The high-efficiency cloning method of BspQI digestion described
elsewhere (Galloway et al., 2013, VanDrisse and Escalante-Semerena,
2016) was used to clone pagR into plasmids pCV1 and pTEV19, andtktD-tktE , tktA and tktB into pCV1. All genes
cloned were PCR amplified from S. Typhimurium strain JE6583
genomic DNA using PFU Ultra II Fusion DNA polymerase and PCR products
were visualized as described above. PCR products were cleaned using the
Promega Wizard SV gel and PCR clean up kit, and cloning using the
method cited above. After transformation into E. coli DH5α cells
and colony PCR screening of correctly ligated plasmids, plasmids were
isolated using the Wizard Plus SV miniprep kit (Promega). Sanger DNA
sequencing was performed by Eton Bioscience to rule out mutations and
confirm insertions.
Culture media, chemicals, and in vivo growth analyses.Growth behavior studies were conducted as follows: i) starter bacterial
cultures were grown from a single colony in nutrient broth (NB, Difco)
with overnight shaking at 180 rpm at 37 ºC; ii) after
~16 h of growth, cells
(~108 cfu) were sub-cultured into
no-carbon essential (NCE) minimal medium (Berkowitz et al., 1968)
containing MgSO4 (1 mM), Wolfe’s trace minerals (Balch
and Wolfe, 1976), ampicillin (100 µg/mL), L-(+)-arabinose (concentration
stated in figure legends), with sodium succinate (30 mM) as the sole
carbon and energy source. Inoculum used was routinely 1% (v/v) of the
final volume of the culture. Growth studies were performed in 96-well
polystyrene (Falcon) microtiter plates with each well containing 198 µL
of fresh medium and a 2-µL inoculum. Microtiter plates were incubated at
37 ºC shaking continuously inside a PowerWave microtiter plate reader
(Bio-Tek Instruments). Density of cells was monitored at 630 nm and data
were analyzed using Prism 9 (GraphPad).
RNA isolation. Strains JE22070 (pagR +/ vector), JE21566 (pagR1 ::kan+ /
vector), JE21577 (pagR1 ::kan+ / pPagR)
were grown overnight in triplicate in nutrient broth (2 mL; NB, Difco)
with shaking at 37 ºC. After incubation, cultures were diluted 1:100
into 5 mL of fresh lysogeny broth rich medium supplemented with
L-(+)-arabinose (100 µM). Cultures were grown shaking at 37 ºC to an
optical density of 0.5 at 600 nm, then 5 mL of each sample were quickly
centrifuged in 15-mL Falcon tubes at 4000 x g , supernatant was
removed, and pellets were flash-frozen in liquid nitrogen and kept on
dry ice. RNA was isolated following the RNAsnapTMprotocol (Stead et al., 2012). Pellets were re-suspended in 150 µL of
boil solution (ethylenediaminetetraacetic acid (EDTA, 18 mM), SDS
(0.025%, w/v) formamide (95%, v/v; RNA grade), 2-mercaptoethanol (1%
v/v) in RNase-free water) and were vortexed vigorously to break up the
cell pellet. Pellets were incubated at 95 ºC for 7 min and centrifuged
at 16,000 x g for 5 min at room temperature; 100 µL of
supernatant was transferred to a fresh tube. A sodium acetate/ethanol
RNA precipitation was then conducted by the addition of 400 µL of
RNase-free water, 50 µL of sodium acetate (3 M, pH 5.2: final
concentration of 0.3 M), and finally 1.65 mL of ice-cold absolute
ethanol (100%), with mixing briefly before the addition of the next
reagent. The mixture was incubated at -80 ºC for one hour, centrifuged
at 16,000 x g for 30 min at 4 ºC, and ethanol was decanted.
Ethanol (300 µL of cold 70% v/v) was added, and pellets were
centrifuged at 8,000 x g for 5 min at 4 ºC in an Eppendorf 5415D
centrifuge. Ethanol was removed and pellets were allowed to dry. RNA
pellets were re-suspended in RNase-free water. Subsequent RNase-free
DNase I treatment was conducted using the Ambion Turbo DNA-free kit
according to manufacturer’s instructions (ThermoFisher Scientific).
After DNA cleavage, a final sodium acetate/ethanol precipitation was
performed as described above, except using 360 µL of water, 50 µL of 3 M
sodium acetate, and 1.5 mL of cold 100% ethanol. After overnight
incubation at -80 ºC, RNA was centrifuged at 16,000 x g for 30
min, then washed with 300 µL of cold 70% ethanol (v/v). Ethanol was
decanted, and RNA pellets were dried for 20 min at room temperature. RNA
was resuspended in 100 µL of water. A small sample of each preparation
was used for quantification with the RNA Broad Range (BR) Assay kit by
Qubit on a Qubit 4 fluorometer. A small amount of each preparation was
also tested for quality and integrity using the Qubit RNA IQ Assay.
Primers for qPCR were designed using Primer3 (Untergasser et al., 2012,
Koressaar and Remm, 2007, Koressaar et al., 2018)
cDNA synthesis and real-time quantitative polymerase chain
reaction (RT-qPCR). Total RNA (972 ng) from each sample was used for
the synthesis of cDNA using the iScriptTM cDNA
synthesis Kit from Bio-Rad Laboratories according to manufacturer’s
protocol. Each cDNA reaction was then diluted to 7.5 ng/µL and used as
template for PCR. For real-time PCR, 20 µL reactions were prepared with
10 µL of 2X FastSYBR Green master mix (Applied Biosystems), 500 nM of
each gene-specific primer (1 µL of 10 µM primer stock), and 15 ng of
cDNA (2 µL of 7.5 ng/µL cDNA). The real-time PCR reaction was performed
using a 7500 Fast real-time PCR system (Applied Biosystems). The
threshold cycle value of gyrB were checked first to ensure it was
optimal for use as reference genes for these strains under the
conditions chosen for RT-qPCR. Cycle threshold (CT) data
were normalized to the gyrB gene. These normalized values
(∆CT) were transformed using the
2(e-∆CT)/10-6 method (Livak and
Schmittgen, 2001), and were reported as the gene expression ratio
(2^∆∆CT) of the mutant strains/the parent strain
(JE22070 pagR +). Mean 2^∆∆CTvalues were used to calculate the standard error of the mean (SEM) using
Prism9 (GraphPad) from three biological replicates that were each tested
in technical triplicate. Differences in 2^∆∆CTbetween strains were compared using Welch’s t -test with Prism9
software.
β-Galactosidase assays. Plasmids pCV1 and pPagR7 were
independently transformed into JE27072
(pagR ::lacZY+ kan+ ).
Three independent colonies of each strain were grown overnight in 2 mL
of NB plus ampicillin (100 µg/mL), then sub-cultured 1:100 into 5 mL of
LB plus ampicillin (100 µg/mL) and arabinose (100 µM). Cells were grown
shaking at 180 rpm at 37C until an OD600 nm of 0.4-0.6,
and β-galactosidase units were measured as described (Miller and
Hershberger, 1984).
Operon PCR. As described above, total RNA was isolated from
strain JE21107 (pagR1 ::kan+ ) and was
used to generate cDNA. cDNA and genomic DNA isolated from strain JE21107
were used in PCR reactions containing Green GoTaq (Promega) master mix
to amplify overlapping genes within the stm2340 -stm2344operon. Primer pairs are listed in Table S2.
Purification of PagR protein. PagR protein was purified to
homogeneity from plasmid pPagR-8 encoding a PagR protein with a maltose
binding protein-hexahistidine (MBP-H6) tag fused to its N terminus. The
tag was removed after incubation with recombinant tobacco etch virus
(rTEV) protease since the plasmid used to produce
MBP-H6-PagR (pTEV19) contained an rTEV protease cleavage
site (VanDrisse and Escalante-Semerena, 2016). A sample (10 mL) of an
overnight culture of E. coli C41 (λDE3) / pPagR-8 strain was used
to inoculate one liter of lysogeny broth containing 100 µg/mL of
ampicillin. Cells were grown shaking at 125 rpm at 37 °C until the
culture reached an optical density at 600 nm of 0.7. Expression of genes
of interest encoded by the plasmids was induced by the addition of
isopropyl β-D-1-thiogalactopyranoside (IPTG, 0.25 mM) followed by
~12 h of overnight incubation at 37 °C. The next morning
cells were harvested by centrifugation at 6,000 x g for 15 min
using a refrigerated Beckman-Coulter Avanti J-20-XPI centrifuge equipped
with a JLA 8.1 rotor. Cell pellets were resuspended in 20 mL of buffer A
[(4-(2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES) buffer (50
mM, pH 7.5 at 4 ºC) containing NaCl (0.5 M), glycerol (20% v/v), and
imidazole (20 mM)] and were sonicated thrice for 30-s intervals, and
during each interval, sonication was on for 2 s and off for 2 s, at 60%
amplitude. The resulting whole-cell lysates were centrifuged for 30 min
at 40,000 x g and the supernatants were filtered with a 0.45-µm
filter (VWR) to remove large particulates. Each filtered lysate was
applied onto a 1-mL nitrilotriacetic acid (NTA) affinity chromatography
column pre-equilibrated with buffer A. Fractions were collected by
gravity at 4 °C. The purification was performed as follows: After all
the lysate was loaded onto the column, the column was washed with 10
column volumes (CV, i.e., 10 mL) of buffer A, seven CV
(i.e., 7 mL) of buffer A containing 4% elution buffer B [HEPES
buffer (50 mM, pH 7.5 at 4 ºC), NaCl (0.5 M), glycerol (20% v/v), and
imidazole (0.5M)], and finally, MBP-H6-PagR was eluted
in two fractions, first with one CV (i.e., 1 mL) of 100% elution
buffer B, and the second fraction being four CV (i.e., 4 mL) of
100% elution buffer B. Both elution fractions were pooled and
MBP-H6-PagR was cleaved with rTEV protease at a 1 mg
1:100 rTEV:PagR protein ratio while dialyzing at room temperature in
HEPES buffer (50 mM, pH 7.5 at 4 ºC) containing NaCl (0.5 M), glycerol
(10% v/v), and dithiothreitol (DTT, 1 mM). Cleaved protein was dialyzed
twice more at 4 °C in buffer A. Cleaved PagR protein was loaded again
onto a 2-mL NTA column to remove MBP-His and rTEV protease, both of
which were fused to a hexahistidine tag. Tag-less PagR protein did not
interact with the NTA resin and was collected in the flow-through
fraction. To further remove MBP from PagR, flow-through fractions from
the second NTA purification were run over a 1-mL amylose resin to remove
contaminating MBP. Pure, tag-less PagR protein was dialyzed overnight
against HEPES buffer (50 mM, pH 7.5 at 4ºC) containing NaCl (150 mM) and
glycerol (20% v/v) at 4˚C. Fifteen mL of dialyzed PagR solution was
dispensed into Eppendorf microcentrifuge tubes, flash frozen in liquid
nitrogen and stored at -80˚C until used. Protein concentration was
determined using the QubitTM Protein Assay Kit
(ThermoFisher) and the QubitTM 4 fluorometer.
Electrophoretic mobility shift assays (EMSAs). EMSAs were
performed to quantify PagR binding to DNA. EMSAs were performed as
follows: purified PagR protein was incubated at 0, 0.5, 1.0, 2.5, 5.0
and 10.0 pmol of protein with 0.5 pmol of a 5(6)-carboxyfluorescein
(5(6)-FAM) and hexachlorofluorescein (HEX)-labeled DNA probe (1: from
2,457,048 to 2,457,454 nt of the chromosome; 406 nt, 2: from 2,457,048
to 2,457,339 nt of the chromosome; 291 nt). EMSA buffer [HEPES buffer
(50 mM, pH 7.5 at 4ºC) containing NaCl (150 mM), and glycerol (10%
v/v)] was added to the reaction mixture (total volume = 25 µL) and DNA
and protein were incubated at room temperature for 40 min. During
incubation, a 7.5% Tris-Boric acid-EDTA (TBE) polyacrylamide gel was
pre-developed at 100 V for 40 min in 0.5X TBE buffer at 4˚C. After
incubation, 5 µL of glycerol (50% v/v) was added to the reaction
mixtures, and 20 µL of each reaction mixture was resolved by the
polyacrylamide gel. A lane of xylene cyanol and bromophenol blue dye was
added as a tracking indicator, and the gel was run until bromophenol
blue reached the bottom of the gel. The gel was imaged using a Typhoon
Trio Imager (GE Healthcare) at 525 nm with the 488 (Blue) filter.
DNase I footprinting. The promoter region of stm2344 andpagR (from 2,457,048 to 2,457,339 nt of the chromosome; 291 nt)
was PCR amplified using a 5(6)-FAM-labeled primer and a HEX-labeled
primer from JE6583 genomic DNA. The product was purified with the
Wizard SV Gel and PCR Cleanup System (Promega). 7.5 pmol of
5(6)-FAM/HEX-labeled probe was incubated with either no PagR protein or
75 pmol PagR for 40 min at room temperature in 250 µL EMSA Buffer.
Twenty-five ng of DNase I (Sigma) was added to each reaction and
incubated for 5 min at room temperature. DNase was heat inactivated at
80°C for 10 min. Digested fragments were purified with the Wizard SV
Gel and PCR Cleanup System (Promega), eluted from the column with 30 µL
diH2O, and diluted 1:2 in diH2O for
analysis. Fragment analysis by capillary electrophoresis was performed
at the University of Illinois DNA Core Sequencing facility using the
Applied Biosystems 3730xl DNA Analyzer. The results were processed with
GeneMapper6 and aligned to the sequencing results to determine the
protected region(s).
Dideoxy Sanger sequencing. The promoter region ofstm2344 / pagR (from 2,456,991 to 2,457,412 nt of the
chromosome; 421 nt) was PCR amplified from JE6583 genomic DNA and
purified with the Wizard SV Gel and PCR Cleanup System (Promega) to
create a template for dideoxy termination sequencing. This template was
sequenced using the USB® ThermoSequenase Cycle Sequencing Kit
(Affeymetrix) with the HEX and 5(6)-FAM labeled primers used to generate
the digested fragment. Sequencing was performed following the
manufacturer’s instructions for dideoxy termination sequencing using
2pmol of primer, 200ng template, and 60 cycles for each reaction.
Samples were diluted 1:2 in deionized water and analyzed at the
University of Illinois DNA Core Sequencing facility using the Applied
Biosystems 3730xl DNA Analyzer. The resulting electropherograms were
analyzed with GeneMapper™ Software 6 (Thermo Fisher Scientific).
Acknowledgements. This work was supported by NIH grant
R35-GM130399 to J.C.E.-S. The authors thank the DNA Services Lab of the
Roy J. Carver Biotechnology Center of The University of Illinois at
Urbana-Champaign for the performance of the DNAse I Footprinting
Analysis.
Conflict of interest statement. The authors have no conflict of
interest to declare.
Data availability. All the data generated by these studies is
reported in this paper and its Supplementary Material file.