Hundreds of flagellar basal bodies cover the cell surface of the endosymbiotic bacterium Buchnera aphidicola sp. strain APS

Buchnera aphidicola is the endosymbiotic bacterium of the pea aphid. Due to its small genome size, Buchnera lacks many essential genes for autogenous life but obtains nutrients from the host. Although the Buchnera cell is nonmotile, it retains clusters of flagellar genes that lack the late genes necessary for motility, including the flagellin gene. In this study, we show that the flagellar genes are actually transcribed and translated and that the Buchnera cell surface is covered with hundreds of hook-basal-body (HBB) complexes. The abundance of HBB complexes suggests a role other than motility. We discuss the possibility that the HBB complex may serve as a protein transporter not only for the flagellar proteins but also for other proteins to maintain the symbiotic system.     

Molecular characterization of the leucine cluster in Buchnera sp. strain PSY, a primary endosymbiont of the aphid Pemphigus spyrothecae

Buchnera strains from most aphid subfamilies studied to date have been found to carry the leucine gene cluster (leuA, -B, -C, and -D) on a plasmid, an organization unique among bacteria. Here, however, we demonstrate a classical chromosomal location of the cluster in Buchnera sp. strain PSY from the aphid Pemphigus spyrothecae (subfamily Pemphiginae). The genes that flank leuABCD in Buchnera sp. strain PSY appear to be adjacent in the genome of Buchnera sp. strain APS, a strain carrying a leucine plasmid. We propose that the presence of a leucine plasmid predates the diversification of symbiotic Buchnera and that the chromosomal location observed in Buchnera sp. strain PSY arose by a transfer of the leucine genes from a plasmid to the chromosome.     

A strong effect of AT mutational bias on amino acid usage in Buchnera is mitigated at high-expression genes

The advent of full genome sequences provides exceptionally rich data sets to explore molecular and evolutionary mechanisms that shape divergence among and within genomes. In this study, we use multivariate analysis to determine the processes driving genome-wide patterns of amino usage in the obligate endosymbiont Buchnera and its close free-living relative Escherichia coli. In the AT-rich Buchnera genome, the primary source of variation in amino acid usage differentiates high- and low-expression genes. Amino acids of high-expression Buchnera genes are generally less aromatic and use relatively GC-rich codons, suggesting that selection against aromatic amino acids and against amino acids with AT-rich codons is stronger in high-expression genes. Selection to maintain hydrophobic amino acids in integral membrane proteins is a primary factor driving protein evolution in E. coli but is a secondary factor in Buchnera. In E. coli, gene expression is a secondary force driving amino acid usage, and a correlation with tRNA abundance suggests that translational selection contributes to this effect. Although this and previous studies demonstrate that AT mutational bias and genetic drift influence amino acid usage in Buchnera, this genome-wide analysis argues that selection is sufficient to affect the amino acid content of proteins with different expression and hydropathy levels.     

Tracing the evolution of gene loss in obligate bacterial symbionts

The gamma-proteobacterial symbionts of insects are a model group for comparative studies of genome reduction. The phylogenetic proximity of these reduced genomes to the larger genomes of well-studied free-living bacteria has enabled reconstructions of the process by which genes and DNA are lost. Three genome sequences are now available for Buchnera aphidicola. Analyses of Buchnera genomes in comparison with those of related enteric bacteria suggest that extensive changes including large deletions, repetitive element proliferation and chromosomal rearrangements occurred initially, followed by extreme stasis in gene order and slow decay of additional genes. This pattern appears to be characteristic of symbiont evolution.     

蚜虫与其胞内共生细菌的相互作用

蚜虫—巴克纳氏菌之间是一种典型的互利共生关系,两者相互依存,缺少一方,另一方便不能生存。研究表明,共生细菌能为寄主蚜虫提供必需氨基酸和维生索,并对寄主具有一些非营养功能,如促进蚜虫传播循环性病毒等。寄主蚜虫则是为共生菌提供一个合适的生存场所,并对共生菌的生长和繁殖进行调控。现代分子生物学技术从基因水平证明了蚜虫与共生菌的相互依赖性。     

Why are the genomes of endosymbiotic bacteria so stable?

The comparative analysis of three strains of the endosymbiotic bacterium Buchnera aphidicola has revealed high genome stability associated with an almost complete absence of chromosomal rearrangements and horizontal gene transfer events during the past 150 million years. The loss of genes involved in DNA uptake and recombination in the initial stages of endosymbiosis probably underlies this stability. Gene loss, which was extensive during the initial steps of Buchnera evolution, has continued in the different Buchnera lineages since their divergence.     

Changing partners in an obligate symbiosis: a facultative endosymbiont can compensate for loss of the essential endosymbiont Buchnera in an aphid

Almost all aphids harbour an endosymbiotic bacterium, Buchnera aphidicola, in bacteriocytes. Buchnera synthesizes essential nutrients and supports growth and reproduction of the host. Over the long history of endosymbiosis, many essential genes have been lost from the Buchnera genome, resulting in drastic genome reduction and the inability to live outside the host cells. In turn, when deprived of Buchnera, the host aphid suffers retarded growth and sterility. Buchnera and the host aphid are often referred to as highly integrated almost inseparable mutualistic partners. However, we discovered that, even after complete elimination of Buchnera, infection with a facultative endosymbiotic gamma-proteobacterium called pea aphid secondary symbiont (PASS) enabled survival and reproduction of the pea aphid. In the Buchnera-free aphid, PASS infected the cytoplasms of bacteriocytes that normally harbour Buchnera, establishing a novel endosymbiotic system. These results indicate that PASS can compensate for the essential role of Buchnera by physiologically and cytologically taking over the symbiotic niche. By contrast, PASS negatively affected the growth and reproduction of normal host aphids by suppressing the essential symbiont Buchnera. These findings illuminate complex symbiont-symbiont and host-symbiont interactions in an endosymbiotic system, and suggest a possible evolutionary route to novel obligate endosymbiosis by way of facultative endosymbiotic associations.     

Expression and control of an operon from an intracellular symbiont which is homologous to the groE operon.

Members of the genus Buchnera are intracellular symbionts harbored by the aphid bacteriocyte which selectively synthesize symbionin, a homolog of the Escherichia coli GroEL protein, in vivo. Symbionin and SymS, a GroES homolog, are encoded in the symSL operon. Northern blotting and primer extension analyses revealed that the symSL operon invariably gives rise to a bicistronic mRNA under the control of a heat shock promoter, though the amount of the symSL mRNA in the isolated symbiont did not increase in response to heat shock. The sigma32 protein that recognizes the heat shock promoter in E. coli was scarcely detected in Buchnera cells even after heat shock. Although the functionally essential regions of the Buchnera sigma32 protein were well conserved, the Buchnera rpoH gene did not complement an E. coli delta rpoH mutant. On the one hand, the A-T evolutionary pressure imposed on the Buchnera genome may have not only decreased the activity of its sigma32 but also ruined the nucleotide sequences necessary for the expression of rpoH; on the other hand, it may have facilitated expression of the symSL operon without activation by sigma32.     

The role of mutational dynamics in genome shrinkage

Genome shrinkage occurs after whole genome duplications (WGDs) and in the evolution of parasitic or symbiotic species. The dynamics of this process, whether it occurs by single gene deletions or also by larger deletions are however unknown. In yeast, genome shrinkage has occurred after a WGD. Using a computational model of genome evolution, we show that in a random genome single gene deletions cannot explain the observed pattern of gene loss in yeast. The distribution of genes deleted per event can be very well described by a geometric distribution, with a mean of 1.1 genes per event. In terms of deletions of a stretch of base pairs, we find that a geometric distribution with an average of 500-600 base pairs per event describes the data very well. Moreover, in the model, as in the data, gene pairs that have a small intergenic distance are more likely to be both deleted. This proves that simultaneous deletion of multiple genes causes the observed pattern of gene deletions, rather than deletion of functionally clustered genes by selection. Furthermore, we found that in the bacterium Buchnera aphidicola larger deletions than in yeast are necessary to explain the clustering of deleted genes. We show that the excess clustering of deleted genes in B. aphidicola can be explained by the clustering of genes in operons. Therefore, we show that selection has little effect on the clustering of deleted genes after the WGD in yeast, while it has during genome shrinkage in B. aphidicola.     

Mutualists and parasites: how to paint yourself into a (metabolic) corner.

Eukaryotes have developed an elaborate series of interactions with bacteria that enter their bodies and/or cells. Genome evolution of symbiotic and parasitic bacteria multiplying inside eukaryotic cells results in both convergent and divergent changes. The genome sequences of the symbiotic bacteria of aphids, Buchnera aphidicola, and the parasitic bacteria of body louse and humans, Rickettsia prowazekii, provide insights into these processes. Convergent genome characteristics include reduction in genome sizes and lowered G+C content values. Divergent evolution was recorded for amino acid and cell wall biosynthetic genes. The presence of pseudogenes in both genomes provides examples of recent gene inactivation events and offers clues to the process of genome deterioration and host-cell adaptation.     

New clues about the evolutionary history of metabolic losses in bacterial endosymbionts, provided by the genome of Buchnera aphidicola from the aphid Cinara tujafilina

The symbiotic association between aphids (Homoptera) and Buchnera aphidicola (Gammaproteobacteria) started about 100 to 200 million years ago. As a consequence of this relationship, the bacterial genome has undergone a prominent size reduction. The downsize genome process starts when the bacterium enters the host and will probably end with its extinction and replacement by another healthier bacterium or with the establishment of metabolic complementation between two or more bacteria. Nowadays, several complete genomes of Buchnera aphidicola from four different aphid species (Acyrthosiphon pisum, Schizaphis graminum, Baizongia pistacea, and Cinara cedri) have been fully sequenced. C. cedri belongs to the subfamily Lachninae and harbors two coprimary bacteria that fulfill the metabolic needs of the whole consortium: B. aphidicola with the smallest genome reported so far and "Candidatus Serratia symbiotica." In addition, Cinara tujafilina, another member of the subfamily Lachninae, closely related to C. cedri, also harbors "Ca. Serratia symbiotica" but with a different phylogenetic status than the one from C. cedri. In this study, we present the complete genome sequence of B. aphidicola from C. tujafilina and the phylogenetic analysis and comparative genomics with the other Buchnera genomes. Furthermore, the gene repertoire of the last common ancestor has been inferred, and the evolutionary history of the metabolic losses that occurred in the different lineages has been analyzed. Although stochastic gene loss plays a role in the genome reduction process, it is also clear that metabolism, as a functional constraint, is also a powerful evolutionary force in insect endosymbionts.     

Comparative analysis of two genomic regions among four strains of Buchnera aphidicola, primary endosymbiont of aphids

Preliminary analysis of two selected genomic regions of Buchnera aphidicola BCc, the primary endosymbiont of the cedar aphid Cinara cedri, has revealed a number of interesting features when compared with the corresponding homologous regions of the three B. aphidicola genomes previously sequenced, that are associated with different aphid species. Both regions exhibit a significant reduction in length and gene number in B. aphidicola BCc, as it could be expected since it possess the smallest bacterial genome. However, the observed genome reduction is not even in both regions, as it appears to be dependent on the nature of their gene content. The region fpr-trxA, that contains mainly metabolic genes, has lost almost half of its genes (45.6%) and has reduced 52.9% its length. The reductive process in the region rrl-aroK, that contains mainly ribosomal protein genes, is less dramatic, since it has lost 9.3% of genes and has reduced 15.5% of its length. Length reduction is mainly due to the loss of protein-coding genes, not to the shortening of ORFs or intergenic regions. In both regions, G+C content is about 4% lower in BCc than in the other B. aphidicola strains. However, when only conserved genes and intergenic regions of the four B. aphidicola strains are compared, the G+C reduction is higher in the fpr-trxA region.     

Serratia symbiotica from the aphid Cinara cedri: a missing link from facultative to obligate insect endosymbiont

The genome sequencing of Buchnera aphidicola BCc from the aphid Cinara cedri, which is the smallest known Buchnera genome, revealed that this bacterium had lost its symbiotic role, as it was not able to synthesize tryptophan and riboflavin. Moreover, the biosynthesis of tryptophan is shared with the endosymbiont Serratia symbiotica SCc, which coexists with B. aphidicola in this aphid. The whole-genome sequencing of S. symbiotica SCc reveals an endosymbiont in a stage of genome reduction that is closer to an obligate endosymbiont, such as B. aphidicola from Acyrthosiphon pisum, than to another S. symbiotica, which is a facultative endosymbiont in this aphid, and presents much less gene decay. The comparison between both S. symbiotica enables us to propose an evolutionary scenario of the transition from facultative to obligate endosymbiont. Metabolic inferences of B. aphidicola BCc and S. symbiotica SCc reveal that most of the functions carried out by B. aphidicola in A. pisum are now either conserved in B. aphidicola BCc or taken over by S. symbiotica. In addition, there are several cases of metabolic complementation giving functional stability to the whole consortium and evolutionary preservation of the actors involved.     

Sequence evolution in bacterial endosymbionts having extreme base compositions.

A major limitation on ability to reconstruct bacterial evolution is the lack of dated ancestors that might be used to evaluate and calibrate molecular clocks. Vertically transmitted symbionts that have cospeciated with animal hosts offer a firm basis for calibrating sequence evolution in bacteria, since fossils of the hosts can be used to date divergence events. Sequences for a functionally diverse set of genes have been obtained for bacterial endosymbionts (Buchnera) from two pairs of aphid host species, each pair diverging 50-70 MYA. Using these dates and estimated numbers of Buchnera generations per year, we calculated rates of base substitution for neutral and selected sites of protein-coding genes and overall rates for rRNA genes. Buchnera shows homogeneity among loci with regard to synonymous rate. The Buchnera synonymous rate is about twice that for low-codon-bias genes of Escherichia coli-Salmonella typhimurium on an absolute timescale, and fourfold higher on a generational timescale. Nonsynonymous substitutions show a greater rate disparity in favor of Buchnera, a result consistent with a genomewide decrease in selection efficiency in Buchnera. Ratios of synonymous to nonsynonymous substitutions differ for the two pairs of Buchnera, indicating that selection efficiency varies among lineages. Like numerous other intracellular bacteria, such as Rickettsia and Wolbachia, Buchnera has accumulated amino acids with codons rich in A or T. Phylogenetic reconstruction of amino acid replacements indicates that replacements yielding increased A + T predominated early in the evolution of Buchnera, with the trend slowing or stopping during the last 50 Myr. This suggests that base composition in Buchnera has approached a limit enforced by selective constraint acting on protein function.     

Tempo and mode of early gene loss in endosymbiotic bacteria from insects

Background  

Understanding evolutionary processes that drive genome reduction requires determining the tempo (rate) and the mode (size and types of deletions) of gene losses. In this study, we analysed five endosymbiotic genome sequences of the gamma-proteobacteria (three different Buchnera aphidicola strains, Wigglesworthia glossinidia, Blochmannia floridanus) to test if gene loss could be driven by the selective importance of genes. We used a parsimony method to reconstruct a minimal ancestral genome of insect endosymbionts and quantified gene loss along the branches of the phylogenetic tree. To evaluate the selective or functional importance of genes, we used a parameter that measures the level of adaptive codon bias in E. coli (i.e. codon adaptive index, or CAI), and also estimates of evolutionary rates (Ka) between pairs of orthologs either in free-living bacteria or in pairs of symbionts.     

Decay of mutualistic potential in aphid endosymbionts through silencing of biosynthetic loci: Buchnera of Diuraphis

Buchnera, the primary bacterial endosymbiont of aphids, is known to provision essential amino acids lacking in the hosts' diet of plant sap. The recent discovery of silenced copies of genes for tryptophan biosynthesis (trpEG) in certain Buchnera lineages suggests a decay in symbiotic functions in some aphid species. However, neither the distribution of pseudogenes among lineages nor the impact of this gene silencing on amino-acid availability in hosts has been assessed. In Buchnera of the aphid Diuraphis noxia, tandem repeats of these pseudogenes have persisted in diverse lineages, and thpEG pseudogenes have originated at least twice within this aphid genus. Measures of amino-acid concentrations in Diuraphis species have shown that the presence of the pseudogene is associated with a decreased availability of tryptophan, indicating that gene silencing decreases nutrient provisioning by symbionts. In Buchnera of Diuraphis, rates of nonsynonymous substitutions are elevated in functional trpE copies, supporting the hypothesis that pseudogene origin and persistence reflect a reduced selection for symbiont biosynthetic contributions. The parallel evolution of trpEG pseudogenes in Buchnera of Diuraphis and certain other aphid hosts suggests that either selection at the host level is not effective or that fitness in these aphids is not limited by tryptophan availability.