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Supplementary Materials

Description and genome analysis of Microvirga antarctica sp. nov., a novel pink- pigmented psychrotolerant bacterium isolated from Antarctic soil

Antonie van Leeuwenhoek

Lin Zhu, Weiwei Ping, Siyue Zhang, Ya Chen, Ying Zhang and Jianli Zhang*

Corresponding author: [email protected]

Key Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, Beijing 100081, PR China T Microvirga aerilata 5420S-16 (GQ421849)

T ( ) 60 Microvirga zambiensis WSM3693 HM362433

T Microvirga ossetica V5/3M CL (CP016616)

54 T Microvirga arabica SV2184P (JN989301)

T Microvirga flocculans ATCC BAA-817 (JAEA01000030)

T Microvirga soli R491 (KX247636) 77 T 69 Microvirga lotononidis WSM3557 (AJUA01000055)

T Microvirga pakistanensis NCCP-1258 (LC065285)

T Microvirga lupini Lut6 (KI912026)

T Microvirga vignae BR3299 (LCYG01000082) T ( ) 77 Microvirga makkahensis SV1470 JN989300 T 54 Microvirga arsenatis 3D203 (MN879271)

T Microvirga subterranea DSM 14364 ( FR733708)

T Microvirga indica S-MI1b (KM588957)

58 T Microvirga guangxiensis 25B (EU727176)

T 95 Microvirga antarctica 3D7 (MH561859)

T ‘Microvirga brassicacearum’CDVBN77 (MK938302)

T Microvirga aerophila DSM 21344 (QOIO01000123)

T 75 Microvirga flavescens c27j1 (MH542628)

T Methylobacterium oryzihabitans TER-1 (MF980971) 61 T Microvirga massiliensis JC119 (LN811386)

T 76 Salinarimonas rosea DSM 21201 (AUBC01000058)

T Bosea massiliensis 63287 (AF288309)

T 100 Bosea vaviloviae Vaf-18 (KJ721000)

72 T Bosea caraganae RCAM04680 (MH633716)

T Chelatococcus asaccharovorans TE2 (AJ294349)

99 T Chelatococcus reniformis B2974 ( KJ469373)

T Phyllobacterium salinisoli LLAN61 (LT614649)

0.01

Fig. S1. Minimum-evolution phylogenetic tree based on 16S rRNA gene sequences showing the relationships between strain 3D7T and the closely related species of the genus Microvirga. Numbers at nodes represent bootstrap percentages (>50 %) based on 1000 replicates. Phyllobacterium salinisoli LLAN6T was chosen as an outgroup. Bar represents 0.01 substitutions per nucleotide position T Microvirga aerilata 5420S-16 (GQ421849)

T Microvirga zambiensis WSM3693 (HM362433)

T Microvirga ossetica V5/3M CL (CP016616)

T 56 Microvirga arabica SV2184P (JN989301)

T Microvirga soli R491 (KX247636)

T Microvirga flocculans ATCC BAA-817 (JAEA01000030)

T Microvirga lupini Lut6 (KI912026)

T Microvirga lotononidis WSM3557 (AJUA01000055)

T 74 Microvirga pakistanensis NCCP-1258 (LC065285)

T 77 Microvirga vignae BR3299 (LCYG01000082)

T Microvirga makkahensis SV1470 (JN989300)

T Microvirga arsenatis 3D203 (MN879271)

T Microvirga indica S-MI1b (KM588957) 55 T Microvirga guangxiensis 25B (EU727176) 50 T Microvirga subterranea DSM 14364 (FR733708)

T Microvirga aerophila DSM 21344 (QOIO01000123) Microvirga antarctica 3D7T (MH561859) 88 T ‘Microvirga brassicacearum’CDVBN77 (MK938302) 77 T Microvirga flavescens c27j1 (MH542628)

T Microvirga massiliensis JC119 (LN811386)

T Methylobacterium oryzihabitans TER-1 (MF980971)

T 73 Bosea caraganae RCAM04680 (MH633716)

T 100 Bosea vaviloviae Vaf-18 (KJ721000)

T Bosea massiliensis 63287 (AF288309)

T Salinarimonas rosea DSM 21201 (AUBC01000058)

T 85 Chelatococcus asaccharovorans TE2 (AJ294349)

T Chelatococcus reniformis B2974 (KJ469373)

64 T Phyllobacterium salinisoli LLAN61 (LT614649)

0.02

Fig. S2. Maximum-likehood phylogenetic tree based on 16S rRNA gene sequences showing the relationships between strain 3D7T and the closely related species of the genus Microvirga. Numbers at nodes represent bootstrap percentages (>50 %) based on 1000 replicates. Phyllobacterium salinisoli T LLAN61 was chosen as an outgroup. Bar represents 0.02 substitutions per nucleotide position

Fig. S3. Phylogenomic tree constructed with the Composition Vector (CV) approach based on whole genome sequences of strain 3D7T and other Microvirga species, showing the position of strain 3D7Tamong its phylogenomic neighbors. Phyllobacterium salinisoli LLAN61T was chosen as an outgroup. The K-tuple length is 12

T Fig. S4. Distribution of the predicted genes of strain 3D7 to different GO functional categories Fig. S5. Genes associated with general COG functional category prediction of strain 3D7T Fig. S6. Genes associated with general KEEG functional category prediction of strain 3D7T

Fig. S7. Transmission electron micrograph showing the morphology of strain 3D7T. Bar, 0.5 μm

a b c

Fig. S8. Two-dimensional TCL (thin-layer chromatograms) of polar lipids of strain 3D7T. Total polar lipids were stained with molybdatophosphoric acid (a); Phospholipids were stainedwith the reagent of the Dittmer and Lester (b); Aminolipids were stained with ninhydrin (c). PC, phosphatidylcholine; PE, phosphatidylethanolamine; APL, unidentified aminophospholipid; AL,unidentified amino lipid; L, unidentified lipid

Table S1. Predicted gene modules for carbohydrate-active (CAZymes) families in the genome of strain 3D7T

CAZyme Family Gene Count Known CAZyme Activities GH109 4 alpha-N-acetylgalactosaminidase (EC 3.2.1.49)

GH16 3 xyloglucan:xyloglucosyltransferase (EC 2.4.1.207); keratan-sulfate endo-1,4-beta-galactosidase (EC 3.2.1.103); endo-1,3-beta-glucanase (EC 3.2.1.39); endo-1,3(4)-beta-glucanase (EC 3.2.1.6); licheninase (EC 3.2.1.73); beta- (EC 3.2.1.81); kappa;-carrageenase (EC 3.2.1.83); xyloglucanase (EC 3.2.1.151); endo-beta-1,3-galactanase (EC 3.2.1.181); beta-porphyranase (EC 3.2.1.178); (EC 3.2.1.35); endo-beta-1,4-galactosidase (EC 3.2.1.-); chitin beta-1,6-glucanosyltransferase (EC 2.4.1.-); endo-beta-1,4-galactosidase (EC 3.2.1.-)

GH23 2 type G (EC 3.2.1.17); peptidoglycan (EC 4.2.2.n1) also known in the literature as peptidoglycan lytic transglycosylase; (EC 3.2.1.14)

GH103 4 peptidoglycan lytic transglycosylase (EC 3.2.1.-) GH25 1 lysozyme (EC 3.2.1.17)

GH1 3 beta-glucosidase (EC 3.2.1.21); beta-galactosidase (EC 3.2.1.23); beta- (EC 3.2.1.25); beta-glucuronidase (EC 3.2.1.31); beta-xylosidase (EC 3.2.1.37); beta-D-fucosidase (EC 3.2.1.38); phlorizin (EC 3.2.1.62); exo-beta-1,4-glucanase (EC 3.2.1.74); 6-phospho-beta-galactosidase (EC 3.2.1.85); 6-phospho-beta-glucosidase (EC 3.2.1.86); strictosidine beta-glucosidase (EC 3.2.1.105); (EC 3.2.1.108); amygdalin beta-glucosidase (EC 3.2.1.117); prunasin beta-glucosidase (EC 3.2.1.118); vicianin hydrolase (EC 3.2.1.119); raucaffricine beta-glucosidase (EC 3.2.1.125); thioglucosidase (EC 3.2.1.147); beta-primeverosidase (EC 3.2.1.149); isoflavonoid 7-O-beta-apiosyl-beta-glucosidase (EC 3.2.1.161); ABA-specific beta-glucosidase (EC 3.2.1.175); DIMBOA beta- glucosidase (EC 3.2.1.182); beta-glycosidase (EC 3.2.1.-); hydroxyisourate hydrolase (EC 3.-.-.-)

GH108 2 N-acetylmuramidase (EC 3.2.1.17) GH31 1 alpha-glucosidase (EC 3.2.1.20); alpha-galactosidase (EC 3.2.1.22); alpha-mannosidase (EC 3.2.1.24); alpha-1,3-glucosidase (EC 3.2.1.84); -isomaltase (EC 3.2.1.48) (EC 3.2.1.10); alpha- xylosidase (EC 3.2.1.177); alpha-glucan lyase (EC 4.2.2.13); isomaltosyltransferase (EC 2.4.1.-); oligosaccharide alpha-1,4- (EC 2.4.1.161); sulfoquinovosidase (EC 3.2.1.-)

GH15 1 glucoamylase (EC 3.2.1.3); glucodextranase (EC 3.2.1.70); alpha,alpha- (EC 3.2.1.28); dextran dextrinase (EC 2.4.1.2)

GH2 1 beta-galactosidase (EC 3.2.1.23) ; beta-mannosidase (EC 3.2.1.25); beta-glucuronidase (EC 3.2.1.31); alpha-L-arabinofuranosidase (EC 3.2.1.55); mannosylglycoprotein endo-beta-mannosidase (EC 3.2.1.152); exo-beta-glucosaminidase (EC 3.2.1.165)

GH113 1 beta-mannanase (EC 3.2.1.78)

GH8 2 (EC 3.2.1.132); (EC 3.2.1.4); licheninase (EC 3.2.1.73); endo-1,4-beta- (EC 3.2.1.8); reducing-end- releasing exo-oligoxylanase (EC 3.2.1.156) GH3 1 beta-glucosidase (EC 3.2.1.21); xylan 1,4-beta-xylosidase (EC 3.2.1.37); beta-glucosylceramidase (EC 3.2.1.45); beta-N-acetylhexosaminidase (EC 3.2.1.52); alpha-L-arabinofuranosidase (EC 3.2.1.55); glucan 1,3-beta-glucosidase (EC 3.2.1.58); glucan 1,4-beta-glucosidase (EC 3.2.1.74); isoprimeverose-producing oligoxyloglucan hydrolase (EC 3.2.1.120); coniferin beta-glucosidase (EC 3.2.1.126); exo-1,3-1,4-glucanase (EC 3.2.1.-); beta-N-acetylglucosaminide (EC 2.4.1.-)

GH102 1 peptidoglycan lytic transglycosylase (EC 3.2.1.-)

CE10 6 arylesterase (EC 3.1.1.-); carboxyl (EC 3.1.1.3); (EC 3.1.1.7); (EC 3.1.1.8); sterol esterase (EC 3.1.1.13); brefeldin A esterase (EC 3.1.1.-). CE1 8 acetyl xylan esterase (EC 3.1.1.72); cinnamoyl esterase (EC 3.1.1.-); feruloyl esterase (EC 3.1.1.73); carboxylesterase (EC 3.1.1.1); S-formylglutathione hydrolase (EC 3.1.2.12); diacylglycerol O- acyltransferase (EC 2.3.1.20); trehalose 6-O-mycolyltransferase (EC 2.3.1.122)

CE10 1 arylesterase (EC 3.1.1.-); carboxyl esterase (EC 3.1.1.3); acetylcholinesterase (EC 3.1.1.7); cholinesterase (EC 3.1.1.8); sterol esterase (EC 3.1.1.13); brefeldin A esterase (EC 3.1.1.-).

CE4 14 acetyl xylan esterase (EC 3.1.1.72); chitin deacetylase (EC 3.5.1.41); chitooligosaccharide deacetylase (EC 3.5.1.-); peptidoglycan GlcNAc deacetylase (EC 3.5.1.-); peptidoglycan N-acetylmuramic acid deacetylase (EC 3.5.1.-).

CE11 1 UDP-3-0-acyl N-acetylglucosamine deacetylase (EC 3.5.1.-).

CE9 2 N-acetylglucosamine 6-phosphate deacetylase (EC 3.5.1.25); N-acetylglucosamine 6-phosphate deacetylase (EC 3.5.1.80)

CE3 3 acetyl xylan esterase (EC 3.1.1.72).

CE7 1 acetyl xylan esterase (EC 3.1.1.72); cephalosporin-C deacetylase (EC 3.1.1.41).

GT4 21 (EC 2.4.1.13); sucrose-phosphate synthase (EC 2.4.1.14); alpha-glucosyltransferase (EC 2.4.1.52); lipopolysaccharide N-acetylglucosaminyltransferase (EC 2.4.1.56); phosphatidylinositol alpha- (EC 2.4.1.57); GDP-Man: Man1GlcNAc2-PP-dolichol alpha-1,3-mannosyltransferase (EC 2.4.1.132); GDP-Man: Man3GlcNAc2-PP-dolichol/Man4GlcNAc2-PP-dolichol alpha-1,2-mannosyltransferase (EC 2.4.1.131); digalactosyldiacylglycerol synthase (EC 2.4.1.141); 1,2-diacylglycerol 3-glucosyltransferase (EC 2.4.1.157); diglucosyl diacylglycerol synthase (EC 2.4.1.208); trehalose (EC 2.4.1.231); NDP-Glc: alpha- alpha-glucosyltransferase / alpha,alpha-trehalose synthase (EC 2.4.1.245); GDP-Man: Man2GlcNAc2-PP-dolichol alpha-1,6- mannosyltransferase (EC 2.4.1.257); UDP-GlcNAc: 2-deoxystreptamine alpha-N-acetylglucosaminyltransferase (EC 2.4.1.283); UDP-GlcNAc: ribostamycin alpha-N-acetylglucosaminyltransferase (EC 2.4.1.285); UDP-Gal alpha- (EC 2.4.1.-); UDP-Xyl alpha-xylosyltransferase (EC 2.4.2.-); UDP-GlcA alpha-glucuronyltransferase (EC 2.4.1.-); UDP-Glc alpha-glucosyltransferase (EC 2.4.1.-); UDP-GalNAc: GalNAc-PP-Und alpha-1,3-N-acetylgalactosaminyltransferase (EC 2.4.1.306); UDP-GalNAc: N,N'-diacetylbacillosaminyl-PP-Und alpha-1,3-N- acetylgalactosaminyltransferase (EC 2.4.1.290); ADP-dependent alpha-maltose-1-phosphate synthase (2.4.1.342)

GT2_Glycos_transf_2 8

GT28 1 1,2-diacylglycerol 3-beta-galactosyltransferase (EC 2.4.1.46); 1,2-diacylglycerol 3-beta-glucosyltransferase (EC 2.4.1.157); UDP-GlcNAc: Und-PP-MurAc-pentapeptide beta-N- acetylglucosaminyltransferase (EC 2.4.1.227); digalactosyldiacylglycerol synthase (EC 2.4.1.241)

GT20 1 alpha,alpha-trehalose-phosphate synthase [UDP-forming] (EC 2.4.1.15); Glucosylglycerol-phosphate synthase (EC 2.4.1.213); trehalose-6-P (EC 3.1.3.12); [retaining] GDP-valeniol: validamine 7-phosphate valeniolyltransferase (EC 2.-.-.-)

GT2_Glyco_tranf_2_3 4

GT83 2 undecaprenyl phosphate-alpha-L-Ara4N: 4-amino-4-deoxy-beta-L- (EC 2.4.2.43); dodecaprenyl phosphate-beta-galacturonic acid: lipopolysaccharide core alpha-galacturonosyl (EC 2.4.1.-)

GT2_Glyco_trans_2_3 2 GT51 1 murein polymerase (EC 2.4.1.129).

GT30 1 CMP-beta-KDO: alpha-3-deoxy-D-manno-octulosonic-acid (KDO) transferase (EC 2.4.99.-).

GT1 3 UDP- (EC 2.4.1.17); zeatin O-beta-xylosyltransferase (EC 2.4.2.40); 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (EC 2.4.1.45); N-acylsphingosine galactosyltransferase (EC 2.4.1.47); flavonol 3-O-glucosyltransferase (EC 2.4.1.91); anthocyanidin 3-O-glucosyltransferase (EC 2.4.1.115); sinapate 1-glucosyltransferase (EC 2.4.1.120); indole-3-acetate beta- glucosyltransferase (EC 2.4.1.121); flavonol L-rhamnosyltransferase (EC 2.4.1.159); sterol glucosyltransferase (EC 2.4.1.173); UDP-Glc: 4-hydroxybenzoate 4-O-beta-glucosyltransferase (EC 2.4.1.194); zeatin O-beta-glucosyltransferase (EC 2.4.1.203); limonoid glucosyltransferase (EC 2.4.1.210); UDP-GlcA: baicalein 7-O-beta-glucuronosyltransferase (EC 2.4.1.253); UDP-Glc: chalcone 4′-O-beta- glucosyltransferase (EC 2.4.1.286); ecdysteroid UDP-glucosyltransferase (EC 2.4.1.-); salicylic acid beta-glucosyltransferase (EC 2.4.1.-); anthocyanin 3-O-galactosyltransferase (EC 2.4.1.-); anthocyanin 5-O-glucosyltransferase (EC 2.4.1.-); dTDP-beta-2-deoxy-L-fucose: alpha-L-2-deoxyfucosyltransferase (EC 2.4.1.-); UDP-beta-L-rhamnose: alpha-L-rhamnosyltransferase (EC 2.4.1.-); zeaxanthin glucosyltransferase (EC 2.4.1.-)

GT41 4 UDP-GlcNAc: peptide beta-N-acetylglucosaminyltransferase (EC 2.4.1.255); UDP-Glc: peptide N-beta-glucosyltransferase (EC 2.4.1.-)

GT19 1 lipid-A-disaccharide synthase (EC 2.4.1.182).

GT27 1 polypeptide alpha-N-acetylgalactosaminyltransferase (EC 2.4.1.41)

AA7 3 glucooligosaccharide oxidase (EC 1.1.3.-); chitooligosaccharide oxidase (EC 1.1.3.-)

AA4 5 vanillyl-alcohol oxidase (EC 1.1.3.38)

AA3 3 cellobiose dehydrogenase (EC 1.1.99.18); glucose 1-oxidase (EC 1.1.3.4); aryl alcohol oxidase (EC 1.1.3.7); alcohol oxidase (EC 1.1.3.13); pyranose oxidase (EC 1.1.3.10)

AA6 2 1,4-benzoquinone reductase (EC. 1.6.5.6)

AA3_2 5 AA3's subfamily

AA1 2 Laccase / p-diphenol:oxygen / ferroxidase (EC 1.10.3.2); ; ferroxidase (EC 1.10.3.-); Laccase-like multicopper oxidase (EC 1.10.3.-)

CBM50 1 Modules of approx. 50 residues found attached to various enzymes from families GH18, GH19, GH23, GH24, GH25 and GH73, i.e. enzymes cleaving either chitin or peptidoglycan. Binding to chitopentaose demonstrated in the case of Pteris ryukyuensis chitinase A [Ohnuma T et al. (2008) J. Biol. Chem. 283:5178-87 (PMID: 18083709)]. CBM50 modules are also found in a multitude of other enzymes targeting the petidoglycan such as peptidases and amidases. These enzymes are not reported in the list below.

PL20 2 endo-beta-1,4-glucuronan lyase (EC 4.2.2.14)