Supplementary material
Chance and necessity in the evolution of minimal metabolic networks
Csaba Pál, Balázs Papp, Martin J. Lercher, Péter Csermely, Stephen G. Oliver and Laurence D. Hurst
Table of Contents
Supplementary Methods...........................................................................................................................2
I. Finding homologues across species. ................................................................................................2
II. Identification of horizontally transferred genes..............................................................................3
III. Impact of block deletions on minimal reaction sets .......................................................................5
IV. Receiver Operating Characteristic Curve (ROC) method .............................................................6
V. Systematic identification of cofactors synthesized by Wigglesworthia...........................................7
Modelling conditions used for simulations of minimal metabolic networks under nutrient-rich
conditions .................................................................................................................................................8
Supplementary Table 1.a List of major biosynthetic components and their contributions to the
biomass of E. coli.................................................................................................................................8
Supplementary Table 1.b List of metabolites available for uptake ......................................................9
Modelling conditions used in the simulations of Buchnera gene content evolution ..............................10
Supplementary Table 2.List of major biosynthetic components and their contributions to the biomass
of Buchnera........................................................................................................................................10
Metabolites available for uptake .......................................................................................................11
Modelling conditions used in the simulations of Wigglesworthia gene content evolution.....................12
Supplementary Table 3a.List of major biosynthetic components and their contributions to the
biomass of Wigglesworthia................................................................................................................12
Supplementary Table 3b List of metabolites available for uptake .....................................................14
Supplementary Figure 2. Comparison of reaction content of simulated and Wigglesworthia glossinidia
metabolic networks.................................................................................................................................15
Supplementary Table 4. The impact of block deletions on the properties of minimal minimal networks
................................................................................................................................................................17
Supplementary Table 5. Accuracy of reaction content predictions ........................................................18
Supplementary Table 6. Accuracy of reaction content prediction in different metabolic subsystems ...19
Table 6a Buchnera.............................................................................................................................19
Table 6b Wigglesworthia...................................................................................................................20
Metabolic pathways showing variability in retention across simulations...............................................21
Supplementary Table 7. .....................................................................................................................21
Supplementary Table 8. Examples of alternative metabolic solutions during reductive evolution of
Buchnera ................................................................................................................................................22
Supplementary Table 9. Cross comparison of Buchnera and Wigglesworthia reaction content
predictions ..............................................................................................................................................23
Supplementary Table 10. Physiologically coupled reactions are frequently lost together in
endosymbionts........................................................................................................................................24
Supplementary Table 11. List of 140 bacterial species investigated and their lifestyles........................26
Supplementary Table 12. Endosymbiont genes without orthologues in E. coli K-12 ............................28
References ..............................................................................................................................................32
1
Supplementary Methods
I. Finding homologues across species.
Homologues of E. coli genes present in the metabolic network were identified for 140 completely
sequenced bacterial species using STRING1. Lifestyle information was compiled from
http://www.genomenewsnetwork.org/resources/sequenced_genomes/genome_guide_index.shtml and
from the corresponding genome articles (Supplementary Table 11). For detailed comparisons of model
predictions with gene content of endosymbiotic genomes, we identified orthologues by reciprocal best
Blast hits between each endosymbiont and E. coli. This resulted in orthology assignments to all but a
handful of endosymbiont genes. Of the genes without orthology assignment, most had no gene with
significant sequence similarity in E. coli K-12 (mostly hypothetical proteins), while a few were
apparent duplicates (Supplementary Table 12). The presence of reactions in endosymbionts is inferred
from gene orthology in combination with known enzymatic functions in E.coli. Detailed results of all
simulations are in Supplementary Table 13.
2
II. Identification of horizontally transferred genes
Here we briefly describe how horizontally transferred genes were identified. For more details, see 2.
Inference of phylogenetic genome tree
Putative orthologous as well as paralogous proteins were identified for 56 completely sequenced
proteobacteria species (Supplementary figure 1) using STRING3, a database that integrates and extends
the widely used clusters of orthologous groups (COG)4. To exclude paralogues, only genes present as a
single copy in all investigated genomes were retained. Each of the remaining 47 protein families was
aligned using MUSCLE5 with default settings. Highly variable parts were excluded from the
alignments through GBLOCKS6. We constructed maximum-likelihood phylogenies from the
concatenated protein sequences with PHYML7 under an empirical substitution matrix8 and with a Γmodel to account for among-site variation in evolutionary rates. Comparison with independent
phylogenetic studies9-12 confirmed the branching order of all previously investigated species sets.
Below, we investigate 51 enterobacteria species, using the Helicobacter/Wolinella/Campylobacter
clade as outgroups (see Supplementary Figure 1).
Parsimony analysis of gene acquisition and loss
Presence and absence calls for gene families across all 51 proteobacterial species were obtained from
STRING3. Using the protocol of Bousseau and colleagues9, the most parsimonious scenarios for geneloss and horizontal transfer events (gene gains) on the rooted phylogeny were reconstructed from these
data using generalized parsimony. We estimated gains and losses under both ACCTRAN (accelerated
transformation) and DELTRAN (delayed transformation) algorithms as implemented in PAUP*
(version 4.0). All results are obtained using relative penalties for horizontal gene transfer and deletions
of 2:1 and the DELTRAN algorithm. The relative penalty value of 2:1 was previously shown to be
biologically realistic13. We found 101 metabolic genes that were putatively transferred since the
separation of the two lineages. Only 2 of them have a significant fitness contribution under the
investigated conditions, confirming the reliability of the method. The transferred genes were excluded
from the statistical analyses as indicated in the main text and in Supplementary Tables 3. Because all
Buchnera genes have unquestionable E. coli orthologues, this reduced network represents a
reconstruction of that part of the ancestral metabolic network that is relevant to the evolution of
Buchnera lineages.
3
2
1
3
4
5
6
100
100
100
100
100
76
100
100
100
100
100
Escherichia coli K12
Shigella flexneri 2a 2457T
100 Escherichia coli EDL933
Escherichia coli O6
100
Salmonella typhimurium
100
100
Salmonella enterica
Yersinia pestis Medievalis
100
Photorhabdus luminescens
Buchnera aphidicola Sg
100
100 Buchnera aphidicola APS
Buchnera aphidicola Bp
100
Wigglesworthia brevipalpis
99
Blochmannia floridanus
Pasteurella multocida
95
100 Haemophilus influenzae
Haemophilus ducreyi
Vibrio vulnificus CMCP6
100
95 Vibrio vulnificus YJ016
100 Vibrio parahaemolyticus
100
Vibrio cholerae
Photobacterium profundum
Shewanella oneidensis
Pseudomonas syringae
100
100 Pseudomonas putida
Pseudomonas aeruginosa
Coxiella burnetii
Xanthomonas axonopodis
100
Xanthomonas campestris
100
Xylella fastidiosa 9a5c
100
Xylella fastidiosa 700964
Neisseria meningitidis A
100
100 Neisseria meningitidis B
Chromobacterium violaceum
98
Bordetella bronchiseptica
100
100 Bordetella parapertussis
100
Ralstonia solanacearum
Nitrosomonas europaea
Brucella suis
100
100 Brucella melitensis
Rhizobium loti
100
Rhizobium meliloti
100
100
Agrobacterium tumefaciens Cereon
Bradyrhizobium japonicum
100
100
Rhodopseudomonas palustris
Caulobacter crescentus
Rickettsia prowazekii
100
100 Rickettsia conorii
Wolbachia sp. wMel
Desulfovibrio vulgaris
95
94 Geobacter sulfurreducens
Bdellovibrio bacteriovorus
Helicobacter pylori 26695
100
100 Helicobacter pylori J99
Outgroup
100 Helicobacter hepaticus
Wolinella succinogenes
Campylobacter jejuni
99
99
}
Supplementary Figure 1. Phylogenetic tree of 56 proteobacterial species
Numbers with arrows indicate the branches on which horizontal gene transfer events leading to E. coli
K12 were considered in the paper. Numbers to the right of internal branches give bootstrap proportions
supporting each branch.
4
III. Impact of block deletions on minimal reaction sets
Deletions may frequently affect genomic regions larger than single genes (especially in the initial stage
of genome reduction)14. To systematically explore the impact of block deletions on minimal reaction
sets, we further developed the basic evolutionary model described in the main text. Specifically, we
examined the effect of block deletions on: i) the size of minimal reaction sets and ii) the similarity in
reaction content across simulations. The annotated E. coli K12 genome was used as a starting point15
(we did not attempt to reconstruct the gene order of ancestral species). The modified algorithm is as
follows:
1) We remove a randomly chosen block of contiguous genes in the genome. Under the
reasonable assumption that deletion size follows an exponential distribution, the probability of
deleting a block with n genes is P(n) = qn, where q (0<q<1) specifies the exact shape of the
distribution.
2) We calculate the impact of deleting these genes on the production rate of major biosynthetic
components. Deletion of genes not present in the reconstructed E. coli metabolic network16
was assumed to have no effect on growth. While this is clearly not the case, this method
allows us not only to ignore unknown genetic interactions among non-metabolic genes, but
(more importantly) it overestimates the possible sizes of deleted blocks.
3) If growth rate is nearly unaffected (in this case less than 1% effect), the deletion is assumed to
be viable, and the genes are considered to be permanently lost (see Methods). Otherwise, the
genes are restored to the genome. This procedure is repeated until no further enzymes in the
metabolic network can be deleted; i.e. all remaining genes are essential for survival of the cell.
4) This simulation is repeated 500 times, with each run providing an independent evolutionary
outcome.
The results of simulations are summarized in Supplementary Table 4.
5
IV. Receiver Operating Characteristic Curve (ROC) method
Two values were assigned to each E. coli metabolic gene: a binary variable representing presence or
absence of the gene in a given Buchnera genome, and the number of occurrences of the gene in 500
simulated reduced genomes. For each cut-off (c) between 1 and 500, we took those genes that are
present in at least c of the simulated genomes as those predicted to be also present in each of the
Buchnera strains. Figure 2a plots the number of true-positive predictions against the number of falsepositive predictions for each cut-off. The empirical area under this ROC curve was then calculated as in
ref 17. The area under the curve gives the probability that a reaction preserved in Buchnera is present in
more simulated reduced networks than a reaction lost since divergence from E. coli. As this statistic is
equivalent to the quantity calculated in Mann-Whitney statistical tests17, we used the Mann-Whitney U
test for the null hypothesis that the area under the ROC curve is 0.5 (i.e. the method has no
discriminatory value).
6
V. Systematic identification of cofactors synthesized by Wigglesworthia
Although physiological studies indicate that Wigglesworthia most likely provides thiamine, pantothenic
acid, pyridoxine, biotin and folic acid to its host18, genomic data suggest that it has also retained the
ability to synthesize some additional cofactors (e.g. protohaeme19, 20, ubiquinone20). To explore
systematically the set of cofactors most likely synthesized by Wigglesworthia, given its gene content,
we sought the biomass composition that maximised the accuracy of the evolutionary simulation.
First, we compiled a list of potentially relevant metabolites from the ‘Cofactor and Prosthetic
Group Biosynthesis’ sub-system of the metabolic network16 (see table below). Note that compounds
already included in the biomass equation of E. coli (NAD, FAD) and the five cofactors known to be
provided to the host18 were treated as fixed components of the biomass equation and not listed here.
Next, we added each of these candidate metabolites to the biomass equation one at a time and repeated
the evolutionary simulations. The metabolite resulting in the best prediction was kept and a new round
of simulations was started, adding again each of the remaining compounds to the biomass one at a time
(greedy algorithm). This iterative procedure resulted in a considerable increase of prediction accuracy
from 0.758 to 0.844 (as measured by the area under the ROC curve) by complementing the biomass
with haeme O, ubiquinone and glutathione (see table below).
Importantly, these results, while showing partial agreement with previous suggestions, shed
some new light on the metabolic capability of Wigglesworthia: Although synthesis of ubiquinone had
been proposed previously20, we report that synthesis of haeme O in addition to protohaeme19, 20
substantially improves the prediction. Haeme O is a prosthetic group of cytochrome o terminal
oxidase21 and is synthesized by transferring of a farnesyl group to protoheme by haeme O synthase22
(protohaeme + farnesyl diphosphate + H2O → haeme O + pyrophosphate). Simulations run by
including haeme O in the biomass equation correctly predict not only the presence of haeme O
synthase in Wigglesworthia, but also the retention of the pathway leading to farnesyl diphosphate.
Furthermore, our evolutionary simulations predict that the ability to synthesise glutathione is also
retained by the symbiont. Moreover, we found that addition of cobalamin, sirohaeme or menaquinone
to the biomass decreased the accuracy of prediction, suggesting that these compounds cannot be
produced by Wigglesworthia.
Compound added to
biomass
Menaquinone 8
Ubiquinone-8
5,6,7,8-Tetrahydrofolate
Protohaeme
Sirohaeme
Haeme O
Riboflavin
Cob(I)alamin
Flavin mononucleotide
Reduced glutathione
Effect on prediction
accuracy
−
+
0
0
−
+
0
−
0
+
7
Modelling conditions used for simulations of minimal metabolic networks
under nutrient-rich conditions
Supplementary Table 1.a List of major biosynthetic components and their contributions to the biomass
of E. coli
Biochemical knowledge on the biomass composition of E. coli is represented in the model as a drain of
precursors and building blocks into biomass, that is a biochemical equation where the stoichiometric
coefficients of the metabolites reflect their cellular concentrations (mmol / g dry weight). The biomass
equation of the E. coli model can be found in the additional data file 1 of ref 16.
contribution
(mmol / g dry
weight)
0.05
5x10-5
0.488
0.0010
0.281
0.229
0.229
45.73
1.29x10-4
6.0x10-6
0.154
0.203
45.56
0.09
0.276
0.428
0.0084
0.326
0.146
0.00215
0.205
0.0070
3.0x10-6
0.241
0.402
component
contribution
(mmol / g dry
weight)
0.126
0.087
0.0247
0.0254
0.0254
0.0247
1.0x10-5
0.25
0.25
0.582
5.0x10-5
1.3x10-4
4.0x10-4
0.001935
0.0276
4.64x10-4
0.176
0.21
5.2x10-5
0.035
0.054
0.131
0.0030
0.136
5-Methyltetrahydrofolate
Acetyl-CoA
L-Alanine
AMP
L-Arginine
L-Asparagine
L-Aspartate
ATP
Cardiolipin
Coenzyme A
Glycogen
GTP
H2O
L-Histidine
L-Isoleucine
L-Leucine
Lipopolysaccharide
L-Lysine
L-Methionine
NAD
L-Serine
Spermidine
Succinyl-CoA
L-Threonine
L-Valine
8
component
CTP
L-Cysteine
dATP
dCTP
dGTP
dTTP
FAD
L-Glutamine
L-Glutamate
Glycine
NADH
NADP
NADPH
Phosphatidylethanolamine
Peptidoglycan
Phospatidylglycerol
L-Phenylalanine
L-Proline
Phosphatidylserine
Putrescine
L-Tryptophan
L-Tyrosine
UDPglucose
UTP
Supplementary Table 1.b List of metabolites available for uptake
The specific uptake rates of individual compounds were not constrained in these simulations, but the
total carbon influx was limited to 60 mmol/g/h.
component
(R)-Pantothenate
(S)-Propane-1,2-diol
1,5-Diaminopentane
2-Dehydro-3-deoxy-Dgluconate
2-Oxoglutarate
3-(3-hydroxyphenyl)propionate
3-hydroxycinnamic acid
4-Aminobutanoate
Acetaldehyde
Acetate
Acetoacetate
Adenine
Adenosine
Allantoin
Ammonium ion
AMP
Butyrate (n-C4:0)
Choline
Citrate
CO2
Cob(I)alamin
Cyanate
Cytidine
Cytosine
Deoxyadenosine
Deoxycytidine
Deoxyguanosine
Deoxyinosine
Deoxyuridine
D-Fructose
D-Galactarate
D-Galactonate
D-Galactose
D-Galacturonate
D-Glucarate
D-Gluconate
D-Glucosamine
D-Glucose
D-Glucose 6-phosphate
D-Glucuronate
D-Glyceraldehyde
Dihydroxyacetone
Dimethyl sulfide
Dimethyl sulfoxide
D-Lactate
D-Mannitol
component
D-Mannose
D-Mannose 6-phosphate
D-Ribose
D-Sorbitol
component
L-Serine
L-Tartrate
L-Threonine
L-Tryptophan
D-Xylose
Ethanol
L-Tyrosine
L-Valine
Fe2+
Formate
Fumarate
Galactitol
gamma-butyrobetaine
Glycerol
Glycerol 3-phosphate
Glycine
Glycine betaine
Glycolate
Guanine
Guanosine
H+
H2O
Hexadecanoate (n-C16:0)
Hypoxanthine
Indole
Inosine
K+
Lactose
L-Alanine
L-Arabinose
L-Arginine
L-Asparagine
L-Aspartate
L-Carnitine
L-Cysteine
L-Fucose
L-Fucose 1-phosphate
L-Glutamate
L-Glutamine
L-Histidine
L-Idonate
L-Isoleucine
L-Leucine
L-Lysine
L-Methionine
L-Phenylalanine
L-Proline
L-Rhamnose
Maltohexaose
Maltopentaose
Maltose
Maltotetraose
Maltotriose
Melibiose
Meso-2,6-Diaminoheptanedioate
N-Acetyl-D-glucosamine
N-Acetyl-D-mannosamine
N-Acetylneuraminate
Nicotinamide adenine dinucleotide
Nicotinate
Nitrate
Nitrite
NMN
O2
Octadecanoate (n-C18:0)
Ornithine
Phenylpropanoate
Phosphate
Putrescine
Pyruvate
Sodium
Spermidine
Succinate
Sucrose
Sulfate
Taurine
Tetradecanoate (n-C14:0)
Thiamin
Thiosulfate
Thymidine
Trehalose
Trimethylamine
Trimethylamine N-oxide
Uracil
Urea
Uridine
Xanthine
Xanthosine
9
Modelling conditions used in the simulations of Buchnera gene content evolution
Biochemical knowledge on the biomass composition of E. coli is represented in the model as a drain of
precursors and building blocks into biomass, that is a biochemical equation where the stoichiometric
coefficients of the metabolites reflect their cellular concentrations (mmol / g dry weight). The biomass
equation of the E. coli model (as can be found in additional data file 1 of ref16) was complemented with
riboflavin, as physiological evidence indicates that Buchnera provides riboflavin23 in addition to
essential amino acids24-28 to the host aphid (the amount of riboflavin was set to the arbitrary value of
0.01). Although the table below presents the biomass composition of E. coli, we also repeated the
evolutionary simulations with a biomass equation containing two-fold higher levels of essential amino
acids, reflecting the fact that these metabolites are also supplied to the host. Importantly, this
modification had virtually no effect on the accuracy of reaction content predictions (for example in the
case of Buchnera aphidicola bp the area under the ROC curve only changed from 0.8016 to 0.8013).
Supplementary Table 2.List of major biosynthetic components and their contributions to the biomass of
Buchnera
contribution
(mmol / g dry
weight)
0.05
5x10-5
0.488
0.0010
0.281
0.229
0.229
45.73
1.29x10-4
6.0x10-6
0.154
0.203
45.56
0.09
0.276
0.428
0.0084
0.326
0.146
0.00215
0.205
0.0070
3.0x10-6
0.241
0.402
component
contribution
(mmol / g dry
weight)
0.126
0.087
0.0247
0.0254
0.0254
0.0247
1.0x10-5
0.25
0.25
0.582
5.0x10-5
1.3x10-4
4.0x10-4
0.001935
0.0276
4.64x10-4
0.176
0.21
5.2x10-5
0.035
0.054
0.131
0.0030
0.136
0.01
5-Methyltetrahydrofolate
Acetyl-CoA
L-Alanine
AMP
L-Arginine
L-Asparagine
L-Aspartate
ATP
Cardiolipin
Coenzyme A
Glycogen
GTP
H2O
L-Histidine
L-Isoleucine
L-Leucine
Lipopolysaccharide
L-Lysine
L-Methionine
NAD
L-Serine
Spermidine
Succinyl-CoA
L-Threonine
L-Valine
10
component
CTP
L-Cysteine
dATP
dCTP
dGTP
dTTP
FAD
L-Glutamine
L-Glutamate
Glycine
NADH
NADP
NADPH
Phosphatidylethanolamine
Peptidoglycan
Phospatidylglycerol
L-Phenylalanine
L-Proline
Phosphatidylserine
Putrescine
L-Tryptophan
L-Tyrosine
UDPglucose
UTP
Riboflavin
Metabolites available for uptake
Based on biochemical evidence26 glucose and glutamate were allowed to enter the system (the total
flux of carbon uptake was limited to 60 mmol/g/h and the ratio of glucose to glutamate was not
specified). In addition, unconstrained uptake of the following compounds was allowed:
CO2, Fe2+, H+, H2O, K+, Na+, NH4, O2, phosphate, SO42- .
11
Modelling conditions used in the simulations of Wigglesworthia gene content
evolution
It has been reported that Wigglesworthia provides thiamine, pantothenic acid, pyridoxine, biotin and
folic acid to its host18. Thus, the biomass equation of E. coli stoichiometric model16 was complemented
with the above metabolites. We slightly modified the previously compiled E. coli network to enable the
synthesis of thiamine and biotin. More specifically, based on the available biochemical evidence29, we
enabled the secretion of 4-Hydroxybenzyl alcohol and S-Adenosyl-4-Methylthio-2-Oxobutanoate.
The early steps of biotin synthesis pathway is unknown. To circumvent this problem, we enabled the
uptake of the first known intermediary product, Pimeloyl-CoA.
Supplementary Table 3a.List of major biosynthetic components and their contributions to the biomass
of Wigglesworthia
contribution
(mmol / g dry
weight)
0.05
5x10-5
0.488
0.0010
0.281
0.229
0.229
45.73
1.29x10-4
6.0x10-6
0.154
0.203
45.56
0.09
0.276
0.428
0.0084
0.326
0.146
0.00215
0.205
0.0070
3.0x10-6
0.241
0.402
0.126
0.087
component
contribution
(mmol / g dry
weight)
0.0247
0.0254
0.0254
0.0247
1.0x10-5
0.25
0.25
0.582
5.0x10-5
1.3x10-4
4.0x10-4
0.001935
0.0276
4.64x10-4
0.176
0.21
5.2x10-5
0.035
0.054
0.131
0.0030
0.136
0.01*
0.01*
0.01*
0.01*
0.01*
5-Methyltetrahydrofolate
Acetyl-CoA
L-Alanine
AMP
L-Arginine
L-Asparagine
L-Aspartate
ATP
Cardiolipin
Coenzyme A
Glycogen
GTP
H2O
L-Histidine
L-Isoleucine
L-Leucine
Lipopolysaccharide
L-Lysine
L-Methionine
NAD
L-Serine
Spermidine
Succinyl-CoA
L-Threonine
L-Valine
CTP
L-Cysteine
component
dATP
dCTP
dGTP
dTTP
FAD
L-Glutamine
L-Glutamate
Glycine
NADH
NADP
NADPH
Phosphatidylethanolamine
Peptidoglycan
Phospatidylglycerol
L-Phenylalanine
L-Proline
Phosphatidylserine
Putrescine
L-Tryptophan
L-Tyrosine
UDPglucose
UTP
Thiamine
(R)-Pantothenate
Pyridoxine
Biotin
7,8-Dihydrofolate
* The amount of these cofactors was set to the arbitrary value of 0.01, which is higher than those
cofactors already present in the E. coli biomass, reflecting the fact that these metabolites are provided
for the host. We note that setting the contribution of these compounds to an alternative value of 0.1
had virtually no effect on the accuracy of predictions (data not shown).
12
Due to lack of physiological or biochemical information on input metabolites consumed by
Wigglesworthia glossinidia, we attempted to compile a list of compounds that are most likely present
inside an insect cell and for which transport reactions are also annotated in the E. coli model16. As an
approximation, we obtained the metabolite list of Drosophila melanogaster from the literature curated
MetaCyc database30 (it is the only annotated insect genome in MetaCyc). As we have no systematic
data on the availability of these compounds on a quantitative scale, we did not constrain their specific
uptake rates, only limited the total carbon influx to 60 mmol/g/h.
Despite the wide range of compounds allowed to enter the simulated network at the outset of
the simulations, we observe that the simulations predict the evolutionary retention of transport
reactions with reasonable accuracy (area under the ROC curve for transport processes is 0.738). For
example, a large fraction (>80%) of simulated minimal metabolic networks import leucine, valine and
isoleucine, which is in agreement with the presence of a branched-chain amino acid transport system in
Wigglesworthia19. Moreover, we also run simulations where nutrient uptake was fixed throughout the
simulations: only those nutrients were allowed for which the appropriate transport genes are retained in
Wigglesworthia. Very similar results were obtained with this modification, even after excluding
transport genes from analysis (area under ROC curve 0.81). This suggests that the model is robust to
modifications of input conditions.
13
Supplementary Table 3b List of metabolites available for uptake
component
component
2-Oxoglutarate
4-Aminobutanoate
Acetaldehyde
Acetate
Acetoacetate
Adenine
Adenosine
Allantoin
Ammonium ion
AMP
Choline
Citrate
CO2
Cytidine
Cytosine
D-Alanine
Deoxyadenosine
Deoxyinosine
D-Fructose
D-Galactose
D-Glucose
D-Glucose 6-phosphate
D-Lactate
D-Mannose 6-phosphate
Ethanol
Fe2+
Formate
Fumarate
Glycerol
Glycerol 3-phosphate
Glycine
Guanine
Guanosine
H+
H2O
Hypoxanthine
Indole
Inosine
K+
Lactose
L-Alanine
L-Arginine
L-Asparagine
L-Aspartate
L-Cysteine
L-Fucose
L-Glutamate
L-Glutamine
L-Histidine
L-Isoleucine
L-Lactate
L-Leucine
L-Lysine
L-Malate
L-Methionine
L-Phenylalanine
L-Proline
L-Serine
L-Threonine
L-Tryptophan
L-Tyrosine
L-Valine
Maltose
Na+
NAD
NMN
O2
Ornithine
Phosphate
Pyruvate
Succinate
Sucrose
Sulfate
Thymidine
Trehalose
Uracil
Urea
Uridine
Xanthine
Xanthosine
14
Supplementary Figure 2. Comparison of reaction content of simulated and
Wigglesworthia glossinidia metabolic networks.
a) Predictive accuracy for all possible cutoffs (ROC curve). Overall accuracy and statistics (area
under curve): biomass composition derived from physiological data only, AUC=0.758 (blue dots),
biomass with optimised co-factor composition, AUC=0.844 (red dots). All results are highly
significant, P<10-25.
15
b) Presence/absence of reactions in Wigglesworthia glossinidia, averaged over genes within defined
ranges of presence/absence in the simulated minimal reaction-sets. Statistics: biomass composition
derived from physiological data only, χ2=222.23, df=4, P<10-46, biomass with optimised co-factor
composition, χ2=317.06, df=4, P<10-66.
16
Supplementary Table 4. The impact of block deletions on the properties of
minimal minimal networks
The Table shows mean ± standard deviation. The fraction of shared simulation reactions across
minimal reaction sets and the number of reactions across simulated sets remain unchanged, regardless
of the mean size of block deletions. However, fewer simulation steps are necessary to reach minimal
reactions sets if block deletions are allowed. For more details, see Supplementary Methods.
Model
Number of
Number of
Fraction of shared
Number of reactions in
metabolic genes
successful
reactions across
simulated minimal
deleted at a
deletion steps to
minimal reaction
reactions sets
single step
reach the
sets
(Mean±Standard
minimal
deviation)
reaction set
Only single
1±0
658.08±6.47
0.78±0.046
288.63±9.63
1.92±0.06
546.44±0.06
0.77±0.045
288.57±9.41
7.18±0.35
345.29±0.34
0.77±0.044
287.74±9.30
gene deletions
Block deletion
(q=0.5)
Block deletion
(q=0.9)
17
Supplementary Table 5. Accuracy of reaction content predictions
When calculating the fraction of positive hits, specificity and sensitivity, reactions absent in some, but
not all simulated networks were excluded. Sensitivity is defined as the fraction of preserved reactions
correctly predicted by the model and specificity is the fraction of lost reactions correctly identified by
the model. Where indicated, reactions horizontally transferred into E. coli were excluded from the
analysis. % of positive hits indicates the fraction of true positives and true negatives. Abbreviations:
Bp, Buchnera aphidicola, endosymbiont of Baizongia pistaciae; APS, Buchnera aphidicola,
endosymbiont of Acyrthosiphon pisum; Sg, Buchnera aphidicola, endosymbiont of Schizaphis
graminum; all – reactions present in all of the three Buchnera genomes. Results for Wigglesworthia
were obtained by using the biomass equation with optimised cofactor composition (see Supplementary
Methods).
Growth-rate
Cut-off used in
the simulations
Number of
reactions
investigated
All reactions*
(N=873)
0.01
No horizontally
transferred
reactions to E.
coli (N=789)
All reactions*
(N=873)
0.1
No horizontally
transferred
reactions to E.
coli (N=789)
Species to which the
simulation results are
compared
Accuracy and
standard error of
prediction17
% of
Sensitivity Specificity
(measured
positive hits
(%)
(%)
analogous to Fig.
2a in main text)
Buchnera aphidicola Bp
Buchnera aphidicola Sg
Buchnera aphidicola APS
0.802 (0.020)
0.800 (0.019)
0.794 (0.019)
76.8
78.1
78.4
85.0
83.0
80.7
74.4
76.4
77.5
Reactions present in all
three Buchnera strains
0.790 (0.022)
74.7
85.0
72.3
0.844 (0.017)
0.787 (0.021)
0.786 (0.020)
0.780 (0.020)
84.4
74.5
76.0
76.3
86.0
85.0
83.0
80.7
83.7
71.1
73.2
74.3
0.774 (0.023)
72.2
85.0
68.7
0.835 (0.017)
0.794 (0.021)
0.794 (0.019)
0.788 (0.019)
83.1
76.3
77.7
78.0
86.0
83.9
81.9
79.5
81.5
74.1
76.3
77.4
0.781 (0.022)
74.1
83.8
71.8
0.825 (0.017)
0.779 (0.021)
0.780 (0.020)
0.774 (0.020)
83.7
73.8
75.4
75.8
87.0
83.9
81.9
79.5
81.8
70.6
72.9
74.1
0.764 (0.023)
71.4
83.8
68.1
0.814 (0.018)
82.1
87.0
78.6
Wigglesworthia glossinidia
Buchnera aphidicola Bp
Buchnera aphidicola Sg
Buchnera aphidicola APS
Reactions present in all
three Buchnera strains
Wigglesworthia glossinidia
Buchnera aphidicola Bp
Buchnera aphidicola Sg
Buchnera aphidicola APS
Reactions present in all
three Buchnera strains
Wigglesworthia glossinidia
Buchnera aphidicola Bp
Buchnera aphidicola Sg
Buchnera aphidicola APS
Reactions present in all
three Buchnera strains
Wigglesworthia glossinidia
* Reactions without annotated genes are not included in the analyses.
18
Supplementary Table 6. Accuracy of reaction content prediction in different
metabolic subsystems
(a) The area under the ROC curve and its standard error17 was calculated for each metabolic subsystem
where at least one reaction was preserved in all Buchnera strains and one reaction was lost in at least
one Buchnera strain. (b) The area under the ROC curve and its standard error17 was calculated for each
metabolic subsystem of Wigglesworthia (prediction is from simulations run with the optimised biomass
composition, Supplementary Methods).
Table 6a Buchnera
Metabolic subsystem
Fractions of
Area under
reactions retained Number of
Standard
the ROC
in Buchnera
reactions
error
curve
genomes
Threonine and Lysine Metabolism
Pyruvate Metabolism
Anaplerotic Reactions
Pentose Phosphate Cycle
Oxidative phosphorylation
Glycine and Serine Metabolism
Folate Metabolism
Transport, Extracellular
Glycolysis/Gluconeogenesis
Alternate Carbon Metabolism
Arginine and Proline Metabolism
Citrate Cycle (TCA)
Cell Envelope Biosynthesis
Cofactor and Prosthetic Group Biosynthesis
Membrane Lipid Metabolism
Methionine Metabolism
Cysteine Metabolism
Tyrosine, Tryptophan, and Phenylalanine
Metabolism
Nucleotide Salvage Pathways
Valine, leucine, and isoleucine metabolism
Methylglyoxal Metabolism
Purine and Pyrimidine Biosynthesis
19
0.923
0.285
0.142
0.777
0.125
0.25
0.5
0.027
0.555
0.031
0.151
0.076
0.121
0.196
0.16
0.285
0.142
13
7
7
9
40
8
6
146
18
127
33
13
74
122
25
7
7
1
1
1
1
0.971
0.833
0.833
0.83
0.825
0.81
0.757
0.75
0.72
0.669
0.667
0.6
0.583
0
0
0
0
0.053
0.199
0.184
0.127
0.1
0.132
0.133
0.299
0.101
0.066
0.161
0.259
0.339
0.7
0.364
0.4
0.333
0.333
20
85
15
3
24
0.583
0.564
0.5
0.5
0.492
0.139
0.066
0.157
0.408
0.127
Table 6b Wigglesworthia
Metabolic subsystem
Threonine and Lysine Metabolism
Pentose Phosphate Cycle
Histidine Metabolism
Alternate Carbon Metabolism
Methionine Metabolism
Cofactor and Prosthetic Group Biosynthesis
Membrane Lipid Metabolism
Tyrosine, Tryptophan, and Phenylalanine
Metabolism
Glycine and Serine Metabolism
Cell Envelope Biosynthesis
Pyruvate Metabolism
Glycolysis/Gluconeogenesis
Glutamate Metabolism
Transport, Extracellular
Purine and Pyrimidine Biosynthesis
Nucleotide Salvage Pathways
Oxidative Phosphorylation
Alanine and Aspartate Metabolism
Anaplerotic Reactions
Folate Metabolism
Methylglyoxal Metabolism
Citrate Cycle (TCA)
Arginine and Proline Metabolism
Fraction of
reactions
reatained in Number of
Wigglesworthia reactions
genome
investigated Area under
the ROC Standard
curve
error
0.692
13
1
0
0.555
9
1
0
0.1
10
1
0
0.031
127
0.958
0.07
0.285
7
0.9
0.164
0.622
122
0.859
0.033
0.6
25
0.85
0.077
0.2
0.25
0.364
0.285
0.555
0.2
0.047
0.916
0.317
0.15
0.444
0.285
0.5
0.333
0.461
0.212
20
20
8
74
7
18
5
146
24
85
40
9
7
6
3
13
33
0.844
0.833
0.827
0.8
0.756
0.75
0.748
0.739
0.731
0.64
0.6
0.6
0.556
0.5
0.452
0.398
0.131
0.199
0.054
0.217
0.115
0.327
0.109
0.159
0.062
0.131
0.201
0.259
0.253
0.408
0.165
0.117
Metabolic pathways showing variability in retention across simulations
To quantify metabolic flexibility, pairwise similarities of reaction contents of 500 simulated minimal
networks were calculated for each functional subsystem of the metabolism (defined as in ref 16). Subsystems where all reactions are lost in either Buchnera or in the simulated genomes were not
considered here. Many subsystems show little or no variability among simulated genomes (e.g. amino
acid synthesis groups, membrane and cofactor metabolism). Most of the variability among genomes lie
within transport processes, alternate carbon metabolism, anapleurotic reactions, pyruvate metabolism
and in nucleotide salvage pathways. The simulations shown result from boundary conditions that
mimic the evolution of Buchnera (Supplementary Table 2).
Supplementary Table 7.
Metabolic subsystem
(as in ref 16)
Alternate Carbon Metabolism (N=127)
Transport, Extracellular (N=146)
Anaplerotic Reactions (N=7)
Pyruvate Metabolism (N=7)
Nucleotide Salvage Pathways (N=85)
Folate Metabolism (N=6)
Arginine and Proline Metabolism (N=33)
Oxidative Phosphorylation (N=40)
Glycine and Serine Metabolism (N=8)
Threonine and Lysine Metabolism (N=13)
Glycolysis/Gluconeogenesis (N=18)
Cysteine Metabolism (N=7)
Citrate Cycle (TCA) (N=13)
Membrane Lipid Metabolism (N=25)
Purine and Pyrimidine Biosynthesis (N=24)
Tyrosine, Tryptophan, and Phenylalanine Metabolism (N=20)
Cofactor and Prosthetic Group Biosynthesis (N=122)
Pentose Phosphate Cycle (N=9)
Cell Envelope Biosynthesis (N=74)
Valine, Leucine, and Isoleucine Metabolism (N=15)
Histidine Metabolism (N=10)
Methionine Metabolism (N=7)
21
Average pairwise similarity of
minimal networks
(Jaccard index)
0.52
0.57
0.58
0.72
0.76
0.87
0.89
0.9
0.91
0.92
0.92
0.94
0.95
0.95
0.96
0.98
0.99
0.99
1
1
1
1
Supplementary Table 8. Examples of alternative metabolic solutions during
reductive evolution of Buchnera
The Table provides examples of alternative solutions (pathways) to three metabolic problems.
Simulated metabolic networks contain one or other of the two alternative solutions, but never both
(data not shown). Importantly, Buchnera strains also retained only one of these pathways (proteins
encoded by Buchnera genomes are underlined in the table). Descriptions are from Ecocyc31.
Metabolic solution #1
(enzymes and reactions
retained by Buchnera)
Metabolic solution #2
(enzymes and reactions lost
by Buchnera)
Description
Ribonucleoside-diphosphate
reductase (NrdA & NrdB)
Ribonucleoside-triphosphate
reductase (NrdD)
NDP + reduced thioredoxin →
dNDP + H2O + oxidized
thioredoxin
NTP + reduced thioredoxin
→ dNTP + H2O + oxidized
thioredoxin
Deoxyribonucleotides are synthetised from
ribonucleotides. It can be achieved by two
ways in E. coli: either NDP is reduced to
dNDP (ribonucleoside-diphosphate
reductase), or NTP is reduced to dNTP. This
latter reaction is catalysed by an oxygen
sensitive enzyme, thus E. coli uses it under
anaerobic conditions.
Buchnera strains retained the oxygen
requiring ribonucleoside-diphosphate
reductase.
Phosphate H+ symporter
(PitA or PitB)
Phosphate ABC transporter
complex (pstA & pstB &
pstC & pstS)
H+ex + Pi ex ↔ H+in + Pi in
ATPin + H2Oin + Pi ex →
ADPin + H+in + 2 Pi in
Step 1: Acetate kinase (AckA
or TdcD or PurT)
Acetyl-CoA synthetase (Acs)
acetate + ATP ↔ acetyl-P +
ADP
acetate + ATP + CoA →
acetyl-CoA + AMP + PPi
Phosphate can be imported by E. coli either
by a low affinity transporter PitA (or PitB)
driven by proton motive force, or by a high
affinity ABC transporter system (pstABCS),
which is ATP-dependent.
Buchnera strains retained the low affinity
PitA transporter.
There are two distinct pathways by which E.
coli activates acetate to acetyl-CoA. AcetylCoA synthetase (ACS) catalyzes one of them.
It is thought that this ACS pathway functions
in a mainly anabolic role, scavenging acetate
present in the extracellular medium.
Buchnera strains retained the acetate kinase phosphotransacetylase pathway (proteins
AckA and Pta).
Step 2: Phosphotransacetylase
(Pta or EutD)
acetyl-P + Coa ↔ acetyl-CoA +
Pi
(ex = extracellular metabolite, in = intracellular metabolite)
22
Supplementary Table 9. Cross comparison of Buchnera and Wigglesworthia
reaction content predictions
Although the metabolic functions of the endosymbionts Buchnera and Wigglesworthia differ
significantly, their reaction contents show considerable overlap (0.38 – 0.40). Even a model that
predicts gene content evolution in Buchnera with 100% accuracy would explain a large fraction of the
gene content evolution in Wigglesworthia as genes lost in these lineages show significant overlap.
Therefore, it is important to investigate whether the differences in the boundary conditions between
Buchnera and Wigglesworthia simulations are substantial enough to specifically predict the reaction
content of Buchnera and Wigglesworthia, respectively.
Our cross-comparisons show that simulations constructed to reflect the lifestyle of Buchnera
predict the reaction content of Buchnera strains with higher accuracy than those of Wigglesworthia.
Similarly, simulations set up for Wigglesworthia predict the reaction content of Wigglesworthia more
successfully than those of Buchnera strains (a biomass equation with optimised cofactor composition
was used for Wigglesworthia simulations, see Supplementary Methods).
Simulation
Genome
Buchnera
Wigglesworthia
Buchnera aphidicola Bp
Buchnera aphidicola Sg
Buchnera aphidicola APS
Wigglesworthia glossinidia
Buchnera aphidicola Bp
Buchnera aphidicola Sg
Buchnera aphidicola APS
Wigglesworthia glossinidia
Accuracy of reaction content
prediction
(area under the ROC curve
and its standard error)
0.802 (0.020)
0.800 (0.019)
0.794 (0.019)
0.708 (0.021)
0.764 (0.022)
0.775 (0.020)
0.756 (0.020)
0.844 (0.017)
23
Supplementary Table 10. Physiologically coupled reactions are frequently lost
together in endosymbionts
To identify physiologically coupled enzyme sets in the E.coli network, we followed a previously
described protocol31.We considered a condition where all external nutrients are available; this scenario
gives a condition-insensitive underestimate of physiologically coupled reactions. Briefly, the analysis is
based on fixing the flux through one reaction, and then maximising and minimizing the flux through all
other reactions in turn. We concentrated on fully coupled reaction pairs. Assuming that endosymbiont
networks are close to minimal, one would expect that such reactions are retained or lost together. We
identified 619 fully coupled reaction pairs by flux coupling analysis32 in E. coli. Fully coupled
reactions have fixed flux ratios and are always used together in steady state flux distributions of the
network (e.g. steps of a linear pathway). We found 619 fully coupled reaction pairs. Having identified
coupled reactions, we counted the number of cases when both members of a coupled reaction pair are
either lost or retained in a given endosymbiont. We found that 74-84% of coupled pairs are lost or
retained together, which is significantly higher than 50-55% expected by chance (P < 10-5 for all
species, calculated by randomising pairings of coupled reactions). This conclusion remains when
horizontally transferred genes are excluded from the analysis (data not shown).
Note however, that as most reactions have more than one fully coupled reaction partner, this
analysis is not suitable for measuring the fraction of compulsory reactions in endosymbionts. One
could get a better view of this process by analysing physiologically coupled reaction sets. In these sets,
all reaction pairs are fully coupled to each other. The 619 fully coupled reaction pairs in E.coli
comprise 100 independent, fully coupled reactions sets. As these sets can only fulfil their function if all
of their constituent reactions are present, one would expect to find either all or none of the coupled
reactions in endosymbionts. Reactions present in a set missing some of their reactions may be
considered superfluous. 13-20% of the reactions in endosymbionts fulfil this requirement (data not
shown). This figure is still likely to be an overestimate, however, if endosymbionts (but not E.coli) can
uptake some of the intermediate metabolites.
Species
% of coupled reaction pairs
where both members are
either lost or retained
% of reaction pairs
expected by chance
Buchnera aphidicola Bp
83.8
55.1
Buchnera aphidicola Sg
73.8
52.6
24
Buchnera aphidicola APS
79.3
51.2
Wigglesworthia glossinidia
76.5
50.4
25
Supplementary Table 11. List of 140 bacterial species investigated and their
lifestyles
Species
lifestyle
Species
lifestyle
Buchnera aphidicola APS
Buchnera aphidicola Bp
Buchnera aphidicola Sg
Blochmannia floridanus
Wigglesworthia brevipalpis
Aeropyrum pernix
Aquifex aeolicus
Bacillus halodurans
Deinococcus radiodurans
Halobacterium sp. NRC-1
Methanobacterium thermautotrophicum
Methanococcus jannaschii
Methanococcus maripaludis
Methanopyrus kandleri
Oceanobacillus iheyensis
Photobacterium profundum
Pyrobaculum aerophilum
Pyrococcus abyssi
Pyrococcus furiosus
Pyrococcus horikoshii
Sulfolobus solfataricus
Sulfolobus tokodaii
Thermoanaerobacter tengcongensis
Thermoplasma acidophilum
Thermoplasma volcanium
Thermotoga maritima
Thermus thermophilus
Nostoc sp. PCC 7120
Bacillus subtilis
Caulobacter crescentus
Chlorobium tepidum
Chromobacterium violaceum
Clostridium acetobutylicum
Corynebacterium efficiens
Corynebacterium glutamicum
Corynebacterium glutamicum 13032
Desulfovibrio vulgaris
Escherichia coli K12
Geobacter sulfurreducens
Gloeobacter violaceus
Lactococcus lactis
Listeria innocua
Methanosarcina acetivorans
Methanosarcina mazei
Pasteurella multocida
Photorhabdus luminescens
endosymbiont
endosymbiont
endosymbiont
endosymbiont
endosymbiont
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
pathogen
pathogen
intracellular
pathogen
Nitrosomonas europaea
Prochlorococcus marinus SS120
Prochlorococcus marinus MIT9313
Prochlorococcus marinus CCMP1378
Pseudomonas putida
Rhodopirellula baltica
Rodopseudomonas palustris
Shewanella oneidensis
Streptomyces avermitilis
Streptomyces coelicolor
Synechococcus elongatus
Synechococcus sp. WH8102
Synechocystis sp. PCC6803
Streptococcus mutans
Agrobacterium tumefaciens Cereon
Agrobacterium tumefaciens WashU
Bacillus anthracis
Bacillus cereus ATCC 10987
Bacillus cereus ATCC 14579
Bordetella bronchiseptica
Bordetella parapertussis
Bordetella pertussis
Borrelia burgdorferi
Brucella melitensis
Brucella suis
Campylobacter jejuni
Clostridium perfringens
Clostridium tetani
Corynebacterium diphtheriae
Enterococcus faecalis
Escherichia coli O157:H7
Escherichia coli EDL933
Escherichia coli O6
Fusobacterium nucleatum
Haemophilus ducreyi
Haemophilus influenzae
Helicobacter hepaticus
Helicobacter pylori 26695
Helicobacter pylori J99
Leptospira interrogans 56601
Leptospira interrogans L1-130
Listeria monocytogenes EGD
Listeria monocytogenes F2365
Mycobacterium bovis
Neisseria meningitidis A
Neisseria meningitidis B
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
free living
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
Ureaplasma parvum
26
Supplementary Table 11. List of 140 bacterial species investigated and their lifestyles (continued).
Species
lifestyle
Porphyromonas gingivalis
Pseudomonas aeruginosa
Pseudomonas syringae
Ralstonia solanacearum
Salmonella enterica
Salmonella typhi
Salmonella typhimurium
Shigella flexneri 2a 301
Shigella flexneri 2a 2457T
Staphylococcus aureus MW2
Staphylococcus aureus Mu50
Staphylococcus aureus N315
Streptococcus agalactiae III
Streptococcus agalactiae V
Streptococcus pneumoniae TIGR4
Streptococcus pneumoniae R6
Streptococcus pyogenes M1
Streptococcus pyogenes MGAS8232
Streptococcus pyogenes MGAS315
Streptococcus pyogenes SSI-1
Treponema denticola
Treponema pallidum
Vibrio cholerae
Vibrio parahaemolyticus
Vibrio vulnificus YJ016
Xanthomonas axonopodis
Xanthomonas campestris
Xylella fastidiosa 9a5c
Xylella fastidiosa 700964
Yersinia pestis CO92
Yersinia pestis KIM
Chlamydia muridarum
Chlamydia pneumoniae AR39
Chlamydia pneumoniae CWL029
Chlamydia pneumoniae J138
Chlamydia trachomatis
Chlamydophila caviae
Chlamydophila pneumoniae TW183
Coxiella burnetii
Mycobacterium leprae
Phytoplasma Onion yellows
Rickettsia conorii
Rickettsia prowazekii
Wolbachia sp. wMel
Mycoplasma genitalium
Mycoplasma penetrans
Mycoplasma pneumoniae
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
pathogen
obligate intracellular
obligate intracellular
obligate intracellular
obligate intracellular
obligate intracellular
obligate intracellular
obligate intracellular
obligate intracellular
obligate intracellular
obligate intracellular
obligate intracellular
obligate intracellular
obligate intracellular
obligate intracellular
obligate intracellular
obligate intracellular
27
Supplementary Table 12. Endosymbiont genes without orthologues in E. coli K12
Supplementary table 12.a Buchnera aphidicola genes with no orthologs in E. coli
Gi number
Gene
Annotation
Blast to E. coli K-12
Best hits from Blast to all of
Genbank exc. Buchnera
hypothetical protein (many
species) / alpha-hemolysin
(Bacillus)
flagellar protein FliJ
(Shewanella)
27904528
yidD
hypothetical protein
no significant hit
27904577
fliJ
flagellar FliJ protein
no significant hit
27904578
fliK
flagellar FliK protein
no significant hit
rpoB (Plasmodium)
27904675
grpE
GrpE protein 2
best hit has other ortholog
(16130533)
GrpE protein/HSP-70
cofactor/heat shock protein
(many species)
27904706
ftsL
cell division protein
FtsL homolog
best hit is cell division
protein (16128076,
e=0.002)
cell division protein FtsL
(Haemophilus)
27904850
aroQ
27904996
trpG
27905000
yba3
28191368
repA2
28191366
repA1
3-dehydroquinate
dehydratase
anthranilate synthase
component II
best hit has other ortholog
(16129224)
hypothetical protein
no significant hit
replication associated
protein
replication associated
protein
no significant hit
3-dehydroquinate dehydratase
(Erwinia)
anthranilate synthase
component II (Shigella)
no significant hit outside
Buchnera
no significant hit
repA1 (Sodalis)
no significant hit
repA (Sodalis)
28
Supplementary Table 12b. Buchnera aphidicola sg genes with no orthologs in E. coli
Gi number
Gene
Annotation
Blast to E. coli K-12
21672309
yidD
hypothetical protein
no significant hit
21672465
grpE
GrpE
best hit has other
ortholog (16130533)
Best hits from Blast to all of
Genbank exc. Buchnera
hypothetical protein (many
species) | alpha-hemolysin
(Bacillus)
GrpE protein/HSP-70 cofactor
(Photorhabdus)
21672467
smpA
small protein A
no significant hit
no significant hit anywhere
21672642
secG
protein-export membrane
protein SecG
no significant hit
21672710
ribD1
riboflavin biosynthesis protein
RibD
best hit has other
ortholog (16128399)
21672822
Yba4
hypothetical protein
no significant hit
paramyosin (Boophilus)
10954447
repA1
replication associated protein
no significant hit
repA1 (Sodalis)
16129145
repA2
replication associated protein
no significant hit
repA1 (Sodalis)
29
no significant hit outside
Buchnera
bifunctional:
diaminohydroxyphosphoribosyla
minopyrimidine deaminase +
uracil reductase (Escherichia
coli K-12)
Supplementary Table 12c. Buchnera aphidicola sp genes with no orthologs in E. coli
Gi
number
Gene
10957102
trpG2
anthranilate synthase small
subunit
best hit has other
ortholog (16129224)
15617003
aroD
type II 3-dehydroquinase
no significant hit
15617059
ribD1
riboflavin deaminase
best hit has other
ortholog (16128399)
10957104
repA1
10957106
repA2
15616700
yba1
15616863
Annotation
replication-associated protein
RepA1
replication-associated protein
RepA2
Blast to E. coli K-12
Best hits from Blast to all of
Genbank exc. Buchnera
anthranilate synthase component II
(Erwinia)
3-dehydroquinate dehydratase
(Erwinia)
bifunctional:
diaminohydroxyphosphoribosylami
nopyrimidine deaminase + uracil
reductase (Escherichia coli K-12)
no significant hit
repA1 protein (Sodalis)
no significant hit
repA protein (Sodalis)
hypothetical protein
no significant hit
no significant hit outside Buchnera
grpE1
heat shock protein GrpE1
best hit has other
ortholog (16130533)
grpE (Wigglesworthia)
15616941
flgN
flagella synthesis protein FlgN
no significant hit
no significant hit outside Buchnera
15616658
cof
Cof protein
best hit has other
ortholog (49176424)
15616984
secG
protein-export membrane
protein SecG
no significant hit
15617175
yba4
hypothetical protein
no significant hit
10957101
trpG
anthranilate synthase small
subunit
best hit has other
ortholog (16129224)
15616800
yba2
hypothetical protein
no significant hit
30
haloacid dehalogenase-like
hydrolase-like protein
(Leishmania)
biotin carboxylase subunit of
acetyl CoA carboxylase, putative
(Plasmodium)
erythrocyte membrane protein
pfemp3 (Plasmodium)
anthranilate synthase component II
(Erwinia)
hypothetical protein (many
species)
Supplementary Table 12d. Wigglesworthia glossinidia genes with no orthologs in E. coli
Gi
number
Gene
Annotation
Blast to E. coli K-12
Best hits from Blast to all of
Genbank
50470479
-
conjugative transfer
surface exclusion
lipoprotein
no significant hit
TraT+ conjugative transfer: surface
exclusion (Salmonella)
32490800
fliO
hypothetical protein
no significant hit
no significant hit anywhere
32491016
recD
hypothetical protein
no significant hit
32490785
flgN
hypothetical protein
no significant hit
32491035
wg003
hypothetical protein
no significant hit
32491341
wg001
hypothetical protein
no significant hit
50470477
repA
replication protein A
no significant hit
31
exodeoxyribonuclease V alpha chain
(Buchera Bp) ; no other hits
flagella synthesis protein (Erwinia;
e=2e-4); no other hits
hypothetical protein (Photorhabdus) |
Ccm2 + Ccm1 (Proteus) | putative
membrane protein (Yersinia)
probable transmembrane protein
(Blochmania) | putative membrane
protein (Erwinia)
replication protein RepA +
replication initiator and transcription
repressor (Erwinia)
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33
Supplementary Table 13. Metabolic gene content of endosymbionts and simulated minimal genomes
Presence (+) or absence (-) in endosymbiontic genomes of 904 genes annotated in the stoichiometric model of E. coli.
Figure 1c in the main text is based on results of the general simulations (column 7).
Four genes (b1416, b1417, b3767, b3768) are not annotated appropriatelyin the NCBI genome database, and therefore these genes were excluded from all analysis.
Presence/Absence of the ortholog in endosymbiont genomes
Blattner names (E.coli ) Gene name (E.coli ) Buchnera aphidicola
b0002
thrA
+
b0003
thrB
+
b0004
thrC
+
b0007
yaaJ
b0008
talB
b0019
nhaA
b0025
ribF
b0029
lytB
+
b0031
dapB
+
b0032
carA
+
b0033
carB
+
b0036
caiD
b0038
caiB
b0040
caiT
b0048
folA
+
b0049
apaH
+
b0052
pdxA
b0061
araD
b0062
araA
b0063
araB
b0066
sfuC
b0067
sfuB
b0068
sfuA
b0071
leuD
+
b0072
leuC
+
b0073
leuB
+
b0074
leuA
+
b0077
ilvI
+
Buchnera aphidicola Sg
+
+
+
+
+
+
+
+
+
+
+
Buchnera sp
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Wigglesworthia brevipalpis
+
+
+
+
+
+
+
+
+
+
-
Fraction of simulated minimal gene sets with the presence of the gene
General (nutrient rich) conditions
0.34
0.342
0.342
0.01
0.494
0.268
1
0
0.704
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0.802
0.802
0.802
0.802
0.368
Wigglesworthia conditions
Buchnera conditions
0.36
0.092
0.092
0.008
0.444
0.004
1
1
1
0
0
0
0
0
1
0
1
0
0
0
0
0
0
0.744
0.744
0.744
0.744
0.406
0.498
0.606
0.606
0
0.512
0
1
0
1
0.012
0.012
0
0
0
1
0
0
0
0
0
0
0
0
1
1
1
1
0.414
b0078
b0085
ilvH
murE
+
+
+
-
+
+
+
0.368
1
0.406
1
0.414
1
b0086
b0087
b0088
b0090
b0091
b0092
b0096
b0099
b0104
b0109
b0112
b0114
b0115
b0116
b0118
b0120
b0121
b0124
b0125
b0126
b0131
b0133
b0134
b0142
b0154
b0158
b0159
b0160
b0166
b0171
b0173
b0174
b0175
b0179
b0180
b0181
b0182
b0185
b0186
murF
mraY
murD
murG
murC
ddlB
lpxC
mutT
guaC
nadC
aroP
aceE
aceF
lpdA
acnB
speD
speE
gcd
hpt
yadF
panD
panC
panB
folK
hemL
yadT
mtn
dgt
dapD
pyrH
dxr
uppS
cdsA
lpxD
fabZ
lpxA
lpxB
accA
ldcC
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
1
1
1
1
1
0.53
1
0.488
0
0.198
0.636
0
0
1
0.478
0.184
0.186
0
0.03
0.514
0.27
0.27
0.27
1
0
0
0.184
0
0.704
0.668
0
0
1
1
1
1
1
1
0
1
1
1
1
1
0.492
1
0.498
0
0.188
0.7
0.002
0.002
0.934
0.458
1
1
0
0.088
0.506
1
1
1
1
1
0
1
0
1
0.6
1
0
1
1
1
1
1
1
0
1
1
1
1
1
0.51
1
0.48
0
1
0
1
1
1
0.484
1
1
0
0.054
0.536
1
1
1
1
0
0
1
0
1
0.618
0
0
1
1
1
1
1
1
0
b0197
b0198
yaeC
yaeE
-
-
-
-
0.858
0.858
0.808
0.808
0
0
b0199
b0200
b0207
b0212
b0221
b0222
b0238
b0242
b0243
b0273
b0312
b0314
b0323
b0331
b0333
b0334
b0335
b0336
b0337
b0339
b0340
b0341
b0343
b0344
b0347
b0348
b0349
b0350
b0351
b0352
b0353
b0356
b0365
b0366
b0367
b0368
b0369
b0381
b0386
abc
yaeD
yafB
gloB
fadF
gmhA
gpt
proB
proA
argF
betB
betT
yahI
prpB
prpC
prpD
prpE
codB
codA
cynT
cynS
cynX
lacY
lacZ
mhpA
mhpB
mhpC
mhpD
mhpF
mhpE
mhpT
adhC
tauA
tauB
tauC
tauD
hemB
ddlA
proC
+
+
+
-
+
+
+
+
-
+
+
+
+
-
+
+
+
+
0.858
1
0
0
0
1
0.032
0.766
0.766
0.4
0
0
0.314
0
0
0
0
0.08
0.094
0.486
0
0
0
0
0
0
0
0
0.02
0
0
0.01
0
0
0
0
0
0.47
0.912
0.808
1
0
0
0
1
0.104
0.728
0.728
0.388
0
0
0.378
0
0
0
0.04
0.088
0.122
0.494
0
0
0
0
0
0
0
0
0
0
0
0.016
0
0
0
0
1
0.508
0.892
0
1
0
0
0
1
0.046
0.388
0.388
0.502
0
0
0.302
0.002
0.002
0.002
0.014
0
0.122
0.464
0
0
0
0
0
0
0
0
0.044
0
0
0.008
0
0
0
0
0
0.49
1
b0388
b0401
aroL
brnQ
-
-
-
+
0.49
0.84
0.514
0.856
0.52
0
b0403
b0414
b0415
b0417
b0418
b0420
b0421
b0423
b0425
b0428
b0429
b0430
b0431
b0432
b0451
b0469
b0474
b0475
b0477
b0480
b0485
b0505
b0507
b0508
b0509
b0511
b0512
b0514
b0516
b0521
b0522
b0523
b0529
b0576
b0583
b0586
b0593
b0594
b0595
malZ
ribD
ribH
thiL
pgpA
dxs
ispA
thiI
panE
cyoE
cyoD
cyoC
cyoB
cyoA
amtB
apt
adk
hemH
gsk
ushA
ybaS
allA
gcl
hyi
glxR
allP
allB
glxK
allC
arcC
purK
purE
folD
pheP
entD
entF
entC
entE
entB
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
0.004
1
1
0
0.524
0
0
0
0.002
0
1
1
1
1
1
0.036
1
0
0.314
1
0
0
0
0
0
0
0
0
0
0.35
0.72
0.72
1
0.038
0
0
0
0
0
0
1
1
0
0.49
1
1
1
0.03
1
1
1
1
1
1
0.186
1
1
0.224
1
0
0
0.012
0
0.006
0
0
0.01
0
0.272
0.846
0.846
1
0.04
0
0
0
0
0
0
1
1
0
0.46
0
0
0
0
0
1
1
1
1
1
0.126
1
0
0
1
0
0
0.014
0.004
0.008
0
0
0.012
0
0.354
1
1
1
0
0
0
0
0
0
b0596
b0612
entA
citT
-
-
-
-
0
0
0
0
0
0
b0615
b0616
b0617
b0621
b0638
b0639
b0652
b0653
b0654
b0655
b0662
b0674
b0677
b0678
b0679
b0688
b0692
b0693
b0696
b0697
b0698
b0720
b0721
b0722
b0723
b0724
b0726
b0727
b0728
b0729
b0733
b0734
b0750
b0751
b0754
b0755
b0757
b0758
b0759
citF
citE
citD
dcuC
phpB
nadD
gltL
gltK
gltJ
gltI
ubiF
asnB
nagA
nagB
nagE
pgm
potE
speF
kdpC
kdpB
kdpA
gltA
sdhC
sdhD
sdhA
sdhB
sucA
sucB
sucC
sucD
cydA
cydB
nadA
pnuC
aroG
gpmA
galK
galT
galE
+
+
+
-
+
+
+
+
-
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
0
0
0
0
0
1
0
0
0
0
0
0.066
0.028
0
0.02
0.504
0.686
0.014
0
0
0
1
1
1
1
1
1
1
0.956
0.956
0
0
0.198
0.546
0.312
0.34
0
0.002
0.002
0
0
0
0
0
1
0
0
0
0
1
0.086
0
0
0
0.548
0.372
0.45
0
0
0
1
1
1
1
1
0.932
0.932
0.908
0.908
0
0
0.188
0.67
0.304
0.362
0
0.002
0.002
0
0
0
0
0
1
0
0
0
0
0
0.306
0
0
0
0.476
0
0.45
0
0
0
1
1
1
1
1
1
1
0.692
0.692
0
0
1
0
0.326
0.32
0
0
0
b0774
b0775
bioA
bioB
+
+
+
+
+
+
+
+
0
0
1
1
0
0
b0776
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?
-
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?
?
-
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?
?
-
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?
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1
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0
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1
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0.312
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1
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1
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1
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1
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1
1
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cysK
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-
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0
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1
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1
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1
0
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1
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?
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?
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?
?
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1
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b4301
b4321
b4322
b4323
b4381
b4382
b4383
b4384
b4388
b4395
b4407
idnD
idnK
sgcE
gntP
uxuA
uxuB
deoC
deoA
deoB
deoD
serB
gpmB
thiS
+
+
-
+
+
-
+
+
-
-
0.09
0
0.494
0
0
0
0.006
0.006
0.252
0.254
1
0.334
0
0.036
0
0.482
0
0
0
0.006
0.004
0.302
0.34
1
0.332
1
0.032
0
0.486
0
0
0
0.55
0.082
0.6
0.352
1
0.36
0