1
Supporting information
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Materials and methods
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Strain isolation and characterization
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The DBHB-degrading strain was isolated from the surface soil collected from a manufacturing facility
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producing halogenated aromatic compounds located in Jiangsu, China. The isolation procedure was
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carried out in MM medium supplemented with 0.2 mM DBHB by the conventional enrichment culture
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technique (Li et al. 2010). The bacterial strain EOB-17 showed the ability to degrade DBHB and was
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isolated and purified.
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Strain EOB-17 was identified based on its morphological, physiological and biochemical properties
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with reference to Bergey’s Manual of Determinative Bacteriology and 16S rRNA gene sequence
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analysis. Cell morphology was examined by light microscopy (Olympus; × 1000) using phase-contrast
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optics and by transmission electron microscopy (H-7650, Hitach) using cells that had been grown for
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16 hours at 30°C on LB medium. The growth of strain EOB-17 under anaerobic conditions was
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determined using the GasPak Anaerobic Systems (BBL) according to the manufacturer’s instructions.
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The genomic DNA of strain EOB-17 was extracted by high-salt precipitation (Miller et al. 1988). The
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16S rRNA gene was amplified by a previously described PCR method (Thompson et al. 1997). The
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nearly complete 16S rRNA gene sequence (1491 bp) was deposited in the GenBank database under the
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accession number KR232560. Phylogeny was analyzed with MEGA version 4.0 software, and distance
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was calculated using the Kimura 2 parameter distance model. An unrooted tree was built by the
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neighbor-joining method (Kumar et al. 2008). The dataset was bootstrapped 1,000 times.
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Supplementary Fig. S1 and Supplementary Fig. S2
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References
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Kumar S, Nei M, Dudley J, Tamura K (2008) MEGA: a biologist-centric software for evolutionary
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analysis of DNA and protein sequences. Brief Bioinform 9:299-306
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Li R, Zheng J, Wang R, Song Y, Chen Q, Yang X, Li SP, Jiang JD (2010) Biochemical degradation
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pathway of dimethoate by Paracoccus sp. Lgjj-3 isolated from treatment wastewater. Int Biodeter
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Biodegr 64:51-57
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Miller SA, Dykes DD, Polesky HF (1988) A simple salting out procedure for extracting DNA from
human nucleated cells. Nucleic Acid Res 16:1215
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows
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interface: flexible strategies for multiple sequence alignment aided by quality analysis
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tools. Nucleic Acid Res 25:4876-4882
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Supplementary Table 1 Sequence comparison of deduced DBHB-degrading genes in Delftia sp.
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EOB-17
Gene (accession
Proposed product
No.)
bhbA3
No. of amino
Homologous protein (GenBank
Identity
acids
accession No.) and host
%
Reductive
BhbA (YP_007878394), Comamonas
1071
(KR232561)
dehalogenase
bhbB3
Extracytoplasmic
99
sp. 7D-2
BhbB (YP_007878395), Comamonas
337
(KR232562)
binding receptor
bhbF3
4-hydroxybenzoate
(KR232563)
3-monooxygenase
99
sp. 7D-2
4-hydroxybenzoate
391
3-monooxygenase (WP_016446365),
99
Delftia acidovorans
protocatechuate
protocatechuate 3,4-dioxygenase
bhbD3
3,4-dioxygenase
121
(WP_027016029),
93
(KR232564)
alpha subunit
Comamonas composti
protocatechuate
protocatechuate 3,4-dioxygenase
bhbE3
3,4-dioxygenase
289
(WP_043822444),
(KR232564)
beta subunit
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64
65
66
67
68
69
70
71
72
73
74
75
Delftia sp. RIT313
99
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Supplementary Fig. S1 Transmission electron micrograph of a negatively stained cell of strain
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EOB-17 shows a rod (0.7-0.9 μm × 1.7-2.2 μm) shape with polar flagella. Bar, 1.0 µm.
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Supplementary Fig. S2 Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences
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shows the relationship of strain EOB-17 and its related taxa. Bootstrap values (expressed as
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percentages of 1,000 replications) > 50% are shown at branching points. Bar, 0.005 substitutions per
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nucleotide position.
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105
106
107
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Supplementary Fig. S1
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Supplementary Fig. S2