CHAPTER 2. Phylogeny Trumps Chemotaxonomy
3.3. Results
(Burkholderia sprentiae LMG 27175T), 78.0% (Burkholderia acidipaludis NBRC 101816T), and 78.0% (Burkholderia tropica LMG 22274T). Those pairwise calculated ANI values were visualized as a dendrogram (Figure 11). The ANI values between strain JC2949T and related type strains were calculated to be 90.4%
(Burkholderia jiangsuensis MP-1TT), 87.4% (Burkholderia zhejiangensis OP-1T), 86.2% (Burkholderia cordobensis YI23), 82.2% (Burkholderia grimmiae R27T), and 80.8% (Burkholderia glathei ATCC 29195T). Those pairwise calculated ANI values were visualized as a dendrogram (Figure 12).
Figure 9. Neighbor-Joining tree based on 16S rRNA gene sequences to describe the taxonomic position of strain JC2948T. Black circles indicate that ML and Bayesian tree-reconstructing methods also supported corresponding groupings. Ralstonia picketti ATCC 27511T was used as outgroup (not shown). Bars stand for nucleotide substitutions per position.
Figure 10. Neighbor-Joining tree based on 16S rRNA gene sequences to describe the taxonomic position of strain JC2949T. Black circles indicate that ML and Bayesian tree-reconstructing methods also supported corresponding groupings. Ralstonia picketti ATCC 27511T was used as outgroup (not shown). Bars stand for nucleotide substitutions per position.
Figure 11. Dendrogram depicting the UPGMA clustering based on ANI values of strain JC2948T and its neighboring strains. GenBank BioProject accessions are written in parentheses.
Figure 12. Dendrogram depicting the UPGMA clustering based on ANI values of strain JC2949T and its neighboring strains. GenBank BioProject accessions are written in parentheses.
The morphological observation indicates that both strains have rod-shaped feature, aerobe, and motile. The strains JC2949T has single, polar flagellum whereas strain JC2948T has no flagella (Figure 13).
Strain JC2948T does not reduce nitrate to nitrite/nitrogen and cannot produce indole. Acid is not produced from glucose. This strain assimilates D-glucose, N- acetylglucosamine and D-mannitol. This strain weakly assimilates malic acid, potassium gluconate, and phenylacetic acid. This strain cannot assimilate beta- galactosidase, arginine dihydrolase and urease (Table 2).
Strain JC2949T showed negative for methyl red test and positive for Voges- Proskauer test, has catalase and oxidase activity. This strain hydrolyzes starch and Tween 80. This strain is positive for glucose fermentation and the PNPG test in the API 20NE kit. This strain also negative for reducing nitrate to nitrite/nitrogen and cannot produce indole, arginine dihydrolase and urease. This strain assimilates D- glucose, D-mannose, D-mannitol, N-acetylglucosamine, potassium gluconate, malic acid and phenylacetic acid. However, this strain does not assimilate L- arabinose, maltose, capric acid, adipic acid or trisodium citrate. In API ZYM tests, strain JC2949T positive for alkaline phosphatase, leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase, but negative for esterase (C4), esterase lipase (C8), lipase (C14), valine arylamidase, crystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase (Table 3).
The major fatty acid composition of JC2948T were C17:0cyclo (22.5%), C16:0
(22.1 %), and C19:0cyclov8c (17.7 %), as mentioned in Table 4. The major fatty acid composition of JC2949T was C18:1v7c (38.6%) and C16:0 (11.7%), as mentioned in
Table 5. The major polar lipids of strain JC2948T were diphosphatidylglycerol (DPG) and phosphatidylethanolamine (PE). The major polar lipids of strain JC2949T were DPG, PE, phosphatidylglycerol (PG) and an unknown aminophospholipid (Figure 14). For both strains, the predominant isoprenoid quinone was ubiquinone UQ-8.
Figure 13. Transmission electron micrograph of the novel strains. Each figure corresponds for (A) JC2948T and (B) JC2949T, respectively. Cells were grown on NA at 30 °C for 2 days. Bar, 0.2μm in (A) and 0.5μm in (B).
Table 2. Major phenotypic characteristics that differentiate strain JC2948T from other species of the genus Burkholderia. Strains: 1, strain JC2948T; 2, B.
sprentiae LMG 27175T; 3, B. tuberum LMG 21444T; 4, B. acidipaludis SA33T; 5, B. tropica LMG 22274T; 6, B. diazotrophica JPY461T. +, Positive; -, negative; ND, not determined; w, weak positive; Rn, root nodule. G+C contents are obtained by genome sequencing except strain 6 (HPLC). Data from this study except strain 4 and 6
* Data from (Aizawa et al., 2010).
† Data from (Sheu et al., 2013).
§ Data from (Reeve et al., 2013)
¶ Data from (Angus et al., 2014)
Characteristic 1 2 3 4* 5† 6†
Isolation source Soil Rn Rn Plant Soil Rn
Nitrate to nitrogen - + - + + +
Motility on soft agar - + + - + +
Oxidase - + + + + +
Assimilation of
Capric acid - - + ND + -
Malic acid w w + ND + +
Trisodium citrate - + + ND - +
Phenyl acetate w + - ND + -
D-Mannitol + + W + + +
D-Mannose - + + + + +
L-Arabinose - - - + + +
G+C (mol %) 63.8 63.2§ 63.2¶ 65.2 64.0 63-65†
Table 3. Major phenotypic characteristics that differentiate strain JC2949T from other species of the genus Burkholderia. Strains: 1, strain JC2949T 2, B. jiangsuensis MP-1T; 3, B. grimmiae R27T; 4, B. zhejiangensis OP-1T; 5, B. cordobensis LMG 27620T; 6, B. glathei ATCC 29195T. +, Positive; -, negative; w, weak or delayed reaction; ND, no data
Characteristic 1 2* 3 4 5† 6
pH Range 5.0-10.0 5.0-9.0 4.5-10.0 4.0-10.0 ND 4.5-10.0 Optimum pH 6.5-7.0 7.0 7.0-7.5 7.0-7.5 ND 5.0-5.5
Voges-Proskauer test + w - + ND +
Tween 80 Hydrolysis + ND + - ND +
Reduction of nitrate to nitrite/nitrogen
-/- +/- +/- -/-
+/N D
+/-
Arginine dihydrolase - - - + - -
PNPG test + - - + - ND
Esterase (C4) - + + + - +
Esterase Lipase (C8) - + - - - +
Valine arylamidase - w - - - -
N-acetyl-β- glucosaminidase
- - - +
Assimilation of:
L-Arabinose - + + + + +
Adipic acid - w - - - -
Capric acid - + - + - +
Trisodium citrate - + + - - -
* Data from (Liu et al., 2014)
† Data from (Draghi et al., 2014)
Table 4. Cellular fatty acid composition of strain JC2948T and related strains of the genus Burkholderia. Strains: 1, strain JC2948T; 2, B.
sprentiae LMG 27175T; 3, B. tuberum LMG 21444T; 4, B. acidipaludis SA33T; 5, B. tropica LMG 22274T; 6, B. diazotrophica JPY461T. Less than 1% of the total fatty acids in all strains were not included. -, Not detected
Fatty acid 1 2* 3† 4† 5 6†
C17:0 cyclo 22.47 10.60 19.70 16.40 21.26 8.40
C16:0 22.09 19.90 24.70 27.80 20.17 18.20
C19:0 cyclo ω8c 17.70 4.10 17.50 11.30 17.61 8.40
C18 : 1 ω7c 8.88 30.50 10.90 15.60 14.30 32.20
Summed Feature 2§ 6.33 8.50 6.10 6.00 5.29 5.20
C16:0 3-OH 5.79 6.70 5.40 5.60 5.18 5.20
C14:0 5.32 4.70 4.10 4.60 4.16 4.50
C16:0 2-OH 2.61 2.10 2.50 3.00 2.69 3.40
Summed Feature 3§ 1.71 13.10 1.70 2.80 2.30 6.20
C16:1 2-OH 1.15 3.30 1.20 1.90 1.89 1.90
C18:1 2-OH 0.98 1.30 - 1.70 1.03 1.20
* Data from (De Meyer et al., 2013).
† Data from (Sheu et al., 2013).
§ Summed feature 2 comprises C14 : 0 3-OH and/or C16: 1 iso I; summed feature 3 comprises C16 : 1 ω7c and/or C16 : 1 ω6c.
Table 5. Cellular fatty acid composition of strain JC2949T and related strains of the genus Burkholderia.Strains: 1, strain JC2949T; 2, B.
jiangsuensis MP-1T; 3, B. grimmiae R27T; 4, B. zhejiangensis OP-1T; 5, B. cordobensis LMG 27620T; 6, B. glathei ATCC 29195T. Fatty acids amounting to less than 1% of the total fatty acids were not included. tr, trace amount. Data from this study
Fatty acid 1 2* 3 4 5† 6
C14:0 3.7 4.6 3.2 3.6 4.2 3.8
C16:0 11.7 16.8 16.7 17.0 17.0 11.9
C16:0 2-OH 2.4 5.1 1.7 3.6 2.5 1.9
C16:0 3-OH 5.2 5.6 4.2 4.8 5.9 5.0
C16:1 2-OH 1.1 tr tr 1.6 tr 2.8
C17:0 cyclo 3.1 15.0 11.9 8.1 5.3 tr
C18 : 1 ω7c 38.6 23.3 37.2 34.8 36.6 40.5
C19:0 10-methyl tr 3.3 tr tr tr tr
C19:0 cyclo ω8c 4.5 8.1 2.4 3.9 1.8 tr
Summed Feature 2§ 5.6 5.7 4.4 5.0 7.2 4.9
Summed Feature 3§ 20.5 8.5 14.7 15.0 17.6 25.9
* Data from (Liu et al., 2014)
† Data from (Draghi et al., 2014)
§ Summed feature 2 comprises C14 : 0 3-OH and/or C16: 1 iso I; summed feature 3 comprises C16 : 1 ω7c and/or C16 : 1 ω6c.
Figure 14. Two-dimensional TLC of polar lipid extracts. Strains: (a) JC2948T and (b) JC2949T. PE: phosphatidyl ethanolamine; PG: phosphatidyl glycerol; DPG:
The phylogenomic tree of Burkholderia sensu lato also driven, and the tree showed that the member genera of Burkholderia sensu lato (Burkholderia, Caballeronia, Mycetohabitans, Paraburkholderia, Robbsia and Trinickia) are clearly separated (Figure 15). It is clearly confirmed that strain JC2948T is located within Paraburkholderia clade, and strain JC2949T is located within Caballeronia clade by the phylogenomic inference.
Figure 15. The compressed and collapsed ML phylogenomic tree of Burkholderia sensu lato based on ubiquitous genes. UBCG software (Na et al., 2018) was used for the tree inference. Ralstonia picketti ATCC 27511T was used as outgroup (not shown). The bar stands for nucleotide substitutions per position.
Numbers at nodes stands for Gene Support Index (GSI) of UBCG software. The newly isolated strains in this study (JC2948T and JC2949T) were written as bold