BACKGROUND
paulinella chromatophora is an amoeba dispensed with phototrophic nutrition that contains blue-green photosynthetic organelles of cyanobacterial origin termed chromatophores  <cit> .
these novel organelles have a monophyletic origin in different strains of photosynthetic paulinella that have been described  <cit>  and were acquired through a primary endosymbiotic event about ~ <dig> to  <dig> mya .
chromatophore genome sequencing from two strains of p. chromatophora , revealed a size of  <dig>  and  <dig>  mbp, respectively.
this represents about 1/ <dig> of the genome size of synechococcus sp.
wh  <dig>  the closest free-living relative cyanobacterium with a sequenced genome.
synechococcus sp.
wh  <dig> has a genome of ~ <dig> mbp and  <dig> protein-coding genes  <cit> .
it indicates that the chromatophore evolved by genome reduction.
however, genome reduction in p. chromatophora is not as extreme as in plastids which rarely exceed  <dig> kbp  <cit> .
chromatophores are genetically integrated with their host.
more than  <dig> nuclear encoded genes of chromatophore origin have been identified  <cit> .
and some of the protein products coded by these genes are imported back into the chromatophore and participate in the photosynthetic apparatus  <cit> .
accordingly, chromatophores have been described as plastids in the making.
p. chromatophora nutrition relies on the reduced-carbon photosynthetically assimilated by the chromatophore  <cit> .
this endosymbiotic-nutrient dependency has been observed in other organisms such as aphids and tsetse flies housing prokaryotic endosymbionts  <cit> .
particularly for aphids, host essential amino acids are provided by an endosymbiotic bacterium called buchnera aphidicola  <cit> .
sequencing of the genome of b. aphidicola revealed a high degree of genetic degradation, while genes necessary for the syntrophic relationship with its host have been retained  <cit> .
prokaryotic endosymbionts evolve small genomes mainly by the combined action of genetic drift and negative selection .
in host-restricted conditions, the endosymbiont experiences a lack of recombination and horizontal gene transfer, as well as recurrent population bottlenecks lowering its effective population size  and a concomitant relaxation of natural selection .
the combined action of these factors allows the accumulation of slightly deleterious mutations through a process called muller’s ratchet  <cit> .
as a consequence, many genes become pseudogenes and are subsequently lost.
in addition, selection at holobiont level by mechanisms like “partner fidelity feedback” have been proposed to promote the evolution of mutualistic interactions  <cit> .
something that should be considered is that, differing from free-living relatives, the metabolism of mutualistic endosymbionts is specialized in the production of metabolites that are released to their host as nutrients  <cit> .
this metabolic specialization is the consequence of metabolic restructuring caused by gene loss and genome reduction  <cit> .
resulting reduced genomes code for fewer genes, however, they are more integrated to the host.
the extreme cases are organelles of endosymbiotic origin such as chloroplasts  <cit> .
therefore, if mutualistic endosymbionts show metabolic adaptations to provide nutrients to their hosts  <cit> , natural selection must have participated in the evolution of these systems.
during early stages of organellogenesis, the cyanobacteria that evolved into the chromatophore, had access to metabolites provided by the host.
it is likely that the availability these metabolites render of some metabolic routes dispensable in the endosymbiont.
the loss of these biosynthetic pathways in the endosymbiont led to restructurings and changes in the remaining metabolic fluxes.
taking into consideration all these modifications experienced by the chromatophore and the nutrient dependency of the holobiont for the photosynthetic function of the chromatophore, we made the analogy of natural selection acting as a “metabolic engineer” directing the strategies for the metabolic specialization of the chromatophore.
in general, the objective of metabolic engineering is the directed improvement of metabolic capabilities through the deletion of metabolic genes or the introduction of new ones  <cit> .
by using these strategies, microorganisms have been engineered for the improvement of the yield and the production and consumption rates of desired metabolites.
for instance, for the of 1-butanol production in cyanobacteria  <cit> , many more examples can be found elsewhere  <cit> .
in this work, we reconstructed the genome based metabolic models of the chromatophore of paulinella chromatophora and the cyanobacteria synechococcus sp.
wh  <dig>  we inquired into the metabolic capabilities of the chromatophore; the possible metabolic interaction of the chromatophore with its host; and in silico simulate the process of metabolic evolution experienced by the chromatophore in host-restricted conditions.
RESULTS
differential gene retention of functional categories in the chromatophore genome
our first objective was to determine to what extent genetic loss affected functional metabolic categories in the chromatophore  when compared to the genome of synechococcus sp.
wh  <dig>  we compared against synechococcus sp.
wh  <dig> because is the closest free-living cyanobacterium with a sequenced genome and it is likely to be similar in gene content to the ancestor of the chromatophore.
to assess the statistical significance we used a hypergeometric distribution.
as is shown in fig.
<dig>  genes belonging to  <dig> functional categories have been less affected by genome erosion.
in particular, photosynthesis and fatty acid biosynthesis categories are less affected.
retention of these  <dig> functional categories in the chromatophore can be attributed to a host-level selection protecting from gene loss.
conserved genes very likely play an adaptive role in the holobiont.fig.
<dig> conservation of functional gene categories in the chromatophore when compared to synechococcus sp.
wh  <dig>  for each functional category we show in green and red the percentage of gene conservation and lost in the chromatophore, respectively.
for instance, if a gene category is completely green, it indicates that all orthologs in synechococcus sp.
wh  <dig> are conserved in the chromatophore.
as shown, gene loss affects differentially each one of the functional categories in the chromatophore.
functional categories particularly well conserved are indicated with asterisks .
statistical significance calculated by using a hypergeometric distribution  <cit> .
genes were classified following kegg database
in silico metabolic reconstruction of the chromatophore of p. chromatophora and synechococcus sp.
wh 5701
to better understand the role in the symbiosis played by remaining genes in the chromatophore, we reconstructed two metabolic models.
one for the chromatophore of p. chromatophora ccac  <dig>  <cit>  and the other for synechococcus sp.
wh  <dig>  the closest free-living cyanobacterium with a sequenced genome.
the rationale behind this is to use synechococcus sp.
wh  <dig> as a proxy of the ancestral cyanobacterium that evolved into the chromatophore.
metabolic model reconstruction of the free-living cyanobacterium synechococcus sp.
wh  <dig> was done by identifying orthologs to those protein-coding genes reported in the metabolic model of synechocystis sp.
pcc  <dig>   <cit> .
the resulting metabolic model of the free-living organism  comprised  <dig> metabolic reactions with  <dig> metabolites and  <dig> protein-coding genes.
metabolic model reconstruction of the chromatophore was done by identifying those genes in the genome of the chromatophore of p. chromatophora ccac  <dig> that are orthologous to the free-living metabolic model .
the metabolic model of the chromatophore  comprised  <dig> reactions,  <dig> metabolites and  <dig> protein-coding genes.
because synechococcus sp.
wh  <dig> is a close free-living relative of the chromatophore, it could be considered that  <dig> reactions were lost along genome reduction in the chromatophore .table  <dig> characteristics of metabolic models of synechococcus sp.
wh  <dig>  and the chromatophore
by using the biomass equation of the cyanobacterium synechocystis sp.
pcc  <dig>  <cit> , we tested the functionality of the icv <dig> and the icv <dig> metabolic models with flux balance analysis .
biomass production was set as objective function.
in silico growth was simulated under autotrophic conditions.
co <dig> and photons uptake were set to  <dig>  mmol × gdw−1 × h− <dig> and 100 mmol × gdw−1 × h− <dig> respectively and set as constraining metabolites as in  <cit> .
in model icv <dig>  almost every metabolic pathway for biomass production is complete.
the exceptions were  <dig> reactions for which no orthologous exist in synechococcus sp.
wh  <dig> when compared to ijn <dig> .
these reactions had to be added to the icv <dig> model in order to produce all the components necessary for the biomass equation.
in this way, icv <dig> showed an in silico growth rate of  <dig>  h− <dig> which is identical to the in silico growth reported for synechocystis sp.
pcc  <dig> metabolic model under autotrophic conditions  <cit> .
under these conditions, the metabolic model of the chromatophore  did not show in silico growth.
this was obviously due to the reduced metabolic capabilities caused by the genomic reduction process experimented by the photosynthetic endosymbiont.
genome reduction has affected the metabolic capabilities of the chromatophore in two ways: a) some biosynthetic pathways were completely lost; while b) some other were partially lost.
for example, in synechocystis sp.
pcc  <dig> riboflavin is synthesized by four genes that perform six reactions by using guanosine 5′-triphosphate  and d-ribulose 5-phosphate  as precursors metabolites  <cit> .
all these genes for riboflavin biosynthesis were lost in the chromatophore.
in this case, we assumed that the host provides riboflavin to the chromatophore.
the possible explanation for this loss is that riboflavin is the main precursor for flavin mononucleotide  and flavin adenine dinucleotide, two main compounds that work as coenzymes for many of the enzymes such as oxidoreductases including nadh dehydrogenase as well as in biological blue-light photo receptors.
this observation is concomitant with the loss in some functional gene categories; as in oxidative phosphorylation .
as the hypothesis is that the metabolic network must preserve its functionality, whenever we found a similar situation, exchange reactions were added to the metabolic model to simulate the incorporation of riboflavin and other metabolites as additional nutrients from the host.
these metabolites included amino acids, cofactors, vitamins and other molecules which are essential for the biomass equation but cannot be produced by the chromatophore .fig.
<dig> nutrients uptake simulation in the chromatophore model .
metabolites that cannot be produced by the chromatophore  include: amino acids , cofactors , vitamins  and others -5-amino-4-imidazolecarboxamide, succoa = succinyl-coa, lipidads = lipid a disaccharide, dahp =2-dehydro-3-deoxy-d-arabino-heptonate 7-phosphate)
some other biosynthetic pathways are truncated in the chromatophore because single gene coding enzymes were lost.
for example, in the biosynthetic pathway of leucine, most gene coding enzymes are present in the chromatophore except for the gene coding for 3-isopropylmalate dehydrogenase.
in this case, we assumed that host encoded enzymes complement the pathway in the endosymbiont.
either by importing host encoded enzymes to the chromatophore or by exchanging intermediated metabolites between the symbionts.
similar situations have been proposed for other host-endosymbiont systems  <cit> .
for this reason, we assumed that the production of these metabolites is shared between the host and the endosymbiont .
in addition, some reactions in the chromatophore model icv <dig> for which no orthologous genes exist with the free-living model icv <dig> but are essential for in silico growth were assumed to be present .
finally, chromatophores lost the ability to store photosynthates as well as the capacity to synthesize sucrose  <cit> .
because of that, glycogen was removed from the biomass equation in icv <dig>  under these conditions, in silico growth of the icv <dig> model was  <dig>  h− <dig>  this is an unrealistic rate because growth of the chromatophores is restricted to host division which is much lower than growth rate reported for free-living cyanobacteria and even other photosynthetic eukaryotes  <cit> .
robustness analysis of metabolic models
we assessed the robustness of the icv <dig> and the icv <dig> models to single gene deletions.
genetic robustness was defined as the capacity of the models to maintain its metabolic capabilities  after a genetic deletion.
under phototrophic conditions, model icv <dig> showed  <dig> genes  to be essential because its deletion decreases the biomass production over 99% .
this result shows that icv <dig> is less robust than the metabolic model of synechocystis sp.
pcc  <dig> where  <dig> % of the genes are essential under these same conditions  <cit> .
in addition, there is a decreasing robustness in the model of the chromatophore where  <dig> of the  <dig> genes  are essential .
this indicates that the genomic reduction experimented by the chromatophore rendered its metabolic network fragile.
the same result has been observed for other metabolic networks of endosymbionts  <cit> .fig.
<dig> genetic robustness analysis of metabolic models of synechocystis sp.
pcc  <dig>   <cit> , synechococcus sp.
wh  <dig>  and the chromatophore of paulinella chromatophora .
percentage of essential genes  for each metabolic model
interestingly, we found that there are  <dig> non-essential genes in the metabolic model icv <dig> whose single deletion decreases in silico growth rate.
these include genes encoding the enzymes acetyl-coa synthetase, malic enzyme  and fumarase.
of these three, the last enzyme is the only one decreasing the in silico growth rate in ijn <dig> when it is deleted .
in the icv <dig> model, all these  <dig> genes were lost.
in addition, the non-essential gene in icv <dig> coding for an enzyme with arginase activity is the only one whose deletion decreases the growth rate in the icv <dig> chromatophore model.
this suggests that genome reduction leading to icv <dig> caused metabolic restructuring because deletion of this enzyme with arginase activity in icv <dig> has no effect.
in silico simulation of metabolic-gene loses in the chromatophore of p. chromatophora
based on the metabolic network of the free living synechococcus sp.
wh  <dig>  we simulated in silico the gene loss.
we evaluated the impact of intracellular conditions  on the evolution of the chromatophore.
in particular, we asked whether the set of metabolites predicted to be provided by the host in the icv <dig> model  determined actual gen content of the chromatophore after genome reduction.
two in silico intracellular conditions were evaluated.
in the first one, we simulated genetic reduction under in silico intracellular conditions where available nutrients were those predicted in the icv <dig> model  .
in the second one, we randomly selected metabolites from the icv <dig> model  and assigned as available nutrients under intracellular conditions .
the algorithm to simulate genome reduction is explained in detail in the methods section.
this algorithm allowed us to obtain in silico evolved chromatophores whose metabolic capabilities regarding the biomass production are equivalent to those of icv265; but differing in their in silico evolutionary history and gene content.
simulations under the proposed nutrients conditions resulted in reduced metabolic networks with  <dig>   genes on average.
in these reduced networks, of the  <dig> genes present in the free-living ancestor ,  <dig> % are strictly conserved in the  <dig> simulations and  <dig> % are dispensable in all of them.
in randomized nutrients simulations, reduced networks have an average size of  <dig>   genes and 54% and  <dig> % are conserved and dispensable in the  <dig> simulations, respectively.
as is shown in fig.
<dig>  the proportion of: i) essential genes ; ii) variable genes ; and iii) dispensable genes , varies between metabolic pathways.
these proportions also vary between treatments .
surprisingly, the most extreme case is that of the genes participating in photosynthetic activity.
in proposed nutrients  <dig> %,  <dig> % and  <dig> % are predicted to be essential, variable and dispensable, respectively.
while for randomized nutrients none was predicted to be essential nor dispensable because 100% of them were variable.fig.
<dig> variation in the proportion of genes classified as essential, variable and dispensable in different metabolic pathways according to two different sets of available nutrients for the chromatophore.
a set of proposed nutrients; b set of randomized nutrients.
aa to bn metabolic pathways: aa* = citrate cycle ; ab = lipopolysaccharide biosynthesis; ac = carotenoid biosynthesis; ad = folate biosynthesis; ae = glycerolipid metabolism; af = hydrogen production; ag = steroid biosynthesis; ah = aminosugars metabolism; ai* = nicotinate and nicotinamide metabolism; aj = nucleotide sugars metabolism; ak = riboflavin metabolism; al = thiamine metabolism; am = carbon fixation; an = glutamate metabolism; ao = lysine metabolism; ap = nitrogen metabolism; aq = terpenoid backbone biosynthesis; ar = fructose and mannose metabolism; as* = pantothenate and coa biosynthesis; at = peptidoglycan biosynthesis; au = ubiquinone and other pterpenoids biosynthesis; av = urea cycle and metabolism of amino groups; aw* = alanine, aspartate and glutamate metabolism; ax = valine leucine and isoleucine biosynthesis; ay = histidine metabolism; az* = pentose phosphate pathway; ba = starch and sucrose metabolism; bb = fatty acid biosynthesis; bc = glyoxylate and dicarboxylate metabolism; bd = sulfur cysteine and methionine metabolism; be = arginine and proline metabolism; bf = pyrimidine metabolism; bg = glycolysis/gluconeogenesis; bh = pyruvate metabolism; bi* = phenylalanine tyrosine and tryptophan biosynthesis; bj* = purine metabolism; bk* = porphyrin and chlorophyll metabolism; bl = oxidative phosphorylation; bm* = photosynthesis and bn = transport.
* metabolic pathways with a difference in categorical genes with p-value <  <dig>
genetic concordance was evaluated between these simulated minimal networks and the real chromatophore model .
this was done by measuring sensitivity and specificity as in  <cit> .
in fig.
<dig>  we show the fraction of true-positives and false-positives for every cutoff .
true-positive and false-positive for every cutoff  form a curve whose area under the curve represents the probability that a gene conserved in icv <dig> is present in more simulations than a gene which has been lost.fig.
<dig> genetic concordance between in silico evolved chromatophores and the chromatophore model .
the area under the curve indicates the predictive accuracy for evolved chromatophores  obtained under proposed and randomized nutrients conditions.
proposed nutrients: simulation with metabolites proposed as nutrients in the icv <dig> model  and randomized nutrients: simulations with randomized metabolites assigned as nutrients .
area under the curve: proposed nutrients = <dig>  ; randomized nutrients = <dig>
the area under the curve shows the contribution of the nutrients available in intracellular conditions explaining the evolutionary history experimented by the chromatophore.
accordingly, the accuracy obtained under the proposed nutrients condition was  <dig> %, while that of the randomized nutrients was  <dig> %.
the difference between the areas under the curve from both conditions is statistically significant .
modeling selection and drift to explain metabolic evolution of the chromatophore
we are interested in understanding the role played by natural selection during the evolution of the metabolic capabilities of the chromatophore.
chromatophores provide the host with reduced-carbon, probably a hexose.
this in analogy to the origin of plastids.
it has been proposed that during the early stages of plastid evolution, the photosynthetic endosymbiont exported reduced-carbon in the form of an hexose-phosphate through an hexose phosphate transporter of bacterial origin   <cit> .
to study how the potential rate of carbon exportation evolved, a hexose export reaction was added to the metabolic models.
this reaction was defined as objective function.
to ensure biomass components production, the biomass reaction was fixed to  <dig>  h− <dig> which, as stated previously, is the growth rate of synechocystis sp.
pcc  <dig> metabolic model under autotrophic growth conditions  <cit> .
under these conditions, there is no exportation of reduced-carbon in the icv <dig> model.
however, in the icv <dig> chromatophore model, the potential rate of hexose exported without affecting the in silico growth rate was  <dig>  mmol × gdw−1 × h− <dig>  in fig.
<dig>  we show the fluxes calculated with fba of the central metabolism of the models of the chromatophore and the free-living cyanobacteria in conditions previously mentioned.fig.
<dig> flux distribution obtained with fba of the central metabolism of icv <dig>  and icv <dig>
fluxes calculated for production of metabolites precursors used to produce biomass components are produced in less quantity in the chromatophore’s model .
this is a consequence of the loss of metabolic capabilities in the metabolism of the chromatophore which allow the redirection of carbon through the gluconeogenic pathway for the production of hexoses as metabolic objective, instead of being used in the production of biomass components.
to analyze the efficiency of the metabolic networks in terms of hexose production at overall metabolism, we calculated the yields.
the yields are parameters that measure the efficiency of the metabolic network and allow the comparison across different microorganisms.
for instance, the yield of the ethanol production is higher in saccharomyces cerevisiae compared to zymomonas mobilis, this was the result of the specialization of the microorganism to produce specific metabolites  <cit> .
therefore, we calculated the yields of carbon, energy and reducing equivalents  required to produce hexose .
these results show that the model of the chromatophore is more efficient for producing hexose from the external carbon than the free-living cyanobacteria.
it means that metabolic restructurings experienced by the chromatophore rendered its metabolism more efficient to produce hexose which can be provided to the host.table  <dig> yields  analysis of extracellular co <dig>  atp and nadph consumed in hexose production for both models
co2
the yields suggest that the loss of some metabolic capabilities in the ancestral cyanobacterium caused a redirection of fixed co <dig> causing changes in metabolic fluxes and consequently increasing the rate of reduced-carbon exported to the extracellular compartment.
we then inquired about the evolutionary forces that determined genetic conservation and metabolic functionality in chromatophores.
specifically, we wanted to infer if these metabolic capabilities of the icv <dig> model  could have been possible under a random model of evolution or were the consequence of natural selection for metabolic specialization of the chromatophore and its positive impact at the holobiont.
to tests this, we simulated the metabolic reduction process with hexose export and biomass production as evolutionary restrictions in a random model where hexose exported must be greater than zero.
it means that every gene affecting the in silico growth rate of  <dig>  h− <dig> and impairing hexose export was considered as essential, while the hexose export rate could always vary while being greater than zero .
minimal networks obtained in silico under these conditions were variable in size and gene content.
of  <dig> simulations, only  <dig>  of the genes conserved in model icv <dig> are conserved in all the simulations.
conversely, there are  <dig> genes predicted to be essential in all these  <dig> simulations which are not conserved in model icv <dig>  the metabolic networks from these  <dig> simulations are different in gene content and show different hexose export rates however they are equivalent in biomass production .
as shown in fig.
<dig>  after in silico metabolic reduction, hexose export rate in minimal networks obtained under these conditions tend to be minimal and close to zero .
on the other hand, only  <dig> % of simulations have a potential rate of hexose exported equal or higher than the metabolic model of the chromatophore .
this suggests that the probability of obtaining a potential rate of hexose exported similar to that of icv <dig> under a random model is less than 5%.
we got a similar result by varying the growth rate constraint of  <dig>  h− <dig> under plausible biological values .fig.
<dig> hexose exportation rate of the chromatophore model  is achieved in only  <dig> % of the simulations under a random model of evolution.
hexose exported rate  for  <dig> independent simulations .
red-dotted line indicates the hexose export rate in the chromatophore model
although variable, our simulations evolved metabolic networks that have approximately the same number of reactions than icv <dig>  the average number of reactions with non-zero fluxes in the reduced metabolic models of the  <dig> simulations is  <dig>  ±  <dig> .
this is slightly less than the number of reactions with non-zero fluxes in the icv <dig> model .
this shows that the small percentage of simulations  showing a potential rate of hexose exported equal or greater than that of the chromatophore  is not due simply to smaller size of the simulated metabolic networks.
these in silico experiments suggest that the potential rate of hexose exported in model icv <dig> is unlikely to be the outcome of only genetic drift and purifying selection .
this suggests that the potential rate of hexose exported was the result of a process of functional specialization in which the increasing rate of hexose exportation was favored by natural selection due to the positive impact at the holobiont level.
interestingly all these  <dig> % of in silico evolved chromatophores have in common the conservation of a phosphate transporter via abc system which is also present in the chromatophore model .
conservation of this phosphate transporter allows the simulated network to get the phosphate necessary to be able to export fixed carbon.
without this transporter most of fixed carbon is oxidized in the pentose phosphate pathway releasing only a small amount to the extracellular compartment .
metabolic integration of the chromatophore to its host
in our previous simulations, we assumed that nutrients  were available simultaneously for the chromatophore since the beginning of the evolutionary process at the onset of the endosymbiosis.
however, it is likely that this has not been the case and transporters for these nutrients were gained  sequentially.
for instance, metabolic transport activity in the chromatophore is reduced due to loss of most transporters in comparison to free-living cyanobacteria  <cit> .
and it has been reported that a large percentage of solute transporters in plastids from plantae have host and bacterial  origin  <cit> .
therefore, we simulated the evolutionary acquisition of transporters and its consequences in gene loss and the capability of the chromatophore to export fixed carbon to its host.
for every simulation, we used icv <dig> as a free-living ancestor of the chromatophore under nutrient-rich conditions .
however, in this experiment, the model icv <dig> did not have access to all nutrients since the beginning of the simulation.
instead, we randomly assigned a transport allowing the uptake of the respective nutrient.
we then randomly deleted one gene at a time from icv <dig>  if the deleted gene affected the growth rate  or impaired hexose exportation, we considered this gene as essential and we restored it to the model.
in this way we analyzed the selective impact caused by gene loss due to the addition of a single transporter and the concomitant relaxation of natural selection for retention of specific biosynthetic pathways.
once we analyzed every gene in the model, we randomly assigned a second transport and then we repeated the gene loss simulation mentioned above.
simulation stops when in silico chromatophore has access to the  <dig> nutrients  and all genes have been evaluated for their essentiality.
as shown in fig.
<dig>  after the incorporation of the  <dig> transporters, the probability of getting a potential rate of hexose exported equal or higher than the metabolic model of the chromatophore  is less than 5% in  <dig> simulations, in agreement with our previous result .fig.
<dig> metabolic integration with the host determines the rate of hexose exported by in silico chromatophores as well as the frequency of simulations that provide higher rates of reduced-carbon to the host.
x axis, hexose export rate; y axis, frequency of simulations; z axis, transporters added to the model
during the process of metabolic integration, it is noted that the maximum rate of hexose exportation becomes greater with every metabolite obtained as nutrient.
however, by inspecting the frequency distribution of simulations with different potential rates of hexose exported, it is obvious that as metabolic integration advances, the probability of getting the maximum rate of hexose exportation decreases .
these changes in frequency distributions during the process of metabolic integration can be interpreted in terms of the functional specialization of the chromatophore.
as metabolic integration proceeds , the chromatophore increased its capacity to provide fixed carbon to its host.
however, continued gene loss led to a simplified metabolic network and a smaller fraction of in silico evolved chromatophores can export as much fixed carbon as icv <dig>  the evolutionary landscape becomes smaller as evolution proceeds.
the metabolism of the chromatophore is specially adapted to produce carbon for its host
as shown above, the potential rate of hexose exported in the chromatophore model  is highly dependent on phosphate consumption.
in addition, the growth rate of the chromatophore is coupled to the host’s growth rate.
as shown above, the potential rate of hexose exported is unlikely to be the outcome of a random evolution.
to test the impact of these two restrictions, we analyzed the metabolic properties of models icv <dig> and icv <dig> in potential rate of hexose exported under growth rate and phosphate uptake restrictions.
as shown in fig.
<dig>  the potential rate of hexose exported in the icv <dig> model is robust with respect to growth rate and phosphate uptake .
this contrasts with the chromatophore model  where, for a given growth rate, only a specific consumption of phosphate is necessary to sustain hexose release.
in addition, the capacity of hexose export in the icv <dig> model for a determined growth rate restriction is greater than the icv <dig> model.fig.
<dig> the metabolic model of the chromatophore  is specially adapted to produce higher rates of reduced-carbon under phosphate and growth restrictions in comparison with synechococcus sp.
wh  <dig> .
a hexose export rate in the free-living model icv <dig>  b hexose export rate in the chromatophore model icv <dig>  y axis, growth rate restriction.
x axis, phosphate uptake restriction.
z axis, potential rate of hexose exported.
colors in axis z are only for drawing purposes and do not represent an extra variable
discussion
in this work, we show that the metabolic network of the chromatophore of p. chromatophora is different to the metabolic network of its free-living relative synechococcus sp.
wh  <dig>  we suggest that these differences evolved by natural selection.
gene-loss and carbon flux redirection guided by natural selection led to metabolic specialization of the chromatophore as a reduced-carbon provider.
purifying selection and the maintenance of the symbiosis
our analysis showed that some metabolic pathways have been preferentially conserved in the chromatophore .
these preserved metabolic pathways  very likely play a prominent role in the symbiosis.
this pattern is analogous to the one observed in many other endosymbionts e.g.
buchnera aphidicola  <cit> .
in this later case, biosynthetic pathways producing essential amino acids for the host  <cit>  are preserved by host-level natural selection.
as mentioned above, differential conservation of gene category functions suggests that purifying selection is preserving relevant symbiotic functions.
accordingly, estimation of the rate of nucleotide substitution in  <dig> dna alignments of protein-coding genes orthologous between chromatophores of two different strains of p. chromatophora  showed that most of them have signals of purifying selection  <cit> .
it has been suggested that host-level selection prevents the fixation of deleterious mutations in endosymbionts thus lowering the chances of a mutational meltdown resulting in extinction  <cit> .
and, of course, this prevents the consequent replacement of non-functional endosymbionts  <cit> .
in addition, selective pressure to maintain functional proteins increases with the time of host-endosymbiont interaction  <cit>  and combined with very strong bottlenecks may help to reduce the accumulation of deleterious mutations.
this has been proposed to explain mitochondrial genome evolution  <cit> .
metabolic integration of the chromatophore to its host
comparison of the metabolic models of the chromatophore and the cyanobacterium synechococcus sp.
wh  <dig> allowed us to inquire into the evolution of the metabolic interaction of the chromatophores with its host.
for example, several metabolic pathways in the chromatophore are incomplete.
it is likely that the host supplies these metabolites as nutrients to the chromatophore.
metabolic pathway sharing is a hallmark of endosymbiotic organisms.
for example wolbachia, which are endosymbionts of many animal species, show a degraded genome  <cit>  whose limited metabolic capabilities are complemented by its host  <cit> .
in turn, the endosymbiont provides the host with nutrients such as riboflavin, positively impacting host fitness  <cit> .
equally remarkable is the likely coupled production of some metabolites between the chromatophore and its host.
as mentioned above, this collaboration in metabolite biosynthesis has been observed in other symbiotic systems .
fragility of a reduced metabolic network
to study the metabolic capabilities of the chromatophores we used fba.
this stoichiometric approach can predict cellular phenotypes in specific environmental conditions.
generally, biomass production is fixed as objective function.
in absence of biomass composition, the use of a biomass equation from a related organism is a valid starting point for metabolic analysis .
in this way, fba has been used to infer the metabolic capabilities of different organisms whose cultivation and experimental management is challenging or not yet possible, as in the case of endosymbionts.
for example, biomass composition and the metabolic model of escherichia coli were used for metabolic analysis of buchnera aphidicola  <cit> , sodalis glossinidius  <cit> , and blattabacterium cuenoti  <cit> .
in the same way, we used the biomass composition and stoichiometric model of synechocystis sp.
pcc  <dig> as a starting point to model the metabolism of the chromatophore and synechococcus sp.
wh  <dig>  <cit> .
we found that the metabolism of the chromatophore is highly fragile to gene deletions.
approximately 84% of the genes in the model are essential when singly deleted in comparison with ~67% of the genes in synechococcus sp.
wh  <dig>  a similar difference in metabolic fragility was found by  <cit>  when comparing the models of b. aphidicola and its free-living relative escherichia coli where 84% and 19% of genes were essential, respectively.
in the same way, the metabolic network of two strains of blattabacterium cuenoti , the obligated primary endosymbiont of cockroaches, were shown to be highly fragile to single gene deletion.
it was found that  <dig> % and  <dig> % were essential genes, respectively  <cit> .
finally, in sodalis glossinidius ,  <dig> % metabolic genes were found to be essential, compared with its ancestral network where only  <dig> % are predicted to be essential  <cit> .
our robustness analyses of the icv <dig> and the icv <dig> models agree with the generalization that metabolic networks of endosymbionts are more fragile than their free-living counterparts.
this metabolic fragility of endosymbionts contrasts with theoretical estimations that suggest that, in general, metabolic systems are robust and complex  <cit> .
however, the metabolic systems of endosymbionts are considered more robust  <cit>  than minimalist metabolic networks  <cit> .
the difference in metabolic fragility of the chromatophore when compared to synechococcus sp.
wh  <dig> reflects the transition from a free-living style to a more stable condition inside paulinella chromatophora.
metabolic environment as a determinant of gene content
it has been shown that retention of metabolic genes in endosymbionts is determined by the metabolic requirements and molecular environment of the host  <cit> .
with the use of fba and the metabolic model of synechococcus sp.
wh  <dig> as a proxy of the ancestor of the chromatophore, we evaluated the impact of the host-metabolic environment in the reduction of the metabolic system of the endosymbiont.
the proposed host-metabolic environment  predicted with  <dig> % of accuracy the actual gene content of the chromatophore.
this is in contrast with the  <dig> % of accuracy obtained when using a randomly set of host-provided metabolites .
this emphasizes the contribution of the intracellular metabolic environment to the evolution of the metabolism in the chromatophore.
similar reductive simulations have been used to predict the set of essential genes of pathogens located in certain environmental niches  within the human body  <cit> .
in the same way, reductive evolution simulations using e. coli as free-living ancestor predicts with 80% of accuracy the metabolic gene content of b. aphidicola and wigglesworthia glossindia  <cit> .
inspection of the proportion of dispensable, variable, and essential genes by in silico reductive simulations  predicts differential gene retention patterns between different metabolic pathways.
for example, in randomized nutrients simulations, photosynthesis pathway  100% of genes are predicted as “variable”  while in proposed nutrients ~78% are essential.
this means that under randomized nutrients, photosynthesis function could be useful but not essential and could have been lost in the chromatophore by chance.
clearly, the set of metabolites comprising randomized nutrients cannot account for the metabolic gene content of extant chromatophores.
maximization of biomass production is regularly used as objective function in fba analysis.
it allows predicting the distribution of fluxes through a metabolic network  <cit> .
the maximization of biomass function is used as a proxy of evolutionary fitness.
however, many other objective functions can be used  <cit> .
for instance, it was estimated that chlorella , releases 57% of its photosynthates to its host  <cit> .
this means that most carbon photosynthetically assimilated is destined to symbiotic interaction instead of biomass production of chlorella itself.
in the same way, p. chromatophora has phototrophic nutrition.
it depends on carbon assimilates which derivate from the endosymbiotic cyanobacterium whose inorganic carbon rate assimilation is the same as a free-living cyanobacteria  <cit> .
but unlike its free-living relatives, its growth rate is restricted by p. chromatophora.
considering the above metabolic analysis of the chromatophore, which predict an in silico growth rate of  <dig>  h− <dig>  it is difficult to consider the biomass as the only objective function in chromatophores.
taking into consideration that chromatophores provide the host with reduced-carbon, a reaction simulating hexose export to extracellular compartment was added.
this reaction was defined as objective function.
and to ensure biomass components production, biomass reaction was fixed to  <dig>  h− <dig> which is the growth rate of a free-living relative cyanobacterium.
interestingly, under these conditions the metabolic model of the chromatophore predicts a potential rate of hexose exportation of  <dig>  mmol × gdw−1 × h− <dig>  as far as we know, this is the first metabolic reductive evolutionary analysis where metabolic functionality  of the endosymbiont is explored as objective function, differing from previous analyses where biomass is set as objective function of mutual endosymbionts as b. aphidicola  <cit> , s. glossinidius  <cit>  and b. cuenoti  <cit> .
abc phosphate transporter is an essential component of the chromatophore
all simulations showing a hexose exportation rate equivalent to that of the chromatophore model  share the abc phosphate transporter.
this pi-dependency in the chromatophore agrees with that observed in isolated spinach chloroplasts  <cit> .
it has been shown that photosynthesis declines dramatically  in chloroplast in the absence of pi in the reaction medium.
also, carbon export from the chloroplast is inhibited  <cit> , with up to 60% of 14c fixed being retained in the chloroplast  <cit> .
as mentioned above, this observation agrees with the more than 95% of simulations which predict that lack of abc phosphate transport favors carbon retention in the chromatophore instead of being released to the host.
therefore, we predict that lack of abc transporter in the genome of the chromatophore of paulinella fk <dig> is compensated by a phosphate transporter coded in the host  <cit> .
the role of natural selection on the evolution of the metabolism of the chromatophore
inspection of fba calculated central metabolic fluxes in the chromatophore and in the free-living cyanobacteria showed that the endosymbiont is better at producing hexose.
this is likely a host related adaptation.
to investigate whether this and other characteristics of the metabolic model of the chromatophore evolved by natural selection, we simulated in silico reductive evolution with a null model not including positive selection.
as a proxy of genome reduction by purifying selection and random genetic drift, we submitted the metabolic model of synechococcus sp.
wh  <dig> with the following algorithm: a) first, we simulated host-level purifying selection by requiring that the rate of hexose exportation of the model must be always greater than  <dig> and biomass is produced at  <dig>  mmol × gdw−1 × h−1; b) next, we performed rounds of single gene deletion until no more genes could be deleted; c) finally, we repeated this process  <dig> times.
by this, we obtained a population of  <dig> reduced metabolic networks all of them capable of producing  <dig>  mmol × gdw−1 × h− <dig> of biomass, but differing in hexose rate exportation.
differences in rates of hexose exportation were due to contingency-dependent loss of alternative pathways  <cit> .
with this experiment, we could determine if the potential rate of hexose exported in icv <dig>  is easily obtained by host-level purifying selection  and contingency-dependent evolution on random gene deletion.
our evolutionary reductive analyses showed that <5% of simulations were predicted to export hexose at a similar rate as the model icv <dig>  this suggests that metabolic functionality of icv <dig> is unlikely to be determined by genetic drift alone.
therefore, we conclude that natural selection at holobiont level may have contributed to shape metabolic functionality of the chromatophore.
natural selection as metabolic engineer
according to the above mentioned, we consider suitable to make the analogy of natural selection as metabolic engineer.
metabolic engineering can be defined as “the directed improvement of product formation or cellular properties through the modification of specific biochemical reactions or introduction of new ones”  <cit> .
one of the objectives of metabolic engineers is to redirect the flux of mass through the metabolism of organisms towards a desired metabolic product.
some genetic strategies to redirect metabolic flux toward production of a desired metabolite include: increasing the precursor supply; altering the regulation  genes; increasing the efficiency of bottleneck enzymes; reducing flux toward unwanted byproducts; or eliminating competing pathways by gene-deletion  <cit> .
it has been proposed that cellular metabolism of free-living microorganisms is primed, through natural selection, for the maximum responsiveness to the history of selective pressures rather than for the overproduction of specific chemical compound  <cit> .
in host-restricted conditions this responsiveness to free-living selective pressures are no longer needed.
instead, new biological objectives are defined now related to holobiont survival.
for instance, it was proposed that the chloroplast metabolic network has improved photosynthetic properties in comparison to free-living cyanobacteria  <cit> .
for example, the metabolic network in chloroplast has: i) a longer average path length; ii) a larger diameter; iii) is calvin cycle-centered; iv) and presents better modular organization when compared with the network of free-living cyanobacteria  <cit> .
in a similar way, the metabolism of the chromatophore  seems to be tailored for the exportation of reduced-carbon; that is, when comparing the export of reduced-carbon between the icv <dig> and the icv <dig> models  we found that icv <dig> shows higher rates of hexose exported than the free-living icv <dig> model at the cost of increased consumption of phosphate .
the evolutionary mechanism outlined above applies when the host benefits from the endosymbiont.
in particular, mechanisms such as “partner fidelity feedback”  promote cooperation between symbionts.
pff requires individuals to be “associated for an extended series of exchanges that last long enough that a feedback operates”  <cit> .
similar mechanisms likely operated in other symbiotic systems.
for example, buchnera  <cit>  and blochmannia  <cit>  overproduce essential amino acids  to its host.
this overproduction of eaas was consequence of metabolic restructurings due to metabolic-gene losses.
for example, the truncation of the purine biosynthesis pathway which allows the endosymbiont to produce histidine at higher rates than free-living relatives  <cit> .
reductive evolutionary simulations carried out by  <cit>  showed that this truncation is an improbable evolutionary event under conditions tested.
CONCLUSIONS
our main objective was to better understand the metabolic changes experienced by the free-living cyanobacteria to become a chromatophore.
in addition, we assessed the evolutionary forces driving organellogenesis.
we found evidence that certain metabolic pathways are preferentially conserved in the chromatophore.
we also found that the pattern of metabolic gene loss strongly depends on the availability of nutrients from its host.
the high fragility of the chromatophore network reflects the transition to a more stable environment and, consequently, its simplification.
the chromatophore is specialized in producing reduced-carbon which could be released to the host.
this specialization was consequence of metabolic restructurings which could not be possible in free-living conditions.
we interpret this specialization as consequence of natural selection acting as a metabolic engineer which modifies intrinsic metabolic properties of the endosymbiont impacting positively at the holobiont level.
our in silico simulations allowed us to determine that metabolic specialization of the chromatophore is an unlikely result of purifying host-level selection and genetic drift alone.
in this way, computational analysis of biological systems allows to obtain new insights on the evolutionary forces shaping metabolic evolution of mutualistic endosymbionts.

 paulinella chromatophora is an amoeba dispensed with phototrophic nutrition that contains blue-green photosynthetic organelles of cyanobacterial origin termed chromatophores. these novel organelles have a monophyletic origin in different strains of photosynthetic paulinella that have been described and were acquired through a primary endosymbiotic event about ~ to  mya. chromatophore genome sequencing from two strains of p. chromatophora, revealed a size of and mbp, respectively. this represents about 1/ of the genome size of synechococcus sp. wh the closest free-living relative cyanobacterium with a sequenced genome. synechococcus sp. wh has a genome of ~ mbp and protein-coding genes. it indicates that the chromatophore evolved by genome reduction. however, genome reduction in p. chromatophora is not as extreme as in plastids which rarely exceed kbp. chromatophores are genetically integrated with their host. more than nuclear encoded genes of chromatophore origin have been identified. and some of the protein products coded by these genes are imported back into the chromatophore and participate in the photosynthetic apparatus. accordingly, chromatophores have been described as plastids in the making. p. chromatophora nutrition relies on the reduced-carbon photosynthetically assimilated by the chromatophore. this endosymbiotic-nutrient dependency has been observed in other organisms such as aphids and tsetse flies housing prokaryotic endosymbionts. particularly for aphids, host essential amino acids are provided by an endosymbiotic bacterium called buchnera aphidicola. sequencing of the genome of b. aphidicola revealed a high degree of genetic degradation, while genes necessary for the syntrophic relationship with its host have been retained. prokaryotic endosymbionts evolve small genomes mainly by the combined action of genetic drift and negative selection. in host-restricted conditions, the endosymbiont experiences a lack of recombination and horizontal gene transfer, as well as recurrent population bottlenecks lowering its effective population size and a concomitant relaxation of natural selection. the combined action of these factors allows the accumulation of slightly deleterious mutations through a process called muller’s ratchet. as a consequence, many genes become pseudogenes and are subsequently lost. in addition, selection at holobiont level by mechanisms like “partner fidelity feedback” have been proposed to promote the evolution of mutualistic interactions. something that should be considered is that, differing from free-living relatives, the metabolism of mutualistic endosymbionts is specialized in the production of metabolites that are released to their host as nutrients. this metabolic specialization is the consequence of metabolic restructuring caused by gene loss and genome reduction. resulting reduced genomes code for fewer genes, however, they are more integrated to the host. the extreme cases are organelles of endosymbiotic origin such as chloroplasts. therefore, if mutualistic endosymbionts show metabolic adaptations to provide nutrients to their hosts, natural selection must have participated in the evolution of these systems. during early stages of organellogenesis, the cyanobacteria that evolved into the chromatophore, had access to metabolites provided by the host. it is likely that the availability these metabolites render of some metabolic routes dispensable in the endosymbiont. the loss of these biosynthetic pathways in the endosymbiont led to restructurings and changes in the remaining metabolic fluxes. taking into consideration all these modifications experienced by the chromatophore and the nutrient dependency of the holobiont for the photosynthetic function of the chromatophore, we made the analogy of natural selection acting as a “metabolic engineer” directing the strategies for the metabolic specialization of the chromatophore. in general, the objective of metabolic engineering is the directed improvement of metabolic capabilities through the deletion of metabolic genes or the introduction of new ones. by using these strategies, microorganisms have been engineered for the improvement of the yield and the production and consumption rates of desired metabolites. for instance, for the of 1-butanol production in cyanobacteria, many more examples can be found elsewhere. in this work, we reconstructed the genome based metabolic models of the chromatophore of paulinella chromatophora and the cyanobacteria synechococcus sp. wh we inquired into the metabolic capabilities of the chromatophore; the possible metabolic interaction of the chromatophore with its host; and in silico simulate the process of metabolic evolution experienced by the chromatophore in host-restricted conditions.  differential gene retention of functional categories in the chromatophore genome our first objective was to determine to what extent genetic loss affected functional metabolic categories in the chromatophore when compared to the genome of synechococcus sp. wh we compared against synechococcus sp. wh because is the closest free-living cyanobacterium with a sequenced genome and it is likely to be similar in gene content to the ancestor of the chromatophore. to assess the statistical significance we used a hypergeometric distribution. as is shown in fig. genes belonging to functional categories have been less affected by genome erosion. in particular, photosynthesis and fatty acid biosynthesis categories are less affected. retention of these functional categories in the chromatophore can be attributed to a host-level selection protecting from gene loss. conserved genes very likely play an adaptive role in the holobiont.fig. conservation of functional gene categories in the chromatophore when compared to synechococcus sp. wh for each functional category we show in green and red the percentage of gene conservation and lost in the chromatophore, respectively. for instance, if a gene category is completely green, it indicates that all orthologs in synechococcus sp. wh are conserved in the chromatophore. as shown, gene loss affects differentially each one of the functional categories in the chromatophore. functional categories particularly well conserved are indicated with asterisks. statistical significance calculated by using a hypergeometric distribution. genes were classified following kegg database in silico metabolic reconstruction of the chromatophore of p. chromatophora and synechococcus sp. wh 5701 to better understand the role in the symbiosis played by remaining genes in the chromatophore, we reconstructed two metabolic models. one for the chromatophore of p. chromatophora ccac and the other for synechococcus sp. wh the closest free-living cyanobacterium with a sequenced genome. the rationale behind this is to use synechococcus sp. wh as a proxy of the ancestral cyanobacterium that evolved into the chromatophore. metabolic model reconstruction of the free-living cyanobacterium synechococcus sp. wh was done by identifying orthologs to those protein-coding genes reported in the metabolic model of synechocystis sp. pcc. the resulting metabolic model of the free-living organism comprised metabolic reactions with metabolites and protein-coding genes. metabolic model reconstruction of the chromatophore was done by identifying those genes in the genome of the chromatophore of p. chromatophora ccac that are orthologous to the free-living metabolic model. the metabolic model of the chromatophore comprised reactions, metabolites and protein-coding genes. because synechococcus sp. wh is a close free-living relative of the chromatophore, it could be considered that reactions were lost along genome reduction in the chromatophore.table characteristics of metabolic models of synechococcus sp. wh and the chromatophore by using the biomass equation of the cyanobacterium synechocystis sp. pcc, we tested the functionality of the icv and the icv metabolic models with flux balance analysis. biomass production was set as objective function. in silico growth was simulated under autotrophic conditions. co and photons uptake were set to  mmol × gdw−1 × h− and 100 mmol × gdw−1 × h− respectively and set as constraining metabolites as in. in model icv almost every metabolic pathway for biomass production is complete. the exceptions were reactions for which no orthologous exist in synechococcus sp. wh when compared to ijn. these reactions had to be added to the icv model in order to produce all the components necessary for the biomass equation. in this way, icv showed an in silico growth rate of  h− which is identical to the in silico growth reported for synechocystis sp. pcc metabolic model under autotrophic conditions. under these conditions, the metabolic model of the chromatophore did not show in silico growth. this was obviously due to the reduced metabolic capabilities caused by the genomic reduction process experimented by the photosynthetic endosymbiont. genome reduction has affected the metabolic capabilities of the chromatophore in two ways: a) some biosynthetic pathways were completely lost; while b) some other were partially lost. for example, in synechocystis sp. pcc riboflavin is synthesized by four genes that perform six reactions by using guanosine 5′-triphosphate and d-ribulose 5-phosphate as precursors metabolites. all these genes for riboflavin biosynthesis were lost in the chromatophore. in this case, we assumed that the host provides riboflavin to the chromatophore. the possible explanation for this loss is that riboflavin is the main precursor for flavin mononucleotide and flavin adenine dinucleotide, two main compounds that work as coenzymes for many of the enzymes such as oxidoreductases including nadh dehydrogenase as well as in biological blue-light photo receptors. this observation is concomitant with the loss in some functional gene categories; as in oxidative phosphorylation. as the hypothesis is that the metabolic network must preserve its functionality, whenever we found a similar situation, exchange reactions were added to the metabolic model to simulate the incorporation of riboflavin and other metabolites as additional nutrients from the host. these metabolites included amino acids, cofactors, vitamins and other molecules which are essential for the biomass equation but cannot be produced by the chromatophore.fig. nutrients uptake simulation in the chromatophore model. metabolites that cannot be produced by the chromatophore include: amino acids, cofactors, vitamins and others -5-amino-4-imidazolecarboxamide, succoa = succinyl-coa, lipidads = lipid a disaccharide, dahp =2-dehydro-3-deoxy-d-arabino-heptonate 7-phosphate) some other biosynthetic pathways are truncated in the chromatophore because single gene coding enzymes were lost. for example, in the biosynthetic pathway of leucine, most gene coding enzymes are present in the chromatophore except for the gene coding for 3-isopropylmalate dehydrogenase. in this case, we assumed that host encoded enzymes complement the pathway in the endosymbiont. either by importing host encoded enzymes to the chromatophore or by exchanging intermediated metabolites between the symbionts. similar situations have been proposed for other host-endosymbiont systems. for this reason, we assumed that the production of these metabolites is shared between the host and the endosymbiont. in addition, some reactions in the chromatophore model icv for which no orthologous genes exist with the free-living model icv but are essential for in silico growth were assumed to be present. finally, chromatophores lost the ability to store photosynthates as well as the capacity to synthesize sucrose. because of that, glycogen was removed from the biomass equation in icv under these conditions, in silico growth of the icv model was  h− this is an unrealistic rate because growth of the chromatophores is restricted to host division which is much lower than growth rate reported for free-living cyanobacteria and even other photosynthetic eukaryotes. robustness analysis of metabolic models we assessed the robustness of the icv and the icv models to single gene deletions. genetic robustness was defined as the capacity of the models to maintain its metabolic capabilities after a genetic deletion. under phototrophic conditions, model icv showed genes to be essential because its deletion decreases the biomass production over 99%. this result shows that icv is less robust than the metabolic model of synechocystis sp. pcc where % of the genes are essential under these same conditions. in addition, there is a decreasing robustness in the model of the chromatophore where of the genes are essential. this indicates that the genomic reduction experimented by the chromatophore rendered its metabolic network fragile. the same result has been observed for other metabolic networks of endosymbionts.fig. genetic robustness analysis of metabolic models of synechocystis sp. pcc, synechococcus sp. wh and the chromatophore of paulinella chromatophora. percentage of essential genes for each metabolic model interestingly, we found that there are non-essential genes in the metabolic model icv whose single deletion decreases in silico growth rate. these include genes encoding the enzymes acetyl-coa synthetase, malic enzyme and fumarase. of these three, the last enzyme is the only one decreasing the in silico growth rate in ijn when it is deleted. in the icv model, all these genes were lost. in addition, the non-essential gene in icv coding for an enzyme with arginase activity is the only one whose deletion decreases the growth rate in the icv chromatophore model. this suggests that genome reduction leading to icv caused metabolic restructuring because deletion of this enzyme with arginase activity in icv has no effect. in silico simulation of metabolic-gene loses in the chromatophore of p. chromatophora based on the metabolic network of the free living synechococcus sp. wh we simulated in silico the gene loss. we evaluated the impact of intracellular conditions on the evolution of the chromatophore. in particular, we asked whether the set of metabolites predicted to be provided by the host in the icv model determined actual gen content of the chromatophore after genome reduction. two in silico intracellular conditions were evaluated. in the first one, we simulated genetic reduction under in silico intracellular conditions where available nutrients were those predicted in the icv model. in the second one, we randomly selected metabolites from the icv model and assigned as available nutrients under intracellular conditions. the algorithm to simulate genome reduction is explained in detail in the methods section. this algorithm allowed us to obtain in silico evolved chromatophores whose metabolic capabilities regarding the biomass production are equivalent to those of icv265; but differing in their in silico evolutionary history and gene content. simulations under the proposed nutrients conditions resulted in reduced metabolic networks with genes on average. in these reduced networks, of the genes present in the free-living ancestor, % are strictly conserved in the simulations and % are dispensable in all of them. in randomized nutrients simulations, reduced networks have an average size of genes and 54% and % are conserved and dispensable in the simulations, respectively. as is shown in fig. the proportion of: i) essential genes; ii) variable genes; and iii) dispensable genes, varies between metabolic pathways. these proportions also vary between treatments. surprisingly, the most extreme case is that of the genes participating in photosynthetic activity. in proposed nutrients %, % and % are predicted to be essential, variable and dispensable, respectively. while for randomized nutrients none was predicted to be essential nor dispensable because 100% of them were variable.fig. variation in the proportion of genes classified as essential, variable and dispensable in different metabolic pathways according to two different sets of available nutrients for the chromatophore. a set of proposed nutrients; b set of randomized nutrients. aa to bn metabolic pathways: aa* = citrate cycle; ab = lipopolysaccharide biosynthesis; ac = carotenoid biosynthesis; ad = folate biosynthesis; ae = glycerolipid metabolism; af = hydrogen production; ag = steroid biosynthesis; ah = aminosugars metabolism; ai* = nicotinate and nicotinamide metabolism; aj = nucleotide sugars metabolism; ak = riboflavin metabolism; al = thiamine metabolism; am = carbon fixation; an = glutamate metabolism; ao = lysine metabolism; ap = nitrogen metabolism; aq = terpenoid backbone biosynthesis; ar = fructose and mannose metabolism; as* = pantothenate and coa biosynthesis; at = peptidoglycan biosynthesis; au = ubiquinone and other pterpenoids biosynthesis; av = urea cycle and metabolism of amino groups; aw* = alanine, aspartate and glutamate metabolism; ax = valine leucine and isoleucine biosynthesis; ay = histidine metabolism; az* = pentose phosphate pathway; ba = starch and sucrose metabolism; bb = fatty acid biosynthesis; bc = glyoxylate and dicarboxylate metabolism; bd = sulfur cysteine and methionine metabolism; be = arginine and proline metabolism; bf = pyrimidine metabolism; bg = glycolysis/gluconeogenesis; bh = pyruvate metabolism; bi* = phenylalanine tyrosine and tryptophan biosynthesis; bj* = purine metabolism; bk* = porphyrin and chlorophyll metabolism; bl = oxidative phosphorylation; bm* = photosynthesis and bn = transport. * metabolic pathways with a difference in categorical genes with p-value <  genetic concordance was evaluated between these simulated minimal networks and the real chromatophore model. this was done by measuring sensitivity and specificity as in. in fig. we show the fraction of true-positives and false-positives for every cutoff. true-positive and false-positive for every cutoff form a curve whose area under the curve represents the probability that a gene conserved in icv is present in more simulations than a gene which has been lost.fig. genetic concordance between in silico evolved chromatophores and the chromatophore model. the area under the curve indicates the predictive accuracy for evolved chromatophores obtained under proposed and randomized nutrients conditions. proposed nutrients: simulation with metabolites proposed as nutrients in the icv model and randomized nutrients: simulations with randomized metabolites assigned as nutrients. area under the curve: proposed nutrients =; randomized nutrients = the area under the curve shows the contribution of the nutrients available in intracellular conditions explaining the evolutionary history experimented by the chromatophore. accordingly, the accuracy obtained under the proposed nutrients condition was %, while that of the randomized nutrients was %. the difference between the areas under the curve from both conditions is statistically significant. modeling selection and drift to explain metabolic evolution of the chromatophore we are interested in understanding the role played by natural selection during the evolution of the metabolic capabilities of the chromatophore. chromatophores provide the host with reduced-carbon, probably a hexose. this in analogy to the origin of plastids. it has been proposed that during the early stages of plastid evolution, the photosynthetic endosymbiont exported reduced-carbon in the form of an hexose-phosphate through an hexose phosphate transporter of bacterial origin. to study how the potential rate of carbon exportation evolved, a hexose export reaction was added to the metabolic models. this reaction was defined as objective function. to ensure biomass components production, the biomass reaction was fixed to  h− which, as stated previously, is the growth rate of synechocystis sp. pcc metabolic model under autotrophic growth conditions. under these conditions, there is no exportation of reduced-carbon in the icv model. however, in the icv chromatophore model, the potential rate of hexose exported without affecting the in silico growth rate was  mmol × gdw−1 × h− in fig. we show the fluxes calculated with fba of the central metabolism of the models of the chromatophore and the free-living cyanobacteria in conditions previously mentioned.fig. flux distribution obtained with fba of the central metabolism of icv and icv fluxes calculated for production of metabolites precursors used to produce biomass components are produced in less quantity in the chromatophore’s model. this is a consequence of the loss of metabolic capabilities in the metabolism of the chromatophore which allow the redirection of carbon through the gluconeogenic pathway for the production of hexoses as metabolic objective, instead of being used in the production of biomass components. to analyze the efficiency of the metabolic networks in terms of hexose production at overall metabolism, we calculated the yields. the yields are parameters that measure the efficiency of the metabolic network and allow the comparison across different microorganisms. for instance, the yield of the ethanol production is higher in saccharomyces cerevisiae compared to zymomonas mobilis, this was the result of the specialization of the microorganism to produce specific metabolites. therefore, we calculated the yields of carbon, energy and reducing equivalents required to produce hexose. these results show that the model of the chromatophore is more efficient for producing hexose from the external carbon than the free-living cyanobacteria. it means that metabolic restructurings experienced by the chromatophore rendered its metabolism more efficient to produce hexose which can be provided to the host.table yields analysis of extracellular co atp and nadph consumed in hexose production for both models co2 the yields suggest that the loss of some metabolic capabilities in the ancestral cyanobacterium caused a redirection of fixed co causing changes in metabolic fluxes and consequently increasing the rate of reduced-carbon exported to the extracellular compartment. we then inquired about the evolutionary forces that determined genetic conservation and metabolic functionality in chromatophores. specifically, we wanted to infer if these metabolic capabilities of the icv model could have been possible under a random model of evolution or were the consequence of natural selection for metabolic specialization of the chromatophore and its positive impact at the holobiont. to tests this, we simulated the metabolic reduction process with hexose export and biomass production as evolutionary restrictions in a random model where hexose exported must be greater than zero. it means that every gene affecting the in silico growth rate of  h− and impairing hexose export was considered as essential, while the hexose export rate could always vary while being greater than zero. minimal networks obtained in silico under these conditions were variable in size and gene content. of simulations, only of the genes conserved in model icv are conserved in all the simulations. conversely, there are genes predicted to be essential in all these simulations which are not conserved in model icv the metabolic networks from these simulations are different in gene content and show different hexose export rates however they are equivalent in biomass production. as shown in fig. after in silico metabolic reduction, hexose export rate in minimal networks obtained under these conditions tend to be minimal and close to zero. on the other hand, only % of simulations have a potential rate of hexose exported equal or higher than the metabolic model of the chromatophore. this suggests that the probability of obtaining a potential rate of hexose exported similar to that of icv under a random model is less than 5%. we got a similar result by varying the growth rate constraint of  h− under plausible biological values.fig. hexose exportation rate of the chromatophore model is achieved in only % of the simulations under a random model of evolution. hexose exported rate for independent simulations. red-dotted line indicates the hexose export rate in the chromatophore model although variable, our simulations evolved metabolic networks that have approximately the same number of reactions than icv the average number of reactions with non-zero fluxes in the reduced metabolic models of the simulations is  ± . this is slightly less than the number of reactions with non-zero fluxes in the icv model. this shows that the small percentage of simulations showing a potential rate of hexose exported equal or greater than that of the chromatophore is not due simply to smaller size of the simulated metabolic networks. these in silico experiments suggest that the potential rate of hexose exported in model icv is unlikely to be the outcome of only genetic drift and purifying selection. this suggests that the potential rate of hexose exported was the result of a process of functional specialization in which the increasing rate of hexose exportation was favored by natural selection due to the positive impact at the holobiont level. interestingly all these % of in silico evolved chromatophores have in common the conservation of a phosphate transporter via abc system which is also present in the chromatophore model. conservation of this phosphate transporter allows the simulated network to get the phosphate necessary to be able to export fixed carbon. without this transporter most of fixed carbon is oxidized in the pentose phosphate pathway releasing only a small amount to the extracellular compartment. metabolic integration of the chromatophore to its host in our previous simulations, we assumed that nutrients were available simultaneously for the chromatophore since the beginning of the evolutionary process at the onset of the endosymbiosis. however, it is likely that this has not been the case and transporters for these nutrients were gained sequentially. for instance, metabolic transport activity in the chromatophore is reduced due to loss of most transporters in comparison to free-living cyanobacteria. and it has been reported that a large percentage of solute transporters in plastids from plantae have host and bacterial origin. therefore, we simulated the evolutionary acquisition of transporters and its consequences in gene loss and the capability of the chromatophore to export fixed carbon to its host. for every simulation, we used icv as a free-living ancestor of the chromatophore under nutrient-rich conditions. however, in this experiment, the model icv did not have access to all nutrients since the beginning of the simulation. instead, we randomly assigned a transport allowing the uptake of the respective nutrient. we then randomly deleted one gene at a time from icv if the deleted gene affected the growth rate or impaired hexose exportation, we considered this gene as essential and we restored it to the model. in this way we analyzed the selective impact caused by gene loss due to the addition of a single transporter and the concomitant relaxation of natural selection for retention of specific biosynthetic pathways. once we analyzed every gene in the model, we randomly assigned a second transport and then we repeated the gene loss simulation mentioned above. simulation stops when in silico chromatophore has access to the nutrients and all genes have been evaluated for their essentiality. as shown in fig. after the incorporation of the transporters, the probability of getting a potential rate of hexose exported equal or higher than the metabolic model of the chromatophore is less than 5% in simulations, in agreement with our previous result.fig. metabolic integration with the host determines the rate of hexose exported by in silico chromatophores as well as the frequency of simulations that provide higher rates of reduced-carbon to the host. x axis, hexose export rate; y axis, frequency of simulations; z axis, transporters added to the model during the process of metabolic integration, it is noted that the maximum rate of hexose exportation becomes greater with every metabolite obtained as nutrient. however, by inspecting the frequency distribution of simulations with different potential rates of hexose exported, it is obvious that as metabolic integration advances, the probability of getting the maximum rate of hexose exportation decreases. these changes in frequency distributions during the process of metabolic integration can be interpreted in terms of the functional specialization of the chromatophore. as metabolic integration proceeds, the chromatophore increased its capacity to provide fixed carbon to its host. however, continued gene loss led to a simplified metabolic network and a smaller fraction of in silico evolved chromatophores can export as much fixed carbon as icv the evolutionary landscape becomes smaller as evolution proceeds. the metabolism of the chromatophore is specially adapted to produce carbon for its host as shown above, the potential rate of hexose exported in the chromatophore model is highly dependent on phosphate consumption. in addition, the growth rate of the chromatophore is coupled to the host’s growth rate. as shown above, the potential rate of hexose exported is unlikely to be the outcome of a random evolution. to test the impact of these two restrictions, we analyzed the metabolic properties of models icv and icv in potential rate of hexose exported under growth rate and phosphate uptake restrictions. as shown in fig. the potential rate of hexose exported in the icv model is robust with respect to growth rate and phosphate uptake. this contrasts with the chromatophore model where, for a given growth rate, only a specific consumption of phosphate is necessary to sustain hexose release. in addition, the capacity of hexose export in the icv model for a determined growth rate restriction is greater than the icv model.fig. the metabolic model of the chromatophore is specially adapted to produce higher rates of reduced-carbon under phosphate and growth restrictions in comparison with synechococcus sp. wh. a hexose export rate in the free-living model icv b hexose export rate in the chromatophore model icv y axis, growth rate restriction. x axis, phosphate uptake restriction. z axis, potential rate of hexose exported. colors in axis z are only for drawing purposes and do not represent an extra variable discussion in this work, we show that the metabolic network of the chromatophore of p. chromatophora is different to the metabolic network of its free-living relative synechococcus sp. wh we suggest that these differences evolved by natural selection. gene-loss and carbon flux redirection guided by natural selection led to metabolic specialization of the chromatophore as a reduced-carbon provider. purifying selection and the maintenance of the symbiosis our analysis showed that some metabolic pathways have been preferentially conserved in the chromatophore. these preserved metabolic pathways very likely play a prominent role in the symbiosis. this pattern is analogous to the one observed in many other endosymbionts e.g. buchnera aphidicola. in this later case, biosynthetic pathways producing essential amino acids for the host are preserved by host-level natural selection. as mentioned above, differential conservation of gene category functions suggests that purifying selection is preserving relevant symbiotic functions. accordingly, estimation of the rate of nucleotide substitution in dna alignments of protein-coding genes orthologous between chromatophores of two different strains of p. chromatophora showed that most of them have signals of purifying selection. it has been suggested that host-level selection prevents the fixation of deleterious mutations in endosymbionts thus lowering the chances of a mutational meltdown resulting in extinction. and, of course, this prevents the consequent replacement of non-functional endosymbionts. in addition, selective pressure to maintain functional proteins increases with the time of host-endosymbiont interaction and combined with very strong bottlenecks may help to reduce the accumulation of deleterious mutations. this has been proposed to explain mitochondrial genome evolution. metabolic integration of the chromatophore to its host comparison of the metabolic models of the chromatophore and the cyanobacterium synechococcus sp. wh allowed us to inquire into the evolution of the metabolic interaction of the chromatophores with its host. for example, several metabolic pathways in the chromatophore are incomplete. it is likely that the host supplies these metabolites as nutrients to the chromatophore. metabolic pathway sharing is a hallmark of endosymbiotic organisms. for example wolbachia, which are endosymbionts of many animal species, show a degraded genome whose limited metabolic capabilities are complemented by its host. in turn, the endosymbiont provides the host with nutrients such as riboflavin, positively impacting host fitness. equally remarkable is the likely coupled production of some metabolites between the chromatophore and its host. as mentioned above, this collaboration in metabolite biosynthesis has been observed in other symbiotic systems. fragility of a reduced metabolic network to study the metabolic capabilities of the chromatophores we used fba. this stoichiometric approach can predict cellular phenotypes in specific environmental conditions. generally, biomass production is fixed as objective function. in absence of biomass composition, the use of a biomass equation from a related organism is a valid starting point for metabolic analysis. in this way, fba has been used to infer the metabolic capabilities of different organisms whose cultivation and experimental management is challenging or not yet possible, as in the case of endosymbionts. for example, biomass composition and the metabolic model of escherichia coli were used for metabolic analysis of buchnera aphidicola, sodalis glossinidius, and blattabacterium cuenoti. in the same way, we used the biomass composition and stoichiometric model of synechocystis sp. pcc as a starting point to model the metabolism of the chromatophore and synechococcus sp. wh. we found that the metabolism of the chromatophore is highly fragile to gene deletions. approximately 84% of the genes in the model are essential when singly deleted in comparison with ~67% of the genes in synechococcus sp. wh a similar difference in metabolic fragility was found by when comparing the models of b. aphidicola and its free-living relative escherichia coli where 84% and 19% of genes were essential, respectively. in the same way, the metabolic network of two strains of blattabacterium cuenoti, the obligated primary endosymbiont of cockroaches, were shown to be highly fragile to single gene deletion. it was found that % and % were essential genes, respectively. finally, in sodalis glossinidius, % metabolic genes were found to be essential, compared with its ancestral network where only % are predicted to be essential. our robustness analyses of the icv and the icv models agree with the generalization that metabolic networks of endosymbionts are more fragile than their free-living counterparts. this metabolic fragility of endosymbionts contrasts with theoretical estimations that suggest that, in general, metabolic systems are robust and complex. however, the metabolic systems of endosymbionts are considered more robust than minimalist metabolic networks. the difference in metabolic fragility of the chromatophore when compared to synechococcus sp. wh reflects the transition from a free-living style to a more stable condition inside paulinella chromatophora. metabolic environment as a determinant of gene content it has been shown that retention of metabolic genes in endosymbionts is determined by the metabolic requirements and molecular environment of the host. with the use of fba and the metabolic model of synechococcus sp. wh as a proxy of the ancestor of the chromatophore, we evaluated the impact of the host-metabolic environment in the reduction of the metabolic system of the endosymbiont. the proposed host-metabolic environment predicted with % of accuracy the actual gene content of the chromatophore. this is in contrast with the % of accuracy obtained when using a randomly set of host-provided metabolites. this emphasizes the contribution of the intracellular metabolic environment to the evolution of the metabolism in the chromatophore. similar reductive simulations have been used to predict the set of essential genes of pathogens located in certain environmental niches within the human body. in the same way, reductive evolution simulations using e. coli as free-living ancestor predicts with 80% of accuracy the metabolic gene content of b. aphidicola and wigglesworthia glossindia. inspection of the proportion of dispensable, variable, and essential genes by in silico reductive simulations predicts differential gene retention patterns between different metabolic pathways. for example, in randomized nutrients simulations, photosynthesis pathway 100% of genes are predicted as “variable” while in proposed nutrients ~78% are essential. this means that under randomized nutrients, photosynthesis function could be useful but not essential and could have been lost in the chromatophore by chance. clearly, the set of metabolites comprising randomized nutrients cannot account for the metabolic gene content of extant chromatophores. maximization of biomass production is regularly used as objective function in fba analysis. it allows predicting the distribution of fluxes through a metabolic network. the maximization of biomass function is used as a proxy of evolutionary fitness. however, many other objective functions can be used. for instance, it was estimated that chlorella, releases 57% of its photosynthates to its host. this means that most carbon photosynthetically assimilated is destined to symbiotic interaction instead of biomass production of chlorella itself. in the same way, p. chromatophora has phototrophic nutrition. it depends on carbon assimilates which derivate from the endosymbiotic cyanobacterium whose inorganic carbon rate assimilation is the same as a free-living cyanobacteria. but unlike its free-living relatives, its growth rate is restricted by p. chromatophora. considering the above metabolic analysis of the chromatophore, which predict an in silico growth rate of  h− it is difficult to consider the biomass as the only objective function in chromatophores. taking into consideration that chromatophores provide the host with reduced-carbon, a reaction simulating hexose export to extracellular compartment was added. this reaction was defined as objective function. and to ensure biomass components production, biomass reaction was fixed to  h− which is the growth rate of a free-living relative cyanobacterium. interestingly, under these conditions the metabolic model of the chromatophore predicts a potential rate of hexose exportation of  mmol × gdw−1 × h− as far as we know, this is the first metabolic reductive evolutionary analysis where metabolic functionality of the endosymbiont is explored as objective function, differing from previous analyses where biomass is set as objective function of mutual endosymbionts as b. aphidicola, s. glossinidius and b. cuenoti. abc phosphate transporter is an essential component of the chromatophore all simulations showing a hexose exportation rate equivalent to that of the chromatophore model share the abc phosphate transporter. this pi-dependency in the chromatophore agrees with that observed in isolated spinach chloroplasts. it has been shown that photosynthesis declines dramatically in chloroplast in the absence of pi in the reaction medium. also, carbon export from the chloroplast is inhibited, with up to 60% of 14c fixed being retained in the chloroplast. as mentioned above, this observation agrees with the more than 95% of simulations which predict that lack of abc phosphate transport favors carbon retention in the chromatophore instead of being released to the host. therefore, we predict that lack of abc transporter in the genome of the chromatophore of paulinella fk is compensated by a phosphate transporter coded in the host. the role of natural selection on the evolution of the metabolism of the chromatophore inspection of fba calculated central metabolic fluxes in the chromatophore and in the free-living cyanobacteria showed that the endosymbiont is better at producing hexose. this is likely a host related adaptation. to investigate whether this and other characteristics of the metabolic model of the chromatophore evolved by natural selection, we simulated in silico reductive evolution with a null model not including positive selection. as a proxy of genome reduction by purifying selection and random genetic drift, we submitted the metabolic model of synechococcus sp. wh with the following algorithm: a) first, we simulated host-level purifying selection by requiring that the rate of hexose exportation of the model must be always greater than and biomass is produced at  mmol × gdw−1 × h−1; b) next, we performed rounds of single gene deletion until no more genes could be deleted; c) finally, we repeated this process times. by this, we obtained a population of reduced metabolic networks all of them capable of producing  mmol × gdw−1 × h− of biomass, but differing in hexose rate exportation. differences in rates of hexose exportation were due to contingency-dependent loss of alternative pathways. with this experiment, we could determine if the potential rate of hexose exported in icv is easily obtained by host-level purifying selection and contingency-dependent evolution on random gene deletion. our evolutionary reductive analyses showed that <5% of simulations were predicted to export hexose at a similar rate as the model icv this suggests that metabolic functionality of icv is unlikely to be determined by genetic drift alone. therefore, we conclude that natural selection at holobiont level may have contributed to shape metabolic functionality of the chromatophore. natural selection as metabolic engineer according to the above mentioned, we consider suitable to make the analogy of natural selection as metabolic engineer. metabolic engineering can be defined as “the directed improvement of product formation or cellular properties through the modification of specific biochemical reactions or introduction of new ones”. one of the objectives of metabolic engineers is to redirect the flux of mass through the metabolism of organisms towards a desired metabolic product. some genetic strategies to redirect metabolic flux toward production of a desired metabolite include: increasing the precursor supply; altering the regulation genes; increasing the efficiency of bottleneck enzymes; reducing flux toward unwanted byproducts; or eliminating competing pathways by gene-deletion. it has been proposed that cellular metabolism of free-living microorganisms is primed, through natural selection, for the maximum responsiveness to the history of selective pressures rather than for the overproduction of specific chemical compound. in host-restricted conditions this responsiveness to free-living selective pressures are no longer needed. instead, new biological objectives are defined now related to holobiont survival. for instance, it was proposed that the chloroplast metabolic network has improved photosynthetic properties in comparison to free-living cyanobacteria. for example, the metabolic network in chloroplast has: i) a longer average path length; ii) a larger diameter; iii) is calvin cycle-centered; iv) and presents better modular organization when compared with the network of free-living cyanobacteria. in a similar way, the metabolism of the chromatophore seems to be tailored for the exportation of reduced-carbon; that is, when comparing the export of reduced-carbon between the icv and the icv models we found that icv shows higher rates of hexose exported than the free-living icv model at the cost of increased consumption of phosphate. the evolutionary mechanism outlined above applies when the host benefits from the endosymbiont. in particular, mechanisms such as “partner fidelity feedback” promote cooperation between symbionts. pff requires individuals to be “associated for an extended series of exchanges that last long enough that a feedback operates”. similar mechanisms likely operated in other symbiotic systems. for example, buchnera and blochmannia overproduce essential amino acids to its host. this overproduction of eaas was consequence of metabolic restructurings due to metabolic-gene losses. for example, the truncation of the purine biosynthesis pathway which allows the endosymbiont to produce histidine at higher rates than free-living relatives. reductive evolutionary simulations carried out by showed that this truncation is an improbable evolutionary event under conditions tested.  our main objective was to better understand the metabolic changes experienced by the free-living cyanobacteria to become a chromatophore. in addition, we assessed the evolutionary forces driving organellogenesis. we found evidence that certain metabolic pathways are preferentially conserved in the chromatophore. we also found that the pattern of metabolic gene loss strongly depends on the availability of nutrients from its host. the high fragility of the chromatophore network reflects the transition to a more stable environment and, consequently, its simplification. the chromatophore is specialized in producing reduced-carbon which could be released to the host. this specialization was consequence of metabolic restructurings which could not be possible in free-living conditions. we interpret this specialization as consequence of natural selection acting as a metabolic engineer which modifies intrinsic metabolic properties of the endosymbiont impacting positively at the holobiont level. our in silico simulations allowed us to determine that metabolic specialization of the chromatophore is an unlikely result of purifying host-level selection and genetic drift alone. in this way, computational analysis of biological systems allows to obtain new insights on the evolutionary forces shaping metabolic evolution of mutualistic endosymbionts. 
by using these metabolites as intracellular conditions, in silico simulations of genome evolution by gene lose recover with 77% accuracy the actual metabolic gene content of the chromatophore. we reconstructed the metabolic networks of the chromatophore of p. chromatophora ccac and a close free-living relative, the cyanobacterium synechococcus sp. genome degradation of host-restricted mutualistic endosymbionts has been attributed to inactivating mutations and genetic drift while genes coding for host-relevant functions are conserved by purifying selection. in addition, our in silico simulations of the evolutionary process leading to the reduced metabolic network of the chromatophore showed that the predicted rate of released reduced-carbon is obtained in less than 5% of the times under a process guided by random gene deletion and genetic drift. also, the metabolic model of the chromatophore allowed us to predict by flux balance analysis a maximum rate of reduced-carbon released by the endosymbiont to the host. as its widely known, the loss of enzyme coding genes leads to metabolic network restructuring sometimes improving the production rates. wh we found that the evolution of free-living to host-restricted lifestyle rendered a fragile metabolic network where >80% of genes in the chromatophore are essential for metabolic functionality. this specialization suggests that natural selection crafted these metabolic adaptations. finally, our model also predicts that the abc phosphate transporter which is conserved in the genome of the chromatophore of p. chromatophora strain ccac is a necessary component to release reduced-carbon molecules to the host. we propose that natural selection acted as a “metabolic engineer” by favoring metabolic restructurings that led to an increased release of reduced-carbon to the host. in this work, we analyzed the evolution of the metabolism of the chromatophore of paulinella chromatophora by in silico modeling. our evolutionary analysis suggests that in the case of paulinella chromatophora natural selection at the holobiont level played a prominent role in shaping the metabolic specialization of the chromatophore. by inspecting the central metabolism of the chromatophore and the free-living cyanobacteria we found that by improvements in the gluconeogenic pathway the metabolism of the endosymbiont uses more efficiently the carbon source for reduced-carbon production. we asked whether genome reduction is driven by metabolic engineering strategies resulted from the interaction with the host. unlike their free-living relatives, the metabolism of mutualistic endosymbionts and endosymbiont-originated organelles is specialized in the production of metabolites which are released to the host. we interpret previous findings as evidence that natural selection at holobiont level shaped the rate at which reduced-carbon is exported to the host.
 genome degradation of host-restricted mutualistic endosymbionts has been attributed to inactivating mutations and genetic drift while genes coding for host-relevant functions are conserved by purifying selection. unlike their free-living relatives, the metabolism of mutualistic endosymbionts and endosymbiont-originated organelles is specialized in the production of metabolites which are released to the host. this specialization suggests that natural selection crafted these metabolic adaptations. in this work, we analyzed the evolution of the metabolism of the chromatophore of paulinella chromatophora by in silico modeling. we asked whether genome reduction is driven by metabolic engineering strategies resulted from the interaction with the host. as its widely known, the loss of enzyme coding genes leads to metabolic network restructuring sometimes improving the production rates. in this case, the production rate of reduced-carbon in the metabolism of the chromatophore.  we reconstructed the metabolic networks of the chromatophore of p. chromatophora ccac and a close free-living relative, the cyanobacterium synechococcus sp. wh we found that the evolution of free-living to host-restricted lifestyle rendered a fragile metabolic network where >80% of genes in the chromatophore are essential for metabolic functionality. despite the lack of experimental information, the metabolic reconstruction of the chromatophore suggests that the host provides several metabolites to the endosymbiont. by using these metabolites as intracellular conditions, in silico simulations of genome evolution by gene lose recover with 77% accuracy the actual metabolic gene content of the chromatophore. also, the metabolic model of the chromatophore allowed us to predict by flux balance analysis a maximum rate of reduced-carbon released by the endosymbiont to the host. by inspecting the central metabolism of the chromatophore and the free-living cyanobacteria we found that by improvements in the gluconeogenic pathway the metabolism of the endosymbiont uses more efficiently the carbon source for reduced-carbon production. in addition, our in silico simulations of the evolutionary process leading to the reduced metabolic network of the chromatophore showed that the predicted rate of released reduced-carbon is obtained in less than 5% of the times under a process guided by random gene deletion and genetic drift. we interpret previous findings as evidence that natural selection at holobiont level shaped the rate at which reduced-carbon is exported to the host. finally, our model also predicts that the abc phosphate transporter which is conserved in the genome of the chromatophore of p. chromatophora strain ccac is a necessary component to release reduced-carbon molecules to the host.  our evolutionary analysis suggests that in the case of paulinella chromatophora natural selection at the holobiont level played a prominent role in shaping the metabolic specialization of the chromatophore. we propose that natural selection acted as a “metabolic engineer” by favoring metabolic restructurings that led to an increased release of reduced-carbon to the host. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords endosymbiontmetabolic evolutionadaptationmetabolic integrationissue-copyright-statement© the author 2017 