Supplementary Information
“What can genome-scale metabolic network reconstructions do for prokaryotic
systematics?”
BISMiS Special issue, AVL
Francisco Barona-Gómez*, Pablo Cruz-Morales and Lianet Noda-García
Supplementary methods.
RpoB-based actinobacterial species-tree (Figures 3 and 4).
The sequences of genes encoding for RpoB were retrieved using BLAST (Altschul et al.
1997). The sequence alignment was performed with ClustalX (Thompson et al. 1994) using
the default parameters, and then edited manually with the Jalview alignment editor
(Waterhouse et al. 2009). The phylogenetic tree was constructed with MrBayes
(Huelsenbeck and Ronquist 2001), using the WAG amino acid substitution model, four
substitution rate categories, and estimating the topology, branch lengths, rate parameters,
the proportion of invariable sites, and the Gamma distribution parameter. Two runs were
performed for one million generations, the runs were sampled every 100 generations, 25 %
of the trees obtained were “burned” and the rest used to obtain the final tree.
Search of enzyme homologs and statistical treatment
For the comparison of in silico reconstructions of the central metabolic pathways (Figure
4), amino acid sequences reported in the GSMR of S. coelicolor (Borodina et al. 2005), M.
tuberculosis (Jamshidi and Palsson 2007) and C. glutamicum (Kjeldsen and Nielsen 2009)
were used as queries (Supplementary Table S2). The amino acid sequences from C.
glutamicum (Kjeldsen and Nielsen 2009) or S. coelicolor (Hiratsuka et al. 2008) were used
as queries for the search of menaquinone biosynthetic enzymes and cardiolipin synthases
homologs (Figure 3), as these were missing genes not included in the original GSMRs. For
short sequences, less than 100 amino acids, a score cut-off equal or superior to 100 was
used. Pathways were curated manually when necessary. These searches were done through
automated BLAST searches (Altschul et al. 1997) in a actinobacterial genomes database
including the 101 organisms shown in figures 3 and 4. The hits were considered as
homologs of a query when the score was equal or superior to 150. For each query reaction
in the metabolic reconstruction, the media (µ) and standard deviation (σ) of the number of
homologs (HN) found among genomes in the database was calculated. Enzymatic
expansions were defined as HN=> µ + σ.
Supplementary Tables.
Table S1. Central Metabolic Pathways analysed.
Pathway
Glycolysis
Pentose Phosphate
Pathway
Citric acid cycle
Amino acids from AKG
Amino acids from THR
and PYR
Amino acids from
Oxalacetate
Amino acids from 3PGA
Amino acids from R5P
Amino acids from E4P
and PEP
Total
Products
Pyruvate, PhosphoenolPyruvate and ATP
Fructose, E-4 Phosphate,
Ribose 5-P
Acetyl CoA, oxalacetate, αKetoglutarate
Glu, Gln, Pro, Arg
Steps
Complexes Querys
10
0
49
8
0
38
8
13
3
1
94
50
Ala,Ile, Leu, Val
14
2
42
Asp, Asn, Thr, Met, Lys
Gly, Ser, Cys
His
18
6
10
0
0
1
64
25
37
Tyr, Phe, Trp
37
17
104
2
9
56
455
Table S2. Comparative analysis of cardiolipin and menaquinone biosynthesis in
Actinobacteria.
PATHWAY
STEP
Bifidobacterium_animalislactisADO11
Bifidobacterium_longumDJO10A
Bifidobacterium_adolescentisATC15703
Tropheryma_whipplei
Tropheryma_whippleiTwist
Leifsonia_xyli_xyliCTCB07
Clavibacter_michiganensis
Brevibacterium_linensBL2
Renibacterium_salmoninarumATCC33209
Arthrobacter_chlorophenolicusA6
Arthrobacter_aurescensTC1
Kocuria_rhizophilaDC2201
Arthrobacter_arilaitensisRe117
Micrococcus_luteusNCTC2665
Citricoccus_CH26A
Kineococcus_radiotoleransSRS30216
Janibacter_HTCC2649
Propionebacterium_acnesKPA171202
Nocardioides_JS614
Catenulispora_acidiphilaDSM44928
Kitasatospora_setaeKM-6054
Streptomyces_hygroscopicusATCC53653
Streptomyces_violaceusnigerTu4113
Streptomyces_bingchenggensis
Streptomyces_clavuligerusATCC27064
Streptomyces_pristinaespiralisATCC25486
Streptomyces_flavogriseusATCC33331
Streptomyces_spACTE
Streptomyces_roseosporusNRRL15998
Streptomyces_roseosporusNRRL11379
Streptomyces_griseus
Streptomyces_griseusXylebKG-1
Streptomyces_spACT1
Streptomyces_venezuelaeATCC10712
Streptomyces_spMG1
Streptomyces_spC
Streptomyces_griseoflavusTu4000
Streptomyces_coelicolorM145
Streptomyces_lividansTK24
Streptomyces_liv1326
Streptomyces_albus
Streptomyces_spSPB74
Streptomyces_spSA3actG
Streptomyces_spTU6071
Streptomyces_spSPB78
Streptomyces_avermitilis
Streptomyces_sviceusATCC29083
Streptomyces_scabiei
Streptomyces_viridochromogenesDSM40736
Streptomyces_ghanaensis
Streptomyces_spE14
Streptomyces_griseoauratiacus
Streptosporangium_roseumDSM43021
Acydothermus_cellulolyticus11B
Thermomonospora_curvataDSM43183
Frankia_alniACN14a
Frankia_EAN1pec
Frankia_SPCcI3
Salinispora_tropica
Salinispora_arenicola
Verrucosispora_marisAB1803
Micromonospora_carbonacea
Micromonospora_aurantiacaATCC27029
Micromonospora_spM42
Micromonospora_spL5
Saccharopolyspora_erythraea
Saccharomonospora_viridisDSM43017
Streptomyces_spAA4
Amycolatopsis_mediterraneiU32
Corynebacterium_amycolatum_SK46
Corynebacterium_kroppenstedtiiDSM44385
Corynebacterium_urealyticumDSM7109
Corynebacterium_jeikeniumK411
Corynebacterium_jeikeiumATCC43734
Corynebacterium_glucuronolyticumATCC51867
Corynebacterium_diphteriae
Corynebacterium_striatumATCC6940
Corynebacterium_accolensATCC49725
Corynebacterium_efficiensYS314
Corynebacterium_glutamicumR
Corynebacterium_glutamicumATCC13032
Nocardia_farcinicaIFM10152
Rhodococcus_equiATCC33707
Rhodococcus_opacusB4
Rhodococcus_jostiiRHA1
Rhodococcus_erythropolisSK121
Rhodococcus_erythropolisPR4
Mycobacterium_vanbaaleniiPYR1
Mycobacterium_gilvumPYRGCK
Mycobacterium_smegmatisMC2155
Mycobacterium_spJLS
Mycobacterium_spMCS
Mycobacterium_spKMS
Mycobacterium_ulceransAgy99
Mycobacterium_marinumM
Mycobacterium_kansasii12478
Mycobacterium_parascrofulaceumATCCBAA614
Mycobacterium_avium104
Mycobacterium_leprae
Mycobacterium_tuberculosisCDC1551
Mycobacterium_bovisAF212297
Chorismate biosynthetic pathway
Futalosine biosynthetic pathway
MenA MenB MenC MenD MenE MenF MenG MenH MqnA
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
0
0
1
0
1
0
0
1
0
0
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
0
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
2
1
1
1
1
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
2
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
1
0
0
0
0
0
1
0
1
0
0
0
0
0
1
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
1
0
0
0
0
1
2
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
1
0
0
0
0
0
1
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
1
0
0
0
0
0
0
0
1
1
1
0
1
1
1
1
1
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
1
1
1
1
1
1
1
0
0
0
0
0
0
0
1
0
1
0
0
0
0
0
1
0
1
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
3
1
1
1
2
0
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
4
1
1
1
2
1
1
1
2
1
1
1
2
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
MqnB
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
0
1
0
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
MqnC
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
0
1
1
1
1
1
1
1
0
0
1
0
1
1
0
1
0
1
1
0
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
MqnD
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
1
3
3
3
3
3
3
3
3
1
3
1
3
3
3
3
3
3
3
3
3
3
1
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
CL Synthase
MK Type
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
0
1
1
1
1
0
1
1
1
1
1
1
1
0
1
0
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
MK-9 (H6 and H8) (1)
MK-9 (H6 and H8) (1)
MK-9 (H6 and H8) (1)
MK-9 (H6 and H8) (1)
MK-9 (H6 and H8) (1)
MK-9 (H6 and H8) (1)
MK-9 (H4) (a) (1)
MK-8 (H2) (2)
MK-8 (H2) (2)
MK-8 (H2) (2)
MK-8 (H2) (2)
MK-8 (H2) (2)
MK-8 (H2) (2)
MK-8 (H2) (2)
MK-8 (H2) (2)
Eukaryotic
1
1
1
0
0
1
0
0
1
0
1
0
0
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Prokaryotic
0
0
0
1
1
1
1
2
0
1
2
1
1
0
1
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
The colours are indicative of: yellow, one ortholog was detected; orange, two or more orthologs were
detected; white or cero, not detected. The MK type data was obteined from (Collins et al. 1977; Collins et al.
1985).
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