Electronic Supplementary Material
Role of a putative tungsten-dependent formylmethanofuran
dehydrogenase in Methanosarcina acetivorans
Nicole Matschiavelli and Michael Rother#
Institut für Mikrobiologie, Technische Universität Dresden
*Corresponding Author:
Michael Rother
Institut für Mikrobiologie
Technische Universität Dresden
D-01062 Dresden, Germany
Phone: ++49-351-46342611
Fax: ++49-351-46337715
e-mail: michael.rother@tu-dresden.de
1
Table S1: Primers used in this study
Nr.
Primer name
Sequence (5`3`)a
Use
1
oMA0832_for
cgcccatgggcCAGCTCGTAAAACT
TTCCCTC
Amplification of ma0832
for Antibody generation
2
oMA0832_rev
tataagcttCTTTATAAGCAAAACGC
CTTTTCC
Amplification of ma0832
for Antibody generation
3
odown0832/5'
cgattggatccGATAAGATCGAAGTA
AACAGAAACTC
Amplification of
downstream region of
ma0832
4
odown0832/3'
cagtatactagtggcgcgccCAAATGGC
AGAGAACCAGAAC
Amplification of
downstream region of
ma0832
5
oup0835/5‘
ctgactcgaggcgcgccGATGGAGTTC
TCAGAATTAAAAGC
Amplification of
upstream region of
ma0835
6
oup0835/3‘
gattacaaagcttGTAACAGAGTTTCG
ATAATGAGTTTC
Amplification of
upstream region of
ma0835
7
opET28aSeq-for
TACGACTCACTATAGGGG
Sequencing of
pETma0832
8
opET28aSeq-rev
TTAGCAGCCGGATCTCAG
Sequencing of
pETma0832
9
oP0835/5’
ggaattcgctagcGCATGAAACACAG
CAGCAAAAGATAAAAAAC
Amplification of promoter
region upstream of
ma0835 (fwdD1)
10
oP0835/3’
gaattcccatatgAATTCCTCATCTGG
GGTGTAACAGAG
Amplification of promoter
region upstream of
ma0835 (fwdD1)
11
oFMD1flank_for
AGGTAGCTTCCTACATGTACGG
C
Amplification of fmdB1
for cloning into pJET1.2
12
oFMD1flank_rev
CTGCGTTTTTCTTCCCTGG
Amplification of fmdB1
for cloning into pJET1.2
13
oFMD1q_for
TTGTGCAGCCTATCTTGCCG
qPCR of fmdB1
14
oFMD1q_rev
CATTCCATGGCGTCAATGACT
qPCR of fmdB1
15
oFMD1cDNA
TTCGTAAACTCGAAGGGC
cDNA-synthesis of
fmdB1
16
oFMD2flank_for
CGTTACAATCCGGGAGAATATA
CG
Amplification of fmdB2
for cloning into pJET1.2
17
oFMD2flank_rev
GGTCCGGGAAATTTCGAGTT
Amplification of fmdB2
for cloning into pJET1.2
2
18
oFMD2q_for
GGTGGGTGCTGACCTTGTAA
qPCR of fmdB2
19
oFMD2q_rev
ACAACTGTAGTCGGACACAGAG
T
qPCR of fmdB2
20
oFMD2cDNA
ATGGCATCGATAACACCC
cDNA-synthesis of
fmdB2
21
oFWD1flank_for
GACTTCGCCAGAGGATATCCAT
A
Amplification of fwdB1
for cloning into pJET1.2
22
oFWD1flank_rev
TCTCTTCAGTTCCCTGACCTTTG
Amplification of fwdB1
for cloning into pJET1.2
23
oFWD1q_for
AAGCAGTCAGGCACCTTGC
qPCR of fwdB1
24
oFWD1q_rev
CCGAGGGGATTACCACGTCT
qPCR of fwdB1
25
oFWD1cDNA
TTCATGCGGAGGGAAATT
cDNA-Synthesis of
fwdB1
26
oFWD2flank_for
TGTAAAGTTTGAAGACGGTGCC
Amplification of fwdB2
for cloning into pJET1.2
27
oFWD2flank_rev
TTGAGAACAGCTTCATCCGAG
Amplification of fwdB2
for cloning into pJET1.2
28
oFWD2q_for
TTTCAAGCCTCCCCAGGTCTAT
qPCR of fwdB2
29
oFWD2q_rev
GCGTTTCTGGAAGTCAGGGT
qPCR of fwdB2
30
oFWD2cDNA
AACAACAGGTTTGAAGTCTACC
cDNA-Synthesis of
fwdB2
31
oMCRbq_for
GAGCAGTCCGGTATCTTTGAGA
TG
qPCR of mcrB
32
oMCRbq_rev
CGACATTGTTGGCGTTGAGAC
qPCR of mcrB
33
oMCRbcDNA
CCGTCCTTTCCATTTTCCT
cDNA-Synthesis of mcrB
pJET1.2forward
CGACTCACTATAGGGAGAGCGG
C
Sequencing of pJET1.2derivates (Thermo
Scientific)
34
Sequencing of pJET1.2derivates (Thermo
Scientific)
a
: lower-case letters correspond to sequences added for cloning purposes; upper-case letters
correspond to the target sequence.
35
pJET1.2reverse
AAGAACATCGATTTTCCATGGC
AG
3
Table S2: Growth of MkoFWD1a. Strains were grown on methanol, acetate or CO, or shifted
from methanol to acetate. Growth was monitored by following the optical density at 578 nm
(OD578).
Methanol
Strain
Wild type
MkoFWD1
td [h]b
8.8 ±
final
OD578
MethanolAcetate
td [d]
1.2 ±
1.8 ±
0.2
0.1
12.8 ±
1.2 ±
2.7 ±
0.6
0.3
0.1
0.8
final
OD578
0.7 ± 0.1
0.7 ± 0.1
Acetate
td [h]b
final
OD578
CO
td [h]b
final
OD578
38.5
0.7 ±
11.0 ±
1.1 ±
± 1.7
0.02
1.6
0.1
40.7
0.7 ±
43.8 ±
0.9 ±
± 2.1
0.01
2.6
0.1
a: shown are average values and standard deviations of six independent cultures for each
strain; the experiment was qualitatively reproduced at least once.
b: doubling time
Table S3: Substrate-dependent metabolite formation from CO. Wild type and MkoFWD1 was
cultivated on methanol, acetate, or CO before cell suspensions were made. The initial
formation rates of methane, acetate and formate from 50 kPa CO were analyzed.
Strain
Wild type
MkoFWD1
a
Growth
substrate
Formation rate [nmol min-1 mg-1]
Methane
Formate
Acetate
Methanol
10.9 ± 2.7
0.4 ± 0.3
0.9 ± 0.1
CO
5.4 ± 1.3
1.2 ± 0.1
9.5 ± 1.3
Acetate
1.0 ± 0.5
4.0 ± 0.4
0.03 ± 0.02
Methanol
6.9 ± 2.9
n.d. a
n.d. a
CO
5.6 ± 1.6
n.d. a
2.6 ± 0.9
Acetate
0.2 ± 0.07
n.d. a
n.d. a
n.d.: not detected (below detection limit of the respective system, ca. 10 µM).
4
b
a
1
2
3
4
kb
1
2
kDa
10
170
6
100
4
55
3
40
35
2
25
1.5
15
Figure S1: Verification of MkoFWD1. A, genotype of MkoFWD1 analyzed by southern
hybridization; a ca. 1000 bp DIG-labeled DNA-fragment corresponding to the region
downstream of the putative fwd1 operon was used to probe genomic DNA from the wild type
(lane 1) and MkoFWD1 (clone #4, lane 2; clone #5, lane3), restricted over night with HpaI; a
DIG-labeled DNA ladder (Roche, lane 4) served as standard; B, immunological analysis of
M. acetivorans wild type (lane 1) and of MkoFWD1 (lane 2), grown on methanol using
FwdC1-specific polyclonal antiserum (1:5000); the arrow indicates the migration position of
FwdC1.
5