92
99
65
66
91
Ochrobactrum cytisi ESC1T (AY776289)
T
98 Ochrobactrum tritici SCII24 (AJ242584)
Ochrobactrum anthropi ATCC49188T (CP000758)
98
Ochrobactrum lupini LUP21T (AY457038)
Brucella ceti NCTC12891T (AM158982)
Brucella melitensis 16MT (AE008918)
99
Brucella microti CCM4915T (CP001578)
Rhizobium daejeonense L61T (AY341343)
Ensifer saheli LMG7837T (X68390)
92
T
99 Sinorhizobium americanum CFNEI156 (AF506513)
Rhizobium etli CFN42T (CP000133)
99
Rhizobium leguminosarum IAM 12609 (D12782)
Rhizobium trifolii ATCC 14480 (AY509900)
87 Rhizobium gallicum R602spT (U86343)
Rhizobium indigoferae CCBAU71042T (AF364068)
Rhizobium soli DS-42T (EF363715)
Rhizobium mesosinicum CCBAU25010T (DQ100063)
Arthrobacter viscosus LMG16473T (AJ639832)
80
Rhizobium alamii GBV016T (AM931436)
Rhizobium oryzae Alt505T (EU056823)
Pleomorphomonas koreensis Y9T (AB127972)
Pleomorphomonas oryzae F-7T (AB159680)
Pleomorphomonas diazotrophica R5-392T (JQ346801)
Blastochloris sulfoviridis DSM729T (D86514)
Methylocystis echinoides IMET10491T (AJ45847)
Prosthecomicrobium pneumaticum ATCC23633T (AB017203)
Agaricicola taiwanensis CC-SBABM117T (FJ594057)
Breoghania corrubedonensis UBF-P1T (GQ272328)
Pannonibacter phragmitetus C6-19T (AJ400704)
Pseudovibrio denitrificans DN34T (AY486423)
Labrenzia aggregata IAM12614T (AAUW01000037)
71
Labrenzia alba CECT5094T (AJ878875)
94
20
Fig. S1. Maximum-parsimony tree showing the phylogenetic position of strain R5-392T based on 16S rRNA gene
sequences. Bar, 20 changes. Bootstrap values of 70 or more from 100 replicates are shown.
M. Madhaiyan, T. Y. Jin, J. J. Roy, S.-J. Kim, H.-Y. Weon, S.-W. Kwon & L. Ji (2013). Pleomorphomonas
diazotrophica sp. nov., an endophytic N-fixing bacterium isolated from the root tissue of Jatropha curcas L.
International Journal of Systematic and Evolutionary Microbiology 63
Azospirillum lipoferum sp59bT (AF216882)
100
Azospirillum brasilense ATCC29145T (M64344)
Sinorhizobium meliloti (V01215)
Methylobacterium sp. 4-46 (NC-010511)
Gluconacetobacter diazotrophicus PA15T (AF030414)
Rhodobacter capsulatus (M15270)
Rhodovulum sulfidophilum W4T (AB079630)
73
Rhodopseudomonas lichen O5 (AB241413)
98
Rhodopseudomonas palustris VA2-2 (AB079623)
Pleomorphomonas oryzae F4 (AB182914)
Pleomorphomonas diazotrophica R5-392T (--------)
Pleomorphomonas oryzae F7 (AB161454)
Pleomorphomonas oryzae B24 (AB182912)
96
Pleomorphomonas oryzae B-4 (AB182910)
98 Pleomorphomonas oryzae B-18 (AB182911)
Pleomorphomonas oryzae B-32 (AB182913)
Beijerinckia indica subsp. indica ATCC21423 (AF296354)
Acidithiobacillus ferrooxidans ATCC33020 (M15238)
Bradyrhizobium japonicum USDA110 (K01620)
Mesorhizobium loti R7A (AL672114)
100
Rhizobium etli CFN42T (NC_004041)
0.1
Fig. S2. Neighbour-joining tree based on nifH gene sequences showing relationships between strain R5-392T and
diazotrophs of the alphaproteobacteria. Bar, 0.1 substitutions per nucleotide position.
M. Madhaiyan, T. Y. Jin, J. J. Roy, S.-J. Kim, H.-Y. Weon, S.-W. Kwon & L. Ji (2013). Pleomorphomonas
diazotrophica sp. nov., an endophytic N-fixing bacterium isolated from the root tissue of Jatropha curcas L.
International Journal of Systematic and Evolutionary Microbiology 63
M. Madhaiyan, T. Y. Jin, J. J. Roy, S.-J. Kim, H.-Y. Weon, S.-W. Kwon & L. Ji (2013). Pleomorphomonas diazotrophica sp. nov., an endophytic N-fixing bacterium isolated
from the root tissue of Jatropha curcas L. International Journal of Systematic and Evolutionary Microbiology 63
Fig. S3. Polar lipid distribution of strain R5-392T visualized with 5% ethanolic molybdatophosphoric acid for total lipids (a); ninhydrin (Sigma) for aminolipids (b); molybdenum
blue (Sigma) for phospholipids (c); Dragendorff reagent for choline-containing lipids (d); and α-naphthol/sulphuric acid reagent for glycolipids (e). The polar lipids were identified
as follows: DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PC, phosphatidylcholine; PME, phosphatidylmonomethylethanolamine; PE, phosphatidylethanolamine; and two
aminophospholipids (AL1 and AL2).
Table S1. Cellular fatty acid profiles of strain R5-392T and its closest relatives
Strains: 1, R5-392T; 2, P. oryzae KCTC 12245T; 3, P. koreensis KCTC 12246T. All data from this study. Strains R5392T and P. oryzae KCTC 12245T were grown on R2A media for 2 days at 30 °C to reach stationary phase but strain P.
koreensis KCTC 12246T reached stationary phase after 4 days. Values are percentages of total fatty acids. Fatty acids
representing less than 0.2% in all strains were omitted.
Fatty acid
1
2
3
C14:0
0.3 0.4
C16:0
11.7 8.3 18.4
C17:0
1.4 0.7
C18:0
3.9 7.8 3.3
C18:0 3-OH
4.0 2.8 2.1
C17:0 cyclo
0.5 0.4 1.3
C19:0 cyclo ω8c
8.2 13.6 40.3
Summed feature 2*
5.6 2.8 3.6
Summed feature 3
1.7 0.7 0.4
Summed feature 8
62.6 61.8 28.3
*Summed features are groups of two or three fatty acids that cannot be separated by GLC with the MIDI system. SF2:
C14:0 3-OH/iso-C16:1 I; SF3: C16:1ω7c/C16:1ω6c; SF8: C18:1ω7c/C18:1ω6c.
M. Madhaiyan, T. Y. Jin, J. J. Roy, S.-J. Kim, H.-Y. Weon, S.-W. Kwon & L. Ji (2013). Pleomorphomonas
diazotrophica sp. nov., an endophytic N-fixing bacterium isolated from the root tissue of Jatropha curcas L.
International Journal of Systematic and Evolutionary Microbiology 63
Table S2. Carbon-source utilization (Biolog) by strain R5-392T and its closest relatives
Strains: 1, R5-392T; 2, P. oryzae KCTC 12245T; 3, P. korensis KCTC 12246T. All data from this study. All strains are
positive for L-arabinose, cellobiose, D-fructose, α-D-glucose, maltose*, D-mannitol, D-mannose, raffinose, sucrose,
trehalose, turanose, pyruvic acid methyl ester, D-gluconic acid* and uridine. All strains are negative for α-cyclodextrin,
glycogen, tween 40, N-acetyl-D-galactosamine, D-arabitol, cis-aconitic acid, citric acid, formic acid, D-galactonic acid
lactone, D-galacturonic acid, D-glucosaminic acid, D-glucuronic acid, α-hydroxybutyric acid, β-hydroxybutyric acid, γhydroxybutyric acid, ρ-hydroxypenylacetic acid, itaconic acid, α-ketovaleric acid, DL-lactic acid, malonic acid,
propionic acid, quinic acid, D-saccharic acid, sebacic acid, succinamic acid, D-alanine, L-alanine, L-alanylglycine, Lasparagine, L-glutamic acid, glycyl-L-aspartic acid, glycyl-L-glutamic acid, L-histidine, hydroxy-L-proline, L-ornithine,
L-phenylalanine, L-pyroglutamic acid, D-serine, L-threonine, DL-carnitine, phenylethylamine, putrescine, 2aminoethanol, 2,3-butanediol, glycerol, DL-α-glycerol phosphate and α-D-glucose-1-phosphate. +, Positive reaction; ,
negative reaction; W, weakly positive reaction. *Discrepancies were found between this study and Im et al. (2006).
Carbon source
1 2 3
Carbon source
Dextrin
+
W
+
Succinic acid monomethyl ester +
W
+
Tween 80
W

W
Acetic acid

W

N-Acetyl-D-glucosamine 
W
+
α-Ketobutyric acid
W W W
Adonitol
W
+ +
α-Ketoglutaric acid
W
 
i-Erythritol
 +
W
Succinic acid

W

L-Fucose
+ 
W
Bromosuccinic acid

W

D-Galactose
+ +
W
Glucuronamide
W
 
Gentiobiose
+ + 
L-Alaninamide
+
W
myo-Inositol
+ +
W
L-Aspartic
 + 
α-Lactose
+ +
W
L-Leucine

W

Lactulose
+ +
W
L-Proline

W

Melibiose
W
+
W
L-Serine

W

C2-β-methyl-D-glucoside +
W
+
γ-Aminobutyric acid
W
 
D-Psicose
+
W
+
Urocanic acid
W

W
L-Rhamnose

W W
Inosine
+ +
W
D-Sorbitol
+
W
Thymidine
+
+
Xylitol
 + +
+
acid
1 2 3
W
+
References
Im, W. T., Kim, S. H., Kim, M. K., Ten, L. N. & Lee, S. T. (2006). Pleomorphomonas koreensis sp. nov., a nitrogenfixing species in the order Rhizobiales. Int J Syst Evol Microbiol 56, 1663–1666. doi:10.1099/ijs.0.63499-0
Medline
M. Madhaiyan, T. Y. Jin, J. J. Roy, S.-J. Kim, H.-Y. Weon, S.-W. Kwon & L. Ji (2013). Pleomorphomonas
diazotrophica sp. nov., an endophytic N-fixing bacterium isolated from the root tissue of Jatropha curcas L.
International Journal of Systematic and Evolutionary Microbiology 63