Supplementary Materials s41

Supplementary materials

Table S1. DNA-DNA relatedness between strain YIM Hb-3T and E. mori LMG 25706T

The two DNAs for DNA-DNA hybridization / DNA as probe / DNA immobilized / Values of three replicates (%) / Average (%) / The result of DNA-DNA hybridization (%)
1 / 2 / 3
1 and 2 / 1
2 / 2
1 / 47.04
32.84 / 52.36
49.82 / 50.9248.60 / 50.11±2.75
43.75±9.47 / 46.93±3.81

1, strain YIM Hb-3T; 2, E. mori LMG 25706T

Table S2 GenBank accessions for the four protein-coding genes gyrB, rpoB, atpD and infB employed in this study. Sequences of strain E. tabaci YIM Hb-3T, E. mori LMG 25706T and E. cloacae subsp. cloacae KCTC 2361T are from this study while the rest are downloaded from http://www.ncbi.nlm.nih.gov.

strain / gyrB / rpoB / atpD / infB
YIM Hb-3T / KT207482 / KT207484 / KT207481 / KT207483
LMG 25706T / KT207478 / KT207480 / KT207477 / KT207479
KCTC 2361T / KT207474 / KT207476 / KT207473 / KT207475
DSM 17506T / JX424989 / JX425248 / JX424859 / JX425118
CCM 2420T / JX424981 / JX425240 / JX424851 / JX425110
LMG 26282T / JX424995 / JX425254 / JX424865 / JX425124
LMG 2683T / JX424979 / JX425238 / JX424849 / JX425108
CCUG 27126T / JX424983 / JX425242 / JX424853 / JX425112
LMG 23768T / JX424985 / JX425244 / JX424855 / JX425114
LMG 7871T / JX425103 / JX425359 / JX424974 / JX425233
LMG 7859T / JX425104 / JX425360 / JX424975 / JX425234
LMG 3246T / JX425089 / JX425345 / JX424960 / JX425219
LMG 5519T / JX425090 / JX425346 / JX424961 / JX425220
CCUG 49023T / JX494750 / JX494753 / JX494748 / JX494751
ATCC 23216T / JX424997 / JX425256 / JX424867 / JX425126
LMG 2094T / JX425098 / JX425354 / JX424969 / JX425228
LMG 23571T / JX425097 / JX425353 / JX424968 / JX425227
LMG 3113T / JX425094 / JX425350 / JX424965 / JX425224
LMG 3184T / JX425096 / JX425352 / JX424967 / JX425226
CCM 7122T / JX425062 / JX425318 / JX424932 / JX425191

Table S3 Sequence similarity indices between the strain YIM Hb-3T and members of the genus Enterobacter for the four concatenated protein-coding genes gyrB, rpoB, atpD and infB as calculated by the Clustal X version 2.1 software.

Sequences of strains 1 to 3 are from this study while the rest are downloaded from http://www.ncbi.nlm.nih.gov.

Gene / Similarities ( % )
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11
gyrB / 100.00 / 99.67 / 92.63 / 93.26 / 92.59 / 89.76 / 92.44 / 90.84 / 92.18 / 97.04 / 93.94
rpoB / 100.00 / 98.88 / 99.72 / 97.96 / 97.17 / 97.02 / 96.55 / 94.35 / 97.65 / 98.12 / 96.55
atpD / 100.00 / 98.87 / 96.16 / 97.20 / 97.20 / 94.39 / 96.57 / 93.93 / 97.35 / 97.66 / 97.20
infB / 100.00 / 99.05 / 90.45 / 93.73 / 89.27 / 88.61 / 94.06 / 88.45 / 93.73 / 95.05 / 93.89
MLSA / 100.00 / 99.52 / 94.22 / 95.31 / 93.86 / 92.20 / 94.79 / 91.72 / 94.95 / 96.85 / 95.27

1, E. tabaci YIM Hb-3T; 2, E. mori LMG 25706T; 3, E. cloacae subsp. cloacae KCTC 2361T; 4, E. hormaechei CCUG 27126T; 5, E. cloacae subsp. dissolvens LMG 2683T; 6, E. aerogenes LMG 2094T; 7, E. soli LMG 26282T; 8, E. kobei CCUG 49203T; 9, E. ludwigii LMG 23768T; 10, E. asburiae DSM 17506T; 11, E. cancerogenus CCM 2420T.

Table S4 MLSA distance based on four concatenated sequences (gyrB, rpoB, infB and atpD) between strain YIM Hb-3T and members of the genus Enterobacter.

The evolutionary distance is determined using MEGA version 5.0 with the Kimura two-parameter model. Sequences of strains 1 to 3 are from this study while the rest are downloaded from http://www.ncbi.nlm.nih.gov.

Strain / MLSA distance (kimura two-parameter)
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11
1
2 / 0.005
3 / 0.060 / 0.062
4 / 0.049 / 0.049 / 0.056
5 / 0.064 / 0.065 / 0.021 / 0.058
6 / 0.083 / 0.084 / 0.093 / 0.076 / 0.091
7 / 0.054 / 0.055 / 0.078 / 0.064 / 0.081 / 0.095
8 / 0.088 / 0.090 / 0.107 / 0.094 / 0.103 / 0.097 / 0.101
9 / 0.053 / 0.053 / 0.061 / 0.045 / 0.061 / 0.081 / 0.057 / 0.090
10 / 0.032 / 0.034 / 0.061 / 0.052 / 0.062 / 0.083 / 0.057 / 0.089 / 0.054
11 / 0.049 / 0.050 / 0.076 / 0.054 / 0.076 / 0.086 / 0.062 / 0.091 / 0.054 / 0.051

Fig. S1 Transmission electron micrograph of strain YIMHb-3T grown on LB medium for 36 h. Bar, 1.0 μm.

(a) (b)

Fig. S2 Polar lipid profiles of strain YIM Hb-3T (a) in comparison with that of the reference strain E. mori LMG 25706T (b). DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PL, phospholipid; AL, aminolipid; APL, aminophospholipid.

The compositions of the polar lipids is analyzed by two-dimensional thin-layer chromatogram and stained with 5% ethanolic molybdophosphoric acid. The chromatographic conditions are as follows: Silica Gel 60 thin-layer plates (10 by 10 cm) are spotted with 20 μl of a whole-cell lipid extract. Chloroform-methanol-water (65:25:4, vol/vol/vol) is used to develop the chromatogram in the first direction, and chloroform-acetic acid-methanol-water (80:18:12:5, vol/vol/vol/vol) is used in the second direction.

Fig. S3 Maximum-Parsimony phylogenetic tree based on 16S rRNA gene sequences showing the relationship between strain YIM Hb-3T and other related strains of the family Enterobacteriaceae. Bootsrap values (expressed as percentages of 1000 replicates) of above 50% are shown at nodes. Xenorhabdus nematophila DSM 3370T (AY278674) is used as the outgroup.

Fig. S4 Maximum-Likelihood phylogenetic tree based on 16S rRNA gene sequences showing the relationship between strain YIM Hb-3T and other related strains of the family Enterobacteriaceae. Bootsrap values (expressed as percentages of 1000 replicates) of above 50% are shown at nodes. Xenorhabdus nematophila DSM 3370T (AY278674) is used as the outgroup. Bar, 0.005 substitutions per nucleotide position.

Fig. S5 Maximum-Parsimon phylogenetic tree between strain YIM Hb-3T and other related strains of the family Enterobacteriaceae based on the concatenated nucleotide sequences of gyrB, rpoB, atpD and infB genes. Percentage bootstrap values based on 1000 replicates are given at nodes. The sequence of Serratia marcescens subsp. sakuensis CCM 7122T is included as the outgroup. Sequences of strains E. tabaci YIM Hb-3T, E. mori LMG 25706T and E. cloacae subsp. cloacae KCTC 2361T are from this study while the rest are downloaded from http://www.ncbi.nlm.nih.gov.

Fig. S6 Maximum-Likelihood phylogenetic tree between strain YIM Hb-3T and other related strains of the family Enterobacteriaceae based on the concatenated nucleotide sequences of gyrB, rpoB, atpD and infB genes. Percentage bootstrap values based on 1000 replicates are given at nodes. Bar, 0.02 substitutions per nucleotide position. The sequence of Serratia marcescens subsp. sakuensis CCM 7122T is included as the outgroup. Sequences of strains E. tabaci YIM Hb-3T, E. mori LMG 25706T and E. cloacae subsp. cloacae KCTC 2361T are from this study while the rest are downloaded from http://www.ncbi.nlm.nih.gov.