Supplementary Materials - Applied and Environmental ......Supplementary Materials Table S1 PCR primers used in this study. Primer Sequence (5´ to 3´)a Purpose A1A2F GATCTGACAAGCTTGCCGGAACCCTAT

Supplementary Materials

Table S1 PCR primers used in this study.

Primer Sequence (5 ́to 3 )́a Purpose

A1A2F GATCTGACAAGCTTGCCGGAACCCTAT

CTGAAGCT

To amplify the sequence of pbaA1A2 gene cluster and

their native promoter

A1A2R GATCTGACGAGCTCCCATCTATCTTTGG

CCGTTTCTT

To amplify the sequence of pbaA1A2 gene cluster and

their native promoter

A1A2BR GATCTGACGAGCTCCCGCCTGAAAGTC

AACGATGTCC

To amplify the sequence of pbaA1A2B gene cluster

and their native promoter

T7F GATCTGACAAGCTTAGATCGATCTCGA

TCCCGCGA

To amplify the sequence of T7 promoter and

ribosome-binding site from pET-29a(+)

T7R ATCTTGCGTTCGATTTGCCATATGTATAT

CTCCTTC

To amplify the sequence of T7 promoter and

ribosome-binding site from pET-29a(+)

A1A2BT7F GAAGGAGATATACATATGGCAAATCGA

ACGCAAGAT


A1A2BT7R GATCTGACGAGCTCGTGTTGCGTCTCA

GGGATGGT


CF GGGACCCATATGAGCTATTATGACGTTC

TG

To amplify the sequence of pbaC gene

CR CTGAAAAAGCTTAGCCAGCCCCACTTC

CTTGAG

To amplify the sequence of pbaC gene

RA2F GGGACCCATATGAGATCAGCGGACGTT

GTC

To amplify the sequence of redA2 gene

RA2R ACATGCGTCGACCTAGACCGGGATCAG

ATCCTT

To amplify the sequence of redA2 gene

R3F GGGACCCATATGACGGATTATGATGTTT

TGATCG

To amplify the sequence of red3 gene

R3R AATCCCAAGCTTTCAGGCCGCAACCAG

TTCCTTC


R4F GGGACCCATATGAACCATTATGACGTT

GTGATCG


R4R CTGAAACTCGAGGGCCAGACCGACTT

CCTTGAGA


R5F GGGACCCATATGGCCCAGTATGACGTT

CTGATCG


R5R AATCCCAAGCTTGGCAGGGAGCAGGG

TCTTCAACGG


R6F GGGACCCATATGAGCTATTATGACGTTC

TGATCG


R6R AATCCCAAGCTTGGCCAGCCCGACTTC

CTTGAGC


RT-A1F CTGTCTGCTGCGCTTGGTGACGT To amplify partialsequence of pbaA1 gene

RT-A1R TATGCGAAATGGCGGCGTGGTAA To amplify partial sequence of pbaA1 gene

RT-A2F AAGCTGGCCGATCCGAAATCCTG To amplify partial sequence of pbaA2 gene

RT-A2R TGTTCCATGCGGGTACGGGTGAA To amplify partial sequence of pbaA2 gene

RT-BF CGTGAATTTACGGTCAATGCCGAGA To amplify partial sequence of pbaB gene

RT-BR TTCCAGCATGGTCTGTTCGTCCG To amplify partial sequence of pbaB gene

RT-CF GGTGCCGTGGTTCTGGTCCAATC To amplify partial sequence of pbaC gene

RT-CR CCTGCACATAGTCCTTCACCGCATT To amplify partial sequence of pbaC gene

RT-16SF GCTTTCGCACCTCAGCGTCAATG To amplify partial sequence of 16S rRNA gene

RT-16SR AGCGCACGTAGGCGGCGATCTAA To amplify partial sequence of 16S rRNA gene

a restriction sites are underlined.

Table S2 Deduced function of each ORF within the catechol-degrading gene cluster

catBCAIJFD in the genome of JZ-1.

Gene name, proposed product Position, product

size (amino acids)

Homologous protein (GenBank accession

no.) and source

%

Identity

catR, Transcriptional regulator 3998-4918, 305 Transcriptional regulator CatR

(YP_007617338), Sphingomonas sp. MM-1

82

catB, Muconate cycloisomerase 5025-6191, 387 Muconate cycloisomerase


87

catC, Muconolactone δ-isomerase 6213-6485, 89 Muconolactone isomerase


87

catA, Catechol-1, 2-dioxygenase 6529-7455, 307 Catechol 1, 2-dioxygenase


77

benA, Benzoate 1, 2-dioxygenase

alpha subunit

7424-8839, 471 Benzoate 1, 2-dioxygenase, large subunit,


79

benB, Benzoate 1, 2-dioxygenase

beta subunit

8836-9318, 159 Benzoate 1, 2-dioxygenase, small subunit


77

benC, Benzoate 1, 2-dioxygenase

electron transfer component

9343-10362, 338 protein BenC (YP_007617332),

Sphingomonas sp. MM-1

72

benD, 1, 6-Dihydroxycyclohexa-2,

4-diene-1-carboxylate

10359-11135, 257 1,6-Dihydroxycyclohexa-2,4-diene-1-carbo

xylate dehydrogenase (YP_007617331),

Sphingomonas sp. MM-1

79

catI, 3-Oxoadipate CoA-transferase

alpha subunit

11150-11875, 240 3-Oxoadipate CoA transferase subunit A


83

catJ, 3-Oxoadipate CoA-transferase

beta subunit

11872-12519, 214 3-Oxoadipate CoA-transferase subunit B


81

catF, β-Ketoadipyl CoA-thiolase 12516-13718, 399 β-Ketoadipyl CoA thiolase


85

catD, 3-Oxoadipate enol-lactone

hydrolase

13715-14515, 265 3-Oxoadipate enol-lactonase


77

A

B

C

D

E

F

Fig. S1 HPLC and MS/MS analyses of 3-phenoxybenzoate transformation by strain

JZ-1. A and B, HPLC spectra of authentic 3-phenoxybenzoate and 3-hydroxybenzoate,

respectively; C, HPLC spectra of the extract obtained from the culture at 36 h; D, MS/MS

analysis of the transformation products; E and F, The second-order MS analysis of m/z

213 [M+H]+ and m/z 137 [M+H]

+ characteristic ion peaks, which were characterized as

3-phenoxybenzoate and 3-hydroxybenzoate, respectively.

Fig. S2 Cell growth of wild type strain JZ-1 and the mutant strain MJZ-1 on LB and

MSM agar. A and C, Wild type strain JZ-1 grown on LB agar and MSM agar

supplemented with 0.5 mM 3-phenoxybenzoate, respectively; B and D, Mutant strain

MJZ-1 grown on LB agar and MSM agar supplemented with 0.5 mM 3-phenoxybenzoate,

respectively.

A

C D

B

Fig. S3 Conserved sequences that characterize the Rieske-type [2Fe-2S] cluster and

Fe (II) binding sites in α subunits of the eleven angular dioxygenases. Well-conserved

fingerprint sequence regions in the N-terminal part of the α subunits are aligned. Shaded

characters represent conserved residues; arrows indicate the amino acids involved in

binding the Rieske-type [2Fe-2S] cluster and the mononuclear iron atom. The

corresponding consensus sequences for the entire family of this type of protein are shown.

Accession numbers in the GenBank database and origins of the large α subunits are

KF879444 for 3-phenoxybenzoate 1′, 2′-dioxygenase from strain JZ-1 (PbaA1. JZ-1),

X72850 for dioxin dioxygenase from Sphingomonas wittichii RW1 (DxnA1. RW1),

AF060489 for carbazole dioxygenase from Sphingomonas sp. CB3 (CarAa. CB3),

22036072 for dibenzofuran dioxygenase from Terrabacter sp. YK3 (DfdA1. YK3),

AB054975 for dibenzofuran dioxygenase from Terrabacter sp. DBF63 (DbfA1. DBF63),

113473718 for carbazole dioxygenase from Sphingomonas sp. KA1 (CarAa1. KA1),

GU123624 for carbazole dioxygenase from Sphingobium yanoikuyae XLDN2-5 (CarAa.

XLDN2-5), AB001723 for carbazole dioxygenase from Pseudomonas stutzeri OM1

(CarAa. OM1), 75765412 for carbazole dioxygenase from Janthinobacterium sp. J3

(CarAa. J3), D89064 for carbazole 1, 9a-dioxygenase from Pseudomonas resinovorans

CA10 (CarAa. CA10), and X78823 for 3-phenoxybenzoate 1, 6-dioxygenase from

Pseudomonas pseudoalcaligenes POB310 (PobA. POB310).

A

B

C

D

E

F

G

H

Fig. S4 3-Phenoxybenzoate transformation by E. Coli BL21(DE3) harboring both

pBBRA1A2BT7 and pETC. A, B and C, HPLC spectra of authentic 3-phenoxybenzoate,

3-hydroxybenzoate and catechol, respectively; D, HPLC spectra of the extract obtained

from the culture at 8 h; E, MS/MS analysis of the transformation products; F, G and H,

The second-order MS analysis of m/z 213 [M+H]+, m/z 137 [M+H]

+ and m/z 109 [M+H]

+

characteristic ion peaks, which were characterized as 3-phenoxybenzoate,

3-hydroxybenzoate and catechol, respectively.

0 2 4 6 8 10 12

0.0

0.1

0.2

0.3

0.4

0.5

Phen

oxyben

zoat

e (m

M)

Time (h)

Fig. S5 Whole-cell transformation of 3-phenoxybenzoate (■) and 4-phenoxybenzoate

(□) by E. coli BL21(DE3) harboring both pBBRA1A2BT7 and pETC. The data are

represented as the mean ± standard deviation for triplicates.

Fig. S6 Phylogenetic tree constructed based on the alignment of PbaAa with the α

subunits of 71 characterized RHOs. The multiple-alignment analysis was performed

with ClustalX 2.1 software. Neighbor-Joining method was used to construct the

phylogenetic unrooted tree through MEGA 5.0. The numbers on some branches refer to

the percentage confidence estimated by a bootstrap analysis with 1000 replications.

Clustering of RHOs according to their respective native substrate is clearly displayed

with the shades of different colors. The yellow, pink, green, and blue colors correspond to

the RHOs that catalyze the following reactions: C-O/C-N bond cleavage; polyaromatic

hydrocarbon and nitroarene hydroxylation; biphenyl, benzene, and substituted benzene

hydroxylation; and benzoate and substituted benzoate hydroxylation, respectively. Type I

represents two-component RHO systems that consist of an oxygenase and an FNRC-type

reductase; Type II represents two-component RHO systems that consist of an oxygenase

and an FNRN-type reductase; Type III represents a group of three-component RHO

systems that consist of an oxygenase, a [2Fe-2S]-type ferredoxin and an FNRN-type

reductase; Type IV represents another three-component systems that consist of an

hetero-oligomeric oxygenase, [2Fe-2S]-type ferredoxin and GR-type reductase; Type V

represents another different three-component systems that consist of an hetero-oligomeric

oxygenase, a [3Fe-4S]-type ferredoxin and a GR-type reductase. The branches of the

eleven α subunits of angular dixoygenases are in red. The proteins abbreviations, species,

and their GI numbers are as follows: PbaA1.JZ-1, strain JZ-1, KF879444; DxnA1.RW1,

Sphingomonas wittichii RW1, 3426122; CarAa.CB3, Sphingomonas sp. CB3, AF060489;

DfdA1.YK3, Terrabacter sp. YK3, 22036072; DbfA1.DBF63, Terrabacter sp. DBF63,

AB054975; CarAa1.KA1, Sphingomonas sp. KA1, 113473718; CarAa.XLDN2-5,

Sphingobium yanoikuyae XLDN2-5, GU123624; CarAa.OM1, Pseudomonas stutzeri

OM1, AB001723; CarAa.J3, Janthinobacterium sp. J3, 75765412; CarAa.CA10,

Pseudomonas resinovorans CA10, 2317678; PobA.POB310, Pseudomonas

pseudoalcaligenes POB310, 473250; Oxo.86, Pseudomonas putida 86, CAA73203.1;

NarAa.NCIMB12038, Rhodococcus sp. NCIMB12038, 78101541; DitA1.BKME-9,

Pseudomonas abietaniphila BKME-9, 4455072; PdoA2.6PY1, Mycobacterium sp. 6PY1,

75446141; PhdA.KP7, Nocardioides sp. KP7, 7619816; NidA3.PYR-1, Mycobacterium

vanbaalenii PYR-1, 68053509; PdoA1.6PY1, Mycobacterium sp. 6PY1, 75446140;

NidA.PYR-1, Mycobacterium vanbaalenii PYR-1, 11038552; PhtAa.PYR-1,

Mycobacterium vanbaalenii PYR-1, 49072886; PhtA1.DBF63, Terrabacter sp. DBF63,

27531093; PhtAa.12B, Arthrobacter keyseri 12B, 13242054; PahAc.OUS82,

Pseudomonas putida OUS82, 391844; NahAc.9816-4, Pseudomonas sp. 9816-4, 1224114;

NagAc.U2, Ralstonia sp. U2, 2828018; DntAc.R34, Burkholderia cepacia R34,

17942397; NtdAc.JS42, Pseudomonas sp. JS42, 1773277; NbzAc.JS765, Comamonas sp.

JS765, 18643025; ArhA1.A4, Sphingomonas sp. A4, 50725019; PhnA1.A5,

Cycloclasticus sp. A5, 75444190; PhnA1a.CHY-1, Sphingomonas sp. CHY-1, 75455648;

HcaE.K-12, Escherichia coli K-12, 81170783; BphA1.TA421, Rhodococcus erythropolis

TA421, 3059209; BphA1.TK102, Comamonas testosteroni TK102, 21624254;

BphA1.KKS102, Pseudomonas sp. KKS102, 391833; BphA.B-356, Comamona

testosteroni B-356, 3023413; IpBAa.RE204, Pseudomonas putida RE204, 2822265;

BphA.LB400, Burkholderia xenovorans LB400, 151084; BphA1.KF707, Pseudomonas

pseudoalcaligenes KF707, 151091; BpdC1.M5, Rhodococcus sp. M5, 927232;

BphA1.RHA1, Rhodococcus sp. RHA1, 510285; BedC1.ML2, Pseudomonas putida ML2,

1168640; TodC1.F1, Pseudomonas putida F1, 151601; TecA1.PS12, Burkholderia sp.

PS12, 2264417; TcbAa.P51, Pseudomonas sp. strain P51, 75429640; CmtAb.F1,

Pseudomonas putida F1, 1263180; PsbAb. No.7, Rhodopseudomonas palustris No.7,

5360700; TdnA1.UCC22, Pseudomonas putida UCC22, 1841362; ORF7Nc.7N, Delftia

acidovorans 7N, 54290080; AtdA.YAA, Acinetobacter sp. YAA, 1395141; AntA.ADP1,

Acinetobacter sp. ADP1, 3511232; AntA.CA10, Pseudomonas sp. CA10, 13094158;

CbdA.2CBS, Burkholderia cepacia 2CBS, 758210; XylX.mt2, Pseudomonas putida mt2,

139861; BenA.ADP1, Acinetobacter sp. ADP1, 17865390; BphA1e.B1, Sphingobium

yanoikuyae B1, 123967465; AhdA1e.P2, Sphingomonas sp. P2, 75444939; NagG.U2,

Ralstonia sp. U2, 2828015; HybB.JB2, Pseudomonas aeruginosa JB2, 3643998;

AhdA1d.P2, Sphingomonas sp. P2, 75389581; PhnA1b.CHY-1, Sphingomonas sp.

CHY-1, 75455645; AhdA1c.P2, Sphingomonas sp. P2, 75444948; DdmC.DI-6,

Pseudomonas maltophilia DI-6, 55584974; TsaM.T-2, Comamonas testosteroni T-2,

1790867; VanA.HR199, Pseudomonas sp. HR199, 1946284; VanA.ATCC19151,

Pseudomonas sp. ATCC19151, 151636; NdmA.CBB5, Pseudomonas putida strain CBB5,

379334189; NdmB.CBB5, Pseudomonas putida CBB53, 379334191; LigX.SYK-6,

Sphingomonas paucimobilis SYK-6, 4062861.

Documents

Supplementary Materials - Applied and Environmental ......Supplementary Materials Table S1 PCR primers used in this study. Primer Sequence (5´ to 3´)a Purpose A1A2F GATCTGACAAGCTTGCCGGAACCCTAT