Electronic Supplementary Material
Organic Cofactors in the Metabolism of Dehalococcoides Strains
Christian J. Schipp, Ernest Marco-Urrea, Jana Seifert and Lorenz Adrian
Figure S1. Concentration of chlorinated metabolites after cultivation of strain CBDB1 with
hexachlorobenzene in the standard defined media lacking specific cofactors. Shown are
concentrations of chlorinated products formed from hexachlorobenzene: DCB dichlorobenzene and TCB - trichlorobenzene. Bars show mean values of five parallel
cultures and standard deviations are indicated. The treatments depicted in the figures are as
follows: V0: negative control without cofactors; V7: positive control containing the seven
vitamins cyanocobalamin (B12), biotin (BIO), thiamine (THI), p-aminobenzoate (PABA),
pantothenate (PAN), nicotinate (NIC), pyridoxine hydrochloride (PDX); V6: V7 without B12.
The symbols minus “–“ or plus “+” preceding a vitamin abbreviation indicate that a specific
cofactor was omitted or added, respectively. DCoB: dicyanocobinamid; Co(2+):
cobalt(II)chloride; DMB: dimethylbenzimidazole; CYA: dicyanocobinamide.
1
Table S1. Proteins with enzyme-bound cofactors and proteins interacting with
tetrahydrofolate encoded in the genome of Dehalococcoides strain CBDB1 and identification
by nanoLC-ESI-MS/MS. Bold locus tags indicate that the enzyme was identified on the basis
of at least 2 peptides in at least 2 different cultures. Frequency refers to the number of
cultures (out of 30) in which the respective protein was found. The mean emPAI rank (MER)
gives an indication for the relative abundance of the respective protein among all proteins of
strain CBDB1. For its calculation all proteins detected in a culture were ranked according to
their emPAI value (see materials an methods) and the ranks of all identifications were
averaged. The mean emPAI rank is a low number for the most abundant proteins and high for
the proteins that were just above the detection threshold.
Cofactor
E.C. no Locus tag
Description
Frequency MER
Cyanocobalamin
cbdbA80
cbdbA84
cbdbA88
cbdbA96
cbdbA187
cbdbA238
cbdbA243
cbdbA1092
cbdbA1453
cbdbA1455
cbdbA1491
cbdbA1495
cbdbA1503
cbdbA1508
cbdbA1535
cbdbA1539
cbdbA1542
cbdbA1546
cbdbA1550
cbdbA1560
cbdbA1563
cbdbA1570
cbdbA1575
cbdbA1578
cbdbA1582
cbdbA1588
cbdbA1595
cbdbA1598
cbdbA1618
cbdbA1624
cbdbA1627
cbdbA1638
cbdbA251
cbdbA287
cbdbA608
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Putative reductive dehalogenase
Ribonucleoside-diphosphate
reductase
Ribonucleoside-diphosphate
reductase
Ribonucleoside-diphosphate
reductase
29
16
3
0
0
0
0
3
0
0
8
0
0
0
0
0
0
0
0
0
0
0
0
0
0
15
0
5
0
0
0
0
9.8
54.1
39.3
66.7
69.4
17.8
114.6
0
4
126.3
0
2
Thiamine
2.2.1.1
cbdbA629
1.2.7.1
cbdbA682
2.2.1.7
cbdbA720
2.2.1.6
cbdbA813
Transketolase
Pyruvic-ferredoxin oxidoreductase,
beta subunit
1-deoxy-D-xylulose-5-phosphate
synthase
Acetolactate synthase, large subunit
4.1.1.3
cbdbA139
pyruvate carboxylase subunit B
6.4.1.2
cbdbA141
pyruvate carboxylase subunit A
2.6.1.42
cbdbA11
4.4.1.2.1.2.1
4.1.1.20
cbdbA257
cbdbA390
cbdbA508
1.12.-.-
cbdbA581
4
173.8
27
90.3
1
139.0
15
72.3
22
90.5
18
119.1
11
96.5
0
24
12
64.3
96.8
11
86.0
Biotin
Pyridoxal
phosphate
cbdbA639
2.6.1.83
cbdbA714
cbdbA730
2.6.1.9
cbdbA825
4.2.1.20
2.6.1.11
2.6.1.1
4.2.1.20
cbdbA1008
cbdbA1181
cbdbA1292
cbdbA1450
branched-chain amino acid
aminotransferase
cysteine desulfurase
serine hydroxymethyltransferase
diaminopimelate decarboxylase
soluble hydrogenase, tritium
exchange subunit
putative threonine-phosphate
decarboxylase (L-threonine-O-3phosphate decarboxylase)
LL-diaminopimelate
aminotransferase
Putative uncharacterized protein
with pyroidoxal phosphate binding
motif
histidinol-phosphate
aminotransferase
tryptophan synthase subunit beta
acetylornithine aminotransferase
aspartate aminotransferase
tryptophan synthase subunit beta
0
10
144.3
0
12
139.8
27
0
8
0
47.9
182.8
Flavin
nucleotides
cbdbA45
1.4.1.13
cbdbA46
cbdbA143
cbdbA203
cbdbA244
cbdbA260
cbdbA383
1.8.1.9
cbdbA516
cbdbA684
cbdbA687
cbdbA744
cbdbA846
cbdbA1059
cbdbA1119
cbdbA1164
ferredoxin--NADP(+) reductase
subunit alpha
putative oxidoreductase
pyridine nucleotide-disulphide
oxidoreductase family protein
hypothetical protein
putative membrane bound
regulatory protein
hypothetical protein
phosphopantothenoylcysteine
synthetase/decarboxylase
thioredoxin reductase
putative [Fe] hydrogenase, HymB
subunit
mercuric reductase
hypothetical protein
hydrogenase subunit HymB
pyridine nucleotide-disulfide
oxidoreductase family protein
dihydroorotate dehydrogenase,
electron transfersubunit
putative iron-sulfur flavoprotein
0
0
25
51.2
0
0
1
137.0
0
2
161.0
22
70.4
0
0
10
96.9
3
139.0
0
1
106.0
3
2.1.1.148
cbdbA1172
cbdbA1263
cbdbA1323
cbdbA1338
cbdbA1389
cbdbA1457
cbdbA1472
cbdbA1688
cbdbA1690
NADPH-dependent FMN reductase
putative iron-sulfur flavoprotein
NADPH-dependent FMN reductase
L-aspartate oxidase
hemolysin-related protein
thymidylate synthase ThyX
flavodoxin
putative membrane bound
regulatory protein,CprC
putative membrane bound
regulatory protein,CprC
0
0
1
1
2
5
0
14
207.0
12.0
186.5
189.0
146.9
0
Molybdopterin
cbdbA118
Molybdopterin oxidoreductase
0
1.2.1.2
cbdbA195
Formate dehydrogenase, major
subunit
30
6.0
2.1.2.1
1.5.1.5/
3.5.4.9
cbdbA390
Serine hydroxymethyltransferase
24
64.3
cbdbA656
Bifunctional protein FolD
6.3.4.3
cbdbA660
Folate
cbdbA1381
2.1.2.9
cbdbA1742
Formate--tetrahydrofolate ligase
(Formyltetrahydrofolate synthetase)
Bifunctional purine biosynthesis
protein
Methionyl-tRNA formyltransferase
0
23
57.8
0
4
Table S2. Expression analysis of cofactor biosynthesis proteins by nLC-MS/MS. Only those
genes are displayed that are involved in cofactor biosynthesis and whose encoded proteins
were identified with at least 2 peptides. Frequency refers to the number of cultures (out of 30)
in which each protein was found. The mean emPAI rank (MER) gives an indication for the
relative abundance of the respective protein among all proteins of strain CBDB1. For its
calculation all proteins detected in a culture were ranked according to their emPAI value (see
materials an methods) and the ranks of all identifications were averaged. The mean emPAI
rank is a low number for the most abundant proteins and high for the proteins that were just
above the detection threshold.
Cofactor
E.C. no
Locus tag
Description
Frequency
MER
2.4.2.21
cbdbA641
3.1.3.73
cbdbA643
6.3.5.10
2.5.1.17
cbdbA890
cbdbA1141
nicotinate-nucleotidedimethylbenzimidazole
phosphoribosyltransferase
alpha-ribazole-5-phosphate
phosphatase
cobyric acid synthase CobQ
cob(I)alamin adenosyltransferase
16
62.8
3
5
2
109.3
172.6
179.0
4.1.99.17
cbdbA749
Phosphomethylpyrimidine
synthase
1
250.0
cbdbA327
pyridoxine biosynthesis protein
12
96.9
2.7.1.26 /
2.7.7.2
4.1.99.12
cbdbA584
5
162.8
7
101.6
3.5.4.25 /
4.1.99.12
3.5.4.26/
1.1.1.193
cbdbA1103
riboflavin biosynthesis protein
RibF
6,7-dimethyl-8-ribityllumazine
synthase
3,4-dihydroxy-2-butanone 4phosphatesynthase
riboflavin biosynthesis protein
RibD
11
138.2
10
101.6
1.5.1.3
cbdbA689
3.5.4.16
cbdbA1120
Oxidoreductase, short chain
oxidoreductase
GTP cyclohydrolase I
2
1
184.0
208.0
2.5.1.6.
cbdbA476
S-adenosylmethionine synthase
29
28.4
2.6.1.42
cbdbA11
1.1.1.86
2.2.1.6
cbdbA811
cbdbA812
11
24
96.5
45.8
17
85.3
2.2.1.6
cbdbA813
4.2.1.9
2.7.1.24
cbdbA815
cbdbA1268
branched-chain amino acid
aminotransferase
ketol-acid reductoisomerase
acetolactate synthase 3
regulatory subunit
acetolactate synthase,
large subunit
dihydroxy-acid dehydratase
dephospho-CoA kinase
15
17
1
72.3
103.9
213.0
2.4.2.19
cbdbA1336
nicotinate-nucleotide
26
46.1
Cyanocobalamin
Thiamine
Pyridoxal
phosphate
Flavines
cbdbA1102
cbdbA1105
Folate
S-adenosylmethionine
Pantothenate
(CoA)
Nicotinic acid
derivates
5
1.4.3.16
2.4.2.11
cbdbA1338
cbdbA1382
2.5.1.72
cbdbA1681
2.1.1.-
cbdbA349
pyrophosphorylase
L-aspartate oxidase
nicotinate
phosphoribosyltransferase
quinolinate synthetase
1
12.0
3
5
168.0
154.2
5
180.6
4
125.3
Menaquinones
cbdbA1683
Putative ubiquinone/menaquinone
biosynthesis methlytransferase
UbiE
SAM-dependent
methyltransferase
6