supplementary matterials soil oligotrophic bacterium ... · 26/03/2013 · supplementary...

Supplementary matterials

Soil oligotrophic bacterium Agromonas oligotrophica (Bradyrhizobium

oligotrophicum) is a nitrogen-fixing symbiont of Aeschynomene indica

as suggested by genome analysis

Takashi Okubo1, Shohei Fukushima1, Manabu Itakura1, Kenshiro Oshima2, Aphakorn Longtonglang3 ,Neung

Teaumroong3, Hisayuki Mitsui1, Masahira Hattori2, Reiko Hattori4, Tsutomu Hattori4, and Kiwamu

Minamisawa1#

Graduate School of Life Sciences, Tohoku University, Katahira, Aoba-ku, Sendai 980-8577, Japan1;

University of Tokyo, Kashiwa-no-ha 5-1-5, Kashiwa, Chiba, 277-8561, Japan2; School of Biotechnology,

Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000,

Thailand3;

Attic Lab, 1-6-2-401 Komegafukuro, Aobaku, Sendai, Miyagi 980-0813, Japan4.

Table S1. Sequences used for phylogenetic analysis

Accession number Genus and Species Strain

16S rRNA gene ITS region

Agromonas oligotrophica S58T AP012603 AP012603

Agromonas oligotrophica S42 AB531473 -




Bradyrhizobium sp. ORS278 AF239255 AF239255

Bradyrhizobium sp. BTAi1 CP000494 CP000494

Bradyrhizobium sp. ORS285 AF230722 CAFH01000247

Bradyrhizobium sp. S23321 AP012279 AP012279

Bradyrhizobium sp. G14130 AB003457 -

Bradyrhizobium sp. HWK12 D89028 -

Bradyrhizobium sp. HW13 D89027 -

Bradyrhizobium japonicum USDA110 BA000040 BA000040

Bradyrhizobium japonicum USDA122 AF208503 AB685466

Bradyrhizobium japonicum USDA124 AF208505 AB100753

Bradyrhizobium japonicum USDA6T AP012206 AP012206

Bradyrhizobium japonicum NC4 AB231917 AB278125

Bradyrhizobium japonicum NC6 AB231918 AB278126

Bradyrhizobium japonicum NK2 AB070567 AB278127

Bradyrhizobium japonicum T7 AB231929 AB278128

Bradyrhizobium japonicum T9 AB231930 AB278129

Bradyrhizobium elkanii USDA76T U35000 -

Bradyrhizobium dinitrificans LMG 8443 S46917 AF338176

Nitrobacter winogradskyi Nb-255 CP000115 -

Rhodopseudomonas palustris CGA009 BX571963 BX571963

Mesorhizobium loti MAFF303099 BA000012 BA000012

Table S2. Genes on S58 genome showing similarity with known product of nodulation (nod) genes.

Agromonas ologotrophica (Bradyrhizobium oligotrophicum) S58 ORS2782) BTAi12) Nod

protein Locus Tag1)

Similarity (%) Closest Organisms Accession No. E value

Query coverage

(%) CDS Similarity

(%) CDS Similarity (%)

NodA Absence Absence Absence

NodB S58_31670 31 Xanthomonas campestris pv. Campestris AAL30085 5.00E-18 65 Brado4564 33 BBta4792 36

NodC Absence Absence Absence NodD S58_55840 70 Bradyrhizobium sp. AAB06562 5.00E-153 98 Brado3695 39 BBta1932 38

NodE S58_41760 38 Mesorhizobium loti MAFF303099 NP_105705 2.00E-86 99 Absence BBta0068 31

NodF Absence

NodG S58_41780 67 Sinorhizobium fredii NGR234 ACP24681 3.00E-113 100 Brado3311 66 BBta3818 65

NodH Absence NodI S58_47400 46 Parabacteroides sp. D13 ZP_05546362 4.00E-61 88 Brado0517 39 BBta7658 39 NodJ Absence Absence Absence NodK Absence NodL Absence Brado2093 42 Absence NodM S58_38120 78 Bradyrhizobium sp. WM9 AF222753 0 100 Brado3763 78 BBta4166 76

NodN S58_44780 97 Bradyrhizobium sp. STM 3843 CCE06638 7.00E-111 100

NodO Absence

NodP S58_03490 86 Bradyrhizobium sp. ORS 375 ZP_09423098 0 100 Brado5192 75 BBta0328 73

NodQ S58_03490 82 Bradyrhizobium sp. ORS 375 ZP_09423099 0 100 Brado5193 66 BBta0327 55

S58_11700 81 Bradyrhizobium japonicum USDA 6 BAL13613 0 100

NodV S58_23810 31 Cupriavidus necator N-1 YP_004688678 5.00E-83 73

NodW S58_51710 43 Xanthomonas axonopodis pv. citri str. 306 NP_642467 5.00E-42 94

1) Absence indicates no hits with >30% amino acid identity and >50% query coverage.

2) Giraud et al. 2007 (2)

Table S3. Conservation of genes between ORS278 and S58 genomes important for nodule development

Locus tag Blastp result

ORS278 S58 Gene annotation Similarity

(%)

Query coverage

(%)

Biblast

hit

Nucleotides biosynthesis

Purines

BRADO0338 S58_06170 bifunctional purine biosynthesis protein purH 95 100 +

BRADO1206 S58_13170 phosphoribosylaminoimidazole carboxylase catalytic subunit

purE 96 100 +

BRADO2779 S58_48410 phosphoribosylformylglycinamidine synthase I purQ 98 100 +

BRADO2785 S58_48310 phosphoribosylformylglycinamidine synthase II purL 96 100 +

BRADO2830 S58_47810 adenylosuccinate lyase purB 96 100 +

BRADO3165 S58_44580 inosine-5'-monophosphate dehydrogenase guaB 97 100 +

BRADO3169 S58_44540 GMP synthase guaA 96 100 +

BRADO3280 S58_42000 amidophosphoribosyltransferase precursor purF 95 100 +

BRADO3344 S58_41450 phosphoribosylformylglycinamidine cyclo-ligase purM 93 99 +

BRADO3345 S58_41440 phosphoribosylglycinamide formyltransferase purN 90 100 +

BRADO6088 S58_41430 phosphoribosylamine--glycine ligase purD 94 100 +

Pyrimidines

BRADO4502 S58_32410 aspartate carbamoyltransferase pyrB 99 95 +

BRADO4503 S58_32400 dihydroorotase pyrC 96 100 +

BRADO6742 S58_32380 dihydro-orotate oxidase pyrD 76 100 +

Amino acids biosynthesis

Leucine

BRADO0353 S58_03610 3-isopropylmalate dehydrogenase leuB 97 100 +

BRADO0365 S58_03730 3-isopropylmalate dehydratase small subunit leuD 97 99 +

BRADO0374 S58_03810 3-isopropylmalate dehydratase large subunit 2 leuC 98 100 +

BRADO5528 S58_20980 2-isopropylmalate synthase leuA 98 100 +

BRADO6039 S58_62120 putative branched-chain amino acid aminotransferase protein

IlvE-like protein 94 100 +

Glutamate

BRADO6238 S58_64110 glutamate synthase large chain gltB 96 99 +

BRADO6240 S58_64120 glutamate synthase small chain gltD 93 100 +

Lysine

BRADO0158 S58_01730 dihydrodipicolinate reductase dapB 94 100 +

Others

BRADO0277 S58_02810 bifunctional: 5,10-methylene-tetrahydrofolate dehydrogenase

folD 95 100 +

BRADO3272 S58_42070 putative transcriptional regulatory protein TetR/AcrR family 75 100 +

BRADO4272 S58_60870 putative sulfonate ABC transporter, periplasmic-binding protein

ssuA 31 89 -

Proteins of unknown function

BRADO0213 S58_02240 conserved hypothetical protein 83 100 +

BRADO2778 no hit conserved hypothetical protein no hit -



Table S4. Conservation of genes between ORS278 and S58 genomes important for symbiotic nitrogen fixation Locus tag Blastp result

ORS278 S58 Gene annotation Similarity (%) Query coverage (%) Biblast hit

Nitrogenase activity BRADO5394 S58_23100 nitrogenase iron protein nifH 97 100 + BRADO5436 S58_22700 nitrogenase reductase-associated ferredoxin nifN 93 100 + BRADO5437 S58_22690 nitrogenase molybdenum-cofactor synthesis protein nifE 98 100 + BRADO5438 S58_22680 nitrogenase molybdenum-iron protein beta chain nifK 93 100 + Nif genes regulators BRADO0113 S58_01220 RNA polymerase sigma-54 factor rpoN 94 100 + BRADO0530 S58_71140 uridylyltransferase glnD 96 100 + BRADO3866 S58_39170 nitrogen assimilation regulatory protein ntrC 99 100 + BRADO3867 S58_39180 nitrogen regulation protein ntrB 96 100 +

BRADO3868 S58_39190 putative tRNA-dihydrouridine synthase putative NifR3-like protein 94 100 +

BRADO5449 S58_22580 Mo/Fe nitrogenase specific transcriptional regulator nifA 94 100 + Electron transfers BRADO2020 S58_55120 putative electron transfer flavoprotein dehydrogenase 94 100 + BRADO2734 S58_48840 cytochrome c-type biogenesis protein cycK 96 100 + BRADO4317 S58_30590 putative pyruvate ferredoxin/flavodoxin oxidoreductase 95 100 + BRADO6491 S58_11210 electron transfer flavoprotein alpha-subunit etfA 97 100 + BRADO6492 S58_11200 electron transfer flavoprotein beta-subunit etfB 96 100 + Central metabolism

BRADO0157 S58_01720 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase gpmA 93 100 +

BRADO0234 S58_02460 succinate dehydrogenase flavoprotein subunit sdhA 97 100 + BRADO0404 S58_04120 succinyl-CoA synthetase, beta subunit 98 100 + BRADO0405 S58_04130 succinyl-CoA synthetase, alpha subunit 98 100 + BRADO0409 S58_04170 dihydrolipoamide dehydrogenase lpD 96 100 + BRADO2034 S58_55000 pyruvate phosphate dikinase ppdK 94 100 + BRADO2682 S58_49330 methylmalonyl-CoA mutase small subunit mutA 84 100 +

BRADO3367 S58_41240 putative bifunctional enzyme: malic oxidoreductase (N-terminal); phosphotransacetylase (C-terminal) dme

97 100 +

BRADO4083 S58_35540 dihydrolipoyl dehydrogenase lpD 95 100 + BRADO4086 S58_35510 pyruvate dehydrogenase E1 component pdhB 96 100 + BRADO4087 S58_35500 pyruvate dehydrogenase E1 component pdhA 97 100 + BRADO4094 S58_35420 enolase eno 97 100 + BRADO5016 S58_26740 fumarase A fumA 96 100 + BRADO5815 S58_18040 putative amylomaltase 88 100 + Calvin cycle BRADO1122 S58_12290 phosphoglycerate kinase cbbK 95 99 + BRADO2274 S58_58790 ribulose bisphosphate carboxylase large chain cbbL 57 98 - BRADO2842 S58_47670 ribulose-phosphate 3-epimerase cbbE 96 100 + Amino acids biosynthesis BRADO0212 S58_02230 imidazoleglycerol-phosphate dehydratase hisB 95 100 + BRADO5973 S58_61440 aspartate aminotransferase A aatA 96 100 + Transporters BRADO1675 S58_58640 high-affinity phosphate transport protein pstC 95 100 + BRADO1677 S58_58620 high-affinity phosphate transport protein pstB 95 100 + BRADO3096 no hit putative ABC transporter no hit - BRADO4782 S58_28930 putative ABC transporter 90 100 + BRADO5117 S58_25640 putative permease 92 98 + BRADO6119 S58_63060 putative ABC transporter 88 98 + Chemotaxis BRADO1477 S58_17090 chemotaxis protein methyltransferase cheR 92 100 + BRADO3225 S58_30990 putative methyl-accepting chemotaxis protein 92 99 + Cofactors biosynthesis BRADO2036 S58_54960 L-aspartate oxidase nadB 80 99 + BRADO2133 S58_53930 putative RibBA-like protein 98 100 + Nucleotides biosynthesis

BRADO1205 S58_13160 phosphoribosylaminoimidazole carboxylase ATPase subunit purK 93 100 +

BRADO2776 S58_48430 phosphoribosylaminoimidazole-succinocarboxamide 99 100 +

synthetase purC BRADO0045 S58_00500 apolipoprotein N-acyltransferase lnt 90 100 + BRADO0589 S58_70520 putative peptidylpropyl isomerase 96 92 + BRADO2179 S58_53450 putative acylCoA dehydrogenase 94 98 + BRADO2938 S58_46790 putative hydrolase 90 99 + BRADO3673 S58_37700 putative cold shock DNA binding protein 96 100 + BRADO3794 S58_38400 putative sulfite reductase 92 101 + BRADO4549 S58_31820 putative D-alanyl-D-alanine carboxypeptidase 89 103 + BRADO5368 S58_23290 putative PhnM protein 85 97 + BRADO6079 S58_62670 DNA mismatch repair protein mutL 92 100 + Various regulators BRADO0560 S58_70830 putative GGDEF/EAL protein 93 99 + BRADO1049 S58_65790 putative PhyR-like protein 98 92 + BRADO3946 S58_39770 putative sensor HK 97 97 + BRADO4470 S58_32720 putative LabA-like protein 94 100 + BRADO4636 S58_30320 putative transcriptional regulator 90 100 + BRADO6832 S58_06370 two-component system regulator 95 100 + Translational processes BRADO0134 S58_01490 translation initiation factor IF-3 infC 96 100 +

BRADO0340 S58_03470 putative Ribosomal RNA small subunit methyltransferase B, putative RsmB-like protein 92 100 +

BRADO0376 S58_03830 tRNA (Guanine-N(1)-)-methyltransferase trmD 92 100 + BRADO0377 S58_03850 16S rRNA processing protein rimM 83 99 + BRADO0571 S58_70730 putative HemK-like protein 77 100 + BRADO3306 S58_41830 50S ribosomal protein L9 93 100 + BRADO3308 S58_41810 30S ribosomal subunit protein S18 rpsR 98 100 + BRADO3359 S58_41310 aspartyl-tRNA synthetase / asparaginyl-tRNA synthetase aspS 95 100 + BRADO3881 S58_39280 valyl-tRNA synthetase valS 95 97 + BRADO6137 S58_63260 50S ribosomal protein L36 rpmJ 100 100 + Proteins of unknown function BRADO0575 S58_70690 conserved hypothetical protein 94 100 + BRADO0665 S58_69770 hypothetical protein 88 100 +

BRADO1364 S58_13890 conserved hypothetical protein 88 100 + BRADO1426 S58_15500 hypothetical protein 59 103 + BRADO2671 S58_49470 conserved hypothetical protein 71 98 + BRADO2937 S58_46800 conserved hypothetical protein 82 99 + BRADO3770 S58_38200 conserved hypothetical protein 88 100 + BRADO4085 S58_35520 conserved hypothetical protein 98 100 + BRADO4683 S58_29910 conserved hypothetical protein 90 100 + BRADO4701 S58_29740 conserved hypothetical protein 83 100 + BRADO5022 S58_26670 conserved hypothetical protein 89 100 + BRADO5546 S58_20740 conserved hypothetical protein 83 97 + BRADO5734 S58_18790 conserved hypothetical protein 96 29 + BRADO5803 S58_18160 hypothetical protein 89 89 + BRADO6852 S58_06170 conserved hypothetical protein 94 99 + BRADO6923 no hit hypothetical protein no hit -

Table S5. Putative type III secretion system of S58 genome

Resuls of blastp against nr database Locus tag

Annotation Organisms Identity (%)

S58_72160 translocation protein in type III secretion system, RhcU Mesorhizobium amorphae CCNWGS0123 38

S58_72170 type III secretion component, RhcT Mesorhizobium alhagi CCNWXJ12-2 37

S58_72180 Y4yM Sinorhizobium fredii 47

S58_72190 type III secretion system protein YscR Mesorhizobium amorphae CCNWGS0123 61

S58_72200 type III secretion system translocation protein, RhcQ Mesorhizobium amorphae CCNWGS0123 35

S58_72210 hypothetical protein Phytophthora sojae 32

S58_72220 type III secretion system ATPase, YscN Hahella chejuensis KCTC 2396 57

S58_72230 NolV protein Bradyrhizobium elkanii 35

S58_72240 NolU protein Bradyrhizobium elkanii 27

S58_72250 Type III secretion bridge between inner and outermembrane

lipoprotein Mesorhizobium amorphae CCNWGS0123 48

S58_72260 prolipoprotein diacylglyceryl transferase Thermobispora bispora DSM 43833 29

S58_72270 NolW protein Sinorhizobium fredii HH103 39

dsRed pBjGroESL4

pBjGroEL4::dsRed2

Fig. S1. Structure of pBjGroEL4::dsRed2 (Kouchi H, Hayahsi M and Umehara Y. unpublished). pBjGroEL4::dsRed2 has three components: pBjGroEL4 (promoter region of the groEL4 gene in Bradyrhizobium japonicum USDA110), dsRed gene, and an Ω cassette in a mini-Tn5 of pUT (1).

Sm/Sp(Ω cassette)

Iend Oend

Fig. S2. Southern hybridization with pufBALM genes from Rhodopseudomonas palustris CGA009. BamHI-digested total DNA was electrophoresed as follows: Bradyrhizobium japonicum USDA110 (lane 1), NC6 (lane 2), USDA122 (lane 3), NC4 (lane 4), NK2 (lane5), USDA6 (lane 6), T7 (lane 7), T9 (lane 8), A. oligotrophica S58 (lane 9), G14130 (lane 10), Bradyrhizobium sp. ORS278 (lane 11), Bradyrhizobium sp. BTAi1 (lane 12), R. palustris CGA009 (lane 13), B. elkanii USDA76 (lane 14), Bradyrhizobium sp. HWK12 (lane 15), and Bradyrhizobium sp. HW13 (lane 16). Hybridization signals were detected in S58 (lane 9), ORS278 (lane 11) and BTAi1 (lane 12).

ORS27

8 BTAi1

S58

CGA00

9

S58 [Mb] S58 [Mb]

OR

S27

8 [M

b]

BTA

i1 [M

b]

Sim

ilarit

y [%

]

0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0 A B

Fig. S3. Synteny plots between Agromonas oligotrophica S58 and two Aeschynomene indica symbionts, Bradyrhizobium sp. ORS278 (A) and Bradyrhizobium sp. BTAi1 (B). Syntenic dot plots represent the results of genome comparisons made with blastn. Dot color indicates percent similarity, as indicated by the color scale on the right.

fixR nifA sufBsufCsufDsufSsufE nifH nifD nifK nifE nifNnifX nifS nifB nifH nifV fixA fixB fixC

ORS278

S58

BTAi1

5640kb 5588kb

2582kb 2632kb

6154kb 6104kb

70 75 80 85 90 95 100

Fig. S4. Comparisons of nitrogen fixation-related gene clusters among Agromonas oligotrophica S58, Bradyrhizobium sp. ORS278, and Bradyrhizobium sp. BTAi1. Nitrogen fixation clusters were highly conserved among the three strains. The color scale indicates the percent similarity.

!" #" $" %" &" '" (" )""cbbS cbbL cbbR

cbbA cbbT cbbP cbbF

*+,-&'./012345-

,$'

6789)

-#--!)).-#-$)(':;3

</=>?04>4;3@5AB

*+,-&'./012345))&&)!%&.)&&($%&:;3

</=>?04>4;3@5AB

%%$$"--.%%%!)(#:;3

%%%)&)#.%%%('&-:;3

Fig. S5. Comparisons of cbb gene clusters among Agromonas oligotrophica S58, Bradyrhizobium sp. ORS278, and Bradyrhizobium sp. BTAi1. The color scale indicates the percent similarity.

!" !# $" $# %" %# &""

!"#$ !"#% #&'( )"*+ ,#)( !"#- !"#. !"#/ !"#0 11*23 !"#4 !"#5 11*26 !7!1#5 189: 189- !"#; !"#< !"#= !"#> "7?+ "7?% "7?@ "7?> "7?/ "7?A !"#@ !"#A #8?B

!"#A!"#@#&'%"7?A"7?/#&'+"7?>"7?@"7?%"7?+!"#>!"#=!"#<!"#;189-189:11*23!"#4!"#511*26!"#0!"#/!"#.!"#-,#)(#&'("!!2

!"#: !"#+ 189(/

18#( !"#+ 189(/

'()*&+,+!"-

+,!.&-

/01.!$&,!&%-

&,!!&-

1#$+,++2- +,!&3-

1.22.&.,"""-

.,"#&-

45)""%&,+!.- &,!.!-

64789:;8;<=>?@A

64789:;8;<=>?@A

64789:;8;<=>?@A

Fig. S6. Comparisons of photosynthetic gene clusters among Agromonas oligotrophica S58, Bradyrhizobium sp. ORS278, Bradyrhizobium sp. BTAi1, Bradyrhizobium sp. S23321, and Rhodopseudomonas palustris CGA009. The photosynthetic gene cluster of S58 was highly conserved with those of ORS278 and BTAi1. The gene arrangement of the photosynthetic gene cluster in the S58 genome was very similar to the arrangements in S23321 and CGA009, but it showed lower similarity with pufMLAB of R. palustris CGA009 (shown in red type), as also analyzed by Southern hybridization (see Fig. S2).

pufMLAB

pufMLAB

Referenceforsupplementarymaterials

1)  WilsonKJ,SessitschA,CorboJC,GillerKE,AkkermansAD,JeffersonRA.1995.beta‐Glucuronidase(GUS)transposonsforecologicalandgeneticstudiesofrhizobiaandothergram‐negativebacteria.Microbiology141:1691–1705.

2)  GiraudE,MoulinL,VallenetD,BarbeV,CytrynE,AvarreJC,JaubertM,SimonD,CartieauxF,PrinY,BenaG,HannibalL,FardouxJ,KojadinovicM,VuilletL,LajusA,CruveillerS,RouyZ,MangenotS,SegurensB,DossatC,FranckWL,ChangWS,SaundersE,BruceD,RichardsonP,NormandP,DreyfusB,PignolD,StaceyG,EmerichD,VerméglioA,MédigueC,SadowskyM.2007.Legumessymbioses:absenceofNodgenesinphotosyntheticbradyrhizobia.Science316:1307‐1312.

supplementary matterials soil oligotrophic bacterium ... · 26/03/2013 · supplementary...

Documents