfish DNA will depend on two factors: the rate of intragenomic proliferation balanced by the rate of spontaneous deletions. Studies of Rickettsia pseudogenes have shown that the mutation rate for small deletions predominates over small insertions (Andersson and Andersson 1999a
, 1999b
, 2001
). Likewise, deletions were found to be more common than insertions in the RPE-tuf sequences, suggesting that these too evolve as neutral sequences. Furthermore, we have shown that RPE-tuf has been lost in two lineages independent of each other, presumably by recombination at short repeated sites. Because insertions into protein-coding genes will impose a stabilizing effect on the element, it may not be surprising that the few remaining RPEs in the R. prowazekii genome are located inside protein-coding genes (Ogata et al. 2000
), with the exception of the partial RPE-fus identified in this study.
If all of the identified RPEs in R. conorii originated before speciation in Rickettsia, as indicated by our phylogenetic analysis, the decay of RPEs must have occurred more rapidly in the TG than in the SFG. This is reminiscent of the finding that gene loss has been much more extensive in R. prowazekii than in R. conorii (Ogata, Audic, and Claverie 2001
). Of the 834 previously identified genes in R. prowazekii, only 30 have been lost from R. conorii, whereas about half of the 552 genes uniquely present in R. conorii have been lost from R. prowazekii, and the remaining 229 are present as highly degraded gene remnants. The extent to which differences in growth properties, population structures, and mode of transmission have influenced the dynamics of gene loss and RPE degradation in the TG and SFG Rickettsia remains to be determined.
Taken together, the observed phylogenetic relationships and the ongoing degradation of RPEs suggest that they are neither modern-day remnants of an ancestral RNA world (Dwyer 2001
) nor the outcome of a recent proliferation within the R. conorii genome (Ogata, Audic, and Claverie 2001
). Although it can not be ruled out that RPEs may in rare cases have conferred novel functional features, the unique presence of RPEs in protein-coding genes of Rickettsia is most likely explained by a reduced selective efficiency of the encoded gene products because of recurrent bottlenecks and small population sizes.
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Acknowledgements |
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Footnotes |
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Keywords: elongation factors
molecular evolution
phylogeny
RPE
Rickettsia
selfish DNA
Address for correspondence and reprints: Siv G. E. Andersson, Department of Molecular Evolution, Norbyvägen 18C, S-752 36 Uppsala, Sweden. siv.andersson{at}ebc.uu.se
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