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 RNAsnap^TMprotocol (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 iScript^TM 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 (C_T) data were normalized to the gyrB gene. These normalized values (∆C_T) were transformed using the 2(e-∆C_T)/10^-6 method (Livak and Schmittgen, 2001), and were reported as the gene expression ratio (2^∆∆C_T) of the mutant strains/the parent strain (JE22070 pagR ⁺). Mean 2^∆∆C_Tvalues 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^∆∆C_Tbetween 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 Qubit^TM Protein Assay Kit (ThermoFisher) and the Qubit^TM 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.