Download A novel three-component system-based regulatory model for

Supplementary Information for:
A novel three-component system-based regulatory model for
D-xylose
sensing and transport in Clostridium beijerinckii
Running title: A novel D-xylose sensing and transport model
Zhe Sun1, Yixiong Chen2, Chen Yang1, Sheng Yang1, Yang Gu1,4*, Weihong Jiang1,3*
1
Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai
200032, China
2
Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant
Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese
Academy of Sciences, Shanghai 200032, China
3
Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130
Meilong Road, Shanghai 200237, China
4
State Key Laboratory of Motor Vehicle Biofuel Technolog, Nanyang 473000, China
To whom correspondence should be addressed to:
Weihong Jiang, 300 Fenglin Road, Shanghai, China. Tel: 86-21-54924172; Fax:
86-21-54924015. E-mail: [email protected]
Yang Gu, 300 Fenglin Road, Shanghai, China. Tel: 86-21-54924178; Fax:
86-21-54924015. E-mail: [email protected]
1
Fig. S1. Confirmation of the gene disruption in C. beijerinckii mutants by PCR.
The lytS, yesN, xylFII, xylF, xylG and xylH genes were disrupted by inserting an
intron. MK, DNA marker; WT, the genome of wild-type C. beijerinckii NCIMB
8052.
2
Fig. S2. The growth and sugar consumption of C. beijerinckii mutants with
disruption in the lytS, yesN (A), xylFII, xylF, xylG and xylH (B) gene. The
experiments were performed in YP2 medium using glucose as the sole carbon source.
Solid lines represent residual glucose in the medium, and dashed lines represent the
growth curves. The vertical bars indicate the standard deviation of the mean for three
independent replicate cultures.
3
Fig. S3. Functional complementation of the xylFII (A), lytS (B), yesN (C), xylF (D)
and xylG (E) mutants. The YP2 medium was used in the fermentation using D-xylose
as the sole carbon source, the residual
D-xylose
in the medium (solid lines) and
growth curves (dashed lines) were monitored. 8052pIMPI, wild-type strain harboring
the plasmid pIMPI; 8052xylFII-pIMPI, xylFII-disrupted strain harboring pIMPI;
8052lytS-pIMPI,
lytS-disrupted
strain
harboring
pIMPI;
8052yesN-pIMPI,
yesN-disrupted strain harboring pIMPI; 8052xylF-pIMPI, xylF-disrupted strain
harboring pIMPI; 8052xylG-pIMPI, xylG-disrupted strain harboring pIMPI;
8052xylFII-ptb-xylFII, xylFII gene-complemented strain; 8052lytS-ptb-lytS, lytS
gene-complemented strain; 8052yesN-ptb-yesN, yesN gene-complemented strain;
8052xylF-ptb-xylF, xylF gene-complemented strain; 8052xylG-ptb-xylG, xylG
gene-complemented strain;. Vertical bars indicate the standard deviation of the mean
for three independent replicate cultures.
4
Fig. S4. Domain organization of LytS and YesN. TM, transmembrane domain;
HAMP, Histidine kinase, Adenylyl cyclase, Methyl-binding protein, Phosphatase
domain; His_kinase, Histidine Kinase A (dimerization/phosphoacceptor) domain;
HATPase_c,
Histidine
kinase-like
ATPase.
REC,
HTH_AraC, helix-turn-helix DNA-binding domain.
5
signal-receiving
domain;
Fig. S5. EMSAs of MBP and YesN proteins binding to the xylFGH promoter
region. (A) The assay was performed using the indicated amounts of purified MBP
protein (nM) and Cy5 fluorescence-labeled xylFGH promoter probe (2 nM). (B)
Binding of either non-phosphorylated or phosphorylated YesN to the xylFGH
promoter. YesN was phosphorylated by addition of 10 mM acetyl phosphate. The
bands were quantified using Quantity One software (Bio-Rad), and the percentage of
migrated DNA was calculated. The dissociation constants (Kd) values were
determined using the GraphPad Prism software.
6
Fig. S6. Mutational analysis of the YesN binding sites in the xylFGH promoter
region. (A) The DNA sequence of wild-type and randomly mutated DNA fragments
of the xylFGH promoter. M11, the 13 bp neighboring the protected region were
randomly mutated as a control; M12, the 42-bp protected region was randomly
mutated. (B) EMSAs of YesN protein binding to the wild-type and mutated xylFGH
promoter regions. The assay was performed using the indicated amounts of purified
YesN protein (nM) and Cy5 fluorescence-labeled xylFGH promoter probe (2 nM).
The 16S rRNA, xylAI, and xylB promoter regions were adopted as negative controls.
7
Fig. S7. Prediction of transmembrane domains in LytS, XylFII, and XylF. The
predictions
were
made
using
(http://www.cbs.dtu.dk/services/TMHMM-2.0).
domain.
8
the
TMHMM
TMhelix,
2.0
transmembrane
server
helix
Fig. S8. ITC studies of
D-xylose
binding by pLytS. The upper panel shows the
calorimetric titration of the binding protein with ligand, and the lower panel displays
the corresponding integrated heat, which was normalized and corrected for the heat of
dilution versus the molar ratio.
9
Table S1. Firmicutes bacteria harboring the “three-component system”.
Bacteria
Class
Order
Clostridium termitidis CT1112
Clostridia
Clostridiales
Clostridium sp. DL-VIII
Clostridia
Clostridiales
Sensor
TCS
ABC transporter
CTER_0909
CTER_0910, CTER_0911
CTER_0912, CTER_0914, CTER_0915,
CDLVIII_5131
CDLVIII_5130,
CDLVIII_5127, CDLVIII_5126,
CDLVIII_5129
CDLVIII_5125
CTER_0916
Clostridium sp. Maddingley
Clostridia
Clostridiales
A370_02150
A370_02149, A370_02148
A370_02147, A370_02146, A370_02145
Clostridia
Clostridiales
Cspa_c23460
Cspa_c23470,
Cspa_c23500, Cspa_c23510, Cspa_c23520
MBC34-26
Clostridium
saccharoperbutylacetonicum
Cspa_c23480
N1-4(HMT)
Clostridium clostridioforme 90A7
Clostridia
Clostridiales
HMPREF1082_03227
HMPREF1082_03226,
HMPREF1082_03224, HMPREF1082_03223,
HMPREF1082_03225
HMPREF1082_03222, HMPREF1082_03221
Clostridium cellulovorans 743B
Clostridia
Clostridiales
Clocel_3840
Clocel_3841, Clocel_3842
Clocel_3839, Clocel_3838, Clocel_3837
Clostridium carboxidivorans P7
Clostridia
Clostridiales
CcarbDRAFT_0061
CcarbDRAFT_0060,
CcarbDRAFT_0058, CcarbDRAFT_0057,
CcarbDRAFT_0059
CcarbDRAFT_0056
Clostridium cellulolyticum H10
Clostridia
Clostridiales
Ccel_1984
Ccel_1983, Ccel_1982
Ccel_1985, Ccel_1986, Ccel_1987
Clostridium phytofermentans
Clostridia
Clostridiales
Cphy_1581
Cphy_1582, Cphy_1583
Cphy_1584, Cphy_1585, Cphy_1586,
ISDg
Clostridium beijerinckii NCIMB
Cphy_1587
Clostridia
Clostridiales
Cbei_2377
Cbei_2378, Cbei_2379
Cbei_2380, Cbei_2381, Cbei_2382
Clostridia
Clostridiales
HMPREF1085_02978
HMPREF1085_02977,
HMPREF1085_02975, HMPREF1085_02974,
HMPREF1085_02976
HMPREF1085_02973, HMPREF1085_02972
CAAU_0343,
CAAU_0341, CAAU_0340, CAAU_0339,
CAAU_0342
CAAU_0338
8052
Clostridium bolteae 90A9
Caloramator australicus RC3
Clostridia
Clostridiales
CAAU_0344
10
Bacteria
Class
Alkaliphilus metalliredigens
Clostridia
Order
Clostridiales
Sensor
Amet_2818
TCS
ABC transporter
Amet_2817, Amet_2816
Amet_2815, Amet_2814, Amet_2813,
QYMF
Amet_2812
Butyrivibrio proteoclasticus B316
Clostridia
Clostridiales
bpr_I1169
bpr_I1170, bpr_I1171
bpr_I1172, bpr_I1173, bpr_I1174
Blautia sp. KLE 1732
Clostridia
Clostridiales
HMPREF1547_00349
HMPREF1547_00348,
HMPREF1547_00346, HMPREF1547_00344,
HMPREF1547_00347
HMPREF1547_00343, HMPREF1547_00342
Desaci_1027, Desaci_1028
Desaci_1029, Desaci_1030, Desaci_1031,
Desulfosporosinus acidiphilus SJ4
Clostridia
Clostridiales
Desaci_1026
Desaci_1032
Thermoanaerobacter
Clostridia
thermohydrosulfuricus WC1
Thermoanaerobacter sp. X514
Thermoanaero
TthWC1_1222
bacterales
Clostridia
Thermoanaero
Teth514_0222
bacterales
Thermoanaerobacter siderophilus
Clostridia
SR4
Thermoanaerobacter wiegelii
Clostridia
Thermoanaero
Thewi_0284
TthWC1_1219, TthWC1_1218,
TthWC1_1220
TthWC1_1217, TthWC1_1216
Teth514_0223,
Teth514_0225, Teth514_0226, Teth514_0227,
Teth514_0224
Teth514_0228
ThesiDRAFT1_0298,
ThesiDRAFT1_0296, ThesiDRAFT1_0295,
ThesiDRAFT1_0297
ThesiDRAFT1_0294, ThesiDRAFT1_0293
Thewi_0285, Thewi_0286
Thewi_0287, Thewi_0288, Thewi_0289,
bacterales
Clostridia
subsp. mathranii str. A3
Thermoanaerobacter italicus Ab9
ThesiDRAFT1_0299
bacterales
Rt8.B1
Thermoanaerobacter mathranii
Thermoanaero
TthWC1_1221,
Thermoanaero
Thewi_0290
Tmath_0325
Tmath_0326, Tmath_0327
Tmath_0328, Tmath_0329, Tmath_0330
Thit_0242
Thit_0243, Thit_0244
Thit_0245, Thit_0246, Thit_0247
TepRe1_1843
TepRe1_1842,
TepRe1_1840, TepRe1_1839, TepRe1_1838
bacterales
Clostridia
Thermoanaero
bacterales
Tepidanaerobacter acetatoxydans
Clostridia
Re1
Thermoanaerobacter
bacterales
Clostridia
tengcongensis MB4
Thermoanaerobacterium
saccharolyticum JW/SL-YS485
Thermoanaero
Thermoanaero
TepRe1_1841
TTE0286
TTE0287, TTE0288
TTE0289, TTE0290, TTE0291, TTE0292
Tsac_0140
Tsac_0139, Tsac_0138
Tsac_0137, Tsac_0136, Tsac_0135, Tsac_0134
bacterales
Clostridia
Thermoanaero
bacterales
11
Bacteria
Class
Order
Sensor
TCS
ABC transporter
Thermoanaerobacterium
Clostridia
Thermoanaeroba
Thexy_2027
Thexy_2026, Thexy_2025
Thexy_2024, Thexy_2023, Thexy_2022,
xylanolyticum LX-11
Thermoanaerobacterium
cterales
Clostridia
Thermoanaeroba
Clostridia
Thermoanaeroba
thermosaccharolyticum M0795
Mahella australiensis 50-1 BON
Thexy_2021
Thethe_02431
cterales
Mahau_2373
cterales
Halanaerobium saccharolyticum
Clostridia
Halanaerobiales
HSACCH_02141
subsp. saccharolyticum DSM
Thethe_02430,
Thethe_02428, Thethe_02427, Thethe_02426,
Thethe_02429
Thethe_02425
Mahau_2372,
Mahau_2370, Mahau_2369, Mahau_2368,
Mahau_2371
Mahau_2367, Mahau_2366
HSACCH_02140,
HSACCH_02138, HSACCH_02137,
HSACCH_02139
HSACCH_02136
6643
Bacillus stratosphericus LAMA
Bacilli
Bacillales
C883_1913
C883_1919, C883_1936
C883_1949, C883_1955, C883_1964
Bacilli
Bacillales
BSONL12_13411
BSONL12_13416,
BSONL12_13431, BSONL12_13436,
BSONL12_13421
BSONL12_13441
BaLi_c04600,
BaLi_c04620, BaLi_c04630, BaLi_c04640
585
Bacillus sonorensis L12
Bacillus licheniformis 9945A
Bacilli
Bacillales
BaLi_c04590
BaLi_c04610
Bacillus sp. M 2-6
Bacilli
Bacillales
BAME_09220
BAME_09210,
BAME_09190, BAME_09180, BAME_09170
BAME_09200
Bacillus bataviensis LMG 21833
Bacilli
Bacillales
BABA_14827
BABA_14832,
BABA_14842, BABA_14847, BABA_14852
BABA_14837
Bacillus pumilus ATCC 7061
Bacilli
Bacillales
BAT_0665
BAT_0664, BAT_0663
BAT_0662, BAT_0661, BAT_0660
Bacillus sp. 1NLA3E
Bacilli
Bacillales
B1NLA3E_05300
B1NLA3E_05305,
B1NLA3E_05315, B1NLA3E_05320,
B1NLA3E_05310
B1NLA3E_05325
B1040_010100007699,
B1040_010100007689,
Bacillus sp. 10403023
Bacilli
Bacillales
B1040_0101000077
04
B1040_010100007694
B1040_010100007684, B1040_010100007679
Bacillus sp. HYC-10
Bacilli
Bacillales
BA1_08141
BA1_08136, BA1_08131
BA1_08126, BA1_08121, BA1_08116
12
Bacteria
Class
Order
Bacillus sp. NRRL B-14911
Bacilli
Bacillales
Sensor
B14911_12712
TCS
ABC transporter
B14911_12707,
B14911_12692, B14911_12687,
B14911_12702,
B14911_12682
B14911_12697
Anoxybacillus flavithermus WK1
Bacilli
Bacillales
Aflv_1307
Aflv_1308, Aflv_1309
Aflv_1310, Aflv_1311, Aflv_1312
Anoxybacillus sp. DT3-1
Bacilli
Bacillales
F510_0891
F510_0890, F510_0889
F510_0888, F510_0887, F510_0886
Anoxybacillus sp. SK3-4
Bacilli
Bacillales
C289_0603
C289_0602, C289_0601
C289_0600, C289_0659, C289_0658
Geobacillus thermoglucosidans
Bacilli
Bacillales
GT20_0220
GT20_0221, GT20_0222
GT20_0223, GT20_0224, GT20_0225
Geobacillus kaustophilus HTA426
Bacilli
Bacillales
GK3210
GK3209, GK3208
GK3207, GK3206, GK3205
Geobacillus thermodenitrificans
Bacilli
Bacillales
GTNG_3129
GTNG_3128,
GTNG_3126, GTNG_3125, GTNG_3124
TNO-09.020
NG80-2
Geobacillus thermoleovorans
GTNG_3127
Bacilli
Bacillales
CCB_US3_UF5
GTCCBUS3UF5_3610
GTCCBUS3UF5_36090,
GTCCBUS3UF5_36060,
0
GTCCBUS3UF5_36080
GTCCBUS3UF5_36050,
GHH_c33010
GHH_c33000,
GHH_c32280, GHH_c32270, GHH_c32260
GTCCBUS3UF5_36040
Geobacillus sp. GHH01
Bacilli
Bacillales
GHH_c32290
Geobacillus sp. C56-T3
Geobacillus sp. JF8
Bacilli
Bacilli
Bacillales
Bacillales
GC56T3_3222
M493_16640
GC56T3_3221,
GC56T3_3219, GC56T3_3218,
GC56T3_3220
GC56T3_3217
M493_16635,
M493_16625, M493_16620, M493_16615
M493_16630
Geobacillus sp. Y4.1MC1
Bacilli
Bacillales
GY4MC1_0228
GY4MC1_0229,
GY4MC1_0231, GY4MC1_0232,
GY4MC1_0230
GY4MC1_0233
Paenibacillus sp. JDR-2
Bacilli
Bacillales
Pjdr2_1852
Pjdr2_1853, Pjdr2_1854
Pjdr2_1855, Pjdr2_1856, Pjdr2_1857
Paenibacillus sp. Y412MC10
Bacilli
Bacillales
GYMC10_1382
GYMC10_1383,
GYMC10_1386, GYMC10_1387,
GYMC10_1384
GYMC10_1388
13
Bacteria
Class
Paenibacillus dendritiformis C454
Bacilli
Order
Bacillales
Sensor
TCS
ABC transporter
PDENDC454_15944,
PDENDC454_15954, PDENDC454_15959,
PDENDC454_15949
PDENDC454_15964
C812_02866
C812_02865, C812_02864
C812_02863, C812_02862, C812_02861
PDENDC454_15939
Paenibacillus barengoltzii G22
Bacilli
Bacillales
C812_03378
C812_03379, C812_03380
C812_03381, C812_03382, C812_03383
Paenibacillus mucilaginosus K02
Bacilli
Bacillales
B2K_31410
B2K_31405, B2K_31400
B2K_31395, B2K_31390, B2K_31385
Paenibacillus curdlanolyticus
Bacilli
Bacillales
PaecuDRAFT_0108
PaecuDRAFT_0109,
PaecuDRAFT_0111, PaecuDRAFT_0112
YK9
PaecuDRAFT_0110
14
Table S2. Transcriptional fold-changes of C. beijerinckii genes xylFII, xylG and
xylH in E. coli mutant strains.
Strains
Transcriptional level (mean fold change±SD)
xylFII
xylG
xylH
K12xylEFGH-pUC118
1.00±0.69
1.00±0.20
1.00±0.08
K12xylEFGH-pUC118-cxyl 0.52±0.68
1.72E+04±1.99E 3.03E+04±3.31E
GH
+03
+03
K12xylEFGH-pUC118-cxyl 1.13E+04±5.28E 1.02E+04±8.90E 1.38E+04±3.77E
FIIGH
+02
+02
+02
Samples were harvested after 4 h incubation in LB medium. The rRNA 16S gene (rrsB) was used
as the internal control.
15
Table S3. Strains and plasmids used in this study.
Strain or plasmid
Relevant genotype or description
Source or reference
8052
Wild type
NCIMB
8052lytS
lytS::intron/pWJ1-lytS
This study
8052yesN
yesN::intron/pWJ1-yesN
This study
8052xylFII
xylFII::intron/pWJ1-xylFII
This study
8052xylF
xylF::intron/pWJ1-xylF
This study
8052xylG
xylG::intron/pWJ1-xylG
This study
C. beijerinckii
8052xylH
xylH::intron/pWJ1-xylH
This study
8052lytS-ptb-lytS
8052lytS complemented with plasmid ptb-lytS
This study
8052yesN-ptb-yesN
8052yesN complemented with plasmid ptb-yesN
This study
8052xylFII-ptb-xylFII
8052xylFII complemented with plasmid ptb-xylFII
This study
Top 10
General cloning host strain
Invitrogen
K12
Wild type
Takara
K12xylE
E. coli K12 with xylE inactivated
CGSC
E. coli K12 with xylEFGH inactivated
This study
E. coli
K12xylEFGH
Rosetta (DE3)
-
-
-
F ompT hsdSB(RB mB ) gal dcm λ(DE3 [lacI lacUV5-T7
Novagen
gene 1 ind1 sam7 nin5]) pLysSRARE (CamR)
Validation reporter strain
Host strain for bacterial two-hybrid analysis
Stratagene
pMD-18T
TA-cloning vector
Takara
pUC118
Expression vector in E. coli
Novagen
pUC118-cxylGH
Vector for overexpressing xylGH from C. beijerinckii
This study
pUC118-cxylFGH
Vector for overexpressing xylFGH from C. beijerinckii
This study
pUC118-cxylFIIGH
Vector for overexpressing xylFIIGH from C. beijerinckii
This study
Plasmids
pWJ1
37
Derived from pSY6 , with pCB102 replicon to replace
This study
pIM13 replicon, for gene inactivation by Targetron method
pWJ1-lytS
Vector for intron insertion in lytS at 821/822 nt
This study
pWJ1-yesN
Vector for intron insertion in yesN at 285/286 nt
This study
pWJ1-xylFII
Vector for intron insertion in xylFII at 468/469 nt
This study
pWJ1-xylF
Vector for intron insertion in xylF at 492/493 nt
This study
pWJ1-xylG
Vector for intron insertion in xylG at 711/712 nt
This study
pWJ1-xylH
Vector for intron insertion in xylH at 461/462 nt
This study
pQ8
Vector for overexpressing proteins with a N-terminal
(Sun et al., 2013)
MBP-tag in E. coli
pQ8-yesN
Vector for overexpressing YesN protein in E. coli
This study
pET-32a
Vector for overexpressing proteins in E. coli
Invitrogen
pET-32a-xylR
Vector for overexpressing XylR protein in E. coli
This study
pET-32a-mbp
Vector for overexpressing MBP protein in E. coli
This study
pET-32a-pxylFII
Vector for overexpressing XylFII(25-326) protein in E. coli
This study
pET-32a-plytS
Vector for overexpressing LytS(1-134) protein in E. coli
This study
16
Strain or plasmid
Relevant genotype or description
Source or reference
pET-32a-pxylFII-HA
Vector for overexpressing XylFII(25-326) protein with a
This study
C-terminal HA-tag in E. coli
pET-32a-Flag-plytS
Vector for overexpressing LytS(1-134) protein with a
This study
N-terminal Flag-tag in E. coli
pET-32a-xylF-HA
Vector for overexpressing XylFII protein with a C-terminal
This study
HA-tag in E. coli
placZFT
lacZ reporter gene fusion vector
Offered by Prof.
Peter Dürre
(Feustel et al., 2004)
pIMPI-lacZ
Derived from placZFT to determine β-Galactosidase activity
This study
pIMP1-lacZxylFII
lacZ reporter gene driven by xylFII promoter
This study
pIMP1-lacZlytS
lacZ reporter gene driven by the intergenic region between
This study
xylFII and lytS
pIMP1-lacZxylF
lacZ reporter gene driven by xylFGH promoter
This study
pIMP1
Expression vector in C. beijerinckii with pIM13 replicon and
Offered by Prof.
ptb promoter
Papoutsakis E.T.
(Mermelstein and
Papoutsakis, 1993)
ptb-lytS
lytS overexpression vector derived from pIMP1
This study
ptb-yesN
yesN overexpression vector derived from pIMP1
This study
ptb-xylFII
xylFII overexpression vector derived from pIMP1
This study
ptb-xylFGH
xylFGH overexpression vector derived from pIMP1
This study
ptb-xylFIIGH
xylFIIGH overexpression vector derived from pIMP1
This study
pXY1
Expression vector in C. beijerinckii derived from pIMP1, with
This study
pCB102 replicon to replace pIM13 replicon
pXY1-Flag-lytS
Vector for subcellular location of LytS protein with a
This study
N-terminal Flag-tag
pXY1-xylFII-HA
Vector for subcellular location of XylFII protein with a
pXY1-xylF-HA
Vector for subcellular location of XylF protein with a
This study
C-terminal HA-tag
This study
C-terminal HA-tag
pBT
Bacterial two-hybrid system bait plasmid
Stratagene
pTRG
Bacterial two-hybrid system target plasmid
Stratagene
Positive control bait plasmid for bacterial two-hybrid system
Stratagene
Positive control target plasmid for bacterial two-hybrid system
Stratagene
LytS(1-134) expression plasmid for bacterial two-hybrid
This study
pBT-LGF2
pTRG-Gal11
p
pBT-plytS
system
pTRG-pxylFII
XylFII(25-326) expression plasmid for bacterial two-hybrid
This study
system
pTRG-xylF
XylF expression plasmid for bacterial two-hybrid system
17
This study
Table S4. Co-transcription analysis and qRT-PCR primers used in this study.
Primer name
Sequence (5’→3’)
Description
xylFII-lytS co-s
AAGCGAGGAGATGATAAA
Forward primer for xylFII-lytS co-transcription analysis
xylFII-lytS co-a
ATTCTTGGCTGCTACAAA
Reverse primer for xylFII-lytS co-transcription analysis
lytS-yesN co-s
GAAGGCAAAGGGACTAAA
Forward primer for lytS-yesN co-transcription analysis
lytS-yesN co-a
CAAGCACAGGCAATAAAG
Reverse primer for lytS-yesN co-transcription analysis
yesN-xylF co-s
TGAAGGTGTTAGTCCTG
Forward primer for yesN-xylF co-transcription analysis
yesN-xylF co-a
CATTGCTAGATGTTTGTG
Reverse primer for yesN-xylF co-transcription analysis
xylF-xylG co-s
AGATGGCTAAAGGTGAA
Forward primer for xylF-xylG co-transcription analysis
xylF-xylG co-a
TTTCCAGAGTAAGACCC
Reverse primer for xylF-xylG co-transcription analysis
xylG-xylH co-s
GAAATAAAGGCAAGTCT
Forward primer for xylG-xylH co-transcription analysis
xylG-xylH co-a
TACCACTGTTGGGATAG
Reverse primer for xylG-xylH co-transcription analysis
xylFII rt-s
AGAGCTGCTAAAGAAAGA
Forward qRT-PCR primer for xylFII-lytS-yesN
xylFII rt-a
TAACTACTGGAATACCCT
Reverse qRT-PCR primer for xylFII-lytS-yesN
xylF rt-s
TATGGACGACCTAAGACT
Forward qRT-PCR primer for xylFGH
xylF rt-a
GCTATTGATTCACCGTTAT
Reverse qRT-PCR primer for xylFGH
xylAI rt-s
CCTTATCTTATTGGCACA
Forward qRT-PCR primer for xylAI
xylAI rt-a
AACGCAACTATCTCATCT
Reverse qRT-PCR primer for xylAI
xylB rt-s
GTATTGGATTAAGTGGGC
Forward qRT-PCR primer for xylB
xylB rt-a
GTTAATGCAGGATTACCA
Reverse qRT-PCR primer for xylB
xylR rt-s
ATCGTTGGAATCTACAG
Forward qRT-PCR primer for xylR
xylR rt-a
GTCTAACTCGCATACTT
Reverse qRT-PCR primer for xylR
r16s rt-s
TAAAGGAGTAATCCGCTATG
Forward qRT-PCR primer for 16S rRNA
r16s rt-a
TTATCGTCCCTGAAGACAG
Reverse qRT-PCR primer for 16S rRNA
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Supplementary References
Feustel, L., Nakotte, S., and Durre, P. (2004) Characterization and development of
two reporter gene systems for Clostridium acetobutylicum. Appl Environ Microbiol
70: 798-803.
Mermelstein, L. D., and Papoutsakis, E. T. (1993) In vivo methylation in Escherichia
coli by the Bacillus subtilis phage phi 3T I methyltransferase to protect plasmids
from restriction upon transformation of Clostridium acetobutylicum ATCC 824.
Appl Environ Microbiol 59: 1077-1081.
Sun, P., Zhao, Q., Yu, F., Zhang, H., Wu, Z., Wang, Y., et al. (2013) Spiroketal
formation and modification in avermectin biosynthesis involves a dual activity of
AveC. J Am Chem Soc 135: 1540-1548.
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