Metabolic Complementation Research Articles

Characterizing and Tailoring the Substrate Profile of a γ-Glutamyltransferase Variant.

Xenobiology is an emerging field that focuses on the extension and redesign of biological systems through the use of laboratory-derived xenomolecules, which are molecules that are new to the metabolism of the cell. Despite the enormous potential of using xenomolecules in living organisms, most noncanonical building blocks still need to be supplied externally, and often poor uptake into cells limits wider applicability. To improve the cytosolic availability of noncanonical molecules, a synthetic transport system based on portage transport was developed, in which molecules of interest "cargo" are linked to a synthetic transport vector that enables piggyback transport through the alkylsulfonate transporter (SsuABC) of Escherichia coli. Upon cytosolic delivery, the vector-cargo conjugate is enzymatically cleaved by GGTxe, leading to the release of the cargo molecule. To deepen our understanding of the synthetic transport system, we focused on the characterization and further development of the enzymatic cargo release step. Hence, the substrate scope of GGTxe was characterized using a library of structurally diverse vector-cargo conjugates and MS/MS-based quantification of hydrolysis products in a kinetic manner. The resulting substrate tolerance characterization revealed that vector-amino acid conjugates were significantly unfavored. To overcome this shortcoming, a selection system based on metabolic auxotrophy complementation and directed evolution of GGTxe was established. In a directed evolution campaign, we improved the enzymatic activity of GGTxe for vector-amino acid conjugates and revealed the importance of residue D386 in the cargo unloading step.

Rescue of pyrimidine-defective Pseudomonas aeruginosa through metabolic complementation.

This research holds crucial implications for combating chronic infections, where multiple pathogens coexist and interact within the same environment. By uncovering the dynamic exchange of pyrimidines between Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA), our study reveals a previously unrecognized aspect of interspecies cooperation. The observed enhanced growth of a pyrimidine-deficient PA strain when co-cultured with SA suggests potential avenues for understanding and disrupting bacterial metabolic interdependencies in chronic infection settings. Furthermore, our findings highlight the mechanisms involved in metabolic exchange, emphasizing the importance of cell-to-cell contact. This research explored essential metabolic interactions to address the challenges posed by difficult-to-treat chronic infections.

Proximity ligation strategy for the genomic reconstruction of microbial communities associated with the ectoparasite Caligus rogercresseyi

The sea louse Caligus rogercresseyi has become one of the main constraints for the sustainable development of salmon aquaculture in Chile. Although this parasite's negative impacts are well recognized by the industry, some novel potential threats remain unnoticed. The recent sequencing of the C. rogercresseyi genome revealed a large bacterial community associated with the sea louse, however, it is unknown if these microorganisms should become a new focus of sanitary concern. Herein, chromosome proximity ligation (Hi-C) coupled with long-read sequencing were used for the genomic reconstruction of the C. rogercresseyi microbiota. Through deconvolution analysis, we were able to assemble and characterize 413 bacterial genome clusters, including six bacterial genomes with more than 80% of completeness. The most represented bacterial genome belonged to the fish pathogen Tenacibacullum ovolyticum (97.87% completeness), followed by Dokdonia sp. (96.71% completeness). This completeness allowed identifying 21 virulence factors (VF) within the T. ovolyticum genome and four antibiotic resistance genes (ARG). Notably, genomic pathway reconstruction analysis suggests putative metabolic complementation mechanisms between C. rogercresseyi and its associated microbiota. Taken together, our data highlight the relevance of Hi-C techniques to discover pathogenic bacteria, VF, and ARGs and also suggest novel host-microbiota mutualism in sea lice biology.

Interspecies Metabolic Complementation in Cystic Fibrosis Pathogens via Purine Exchange

Cystic fibrosis (CF) is a genetic disease frequently associated with chronic lung infections caused by a consortium of pathogens. It is common for auxotrophy (the inability to biosynthesize certain essential metabolites) to develop in clinical isolates of the dominant CF pathogen Pseudomonas aeruginosa, indicating that the CF lung environment is replete in various nutrients. Many of these nutrients are likely to come from the host tissues, but some may come from the surrounding polymicrobial community within the lungs of CF patients as well. To assess the feasibility of nutrient exchange within the polymicrobial community of the CF lung, we selected P. aeruginosa and Staphylococcus aureus, two of the most prevalent species found in the CF lung environment. By comparing the polymicrobial culture of wild-type strains relative to their purine auxotrophic counterparts, we were able to observe metabolic complementation occurring in both P. aeruginosa and S. aureus when grown with a purine-producing cross-species pair. While our data indicate that some of this complementation is likely derived from extracellular DNA freed by lysis of S. aureus by the highly competitive P. aeruginosa, the partial complementation of S. aureus purine deficiency by P. aeruginosa demonstrates that bidirectional nutrient exchange between these classic competitors is possible.

Choreography of methylation: KDM4C histone demethylation enables ALKBH5 m6A RNA demethylation to sustain AML stem cells.

Choreography of methylation: KDM4C histone demethylation enables ALKBH5 m6A RNA demethylation to sustain AML stem cells.

Performance and Mechanisms of Advanced Nitrogen Removal via FeS-driven Autotrophic Denitrification Coupled with ANAMMOX

Synergy among members of complex microbial communities in the transformation of elements is a key ecological regulation strategy in nature. Making full use of this phenomenon and achieving functional combinations of different microorganisms may have a significant effect on developing new wastewater treatment processes. In this study, nitrogen-containing pollutants were applied in a static batch experiment. The dosage of FeS, the ratio of NO3--N/NO2--N, and the ratio of ANAMMOX (AN) to autotrophic denitrification (AD) biomass were the controlled reaction conditions. The cooperation mechanism resulting from the metabolic complementation of AN and AD is discussed, and the concept of a (AN+AD)TN 0 nitrogen removal process is proposed. This study showed that the excessive dosage of FeS could ensure the more thorough reaction of AD without significantly affecting the metabolic activity of AN bacteria. A complex microbial community was involved in the competition for metabolic substrates when the proportion of NO2--N in the electron acceptor was increased, resulting in a negative impact on the removal of TN. The increase of AN biomass contributed to the strengthening of the cooperation between AN and AD. When the stoichiometric ratio of NH4+-N to NO3--N was less than 0.85, TN could be completely removed. The results showed that a more effective wastewater treatment process may be established by understanding the interactions between microorganisms, and by manipulating or regulating complex microbial communities. This could achieve the efficient removal of pollutants under low material consumption conditions.

Insights into the ecology, evolution, and metabolism of the widespread Woesearchaeotal lineages

BackgroundAs a recently discovered member of the DPANN superphylum, Woesearchaeota account for a wide diversity of 16S rRNA gene sequences, but their ecology, evolution, and metabolism remain largely unknown.ResultsHere, we assembled 133 global clone libraries/studies and 19 publicly available genomes to profile these patterns for Woesearchaeota. Phylogenetic analysis shows a high diversity with 26 proposed subgroups for this recently discovered archaeal phylum, which are widely distributed in different biotopes but primarily in inland anoxic environments. Ecological patterns analysis and ancestor state reconstruction for specific subgroups reveal that oxic status of the environments is the key factor driving the distribution and evolutionary diversity of Woesearchaeota. A selective distribution to different biotopes and an adaptive colonization from anoxic to oxic environments can be proposed and supported by evidence of the presence of ferredoxin-dependent pathways in the genomes only from anoxic biotopes but not from oxic biotopes. Metabolic reconstructions support an anaerobic heterotrophic lifestyle with conspicuous metabolic deficiencies, suggesting the requirement for metabolic complementarity with other microbes. Both lineage abundance distribution and co-occurrence network analyses across diverse biotopes confirmed metabolic complementation and revealed a potential syntrophic relationship between Woesearchaeota and methanogens, which is supported by metabolic modeling. If correct, Woesearchaeota may impact methanogenesis in inland ecosystems.ConclusionsThe findings provide an ecological and evolutionary framework for Woesearchaeota at a global scale and indicate their potential ecological roles, especially in methanogenesis.

Standardized cloning vectors for protein production and generation of large gene libraries in Escherichia coli.

Here, we modified the multiple cloning sites from commonly used expression vectors to create a new suite of cloning plasmids that simplify and speed up cloning procedures in Escherichia coli. Each of our standardized plasmids contains two BsaI restriction sites, allowing for highly efficient cloning of genes and bringing their expression under control of either a T7 (pET21a_BsaI, pET28a_BsaI, and pMAL-c5T_BsaI) or T5 promoter (pUR22 and pUR23). Another plasmid in our suite (pTNA_BsaI) allows for generation of large gene libraries containing >108 variants, which can be constitutively expressed in selection experiments using metabolic complementation of auxotrophic E. coli strains. Coupling restriction and ligation with the BsaI restriction enzyme minimizes hands-on time, while the need for only three different primers to clone a target gene into the six different vectors keeps overall cloning costs low.

Determinism and Contingency Shape Metabolic Complementation in an Endosymbiotic Consortium.

Bacterial endosymbionts and their insect hosts establish an intimate metabolic relationship. Bacteria offer a variety of essential nutrients to their hosts, whereas insect cells provide the necessary sources of matter and energy to their tiny metabolic allies. These nutritional complementations sustain themselves on a diversity of metabolite exchanges between the cell host and the reduced yet highly specialized bacterial metabolism—which, for instance, overproduces a small set of essential amino acids and vitamins. A well-known case of metabolic complementation is provided by the cedar aphid Cinara cedri that harbors two co-primary endosymbionts, Buchnera aphidicola BCc and Ca. Serratia symbiotica SCc, and in which some metabolic pathways are partitioned between different partners. Here we present a genome-scale metabolic network (GEM) for the bacterial consortium from the cedar aphid iBSCc. The analysis of this GEM allows us the confirmation of cases of metabolic complementation previously described by genome analysis (i.e., tryptophan and biotin biosynthesis) and the redefinition of an event of metabolic pathway sharing between the two endosymbionts, namely the biosynthesis of tetrahydrofolate. In silico knock-out experiments with iBSCc showed that the consortium metabolism is a highly integrated yet fragile network. We also have explored the evolutionary pathways leading to the emergence of metabolic complementation between reduced metabolisms starting from individual, complete networks. Our results suggest that, during the establishment of metabolic complementation in endosymbionts, adaptive evolution is significant in the case of tryptophan biosynthesis, whereas vitamin production pathways seem to adopt suboptimal solutions.

Genetic metabolic complementation establishes a requirement for GDP-fucose in Leishmania

To survive in its sand fly vector, the trypanosomatid protozoan parasite Leishmania first attaches to the midgut to avoid excretion, but eventually it must detach for transmission by the next bite. In Leishmania major strain Friedlin, this is controlled by modifications of the stage-specific adhesin lipophosphoglycan (LPG). During differentiation to infective metacyclics, d-arabinopyranose (d-Arap) caps the LPG side-chain galactose residues, blocking interaction with the midgut lectin PpGalec, thereby leading to parasite detachment and transmission. Previously, we characterized two closely related L. major genes (FKP40 and AFKP80) encoding bifunctional proteins with kinase/pyrophosphorylase activities required for salvage and conversion of l-fucose and/or d-Arap into the nucleotide-sugar substrates required by glycosyltransferases. Whereas only AFKP80 yielded GDP-d-Arap from exogenous d-Arap, both proteins were able to salvage l-fucose to GDP-fucose. We now show that Δafkp80− null mutants ablated d-Arap modifications of LPG as predicted, whereas Δfkp40− null mutants resembled wild type (WT). Fucoconjugates had not been reported previously in L. major, but unexpectedly, we were unable to generate fkp40−/afkp80− double mutants, unless one of the A/FKPs was expressed ectopically. To test whether GDP-fucose itself was essential for Leishmania viability, we employed “genetic metabolite complementation.” First, the trypanosome de novo pathway enzymes GDP-mannose dehydratase (GMD) and GDP-fucose synthetase (GMER) were expressed ectopically; from these cells, the Δfkp40−/Δafkp80− double mutant was now readily obtained. As expected, the Δfkp40−/Δafkp80−/+TbGMD-GMER line lacked the capacity to generate GDP-Arap, while synthesizing abundant GDP-fucose. These results establish a requirement for GDP-fucose for L. major viability and predict the existence of an essential fucoconjugate(s).

Metabolic Complementation in Bacterial Communities: Necessary Conditions and Optimality.

Bacterial communities may display metabolic complementation, in which different members of the association partially contribute to the same biosynthetic pathway. In this way, the end product of the pathway is synthesized by the community as a whole. However, the emergence and the benefits of such complementation are poorly understood. Herein, we present a simple model to analyze the metabolic interactions among bacteria, including the host in the case of endosymbiotic bacteria. The model considers two cell populations, with both cell types encoding for the same linear biosynthetic pathway. We have found that, for metabolic complementation to emerge as an optimal strategy, both product inhibition and large permeabilities are needed. In the light of these results, we then consider the patterns found in the case of tryptophan biosynthesis in the endosymbiont consortium hosted by the aphid Cinara cedri. Using in-silico computed physicochemical properties of metabolites of this and other biosynthetic pathways, we verified that the splitting point of the pathway corresponds to the most permeable intermediate.

Reinventing the Wheel and Making It Round Again: Evolutionary Convergence in Buchnera-Serratia Symbiotic Consortia between the Distantly Related Lachninae Aphids Tuberolachnus salignus and Cinara cedri.

Virtually all aphids (Aphididae) harbor Buchnera aphidicola as an obligate endosymbiont to compensate nutritional deficiencies arising from their phloem diet. Many species within the Lachninae subfamily seem to be consistently associated also with Serratia symbiotica. We have previously shown that both Cinara (Cinara) cedri and Cinara (Cupressobium) tujafilina (Lachninae: Eulachnini tribe) have indeed established co-obligate associations with both Buchnera and S. symbiotica. However, while Buchnera genomes of both Cinara species are similar, genome degradation differs greatly between the two S. symbiotica strains. To gain insight into the essentiality and degree of integration of S. symbiotica within the Lachninae, we sequenced the genome of both Buchnera and S. symbiotica endosymbionts from the distantly related aphid Tuberolachnus salignus (Lachninae: Tuberolachnini tribe). We found a striking level of similarity between the endosymbiotic system of this aphid and that of C. cedri. In both aphid hosts, S. symbiotica possesses a highly reduced genome and is found exclusively intracellularly inside bacteriocytes. Interestingly, T. salignus’ endosymbionts present the same tryptophan biosynthetic metabolic complementation as C. cedri’s, which is not present in C. tujafilina’s. Moreover, we corroborate the riboflavin-biosynthetic-role take-over/rescue by S. symbiotica in T. salignus, and therefore, provide further evidence for the previously proposed establishment of a secondary co-obligate endosymbiont in the common ancestor of the Lachninae aphids. Finally, we propose that the putative convergent split of the tryptophan biosynthetic role between Buchnera and S. symbiotica could be behind the establishment of S. symbiotica as an obligate intracellular symbiont and the triggering of further genome degradation.

Discoveries, metabolic roles and diseases of mitochondrial carriers: A review

Mitochondrial carriers (MCs) are a superfamily of nuclear-encoded proteins that are mostly localized in the inner mitochondrial membrane and transport numerous metabolites, nucleotides, cofactors and inorganic anions. Their unique sequence features, i.e., a tripartite structure, six transmembrane α-helices and a three-fold repeated signature motif, allow MCs to be easily recognized. This review describes how the functions of MCs from Saccharomyces cerevisiae, Homo sapiens and Arabidopsis thaliana (listed in the first table) were discovered after the genome sequence of S. cerevisiae was determined in 1996. In the genomic era, more than 50 previously unknown MCs from these organisms have been identified and characterized biochemically using a method consisting of gene expression, purification of the recombinant proteins, their reconstitution into liposomes and transport assays (EPRA). Information derived from studies with intact mitochondria, genetic and metabolic evidence, sequence similarity, phylogenetic analysis and complementation of knockout phenotypes have guided the choice of substrates that were tested in the transport assays. In addition, the diseases associated to defects of human MCs have been briefly reviewed. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.

Systems-level response to point mutations in a core metabolic enzyme modulates genotype-phenotype relationship.

Linking the molecular effects of mutations to fitness is central to understanding evolutionary dynamics. Here, we establish a quantitative relation between the global effect of mutations on the E. coli proteome and bacterial fitness. We created E. coli strains with specific destabilizing mutations in the chromosomal folA gene encoding dihydrofolate reductase (DHFR) and quantified the ensuing changes in the abundances of 2,000+ E. coli proteins in mutant strains using tandem mass tags with subsequent LC-MS/MS. mRNA abundances in the same E. coli strains were also quantified. The proteomic effects of mutations in DHFR are quantitatively linked to phenotype: the SDs of the distributions of logarithms of relative (to WT) protein abundances anticorrelate with bacterial growth rates. Proteomes hierarchically cluster first by media conditions, and within each condition, by the severity of the perturbation to DHFR function. These results highlight the importance of a systems-level layer in the genotype-phenotype relationship.

Genome reduction and potential metabolic complementation of the dual endosymbionts in the whitefly Bemisia tabaci

BackgroundThe whitefly Bemisia tabaci is an important agricultural pest with global distribution. This phloem-sap feeder harbors a primary symbiont, “Candidatus Portiera aleyrodidarum”, which compensates for the deficient nutritional composition of its food sources, and a variety of secondary symbionts. Interestingly, all of these secondary symbionts are found in co-localization with the primary symbiont within the same bacteriocytes, which should favor the evolution of strong interactions between symbionts.ResultsIn this paper, we analyzed the genome sequences of the primary symbiont Portiera and of the secondary symbiont Hamiltonella in the B. tabaci Mediterranean (MED) species in order to gain insight into the metabolic role of each symbiont in the biology of their host. The genome sequences of the uncultured symbionts Portiera and Hamiltonella were obtained from one single bacteriocyte of MED B. tabaci. As already reported, the genome of Portiera is highly reduced (357 kb), but has kept a number of genes encoding most essential amino-acids and carotenoids. On the other hand, Portiera lacks almost all the genes involved in the synthesis of vitamins and cofactors. Moreover, some pathways are incomplete, notably those involved in the synthesis of some essential amino-acids. Interestingly, the genome of Hamiltonella revealed that this secondary symbiont can not only provide vitamins and cofactors, but also complete the missing steps of some of the pathways of Portiera. In addition, some critical amino-acid biosynthetic genes are missing in the two symbiotic genomes, but analysis of whitefly transcriptome suggests that the missing steps may be performed by the whitefly itself or its microbiota.ConclusionsThese data suggest that Portiera and Hamiltonella are not only complementary but could also be mutually dependent to provide a full complement of nutrients to their host. Altogether, these results illustrate how functional redundancies can lead to gene losses in the genomes of the different symbiotic partners, reinforcing their inter-dependency.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1379-6) contains supplementary material, which is available to authorized users.

Mitochondrial fusion provides an 'initial metabolic complementation' controlled by mtDNA.

Heteroplasmic cells, harboring both mutant and normal mitochondrial DNAs (mtDNAs), must accumulate mutations to a threshold level before respiratory activity is affected. This phenomenon has led to the hypothesis of mtDNA complementation by inter-mitochondrial content mixing. The precise mechanisms of heteroplasmic complementation are unknown, but it depends both on the mtDNA nucleoid dynamics among mitochondria as well as the mitochondrial dynamics as influenced by mtDNA. We tracked nucleoids among the mitochondria in real time to show that they are shared after complete fusion but not 'kiss-and-run'. Employing a cell hybrid model, we further show that mtDNA-less mitochondria, which have little ATP production and extensive Opa1 proteolytic cleavage, exhibit weak fusion activity among themselves, yet remain competent in fusing with healthy mitochondria in a mitofusin- and OPA1-dependent manner, resulting in restoration of metabolic function. Depletion of mtDNA by overexpression of the matrix-targeted nuclease UL12.5 resulted in heterogeneous mitochondrial membrane potential (ΔΨm) at the organelle level in mitofusin-null cells but not in wild type. In this system, overexpression of mitofusins or application of the fusion-promoting drug M1 could partially rescue the metabolic damage caused by UL12.5. Interestingly, mtDNA transcription/translation is not required for normal mitochondria to restore metabolic function to mtDNA-less mitochondria by fusion. Thus, interplay between mtDNA and fusion capacity governs a novel 'initial metabolic complementation'.

Evolution of small prokaryotic genomes.

As revealed by genome sequencing, the biology of prokaryotes with reduced genomes is strikingly diverse. These include free-living prokaryotes with ∼800 genes as well as endosymbiotic bacteria with as few as ∼140 genes. Comparative genomics is revealing the evolutionary mechanisms that led to these small genomes. In the case of free-living prokaryotes, natural selection directly favored genome reduction, while in the case of endosymbiotic prokaryotes neutral processes played a more prominent role. However, new experimental data suggest that selective processes may be at operation as well for endosymbiotic prokaryotes at least during the first stages of genome reduction. Endosymbiotic prokaryotes have evolved diverse strategies for living with reduced gene sets inside a host-defined medium. These include utilization of host-encoded functions (some of them coded by genes acquired by gene transfer from the endosymbiont and/or other bacteria); metabolic complementation between co-symbionts; and forming consortiums with other bacteria within the host. Recent genome sequencing projects of intracellular mutualistic bacteria showed that previously believed universal evolutionary trends like reduced G+C content and conservation of genome synteny are not always present in highly reduced genomes. Finally, the simplified molecular machinery of some of these organisms with small genomes may be used to aid in the design of artificial minimal cells. Here we review recent genomic discoveries of the biology of prokaryotes endowed with small gene sets and discuss the evolutionary mechanisms that have been proposed to explain their peculiar nature.

Widespread expression of conserved small RNAs in small symbiont genomes.

Genome architecture of a microbe markedly changes when it transitions from a free-living lifestyle to an obligate symbiotic association within eukaryotic cells. These symbiont genomes experience numerous rearrangements and massive gene loss, which is expected to radically alter gene regulatory networks compared with those of free-living relatives. As such, it remains unclear whether and how these small symbiont genomes regulate gene expression. Here, using a label-free mass-spec quantification approach we found that differential protein regulation occurs in Buchnera, a model symbiont with a reduced genome, when it transitions between two distinct life stages. However, differential mRNA expression could not be detected between Buchnera life stages, despite the presence of a small number of putative transcriptional regulators. Instead a comparative analysis of small RNA expression profiles among five divergent Buchnera lineages, spanning a variety of Buchnera life stages, reveals 140 novel intergenic and antisense small RNAs and 517 untranslated regions that were significantly expressed, some of which have been conserved for ∼65 million years. In addition, the majority of these small RNAs exhibit both sequence covariation and thermodynamic stability, indicators of a potential structural RNA role. Together, these data suggest that gene regulation at the post-transcriptional level may be important in Buchnera. This is the first study to empirically identify Buchnera small RNAs, and we propose that these novel small RNAs may facilitate post-transcriptional regulation through translational inhibition/activation, and/or transcript stability. Ultimately, post-transcriptional regulation may shape metabolic complementation between Buchnera and its aphid host, thus impacting the animal's ecology and evolution.

Mealybugs nested endosymbiosis: going into the ‘matryoshka’ system in Planococcus citri in depth

BackgroundIn all branches of life there are plenty of symbiotic associations. Insects are particularly well suited to establishing intracellular symbiosis with bacteria, providing them with metabolic capabilities they lack. Essential primary endosymbionts can coexist with facultative secondary symbionts which can, eventually, establish metabolic complementation with the primary endosymbiont, becoming a co-primary. Usually, both endosymbionts maintain their cellular identity. An exception is the endosymbiosis found in mealybugs of the subfamily Pseudoccinae, such as Planococcus citri, with Moranella endobia located inside Tremblaya princeps.ResultsWe report the genome sequencing of M. endobia str. PCVAL and the comparative genomic analyses of the genomes of strains PCVAL and PCIT of both consortium partners. A comprehensive analysis of their functional capabilities and interactions reveals their functional coupling, with many cases of metabolic and informational complementation. Using comparative genomics, we confirm that both genomes have undergone a reductive evolution, although with some unusual genomic features as a consequence of coevolving in an exceptional compartmentalized organization.ConclusionsM. endobia seems to be responsible for the biosynthesis of most cellular components and energy provision, and controls most informational processes for the consortium, while T. princeps appears to be a mere factory for amino acid synthesis, and translating proteins, using the precursors provided by M. endobia. In this scenario, we propose that both entities should be considered part of a composite organism whose compartmentalized scheme (somehow) resembles a eukaryotic cell.

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.