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Title: Evidence of Selection upon Genomic GC-Content in Bacteria}
Article:
Abstract:
The genomic GC-content of bacteria varies dramatically, from less than 20% to more than 70%. This variation is generally ascribed to differences in the pattern of mutation between bacteria. Here we test this hypothesis by examining patterns of synonymous polymorphism using datasets from 149 bacterial species. We find a large excess of synonymous GC→AT mutations over AT→GC mutations segregating in all but the most AT-rich bacteria, across a broad range of phylogenetically diverse species. We show that the excess of GC→AT mutations is inconsistent with mutation bias, since it would imply that most GC-rich bacteria are declining in GC-content; such a pattern would be unsustainable. We also show that the patterns are probably not due to translational selection or biased gene conversion, because optimal codons tend to be AT-rich, and the excess of GC→AT SNPs is observed in datasets with no evidence of recombination. We therefore conclude that there is selection to increase synonymous GC-content in many species. Since synonymous GC-content is highly correlated to genomic GC-content, we further conclude that there is selection on genomic base composition in many bacteria.
Introduction:
Bacteria show an astonishing diversity of genomic GC-contents, from species such as the endosymbiont Carsonella ruddii, which has a GC-content of 16. 5%[1], to Anaeromyxobacter dehalogens, which has a GC-content of 75%. This is particularly surprising given that bacteria generally have little intergenic DNA, so the extreme variation in base composition must affect both coding and non-coding sites; this is evident in the correlation between the GC-content of the first two codon positions of protein coding genes, and genomic GC-content[2]–[4]( Figure S1). The variation in base composition is not restricted to particular groups of bacteria. Although, genomic GC content shows a fair level of phylogenetic inertia[5], most classes of bacteria show substantial variation in genomic GC content; one of the most dramatic examples is afforded by the α-proteobacteria, which have genomic GC-contents ranging from less than 30% to greater than 60%[6]. The reasons for the variation in genomic GC-content are controversial. In the first example of a “neutral” theory being used to explain a phenomenon at the molecular level, Sueoka[7] and Freese[8] independently suggested that the extreme variation in genomic base composition between bacteria was a consequence of differences in the mutation pattern. Fifty years on and there is little evidence to contradict this view. It has been shown that genomic GC-content is correlated with a number of factors including genome size[6], whether the bacterium is free-living or not[9],[10], the environment[11], aerobiosis[12], nitrogen utilization[13] and possibly temperature[14]( but see[15]–[17]). However, it is still unknown whether these correlations are due to the influence of these factors on the process of mutation, or whether natural selection is involved. For a few bacteria there is evidence that the genomic GC-content is not a simple consequence of mutation bias. The mutation pattern has been directly measured in Escherichia coli and found to be AT-biased[18],[19]. If the genomic GC-content was simply determined by mutation bias it would be expected to decline from its current value of 0. 5 to 0. 32[20]. The fact that the genomic GC-content is higher than this value can be explained by functional constraints. However, even sites that appear to be under little selection, such as synonymous sites in lowly expressed genes, have a GC-content of ∼57%[21], which is considerably above the equilibrium inferred from the mutation studies. Of course, it is likely that the mutation pattern measured in the laboratory is not identical to that experienced by the bacterium in the wild. However, a phylogenetic analysis of substitutions between closely related E. coli strains also shows the same tendency for an excess of GC→AT mutations[22]. Furthermore this bias towards GC→AT mutations is less acute amongst common polymorphisms, consistent with natural selection acting against GC→AT mutations[22]. A similar excess of GC→AT substitutions is seen in the pseudogenes between strains of Mycobacterium leprae[23], again suggesting that the mutation pattern is AT biased relative to the current genomic GC-content, and that it will, given the opportunity, decrease the GC-content[20]. However, M. leprae is an intra-cellular parasite and this has led to the loss of many genes; it is possible that one of these is a DNA repair enzyme that has shifted the mutation bias towards being AT-rich. Here we test whether genomic GC-content is a simple consequence of mutation bias by investigating the pattern of synonymous genetic variation in 149 bacterial species.
Discussion:
We have shown that there is a large excess of GC→AT synonymous SNPs segregating at 4-fold degenerate sites in GC-rich bacteria, with AT-rich bacteria showing the opposite pattern. These patterns are found across different phyla and classes of bacteria suggesting that these patterns are not restricted to select groups of bacteria. We have shown that the excess in GC-rich bacteria is probably not due to sequencing error, a violation of the infinite sites assumption, translational selection, biased gene conversion or horizontal gene transfer. In contrast the excess of AT→GC SNPs in AT-rich species may be due to either a violation of the infinite sites assumption, translational selection, or selection for low GC-content. The excess of GC→AT SNPs in GC-rich species is consistent with selection[24]; under selection in favour of increased GC we expect to see an excess of GC→AT SNPs, even when the direction is inferred from the allele frequency( Figure 5). We therefore conclude that there is selection to increase GC4 in GC-rich species. Since there is a strong correlation between GC4 and genomic GC-content[2],[4]( Figure S1) we furthermore infer that selection is acting to increase not only GC4, but also the genomic GC-content in GC-rich species, as others have suggested before based on less extensive data[20],[22]. Our results are in accord with those in an accompanying paper in this journal by Hershberg and Petrov[42]. They have investigated the pattern of mutation in five clonal pathogens using a phylogenetic analysis of closely related strains and an outgroup. They infer the pattern of mutation to be AT biased in all species, even those that are GC-rich. This suggests that there is a universal mutation bias towards AT, as Lynch has suggested previously based on more limited data[43],[44]. This in turn suggests that there must be some pressure maintaining high GC content in those species that have a high genomic GC content. We have investigated whether the bias towards GC→AT SNPs, in GC-rich species, is due to biased gene conversion by removing all datasets which fail the four gamete test, and testing whether GC-content and the bias towards GC→AT SNPs is correlated to measures of recombination. Biased gene conversion is a process that drives mutations through a population; since it is not expected to affect the frequency of mutations at linked sites it is expected to generate four gametes during this process. Nevertheless the FGT may miss some datasets that are undergoing gene conversion and we cannot completely rule out biased gene conversion as an explanation. Intriguingly, it has recently been shown that the GC-content across the E. coli genome is correlated to the rate of recombination; this is consistent with biased gene conversion[45]. However, both the rate of recombination and GC content are correlated to the distance from the origin and terminus of replication, so it is unclear whether there is a causal relationship between the two. It has been suggested that there is a universal mutational bias in both prokaryotes and eukaryotes towards AT[42]–[44]. Our analysis provides some limited support for this hypothesis. To a first approximation we can infer whether the mutation pattern is AT-biased by assuming there is no selection and estimating the GC4 to which the sequences would evolve, an AT-biased mutation pattern being one that will give an equilibrium GC4<0. 5. This method is slightly liberal, because under selection there will be an excess of deleterious mutations segregating which will slightly exaggerate the apparent mutation bias. Almost all species with GC4<0. 6 are predicted to evolve to GC4<0. 5 if selection was relaxed( Figure 3), however for many species with GC4>0. 6 they are predicted to evolve a lower GC4, but not one which would indicate an AT-biased mutation pattern. This may be because, as the true bias increases, so the level of mis-inference increases. This can be seen by considering an extreme example; imagine that all mutations are GC→AT and the predicted GC4 is therefore zero. Some of the SNPs will be mis-inferred as AT→GC and hence the predicted GC4 will be greater than zero. The bias towards AT seems to be largely due to a bias in transitions; across the whole dataset there are 9162 GC→AT and 6694 AT→GC transitions at 4-fold degenerate sites, and 1883 GC→TA and 1615 TA→GC transversions. Endosymbiotic bacteria typically have low AT-contents[46]. This is often ascribed to a loss of DNA repair genes due to their small effective population size and strict clonality, which leads to the accumulation of deleterious mutations[46], although selective explanations have also been proposed[9]. However, the low GC content of endosymbionts might in part be due to a more direct effect of their small effective population size[22],[42]; there might be selection for increased GC content but this is ineffective in endosymbionts, leading to low GC content. In support of this hypothesis, the pattern of substitution between and within Buchnera species suggests that they are equilibrium and that there is no selection acting upon base composition[47],[48]( Table S1). Although, most obligate endosymbionts have low GC content, Candidatus Hodgkinia cicadicola is an exception with a genomic GC content of 58%[49]. This is a challenge to any selective explanation of GC-content since selection appears to be less effective in endosymbionts because of their small effective population size[10],[46]. There are a number of possible explanations. First, it might be that selection is sufficiently strong to overcome the decreased efficiency of selection. Second, it might be that a mutation conferring a GC-biased mutation pattern has fixed in Hodgkinia. Neither of these explanations is very satisfactory and it will be of great interest to see what pattern of SNPs is present within this species. Although, we have evidence of selection on GC-content in GC-rich bacteria, the nature of the selective agent is unclear. Recently Foerstner et al.[11] showed that bacterial communities from particular environments have surprisingly narrow GC-content distributions and that these distributions differ between environments; for example the bacteria from a sample of surface seawater had a median GC-content of 34%, while a soil sample had a median of 61%. This difference was not due to different phyla being present in the different environments. This suggests, in association with our results, that certain GC-contents are favoured in particular environments. But why this is so, remains a mystery.
Shortly after it was proved that DNA was the genetic material it became apparent that organisms , and in particular bacteria , use the four letters of the genetic code to very different extents; the use of G and C varies from less than 20% in some species , to more than 70% in others . This variation in the use of G and C is usually attributed to differences in the pattern of mutation between species . Here we test this hypothesis , and show that , on the contrary , there seems to be pervasive selection on the base composition of the bacterial genome , particularly in GC-rich species . This suggests that many , if not all , sites may be subject to natural selection in many bacteria . Unfortunately , the reason why some bacteria should be selected for high GC-content remains unclear .