Laboratoire de Biométrie et Biologie Évolutive, Université Claude Bernard Lyon 1, Villeurbanne, France
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Abstract |
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Key Words: complete genome bacteria mutation pressure G+C content lateral transfer evolutionary rate
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Introduction |
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A first analysis performed on Escherichia coli genome already showed that the G+C composition at the third codon position (G+C3) varies along the genome in relation to the proximity of the replication terminus (Guindon and Perrière 2001). In the present work, we have completed and extended this analysis to 48 bacterial and 11 archaeal genomes. We show that the majority of bacterial and archaeal species display a significant structuring for some of the factors generally used to detect lateral transfers. Among bacterial structured genomes, two categories are identifiable: those concordant with the pattern observed in E. coli, which seem to have representatives in most bacterial phyla, and those showing mosaic structures that require other explanations involving lateral transfers and genome rearrangements. We also show that the evolutionary rates vary significantly along several bacterial genomes with a tendency for genes close to the replication terminus to evolve more rapidly. Taken together, these two observations suggest that a previously neglected evolutionary constraint may be responsible for A+T enrichment and increasing of evolutionary rates in a large chromosomal region around the replication terminus of bacteria.
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Materials and Methods |
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Divergence Calculation
Divergence of gene sequences was estimated for several pairs of genomes (fig. 2). Homologous genes were identified using BLASTP2 (Altschul et al. 1997) searches between closely related genomes. Only proteins having E values less than 10-20 were considered as significant matches. Nucleotide sequences were then aligned with respect to the protein alignment. Ka and Ks values were calculated using JaDis (Gonçalves et al. 1999) and PAML (Yang 1997) for verification.
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Results |
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The two cyanobacteria (i.e., Nostoc sp. and Synechocystis sp.) only show a weak structuring (P = 5.10-2), while the hyperthermophilic bacteria A. aeolicus does not show a significant structuring of its genome. Their case, and especially the one of A. aeolicus, is very puzzling, and we lack information on the replication process in these bacteria to understand this peculiar feature. Interestingly, contrarily to most bacterial species, Synechocystis, Nostoc, and Aquifex do not present the very common G+C skew between strands allowing prediction of the replication origin (Lobry 1996; Karlin, Campbell, and Mràzek 1998; Lopez et al. 1999). This suggests that their replication mechanisms may present some important differences from other bacteria. Since the position of the replication terminus is still unknown in cyanobacteria, it was impossible to link the weak structuring with replication landmarks.
The Structuring of G+C3: Variations on a Common Theme
All other bacteria or archaea tested showed a strong structuring for their G+C3 at the scale of their complete genome. Among them, we find species from most large taxonomic groups of bacteria: low G+C gram positives, high G+C gram positives, Proteobacteria, Chlamydiales, Thermotogales, Spirochaetes, and Deinococcales (table 1). Only Aquificales and Cyanobacteria do not present evidence of such a structuring, possibly because of a sampling effect, since only a few bacteria of these phyla have been presently sequenced. Several species showing a structuring are endoparasites (like Chlamydiales), suggesting that the parasitic way of life is not a sufficient reason for the lack of structuring observed in other species. The structuring of the G+C3 is often accompanied by a structuring of the CAI. This index can be either positively or negatively correlated to G+C3, depending on the optimal codons (G+C-rich or A+T-rich) used in the species (fig. 1). The use of other codon usage indices such as
2-weighted by length (Shields and Sharp 1987) yields results very similar to CAI structuring (results not shown).
Two types of genome structuring were identified in our analysis. The first type corresponds to the one described by Guindon and Perrière (2001) in E. coli K12. It is characterized by enrichment in A+T near the replication terminus of the chromosome. Bacteria phylogenetically close to E. coli K12, such as the pathogenic strain O157:H7 and the two Salmonella species, show very similar patterns. However, bacteria as diverse as Pasteurella multocida, Sinorhizobium meliloti, Brucella melitensis, R. prowazeki, C. trachomatis, Bacillus subtilis, Staphylococcus aureus, L. monocytogenes, and M. tuberculosis have the same typical A+T enrichment near the terminus region. The G+C content of intergenic regions follows exactly the same pattern (results not shown). As well, when considering only genes having conserved their position relative to the origin and terminus between E. coli and S. typhimurium (i.e., genes already present in the common ancestor), the same pattern is found (fig. 4).
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Since the structuring is very common in bacteria, we also investigated the case of archaeal species. All of the 11 species tested present several regions of contrasting G+C content. However, although evidence suggests that archaea, like bacteria, possess a unique origin from which replication initiates bidirectionally (Lopez et al. 1999; Myllykallio et al. 2000; McNeill 2001), the majority of archaeal species still have no confirmed position for their replication origin and terminus. Recently, the position of the origin of P. horikoshii, P. abyssi, Methanobacterium thermoautotrophicum, and S. solfataricus have been predicted using cumulative skew (Lopez et al. 1999; She et al. 2001) and experimentally confirmed for one of these species (P. abyssi) (Myllykallio et al. 2000). The identified origins always coincide with a cdc6/orc1 locus, which is known to play a role in eukaryotic replication. In Pyrococcus and Sulfolobus, if we suppose that the terminus is located oppositely from the origin on the circular chromosome, the corresponding region is an A+T-rich one, although relatively short compared with the analogous regions observed in bacteria (fig. 1). Methanobacterium, on the other hand, do not show such patterns. However, this result must be confirmed since no clear archaeal terminus has yet been identified. In other archaeal species, placing the replication terminus seems even more risky, since it has been proposed for example that Halobacterium species could have several replication origins (Ng et al. 2000).
Evolutionary Rate Variation Along the Genome
Sharp et al. (1989) have observed that evolutionary rates tend to increase with the distance to the replication origin in enterobacteria. Since their work was based on a few genes, we have repeated the experiment using genes having conserved their distance to the replication origin between E. coli K12 and Salmonella typhymurium and the other pairs listed in the Material and Methods section.
The results are shown in figure 2. In three of the seven pairs tested, we found a significant increase of the synonymous evolutionary rate (Ks) with the distance to the replication origin. The same is observed for Ka (fig. 3) in Chlamydia and enterobacteria. Chlamydia, Neisseria, Helicobacter, and Pyrococcus pairs do not show a significant tendency for Ks increase with the distance to the replication origin. It is interesting to note that genomes showing a clear A+T enrichment near the terminus region (i.e., enterobacteria, Rickettsia, Listeria, and Chlamydia) also display structuring of at least one of the two evolutionary rates tested. However, Neisseria and Helicobacter strains have diverged recently as witnessed by their low Ks values. The comparison of E. coli K12 with both E. coli O157:H7 and S. typhymurium shows that the effect of the distance to the origin needs a divergence time large enough to be observed. Thus, one cannot exclude that the low increase of evolutionary rate with distance from the origin in Helicobacter could become significant with time. Neisseria species do not show such tendency. Remarkably, the most divergent pairs (i.e., Chlamydia and enterobacteria) also present an increase of Ka with the distance to the origin (fig. 3). This is particularly surprising for the Chlamydia pair since it shows no significant increase of the Ks. However, the shape of the graph suggests that it may be due to a peculiar set of genes having high synonymous evolutionary rate in the region of the origin in these species.
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Discussion |
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The pattern of the first chromosome of Mesorhizobium loti (fig. 1) seems to be due to the same kind of event since the region of low G+C3 content contains homologs of a plasmid present in closely related -Proteobacteria (Kaneko et al. 2001). However, for the other bacteria, the reason for the nonrandom repartition of genes with respect to their G+C3 remains under question.
The case of the two close species Mycoplasma genitalium and M. pneumoniae is particularly interesting. The first one shows a very intriguing profile already noted by Kerr, Peden, and Sharp (1997). There is indeed a peculiar variation of the G+C content along the chromosome, but contrarily to most bacteria showing a similar pattern, it is clearly not related to the position of the replication origin and terminus (Kerr, Peden, and Sharp 1997). Surprisingly, the origin takes place in a rather A+T-rich region (18.4% G+C3), but the terminus is in a region where the G+C content is close to the mean of the genome (23.3% G+C3). The second has a very perturbed pattern. Though they both are obligate parasites, it seems that the dependence of M. genitalium on its host is stronger, since it is much more difficult to grow in artificial media (Jensen, Hansen, and Lind 1996). Interestingly, M. genitalium has a more reduced genome and a lower G+C3 content than M. pneumoniae. However, Himmelreich et al. (1997) have compared the two genomes and showed that the gene order was highly conserved within six fragments, concluding that these rearrangements occurred in the lineage of M. genitalium. The rearrangements do not correspond, contrarily to what is observed in most bacterial genomes (Eisen et al. 2000), to inversions around the origin and terminus. Himmelreich et al. (1997) have shown that these rearrangement events are due to the presence of repeats that are completely conserved only in M. pneumoniae. It has been proposed that rearrangements play a significant role in these bacteria for the mechanism of virulence (Rocha and Blanchard 2002). Himmelreich et al. (1997) have proposed that the rearrangements events observed here took place in M. genitalium. This suggests that the regular pattern found in this species has shaped rapidly after the speciation of the two Mycoplasma organisms, simultaneously with the reduction in G+C content. The analysis of the differences in G+C3 between the two species indeed suggests clearly that the reduction in G+C content has been much stronger in the A+T-rich region of the M. genitalium genome (fig. 5). The mechanism explaining this feature remains mysterious. However, the peculiar structuring as well as the rearrangement pattern suggests that the replication mechanism in M. genitalium possesses atypical properties in bacteria.
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Evolutionary Rate Heterogeneity: Differential Selection or Mutation?
The observed increasing evolutionary rate and A+T-richness in the region of the terminus can be explained by weaker selection efficiency in this region or a higher mutational pressure. Since bacterial codon usage is more or less biased, the Ks heterogeneity could be due to differences in codon usage bias among genes. Thus, a biased location of highly expressed genes would explain our results. However, Sharp et al. (1989, p. 809) noted that there was "no significant linear relation between codon bias and distance from the origin, reflecting the fact that highly expressed genes are not predominantly clustered near OriC." They concluded that map position has no effect on selective constraints in enterobacteria. Although the structuring of CAI suggests an opposite explanation, the more reproducible shape of G+C3c cumulative curves shows that constraints on G+C content are more likely responsible for this. In several species, including enterobacteria, B. melitensis, Vibrio cholerae, S. meliloti, and M. tuberculosis, in which the CAI is positively correlated to the G+C3, CAI values for the genes near the terminus are lower than in the remainder of the chromosome. In contrast, this trend is inverted for species in which optimal codons are A+T-rich (e.g., B. subtilis, L. monocytogenes, and S. aureus). Thus, the main explanation for CAI structuring is clearly the G+C3 content. Indeed, favoring a structuring of highly expressed genes would require hypothesizing an opposite strategy of gene organization in A+T-rich and G+C-rich genomes. Indeed, in a genome, genes may display high CAI values because they undergo high selection on their codon usage or because the mutational bias enriches them in preferred codons. It seems probable that in A+T-rich genomes, the high values of CAI in the terminus region are due to the second mechanism. Conversely, it is interesting to note that some bacteria (i.e., P. aeruginosa or T. maritima), which do not present a structuring of G+C3 related to the position of the terminus, display a strong decrease of CAI values in this region.
To test whether the observed increasing of Ks values can be attributed to a clustering of highly expressed genes in the origin region, we have divided genes having low and high CAI values and found that the increasing of Ks was significant (P < 10-4) within each class for enterobacteria, Listeria, and Rickettsia. This excludes the hypothesis of a pattern due to higher codon bias in the origin region and confirms the existence of differential mutation rates along genomes of several bacterial species. The particularly high values of Ks in the terminus region of the four pairs showing an A+T-enrichment of this region (i.e., enterobacteria, Listeria, Rickettsia, and Chlamydia) suggests that this region is subject to particularly high mutation rates. This is comforted by the Ka variations for enterobacteria and Chlamydia (fig. 3), since every region of the chromosome display similar Ka values except this region. In Chlamydia, the increase of Ks with distance to the origin is not significant, probably because of the high values found near the origin.
Why Peculiar Patterns for Genes Near the Terminus?
Several hypotheses may explain the pattern of decreasing G+C3 content observed near the replication terminus. One may consider, for instance, that the low G+C3 content of this region is due to a higher frequency of insertions of laterally transferred genes in this region. Indeed, Lawrence and Ochman (1998) have found that the genes they predicted as laterally transferred in E. coli were preferentially located in this region and had a low G+C content. Moreover, in the case of B. subtilis, several prophages, including a large one (SPß) are known to be inserted in the terminus region (Kunst et al. 1997) and confers to the G+C3c cumulative curve its particularly high slope. However, even when removing the sequences from these prophages, the decrease in G+C3 content is still visible. Furthermore, when considering only genes conserved between E. coli and S. typhimurium, the same pattern is observed in both species (fig. 4). It seems surprising that genes transferred to the common ancestor of these enterobacteria have retained such a strong bias of nucleotide composition, despite the particularly high Ks values observed in this region. This rather suggests that an intrinsic enrichment of this region could be responsible for a misleading of methods based on nucleotide content to predict recently acquired genes. Moreover, the case of C. trachomatis hardly fit with the hypothesis of alien genes since few or no candidates for recently acquired genes have been found in this species (Garcia-Vallvé, Romeu, and Pallau 2000; Ochman, Lawrence, and Groisman 2000).
Another hypothesis is that structural constraints due either to peculiar supercoiling of the terminus region or to the resolution of chromosome dimers at the end of the replication gives this region a peculiar composition. First, Capiaux et al. (2001) have shown that some oligomers tend to increase in frequency near the terminus in E. coli, suggesting peculiar constraints acting in this region. Also, Ussery et al. (2001) have found that Fis, a very abundant and pleiotropic architectural protein involved in the structure of bacterial chromatin and the regulation of several genes (Finkel and Johnson 1992; Schneider et al. 2001; Travers, Schneider, and Muskhelishvili 2001), possesses a high density of binding sites in a large region of about 1 Mb around the E. coli terminus corresponding to the A+T-rich part of the chromosome. This region has also been found to be highly enriched in sequences favoring DNA curvature in both E. coli and B. subtilis (Pedersen et al. 2000), which may favor the fixation of other proteins involved in chromosome condensation, such as H-NS in E. coli (Ussery et al. 2001). The resulting structure of this large region encompassing terminus sites may play a role in the segregation of neosynthesized chromosomes in E. coli (Tsai and Sun 2001) and/or the resolution of chromosome dimers at the dif site. These processes have been shown to be highly dependent on flanking sequences (Perals et al. 2000). The mechanisms of chromosome replication termination in E. coli and B. subtilis are thought to have evolved independently (Hill 1992; Wake 1997) but occur via extremely similar mechanisms (Wake 1997; Bussière and Bastia 1999). Although no homologue of fis has been found in B. subtilis, a protein, AbrB, seems to share very similar characteristics in terms of size, DNA binding, expression pattern, and control on gene expression, which suggests that it could play an equivalent role (O'Reilly and Devine 1997). Thus, the A+T richness of the terminus region could have a functional interest for the replication process by facilitating protein binding and loop formation at least in E. coli and B. subtilis. Hence, the peculiar characteristics of this region would reveal a conflict between different levels of selection (i.e., at the gene level and at the chromosome level). Relatively little is known about the termination mechanism in other bacterial and archaeal species. Though the ter siteswhich inhibit the action of helicases ahead of the fork in a polar manner, precluding the replication forks to exit the terminus regionare believed to play a significant evolutionary role, it is worth noting that their deletion in E. coli and B. subtilis have no detectable effect on fitness in laboratory conditions (Bierne and Michel 1994). This suggests that the mechanism is dispensable and that some species might not possess such systems.
Several constraints apply at the replication terminus: (1) the two forks have to meet simultaneously at the dif site, sometimes necessitating the arrest of one of the forks in the terminus region by ter sites (Wake 1997; Bussière and Bastia 1999); (2) structural constraints may arise from the meeting of these forks (Lewis 2001); (3) chromosomes catenates and dimers have to be resolved (Lemon, Kuster, and Grossman 2001; Lewis 2001; Perals et al. 2001); and (4) the terminus region may also play a role in the segregation of chromosomes notably by interacting with XerCD and FtsK (Perals et al. 2001). These different constraints could be responsible both for an A+T-enrichment and for an enhancement of genes mutation rates. For example, the presence of DNA ends and persistent single-stranded DNA regions characterize an arrested replication fork. Therefore, it may be more sensitive to mutation or recombination processes (Bierne, Ehrlich, and Michel 1997).
On the other hand, the terminus region may also differ in its base composition due to the preferential use of specific repair systems. Sharp et al. (1989) have proposed such a hypothesis to explain the correlation between the distance between the replication terminus and the evolutionary rate in enterobacteria. They hypothesized that the presence of multiple forks during chromosome replication allows more frequent recombination events in the region of the replication origin and consequently provides a more efficient mutation repair. Under this model, sequences close to the replication terminus are in single copy for a longer part of the cell cycle than are the origin-linked genes, so they have fewer opportunities to engage in repair via homologous recombination. However, this hypothesis hardly fit with recent observations showing that the origins are segregated at opposites poles of the cell during replication, precluding contacts between these sequences (Sawitzke and Austin 2001). We rather propose another possible difference between the origin and terminus in replication-correlated repair mechanisms. The region of the replication terminus in E. coli contains at least 10 ter sites that, by combining to Tus proteins, inhibit the action of helicases ahead of the replication complex in a polar manner (Bussière and Bastia 1999). This allows the forks to meet close to the dif site, where chromosome dimers and catenates are resolved. When the replication complex meets a DNA lesion, the fork is stalled. This lesion must be repaired or bypassed by the replication machinery (Cox et al. 2000). This requires a regression of the fork, that is, the melting of the neosynthesized strands from their matrices and the formation of a Holliday junction through the action of the specific helicases RecG and PriA in a direction opposite to replication (McGlynn and Lloyd 2001; Gregg et al. 2002). However, it is possible that after the fork has entered the terminus region, the presence of ter/Tus complexes precludes this fork regression by inhibiting the PriA and/or RecG helicases. These complexes have indeed been shown to inhibit the action of PriA in vitro, although their action on RegG is still unknown (Hiasa and Mirans 1992). The DNA synthesis has then to be completed by polymerases from the translesion pathway. This mechanism is both error-prone and biased toward A+T nucleotides. The polymerases involved in the SOS system (notably polII and polV), which undertake "translesion" synthesis, are known to follow the "A-rule" (Strauss 1991; Ide et al. 1995), that is, the preferential incorporation of a dAMP at abasic sites. Since these lesions can arise from several spontaneous and induced mechanisms and are therefore very frequent (Ide et al. 1995), the A-rule could be responsible for the A+T enrichment of a region lacking recombination-dependent repair.
The mechanism proposed above might seem to be in contradiction with recent work by Hudson et al. (2002). These authors have inserted a nonfunctional lacZ gene in different locations of the Salmonella enterica chromosome and measured the frequency of reversion. They found no significant difference in reversion rates between genes inserted near the replication origin and genes inserted near the terminus. They examined several types of substitutions, including transition and transversion. However, it is interesting to note that all the possible reversions were mutation from A or T to C or G nucleotides. Thus, if the rate of mutations toward A+T increases along the chromosome, this study would not have detected it.
Implications for Lateral Transfer Detection
As shown by Ragan (2001), different intrinsic methods of lateral transfer detection fail to give consistent results in E. coli. We observe a structuring for CAI and 2 values along bacterial chromosomes, which shows that different parts of the genome may have different codon usage. The pattern observed in E. coli, which might be the result of a replication correlated mechanism, seems to apply to virtually every bacterial phylum. Thus, the underlying hypothesis of laterally transferred genes detection methods (i.e., the weak intrinsic heterogeneity of base content among genes of a genome) might not be applicable for most species. Although the lateral transfer of large DNA fragments remains a valuable hypothesis for explaining the structuring observed in some genomes, the natural tendency of genomes to be structured is a potential source of overestimation of alien genes. This implies potential bias on codon composition approaches, which are often used to detect alien genes (Karlin, Campbell, and Mràzek 1998; Lawrence and Ochman 1998; Garcia-Vallvé, Romeu, and Pallau 2000; Ochman, Lawrence, and Groisman 2000). Especially, it seems unjustified to suppose a priori that the G+C3 of genes follows a normal distribution in a genome (Lawrence and Ochman 1998). As we already mentioned, Lawrence and Ochman (1997, 1998) noted in their work that the hypothetically transferred genes they detected were significantly more represented in the terminus region. However, this possible overestimation does not only concern species in which the genome presents a structuring resembling the E. coli genome. Indeed, rearrangement of a structured genome will still produce a structured genome since it will take a lot of events to randomize the distribution of genes.
Conclusion
It is now well known that both G+C content and evolutionary rates of genes are under the control of several factors, including gene expression level, codon bias, protein hydrophobicity, and strand location. We have presented here evidence that another factor may contribute to gene heterogeneity in several bacterial genomes: the proximity to the replication terminus. This may correspond to constraints at the chromosome level. We retained two hypotheses that may explain the peculiar features of the region of the replication terminus: (1) processes that may lead to a conflict between different levels of selection (i.e., the gene level and the chromosome level) in this region and (2) constraints due to a difference in mutation frequency and/or repair mechanism. These two hypotheses are not mutually exclusive and may both play a role. Interestingly, a few species do not present any structuring of their G+C content. A better understanding of the replication process in these species may highlight their peculiarities and allow a choice between these hypotheses.
The tendency for the terminus region to undergo mutational pressure biased toward A+T nucleotides could be ultimately demonstrated by a parsimony analysis of sets of three completely sequences genomes. This would allow orientating mutations with respect to an outgroup and testing whether mutation toward A+T nucleotides are more frequent in the terminus region. However, such an analysis is not presently possible with available data, because, for instance, E. coli 0157:H7 strains are too closely related, and Ks values currently exceed 1 between E. coli and Salmonella, precluding a parsimony hypothesis. New completely sequenced genomes should allow testing of our model of genome evolution.
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Acknowledgements |
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Footnotes |
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William Martin, Associate Editor
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