Figures and figure supplements
Phylogenomic analysis supports the ancestral presence of LPS-outer membranes inthe Firmicutes
Luisa CS Antunes et al
Antunes et al. eLife 2016;5:e14589. DOI: 10.7554/eLife.14589 1 of 18
RESEARCH ARTICLE
Figure 1. Transmission electron microscopy of a member of Negativicutes and a member of Halanaerobiales. Ultrathin sections of high-pressure frozen
cells of the Negativicutes member Megamonas rupellensis (A,C), and the Halanerobiales member Halanaerobium saccharolyticum (B,D). A Gram-
negative like cell wall architecture is visible for both taxa (A,B): a cytoplasmic membrane (CM) surrounding the cytoplasm (C), a thin peptidoglycan layer
(PG), and an outer membrane (OM). Pili-like structures (P) are also visible in M. rupelllensis. In some cases and due to a preparation artifact caused by
swelling of the cells, the OM detaches from the IM creating an enlarged periplasmic space (PP) between two dividing cells (C,D). In these cases, the
peptidoglycan becomes more apparent as it is also the case for an electron dense surface coat (SC), which might represent lipopolysaccharide (LPS) or
a potential S-layer. Scale bars: 200 nm (A,C) and 100 nm (B,D).
DOI: 10.7554/eLife.14589.003
Antunes et al. eLife 2016;5:e14589. DOI: 10.7554/eLife.14589 2 of 18
Research article Cell Biology Genomics and Evolutionary Biology
0.5
Negativicutes
Clostridiales
Thermoanaerobacterales
Lactobacillales
Bacillales
Natranaerobiales
Halanaerobiales
1
1
1
1
1
1
1
1
0.98
1
0.96
1
1
1
1
0.93
1
1
1
1
1
0.67
1
1
0.53
1
1
1
1
1
11
1
1
1
1
1
1
1
1
0.65
1
0.98
1
1
1
1
1
1
1
0.96
1
1
1
1
1
1
1
1
1
0.99
1
1
1
1
1
1
0.99
1
1
1
1
0.99
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0.99
1
1
1
0.78
1
1
1
1
1
0.99
0.97
0.61
1
1
1
1
1
1
0.99
0.99
1
1
1
1
1
1
1
1
1
1
0.83
1
1
1
1
0.96
0.99
0.87
0.88
1
0.99
1
1
0.86
1
1
1
1
1
1
1
0.57
1
1
1
1
1
1
0.82
1
1
1
1
1
1
0.79
1
1
1
1
0.96
1
1
1
1
1
1
1
1
1
0.73
1
1
1
1
1
1
1
1
1
0.99
1
1
1
1
1
1
1
1
1
1
1
1
1
0.57
1
1
1
1
1
1
1
1
1
1
1
1
1
Selenomonas noxia ATCC 43541
Bacillus atrophaeus 1942
Chlamydia trachomatis D_UW-3_CX
Listeria monocytogenes 07PF0776
Amphibacillus xylanus NBRC 15112
Lactobacillus sanfranciscensis TMW 1.1304
Lactobacillus plantarum JDM1
Enterococcus faecalis D32
Bacillus halodurans C-125
Nostoc punctiforme PCC 73102
Streptomyces coelicolor A32
Selenomonas sputigena ATCC 35185
Clostridium perfringens str. 13
Megasphaera sp. UPII 135-E
Ruminococcus albus 7
Bacillus pseudofirmus OF4
Paenibacillus sp. Y412MC10
Anaerococcus prevotii DSM 20548
Paenibacillus mucilaginosus 3016
Orenia marismortui DSM 5156
Selenomonas infelix ATCC 43532
Streptococcus sanguinis SK36
Natranaerobius thermophilus JW_NM-WN-LF
Veillonella ratti ACS-216-V-Col6b
Eubacterium siraeum 70_3
Syntrophomonas wolfei subsp. wolfei str. Goettingen
Megamonas rupellensis DSM 19944
Megasphaera genomosp. type_1 str. 28L
Caldicellulosiruptor obsidiansis OB47
Lactobacillus delbrueckii subsp. bulgaricus 2038
Megasphaera micronuciformis F0359
Ethanoligenens harbinense YUAN-3
Thermosediminibacter oceani DSM 16646
Centipeda periodontii DSM 2778
Deinococcus radiodurans R1
Geobacillus sp. WCH70
Streptococcus pneumoniae 670-6B
Streptococcus salivarius CCHSS3
Thermosinus carboxydivorans Nor1
Selenomonas sp. oral taxon 138 str. F0429
Acidaminococcus fermentans DSM 20731
Selenomonas bovis DSM 23594
Thermobacillus composti KWC4
Thermoanaerobacter wiegelii Rt8.B1
Veillonella atypica KON
Eubacterium infirmum F0142
Veillonella parvula DSM 2008
Thermoanaerobacter italicus Ab9
Halanaerobium hydrogeniformans
Eubacterium rectale ATCC 33656
Solibacillus silvestris StLB046
Lactococcus garvieae ATCC 49156
Clostridium lentocellum DSM 5427
Dialister succinatiphilus YIT 11850
Selenomonas ruminantium subsp. lactilytica TAM6421
Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130
Selenomonas artemidis F0399
Staphylococcus epidermidis ATCC 12228
Bacillus sp. 1NLA3E
Streptococcus oralis Uo5
Ruegeria pomeroyi DSS-3
Oceanobacillus iheyensis HTE831
Candidatus Arthromitus sp. SFB-mouse-Japan
Clostridium beijerinckii NCIMB 8052
Clostridium botulinum A2 str. Kyoto
Lactobacillus buchneri NRRL B-30929
Bacillus licheniformis 9945A
Clostridium saccharolyticum WM1
Streptococcus pyogenes A20
Desulfitobacterium hafniense DCB-2
Desulfotomaculum kuznetsovii DSM 6115
Bacillus pumilus SAFR-032
Desulfotomaculum gibsoniae DSM 7213
Butyrivibrio fibrisolvens 16_4
Clostridium difficile 630
Streptococcus mutans GS-5
Bacteroides thetaiotaomicron VPI-5482
Dialister invisus DSM 15470
Coprococcus catus GD_7
Desulfotomaculum carboxydivorans CO-1-SRB
Mitsuokella sp. oral taxon 131 str. W9106
Halobacillus halophilus DSM 2266
Carboxydothermus hydrogenoformans Z-2901
Clostridium ljungdahlii DSM 13528
Lactococcus lactis subsp. cremoris A76
Clostridium pasteurianum BC1
Moorella thermoacetica ATCC 39073
Lactobacillus casei ATCC 334
Listeria ivanovii subsp. ivanovii PAM 55
Bacillus cereus 03BB102
Aerococcus urinae ACS-120-V-Col10a
Roseburia intestinalis M50_1
Phascolarctobacterium sp. CAG:266
Thermoanaerobacter tengcongensis MB4
Enterococcus casseliflavus EC20
Oenococcus oeni PSU-1
Geobacillus kaustophilus HTA426
Bacillus selenitireducens MLS10
Carnobacterium maltaromaticum LMA28
Macrococcus caseolyticus JCSC5402
Thermaerobacter marianensis DSM 12885
Lactobacillus reuteri DSM 20016
Filifactor alocis ATCC 35896
Lactobacillus johnsonii DPC 6026
Acetobacterium woodii DSM 1030
Candidatus Desulforudis audaxviator MP104C
Clostridium thermocellum ATCC 27405
Thermincola potens JR
Bacillus cellulosilyticus DSM 2522
Desulfotomaculum ruminis DSM 2154
Lactobacillus helveticus CNRZ32
Syntrophobotulus glycolicus DSM 8271
Desulfotomaculum reducens MI-1
Bacillus clausii KSM-K16
Desulfotomaculum acetoxidans DSM 771
Gemella bergeriae ATCC 700627
Alkaliphilus metalliredigens QYMF
Clostridium novyi NT
Bacillus coagulans 2-6
Sporolactobacillus inulinus CASD
Anaeromusa acidaminophila DSM 3853
Listeria innocua Clip11262
Veillonella dispar ATCC 17748
Eubacterium limosum KIST612
Desulfosporosinus acidiphilus SJ4
Clostridium cellulolyticum H10
Alkaliphilus oremlandii OhILAs
Mahella australiensis 50-1 BON
Halanaerobium saccharolyticum subsp. saccharolyticum DSM 6643
Lysinibacillus sphaericus C3-41
Flavobacterium johnsoniae UW101
Caldicellulosiruptor bescii DSM 6725
Succinispira mobilis DSM 6222
Zymophilus raffinosivorans DSM 20765
Faecalibacterium prausnitzii L2-6
Heliobacterium modesticaldum Ice1
Paenibacillus polymyxa E681
Lactobacillus sakei subsp. sakei 23K
Clostridium sticklandii DSM 519
Lactobacillus ruminis ATCC 27782
Thermoanaerobacterium xylanolyticum LX-11
Acetonema longum DSM 6540
Ruminococcus bromii L2-63
Bacillus subtilis subsp. subtilis str. 168
Anaeroglobus geminatus F0357
Pediococcus pentosaceus ATCC 25745
Clostridium cellulovorans 743B
Desulfitobacterium dehalogenans ATCC 51507
Sporomusa ovata DSM 2662
Bacillus cytotoxicus NVH 391-98
Tepidanaerobacter acetatoxydans Re1
Pediococcus claussenii ATCC BAA-344
Rhodopirellula baltica SH 1
Lactobacillus brevis ATCC 367
Anoxybacillus flavithermus WK1
Bacillus megaterium DSM 319
Alicyclobacillus acidocaldarius LAA1
Enterococcus hirae ATCC 9790
Clostridium phytofermentans ISDg
Bacillus anthracis str. Ames Ancestor
Staphylococcus haemolyticus JCSC1435
Exiguobacterium sibiricum 255-15
Mitsuokella multacida DSM 20544
Staphylococcus aureus 04-02981
Brevibacillus brevis NBRC 100599
Megasphaera elsdenii DSM 20460
Halothermothrix orenii H 168
Enterococcus sp. 7L76
Cohnella laeviribosi DSM 21336
Leuconostoc gasicomitatum LMG 18811
Pelosinus fermentans DSM 17108
Bacillus weihenstephanensis KBAB4
Halobacteroides halobius DSM 5150
Ammonifex degensii KC4
Exiguobacterium sp. AT1b
Pelotomaculum thermopropionicum SI
Clostridium tetani E88
Butyrivibrio proteoclasticus B316
Halanaerobium praevalens DSM 2228
Bacillus thuringiensis str. Al Hakam
Finegoldia magna ATCC 29328
Thermoanaerobacter brockii subsp. finnii Ako-1
Phascolarctobacterium succinatutens YIT 12067
Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293
Sulfobacillus acidophilus DSM 10332
Clostridium sp. SY8519
Roseburia hominis A2-183
Lactobacillus fermentum IFO 3956
Tetragenococcus halophilus NBRC 12172
Weissella koreensis KACC 15510
Clostridium saccharoperbutylacetonicum N1-4HMT
Dialister micraerophilus DSM 19965
Thermoanaerobacterium thermosaccharolyticum DSM 571
Bacillus amyloliquefaciens DSM 7
Clostridium acetobutylicum ATCC 824
Enterococcus faecium Aus0004
Syntrophothermus lipocalidus DSM 12680
Megamonas funiformis YIT 11815
Acetohalobium arabaticum DSM 5501
Desulfitobacterium dichloroeliminans LMG P-21439
Phascolarctobacterium sp. CAG:207
Veillonella sp. oral taxon 780 str. F0422
Desmospora sp. 8437
Chlorobium limicola DSM 245
Symbiobacterium thermophilum IAM 14863
Melissococcus plutonius ATCC 35311
Corynebacterium glutamicum ATCC 13032
Geobacillus thermoglucosidasius C56-YS93
Clostridium kluyveri DSM 555
Acidaminococcus intestini RyC-MR95
Oscillibacter valericigenes
Selenomonas flueggei ATCC 43531
Desulfosporosinus meridiei DSM 13257
Trichodesmium erythraeum IMS101
Desulfosporosinus orientis DSM 765
Lactobacillus salivarius CECT 5713
Vibrio fischeri ES114
Eubacterium eligens ATCC 27750
CLASS CLOSTRIDIA
CLASS BACILLI
CLASS CLOSTRIDIA
OUTGROUP
Peptococcaceae
Figure 2. Phylum-level phylogeny of the Firmicutes. Bayesian phylogeny of the Firmicutes based on a concatenation of 47 orthologous ribosomal
proteins comprising 5551 amino acid positions and the CAT+GTR+G4 model. Values at nodes represent Bayesian posterior probabilities. The scale bar
represents the average number of substitutions per site. For details on analyses, see Materials and methods.
DOI: 10.7554/eLife.14589.004
Antunes et al. eLife 2016;5:e14589. DOI: 10.7554/eLife.14589 3 of 18
Research article Cell Biology Genomics and Evolutionary Biology
0.5
Negativicutes
Clostridiales
Thermoanaerobacterales
Lactobacillales
Bacillales
Natranaerobiales
Halanaerobiales
1
1
1
1
1
1
1
1
0.98
1
0.96
1
1
1
1
0.93
1
1
1
1
1
0.67
1
1
0.53
1
1
1
1
1
11
1
1
1
1
1
1
1
1
0.65
1
0.98
1
1
1
1
1
1
1
0.96
1
1
1
1
1
1
1
1
1
0.99
1
1
1
1
1
1
0.99
1
1
1
1
0.99
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0.99
1
1
1
0.78
1
1
1
1
1
0.99
0.97
0.61
1
1
1
1
1
1
0.99
0.99
1
1
1
1
1
1
1
1
1
1
0.83
1
1
1
1
0.96
0.99
0.87
0.88
1
0.99
1
1
0.86
1
1
1
1
1
1
1
0.57
1
1
1
1
1
1
0.82
1
1
1
1
1
1
0.79
1
1
1
1
0.96
1
1
1
1
1
1
1
1
1
0.73
1
1
1
1
1
1
1
1
1
0.99
1
1
1
1
1
1
1
1
1
1
1
1
1
0.57
1
1
1
1
1
1
1
1
1
1
1
1
1
Selenomonas noxia ATCC 43541
Bacillus atrophaeus 1942
Chlamydia trachomatis D_UW-3_CX
Listeria monocytogenes 07PF0776
Amphibacillus xylanus NBRC 15112
Lactobacillus sanfranciscensis TMW 1.1304
Lactobacillus plantarum JDM1
Enterococcus faecalis D32
Bacillus halodurans C-125
Nostoc punctiforme PCC 73102
Streptomyces coelicolor A32
Selenomonas sputigena ATCC 35185
Clostridium perfringens str. 13
Megasphaera sp. UPII 135-E
Ruminococcus albus 7
Bacillus pseudofirmus OF4
Paenibacillus sp. Y412MC10
Anaerococcus prevotii DSM 20548
Paenibacillus mucilaginosus 3016
Orenia marismortui DSM 5156
Selenomonas infelix ATCC 43532
Streptococcus sanguinis SK36
Natranaerobius thermophilus JW_NM-WN-LF
Veillonella ratti ACS-216-V-Col6b
Eubacterium siraeum 70_3
Syntrophomonas wolfei subsp. wolfei str. Goettingen
Megamonas rupellensis DSM 19944
Megasphaera genomosp. type_1 str. 28L
Caldicellulosiruptor obsidiansis OB47
Lactobacillus delbrueckii subsp. bulgaricus 2038
Megasphaera micronuciformis F0359
Ethanoligenens harbinense YUAN-3
Thermosediminibacter oceani DSM 16646
Centipeda periodontii DSM 2778
Deinococcus radiodurans R1
Geobacillus sp. WCH70
Streptococcus pneumoniae 670-6B
Streptococcus salivarius CCHSS3
Thermosinus carboxydivorans Nor1
Selenomonas sp. oral taxon 138 str. F0429
Acidaminococcus fermentans DSM 20731
Selenomonas bovis DSM 23594
Thermobacillus composti KWC4
Thermoanaerobacter wiegelii Rt8.B1
Veillonella atypica KON
Eubacterium infirmum F0142
Veillonella parvula DSM 2008
Thermoanaerobacter italicus Ab9
Halanaerobium hydrogeniformans
Eubacterium rectale ATCC 33656
Solibacillus silvestris StLB046
Lactococcus garvieae ATCC 49156
Clostridium lentocellum DSM 5427
Dialister succinatiphilus YIT 11850
Selenomonas ruminantium subsp. lactilytica TAM6421
Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130
Selenomonas artemidis F0399
Staphylococcus epidermidis ATCC 12228
Bacillus sp. 1NLA3E
Streptococcus oralis Uo5
Ruegeria pomeroyi DSS-3
Oceanobacillus iheyensis HTE831
Candidatus Arthromitus sp. SFB-mouse-Japan
Clostridium beijerinckii NCIMB 8052
Clostridium botulinum A2 str. Kyoto
Lactobacillus buchneri NRRL B-30929
Bacillus licheniformis 9945A
Clostridium saccharolyticum WM1
Streptococcus pyogenes A20
Desulfitobacterium hafniense DCB-2
Desulfotomaculum kuznetsovii DSM 6115
Bacillus pumilus SAFR-032
Desulfotomaculum gibsoniae DSM 7213
Butyrivibrio fibrisolvens 16_4
Clostridium difficile 630
Streptococcus mutans GS-5
Bacteroides thetaiotaomicron VPI-5482
Dialister invisus DSM 15470
Coprococcus catus GD_7
Desulfotomaculum carboxydivorans CO-1-SRB
Mitsuokella sp. oral taxon 131 str. W9106
Halobacillus halophilus DSM 2266
Carboxydothermus hydrogenoformans Z-2901
Clostridium ljungdahlii DSM 13528
Lactococcus lactis subsp. cremoris A76
Clostridium pasteurianum BC1
Moorella thermoacetica ATCC 39073
Lactobacillus casei ATCC 334
Listeria ivanovii subsp. ivanovii PAM 55
Bacillus cereus 03BB102
Aerococcus urinae ACS-120-V-Col10a
Roseburia intestinalis M50_1
Phascolarctobacterium sp. CAG:266
Thermoanaerobacter tengcongensis MB4
Enterococcus casseliflavus EC20
Oenococcus oeni PSU-1
Geobacillus kaustophilus HTA426
Bacillus selenitireducens MLS10
Carnobacterium maltaromaticum LMA28
Macrococcus caseolyticus JCSC5402
Thermaerobacter marianensis DSM 12885
Lactobacillus reuteri DSM 20016
Filifactor alocis ATCC 35896
Lactobacillus johnsonii DPC 6026
Acetobacterium woodii DSM 1030
Candidatus Desulforudis audaxviator MP104C
Clostridium thermocellum ATCC 27405
Thermincola potens JR
Bacillus cellulosilyticus DSM 2522
Desulfotomaculum ruminis DSM 2154
Lactobacillus helveticus CNRZ32
Syntrophobotulus glycolicus DSM 8271
Desulfotomaculum reducens MI-1
Bacillus clausii KSM-K16
Desulfotomaculum acetoxidans DSM 771
Gemella bergeriae ATCC 700627
Alkaliphilus metalliredigens QYMF
Clostridium novyi NT
Bacillus coagulans 2-6
Sporolactobacillus inulinus CASD
Anaeromusa acidaminophila DSM 3853
Listeria innocua Clip11262
Veillonella dispar ATCC 17748
Eubacterium limosum KIST612
Desulfosporosinus acidiphilus SJ4
Clostridium cellulolyticum H10
Alkaliphilus oremlandii OhILAs
Mahella australiensis 50-1 BON
Halanaerobium saccharolyticum subsp. saccharolyticum DSM 6643
Lysinibacillus sphaericus C3-41
Flavobacterium johnsoniae UW101
Caldicellulosiruptor bescii DSM 6725
Succinispira mobilis DSM 6222
Zymophilus raffinosivorans DSM 20765
Faecalibacterium prausnitzii L2-6
Heliobacterium modesticaldum Ice1
Paenibacillus polymyxa E681
Lactobacillus sakei subsp. sakei 23K
Clostridium sticklandii DSM 519
Lactobacillus ruminis ATCC 27782
Thermoanaerobacterium xylanolyticum LX-11
Acetonema longum DSM 6540
Ruminococcus bromii L2-63
Bacillus subtilis subsp. subtilis str. 168
Anaeroglobus geminatus F0357
Pediococcus pentosaceus ATCC 25745
Clostridium cellulovorans 743B
Desulfitobacterium dehalogenans ATCC 51507
Sporomusa ovata DSM 2662
Bacillus cytotoxicus NVH 391-98
Tepidanaerobacter acetatoxydans Re1
Pediococcus claussenii ATCC BAA-344
Rhodopirellula baltica SH 1
Lactobacillus brevis ATCC 367
Anoxybacillus flavithermus WK1
Bacillus megaterium DSM 319
Alicyclobacillus acidocaldarius LAA1
Enterococcus hirae ATCC 9790
Clostridium phytofermentans ISDg
Bacillus anthracis str. Ames Ancestor
Staphylococcus haemolyticus JCSC1435
Exiguobacterium sibiricum 255-15
Mitsuokella multacida DSM 20544
Staphylococcus aureus 04-02981
Brevibacillus brevis NBRC 100599
Megasphaera elsdenii DSM 20460
Halothermothrix orenii H 168
Enterococcus sp. 7L76
Cohnella laeviribosi DSM 21336
Leuconostoc gasicomitatum LMG 18811
Pelosinus fermentans DSM 17108
Bacillus weihenstephanensis KBAB4
Halobacteroides halobius DSM 5150
Ammonifex degensii KC4
Exiguobacterium sp. AT1b
Pelotomaculum thermopropionicum SI
Clostridium tetani E88
Butyrivibrio proteoclasticus B316
Halanaerobium praevalens DSM 2228
Bacillus thuringiensis str. Al Hakam
Finegoldia magna ATCC 29328
Thermoanaerobacter brockii subsp. finnii Ako-1
Phascolarctobacterium succinatutens YIT 12067
Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293
Sulfobacillus acidophilus DSM 10332
Clostridium sp. SY8519
Roseburia hominis A2-183
Lactobacillus fermentum IFO 3956
Tetragenococcus halophilus NBRC 12172
Weissella koreensis KACC 15510
Clostridium saccharoperbutylacetonicum N1-4HMT
Dialister micraerophilus DSM 19965
Thermoanaerobacterium thermosaccharolyticum DSM 571
Bacillus amyloliquefaciens DSM 7
Clostridium acetobutylicum ATCC 824
Enterococcus faecium Aus0004
Syntrophothermus lipocalidus DSM 12680
Megamonas funiformis YIT 11815
Acetohalobium arabaticum DSM 5501
Desulfitobacterium dichloroeliminans LMG P-21439
Phascolarctobacterium sp. CAG:207
Veillonella sp. oral taxon 780 str. F0422
Desmospora sp. 8437
Chlorobium limicola DSM 245
Symbiobacterium thermophilum IAM 14863
Melissococcus plutonius ATCC 35311
Corynebacterium glutamicum ATCC 13032
Geobacillus thermoglucosidasius C56-YS93
Clostridium kluyveri DSM 555
Acidaminococcus intestini RyC-MR95
Oscillibacter valericigenes
Selenomonas flueggei ATCC 43531
Desulfosporosinus meridiei DSM 13257
Trichodesmium erythraeum IMS101
Desulfosporosinus orientis DSM 765
Lactobacillus salivarius CECT 5713
Vibrio fischeri ES114
Eubacterium eligens ATCC 27750
CLASS CLOSTRIDIA
CLASS BACILLI
CLASS CLOSTRIDIA
OUTGROUP
Peptococcaceae
Antunes et al. eLife 2016;5:e14589. DOI: 10.7554/eLife.14589 4 of 18
Research article Cell Biology Genomics and Evolutionary Biology
Figure 2—figure supplement 1. Results of IC congruence test for the 47 ribosomal proteins. IC values were mapped onto the ribosomal protein
concatenation phylogeny shown in Figure 2. Branches in red indicate congruence among markers according to IC tests. Raw results of the test are
provided as Additional Data in Dryad (Antunes et al., 2016).
DOI: 10.7554/eLife.14589.005
Antunes et al. eLife 2016;5:e14589. DOI: 10.7554/eLife.14589 5 of 18
Research article Cell Biology Genomics and Evolutionary Biology
0.3
Ruminococcus bromii L2-63@fusion
Streptococcus oralis Uo5@fusionLactobacillus casei ATCC 334@fusion
Enterococcus casseliflavus EC20@fusion
Clostridium pasteurianum BC1@fusion
Acetonema longum DSM 6540@fusion
Desulfotomaculum reducens MI-1@fusion
Gemella bergeriae ATCC 700627@fusion
Veillonella atypica KON@fusion
Bacillus selenitireducens MLS10@fus
Faecalibacterium prausnitzii L2-6@fusion
Clostridium cellulovorans 743B@fusion
Thermoanaerobacterium xylanolyticum LX-11@fusion
Brevibacillus brevis NBRC 100599@fusion
Enterococcus faecalis D32@fusion
Centipeda periodontii DSM 2778@fusion
Carnobacterium maltaromaticum LMA28@fusi
Selenomonas flueggei ATCC 43531@fusion
Ruegeria pomeroyi DSS-3@fusion
Halothermothrix orenii H 168@fusion
Anaerococcus prevotii DSM 20548@fusion
Orenia marismortui DSM 5156@fusion
Bacillus pseudofirmus OF4@fusion
Candidatus Arthromitus sp. SFB-mouse-Japan@fusion
Pelosinus fermentans DSM 17108@fusion
Zymophilus raffinosivorans DSM 20765@fus
Clostridium lentocellum DSM 5427@fusion
Tepidanaerobacter acetatoxydans Re1@fusion
Clostridium beijerinckii NCIMB 8052@fusion
Listeria innocua Clip11262@fusion
Lactobacillus johnsonii DPC 6026@fusion
Megamonas rupellensis DSM 19944@fusion
Mitsuokella multacida DSM 20544@fusion
Halanaerobium praevalens DSM 2228@fusion
Staphylococcus haemolyticus JCSC1435@fusion
Lactococcus garvieae ATCC 49156@fusion
Candidatus Desulforudis audaxviator MP104C@fusion
Bacillus coagulans 2-6@fusion
Natranaerobius thermophilus JW_NM-WN-LF@fusion
Trichodesmium erythraeum IMS101@fusion
Veillonella parvula DSM 2008@fusion
Acetohalobium arabaticum DSM 5501@fusion
Ethanoligenens harbinense YUAN-3@fusion
Acetobacterium woodii DSM 1030@fusion
Bacillus sp. 1NLA3E@fusion
Alkaliphilus metalliredigens QYMF@fusi
Clostridium acetobutylicum ATCC 824@fusio
Desulfitobacterium dichloroeliminans LMG P-21439@fu
Geobacillus sp. WCH70@fusion
Bacillus licheniformis 9945A@fusion
Vibrio fischeri ES114@fusion
Streptococcus sanguinis SK36@fusion
Leuconostoc gasicomitatum LMG 18811@fusion
Amphibacillus xylanus NBRC 15112@fusion
Halobacillus halophilus DSM 2266@fusion
Moorella thermoacetica ATCC 39073@fusion
Bacillus halodurans C-125@fusion
Bacteroides thetaiotaomicron VPI-5482@fusio
Carboxydothermus hydrogenoformans Z-2901@fus
Selenomonas artemidis F0399@fusion
Lactobacillus reuteri DSM 20016@fusion
Eubacterium siraeum 70_3@fusion
Paenibacillus mucilaginosus 3016@fusio
Caldicellulosiruptor bescii DSM 6725@fusion
Lactobacillus brevis ATCC 367@fusionPediococcus pentosaceus ATCC 25745@fusion
Selenomonas sp. oral taxon 138 str. F0429@fusion
Dialister succinatiphilus YIT 11850@fusi
Succinispira mobilis DSM 6222@fusion
Chlorobium limicola DSM 245@fusion
Heliobacterium modesticaldum Ice1@fusion
Desulfosporosinus acidiphilus SJ4@fusion
Geobacillus kaustophilus HTA426@fusion
Caldicellulosiruptor obsidiansis OB47@fusion
Clostridium kluyveri DSM 555@fusion
Pelotomaculum thermopropionicum SI@fu
Pediococcus claussenii ATCC BAA-344@fusion
Clostridium saccharolyticum WM1@fusi
Lactobacillus sakei subsp. sakei 23K@fusion
Mahella australiensis 50-1 BON@fusion
Clostridium perfringens str. 13@fusion
Anoxybacillus flavithermus WK1@fusion
Clostridium sp. SY8519@fusion
Roseburia intestinalis M50_1@fusion
Lactobacillus salivarius CECT 5713@fusion
Syntrophothermus lipocalidus DSM 12680@fusion
Thermosediminibacter oceani DSM 16646@fusion
Selenomonas infelix ATCC 43532@fusion
Staphylococcus epidermidis ATCC 12228@fusion
Desulfotomaculum carboxydivorans CO-1-SRB@fusi
Thermoanaerobacter brockii subsp. finnii Ako-1@fusion
Exiguobacterium sibiricum 255-15@fusion
Lactococcus lactis subsp. cremoris A76@fusion
Bacillus anthracis str. Ames Ancestor@fusion
Dialister micraerophilus DSM 19965@fusio
Bacillus pumilus SAFR-032@fusion
Lactobacillus delbrueckii subsp. bulgaricus 2038@fusion
Megasphaera micronuciformis F0359@fusi
Clostridium novyi NT@fusion
Thermoanaerobacterium thermosaccharolyticum DSM 571@fusion
Filifactor alocis ATCC 35896@fusion
Thermobacillus composti KWC4@fusion
Lactobacillus buchneri NRRL B-30929@fusion
Alkaliphilus oremlandii OhILAs@fusion
Selenomonas bovis DSM 23594@fusion
Clostridium cellulolyticum H10@fusio
Paenibacillus polymyxa E681@fusion
Bacillus cytotoxicus NVH 391-98@fusion
Clostridium ljungdahlii DSM 13528@fusion
Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293@fusion
Oscillibacter valericigenes@fusion
Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130@fusion
Desulfotomaculum ruminis DSM 2154@fusion
Eubacterium infirmum F0142@fusion
Nostoc punctiforme PCC 73102@fusion
Listeria monocytogenes 07PF0776@fusio
Megasphaera genomosp. type_1 str. 28L@fusion
Sporolactobacillus inulinus CASD@fusion
Thermoanaerobacter italicus Ab9@fusion
Flavobacterium johnsoniae UW101@fusion
Corynebacterium glutamicum ATCC 13032@fusion
Veillonella ratti ACS-216-V-Col6b@fusion
Butyrivibrio fibrisolvens 16_4@fusion
Sporomusa ovata DSM 2662@fusionAnaeromusa acidaminophila DSM 3853@fusi
Thermoanaerobacter tengcongensis MB4@fusion
Desulfitobacterium dehalogenans ATCC 51507@fusion
Bacillus megaterium DSM 319@fusion
Eubacterium limosum KIST612@fusion
Oceanobacillus iheyensis HTE831@fusion
Bacillus weihenstephanensis KBAB4@f
Streptococcus salivarius CCHSS3@fusion
Enterococcus faecium Aus0004@fusion
Clostridium tetani E88@fusion
Sulfobacillus acidophilus DSM 10332@fusion
Desulfotomaculum gibsoniae DSM 7213@fusion
Selenomonas sputigena ATCC 35185@fusion
Lactobacillus plantarum JDM1@fusion
Bacillus amyloliquefaciens DSM 7@fu
Bacillus thuringiensis str. Al Hakam@fusion
Ammonifex degensii KC4@fusion
Deinococcus radiodurans R1@fusion
Desulfotomaculum kuznetsovii DSM 6115@fusion
Roseburia hominis A2-183@fusion
Coprococcus catus GD_7@fusion
Thermaerobacter marianensis DSM 12885@fusion
Chlamydia trachomatis 434_Bu@fusion
Megasphaera elsdenii DSM 20460@fusion
Acidaminococcus fermentans DSM 20731@fusion
Mitsuokella sp. oral taxon 131 str. W9106@fusion
Selenomonas noxia ATCC 43541@fusion
Enterococcus hirae ATCC 9790@fusion
Lactobacillus fermentum IFO 3956@fusion
Macrococcus caseolyticus JCSC5402@fusion
Thermoanaerobacter wiegelii Rt8.B1@fusion
Syntrophomonas wolfei subsp. wolfei str. Goettingen@fusion
Megamonas funiformis YIT 11815@fusion
Veillonella dispar ATCC 17748@fusion
Solibacillus silvestris StLB046@fusion
Butyrivibrio proteoclasticus B316@fusi
Bacillus subtilis subsp. subtilis str. 168@fusion
Aerococcus urinae ACS-120-V-Col10a@fusion
Halanaerobium hydrogeniformans@fus
Paenibacillus sp. Y412MC10@fusion
Alicyclobacillus acidocaldarius LAA1@fusioDesmospora sp. 8437@fusion
Desulfosporosinus meridiei DSM 13257@fusion
Megasphaera sp. UPII 135-E@fusion
Rhodopirellula baltica SH 1@fusion
Bacillus clausii KSM-K16@fusion
Desulfitobacterium hafniense DCB-2@fusion
Phascolarctobacterium sp. CAG 207@fusion
Anaeroglobus geminatus F0357@fusion
Lysinibacillus sphaericus C3-41@fusion
Weissella koreensis KACC 15510@fusion
Finegoldia magna ATCC 29328@fusion
Lactobacillus sanfranciscensis TMW 1.1304@fus
Enterococcus sp. 7L76@fusion
Dialister invisus DSM 15470@fusion
Eubacterium eligens ATCC 27750@fusion
Veillonella sp. oral taxon 780 str. F0422@fusion
Listeria ivanovii subsp. ivanovii PAM 55@fusion
Clostridium phytofermentans ISDg@fusi
Clostridium sticklandii DSM 519@fusion
Bacillus atrophaeus 1942@fusion
Streptomyces coelicolor A32@fusion
Exiguobacterium sp. AT1b@fusion
Streptococcus pneumoniae 670-6B@fusion
Lactobacillus helveticus CNRZ32@fusion
Clostridium thermocellum ATCC 27405@fusion
Bacillus cereus 03BB102@fusion
Streptococcus mutans GS-5@fusion
Phascolarctobacterium succinatutens YIT 12067@fusion
Desulfosporosinus orientis DSM 765@fusion
Bacillus cellulosilyticus DSM 2522@fus
Thermosinus carboxydivorans Nor1@fusi
Melissococcus plutonius ATCC 35311@fusion
Halanaerobium saccharolyticum subsp. saccharolyticum DSM 6643@fus
Cohnella laeviribosi DSM 21336@fusion
Clostridium saccharoperbutylacetonicum N1-4HMT@fusion
Selenomonas ruminantium subsp. lactilytica TAM6421@fusion
Eubacterium rectale ATCC 33656@fusion
Acidaminococcus intestini RyC-MR95@fusion
Tetragenococcus halophilus NBRC 12172@fusion
Lactobacillus ruminis ATCC 27782@fusion
Clostridium difficile 630@fusion
Thermincola potens JR@fusion
Halobacteroides halobius DSM 5150@fusion
Geobacillus thermoglucosidasius C56-YS93@
Oenococcus oeni PSU-1@fusion
Phascolarctobacterium sp. CAG 266@fusion
Clostridium botulinum A2 str. Kyoto@fusion
Ruminococcus albus 7@fusion
Staphylococcus aureus 04-02981@fusion
Streptococcus pyogenes A20@fusion
Syntrophobotulus glycolicus DSM 8271@fusion
Desulfotomaculum acetoxidans DSM 771@fusion
Symbiobacterium thermophilum IAM 14863@fusion
70
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Research article Cell Biology Genomics and Evolutionary Biology
Figure 2—figure supplement 2. Maximum likelihood phylogeny of the Firmicutes. The tree was obtained by PhyML 3.0 based from the same
concatenation of 47 orthologous ribosomal proteins as the Bayesian tree in Figure 2 and the LG+G4 model. Values at nodes represent non-parametric
bootstrap values calculated on 100 replicates of the original dataset. The scale bar represents the average number of substitutions per site.
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Research article Cell Biology Genomics and Evolutionary Biology
Figure 2—figure supplement 3. Results of AU test for 12 alternative topologies. Full results of the test are provided as Additional Data in
Dryad (Antunes et al., 2016).
Figure 2—figure supplement 3 continued on next page
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Research article Cell Biology Genomics and Evolutionary Biology
Figure 2—figure supplement 3 continued
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Research article Cell Biology Genomics and Evolutionary Biology
Perm
ease
ATPas
e
kdsB
kdsA
kdsD
kdsC
waa
M
lptC
lptA
lptB
lptF
G
lpxA
lpxC
waa
M
skp
skp
lepB
fabZ
hypoth
etic
al
hypoth
etic
al (C
OG08
16)
hypoth
etic
al (C
OG43
72)
mre
B-li
ke
murA
tolC
Perm
ease
Hyp
othet
ical
(COG 0
750)
carb
oxyla
se
epsL
tam
B
bamA/ta
mA
skp
lpxD
lpxB
msb
A
lpxK
lpxI
waa
A
ompM
ompM
ompM
skp
skp
spoIID
murA
Peptid
ase
M23
tam
Bsk
plp
xDlp
xB
msb
Alp
xK
kdsB
kdsA
kdsD
kdsC
waa
Mlp
tClp
tAlp
tB
lptF
Glp
xAlp
xCfa
bZ
lpxI
waa
A
hypoth
etic
alflg
Fflg
Aflg
I
hypoth
etic
al
flgG
Hyp
othet
ical
(COG 0
750)
flgH
flgJ
spoIII
Dgln
Qsm
c
hypoth
etic
al (C
OG43
72)
hypoth
etic
al (C
OG08
16)
YkuD
Peptid
ase
S8lp
tGlp
tF
ostA d
omai
n
conta
inin
gprote
in lptC
ostA
HALANAEROBIALESAcetohalobium arabaticum DSM 5501
NEGATIVICUTESAcidaminococcus fermentans DSM 20731
NEGATIVICUTESVeillonella parvula DSM 2008
HALANAEROBIALESHalobacteroides halobius DSM 5150CP003359 REGION: 2416970..246956
NC_014378 REGION: 2287090..2329181
NC_013740 REGION: 1843921..1889918
NC_013520 REGION: 631051..677624
ompM
ompM
bamA/ta
mA
Figure 3. Conserved genomic locus for cell envelope components. Co-localization of the genes coding for LPS synthesis and transport, OMP assembly
and structural OMPs in the Negativicutes and the Halanaerobiales. Representatives of the 2 families of Negativicutes and the 2 families of
Halanaerobiales are shown (for full distribution and accession numbers see Supplementary file 1). Genes are colored according to their functional
class: LPS synthesis and transport (green), OMP assembly (blue), flagellum (light pink), OM-PG attachment (red), hypothetical (brown), efflux (purple)
(see text for discussion). White boxes indicate proteins not known to being related to the OM or non-conserved proteins whose connection with the
OM is unclear. The figure was obtained by EasyFig (Sullivan et al., 2011), where vertical lines represent BLAST hits with a cutoff of 0.0001.
DOI: 10.7554/eLife.14589.008
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Research article Cell Biology Genomics and Evolutionary Biology
A
Negativicutes
Halanaerobiales
Cyanobacteria
Armatimonadetes
Synergistetes
Atribacteria
Dictyoglomi
Deinococcus -Thermus
Spirochaetes
Fusobacteria
Epsilonproteobacteria
Gammaproteobacteria / Betaproteobacteria
Alphaproteobacteria
Chrysiogenetes
Deltaproteobacteria
Thermodesulfobacteria
Deltaproteobacteria
Acidobacteria
Nitrospirae
Deferribacteres
Aquificae
Bacteroidetes/Chlorobi group
Planctomycetes
Chlamydiae/Verrucomicrobia group
Halanaerobiales
Natranaerobiales
Lactobacillales Bacillales
Other Thermoanaerobacterales Other Clostridiales
Clostridiales Family XVIII. Incertae Sedis Clostridiales Family XVII. Incertae Sedis
Negativicutes
Peptococcaceae Thermoanaerobacteraceae
B
0.5
0.91
0.99
0.63
0.92
0.53
1
1
0.83
0.65
1
1
0.63
0.99
1
0.58
1
0.83
1
0.76
1
0.62
1
1
1
0.96
0.99
1
1
1
1
1
0.93
0.52
1
0.99
1
1
0.95
0.99
1
0.95
1
0.93
1
1
1
1
1
1
1
0.53
1
1
1
0.83
0.95
1
1
1
1
1
0.94
1
1
0.59
0.99
1
1
1
0.57
1
1
0.99
0.99
1
0.91
0.7
0.98
0.66
1
0.99
1
1
0.94
0.66
0.99
0.86
0.73
1
0.73
0.53
1
1
1
0.89
0.51
0.62
0.72
0.99
1
1
1
1
1
1
0.98
1
0.99
1
0.99
1
1
1
1
0.88
1
1
1
1
0.99
0.56
0.67
1
0.72
1
0.95
1
1
1
1
Gemmatimonas aurantiaca T-27-Gemmatimonadetes
Leptotrichia buccalis C-1013-b
Leptospirillum ferrooxidans C2-3
Orenia marismortui DSM 5156
Candidatus Protochlamydia amoebophila UWE25
Thiobacillus denitrificans ATCC 25259
Desulfurispirillum indicum S5
Halobacteroides halobius DSM 5150
Propionispira raffinosivorans DSM 20765
Phascolarctobacterium CAG207
Anabaena variabilis ATCC 29413
Cytophaga hutchinsonii ATCC 33406
Halanaerobium hydrogeniformans
Thermanaerovibrio acidaminovorans DSM 6589
Pelodictyon phaeoclathratiforme BU-1
Mitsuokella multacida DSM 20544
Rubinisphaera brasiliensis DSM 5305
Chlamydia muridarum str. Nigg
Hahella chejuensis KCTC 2396
Megasphaera UPII 135
Dictyoglomus turgidum DSM 6724
Aquifex aeolicus VF5
Acetobacter pasteurianus IFO 3283-01
Thermodesulfobacterium geofontis OPF15
Acaryochloris marina MBIC11017
Truepera radiovictrix DSM 17093
Candidatus Solibacter usitatus Ellin6076
Halanaerobium saccharolyticum subsp. saccharolyticum DSM 6643
Megamonas rupellensis DSM 19944
Marinithermus hydrothermalis DSM 14884
Pseudomonas aeruginosa PAO1
Melioribacter roseus P3M-2
Microcystis aeruginosa NIES-843
Selenomonas artemidis F0399
Simkania negevensis Z
Megamonas funiformis YIT 11815
Veillonella dispar ATCC 17748
Rickettsia prowazekii str. Madrid E
Selenomonas ruminantium subsp. lactilytica TAM6421
Chromobacterium violaceum ATCC 12472
Nitrospira defluvii
Prosthecochloris aestuarii DSM 271
Fimbriimonas ginsengisoli Gsoil 348
Bartonella bacilliformis KC583
Thermosinus carboxydivorans Nor1
Trichodesmium erythraeum IMS101
Syntrophobacter fumaroxidans MPOB
Opitutus terrae PB90-1
Phascolarctobacterium CAG266
Akkermansia muciniphila ATCC BAA-835
Nautilia profundicola AmH
Granulicella mallensis MP5ACTX8
Vibrio cholerae O1 biovar El Tor str. N16961
Desulfurobacterium thermolithotrophum DSM 11699
Selenomonas sputigena ATCC 35185
Veillonella parvula DSM 2008
Acidaminococcus fermentans DSM 20731
Cyanothece sp. PCC 7424
Phycisphaera mikurensis NBRC 102666
Anaeroglobus geminatus F0357
Dictyoglomus thermophilum H-6-12
Porphyromonas gingivalis ATCC 33277
Chthonomonas calidirosea T49
Pelobacter carbinolicus DSM 2380
Megasphaera elsdenii DSM 20460
Candidatus Caldatribacterium saccharofermentans OP9-77CS
Chitinivibrio alkaliphilus ACht1-Unclassified Bacteria
Selenomonas infelix ATCC 43532
Ignavibacterium album JCM 16511
Dinoroseobacter shibae DFL 12 = DSM 16493
Sporomusa ovata DSM 2662
Synechococcus elongatus PCC 6301
Leptospira biflexa serovar Patoc strain Patoc 1 Ames
Desulfobulbus propionicus DSM 2032
Acidaminococcus intestini RyC-MR95
Selenomonas flueggei ATCC 43531
Candidatus Caldatribacterium californiense OP9-cSCG
Pelosinus fermentans DSM 17108
Dialister succinatiphilus YIT 1185
Myxococcus xanthus DK 1622
Allochromatium vinosum DSM 180
Phascolarctobacterium succinatutens YIT 12067
Selenomonas noxia ATCC 43541
Pedobacter heparinus DSM 2366
Waddlia chondrophila WSU 86-1044
Nitrospina gracilis 3_211 - Nitrospinae
Acetonema longum DSM 6540
Isosphaera pallida ATCC 43644
Fusobacterium nucleatum subsp. nucleatum ATCC 25586
Arcobacter butzleri ED-1
Meiothermus ruber DSM 1279
Deferribacter desulfuricans SSM1Flexistipes sinusarabici DSM 4947
Anaeromusa acidaminophila DSM 3853
Veillonella oral taxon 780
Acinetobacter baumannii 1656-2
Halanaerobium praevalens DSM 2228
Selenomonas sp. oral taxon 138 str. F0429
Thermodesulfatator indicus DSM 15286
Veillonella atypica KONVeillonella ratti ACS-216-V-Col6b
Megasphaera genomosp type_1 28L
Burkholderia ambifaria AMMD
Rhodopirellula baltica SH 1
Succinispira mobilis DSM 6222
Chlorobaculum parvum NCIB 8327
Bacteroides fragilis 638R
Helicobacter pylori 26695
Stigmatella aurantiaca DW4_3-1
Helicobacter acinonychis str. Sheeba
Halothermothrix orenii H 168
Chrysiogenes arsenatis DSM 11915
Calditerrivibrio nitroreducens DSM 19672
Agrobacterium radiobacter K84
Escherichia coli str. K-12 substr. MG1655
Geobacter uraniireducens Rf4
Alteromonas macleodii str. Black Sea 11
Anaerobaculum mobile DSM 13181
Campylobacter jejuni subsp. jejuni NCTC 11168
Thermodesulfovibrio yellowstonii DSM 11347
Methylacidiphilum infernorum V4
Dialister invisus DSM 15470
Candidatus Cloacimonas acidaminovorans str. Evry-Cloacimonetes
Centipeda periodontii DSM 2778
Selenomonas bovis DSM 23594
Brachyspira hyodysenteriae WA1
Bordetella parapertussis Bpp5
Acetohalobium arabaticum DSM 5501
Aminobacterium colombiense DSM 12261
Acidobacterium capsulatum ATCC 51196
Megasphaera micronuciformis F0359
Dialister micraerophilus DSM 19965
Turneriella parva DSM 21527
Legionella longbeachae NSW150
Coraliomargarita akajimensis DSM 45221
Persephonella marina EX-H1
Gloeobacter violaceus PCC 7421
Mitsuokella sp. oral taxon 131 str. W9106
Ilyobacter polytropus DSM 2926
Figure 4. Phylogenetic tree of core LPS components. (A) Bayesian phylogeny based on a concatenation of orthologs of the four core components of
the LPS biosynthesis pathway (lpxABCD), comprising 898 amino acid positions and the CAT+GTR+G4 model. Values at nodes represent Bayesian
Figure 4 continued on next page
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Research article Cell Biology Genomics and Evolutionary Biology
Figure 4 continued
posterior probabilities. The scale bar represents the average number of substitutions per site. For details on analyses, see Materials and methods. (B)
Schematic representation of the Firmicutes phylum-level phylogeny from Figure 2, onto which putative losses of the OM are mapped (red crosses). See
text for discussion.
DOI: 10.7554/eLife.14589.009
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Research article Cell Biology Genomics and Evolutionary Biology
A
Negativicutes
Halanaerobiales
Cyanobacteria
Armatimonadetes
Synergistetes
Atribacteria
Dictyoglomi
Deinococcus -Thermus
Spirochaetes
Fusobacteria
Epsilonproteobacteria
Gammaproteobacteria / Betaproteobacteria
Alphaproteobacteria
Chrysiogenetes
Deltaproteobacteria
Thermodesulfobacteria
Deltaproteobacteria
Acidobacteria
Nitrospirae
Deferribacteres
Aquificae
Bacteroidetes/Chlorobi group
Planctomycetes
Chlamydiae/Verrucomicrobia group
Halanaerobiales
Natranaerobiales
Lactobacillales Bacillales
Other Thermoanaerobacterales Other Clostridiales
Clostridiales Family XVIII. Incertae Sedis Clostridiales Family XVII. Incertae Sedis
Negativicutes
Peptococcaceae Thermoanaerobacteraceae
B
0.5
1
0.99
0.62
1
0.52
0.95
1
1
1
1
1
0.57
1
1
0.99
1
0.7
1
1
1
1
1
1
0.88
1
1
0.99
1
1
1
Gemmatimonas aurantiaca T-27-Gemmatimonadetes
Leptotrichia buccalis C-1013-b
Leptospirillum ferrooxidans C2-3
Orenia marismortui DSM 5156
Candidatus Protochlamydia amoebophila UWE25
Thiobacillus denitrificans ATCC 25259
Desulfurispirillum indicum S5
Halobacteroides halobius DSM 5150
Propionispira raffinosivorans DSM 20765
Phascolarctobacterium CAG207
Anabaena variabilis ATCC 29413
Cytophaga hutchinsonii ATCC 33406
Halanaerobium hydrogeniformans
Thermanaerovibrio acidaminovorans DSM 6589
Pelodictyon phaeoclathratiforme BU-1
Mitsuokella multacida DSM 20544
Rubinisphaera brasiliensis DSM 5305
Chlamydia muridarum str. Nigg
Hahella chejuensis KCTC 2396
Megasphaera UPII 135
Dictyoglomus turgidum DSM 6724
Aquifex aeolicus VF5
Acetobacter pasteurianus IFO 3283-01
Thermodesulfobacterium geofontis OPF15
Acaryochloris marina MBIC11017
Truepera radiovictrix DSM 17093
Candidatus Solibacter usitatus Ellin6076
Halanaerobium saccharolyticum subsp. saccharolyticum DSM 6643
Megamonas rupellensis DSM 19944
Marinithermus hydrothermalis DSM 14884
Pseudomonas aeruginosa PAO1
Melioribacter roseus P3M-2
Microcystis aeruginosa NIES-843
Selenomonas artemidis F0399
Simkania negevensis Z
Megamonas funiformis YIT 11815
Veillonella dispar ATCC 17748
Rickettsia prowazekii str. Madrid E
Selenomonas ruminantium subsp. lactilytica TAM6421
Chromobacterium violaceum ATCC 12472
Nitrospira defluvii
Prosthecochloris aestuarii DSM 271
Fimbriimonas ginsengisoli Gsoil 348
Bartonella bacilliformis KC583
Thermosinus carboxydivorans Nor1
Trichodesmium erythraeum IMS101
Syntrophobacter fumaroxidans MPOB
Opitutus terrae PB90-1
Phascolarctobacterium CAG266
Akkermansia muciniphila ATCC BAA-835
Nautilia profundicola AmH
Granulicella mallensis MP5ACTX8
Vibrio cholerae O1 biovar El Tor str. N16961
Desulfurobacterium thermolithotrophum DSM 11699
Selenomonas sputigena ATCC 35185
Veillonella parvula DSM 2008
Acidaminococcus fermentans DSM 20731
Cyanothece sp. PCC 7424
Phycisphaera mikurensis NBRC 102666
Anaeroglobus geminatus F0357
Dictyoglomus thermophilum H-6-12
Porphyromonas gingivalis ATCC 33277
Chthonomonas calidirosea T49
Pelobacter carbinolicus DSM 2380
Megasphaera elsdenii DSM 20460
Candidatus Caldatribacterium saccharofermentans OP9-77CS
Chitinivibrio alkaliphilus ACht1-Unclassified Bacteria
Selenomonas infelix ATCC 43532
Ignavibacterium album JCM 16511
Dinoroseobacter shibae DFL 12 = DSM 16493
Sporomusa ovata DSM 2662
Synechococcus elongatus PCC 6301
Leptospira biflexa serovar Patoc strain Patoc 1 Ames
Desulfobulbus propionicus DSM 2032
Acidaminococcus intestini RyC-MR95
Selenomonas flueggei ATCC 43531
Candidatus Caldatribacterium californiense OP9-cSCG
Pelosinus fermentans DSM 17108
Dialister succinatiphilus YIT 1185
Myxococcus xanthus DK 1622
Allochromatium vinosum DSM 180
Phascolarctobacterium succinatutens YIT 12067
Selenomonas noxia ATCC 43541
Pedobacter heparinus DSM 2366
Waddlia chondrophila WSU 86-1044
Nitrospina gracilis 3_211 - Nitrospinae
Acetonema longum DSM 6540
Isosphaera pallida ATCC 43644
Fusobacterium nucleatum subsp. nucleatum ATCC 25586
Arcobacter butzleri ED-1
Meiothermus ruber DSM 1279
Deferribacter desulfuricans SSM1Flexistipes sinusarabici DSM 4947
Anaeromusa acidaminophila DSM 3853
Veillonella oral taxon 780
Acinetobacter baumannii 1656-2
Halanaerobium praevalens DSM 2228
Selenomonas sp. oral taxon 138 str. F0429
Thermodesulfatator indicus DSM 15286
Veillonella atypica KONVeillonella ratti ACS-216-V-Col6b
Megasphaera genomosp type_1 28L
Burkholderia ambifaria AMMD
Rhodopirellula baltica SH 1
Succinispira mobilis DSM 6222
Chlorobaculum parvum NCIB 8327
Bacteroides fragilis 638R
Helicobacter pylori 26695
Stigmatella aurantiaca DW4_3-1
Helicobacter acinonychis str. Sheeba
Halothermothrix orenii H 168
Chrysiogenes arsenatis DSM 11915
Calditerrivibrio nitroreducens DSM 19672
Agrobacterium radiobacter K84
Escherichia coli str. K-12 substr. MG1655
Geobacter uraniireducens Rf4
Alteromonas macleodii str. Black Sea 11
Anaerobaculum mobile DSM 13181
Campylobacter jejuni subsp. jejuni NCTC 11168
Thermodesulfovibrio yellowstonii DSM 11347
Methylacidiphilum infernorum V4
Dialister invisus DSM 15470
Candidatus Cloacimonas acidaminovorans str. Evry-Cloacimonetes
Centipeda periodontii DSM 2778
Selenomonas bovis DSM 23594
Brachyspira hyodysenteriae WA1
Bordetella parapertussis Bpp5
Acetohalobium arabaticum DSM 5501
Aminobacterium colombiense DSM 12261
Acidobacterium capsulatum ATCC 51196
Megasphaera micronuciformis F0359
Dialister micraerophilus DSM 19965
Turneriella parva DSM 21527
Legionella longbeachae NSW150
Coraliomargarita akajimensis DSM 45221
Persephonella marina EX-H1
Gloeobacter violaceus PCC 7421
Mitsuokella sp. oral taxon 131 str. W9106
Ilyobacter polytropus DSM 2926
0.92
0.66
1
1
1
10.99
0.76
0.56 1
1
11
1
1
0.67
0.95
0.95
1
1
0.94
1
1
0.99
0.99
1
1
0.99
0.83
1
1
1
1
0.99
0.99
1
0.73
0.72
1
0.99
1
1
0.53
1
11
1
0.98
0.530.65
11
0.991
1
11
1
1
1
0.83
0.86
0.58
0.89
1
1
0.63
1
0.73
1
0.53
1
0.94
0.95
10.93
0.66
0.93
0.99
0.62
0.83
0.63
1
0.51
1
0.91
1
1
1
0.99
0.720.99
1
0.591
1
0.98
1
1
Antunes et al. eLife 2016;5:e14589. DOI: 10.7554/eLife.14589 13 of 18
Research article Cell Biology Genomics and Evolutionary Biology
Figure 4—figure supplement 1. Results of IC congruence test for the 4 LPS core proteins. IC values were mapped onto the LPS core proteins
concatenation phylogeny shown in Figure 3. Branches in red indicate congruence among markers (IC values>1). Full results of the test are provided as
Additional Data in Dryad (Antunes et al., 2016).
DOI: 10.7554/eLife.14589.010
Antunes et al. eLife 2016;5:e14589. DOI: 10.7554/eLife.14589 14 of 18
Research article Cell Biology Genomics and Evolutionary Biology
LptE
LptA
LptF MsbA
LptCLolA
LolB
LolELolDLolD
LolC
ATP
ADP+Pi
ATP
ADP+Pi
ATP
ADP+Pi
ATP
ADP+Pi
LptB
LptG
LptB
LspALgt Lnt
LpxALpxC/FabZLpxD
ATP
ADP+Pi
UDP-GlcNAc UDP-2,3-
diacylglucosamine LpxILipid X
LpxB
Disaccharide
-1-phosphate
LpxK
Lipid A
UDPUMP
D-Arabinose-5PKdsDKdsAKdsCKdsB
CMP-3-deoxy-D-manno-octulosonate (KDO)
WaaA
Lauroyl-(KDO)2-Lipid
(KDO)2-Lipid A
WaaMLauroyl-ACP
ACP
Skp
Sec
PilQ
LptD
BamDBamE
BamC
BamA
BamB
TamB
OmpM
ATP
ADP+PiFlgH FlgI
FlgA
PilQ
Figure 5. Sketched diagram of inferred characteristics of the diderm Firmicutes cell envelope. The main processes discussed in the text are shown
schematically. Components that were not detected in the genomes of Negativicutes and Halanaerobiales are indicated with a dashed outline and grey
font.
DOI: 10.7554/eLife.14589.011
Antunes et al. eLife 2016;5:e14589. DOI: 10.7554/eLife.14589 15 of 18
Research article Cell Biology Genomics and Evolutionary Biology
NEGATIVICUTES(Pelosinus fermentans
DSM 17108)
NEGATIVICUTES(Selenomonas ruminantium
subs. lactilytica TAM6421)
HALANAEROBIALES(Acetohalobium
arabaticum DSM 5501)
HALANAEROBIALES(Halothermothrix
orenii H 168)
hslV
hsiU
codY
flgB
flgCfli
Efli
Ffli
Gfli
Hfli
Ifli
J
hslV
hsiU
codY
flgB
flgC
fliE
fliF
fliG
fliH
fliI
fliJ
hslV
hsiU
codY
flgB
fliE
fliH
fliI
fliJ
flgB
flgC
fliE
fliF
fliG
fliH
fliI
fliJ
mgtE
fliF
fliG
mgtE
mgtE
mgtE
flgE
flgE
flgE
flgD
fliK
flgD
fliK
flgD
fliK
flbD
flbD
flbD
flbD
amiC
norM
fliL
fliM
fliY
cheY
fliO
fliP
fliQ
fliR
flhB
fliL
fliM
fliY
cheY
fliO
flhB
fliL
fliM
fliY
fliO
fliP
fliQ
fliR
flhB
fliL
fliM
fliY
fliO
fliP
fliQ
fliR
flhB
motA
motB
motA
motB
flhA
flhF
flhG
pilZ
flhA
flhF
flhG
pilZ
flhA
flhF
flhG
pilZ
flhA
flhF
flhG
pilZ
cheB
cheA
cheB
cheA
cheW
cheC
cheD
cheW
cheC
cheD
cheC
cheD
fliA
fliA
BACILLALES
(Bacillus subtilis
subs. subtilis 6051 HGW)
GAMMA-PROTEOBACTERIA(Escherichia coli str. K-12
MG1655)
fliE
fliF
fliG
fliH
fliI
fliJ
mgtE
flgC
yecT
argSch
eY
cheZ
cheB
cheR
tap
tar
cheW
cheA
motA
flhD
motB
flhC
insA
insBfli
Ffli
Gfli
Hfli
I fliJ
emrE
ybcM
ybcL
dsrB
codY
fliA
Hyp
othet
ical
Hyp
othet
ical
Hyp
othet
ical
fliA
Hyp
othet
ical
Hyp
othet
ical
Polym
eras
e
Polym
eras
e
neuB
Hyp
othet
ical
Hyp
othet
ical
Hyp
othet
ical
Hyp
othet
ical
Hyp
othet
ical
Hyp
othet
ical
Hyp
othet
ical
Hyp
othet
ical
Hyp
othet
ical
flgC
Hyp
othet
ical
Hyp
othet
ical
fliP
fliQ
fliR
Hyp
othet
ical
Hyp
othet
ical
Hyp
othet
ical
flgB
hsiU
hsiV
flgE
flgD
fliK
flbDfli
Lfli
Mfli
Ych
eY
fliO
fliP
fliQ
fliR
flhB
flhA
flhF
flhG
cheB
cheA
cheW
cheC
cheD
fliA
ylxL
rpsB
fliK fli
Lfli
Mfli
Nfli
Zrc
sAfli
Pfli
Qfli
R
fliE flh
Bflh
Aflh
E
CLOSTRIDIALES(Thermincola potens JR)
hsiU
codY
fliE
fliF
fliG
fliH
fliI
fliJ
mgtE
flgE
flgD
fliK
flbDfli
Lfli
Mfli
Ych
eY
fliO
fliP
fliQ
fliR
flhB
flhA
flhF
flhG
pilZ cheB
cheA
cheW
cheC
cheD
fliA
napC
hslV
Hyp
othet
ical
Hyp
othet
ical
Hyp
othet
ical
flgBflg
C
NATRANAEROBIALES
(Natranaerobius
thermophilus JW/NM-WN-LF)
flgB
flgC
fliE
fliF
fliG
fliH
fliI
fliJ
mgtE
flgE
flgD
fliK
Hyp
othet
ical
flbD
fliL
fliM
fliY
cheY
fliO
fliP
fliQ
fliR
flhB
motA
motB
flhA
flhF
flhG
pilZ cheB
cheA
cheW
cheC
cheD
codY
Polym
eras
e
fliA
Hyp
othet
ical
Hyp
othet
ical
Figure 5—figure supplement 1. Flagellar gene cluster of Negativicutes and Halanaerobiales. Structure of the region coding for flagellar components
in representative members of Negativicutes and Halanaerobiales, and its conservation with respect to their closely related monoderm relatives
Therminicola potens, and Natranaerobius thermophiles, respectively. By comparison is shown the structure of the operon in Escherichia coli as
representative of a classical diderm. Colors are only meant to highlight synteny.
DOI: 10.7554/eLife.14589.012
Antunes et al. eLife 2016;5:e14589. DOI: 10.7554/eLife.14589 16 of 18
Research article Cell Biology Genomics and Evolutionary Biology
HALANAEROBIALES(Halothermothrix orenii H 168)
HALANAEROBIALES(Acetohalobium arabaticum DSM 5501)
flgH
flgI
flgJ
flgF
flgG
flgE
flgA
flgD
flgB
flgC
flgN
flgM
mvi
M
mvi
Nflg
Kflg
L
rne
pilZ fliA
Polym
eras
e
GAMMA-PROTEOBACTERIA(Escherichia coli str. K-12 substr. MG1655)
csaB
spoVT
flgJ
mre
B
flgG
Hyp
othet
ical
atpD
spoIII
D
spoIID
atpG
atpB
CLOSTRIDIALES(Thermincola potens JR)
flgF
flgH
flgI
flgJ
flgF
flgG
flgA
murA
NEGATIVICUTES(Pelonsinus fermentans DSM 17108)
mre
BPepitd
ase
M23
spoIII
D
spoIID
spoIV
B
ldh
sucD
sucC
Putativ
e
Transp
onase
flgH
flgI
flgJ
flgF
flgG
flgA
NEGATIVICUTES(Selonomonas ruminantium subsp. lactilytica TAM6421)
mre
B
spoIV
B
murA
Acr
iflav
in re
sist
ance
prote
in
Inte
grase
cat
alyt
ic re
gion
Transp
osase
flgF
flgG
murA
NATRANAEROBIALES(Natranaerobius thermophilus JW/NM-WN-LF)
mre
BPepitd
ase
M23
spoIII
D
spoIID
flgJ
M
g
tran
sport
er
Rev
erse
Transc
ripta
se
flgH
flgI
flgJ
flgF
flgG
flgA
murA Pep
itdas
e
M23
spoIII
D
spoIID
spoIV
B
atpD
Hyp
othet
ical
atpB
flgH
flgI
flgJ
flgF
flgG
flgA
rapD fa
bZm
scL
flgF
flgG
mbl
Hyp
othet
ical
ywoH usd
ywoGpucI
ywoF
ywpD yw
pE
ywpFyw
pG
ssbB
BACILLALES(Bacillus subtilis subs subtilis 6051 HGW)
Hyp
othet
ical
S-laye
r pro
tein
lytR
Hyp
othet
ical
Hyp
othet
ical
Hyp
othet
ical
Am
inodeo
xy-
choris
mat
e ly
ae
Hyp
othet
ical
pfkB
Hyp
othet
ical
Hyp
othet
ical
Hyp
othet
ical
Hyp
othet
ical
Hyp
othet
ical
Hyp
othet
ical
Hyp
othet
ical
Figure 5—figure supplement 2. Genomic context of the genes coding for flagellar rings in Halanaerobiales and Negativicutes. Structure of the region
coding for components of the flagellar rings (flgA, flgH, flgI) in representative members of Negativicutes and Halanaerobiales, in comparison with their
closely related monoderm relatives Therminicola potens, and Natranaerobius thermophiles, respectively. The genomic structures in Bacillus subtilis and
Escherichia coli are also shown as the most studied models for monoderm and diderm flagella. Colors are only meant to highlight synteny.
DOI: 10.7554/eLife.14589.013
Antunes et al. eLife 2016;5:e14589. DOI: 10.7554/eLife.14589 17 of 18
Research article Cell Biology Genomics and Evolutionary Biology
pilC pilA pilDpilB pilT pfkA
Phosphogly
cero
muta
se
aroK
Hyp
othet
ical
Pro
tein
Enolase
Hyp
othet
ical
Pro
tein
Hyp
othet
ical
Pro
tein
Phage
Inte
grase
pilMTriosp
hosphat
e is
omer
ase
pilB pilT pilC pilDNucl
eosi
der R
ecogniti
on
prote
in
aroE
rhaT
pilV
pilTpilB pilC pilA pilApilD
O-a
ntigen
Poly
mer
ase
pilT pilW pilin li
ke h
ypoth
etic
al
pilMpilNpilOmre
R
aroE
pilTpilB pilC pilA pilD pilin li
ke h
ypoth
etic
al
Hyp
othet
ical
Pro
tein
HAD h
ydro
lase
HAD h
ydro
lase
aroE
pilQ pilW li
ke p
rote
in
com
F
pilM pilNpilT
pilTpilB pilC pilM pilN pilO pilA pilA pilin li
ke h
ypoth
etic
al
pilin li
ke h
ypoth
etic
al
ATPas
e
Hyp
othet
ical
Pro
tein
Hyp
othet
ical
Pro
tein
Cu a
min
e oxi
dase
Hyp
othet
ical
Pro
tein
pilTpilB pilC pilA pilDaroE
rhaT
Car
DHyp
othet
ical
Pro
tein
pilM pilN pilO pilQ aroK
dhqS
pilin li
ke h
ypoth
etic
al
pilP
aroK
dhqS
aroC
aroC
pilM pilN pilO pilQpilP aroK
dhqS
aroC
pilin li
ke h
ypoth
etic
al
pilin li
ke h
ypoth
etic
al
aroK
aroC
pilDpilin li
ke h
ypoth
etic
al
pilO
Di-G
MP c
ycla
se
Ferrit
in
Hyp
othet
ical
Pro
tein
Hyp
othet
ical
Pro
tein
galE
pilQ Sulfate
bin
ding p
rote
in
Hyp
othet
ical
Pro
tein
uup
GAMMA-PROTEOBACTERIA(Pseudomonas aeruginosa PA01)
pilB pilC
pilApilD
Hyp
othet
ical
Pro
tein
coaE
nadC
Hyp
othet
ical
Pro
tein
pilM pilN pilO pilQpilP aroK
aroB
ponA
HALANAEROBIALES(Halothermothrix orenii H 168)
NATRANAEROBIALES(Natranaerobius thermophilus JW/NM-WN-LF)
pilin li
ke h
ypoth
etic
al
NEGATIVICUTES(Pelonsinus fermentans DSM 17108)
pilin li
ke h
ypoth
etic
al
CLOSTRIDIALES(Thermincola potens JR)
pilin li
ke h
ypoth
etic
al
pilin li
ke h
ypoth
etic
al
pilA pilA
HALANAEROBIALES(Acetohalobium arabaticum DSM 5501)
pilin li
ke h
ypoth
etic
al
NEGATIVICUTES(Veillonella parvula DSM 2008)
Figure 5—figure supplement 3. Structure of the main Type IV pilus cluster in Negativicutes and Halanaerobiales. Structure of the region coding for
components of the type IV pilus in representative members of Negativicutes and Halanaerobiales, in comparison with their closely related monoderm
relatives Therminicola potens, and Natranaerobius thermophiles, respectively. The genomic structure in Pseudomonas aeruginosa is also shown as the
most studied model for diderm type IV pili. Colors are only meant to highlight synteny.
DOI: 10.7554/eLife.14589.014
Antunes et al. eLife 2016;5:e14589. DOI: 10.7554/eLife.14589 18 of 18
Research article Cell Biology Genomics and Evolutionary Biology