Appendix S1
Plasmid construction. All plasmids used in this study are listed in Table 2. Oligo
sequences are provided in supplementary Table A1. Plasmid pAT1379 encoding a
mTn5lacZ transposon was constructed by amplifying the E. coli lacZ gene with primers
TnLacF and TnLacR and ligating into NotI-digested pUTmTn5Km2-STM (Hensel et al.,
1995). The TnLacF primer was designed with stop codons in all three reading frames
before the lacZ start and encodes a modified ribosome binding site with one randomized
base. The ribosome binding site in pAT1379 is wild type (G) at this position.
Plasmids pAT1337 and pAT1338, which encode arabinose-inducible vieA and
vieB, respectively, were constructed by PCR-amplification of the vieA and vieB genes
from O395 genomic DNA with primer pairs VieAF2/VieAR and VieBF2/VieBR. Each
forward primer contains a ribosome binding site followed by a 9 base spacer and an ATG
start. The vieA and vieB PCR products were cloned into PstI/SacI and PstI/HindIII
digested pBAD33 (Guzman et al., 1995), respectively.
Plasmids for generating in-frame deletions or point mutations in V. cholerae were
constructed in the allelic exchange vector pCVD442, which encodes the sacB counterselectable marker (Donnenberg and Kaper, 1991). Splicing by overlap extension (SOE)
PCR (Senanayake and Brian, 1995), was used to generate all deletion and point
mutations. DNA fragments upstream and downstream of each mutation were amplified
by PCR from O395 genomic DNA, annealed together by complementary sequences in
the R1 and F2 primers, and PCR-amplified with the F1 and R2 primers. The final PCR
product was ligated into either pGEM-T or pCR-Script, and subcloned into pCVD442 by
restriction enzyme digestion with either SalI/SphI (pGEM-T) or SalI/SacI (pCR-Script).
The respective F1/R1 primers, F2/R2 primers, and plasmid used for cloning the SOE
product were: pAT1385 - PVAF2/DVAR1, DVAF2/AHTHR2, pGEM-T; pAT1486 PVAF2/D52AR1, D52AF2/D52AR2, pCR-Script; pAT1542 - D403F1/D403R1,
D403F2/D403R2, pGEM-T; pAT1591 - RPAVA2/DDUFR1, DDUFF2/VAR1, pCRScript; pAT1594 - RPAVA2/E170AR, E170AF/D430R1, pGEM-T; pAT1595 HVAF1/D286AR, D286AF/CHVAR1, pGEM-T.
Plasmids containing arabinose-inducible truncations of VieA or other
GGDEF/EAL domains were constructed by PCR-amplifying the desired gene segment
with a forward primer containing a ribosome binding site, 9 base spacer, and ATG start,
and a reverse primer encoding a His6 tag, and cloning into appropriately digested
pBAD33. The following plasmids were constructed by PCR amplification of the vieA
gene with the indicated primer pairs using genomic DNA from O395 as template and
ligating into PstI/SacI-digested pBAD33: pAT1535 - VieAF2/CHVAR1; pAT1536 VieAF2/CHVAR2; pAT1564 - VieAF2/NTVAR1; pAT1567 - NTVAF2/CHVAR2;
pAT1568 - NTVAF3/CHVAR2; pAT1575 - NTVAF4/CHVAR2; pAT1581 NTF5/CHVAR2; pAT1582 - NTF1/CHVAR2; pAT1583 - NTF6/CHVAR2. The
plasmids pAT1561, pAT1615, and pAT1616 encoding truncated versions of VieA with
point mutations were constructed by amplifying vieA from V. cholerae point mutant
strains AC-V1487, AC-V1596, and AC-V1597, respectively, using primer pairs
VieAF2/CHVAR2 or NTVAF3/CHVAR2 and ligating into PstI/SacI-digested pBAD33.
Plasmids pAT1645, pAT1646, and pAT1647, encoding portions of the VC0653 GGDEFEAL protein were generated by PCR amplification with primers 0653F1/0653R1,
0653F2/0653R2, and 0653F1/0653R2, respectively, and cloning into XbaI/HindIII
digested pBAD33. The plasmids pAT1648 and pAT1662 were constructed by cloning
the VCA0785 EAL domain amplified with primers A0785F/A0785R and VCA0956
amplified with primers A0956F/A0956R, respectively, into XbaI/SphI digested pBAD33.
Plasmids for generation of RPA riboprobes were constructed in pGEM-T
(Promega). O395 genomic DNA was amplified by PCR using Taq polymerase and
primer pairs: 928F/928R (274 bp), 917F/917R (353 bp), 934F/934R (293 bp),
vpsRF/vpsRR (336 bp), and HVAF1/DDUFR1 (386 bp) to generate plasmids pAT1455,
pAT1454, pAT1456, pAT1509, and pAT1537, respectively. Insert orientation was
determined by PCR using the appropriate forward primer and either the SP6 or T7
primer.
Strain construction. All strains used in this study are listed in Table 2. V.
cholerae strains AC-V1101, AC-V1198, AC-V1200, AC-V1212, AC-V1386, ACV1487, AC-V1543, AC-V1544, AC-V1728, AC-V1596, and AC-V1597 containing inframe deletions or point mutations in the vieSAB locus were generated by allelic
exchange in the O395 or AC-V61 background. Strains AC-V1729, AC-V1598, and ACV1614 were generated by allelic exchange with plasmid pAT1591, pAT1615, and
pAT1616, respectively, in the AC-V1417 strain background. The ∆flaA mutation in
strain AC-V1539 was constructed by allelic exchange with plasmid pSL140 in the ACV61 background. Plasmids were conjugated into O395, AC-V61, or AC-V1417 from E.
coli SM10pir as previously described (Lee et al., 1998). After one passage in LB broth
in the absence of antibiotics, sucrose-resistant colonies were seleceted and were
subsequently screened for deletion or point mutations by colony PCR and DNA
sequencing.
The mTn5-lacZ insertions in VCA0707 and VC0928 isolated in the screen for
VieA-regulated genes were transduced into strain backgrounds AC-V1352, AC-V1351,
and AC-V1353 by CP-T1ts phage transduction as previously described (Hava and
Camilli, 2001), to generate strains AC-V1409, AC-V1420, AC-V1421, AC-V1407, ACV1415, and AC-V1416. The VC0928::lacZ fusion was also transduced into the AC-V61,
AC-V1386, and AC-V1200 strain backgrounds to generate strains AC-V1417, ACV1418, and AC-V1419, respectively. V. cholerae strains carrying the pBAD33 vector
and its derivatives were created by electroporation of the appropriate strains as previously
described (Tischler et al., 2002). Transformants were selected on LB agar containing 5
g/ml Cm.
His6 Immunoblotting. Bacteria grown overnight in LB ± 0.2% arabinose were pelleted
by centrifugation, resuspended in 1x sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) sample buffer, and boiled for 5 minutes. Samples were
separated on 10% pre-cast SDS-PAGE gels (Biorad) and transferred electrophoretically
to nitrocellulose membranes (Hybond). The His6 epitope was detected using mouse
penta-His antibody (Qiagen) and the ECL enhanced chemiluminescence detection system
(Amersham). Reactive bands were visualized by exposure to X-ray film (Kodak).
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