Three globin lineages belonging to two structural classes in genomes from the three kingdoms of life -- Vinogradov et al. 102 (32): 11385 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Vinogradov et al. 10.1073/pnas.0502103102.

Supporting Information

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Supporting Table 1
Supporting Table 2
Supporting Materials and Methods
Supporting Table 3
Supporting Table 4
Supporting Figure 3
Supporting Table 5
Supporting Table 6
Supporting Figure 4
Supporting Figure 5
Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
Supporting Figure 10
Supporting Figure 11

Supporting Figure 3

Fig. 3. Alignment of globins based on the myoglobin (Mb)-fold: 44 bacterial flavohemoglobins (FHbs), 21 bacterial 3/3 single-domain globins (SDgbs) (marked by an asterisk), 31 eukaryote FHbs, including those from Giardia lamblia and Dictyostelium discoideum, the Cyanidioschyzon merolae and Thalassiosira pseudonana SDgbs, 106 bacterial 2/2 Hbs (22 2/2 Hb1s, 59 2/2 Hb2s, and 25 2/2 Hb3s), 3 Chlamydomonas reinhardtii, 6 ciliate 2/2 Hbs, 7 plant 2/2 Hbs, and one Thalassiosira pseudonana 2/2 Hb, 42 bacterial globin-coupled sensors (GCSs), and 6 protoglobins (Pgbs).

Supporting Figure 4

Fig. 4. Bayesian phylogenetic tree of all 3/3 single-domain globins (SDgbs). Each sequence is identified by the first three letters of the binomial name, provided in the alignment of sequences shown in Fig. 3. The numbers at the various nodes indicate Bayesian probabilities ´100. The Cyanidioschyzon merolae and Thalassiosira pseudonana SDgbs are shaded in blue.

Supporting Figure 5

Fig. 5. Bayesian phylogenetic tree of all flavohemoglobins, including 3/3 single-domain globins. Each sequence is identified by the first three letters of the binomial name provided in the alignment of sequences shown in Fig. 3. The numbers at the various nodes indicate Bayesian probabilities ´100. Eukaryotes are shaded in blue, Bacteria are shaded in green, 3/3 single-domain globins are labeled with asterisks. The red circles labeled 1-3 indicate putative horizontal gene transfer events.

Supporting Figure 6

Fig. 6. Bayesian phylogenetic tree of all flavohemoglobins based on their complete sequences. Each sequence is identified by the first three letters of the binomial name provided in the alignment of sequences shown in Fig. 3. The numbers at the various nodes indicate Bayesian probabilities ´100. Eukaryotes are shaded in blue, Bacteria are shaded in green. The red circles indicate putative horizontal gene transfer events.

Supporting Figure 7

Fig. 7. Bayesian phylogenetic tree based on the small subunit rRNA sequences of all of the flavohemoglobin-containing organisms. Each sequence is identified by the first three letters of the binomial name. The eukaryotic sequences are shaded.

Supporting Figure 8

Fig. 8. Bayesian phylogenetic tree of all 2/2 Hbs. Each sequence is identified by the first three letters of the binomial name, provided in the alignment of sequences shown in Fig. 3. The numbers at the various nodes indicate Bayesian probabilities ´100. Bacterial 2/2 Hb1s, 2/2 Hb2s, and 2/2 Hb3s are colored red, green, and blue, respectively. Eukaryotic 2/2 Hbs are colored black.

Supporting Figure 9

Fig. 9. The Bayesian phylogenetic tree based on the alignment of 150 sequences (shown in Fig. 10) representing the following groups of globins: 10 plant 3/3 nonsymbiotic plant Hb (light green), 5 plant 3/3 symbiotic Hbs (dark green), 15 bacterial globin-coupled sensors (red), 4 protoglobins (pink), 39 bacterial 2/2 Hbs (purple), 2 Chlamydomonas reinhardtii (orange), 2 ciliate 2/2 Hbs (yellow), 3 plant 2/2 Hbs (brown), Thalassiosira pseudonana 2/2 Hb (black), 20 bacterial flavohemoglobins (blue), 19 bacterial 3/3 single-domain Hbs (blue), 9 eukaryote flavohemoglobins (blue), 1 diplomonad Giardia lamblia, and 1 mycetozoan Dictyostelium discoideum (both blue and boxed), the Cyanidioschyzon merolae and Thalassiosira pseudonana 3/3 single-domain globins (black), and 3 vertebrate (human, bird, and fish) 3/3 neuroglobins, cytoglobins, a- and b-globins and myoglobins (all in gray), and 2 urochordate Hbs.

Supporting Figure 10

Fig. 10. Alignment of 150 globin sequences representing the various globin groups, used in the construction of Bayesian phylogenetic trees: The full version shown is in Fig. 9 and the condensed version is shown in Fig. 1. In order of appearance: 10 plant 3/3 nonsymbiotic plant Hbs, 5 plant 3/3 symbiotic Hbs, 15 bacterial globin-coupled sensors, 4 protoglobins, 9 2/2 Hb1s, 19 2/2 Hb2s, 102/2 Hb3s, 2 Chlamydomonas reinhardtii, 2 ciliate, 3 plant and Thalassiosira pseudonana 2/2 Hbs, 20 bacterial flavohemoglobins, 19 bacterial 3/3 single-domain globins (marked by asterisks), 11 eukaryote flavohemoglobins, including Giardia lamblia and Dictyostelium discoideum, the Cyanidioschyzon merolae and Thalassiosira pseudonana 3/3 single-domain globin, 3 each of vertebrate (human, bird, and fish) 3/3 neuroglobins, cytoglobins, a- and b-globins and myoglobins, and 2 urochordate Hbs.

Supporting Figure 11

Fig. 11. A comparison of 2/2 and 3/3 globin structures. (A) Shows the stereo view of the tertiary structure of Paramecium caudatum (Protein Data Bank ID code 1DLW) 2/2 Hb, the heme (red) and the proximal histidine residue. Helices are labeled according to the conventional globin fold nomenclature (A–H). To highlight the 2/2 structural motif, orange and cyan helix colors were used. (B) Shows the stereo view of the tertary structure of sperm whale myoglobin (Protein Data Bank ID code 1A6M) in an orientation comparable to that of A and with the same color conventions. All structural elements (helices and loops) additional to the 2/2 or 3/3 a-helical sandwich motifs are highlighted in gray. The structures were drawn with MOLSCRIPT (1).

1. Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946–950.

Table 1. Eukaryote genomes

Eukaryote name	State of genome work
Anopheles gambiae	Unfinished
Arabidopsis thaliana	Completed
Aspergillus nidulans	Unfinished
Brachydanio (Danio) rerio	Unfinished
Candida albicans	Unfinished
Caenorhabditis briggsae	Unfinished
Caenorhabditis elegans	Completed
Chlamydomonas reinhardtii	Unfinished
Ciona intestinalis	Completed
Cryptococcus neoformans	Unfinished
Cryptosporidium parvum	Unfinished
Cyanidioschyzon merolae	Completed
Drosophila melanogaster	Unfinished
Encephalitozoon cuniculi	Completed
Entamoeba histolytica	Unfinished
Eremothecium gossypii	Completed
Giardia lamblia	Unfinished
Gibberella zeae	Unfinished
Homo sapiens	Completed
Kluyveromyces lactis	Completed
Leishmania major	Completed
Magnaporthe grisea	Unfinished
Mus musculus	Completed
Neurospora crassa	Unfinished
Oryza sativa	Completed
Plasmodium falciparum	Completed
Plasmodium yoelii yoelii	Unfinished
Rattus norvegicus	Completed
Saccharomyces cerevisiae	Completed
Schizosaccharomyces pombe	Completed
Takifugu (Fugu) rubripes	Completed
Tetraodon nigroviridis	Unfinished
Thalassiosira pseudonana	Completed
Trypanosoma brucei	Completed
Trypanosoma cruzi	Unfinished
Ustilago maydis	Unfinished

Table 2. Microbial database

Organism name	State of genome work
Archea
Aeropyrum pernix	Completed
Haloarcula marismortui	Completed
Halobacterium sp. NRC-1	Completed
Halobacterium salinarum	Completed
Methanocaldococcus jannaschii	Completed
Methanococcus maripaludis	Completed
Methanopyrus kandleri	Completed
Methanococcoides burtonii	Unfinished
Methanosarcina acetivorans	Completed
Methanosarcina barkeri	Completed
Methanosarcina mazei	Completed
Methanothermobacter thermoautotrophicum	Completed
Nanoarchaeum equitans	Completed
Picrophilus torridus	Completed
Pyrobaculum aerophilum	Completed
Pyrococcus abyssi	Completed
Pyrococcus furiosus	Completed
Pyrococcus horikoshii	Completed
Sulfolobus solfataricus	Completed
Sulfolobus tokodaii	Completed
Thermoplasma acidophilum	Completed
Thermoplasma volcanium	Completed
Eubacteria
Actinobacillus pleuropneumoniae	Completed
Agrobacterium tumefaciens	Completed
Anabaena variabilis	Unfinished
Archaeoglobus fulgidus	Completed
Aquifex aeolicus	Completed
Azotobacter vinelandii	Completed
Bacillus anthracis	Completed
Bacillus cereus	Completed
Bacillus halodurans	Completed
Bacillus subtilis	Completed
Bacillus thuringiensis	Completed
Bacteroides thetaiotaomicron	Completed
Bartonella henselae	Completed
Bartonella quintana	Completed
Bdellovibrio bacteriovorus	Completed
Bifidobacterium longum	Completed
Bordetella bronchiseptica	Completed
Bordetella parapertussis	Completed
Bordetella pertussis	Completed
Borrelia burgdorferi	Completed
Bradyrhizobium japonicum	Completed
Brucella melitensis	Completed
Brucella suis	Completed
Buchnera aphidicola	Completed
Burkholderia cepacia	Unfinished
Burkholderia fungorum	Completed
Campylobacter jejuni	Completed
Candidatus Blochmannia floridanus	Completed
Caulobacter crescentus	Completed
Chlamydia muridarum	Completed
Chlamydia trachomatis	Completed
Chlamydophila caviae	Completed
Chlamydophila pneumoniae	Completed
Chlorobium tepidum	Completed
Chloroflexus aurantiacus	Completed
Chromobacterium violaceum	Completed
Clostridium acetobutylicum	Completed
Clostridium perfringens	Completed
Clostridium tetani	Completed
Clostridium thermocellum	Completed
Corynebacterium diphtheriae	Completed
Corynebacterium efficiens	Completed
Corynebacterium glutamicum	Completed
Coxiella burnetii	Completed
Crocosphaera watsonii	Unfinished
Cytophaga hutchinsonii	Completed
Dechloromonas aromatica	Unfinished
Deinococcus radiodurans	Completed
Desulfitobacterium hafniense	Completed
Desulfovibrio desulfuricans	Unfinished
Desulfovibrio vulgaris	Completed
Ehrlichia canis	Unfinished
Ehrlichia ruminantium	Unfinished
Enterococcus faecalis	Completed
Enterococcus faecium	Completed
Escherichia coli	Completed
Exiguobacterium sp. 255-15	Unfinished
Ferroplasma acidarmanus	Completed
Fusobacterium nucleatum	Completed
Fusobacterium nucleatum	Unfinished
Gloeobacter violaceus	Completed
Geobacter metallireducens	Completed
Geobacter sulfurreducens	Completed
Haemophilus ducreyi	Completed
Haemophilus influenzae	Unfinished
Haemophilus somnus	Completed
Helicobacter hepaticus	Completed
Helicobacter pylori	Completed
Kineococcus radiotolerans	Unfinished
Lactobacillus gasseri	Completed
Lactobacillus johnsonii	Completed
Lactobacillus plantarum	Completed
Lactococcus lactis	Completed
Leuconostoc mesenteroides	Completed
Listeria innocua	Completed
Listeria monocytogenes	Completed
Magnetococcus sp. MC-1	Completed
Magnetospirillum magnetotacticum	Completed
Mesorhizobium loti	Completed
Mesorhizobium sp. BNC1	Unfinished
Methylobacillus flagellatus	Unfinished
Microbulbifer degradans	Completed
Moorella thermoacetica	Unfinished
Mycobacterium avium	Completed
Mycobacterium bovis	Completed
Mycobacterium leprae	Completed
Mycobacterium tuberculosis	Completed
Mycoplasma gallisepticum	Completed
Mycoplasma genitalium	Completed
Mycoplasma mobile	Completed
Mycoplasma mycoides	Completed
Mycoplasma penetrans	Completed
Mycoplasma pneumoniae	Completed
Mycoplasma pulmonis	Completed
Neisseria meningitidis	Completed
Nitrosomonas europaea	Completed
Nostoc sp. PCC 7120	Completed
Nostoc punctiforme	Completed
Novosphingobium aromaticivorans	Completed
Oceanobacillus iheyensis	Completed
Oenococcus oeni	Completed
Onion yellows phytoplasma	Completed
Parachlamydia sp. UWE25	Completed
Pasteurella multocida	Completed
Pasteuria nishizawae	Unfinished
Pediococcus pentosaceus	Unfinished
Photorhabdus luminescens	Completed
Pirellula sp. 1	Completed
Porphyromonas gingivalis	Completed
Prochlorococcus marinus	Completed
Pseudomonas aeruginosa	Completed
Pseudomonas fluorescens	Completed
Pseudomonas putida	Completed
Pseudomonas syringae	Completed
Psychrobacter sp. 273-4	Unfinished
Rhodobacter sphaeroides	Completed
Rhodopseudomonas palustris	Completed
Rhodospirillum rubrum	Completed
Sinorhizobium meliloti	Completed
Rickettsia conorii	Completed
Rickettsia prowazekii	Completed
Rickettsia rickettsii	Unfinished
Rickettsia sibirica	Unfinished
Ralstonia eutropha;	Unfinished
Ralstonia metallidurans	Completed
Ralstonia solanacearum	Completed
Rubrivivax gelatinosus	Unfinished
Rubrobacter xylanophilus	Unfinished
Salmonella enterica s	Completed
Salmonella typhimurium	Completed
Shewanella oneidensis	Completed
Shigella flexneri	Completed
Staphylococcus aureus	Completed
Staphylococcus epidermidis	Completed
Streptomyces avermitilis	Completed
Streptomyces coelicolor	Completed
Streptococcus agalactiae	Completed
Streptococcus mutans	Completed
Streptococcus pneumoniae	Completed
Streptococcus pyogenes	Completed
Synechococcus elongatus	Unfinished
Synechococcus sp. WH 8102	Completed
Synechocystis sp. PCC 6803	Completed
Thermoanaerobacter tengcongensis	Completed
Thermobifida fusca	Completed
Thermosynechococcus elongatus	Completed
Thermotoga maritima	Completed
Thermus thermophilus	Completed
Treponema denticola	Completed
Treponema pallidum	Completed
Trichodesmium erythraeum	Completed
Tropheryma whipplei	Completed
Ureaplasma parvum	Completed
Vibrio cholerae	Completed
Vibrio parahaemolyticus	Completed
Vibrio vulnificus	Completed
Wigglesworthia glossinidia	Completed
Wolbachia endosymbiont of
Drosophila melanogaster	Completed
Wolinella succinogenes	Completed
Xanthomonas axonopodis	Completed
Xanthomonas campestris	Completed
Xylella fastidiosa	Completed
Yersinia pestis	Completed

Table 3. Expect (E) values for the first hits obtained from BLASTP searches using Thalassiosira pseudonana Scaff_137 and Cyanidioschyzon merolae CMR319C as queries, and from PSI-BLAST searches based on all the members of bacterial 3/3 single-domain globins (SDgbs) and flavohemoglobins (FHbs) and of vertebrate 3/3 neuroglobin (Ngbs)

	C. merolae CMR319C	T. pseudonana Scaffold_137	Bacterial SDgbs	Bacterial FHbs	Vertebrate Ngbs
	C. merolae CMR319C	T. pseudonana Scaffold_137	Bacterial SDgbs	Bacterial FHbs	Vertebrate Ngbs
No. of hits, above threshold/total	146/274 (1st iteration)	131/430 (1st iteration)	864/1,040 (4th iteration)	455/1,022 (3rd iteration)	690/1,040 (3rd iteration)
Bacterial SDgbs^a
Bacterial FHbs	e ^–10	e ^–15	e ^–42	e ^–64	e ^–8
Eukaryote FHbs^b	e ^–7	e ^–10	e ^–37	e ^–53	e ^–9
Vertebrate Ngbs^c	e ^–11	e ^–11	e ^–13	e ^–12	e ^–78
Vertebrate Cygbs^d	—	0.04	e ^–5	0.02	e ^–4
Vertebrate a	—	0.06	e ^–8	e ^–7	e ^–10
Vertebrate b	—	—	0.003	e ^–3	e ^–8
Vertebrate Mbs	—	—	—	—	e ^–8
Urochordate Hbs^e	0.6	0.002	e ^–13	e ^–9	e ^–11
Echinoderm Hbs^f	0.06	0.5	e ^–6	e ^–6	e ^–8
Plant SHbs^g	0.006	e ^–4	e ^–9	e ^–11	e ^–8
Plant NsHbs^h	e ^–4	e ^–6	e ^–12	e ^–8	e ^–6
Annelid Hbsⁱ	0.001	e ^–4	e ^–5	e ^–5	e ^–5
Crustacean Hbs^j	0.001	e ^–5	e ^–7	e ^–5	e ^–10
Insect Hbs^k	—	—	e ^–5	—	e ^–4
Mollusc Hbs^l	—	0.05	e ^–7	0.04	e ^–5
Nematode Hbs^m	—	e ^–4	e ^–4	e ^–4	e ^–6

Searchs were conducted against all organisms, using default settings, the BLOSUM62 scoring matrix, and threshold E = 0.005. The E values rounded off to the nearest integer are provided for the first hit, i.e. the first member of a globin group recognized by the query sequence(s). E > 0.005 are given only for cases where the alignment is acceptable (see Discussion). The eukaryote FHbs were not included because the results were similar to those obtained with the bacterial FHbs. Cygbs, cytoglobins; SHbs, 3/3 symbiotic plant Hb; NsHbs, 3/3 nonsymbiotic plant Hb; Mbs, myoglobins.

^aBacterial SDgbs, with accession nos. in parentheses: Acinetobacter sp. ADP (YP_047731), Aquifex aeolicus (NP_21346), Bradyrhizobium japonicum (NP_769447 and NP_771523), Campylobacter coli (AAP86201), Campylobacter jejuni (NP_282714), Clostridium perfringens (NP_562869), Chromobacterium violaceum (NP_562869), Desulfitobacterium hafniense (ZP_00102525), Gloebacter violaceus (NP_924518), Nostoc punctiforme (ZP_00111020), Novosphingobium aromaticivorans (ZP_00304525), Photobacterium profundun (CAG22319), Pseudomonas aeruginosa (NP_252656), Rhodopirellula baltica (NP_864649), Rhodopseudomonas palustris (NP_949046 and NP_949047), Silicibacter sp. (ZP_00336820), Thermobifida fusca (ZP_00292898), Vibrio parahaemolyticus (NP_800617), and Vitreoscilla stercoraria (AAT01097). ^bEukaryote FHbs: fungal, diplomonad (Giardia lamblia), and mycetozoan (Dictyostelium discoideum) FHbs. ^cVertebrate Ngbs, with accession nos. in parentheses: Bos taurus (NP_001001853), Canis familiaris (NP_001003356), Cavia porcellus (CAH03122), Danio rerio (NP_571928), Gallus gallus (CAG25721), Homo sapiens (NP_067080), Macaca mulatta (AAP92374), Mus musculus (NP_071859), Ochotona curzoniae (AAT28324), Oncorhynchus mykiss (P59742 and P59743), Oryctolagus cuniculus (CAH-3123), Rattus norvegicus (AAK1576), Sus scrofa (CAG25617), and Tetraodon nigroviridis (CAC59975). ^dVertebrate Cygbs, with accession nos. in parentheses: Danio rerio (NP_694484), Gallus gallus (XP_425373), Homo sapiens (NP_599030), Mus musculus (AAH55040), Onncorhynchus mykiss (CAD68070), Rattus norvegicus (NP_570100), Tetraodon nigroviridis (CAG02681), and Xenopus tropicalis (AAH76983). ^eUrochordate Hbs, with accession nos. in parentheses: Ciona intestinalis (CAD89600, CAD68145, CAD68146, CAD68147). ^fEchinoderm Hbs, with accession nos. in parentheses: Caudina arenicola (P80017, P80018, AAB19247, and AAB23119) and Paracaudina chilensis (S06134). ^gPlant SHbs, with accession nos. in parentheses: Canavalia lineata (P42511), Glycine max (P02235, P02236, CAA23729, CAA23731, and CAA23732); Lupinus luteus (O607193A and CAA54331), Medicago sativa (AAA32659, CAA32491, CAA32491, CAA31750, CAA38408, and CAA38409), Medicago truncatula (CAA40899 and CAA40900), Phaseolus vulgaris (P02234, AAA33767, and O04939), Pisum sativum (O80405, O48665, P02233, O9SAZ0, and O9SAZ1), Psophocarpus tetragonolobus (CAA46704), Sesbania rostrata (AAA03002, AAA03005, and CAA32044), and Vigna unguiculata (T11573). ^hPlant NsHbs, with accession nos. in parentheses: Arabidopsis thaliana (AAP21213 and AAF76353), Brassica napus (AAK07741), Ceratodon purpureus (AAG22831), Glycine max (AAA97887), Gossypium hirsutum (AAL09463 and AAK21604), Hordeum vulgare (AAB70097), Lycopersicon esculentum (AAK07676 and AAK07677), Marchantia polymorpha (AAK07743), Medicago sativa (AAG29748), Oryza sativa (AAK72228 and AAK72229), (AAK72230 and AAK72231), Physcomitrella patens (AAF66104), Raphanus sativus (AAP37043), Solanum tuberosum (AAN85431), Triticum aestivum (AAN85432), and Zea mays (AAG01375). ⁱAnnelid extracellular Hbs, with accession nos. in parentheses: Lamellibrachia (P15469), Lumbricus terrestris (CAA09958, C28151, AAC14535, and P08924), Macrobdella decora (BAC82445), Oligobrachia mashikoi (BAD86542 and O7M413), Pheretima hilgendorfi (P83122), Pheretima sieboldi (P11740), Riftia pachyptila (P80592 and CAD29155), Sabella spallanzanii (CAC37410 and CAC37412); Tubifex tubifex (P18202), Tylorrhynchus heterochaetus (P13578). Annelid intracellular Hbs: Glycera dibranchiata monomer (A33420 and S13157), polymer P1 (P23216), P2 (A36529), and P3 (B36529). ^jCrustacean extracellular Hbs, with accession nos. in parentheses: Artemia salina (AAC96001 and AAC96002), Daphnia magna (AAC47544, AAU44972, BAA76874, and BAA76875), Daphnia spinulata (AAM22870, AAM22873, AAM22874, AAM22875, AAM22876, AAM22878, AAM22880, AAM22882, AAM22883, AAM22884, AAM22886, and AAM22887), Moina macrocopa (BAB62537, BAB62536, and BAB62535), and Parartemia zietsiana (AAF72633). ^kInsect Hbs, with accession nos. in parentheses: Chironomus tentans (CAA39721, CAA39724, and CAA39725), Chironomus thummi piger (S04499, S04500, S21641, and S21633), Chironomus thummi thummi (AAB58931, AAF87695, A30477, GGICE9, JT0349, JT0423, P02224, P12548, P12550, P29245, O23761, O23762, and O23763), Drosophila melanogaster (NP_732083), and Kiefferulus cornishi (AAC47976, AAC46978, AAC48979, AAC46980, AAC46981, and AAC46982). ^lMollusc Hbs, with accession nos. in parentheses: Anadara trapezia (S06503), Aplysia juliana (BAA19794), Aplysia kurodai (P02211), Barbatia lima (BAA09588, S61518, S61519, and S61520), Barbatia virescens (BAA09587), Biomphalaria glabrata (AAC24318 and CAH23232) Buccinum undatum (Q7M424), Calyptogena nautilei (BAD30645), Lucina pectinata (AAO89499, P41261, and P41262) Nassa mutabilis (P31331), and Scapharca inaequivalvis (AAL96376, AAL96375, P14822, and S09068). ^mNematode Hbs, with accession nos. in parentheses: Caenorhabditis briggsae, CAE63297 (CBG07681), CAE60743 (CBG04428), CAE64614 (CBG09371), CAE65561 (CBG10551), CAE62918 (CBG07112), CAE66591 (CBG11915), CAE71628, and CAE75175; Caenorhabditis elegans CAB60330 (Y15E3A.2), CAE17840 (F56C4.3), NP_501142 (4H982), NP_492107 (C26C6.7), NP_504466, CAB04152.2 (F21A3.6); AAK52176 (C18C4), and AAP82661 (C23H5.2); Mermis nigrescens (AAF35435); and Syngamus trachea (AAL56426 and AAL56427).

Table 4. Expect (E) values for the first hit obtained from

BLASTP searches using Thalassiosira pseudonana Scaff_18 and Cyanidioschyzon reinhardtii C160981 as queries, and from PSI-BLAST searches based on all the members of bacterial, plant and ciliate 2/2Hbs

	T.pseudonana Scaff_18	C. reinhardtii C160981	Bacterial 2/2 Hbs	Plant 2/2 Hbs	Ciliate 2/2 Hbs	All 2/2 Hbs
No. of hits, above threshold/total	28/61	35/72	138/211 (4^th iteration)	68/87 (3^rd iteration)	52/111 (2^nd iteration)	144/201 (6^th iteration)
Bacterial 2/2 Hbs	e ^–9	e ^–23	e ^–37	e ^–14	e ^–30	e ^–33
Plant 2/2 Hbs*	e ^–25 to e^–20	—	e ^–15	e ^–87 to e^–89	—	e ^–23
Ciliate 2/2 Hbs^†	—	e ^–15	e ^–26	—	e ^–51	e ^–23
Bacterial and eukaryotic FHbs	—	—	e ^–4	—	—	e ^–4

Searches were conducted against all organisms, using default settings, the BLOSUM62 scoring matrix and threshold E = 0.005. The E values, rounded off to the nearest integer, are provided for the first hit, i.e. the first member of a globin group recognized by the query sequence(s). E > 0.005 are given only for cases where the alignment is acceptable (see Discussion). The results obtained with the other C. reinhardtii globins were similar. FHbs, flavohemoglobins.

*Plant 2/2 Hbs, with accession nos. in parentheses: Arabidopsis thaliana (AAK55409), Datisca glomerata (CAD33536), Glycine max (AAS48191), Hordeum vulgare (AAK55410), Oryza sativa (BAD32857), and Triticum aestivum (AAN85433).

^†Ciliate 2/2 Hbs, with accession nos. in parentheses: Paramecium caudatum (S27185), P. multimicronucleatum (S60031), P. triaurelia (S60030), Tetrahymena pyriformis (A36270), and Tetrahymena thermophila (pir|S32556).

Table 5. Expect (E) values for the first hit obtained from

PSI-BLAST searches using as queries Mycobacterium tuberculosis 2/2Hb1 (HbN) and 2/2Hb2 (HbO) and Desulfitobacterium hafniense 2/2Hb3

	M. tuberculosis HbN NP_216058 2/2Hb1	M. tuberculosi s HbO NP_216986 2/2Hb2	D. hafniense ZP_00102524 2/2Hb3
No. of hits, above threshold/total	36/70 (1st iteration)	72/91 (1st iteration)	25/36 (1st iteration)
Bacterial 2/2Hbs 1s/2s/3s	e ^–54	e ^–56	e ^–21
Plant 2/2Hbs*	—	e ^–9	—
Ciliate 2/2Hbs^†	e ^–16	—	—
No. of hits, above threshold/total	62/113 (3^rd iteration)	109/138 (6th iteration)	44/86 (3rd iteration)
Bacterial 2/2Hb1s/2s/3s	20 e ^–51 to e^–34	64 e ^–43 to e^–19	23 e ^–43 to e^–29
Plant 2/2Hbs*	—	e ^–21 to e^–18	—
Ciliate 2/2Hbs^†	e ^–29	e ^–11	0.01

Searches were conducted against all organisms, using default settings, the BLOSUM62 scoring matrix, and threshold E = 0.005. The E values, rounded off to the nearest integer, are provided for the first hit, i.e., the first member of a globin group recognized by the query sequence(s). E > 0.005 are given only for cases where the alignment is acceptable (see Discussion).

*Plant 2/2Hbs, with accession nos. in parentheses: Arabidopsis thaliana (AAK55409), Datisca glomerata (CAD33536), Glycine max (AAS48191), Hordeum vulgare (AAK55410), Oryza sativa (BAD32857), and Triticum aestivum (AAN85433).

^†Ciliate 2/2Hbs, with accession nos. in parentheses: Paramecium caudatum (S27185), P. multimicronucleatum (S60031), P. triaurelia (S60030), Tetrahymena pyriformis (A36270), and T. thermophila (S32556).

Table 6. Expect (E) values for the first hit obtained from psiblast searches using all the members of bacterial globin-coupled sensors (GCSs) and protoglobins (Pgbs) as queries

	Bacterial GCSs	Bacterial Pgbs
No. of hits (above threshold/total)	49/206 (4th iteration)	23/54 (4th iteration)
Bacterial GCSs *	e ^–45 to e^–11	e ^–9
Bacterial Pgbs^†	e ^–18	e ^–91 to e^–42
Bacterial FHbs	e ^–4	—
Eukaryotic FHbs	e ^–4	—

*Bacterial GCSs, with accession nos. and lengths in parentheses: Agrobacterium tumefaciens [NP_354049 (499 aa) and NP_531216 (568 aa)], Azoarcus sp. (EbN1 YP_158656, 678 aa), Azotobacter vinelandii (ZP_00090857, 434 aa), Bacillus anthracis (YP_031514, 433 aa), Bacillus cereus (ZP_00239241, 433 aa), Bacillus clausii (YP_176973, 449 aa), Bacillus halodurans (NP_241371, 439 aa), Bacillus licheniformis (YP_90716, 430 aa), Bacillus subtilis (NP_388919, 432aa), Bacillus thuringiensis (YP_039413, 433 aa), Bordetella bronchiseptica (NP_888505, 475 aa), Bordetella parapertussis (NP_884745, 475 aa), Bordetella pertussis (NP_882025, 475 aa), Burkholderia fungorum (ZP_00277651, 723 aa), Caulobacter crescentus [NP_419247 (537 aa) and NP_421120 (555 aa)], Chromobacterium violaceum [NP_900548 (295 aa) and NP_899909 (375 aa)], Desulfotalea psychrophila (YP_063937, 363 aa), Desulfitobacterium hafniense (ZP_00102172, 157 aa), Erwinia carotovara (YP_049782, 442 aa), Escherischia colli (NP_416007, 460 aa), Exiguobacterium [ZP_00183969 (430aa) and ZP00182337 (425 aa)], Geobacter metallireducens (ZP_00298606, 300 aa), Geobacter sulfurreducens (NP_954351, 300 aa), Gluconobacter oxydans (YP_191196, 458 aa), Haloarcula marismortui (AAV45247, 497 aa), Halobacterium salinarum (T44978, 489 aa), Magnetococcus sp. [ZP_00289561 (453 aa) and ZP_00289126 (507aa)], Magnetospirillum magnetotacticum [ZP_00054075 (721 aa) and ZP_00054774 (443 aa)], Moorella thermoacetica (ZP_00330036, 245 aa), Novosphingobium aromaticivorans (ZP_00304036, 481 aa), Rhizobium meliloti (S61831, 533 aa), Rhodobacter sphaeroides (ZP_00207372, 341 aa), Rhodospirillum rubrum [ZP_00268251 (442 aa) and ZP_00270832 (445 aa)], Shigella flexneri (NP_707605, 381 aa), Silicibacter sp. [ZP_0038974 (308 aa), ZP_00336325 (485 aa), and ZP00336080 (487 aa)], Sinorhizobium meliloti (NP_384742, 533 aa), Thermosynechococcus elongatus (NP_682779, 194 aa), Thermus thermophilus (YP_005074, 203 aa), Vibrio vulnificus (NP_936636, 306 aa), and Zymomonas mobilis (AAV_89506, 467 aa).

^†Bacterial Pgbs, with accession nos. and lengths in parentheses: Aeropyrum pernix (NP_147118, 195 aa), Chloroflexus aurantiacus (ZP_00359040, 227 aa), Methanosarcina acetivorans (NP_617780, 195 aa), Methanosarcina barkeri (ZP_000294773, 195 aa), Rubrobacter xylanophilus (ZP_00200180, 196 aa), and Thermobifida fusca (ZP_00293478, 197aa).

Supporting Materials and Methods

All of the putative globin sequences were aligned manually by following the procedure used earlier in the alignment of >700 globins (1) designed to fit the sequences to the myoglobin fold (2, 3), the pattern of predominantly hydrophobic residues at 37 conserved, mostly solvent-inaccessible positions with mean solvent-accessible areas of <15 Å² (4), including 33 intrahelical residues at positions A8, A11, A12, A15, B6, B9, B10, B13, B14, C4, E4, E7, E8, E11, E12, E15, E18, E19, F1, F4, G5, G8, G11, G12, G13, G15, G16, H7, H8, H11, H12, H15, and H19; the three interhelical residues at CD1, CD4, and FG4; and the invariant His at F8. The alignment was based on the crystal-structure-based alignment of the 58 known globin crystal structures available from the Protein Data Bank: 1A6M, 1IRDA, 1IRDB, 1I3DA, 1A9W, 1OJ6, 1UMO, 1HDSA, 1HDSB, 1A4FA, 1A4FB, 1HBRA, 1HBRB, 1CG5A, 1CG5B, 1SPGA, 1GCVA, 1GCVB, 1OUTA, 1OUTB, 1LA6A, 1LA6B, 1V75A, 1V75B, 2MM1, 1LHT, 1EMY, 1IT2, 2LHB, 1EW6, 1MBA, 1ASH, 1HLB, 1HLM, 1ECO, 2HBG, 1BOB, 1H97, 3SDH, 1SCTA, 1SCTB, 1ITH, 1FSL, 2GDM, 1D8U, 1KR7, 1DLY, 1IDR, 1NGK, 1UX8, 1DLW, 1MWB, 1VHB, 1GVH, 1CQX, 1OR4, and 1TU9. Although an earlier alignment of 700 globins had suggested that globins were characterized by two invariant residues, F8His and CD1Phe (1), members of the recently discovered 2/2 Hb family (5-8), can accomodate other hydrophobic residues, such as Tyr/Met/Leu/Ile/Val at the CD1 position and Ala/Ser/Thr/Leu at the distal E7 position, in addition to His and Gln. Moss 3/3 nonsymbiotic plant Hb also has a Tyr at CD1 (9). Thus, we required only that the putative globin sequence alignment be such as to provide a hydrophobic residue at CD1 in the order of preference Phe>Tyr>Leu>Met>Ile>Val, the residues at the distal E7 in the order of preference His>Gln>Leu≈Thr>Ala≈Val≈Ser≈Tyr, a candidate His at the proximal F8 position, and at the remaining 32 positions an amino acid already encountered in the crystal structure alignment. Deletions in the helical regions were avoided, and interhelical regions were unrestricted to obtain the optimum alignment in the flanking helical regions. The C-terminal FAD reductase sequences and small subunit rRNA sequences were aligned with CLUSTALW and manually improved using GENEDOC.

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