Supplementary material
Methods
Primer extension analyses
V. cholerae strains were grown in LB to an OD600 of 0.6-0.7. After harvesting cells, total
RNA was extracted and purified using Trizol Reagent (Invitrogen) according to
manufacturer’s instructions. To initiate primer extension, 10pmol of 5’ FAM-labeled
primer (CMW234) was first hybridized with 15g of purified RNA at 70°C for 5 minutes,
ice for 10 minutes, 58°C for 20 minutes, and room temperature for 15 min. To catalyze
primer elongation, SuperscriptIII reverse transcriptase (Invitrogen) along with reagent
components were added to the primer-mRNA hybrid as specified by the manufacturer
and incubated at 55°C for one hour. The resulting FAM-labeled cDNA was purified
using phenol extraction and ethanol precipitation. After resuspension of the FAMlabeled cDNA in 10 mL of ddH2O, 5 mL of digested fragments were mixed with 4.9 L
HiDi formamdide and 0.1 L 500 LIZ size standards prior to submission using the ABI
Prism 3130xl Genetic analyzer (MSU Research Technology Support Facility).
Chromatograms were aligned using PeakScanner Software v1.0. This experiment was
repeated three times and a representative chromatogram is shown.
.
Figures
Fig.S1. Identification of the flrBC transcription start site of flrBC promoter. (A)
Nucleotide sequence of template strand flrBC promoter. Primer extension analysis was
performed with 6-FAM labeled primer which hybridizes 15 to 32 bp downstream of the
flrBC translation start site. Total RNA was isolated from pDS49(flrBC-lux) in vpsLflrA
(B) and pDS49 in vpsL (C). One major transcript, boxed in black, is produced from
pDS49 when FlrA is present. This transcript corresponds to 23 bp upstream from the
translation start site of flrBC.
Fig. S2: Architecture of flrBC promoter. flrBC promoter schematic showing the +1
transcription start site and FlrA binding site as deciphered from primer extension and
DNAseI footprinting assays respectively.
Fig. S3: FlrA does not regulate vps gene expression. The expression of vpsR, vpsT
and vpsL. Luciferase production from vpsR-lux, vpsT-lux, vpsL-lux in WT and flrA V.
cholerae strains. High (black bars) and low (gray bars) intracellular levels of c-di-GMP
were generated by overexpression of the DGC QrgB or its active site variant, QrgB*,
respectively.
Fig. S4: FlrA does not co-purify with any proteins. Purified FlrA (WT, FlrA (R176H)
and FlrA (R135H) run on a 12.5% SDS PAGE gel and stained with Commassie Brilliant
blue R250.
Table S1: Plasmids and primers
Plasmid
Description/Primers (5’-3’)
Reference
pBH629
vpsL promoter driving lux operon
pKAS32
Suicide plasmid for generation of mutants
pBBRlux
lux operon containing reporter plasmid
pBRP1
qrgB* (inactive GGDEF mutant) IPTG inducible overexpression
vector
qrgB (active GGDEF) IPTG inducible overexpression vector
(Hammer
Bassler, 2009)
(Skorupski
Taylor, 1996)
(Hammer
Bassler, 2007)
this work
pBRP2
pDS1
pDS22
pDS54
pEVS143
pLLP15
FlrA overexpression construct in pTXB1
FP: ATA CAT ATG CAG AGT TTT AGC GAA ACT
RP: GCG TTG CAT GTT GTA TTT GC
YcgR overexpression construct in pTXB1
pKAS32 derivative for flrA mutant construction
FP up: ATA GCA TGC CAG TTA AAA ACG GCG GCG AT
RP up: ATA TCT AGA AGG TGA GAT TAT TTG CCT TT
FP ds: ATA TCT AGA TAG GGA AAC CAT AGT CAA TA
RP ds: ATA GTC GAC TCG TGC AGT CGG TCA ACC AA
Overexpression vector with pTac promoter
flrA overexpression cloned in pEVS143
FP:GGTAGGCCTAGGAGCTAAGGAAGCTAAAATGCAGAGTTTAG
CGAAAC
&
&
&
this work
NEB
IMPACT
system TM
this work
(Srivastava et al.,
2011)
this work
(Dunn et al., 2006)
this work
RP: GGTGGATCCGCGTTGCATGTTGTATTTGCG
pDS49
flrBC promoter in pBBRlux
FP:GGTACTAGTCGCAAATACAAC
this work
RP:GGTGGATCCGAATGCTGCTCTT
EMSA and
DNaseI
footprinting
primers
CMW234
/56-FAM/ATTTTGCGGCCGCAACTGA
(Srivastava et al.,
2011)
CMW235
/56-FAM/CCGCGGTGGCGGCCGCTCTA
CMW1834
CCGCGGTGGCGGCCGCTCTA
pDS72
flaA promoter in pBBRlux
FP:GGTACTAGTAATGGGTGTCAA
RP: GGTGGATCCACACGTTGGTAT
this work
pDS73
flgB promoter in pBBRlux
FP: GGTACTAGTGTTGTAACCCTG
RP: GGTGGATCCAGGGCTCTGTCA
this work
pDS74
flgM promoter in pBBRlux
FP:GGTACTAGTCAAGTCAGTGGT
RP: GGTGGATCCTTAACGACTGTC
this work
1:B8
fragment upstream of VCA0213 cloned in pBBRlux identified in
screen
(Srivastava et al.,
2011)
1:F6
fragment upstream of VC2647 (aphA) cloned in pBBRlux identified in
screen
(Srivastava et al.,
2011)
2:G12
fragment upstream of VC2610 cloned in pBBRlux identified in screen
4:H4
fragment upstream of VC2108 cloned in pBBRlux identified in screen
5:A6
fragment upstream of VC1899 cloned in pBBRlux identified in screen
6:C9
fragment in the ORF of VC1673 cloned in pBBRlux identified in
screen
(Srivastava
2011)
(Srivastava
2011)
(Srivastava
2011)
(Srivastava
2011)
9:C11
fragment upstream of VCA0055 cloned in pBBRlux identified in
screen
(Srivastava et al.,
2011)
pDS82
G168A/T169A/G170A mutation on pLLP15
Mutagenesis primer:
GGAAGCCAACGTGCTGATCCTCGGTGAGTCGGCCGCGGCTAAA
GAAGTGGTTGCGCGTAACATTCACT
this work
pDS83
K171A/E172A/V173A mutation on pLLP15
Mutagenesis primer:
GTGCTGATCCTCGGTGAGTCGGGCACGGGTGCAGCAGCGGTTG
CGCGTAACATTCACTACCATTCAGG
this work
pDS84
V174A/R176A mutation on pLLP15
Mutagenesis primer:
TCGGTGAGTCGGGCACGGGTAAAGAAGTGGCTGCGGCTAACAT
TCACTACCATTCAGGACGCCGTAA
this work
pDS88
G351A/N352A mutation on pLLP15
Mutagenesis primer:
TCAATTCGATGATGGAGCATGACTGGCCGGCTGCTGTGCGTGA
ACTTGCCAACTTGGTTGAGCG
this work
pDS89
V353A/R354A mutation on pLLP15
Mutagenesis primer:
CGATGATGGAGCATGACTGGCCGGGTAATGCGGCTGAACTTGC
CAACTTGGTTGAGCGTATGGT
this work
et al.,
et al.,
et al.,
et al.,
pDS90
G165A/E166A/S167A mutation on pLLP15
Mutagenesis primer:
TACGCGCAACCACTTCTTTACCCGTGCCCGCCGCAGCGAGGAT
CAGCACGTTGGCTTCCGTGGTCGAG
this work
pDS91
L162A/I163A mutation on pLLP15
Mutagenesis primer:
GAGCAAGTCTCGACCACGGAAGCCAACGTGGCGGCCCTCGGTG
AGTCGGGCACGGGTAAAGAAGT
this work
pDS92
W349A/P350A mutation on pLLP15
Mutagenesis primer:
CGCGTGCGATCAATTCGATGATGGAGCATGCCTGGGCGGGTAA
TGTGCGTGAACTTGCCAACTTGG
this work
pDS136
E355A/L356A
Mutagenesis primer:
TGGAGCATGACTGGCCGGGTAATGTGCGTGGCCGCGCCAACTT
GGTTGAGCGTATGGTCATCCTG
this work
pDS93
R176A mutation on pLLP15
Mutagenesis primer:
GAGTCGGGCACGGGTAAAGAAGTGGTTGCGGCTAACATTCACT
ACCATTCAGGACGCCGTAAT
this work
pDS101
V174A mutation on pLLP15
Mutagenesis primer:
CTCGGTGAGTCGGGCACGGGTAAAGAAGTGGCTGCGCGTAACA
TTCACTACCATTCAGGACGC
this work
pDS103
R176H mutation on pLLP15
Mutagenesis primer:
GAGTCGGGCACGGGTAAAGAAGTGGTTGCGCATAACATTCACTA
CCATTCAGGACGCCGTAA
this work
pDS104
R176K mutation on pLLP15
Mutagenesis primer:
GAGTCGGGCACGGGTAAAGAAGTGGTTGCGAAAAACATTCACTA
CCATTCAGGACGCCGTAAT
this work
pDS105
R176E mutation on pLLP15
Mutagenesis primer:
GAGTCGGGCACGGGTAAAGAAGTGGTTGCGGAGAACATTCACT
ACCATTCAGGACGCCGTAAT
this work
pDS110
R176H mutation in pDS1
Mutagenesis
primer:
GAGTCGGGCACGGGTAAAGAAGTGGTTGCGCATAACATTCACTA
CCATTCAGGACGCCGTAA
this work
pDS123
R135H mutation in pLLP15
Mutagenesis primer:
this work
TTGCGACCGCACGCAAAAACACCCTGTTTCATAGCCTTGTTGGG
CAAAGCATGGGG
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