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Supporting Information
Identification and Characterization of naturally occurring DSF-Family
Quorum Sensing Signal Turnover System in the Phytopathogen
Xanthomonas
Lian Zhou1¶，Xing-Yu Wang1¶, Ming Li1, Li-Chao Yang2, Bo-Le Jiang2, Ya-Wen He1
Contents
Fig. S1
Fig. S2
Fig. S3
Fig. S4
Fig. S5
Fig. S6
Fig. S7
Table S1
Table S2
Table S3
Table S4
Table S5
Fig. S1
A
-ESI EIC(211.1704) DSF 5µM
-ESI EIC(197.1547) BDSF 5µM
BDSF
DSF
50
50
40
40
30
20
BDSF (µM) =
9.6010-7PI0.53
10
0
0.0E+00
R2= 0.998
2.0E+07
4.0E+07
6.0E+07
Peak Intensity
DSF (µM)
BDSF (µM)
B
30
20
DSF (µM) =
9.8310-7 PI0.20
10
0
0.0E+00
R2= 0.996
2.0E+07
4.0E+07
6.0E+07
Peak Intensity
C
XC1 (200 ), BDSF
ΔrpfF (200 ), BDSF
ΔrpfC (2  ), BDSF
XC1 (200 ), DSF
ΔrpfF (200  ), DSF
ΔrpfC (2  ), DSF
Supporting Information Fig. S1 Quantitative assay of DSF and BDSF levels in the
supernatant of Xcc cultures using liquid chromatography-mass spectrometry (LCMS). (A) Mass spectrometry of DSF and BDSF; (B) Standard curves showing the peak
intensities of different concentrations of DSF and BDSF; (C) Peak intensities of the
extracts of WT, rpfC, rpfG, clp cultures. To quantify DSF and BDSF production in
rpfC culture, 0.2 mL of the supernatant was collected. Its crude ethyl acetate extract
was passed through a 0.45-µm Minisart filter unit and was then condensed to 0.1 mL
for LC-MS analysis. To quantify DSF and BDSF production in the wild-type XC1
culture, 20.0 mL of the supernatant was collected. The crude ethyl acetate crude
extracts were then condensed to a volume of 0.1 mL for LC-MS analysis. Three
microliters of the condensed samples were applied to an Ultra Performance Liquid
Chromatographic system (UPLC, Agilent 1290 Infinity) on a Zorbax XDB C18 reverse
phase column (4.6  150 mm, temperature-controlled at 30C), and eluted with
methanol-water (80:20, v/v) at a flow rate of 0.4 mL/min in a diode array detector
(Agilent G4212A). Data was acquired in the centroid mode using the Agilent
MassHunter Workstation Data Acquisition Software (revision B.04). BDSF and DSF
levels in the culture supernatant were quantified using Peak Intensity (PI) in the
extracted ion chromatogram according to the following formula: BDSF (µM) = 9.60 
10-7  PI-0.53, DSF (µM) = 9.83  10-7  PI-0.20. The formula was derived from a
dose–PI plot in MS chromatogram using various dilutions of synthetic BDSF and DSF
signals, with a correlation coefficient (R2) of 0.998 and 0.996, respectively.
A
3.5
rpfC
rpfCrpfB
rpfCrpfB::rpfB
OD600
2.8
2.1
1.4
rpfCrpfC (rpfB)
rpfCrpfB (fadD)
rpfCrpfB (E365A)
rpfCrpfB (E361A)
0.7
0.0
0
12
24
36
48
60
Hours after inoculation (H)
B
60
DSF(M)
45
30
15
7.5
5.0
2.5
0.0
12
C
24
36
48
60
Hours after inoculation (H)
10
BDSF(M)
8
6
4
2
2
1
0
12
24
36
48
60
Hours after inoculation (H)
Supporting Information Fig. S2 Fig. Time course of growth, DSF
and BDSF production in Xcc strains derived from the strain
rpfC. (A) Time course of bacterial growth in NA liquid medium;
(B) DSF production; (C) BDSF production. Data are expressed as the
means  one standard deviation of three independent assays.
Fig. S3
A
B
Abundance
Standard Oleic acid (35.5 µM)
2.2E+6
C16
18.937
1.8E+6
Abundance
Oleic acid (C18:1)
73.1
21.386
1.4E+6
1.0E+5
C18
21.843
1.0E+6
339.3
117.1
0.6E+6
0.8E+5
0.2E+6
17.50
Time-->
18.50
19.50
20.50
21.50
22.50
0.6E+5
C
40
Oleic acid (µM)
35
0.4E+5
145.1
30
25
20
10
R2=
5
95.1
0.2E+5
Oleic acid (µM) =
9.0910-7PI1.03
15
185.1
222.2
264.3
0.995
311.2
286.3
50.9
0
0
-1.0E+07
1.0E+07
3.0E+07
5.0E+07
m/z--> 60 80 100120140160180200220240260280300320340360
Area
D
Abundance
Abundance
2.2E+6
18.943
2.2E+6
18.937
1.8E+6
1.8E+6
1.4E+6
1.4E+6
17.672
1.0E+6
0.6E+6
20.116
21.386
21.844
1.0E+6
17.667
0.6E+6
0.2E+6
Time-->
20.116
18.50
19.50
20.50
21.50
22.50
XC1
Time-->
17.50
18.50
2.2E+6
20.50
21.50
22.50
ΔrpfB (rpfB)
60 min
18.942
2.2E+6
1.8E+6.
1.8E+6
1.4E+6
1.4E+6
1.0E+6
17.672
21.844
20.121 21.386
0.6E+6
19.50
Abundance
60 min
0.2E+6
Time-->
21.844
21.386
0.2E+6
17.50
Abundance
1.0E+6
ΔrpfB
60 min
0 min
18.937
17.666
21.843
0.6E+6
20.121
0.2E+6
17.50
18.50
19.50
20.50
21.50
22.50
Time-->
17.50
18.50
19.50
20.50
21.50
22.50
Supporting information S3 Quantitative assay of oleic acid level in the supernatant
of Xcc cultures using gas chromatography-mass spectrometry (GC-MS). (A) Gas
chromatography of oleic acid. (B) Mass spectrometry of oleic acid; (C) Standard curve
showing peak intensities of different concentrations of oleic acid in gas chromatogram;
(D) Gas chromatogram of oleic acid extracted from the culture supernatants of Xcc
strains. The Xcc strains were grown in NA liquid medium till an OD600 of 0.5. Sodium
oleate was exogenously added to Xcc cultures at the final concentration of 15.0 µM. After
incubation for 60 min, 1 mL of Xcc cultures were collected for oleic acid extraction and
quantification. Briefly, 5 µL of 6 M HCl was added to 1 mL of the culture supernatant
followed by extraction by 2 volumes of ethyl acetate. The ethyl acetate extracts were
pooled and the solvent was removed by rotary evaporation at 40 C to dryness. The crude
extract was re-dissolved in 100 µL of n-hexane (UV-IR-HPLC level; CNW®
technologies) and transferred into a Reacti-Vial (Cat. No. 047422-2601, 1 mL, Fisher).
The derivation was conducted in the vial by adding 20 µL of BSTFA:TMCS (99:1, v/v)
(Cat no. 15238; Fluka) and 20 µL of pyridine (Cat no. 270970; Sigma) and by incubating
at 70 C for 40 min. One µL of derivatized sample was injected into a GC-MS system
from Agilent Technologies (AT) (Model: gas chromatograph AT 6850/ mass detector AT
5975C) equipped with split/splitless inlet and a HP-5 MS column (30 cm  250 µm 
0.25 µm ). Inlet temperature was 250 C, the column temperature was initially held at 50
C for 1 min, and increased to 220 C at a temperature gradient of 10 C min-1, the
column temperature was held at 220 C for 4 min, and then increased to a final
temperature of 300 C at a temperature gradient of 30 C min-1, holding for 2 min. Oleic
acid level in the culture supernatant was quantified using peak intensity (PI) of extracted
ion chromatogram according to the following formula: Oleic acid (µM) = 9.09  10-7 
PI-1.03. The formula was derived from a dose–PI plot in MS using various dilutions of
sodium oleate with a correlation coefficient (R2) of 0.995.
150
Conc. (M)
120
RpfB 0 g
RpfB 10 g
RpfB 10 g (heated)
90
60
30
0
DSF
BDSF
Supporting Information S4 In vitro measurement of FCL activity of RpfB on DSF
and BDSF. FCL activity by RpfB was quantitatively analyzed by measuring the
decrease in DSF or BDSF in the reaction mixture. Purified His-tagged RpfB 10 µg or
heat-inactivated His-tagged RpfB (10 µg) was incubated in the reaction mixture
containing 150 mM Tris-HCl (pH 7.2), 10 mM MgCl2, 2 mM EDTA, 5 mM ATP, 0.1%
Triton X-100, 0.5 mM reduced CoA, 150 µM DSF and 120 µM BDSF at 30 C. After
incubating for 5 h, 100 µL of the reaction mixture was sampled for DSF and BDSF
extraction. The levels of DSF and BDSF in the crude extract were quantified using
liquid chromatography-mass spectrometry (LC-MS) as described in S1 Fig.
Sodium Oleate conc. (M)
150
120
RpfB 0 g
RpfB 10 g
RpfB 10 g (heated)
90
60
30
0
Supporting Information S5. In vitro measurement of FCL activity of RpfB on
sodium oleate. FCL activity by RpfB was quantitatively analyzed by measuring
the decrease of sodium oleate in the reaction mixture. Purified His-tagged RpfB
(10 µg) or heat-inactivated His-tagged RpfB (10 µg) was incubated in the
reaction buffer containing 150 mM Tris-HCl (pH 7.2), 10 mM MgCl2, 2 mM
EDTA, 5 mM ATP, 0.1% Triton X-100, 0.5 mM reduced CoA, 100 µM sodium
oleate at 30C. After incubating for 5 h, 100 µL of the reaction mixture was
sampled, and oleic acid was extracted by 200 µL ethyl acetate containing 5 µL of
6M HCl, followed by quantitative analysis using gas chromatography-mass
spectrometry (GC-MS) as described in S2 Fig.
A kDa
M RpfB
C
rpfB
(rpfB)
97.2
B
kDa
80
66.4
RpfB
44.3

M
XC1
ΔrpfB
ΔrpfB
::rpfB
rpfB
(E365A)
His-RpfB
(2.0 ng)
58
46
Supporting Information Fig. S6 Western blotting analysis of RpfB in Xcc strains. (A)
Purified RpfB proteins. (B) Verification of the generated monoclonal antibody against
RpfB. (C) RpfB expression in the rpfB (rpfB) and rpfB(E365A) strains.
XC1 ΔrpfF ΔrpfF
(rpfF)
M
kDa
46
32
A
25
22
XC1 ΔrpfG ΔrpfC
RpfF
B
α
24 h
XC1 Δclp
36 h
XC1 Δclp
RpfF
C

Supporting Information Fig. S7 Western blotting analysis of RpfF
expression in Xcc strains. (A) Verification of the generated polyclonal antibody
against RpfF. (B) RpfF expression level in the strains XC1 and rpfC 24 h after
innoculation. (C) RpfF expression level in the strains XC1 and Δclp. The
monoclonal antibody against  subunit of RNA polymerase (NeoClone) was
used as a control for sample loading.
A
RpfB
RpfF
RpfC
RpfH
RpfG
Clp
Xcc
Xcv
XCV1921 (98%)
XCV1920 (97%)
XCV1919(89%)
XCV1918
XCV1917 (95%) XCV0519 (98%)
Xoo
XOO2868 (93%)
XOO2869 (95%)
XAC1880 (52%)
XAC1879 (54%)
DSC9485(72%)
DSC9490(75%)
Xf0287 (85%)
Xf1115 (67%)
XOO2870 (86%)
XOO2871 (94%)
XOO4158 (98%)
Xac
XAC1878 (58%)
XAC1877 (96%)
XAC0483 (98%)
Psx
DSC9495(73%)
DSC9500(81%)
DSC2345(82%)
Xyl
Xf1114 (60%)
Xf1113 (94%)
Xf1540 (86%)
Stm
Smal1772 (79%) Smal1771 (80%)
Smal1770 (63%)
Smal1769 (82%)
Smal3679 (86%)
LF41_153 (80%)
LF41_2177(81%)
Lys
LF41_150(77%)
LF41_151(44%)
LF41_152(55%)
Mfl
Mfla1727 (28%)
Mfla2657 (40%)
Mfla2656 (34%) Mfla2655 (50%)
Mfla1734 (25%)
Tbd
Tbd2597 (40%)
Tbd2672 (40%)
Tbd2671(37%)
Tbd2670 (51%)
Tbd2593 (29%)
B
Xcc:
Xoo:
Xac:
Xff:
Xhc:
AGCCCGACCTGCCACCTCAAAATGCTGCTGCGCATCACGCTTCGCCTTTTCCATCC
ACCACAGGCGACCACGCTTGAATGCTGATCTGCATCACGCTTCGCGTTTTCCATCC
ACCACGCGCCACTGCCCTTGAATGCTGCTCTGCATCACGCTTCGCCTTTTCCATGC
ACCGCGCGCCTCTGCCCTTGAATGCTGCTCTGCATCACGCTTCGCCTTTTCCATGC
CAGTATCGCCATCGCAGTTGAATGCTGCCCTGCATCACACTTCGCCTTTTACATGC
Clp binding motif
Supporting Information Fig. S8. Conservation of the key genes for RpfB-dependent
signal turnover system in the genomes of different bacteria. (A) Genomic organization of
the genes and homology analysis of the products. (B) The putative Clp-binding motif in the
promoter region of rpfB in Xanthomonas. The numbers in brackets indicate the percentages of
identical amino acid compared with those in Xcc strain ATCC 33913.
Symbol: Xac,
Xanthomonas axonopodis pv. citri; Xoo, Xanthomonas oryzae pv. oryzae; Xff, Xanthomonas
fuscans subsp. Fuscans; Xhc, Xanthomonas hortorum pv. carotae strain M081; Psx,
Pseudoxanthomonas spadix BDA-59; Xyl, Xylella fastidiosa; Stm, Stenotrophomonas
maltophilia; Lys, Lysobacter dokdonensis DS-58; Mfl, Methylobacillus flagellatus; Tbd,
Thiobacillus denitrificans. All sequences are retrieved from NCBI Microbial Genomes
Resources. All the amino acid sequences were downloaded from the microbial genome
sequence database of NCBI. Position-Specific Iterated BLAST (PSI-BLAST) was used for
homology analysis.
Supporting Information Table S1. The activities of extracellular
enzymes in Xcc strains. All the Xcc strains were grown in liquid NA
medium until the OD600 of 2.3. The methods for assaying exoenzyme
activity were described in He et al.(Molecular Microbiology, 2006, 59:610622). Data are expressed as the means  one standard deviation of three
independent assays. Different letters indicate significant differences between
treatments (LSD at P=0.05).
Strains
Protease
activity
(% WT)
Cellulase
activity
(% WT)
Amylase
activity
(% WT)
XC1
100a
100a
100a
∆rpfB
121±4.2a
112±7.5a
109±4.6a
ΔrpfB::rpfB
96±5.1a
93±3.9a
96±5.7a
∆rpfB (rpfB)
60±4.5b
70±3.7b
54±4.3b
8004
100a
100a
100a
8004∆rpfB
110±6.5a
99±4.2a
105±4.5a
8004ΔrpfB::
rpfB
94±3.7a
95±4.2a
101±2.8a
8004∆rpfB
(rpfB)
65±3.2b
63±3.7b
62±4.3b
Supporting Information Table S2. Extracellular polysaccharide (EPS)
production in Xcc strains. The method for assaying EPS production was
described in He et al.(Molecular Microbiology, 2006, 59:610-622). All the
Xcc strains were grown in liquid NA medium until the OD600 of 2.3. 10 ml of
cell cultures were used for EPS extraction. Data are expressed as the means
 one standard deviation of three independent assays. Different letters
indicate significant differences between treatments (LSD at P=0.05).
Strains
EPS
(mg / mL)
XC1
5.0±0.5a
∆rpfB
6.3±0.3b
ΔrpfB::rpfB
4.5±0.5a
∆rpfB (rpfB)
2.8±0.2c
8004
3.3±0.2A
8004∆rpfB
4.3±0.3B
8004ΔrpfB::rpfB
3.2±0.3A
8004∆rpfB (rpfB)
2.0±0.2C
Supporting Information Table S3. Bacterial strains and plasmids used in this study.
Strain
Properties / characteristics
Reference / source
8004
Xcc wild-type strain, RifR
Lab stock
XC1
Xcc wild-type strain, RifR
Lab stock
rpfF
The rpfF in-frame deletion mutant, RifR
[1]
rpfC
The rpfC in-frame deletion mutant, RifR
[2]
rpfB
The rpfB in-frame deletion mutant, RifR
This study
rpfBrpfC
The rpfB rpfC double deletion mutant, RifR
This study
clp
The clp in-frame deletion mutant, RifR
[3]
clp::clp
The clp in-frame deletion mutant complemented with a single copy of clp
inserted at the attTn7 site on its chromosome, RifR
This study
rpfCclp
The rpfC clp double deletion mutant, RifR
This study
rpfG
The rpfG in-frame deletion mutant, RifR
[4]
rpfCrpfF
The rpfC rpfF double deletion mutant, RifR
This study
rpfB::rpfB
The rpfB in-frame deletion mutant complemented with a single copy of rpfB
inserted at the attTn7 site on its chromosome, RifR
This study
rpfB(rpfB)
The rpfB in-frame deletion mutant harboring the rpfB expression cosmid
pLAFR3-rpfB, TcR
This study
rpfB(fadD)
The rpfB in-frame deletion mutant harboring the fadD expression cosmid
pLAFR3-fadD, TcR
This study
rpfB(E365A)
The rpfB in-frame deletion mutant harboring the cosmid pLAFR3-rpfB
(E365A) expressing RpfB with a E→A mutation at the 365th amino acid
This study
rpfB(E361A)
The rpfB in-frame deletion mutant harboring the cosmid pLAFR3-fadD
(E361A) expressing FadD with a E→A mutation at the 361st amino acid, TcR
This study
rpfBrpfC::rpfB
The rpfB rpfC double deletion mutant complemented with a single copy of
rpfB inserted at the attTn7 site on its chromosome, RifR
This study
rpfBrpfC(rpfB)
The rpfB rpfC double deletion mutant harboring the rpfB expression cosmid
pLAFR3-rpfB, TcR
This study
rpfBrpfC(fadD)
The rpfB rpfC double deletion mutant harboring the fadD expression cosmid
pLAFR3-fadD, TcR
This study
rpfBrpfC
(E365A)
The rpfB rpfC double deletion mutant harboring the cosmid pLAFR3-rpfB
(E365A) expressing RpfB with a E→A mutation at the 365th amino acid, TcR
This study
rpfBrpfC
(E361A)
The rpfB rpfC double deletion mutant harboring the cosmid pLAFR3-fadD
(E361A) expressing FadD with a E→A mutation at the 361st amino acid, TcR
This study
DH5
E. coli F– Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK–,
mK+) phoA supE44 λ– thi-1 gyrA96 relA1
Lab stock
S17-1
res- pro mod+ integrated copy of RP4, mob+
Lab stock
BL21
E. coli B F- dcm ompT hsdS(rB- mB-) gal [malB+]K-12(λS)
Lab stock
RK2013
Triparental mating helper strain, KanR
Lab stock
Xcc Strains
E. coli strains
Plasmid/Cosmid
Properties / characteristics
Reference / source
pK18mobsacB
A mobilizable vector, allows for selection of double crossover
in Xcc, KanR
[1]
pK18-rpfB
Xcc rpfB deletion cassette in pK18mobscaB, KanR
This study
pK18-PrpfB::AAArpfB380
Xcc PrpfB point mutation
pK18mobscaB, KanR
pLAFR3
A Cosmid derived from pRK290, TetR
[5]
pET-28a
His-tag protein expression vector, KanR
Novagen
pET-14b
His-tag protein expression vector, AmpR
Novagen
pTA2
A high efficient TA cloning vector, AmpR
TOYOBO
mini-Tn7T-Gm
a versatile Mini-Tn7 delivery vector mini-Tn7T-Gm, GmR
[6]
pET-28a-rpfB
pET-28a containing rpfB, KanR
This study
pET-14b-rpfF
pET-14b containing rpfF, KanR
[2]
pGEX-clp
pGEX-6p-1 containing clp, AmpR
[3]
pLAFR3-rpfB
rpfB cloned in pLAFR3, TetR
This study
pLAFR3-rpfB(E365A)
RpfB with a E→A mutation at the 365th amino acid cloned in
pLAFR3, TetR
This study
pLAFR3-fadD
fadD cloned in pLAFR3, TetR
This study
pLAFR3-fadD(E361A)
FadD with a E→A mutation at the 361st amino acid cloned in
pLAFR3, TetR
This study
mini-Tn7T-rpfB
rpfB cloned in mini-Tn7T-Gm under its native promoter, GmR
This study
pTA2-PrpfB
pTA2 containing the promoter region of rpfB, AmpR
This study
pTA2-PrpfB::AAA
The promoter region of rpfB with the TGC→AAA mutation
cloned in pTA2, AmpR
This study
pTA2-PrpfB-rpfB380
Xcc PrpfB-rpfB cassette containing a 431 bp fragment upstream
of the rpfB start codon and the initial 380 bp of rpfB in pTA2,
AmpR
This study
pTA2-PrpfB::AAArpfB380
Xcc PrpfB point mutation (TGC→AAA) cassette in pTA2,
AmpR
This study
(TGC→AAA)
cassette
in
This study
References:
1. He YW, Xu M, Lin K, Ng YJ, Wen CM, Wang LH, et al. Genome scale analysis of diffusible signal factor
regulon in Xanthomonas campestris pv. campestris: identification of novel cell-cell communicationdependent genes and functions. Mol Microbiol. 2006 Jan;59(2):610-22. PMID: 16390454
2. He YW, Wang C, Zhou L, Song H, Dow JM, Zhang LH. Dual signaling functions of the hybrid sensor
kinase RpfC of Xanthomonas campestris involve either phosphorelay or receiver domain-protein
interaction. J Biol Chem. 2006 Nov 3;281(44):33414-21. PMID: 16940295
3. Tao F, He YW, Wu DH, Swarup S, Zhang LH. The cyclic nucleotide monophosphate domain of
Xanthomonas campestris global regulator Clp defines a new class of cyclic di-GMP effectors. J Bacteriol.
2010 Feb;192(4):1020-9. doi: 10.1128/JB.01253-09. PMID: 20008070
4. He YW, Boon C, Zhou L, Zhang LH. Co-regulation of Xanthomonas campestris virulence by quorum
sensing and a novel two-component regulatory system RavS/RavR. Mol Microbiol. 2009
Mar;71(6):1464-76. doi: 10.1111/j.1365-2958.2009.06617.x. PMID: 19220743
5. Staskawicz B, Dahlbeck D, Keen N, Napoli C. Molecular characterization of cloned avirulence genes
from race 0 and race 1 of Pseudomonas syringae pv. glycinea. J. Bacteriol. 1987 Dec;169(12):5789-94.
PMID: 2824447.
6. Choi KH, Schweizer HP. mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas
aeruginosa. Nat Protoc. 2006;1(1):153-61. PMID: 17406227
Supporting Information Table S4. Oligonucleotide primers used in this study
Application
Primer and
application
Sequence (5’ to 3’)
rpfB_del_F1
GgaattcTCACGCTTCGCCTTTTCCATCCAT
rpfB_del_R1
CGGGGTGTAGAGCGGTTGACG
rpfB_del_F2
CCGCTCTACACCCCGAAGGGCCCGCAGGTGATGAA
rpfB_del_R2
CGggatccGCCCGCCCGGGATTGATGG
rpfB(pLA)_F
GgaattcCATGAGTCAGGCACGTCCTTGGTTG
rpfB_R
CGggatccCTATGCCTTGGCCGCATCCC
rpfB_F
CGggatccCGCCGCCGGTCAGCAACA
rpfB(Tn7)_R
CCCaagcttCTATGCCTTGGCCGCATCCC
RpfB(E365A)_F
GCTTACGGCCTGACCGCGACCTCGCCCGCCGCC
RpfB(E365A)_R
GGCGGCGGGCGAGGTCGCGGTCAGGCCGTAAGC
rpfB(pET)_F
GGAATTCcatatgATGAGTCAGGCACGTCCTTGGTTG
rpfB_R
CGggatccCTATGCCTTGGCCGCATCCC
RT_rpfB_F
ACCGCGTCGCCTTGATGATG
RT_rpfB_R
AGCACGCTGGAGCCCGAGTC
PrpfB_F
CGCCGCCGGTCAGCAACA
PrpfB_R
CATCTGCCCCCCCTCCAGGGTATTCGT
PrpfB_PM1
GACCTGCCACCTCAAAAAAATGCTGCGCATCACGC
PrpfB_PM2
GCGTGATGCGCAGCATTTTTTTGAGGTGGCAGGTC
rpfB_F
CGggatccCGCCGCCGGTCAGCAACA
rpfB(380)_R
AGCACGCTGGAGCCCGAGTC
clp(Tn7)_F
GGggtaccGCTCCTTGCCGGCCTGCTTCTTGT
clp(Tn7)_R
CGggatccACCGATCGCCACCCCACGCTTAG
fadD(pLA)_F
GgaattcCTTGAAGAAGGTTTGGCTTAACCGTTATCCC
fadD_R
CGggatccCCAGCGCATCGTCCGTGGTAATCAT
FadD (E361A)
point mutation
FadD(E361A)_F
GGCTATGGCCTTACCGCGTGTGCGCCGCTGGTC
FadD(E361A)_R
GACCAGCGGCGCACACGCGGTAAGGCCATAGCC
qRT-PCR analysis on 16S
rRNA
RT_16S_F
GCGTAAAGCGTGCGTAGGTGGTGGTT
RT_16S_R
CGCTTTCGTGCCTCAGTGTCAGTGTTG
Amplification of Xcc rpfB
deletion cassette
by pLAFR3
RpfB
expression
by miniTn7T-Gm
RpfB (E365A)
point mutation
RpfB protein expression
qRT-PCR analysis on rpfB
Amplification of the
promoter of rpfB (PrpfB)
PrpfB point mutation
Amplification of PrpfB point
mutation (TGC→AAA)
cassette
Clp expression by miniTn7T-Gm
FadD expression by pLAFR3
The sequences in lower cases are the introduced restriction sites