Institut Cavanilles de Biodiversitat i Biologia Evolutiva and Departament de Genètica, Universitat de València, 46071 Valencia, Spain
Correspondence: E-mail: francisco.silva{at}uv.es.
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Abstract |
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Key Words: Buchnera aphidicola DNA loss genome reduction gene disintegration pseudogenes symbiosis
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Introduction |
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The close relationship to other free-living gamma-Proteobacteria such as Escherichia coli, Salmonella spp., Yersinia pestis, or Vibrio spp. has caused the endosymbiont species B. aphidicola to be the subject of many studies on genome degradation (Shigenobu et al. 2000; Mira, Ochman, and Moran 2001; Moran and Mira 2001; Silva, Latorre, and Moya 2001, 2003; Tamas et al. 2002; van Ham et al. 2003). Previous analyses allowed the estimation of the minimal gene content of the genome of the ancestor of E. coli and B. aphidicola in 1,8182,425 genes, indicating that more than 1,000 genes were lost during adaptation to the new lifestyle. Those losses would have occurred by large deletions, simultaneously removing many genes, by gene inactivation and progressive gene disintegration, or by a combination of both processes (Moran and Mira 2001; Silva, Latorre and Moya 2001).
The availability of the genome sequences of three B. aphidicola strains, symbionts of the aphids Acyrthosiphon pisum (BAp; Shigenobu et al. 2000), Schizaphis graminum (BSg; Tamas et al. 2002), and Baizongia pistaciae (BBp; van Ham et al. 2003), has offered the possibility of studying the fate of the DNA of those genes that were lost throughout the evolution of the genome of the three B. aphidicola strains, so becoming part of the nonfunctional DNA. Because B. aphidicola seems only to be transmitted maternally in aphids (Baumann et al. 1995), a parallel evolution of B. aphidicola and aphid lineages has been proposed (Moran et al. 1993). This has permitted an estimation of the divergence times between B. aphidicola strains based on the estimated divergence times of their hosts. Thus, it was estimated that the strains BAp and BSg diverged 50 to 70 MYA (Clark, Moran, and Baumann 1999) due to the fact that their aphid hosts belong to two tribes of the subfamily Aphidinae (Remaudière and Remaudière 1997). On the other hand, the strain BBp, whose host belongs to the subfamily Pemphiginae (Remaudière and Remaudière 1997), probably diverged at the time of the Aphididae family radiation. Recently, it was estimated to have taken place 86 to 164 MYA (von Dohlen and Moran 2000; see fig. 1).
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The aim of this study was the estimation of the rate of nonfunctional DNA loss (pseudogene or intergenic region) in the obligate bacterial endosymbiont B. aphidicola, making use of the estimates of divergent dates for its strains. We have also tried to verify that the ability to lose DNA at a significant rate has been maintained throughout all B. aphidicola lineages.
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Materials and Methods |
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In addition, we established a criterion based on sequence similarity for the cases of pseudogene status in both strains, or pseudogene in one strain and absent gene in the other. To establish the criterion, we try to determine the range of E values reported after a Blast search (Altschul et al. 1997) for those genes inactivated after BAp and BSg divergence. We selected 47 genes with complete confidence because, in one of the two strains, they had the status of gene (case 1, gene in BAp and pseudogene in BSg; case 2, gene in BSg and pseudogene in BAp; case 3 gene in BAp and absent gene in BSg; case 4, gene in BSg and absent gene in BAp; see table 1). We took the E. coli protein as a reference and performed a TBlastN against the genomes of BAp and BSg, searching for the E value of the region of the genome where the remnant DNA of the gene should be located. The E value range for the 33 pseudogenes (cases 1 and 2) was from 0 to nondetected (E value > 7) with a median value of 2 x e74, while no hit was detected for any of the 14 absent genes (cases 3 and 4). These results indicate that the substitution rate in B. aphidicola is so high that the pseudogenes formed prior to BAp and BSg divergence have had time to completely lose any sequence similarity with the functional gene. For that reason the small E values obtained for cvpA, apbE, cmk, bioH, ansA, and hemD in the same conditions (2 x e11, 6 x e80, 5 x e28, 1 x e54, 1 x e126, and 7 x e36, respectively, for the smallest value of BAp or BSg) indicate with high confidence that a functional gene was present at the time of BAp and BSg divergence. Therefore, two convergent gene loss events took place for each of these genes.
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Analysis of Genes, Pseudogenes, and Remnant DNA Sequences
To determine the DNA loss in the inactivated genes, we estimated the length of each gene (Lg) and the length of the disintegrated DNA region after the reductive process (Ld). For single gene losses, Lg was defined as the number of nucleotides (nt) included between the upstream and downstream adjacent genes, minus the length of an average intergenic region. Thus, at the beginning of the disintegration process, this length contained the upstream and downstream intergenic regions plus the gene and, after a complete disintegration, the remnant DNA would correspond to an average intergenic region (fig. 2 is a diagram of the possible situations). We used 55.1 nt as the size of an average intergenic region, as was estimated for ancient spacers, defined as those flanked by the same genes in B. aphidicola and E. coli (Mira, Ochman, and Moran 2001). In a similar way, Ld was estimated as the number of nucleotides between the upstream and downstream adjacent genes minus 55.1 nt. This DNA length represents the average contribution of the two contiguous genes to the final intergenic region. Each original gene would have some upstream and downstream noncoding nucleotides which will be lost together with the coding region during the gene disintegration process. Thus, the new intergenic region will be formed by a few downstream noncoding nucleotides of the upstream gene and by the upstream noncoding nucleotides of the downstream gene. Both DNA segments would form, on average, the 55.1 nt of the new intergenic region (fig. 2).
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When several adjacent genes were simultaneously lost in a lineage we treated them as a block, estimating Ld and Lg for the block (see fig. 2). Independently of the number of lost genes contained in the block, we always considered 55.1 as the final average spacer between two functional genes. For the determination of the number of genes in each disintegrated category, the Ld/Lg ratio of the block was assigned to each of the lost genes in the block. When a block contained one or several pseudogenes, and it was possible to identify their ancestral start and stop codons, the block was divided into the maximum possible number of segments to estimate Ld/Lg. GC contents (GCd and GCg) were estimated in the same DNA segments used for estimating Ld and Lg. Both variables were plotted with a logarithmic transformation for the GCd/GCg ratio. For those genes whose inactivation started after the divergence of BAp and BSg, we estimated the GC content of an active B. aphidicola gene and compared it with the GC content of the remnant DNA region. The ansA and hemD genes were not included in the final analyses because there was not a functional gene in BBp to determine the gene GC content (see table 1).
Although the position on the chromosome of close to 100 genes that were lost after the divergence of E. coli and B. aphidicola and prior to the formation of the LCSA was known (Silva, Latorre, and Moya 2001), they were not taken into account. We consider that the DNA coming from those genes should have practically disappeared, after more than 150 Myr of evolution.
Intergenic Region Size Analysis
A putative shortening of B. aphidicola intergenic regions versus E. coli was studied. Only ancient spacers were analyzed. They were defined according to Mira, Ochman, and Moran (2001) as those with the same flanking genes in E. coli and B. aphidicola. To perform a homogeneous analysis, only those present in all B. aphidicola strains were measured (n = 195). We tested whether each sample comes from a normal distribution and rejected the null hypothesis with the Kolmogorov-Smirnov test with a P value = .000. In addition, because our data were not independent but repeated measures of the sizes of the same intergenic region in four genomes, we applied a nonparametric repeated measures analysis with the Friedman test, the null hypothesis being that there is no difference in mean ranks for the genomes.
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Results |
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One hundred sixty-four gene loss events were identified (table 1). Two of the losses of the BBp lineage were not taken into account because they involved plasmid genes (ibpA and repAC). Thus, at least 94 out of the 629 ancestral chromosomal genes were lost through the 100150 Myr evolution of the BBp lineage chromosome. In contrast, the lineages of BAp and BSg only lost 32 and 44 genes, respectively, during the same period of time. These events only involved 135 different genes because of several convergent losses that occurred during the evolution of the B. aphidicola lineages. The genes ansA and hemD were lost threefold (BBp, and after the split of BAp and BSg, named from this point as late BAp and late BSg). The gene yadF was lost twice (BBp and before the split of BAp and BSg, named from this point as early BAp and early BSg). Nine genes were lost twice in the BBp and late BAp lineages. Eleven genes were lost twice in the BBp and late BSg lineages. Finally, apbE, cmk, cvpA, and bioH were lost twice in the late BAp and late BSg lineages (see table 1).
DNA Loss in the BAp and BSg Lineages
Eight genes (bioC, bioF, mutH, norM, pal, uspA, yqgE, and yadF) were analyzed (table 1 and fig. 3A) whose inactivation occurred before the divergence of BAp and BSg lineages (fig. 1), between 86164 Myr (the estimated age for LCSA) and 5070 Myr (the divergence time of BAp and BSg). Six out of the eight were not adjacent in the LCSA chromosome, while bioC and bioF genes were contiguous and, for that reason, in the DNA loss analysis they were treated as a block. When the length of each gene (Lg) and the length of its homologous disintegrated DNA region (Ld) were compared, in six out of eight cases more than 90% of the nucleotides had been lost, while in the other two genes the remnant DNA was only slightly higher than 10% (fig. 3A). The average Ld/Lg ratio for the eight genes was 0.055, with a range from 0 to 0.13. For each gene, the length of the disintegrated region (Ld) was the mean of the lengths for BAp and BSg. These two lengths were very similar, and the difference in the Ld/Lg value for both strains was not higher than 0.1 in any analyzed gene.
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A few genes not only did not present a reduction but even presented a slight increase in size. They were probably inactivated very recently and their larger size may be due to several causes: (1) nucleotide insertions; (2) the ancestral gene and intergenic sizes might not match exactly with that estimated for the reference B. aphidicola strain; (3) a fraction of the DNA might come from the loss of other nondetectable ancestral genes which, as in the case of yadF, were lost in the three completely sequenced B. aphidicola genomes; and (4) the 5' end of several genes might not be incorrectly annotated.
Because many host-associated bacteria display AT-enriched genomes, it has been proposed that a mutational pressure exists in these genomes toward the increase in A+T content (Moran 2002). In B. aphidicola this shift has caused the genic and intergenic G+C content to be very low: 26% and 15% in the genomes of BAp and BSg, respectively (Tamas et al. 2002). Once a gene is inactivated, the bias in the nucleotide substitutions produces the decrease in the G+C content. We compared the decrease in G+C content with the DNA loss for the gene losses of this period and estimated the correlation coefficient between Ld/Lg and ln (GCd/GCg) to be 0.765 (fig. 5). A parallel decrease in GC and length was observed with an equilibrium for the GC content decrease of around 0.47. This means that on average the final GC content of the analyzed regions is 47% of the initial composition.
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Shortening of Ancient Spacers
A comparison of the sizes of the intergenic regions for ancient spacers between E. coli and each of the three B. aphidicola strains is shown in figure 6. The average size (bp ± standard deviation) for BAp (51.1 ± 70.0), BSg (47.4 ± 63.6), and BBp (55.3 ± 76.1) was only slightly smaller than that for E. coli (67.5 ± 98.2). The Friedman test for the four genomes concluded that there was a difference in the mean ranks (P value = .017). This test was applied exclusively using the three B. aphidicola genomes and, in this case, the null hypothesis was not rejected (P value = .103). Therefore, the size distribution for the intergenic regions is different between B. aphidicola and E. coli.
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Discussion |
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However, a new PFGE including B. aphidicola strains from other aphid subfamilies (Gil et al. 2002), together with the sequencing of the BBp strain with a chromosome of 616 kb (van Ham et al. 2003), revealed a wide range of chromosomal sizes, with 450 kb being the minimum size reported so far for a bacterial species. These results show that the genome of B. aphidicola, at least in some period of the evolution of several lineages, had experienced a process of genome reduction at a nonirrelevant rate.
Once a gene is inactivated, its DNA is affected by two types of changes: (1) a mutational bias towards A+T, which provokes a decrease in the GC content (Moran 2002); and (2) the deletion of some of its nucleotides. Our work has shown that both processes present some degree of correlation and, in general, the DNA of the inactivated genes became shorter and A+T richer with time. The rate of DNA loss of B. aphidicola is sufficiently high to produce the complete or almost complete disintegration of the genes in a short period of time. We have shown that the rate of those genes whose inactivation occurred more than 5070 MYA in the B. aphidicola from the Aphidinae lineage was high enough to almost completely remove them, and the rate of the genes inactivated after this date produced in a few cases a complete loss, and in others a partial loss. Those genes with inappreciable reductions were mainly pseudogenes that were probably inactivated very recently: However, it is possible that some of them were still functional genes. The production of small amounts of a functionally complete protein is possible for some pseudogenes if they are transcribed and if a significant level of ribosomal frameshifting takes place during translation, as has been described for several E. coli genes (Gurvich et al. 2003).
The absence of a date for the divergence of two or more members from the Pemphiginae did not allow us to determine whether the genes lost in an early stage had been completely erased, but it is evident that a large proportion of lost genes have lost a large quantity of their nucleotides. For that reason, we believe that in both lineages the DNA of a gene can be almost completely deleted from the genome in 40 to 60 Myr. Our estimation of the half-life of a pseudogene in B. aphidicola of 23.9 Myr is in the range of the 14.3 Myr estimated for Drosophila (Petrov and Hartl 1998) but is much smaller than the 615 Myr for Laupala (Petrov et al. 2000) or the 884 Myr for mammals (Petrov and Hartl 1998). This estimation was done for an early period of the evolution of the BAp/BSg lineage using the eight genes that became inactivated during this phase. By exclusively using a part of the genes whose disintegration started after BAp/BSg divergence, we obtained a value of the same order (16.248.7 Myr). A similar analysis, although affected by the large period of time used for the estimation, rendered a value from 13.5 to 81.1 for the BBp lineage. These results show that DNA loss is taking place at a nonirrelevant rate during the evolution of all B. aphidicola lineages. The disintegration rate for free-living bacteria or for the initial steps of the adaptation to endosymbiosis was probably higher because several mechanisms, now lost in B. aphidicola, can produce drastic losses of nucleotides. The mechanisms are mainly, on one hand, the loss of an efficient recombinational system (Shigenobu et al. 2000) which, in combination with direct repeats, would produce deletions (Frank, Amiri, and Andersson 2002) and, on the other, the decrease in the close direct repeat frequency in the genome which may be the substrate for DNA polymerase slippage. This is probably the reason why it is very difficult to identify pseudogenes in many bacterial species (Lawrence, Hendrix, and Casjens 2001).
What controls the size of the B. aphidicola genome is the importance, or essentiality, of the function of the different DNA sequences that comprise it, either genes or intergenic regions with some kind of function. Once any of these DNA segments loses its function, a process of gradual DNA loss decreases its length. For that reason, the size of the B. aphidicola chromosome may still continue to be reduced, and the limit for this reduction will be associated with the minimum number of genes required for bacterial cell life and the symbiotic contribution to the life of its insect host. It is worth noticing that the genome of the five completely sequenced bacterial endosymbiont of insects share only 313 genes, 277 of them being protein-coding (Gil et al. 2003). This minimal set would produce genome sizes as small as 300 kb, with one-third of these genes nonessential for a bacterial cell but required for supporting the survival of its host. A slight decrease in the ancient intergenic spacers was also detected, but its contribution to the total chromosomal reduction will be much smaller than the loss and disintegration of the genes.
Genome size in bacteria is a balance between several mechanisms that produce the insertion or deletion of small or large DNA segments. Mechanisms producing the insertion or deletion of hundreds or thousands of nucleotides in a single event have a high impact on total genome size. However, these mechanisms seem to have been lost in the present B. aphidicola genome evolution. The stability of the gene order of its genome is probably due to the lack of elements that disrupt the chromosomal structure, such as transposable elements, phages, large repeats, and probably an efficient homologous recombinational mechanism (Rocha 2003). However, the loss of the ability to acquire foreign DNA fragments by horizontal gene transfer events drastically reduces the impact of the mechanisms that increase the genome size. Therefore, the main mechanism affecting the evolution of the size of the modern B. aphidicola genomes are those mutational events involving a small number of nucleotides (insertions or deletions). In fact, the most frequent polymorphism detected during the sequencing of the BBp and BSg genomes were small indels with an average size of between one and two nucleotides (Tamas et al. 2002; van Ham et al. 2003). Slipped-mispair errors during DNA replication are probably the main cause of these polymorphisms. In addition, the presence of close repeats (Rocha and Blanchard 2002), which are short repeats (> 810 nt) separated by a spacer of several nucleotides, may be important because it generates slightly larger duplications or deletions (up to several hundred nucleotides; Rocha 2003). Furthermore, although these events took place at a lower rate than the single nucleotide indels, their impact will be much stronger. It has been observed that the genes present in BAp or BSg, whose orthologs were lost in the other strain, presented a slightly higher number of close repeats (larger than 9 nt) than the average gene of the genome (Rocha 2003). This would indicate that the probability of inactivation of a nonessential gene is higher when it contains more repeats. However, the density of repeated sequences in B. aphidicola, as well as in other host-associated bacteria, is dramatically decreased when compared with free-living bacteria such as E. coli, Salmonella spp., or Bacillus spp. (Tamas et al. 2002).
On the contrary, the size of the intergenic regions is not greatly affected by the genome reduction process, and ancient spacers are only slightly smaller in B. aphidicola. This difference disappears in BAp if spacers with annotated regulatory regions in E. coli are excluded (Mira, Ochman, and Moran 2001).
Finally, although insertions and deletions are factors contributing to the evolution of genome size, the evolutionary forces that led to its reduction are a matter for discussion. Several authors have proposed that in many bacterial genomes, a bias to the net DNA loss exists based on a higher number of deletion events (vs. insertions) and/or a higher average size of the deleted segments (vs. inserted; Andersson and Andersson 2001; Lawrence, Hendrix, and Casjens 2001; Mira, Ochman, and Moran 2001; Gregory 2004). If this bias is true, genetic drift could contribute to the fixation of the more abundant deletional mutations. The effect of this mechanism would be very important in B. aphidicola, due to the small population sizes and to the special manner of vertical transmission with bottlenecks in each generation (Mira and Moran 2002).
Alternatively or simultaneously, natural selection may be partially or completely responsible for the reduction. If small-size genomes replicate faster, increasing their frequency in the polyploid B. aphidicola cell (Komaki and Ishikawa 2000), and if these cells divide faster, we can expect deletional mutations to become fixed with time, independently of whether they are produced in higher, lower, or equal rates to insertions. Although this hypothesis has been proposed several times for the reduction of the genome sizes of obligate cellular bacteria and mitochondrial genomes (Selosse, Albert, and Godelle 2001; Silva, Latorre, and Moya 2001), there are few examples supporting it. A negative correlation was observed between DNA content and division rate for some ciliate spp. (Wickham and Lynn 1990). However, no such correlation was observed between doubling times in laboratory conditions and genome sizes over bacteria belonging to 10 major taxonomic divisions (Mira, Ochman, and Moran 2001) nor for growth rates of E. coli strains varying in as much as 25% in chromosome size (Bergthorsson and Ochman 1998). Because of the small difference in size that a 1-nt-indel represents, it seems fully reasonable to accept the conclusion that selection does not differentiate between individual small indels (Gregory 2004). However, studies in Drosophila have shown some evidence that deletions larger than 400 bp may be advantageous (Blumenstiel, Hartl, and Lozovsky 2002). Because of the smaller genome size of bacterial endosymbiont chromosomes, it cannot be completely ruled out that chromosomes with small sizes because of one or several small deletions can be selectively advantageous.
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Acknowledgements |
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Footnotes |
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