Institut "Cavanilles" de Biodiversitat i Biologia Evolutiva and Department de Genètica, Universitat de València, Spain
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Abstract |
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Introduction |
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The GroEL protein is a homotetradecamer structured in two rings, each consisting of three structural domains (Braig et al. 1994
; Braig, Adams, and Brünger 1995
). The large equatorial (residues 5132 and 408522) and apical (residues 190375) domains are located at the center and the ends of the tetradecameric complex, respectively, linked by the third smaller intermediate domain (residues 133189 and 376407). According to different detailed studies (Hartl 1996
; Weissman et al. 1996
; Fenton and Horwich 1997
; Rye et al. 1997
), the binding of the peptide-folding intermediates takes place in a hydrophobic groove in the apical domain, where the cofactor GroES also binds (Landry et al. 1993
). The equatorial domain, which is involved in all the inter-ring and most of the intraring interactions, contains the ATP-binding sites that are essential for the GroEL-GroES-polypeptidebinding cycle (Braig et al. 1994
). These three domains do not function independently but have been demonstrated, by means of mutation experiments, to be functionally connected (Kawata et al. 1999
).
The GroEL-homologue protein (Hara et al. 1990
) is abundantly synthesized in several endosymbiotic bacteria studied so far (Ishikawa 1982
, 1984
; Ahn et al. 1994
; Aksoy 1995
; Baumann P, Baumann L, and Clark 1996
; Charles et al. 1997
). GroEL has been reported to be highly expressed in bacteriocyte-harbored endosymbiotic bacteria in aphids (Ishikawa 1982
, 1984
; Hara et al. 1990
), that are vertically transmitted from one generation to the next at an early stage of the host embryogenesis (Buchner 1965, pp. 210332
). In fact, GroEL corresponds to
10% of the total protein produced in Buchnera (Hara et al. 1990
; Baumann, Baumann, and Clark 1996
). Several studies pointed out that GroEL is secreted from Buchnera to the outer space of the bacteriocyte and is able to join viral particles in the hemolymph of the aphid and transmit them between plants (Young and Filichkin 1999
; Hogenhout et al. 2000
; Li et al. 2001
). Two regions of distinct variability distinguish the GroEL of Buchnera from that belonging to their closest free-living relative, the E. coli: the hypervariable serine-rich tract (residues 526538), located at the C-terminus of GroEL, and residues 339347, belonging to the apical domain of this protein, that are involved in substrate polypeptide binding (Fenton et al. 1994
). Precise complementation experiments with the GroE mutants of E. coli showed that both the groES and groEL genes from the endosymbiont codify for the functional molecular chaperones in E. coli (Ohtaka, Nakamura, and Ishikawa 1992
).
The strong bottleneck suffered by endosymbiotic bacteria of aphids during their clonal transmission to the ovaries or during the infection of developing embryos (Buchner 1965
; Hinde 1971
; Moran, von Dohlen, and Baumann 1995
; Baumann et al. 1995
) leads to a strong reduction in their effective population sizes (Funk, Wernegreen, and Moran 2001
). The expected effect of this bottleneck is the accumulation of slightly deleterious mutations by genetic drift (Ohta 1973
), which might reflect on the acceleration of the fixation rate of nucleotide substitutions at nonsynonymous sites in symbiotic bacteria compared with their free-living relatives (Moran 1996
; Wernegreen and Moran 1999
). The difference in the rate of evolution between free-living bacteria and Buchnera was underlined in several studies (Moran, Von Dohlen, and Baumann 1995
; Moran 1996
; Brynnel et al. 1998
). Although all loci examined in Buchnera (Wernegreen and Moran 1999
) have values of nonsynonymous to synonymous rate ratio (Ka/Ks) significantly higher than that in E. coli, groEL showed the lowest nonsynonymous to synonymous rate ratio (Ka/Ks or dN/dS) that was apparently caused by a more effective action of purifying selection on this gene.
In this study our goal is to demonstrate the role played by positive selection in the evolution of groEL in Buchnera and to highlight the specific amino acid regions of this gene that were fixed by positive selection. The assertion of this work is that groEL is an extremely important gene for ensuring the normal function of the endosymbiotic protein system and that only a few beneficial amino acid replacements can be fixed.
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Materials and Methods |
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Aphid Collection and DNA Preparation
Aphids were field collected and stored at 4°C until further use. Several individuals from the same aphid colony were stored in 70% ethanol for species identification, and the remaining were used for DNA isolation.
Isolation of total aphid DNA from single individuals was carried out following the method described in Latorre, Moya, and Ayala (1986)
.
PCR and Sequencing of the groE Operon
Two degenerated primers were designed based on the alignment of the groE operon sequence from E. coli, Sitophilus oryzae endosymbiont, Lactobacillus lactis, Buchnera (S. graminum), Buchnera (A. pisum), Buchnera (M. persicae), and Buchnera (R. padi): groExF15'-(ggaattc)ATGAAWATTCGTCCRTTRCAYGAYCG-3' and groExR1 5'-(ggaattc)TTACATCATKCCRCCATRCCACCCA-3' (van Ham et al. 2000
). The oligo 5' sequences between brackets are EcoRI extensions.
PCR reactions were performed in the GeneAmp 2400 or 9700 System (Perkin-Elmer). Cycling conditions were as follows: 92°C for 2 min; 30 cycles of 92°C for 15 s, 52°C for 30 s, 72°C for 2 min; and a final extension of 4 min at 72°C. PCR conditions were as follows: 200 mM dNTPs (AP Biotech), 20 pmol of each primer, 1 x Taq buffer, 1.5 U of Taq pol (AP Biotech), and 1040 ng of template. PCR products (1.9 kb in length) were excised from a 0.8% agarose gel in 0.5 x TBE and extracted with QIAEX (QIAGEN). PCR products were cloned into pGEM-T Easy (Promega). Plasmid extractions were carried out using CONCERTTM Rapid miniprep system (GIBCO-BRL). Sequencing reactions were performed in Applied Biosystems automatic sequencers/ABI 373, 377, or 3700 using dRhodamine ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer). In order to avoid Taq-mistakes, several clones were sequenced. T7 and SP6 vector primers and internal primers (sequence available upon request) were used to cover the complete length of the PCR product in both the chains.
Phylogenetic Reconstruction and Nucleotide Substitution Models
Alignment of sequences was performed with CLUSTAL X, a Windows version of the CLUSTAL W program (Thompson, Higgins, and Gibson 1994
) and corrected by eye. Phylogenetic analysis of the aligned sequences was performed using different methods: Neighbor-Joining (NJ) (Saitou and Nei 1987
), maximum likelihood (ML) (Felsenstein 1981
), and maximum parsimony (MP) (Fitch 1971
). The MEGA program, version 2.01 (Kumar, Tamura, and Nei 1993
) was used to obtain the NJ trees and the bootstrap support values for 1,000 pseudoreplicates. MP and ML trees were reconstructed using DNPARS and DNAML, respectively, from the PHYLIP package, version 3.5 for Windows (Felsenstein 1993
).
For distance estimation and ML analysis, we determined the appropriate model to explain the evolution of groES/L sequences by using the likelihood ratio test (LRT) (Huelsenbeck and Crandall 1997
). When two models are nested, they can be compared by the LRT; twice the log-likelihood difference follows a chi-square distribution, with the degrees of freedom being the difference between the numbers of free parameters between the models compared. These tests were implemented using the program BASEML from the PAML package, version 3.0 (Yang 2000
). The models tested for a given phylogenetic tree were those described by Jukes and Cantor (1969)
, Kimura (1980)
, Felsenstein (1981
), Hasegawa, Kishino, and Yano (1985)
, Tamura and Nei (1993)
, and Yang (1994)
. In addition, to determine if the substitution rates are equal (Poisson distribution) or unequal (
-distributed rates model) among sites, the LRT was also applied.
Assessing Substitution Rates in Branches Leading to the Different Bacterial Groups
To assess the variation in the substitution rates among different lineages of the phylogenetic tree, we first measured nucleotide and amino acid distances in the different branches of the tree and tested the constancy of the substitution rates, using as an outgroup the groES/L sequence of the gamma proteobacteria Pseudomonas aeruginosa. Nucleotide distances were estimated using Tamura and Nei's model (1993
) under a gamma distribution of substitution rates (Rzhetsky and Nei 1994
) with a shape parameter (
) of 0.2997. This
value was estimated with the PAMP and BASEML programs from the PAML package (Yang 2000
). Accurate amino acid distances were obtained with the Poisson correction. Thereafter, rate distances were assessed using the two-cluster test (Takezaki, Rzhetsky, and Nei 1995
; program LINTREE), which examines the equality of the average substitution rates for two clusters (A and B) linked by a node on the tree, using one or several out-group sequences.
Detection of Branches of the Phylogenetic Tree Under Positive Selection
To test the selective pressures in the different lineages of the phylogenetic tree, we estimated the nonsynonymous to synonymous rate ratio ( = dN/dS). Values of
= 1,
> 1, and
< 1 indicate neutrality, positive selection, and purifying selection, respectively. This is the most accepted and stringent way to detect positively selected changes in a sequence alignment (Sharp 1997
; Akashi 1999
; Crandall et al. 1999
). To perform these analyses, we applied different models of codon evolution implemented in the CODEML program of the PAML package (Yang 2000
). The Goldman and Yang (1994)
model assumes a single
value for all the lineages of the phylogenetic tree and for all codon sites of the sequence alignment. In contrast, the free-ratio model allows the
value to vary among different lineages of the tree. Both the models are nested and hence comparable by the LRT because it can be approached to a chi-square distribution with the degrees of freedom being the total number of estimated rate ratios (
) - 1.
Highlighting Specific Regions Under Selective Constraints
We have developed a new statistical method, based on a sliding-window approach, to detect selective constraints in protein-coding genes (M. A. Fares et al., unpublished data). In brief, the method tests the significance of the deviation of nonsynonymous and synonymous substitutions from that expected under neutrality and allows the testing of different hypotheses, such as saturation of synonymous or nonsynonymous sites, high substitution rates, or the action of selection in a specific region of the protein, that could be important from a structural or functional viewpoint. The fundamentals of the method are similar to those of Suzuki and Gojobori (1999)
, but differ from it in that our method uses a Poisson approach instead of a binomial and enables us to use a statistically appropriate window size to analyze the data. The method is described in the Supplementary Material posted on the MBE web page (http://www.molbiolevol.org).
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Results |
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Detecting Regions Under Positive Selection by the Sliding Window Method
First of all we calculated the probability of nonsynonymous and synonymous nucleotide substitutions following equation (3) (see Supplementary Material) and using a random set of simulated sequence alignments. The estimated probabilities of nonsynonymous and synonymous substitutions, P(N) = 0.1 and P(
S) = 0.9, were used to determine the appropriate window size to analyze the sequence data, which resulted in 4 codons because its lower 5% P(dN) estimated value was higher than 0.05 (fig. 3
). Therefore, we might not expect significant results by chance if we slid a window of 4 codons along the real sequence alignment and if no selective constraints are affecting the evolution of any region of the sequence.
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The significance of these values was tested against chance by calculating the analytical variance of
following the method of Weir (1996)
. The variance of
was estimated to be V(
) = 0.3304, and the probabilities of
values in each region of the groEL alignment sequence applying a normal test are summarized in table 4
.
On the other hand, we estimated the proportion of sites that are under different selective constraints. To do so, we distinguished between three sets of codons: those that are under neutrality (where is not significantly different from 1 or from the average estimate of
), those that are under strong purifying selection (with
values significantly smaller than 1 and very close to 0), and those that evolved under positive selection (with
values significantly higher than the average
values of the alignment and higher than 1). Only 0.67% of codon sites showed
values not significantly different from 1, whereas the vast majority of the codon sites (97.73%) have
values significantly smaller than 1 (ranging between 0.004 and 0.25), which means that GroEL evolved mainly under strong purifying selection. Among the codons analyzed, a small fraction of them (1.6%) showed
values significantly higher than 1 (ranging from 3.53 to 33.94) and than the average
value estimated from the alignment by the sliding windowbased method.
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Discussion |
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In contrast to free-living bacteria, which have large effective population sizes (Selander, Caugant, and Whittam 1987
) and whose evolutionary history is highly controlled by recombination between individuals (Maynard Smith, Dowson, and Spratt 1991
; Maynard Smith et al. 1993
; Dykhuizen and Green 1993
), strictly intracellular symbiotic bacteria have very small effective population sizes, apparently lack recombination, and are maternally transmitted in a clonal manner (Funk, Wernegreen, and Moran 2001
). All these characteristics make endosymbiotic bacteria an ideal model for studying the effect of genetic drift in the accumulation of mutations on their genomes and to determine how this accumulation is biased to the fixation of deleterious mutations because of their asexuality (Muller 1964
; Lynch 1996
, 1997
).
Several previous studies showed an accelerated fixation rate of amino acid replacements in Buchnera aphidicola genome, the primary symbiont of the aphids (Moran 1996
; Brynel et al. 1998
; Lambert and Moran 1998
; Clark, Moran, and Baumann 1999
; Wernegreen and Moran 1999
). In agreement with the data obtained by Wernegreen and Moran (1999)
, the relative rate test evidences 1.3 to 6.9 times higher rates of evolution in Buchnera genes compared with those from E. coli. When the authors examined the rates of nonsynonymous to synonymous nucleotide substitutions (Ka/Ks or the corrected
= dN/dS) in different genes, they realized that, in a vast majority of cases, the Ka/Ks of Buchnera genes significantly exceeded that obtained when the out-group sequence S. typhimurium was compared with one of the closest free-living relatives of Buchnera, E. coli. Because the distribution of amino acid replacements was random along the entire protein-coding gene, they asserted that one cannot expect that these changes have been fixed by positive selection, and hence, the best explanation is that amino acid changes in the lineage of Buchnera were slightly deleterious mutations fixed by genetic drift. This accumulation of slightly deleterious mutations has previously been attributed to mutational bias, not buffered because of the absence of recombination, and to small population sizes (Moran 1996
; Wernegreen and Moran 1999
; Funk, Wernegreen, and Moran 2001
). However, an interesting result was that the difference between the two rate ratios was slightly smaller when the heat-shock protein GroEL was examined; thus, it can be concluded that compared with other genes in Buchnera, purifying selection is apparently more effective against nonsynonymous substitutions in this gene. Our results demonstrate that groEL is subjected to a strong purifying selection (97.73% of the codon sites have
< 1 and close to 0), and only very few codons (0.67%) have
values not significantly different than 1, which means that groEL is not allowed to accumulate nonsynonymous substitutions even under the strong bottleneck to which Buchnera is subjected. This result suggests that, in contrast to the rest of the protein-coding genes that accumulated nonsynonymous changes because of the effect of genetic drift, groEL cannot accumulate slightly deleterious amino acid replacements by genetic drift because this protein may be functionally important to buffer the conformation loss of the rest of the proteins and any amino acid replacement could affect its function, and hence, disable groEL from folding the damaged proteins.
Our results suggest that an acceleration of nonsynonymous (amino acid) substitution rates occurred during the evolution of the symbiotic lineage but also showed acceleration among lineages within the cluster of Buchnera. This acceleration of rates affected nonsynonymous sites in groEL, which is consistent with the results obtained by Moran (1996)
.
However, ML codon-based models applied in this study revealed that, although purifying selection explains the evolution of groEL in free-living bacteria and within each aphid family, positive selection is the alternative explanation for the fixation of amino acid replacements in some branches of the tree connecting the aphid endosymbionts. Thus, ML analyses revealed that positive selection is the most likely explanation for the fixation of the amino acid changes in the lineage leading to T. suberi and T. caerulescens aphid symbionts and the branch connecting Aphididae symbionts. These results agree partially with the results obtained by the analysis of the constancy of rates using the two-cluster test. In this way, accelerated amino acid changes were detected in the same branch leading to the cluster of T. salignus-T. suberi symbionts. This amino acid replacement acceleration is compatible with the fixation of amino acid changes by positive selection as indicated by the ML analyses. Furthermore, although no accelerated rates were evidenced in the branch leading to the symbionts of the Aphididae family, fixation of amino acid replacements by positive selection was also detected in this lineage. These results agree with the idea that, on average, primary symbiotic bacteria of aphids have accumulated amino acid replacements mainly by their fixation because of genetic drift. However, the fixation of amino acid replacements in GroEL in the lineages leading to endosymbionts cannot be explained under the genetic drift hypothesis but by positive selection.
In agreement with these results, the application of the same analyses to the different groEL regions, coding for different functional and structural regions of the GroEL protein, consistently detected positive selection in the same branches as in the case of the complete groEL alignment. In addition, the analysis of the equatorial and intermediate domains did not show any branch of the phylogenetic tree to be under positive selection.
The main result obtained when ML codon-based models were applied to detect lineages with positively selected amino acid changes is that all the amino acids are located within GroEL regions involved in peptide and GroES binding. Moreover, five out of the 11 replacements detected in these branches constitute important changes in the nature of the amino acids. Three of them (E340, H344 and Q346) are located within the domain 339347 of the apical region involved in substrate polypeptide binding (Fenton et al. 1994
). Site-directed mutation experiments demonstrated that changes in these amino acid positions resulted in the loss of GroEL function. Also, position 191 (EK) was detected as fixed in the Buchnera lineage by positive selection. This amino acid is conformationally important because it is located next to residue G192 that controls the block massive movements of the GroEL apical domain (Braig et al. 1994
; Xu, Horwich, and Sigler 1997
).
From these results we can conclude that strong purifying selection prevents deleterious amino acid fixation in groE by genetic drift even during the strong bottlenecks caused by the maternal transmission of the symbiont to the next generation and the absence of recombination. Most of the amino acid replacements observed in the endosymbiotic groEL were fixed by positive selection as indicated by our results. A second conclusion derived from our analysis is that the majority of the amino acid sites are extremely conservative (97.73% of codons are under a strong purifying selection), which indicates that any nonsynonymous substitution in groEL has a strong antagonistic effect on its chaperonin function; hence, it can lead to a loss of in cell viability.
An objection to the use of ML methods based on a phylogenetic tree (Yang 1999) to detect positive selected branches is that these methods rely on the assumption that all amino acid sites are under the same selective constraints, and hence, have the same rate of amino acid fixation. Far from realistic, this assumption does not collect the information to analyze selective constraints in a specific region of the sequence alignment.
Our new method allowed us to answer if the amino acid replacement acceleration observed is mainly caused by the action of positive selection, or, alternatively, is randomly distributed along the sequence and can be explained by chance (genetic drift). According to our results, the nonsynonymous substitutions causing amino acid replacements were not randomly distributed along the protein-coding gene groEL but occurred in key positions for the peptide- and GroES-binding function. All the regions under positive selection showed higher amino acid replacements than that expected by chance, and their values were always significantly higher than the expected value under neutrality. However, in contrast to the results obtained using the ML method, we also detected with the new method several groEL regions coding for parts of the equatorial domain and the intermediate domain as subjected to positive selection. When these regions were examined, we realized that they are located in the hypervariable region consisting in a serine-rich tract.
The main conclusion from our study is that the heat-shock protein (chaperonin) GroEL appears to have been subjected to different selective constraints along the evolution of the symbiotic lineages. Our results indicate that, with the exception of a very small proportion of amino acid replacements in the lineage leading to primary symbiotic bacteria of aphids that were fixed by genetic drift, a vast majority of amino acid substitutions were fixed by positive selection in the lineages leading to the symbionts of the Aphididae familiy and to the T. salignus and T. suberi symbiont cluster.
These results, together with the fact that GroEL has been reported as having the highest expression levels in Buchnera, suggest a very important role of GroEL in the functional maintenance of the endosymbiont proteome. In accordance with the hypothesis postulated by Moran (1996)
, a plausible explanation for the different pattern of protein expression and evolution of GroEL is that this protein may act by buffering the effect of the accumulation of mildly deleterious amino acid replacements in the symbiont proteins caused by genetic drift, during the strong bottlenecks suffered by these symbionts, maintaining the appropriate functional protein folding.
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Acknowledgements |
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Footnotes |
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Abbreviations: PCR, polymerase chain reaction; LRT, likelihood ratio test.
Keywords: aphid endosymbionts
Buchnera aphidicola
groEL
positive selection
rates of evolution
Address for correspondence and reprints: Andrés Moya, Institut "Cavanilles" de Biodiversitat i Biologia Evolutiva, Universitat de València, Edifici d'Instituts del Campus de Paterna, P.O. Box 2085, E-46071 València, Spain. andres.moya{at}uv.es
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