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Two distinct regulatory systems control pulcherrimin biosynthesis in Bacillus subtilis [1]

['Nicolas L. Fernandez', 'Department Of Molecular', 'Cellular', 'Developmental Biology', 'University Of Michigan', 'Ann Arbor', 'Michigan', 'United States Of America', 'Lyle A. Simmons']

Date: 2024-05

Regulation of transcription is a fundamental process that allows bacteria to respond to external stimuli with appropriate timing and magnitude of response. In the soil bacterium Bacillus subtilis, transcriptional regulation is at the core of developmental processes needed for cell survival. Gene expression in cells transitioning from exponential phase to stationary phase is under the control of a group of transcription factors called transition state regulators (TSRs). TSRs influence numerous developmental processes including the decision between biofilm formation and motility, genetic competence, and sporulation, but the extent to which TSRs influence bacterial physiology remains to be fully elucidated. Here, we demonstrate two TSRs, ScoC and AbrB, along with the MarR-family transcription factor PchR negatively regulate production of the iron chelator pulcherrimin in B. subtilis. Genetic analysis of the relationship between the three transcription factors indicate that all are necessary to limit pulcherrimin production during exponential phase and influence the rate and total amount of pulcherrimin produced. Similarly, expression of the pulcherrimin biosynthesis gene yvmC was found to be under control of ScoC, AbrB, and PchR and correlated with the amount of pulcherrimin produced by each background. Lastly, our in vitro data indicate a weak direct role for ScoC in controlling pulcherrimin production along with AbrB and PchR. The layered regulation by two distinct regulatory systems underscores the important, role for pulcherrimin in B. subtilis physiology.

Regulation of gene expression is important for survival in ever changing environments. In the soil bacterium Bacillus subtilis, key developmental processes are controlled by overlapping networks of transcription factors, some of which are termed transition state regulators (TSRs). Despite decades of research, the scope of how TSRs influence B. subtilis physiology is still unclear. We found that three transcription factors, two of which are TSRs, converge to inhibit production of the iron-chelator pulcherrimin. Only when all three are missing is pulcherrimin production elevated. Finally, we demonstrate that expression of pulcherrimin biosynthesis genes occurs via direct and indirect regulation by the trio of transcription factors. Due to its iron chelating ability, pulcherrimin has been characterized as a modulator of niche development with antioxidant properties. Thus, our findings that TSRs control pulcherrimin, concurrently with other developmental phenotypes, provides new insight into how TSRs impact B. subtilis and its interaction with the environment.

In this study, we provide evidence for a multi-layered regulation of pulcherrimin biosynthesis by the TSRs ScoC and AbrB as well as the pulcherrimin regulator PchR. We explore the kinetics of pulcherrimin production throughout the transition state and found that ScoC, AbrB, and PchR control the timing, rate, and amount of pulcherrimin produced by modulating expression of the pulcherrimin biosynthetic gene cluster yvmC-cypX. We further establish the roles of PchR and AbrB in direct regulation of gene expression utilizing in vitro DNA binding assays and provide evidence that ScoC can bind directly to the yvmC promoter in vitro. Together our results establish a model where pulcherrimin biosynthesis is regulated by nutrient levels during the transition from exponential phase to stationary phase in addition to input from PchR, linking stationary phase with extracellular iron sequestration.

Pulcherrimin biosynthesis is negatively regulated by the MarR-family transcription factor PchR, which is found in a cluster of two divergently transcribed gene-pairs encoding pulcherrimin biosynthesis (yvmC and cypX), regulation and transporter (pchR and yvmA) ( Fig 1B ) [ 12 , 13 ]. Interestingly, AbrB binding sites have been identified in the promoter for pulcherrimin biosynthesis and regulatory genes, suggesting AbrB was involved in pulcherrimin biosynthesis, however genetic analysis of AbrB regulation of pulcherrimin was not demonstrated [ 9 , 11 ]. Further, whether other TSRs are involved in the regulation of pulcherrimin production is not known.

A) Pulcherriminic acid biosynthesis by the cyclization of tRNA-charged leucines to form cyclo(L-leucine-leucine) and the subsequent oxidation by CypX to form water-soluble pulcherriminic acid. Pulcherriminic acid is then transported out of the cell by YvmA, where it can form the insoluble pulcherrimin complex with iron, which forms a red color and has a peak absorbance at 410 nm. B) Genetic architecture of the pulcherrimin cassette. PchR, the MarR-family regulator encoded within the cassette, negatively regulates two promoters controlling pchR-yvmA and yvmC-cypX expression [ 13 ].

Pulcherrimin is a secreted iron chelating molecule that has been a topic of research in B. subtilis and other microorganisms [ 12 – 16 ]. Pulcherrimin is synthesized by first, cyclization of two tRNA-charged leucines to form cyclo-L (leucine-leucine) (cLL) and second, oxidation to form water-soluble pulcherriminic acid ( Fig 1A ). Pulcherriminic acid is then transported outside of the cell where it can bind to free ferric iron to form the water-insoluble pulcherrimin ( Fig 1A ). While many microorganisms harbor the genes to produce pulcherrimin, the purpose of such a system to sequester iron is still unclear. Arnaouteli and coworkers found that pulcherrimin production contributed to growth arrest during B. subtilis biofilm formation through its ability to precipitate available extracellular iron [ 12 ]. Additionally, the ability of pulcherriminic acid to strongly sequester iron contributes to its anti-oxidative effects by limiting Fenton chemistry, providing evidence as an important antioxidant in cells [ 17 , 18 ].

Originally defined in the context of sporulation, TSRs are regulators that inhibit expression of genes involved in developmental processes but do not result in a sporulation null mutation when deleted [ 2 , 3 ]. Notably, mutants in TSRs are still able to carry out post-exponential phenotypes, however the magnitude and timing of these phenotypes are disrupted [ 4 ]. ScoC and AbrB represent two well-studied TSRs in B. subtilis. ScoC is a MarR-family winged helix-turn-helix transcription factor first identified in hyper-protease mutants [ 5 , 6 ]. Microarray analysis between WT and scoC mutants identified 560 genes with differential gene expression involved in motility and genetic competence as well as protease production and peptide transport [ 7 ]. The smaller AbrB (10.8 kDa) is part of a large family of transcription factors with a beta-alpha-beta DNA binding N-terminal domain [ 8 ]. Mutants of abrB have pleiotropic effects and some regulatory overlap with ScoC [ 9 , 10 ]. ChIP-seq analyses of AbrB identified many binding sites with a bipartite TGGNA motif [ 9 , 11 ].

In the soil bacterium Bacillus subtilis, complex arrays of gene networks function together to precisely time the expression of gene products. A prime example is the series of decisions made as cells transition from exponential growth to stationary phase upon nutrient limitation [ 1 ]. This phase, termed the transition state, is where cells in the population use environmental cues to inform the next course of action to survive in the new environment, specifically whether to engage in competence, biofilm formation, motility, secondary metabolism, and/or acquisition of nutrients [ 1 ]. While there are many transcription factors controlling these processes, an important set among these are called transition state regulators (TSRs).

Methods

Bacterial strains and culturing A derivative of the wild-type strain 3610 B. subtilis harboring an amino acid substitution in the competence inhibitor ComI (Q12I) was used as the background strain in these studies [19]. Gene replacements and deletions were constructed as described [20]. Gel purified gene-targeting antibiotic resistance cassettes and non-replicative plasmids (see Cloning for construction details) were used to transform B. subtilis by natural transformation. Briefly, single colonies of the strain of interest were used to inoculate 1 mL LB supplemented with 3 mM MgSO 4 and grown to mid-exponential phase while shaking at 230 RPM at 37°C. The cultures were then back diluted 1:50 into 2 mL MD media (1X PC buffer [10X PC—10.7 g K 2 HPO 4 , 6 g KH 2 PO 4 , 1.18 g trisodium citrate dehydrate, deionized water to 100 ml, filter sterilize], 2% glucose, 0.05 mg/mL tryptophan, 0.05 mg/mL phenylalanine, 0.01 mg/mL ferric ammonium citrate, 2.5 mg/ml potassium aspartate, 3 mM MgSO 4, water up to 2 mL) and grown for 3–5 hours, until early stationary phase. 10 μL of purified gene-targeting antibiotic resistance cassettes (~200–400 ng total) were added to 0.2 mL competent B. subtilis, were further incubated one hour, and plated on LB agar plates supplemented with either erythromycin (50 μg/mL), kanamycin (10 μg/mL), chloramphenicol (5 μg/mL), and/or spectinomycin (100 μg/mL). Antibiotic resistance clones were restruck on selection and insertions were verified by colony PCR using the US forward and DS reverse primers (See S1 Table). To remove the antibiotic resistance cassette, plasmid pDR224 was used to transform the appropriate strain with transformants selected for on LB supplemented with spectinomycin (100 μg/mL). Spectinomycin resistant clones were struck out on LB and incubated at the non-permissive temperature of 42°C; this process was repeated twice. Clones were then rescreened for sensitivity of spectinomycin and the absence of the integrated antibiotic resistance cassette using PCR.

Cloning To generate gene disruptions, oligos were designed to amplify upstream (US) and downstream (DS) of the gene of interest with appropriate overhangs to fuse either an erythromycin or kanamycin resistance cassette (AbR) flanked by CRE recombinase recognition sites [20]. Oligonucleotides were designed using NEBuilder (NEB) with the default parameters except minimum overlap length was changed from 20 nucleotides to 30 nucleotides. Q5 polymerase (NEB) was used to amplify the appropriate PCR amplicon. All amplicons were gel extracted prior to assembly reactions (Qiagen). US, DS, and AbR fragments were assembled by splice by overlap extension (SOE) PCR (adapted from [21]). First, 0.5 pmol each of the US, DS, and AbrB amplicons were mixed with 18 μL Q5 5X buffer, 0.25 mM dNTPs, and water up to 89 μL. 1 μL Q5 (2U) was added, and PCR was carried out with the following parameters: 1 cycle of 98°C– 10s, 10 cycles of 98°C– 10s, 55°C– 30s, 72°C– 2 minutes, 1 cycle of 72°C– 10 minutes. After completion of the PCR, 5 μL of US forward prime and 5 μL of the DS reverse primer were added and PCR was set under the following conditions: 1 cycle of 98°C– 2 minutes, 15 cycles of 98°C– 10s, 55°C– 30s, 72°C– 3 minutes, 1 cycle of 72°C– 10 minutes. Following PCR, spliced amplicons were analyzed on a gel and 10 μL used directly for transformation into competent B. subtilis. Vectors for protein purification (pNF039, pNF040, and pTMN007) and homologous recombination (pNF038) in B. subtilis were constructed using Gibson Assembly (NEB) following the manufacturer’s protocol. Protein expression vectors were used to transform E. coli DH5alpha and homologous recombination vectors were used to transform E. coli MC1061 and clones were verified via sanger sequencing (Azenta) or whole plasmid sequencing (Eurofins). All primers and assembly methods are included in S1 Table. To generate a pyvmC-GFP transcriptional fusion (pNF047), plasmid pYFP-STAR was amplified with the primer pair oNLF554-oNLF555 and gel extracted. The ORF for sfGFP was amplified from plasmid pDR110-GFP(Sp) using the primer pair oLVG035A-oLVG035B and gel extracted. The pyvmC locus was amplified in two fragments: 1) oNLF524-525 and 2) oNLF526-527. The resulting fragments were then assembled by Gibson Assembly (NEB), and used to transform MC1061 E. coli cells by heat shock. Transformants were then selected for by plating on LB Amp plates. The assembled plasmid consisted of the pyvmC promoter lacking 17 nucleotides upstream of the ATG start codon of yvmC, deleting the native ribosome binding site which can contribute to spurious translation and high GFP background [22].

Media WT and derived B. subtilis strains were either grown in lysogeny broth (LB) or Tris-Spizizen salts (TSS) [Reagents added in order: 50 mM Tris pH 7.5, 136 μM trisodium citrate dihydrate, water up to final volume, 2.5 mM dibasic potassium phosphate, 811 μM MgSO 4 , 1X FeCl 3 from a 100X stock solution [150 μM FeCl 3 , 0.1 g trisodium citrate dihydrate, 100 mL deionized water, filter sterilized], 0.5% glucose [25% stock solution, filter sterilized], and 0.2% ammonium chloride [20% stock solution, filter sterilized]. For liquid TSS, all components were mixed, filter sterilized, stored in the dark, and used within a week. For TSS agar plates, all reagents, except the 1X FeCl 3 solution, glucose, and ammonium chloride, were mixed with agar at 1.5% w/v and autoclaved. After the agar solution cooled to approximately 55°C, filter sterilized FeCl 3 , glucose, and ammonium chloride were added, approximately 20–25 mL were added to sterile petri plates, and plates were allowed to dry overnight. TSS agar plates were stored at 4°C and were used within 6 months.

Spot plating and liquid culture imaging One day prior to the spotting, TSS plates with varying concentrations of FeCl 3 (final concentrations: 0.15, 1.5, 15, and 150 μM FeCl 3 ) were poured and dried overnight at room temperature. Spots for Fig 1 were on TSS plates supplemented with 150 μM FeCl 3 . Strains were inoculated from frozen stocks into 1 mL TSS media and grown overnight at 37°C while shaking at 250 RPM. The next day, the turbidity of each culture was measured and adjusted to an OD 600 of 1.0 in fresh TSS media. 10 μL of each culture were spotted 15 mm apart on the same TSS plate and incubated for 24hr at 30°C. The next day, plates were imaged using an imaging box [23] and an iPhone 7 running iOS 15.7.5. Images were cropped and arranged using Adobe Photoshop and Illustrator. Each experiment included two technical spotting replicates and was repeated at least twice on separate days. For liquid cultures, 2 mL overnights were started from frozen stocks in TSS media and grown overnight at 37°C while shaking at 250 RPM. Overnight cultures were diluted to a starting OD 600 of 0.05 in 40 mL TSS in 125 mL flasks and grown for 20–24 hours at 37°C while shaking at 250 RPM. Images of the flasks were taken as stated above.

LC/MS for cyclo-dileucine measurement WT and yvmC::erm were struck out onto TSS plates from frozen stocks and grown overnight at 37°C for 16 hours. The next day, the strains were washed from the plate into fresh TSS and the resulting culture was used to inoculate 16 mL TSS in 50 mL flask at a starting OD 600 of 0.050. Cultures were grown for 6 hours while shaking at 250 RPM at 37°C. After six hours (OD 600 ~ 1.0), 15 mL of culture was collected by centrifugation in a 15-mL falcon tube (3 minutes, 4200xg) and the resulting pellets were resuspended in 200 μL cold extraction buffer (acetonitrile:methanol:water, 40:40:20, [24]). The resulting cellular mixture was centrifuged in a microcentrifuge (30s, 15,000 x g) and the supernatant containing the extracted metabolites were moved to a new 1.5 mL microcentrifuge tube and frozen at -80C. Samples were sent to the Michigan State University Research Technology Support Facility for LC-MS analysis of cyclo-dileucine. Two hundred microliters of the extract were evaporated to dryness using a SpeedVac and resuspended with an equal volume of methanol: water, 1:9 (v/v). Ten microliters of the sample was injected onto an Acquity Premier HSS T3 column (1.8 μm, 2.1 x 100 mm, Waters, Milford, MA) and separated using a 10 min gradient as follows: 0 to 1 min were 100% mobile phase A (0.1% formic acid in water) and 0% mobile phase B (acetonitrile); linear ramp to 99% B at 6 min, hold at 99% B until 8 min, return to 0% B at 8.01 min and hold at 0% B until 10 min. The column was held at 40°C and the flow rate was 0.3 mL/min. The mass spectrometer (Xevo G2-XS QToF, Waters, Milford, MA) was equipped with an electrospray ionization source and operated in positive-ion and sensitivity mode. Source parameters were as follows: capillary voltage 3000 V, cone voltage 30V, desolvation temperature 350°C, source temperature 100°C, cone gas flow 40 L/hr, and desolvation gas flow 600 L/Hr. Mass spectrum acquisition was performed in positive ion mode with a range of m/z 50 to 1500 with the target enhancement option tuned for m/z 227. A calibration curve was made using cyclo-dileucine standard (Santa Cruz Biotechnology). The peak area for cyclo-dileucine was integrated based on the extracted ion chromatogram of m/z 227.18 with an absolute window of 0.05 Da. Peak processing was performed using the Targetlynx tool in the Waters Masslynx software.

Pulcherrimin isolation and measurement Strains of interest were struck out on TSS agar plates from frozen stocks and incubated overnight (16–20 hours) at 37°C. The next day, cells were collected by adding 1 mL fresh TSS media to the plates and gently swirled to remove bacteria adhered to the agar. The resulting bacterial suspension was then moved to microcentrifuge tubes, the OD 600 recorded, and diluted to a starting OD 600 of 0.05 in 40 mL TSS media in 125 mL round bottom flasks. At the time indicated, 1.5 mL of culture was aliquoted into a microcentrifuge tube. 0.1 mL were used for OD 600 measurement while the remaining cells were collected by centrifugation (10,000 x g, 30s). The supernatant was removed and the cell pellet and insoluble pulcherrimin were resuspended in 0.1 mL 2M NaOH by pipetting the solution until completely resuspended. The samples were then centrifuged again (10,000 x g, 1 minute) and the supernatant were moved to clean wells in a 96-well plate and absorbance at 410 nm was measured using a Tecan M200 plate reader. To determine the time of entry into stationary phase, the R package growthrates was used to fit a linear growth model for every strain and replicate [25]. The time in which the growth data deviated from exponential growth was used as time 0 for Fig 2. The grow_gompertz3 function was used to model the change in absorbance at 410 nm over time using the growthrates package [25]. Each strain and replicate (n = 3) were modeled individually and the resulting parameters (maximum growth rate [mumax] and carrying capacity (abs. 410 nm) [K]) were summarized by taking the average and standard deviation plotted in Fig 1B. For the production start time, curves were analyzed manually to determine when the predicted A 410 from the mutant strain deviated from the predicted A 410 value from the WT background during exponential phase. For the duration of pulcherrimin production, the x-axis distance between the beginning of the exponential phase of pulcherrimin production and the start of the stationary phase of pulcherrimin production were measured manually. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Pulcherrimin production in liquid (top) and solid (bottom) TSS media. WT (DK1042) and isogenic mutants were grown in liquid TSS media or spotted (10 μL) onto solid TSS media and grown overnight at 30°C. The black scale marker corresponds to 5 mm. https://doi.org/10.1371/journal.pgen.1011283.g002

Fluorescence reporter assay WT and isogenic mutants harboring the pyvmC-GFP transcriptional fusion at the amyE locus were struck out on TSS agar plates and grown overnight for ~ 16 hours at 37°C. The next day, the strains were plate washed into 1 mL TSS media and the OD 600 was recorded. 40 mL TSS in 125 mL Erlenmeyer flasks were inoculated with the plate washed cells at a starting OD 600 of 0.050. The cultures were incubated at 37°C while shaking at 250 RPM until the cultures reached mid-exponential phase. Fluorescence was measured from a 1 mL sample using an Attune NxT Acoustic Focusing Cytometer (ThermoFisher Scientific) using the following settings: Flow rate, 25 μl/min; FSC voltage, 200; SSC voltage, 250; BL1 voltage, 250 [26]. A WT strain not harboring GFP was used as a control to assess background fluorescence (grey bars in Fig 3A). Percent GFP positive was calculated by dividing the number of fluorescent events with a signal above the maximum fluorescence of the negative control (No GFP) by the total number of fluorescent events of a given strain and multiplying by 100. This was repeated for each replicate for each strain and the mean +/- standard deviation of the percent positive values are provide to the right of each panel in Fig 3A. PPT PowerPoint slide

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TIFF original image Download: Fig 3. ScoC, ArbB, and PchR Control Timing and Rate of Pulcherrimin Production. A) Pulcherrimin production was measured as a function of growth phase, where T0 marks the transition from the exponential growth to stationary phase. Each panel represents the average A 410 for a given strain compared to the A 410 from WT (white squares). Error bars represent +/- the standard deviation. Lines running through the points are modeled using the drm function from the drc package in R (see Methods and Materials). B) Pulcherrimin production parameters as a function of genetic background: i) start of pulcherrimin production time relative to the transition phase of growth (T0), ii) the duration of pulcherrimin production, iii) the maximum estimated production rate, and iv) the maximum absorbance at 410 nm. For panels i-iii, brackets and asterisks indicate significant comparisons. For panel iv, brackets and “ns” indicate non-significant comparisons, where every other comparison had an adjusted p-value less than 0.05 as determined by T-test corrected for multiple comparisons with the Bonferroni correction. https://doi.org/10.1371/journal.pgen.1011283.g003

Protein purification ScoC purification was carried out as previously described with some minor amendments [26]. Plasmid pTMN007 harboring ScoC in a T7 expression vector pE-SUMO was used to transform BL21-DE3 E. coli and plated on LB supplemented with kanamycin (25 μg/mL). The next day, a single colony was inoculated into 1 mL LB Kan 25 , grown to mid-exponential phase at 37°C while shaking at 230 RPM, diluted 1:5 in 5 mL LB Kan 25 , and grown overnight at 37°C while shaking at 37°C. After overnight growth, the culture was diluted to a starting OD 600 of 0.05 in 400 mL LB Kan 25 at 37°C shaking at 230 RPM and grown until the OD 600 reached between 0.7, after which 1 mM final concentration of Isopropyl ß-D-1-thiogalactopyranoside (IPTG) was added to induce protein expression for three hours at 37°C. After induction, cells were collected by centrifugation (5 minutes at 7,500 RPM using SLA-1500 rotor in a Sorvall RC 5B plus centrifuge) and cell pellets were stored at -20°C until use. Cell pellets were thawed at room temperature and resuspended in 40 mL lysis buffer (50 mM Tris pH 8, 300 mM NaCl, 10% sucrose, 10 mM imidazole, and 1 EDTA-free protease inhibitor tablet added the day of purification (Roche)). The cell solution was moved to a beaker in an ice-water bath and sonicated (15s ON, 25s OFF, 24 cycles, 50% amplitude, Fisher Scientific Model 505 Sonic Dismembrator). The lysate was cleared by centrifugation (10 minutes, 12,000 RPM, using an SS-34 rotor in a Sorvall RC 5B plus centrifuge). The clarified lysate was loaded onto a 3 mL Ni-NTA column pre-equilibrated with lysis buffer and the flow through was discarded. The column was washed three times with 20 mL wash buffer (50 mM Tris pH 8, 2M NaCl, 25 mM imidazole, and 5% glycerol) and ScoC-SUMO-His was eluted with 15 mL elution buffer (50 mM Tris pH 8, 150 mM NaCl, 200 mM imidazole). The protein solution was dialyzed at 4°C into dialysis buffer (50 mM Tris pH 8, 150 mM NaCl, 5% glycerol). The next day, DTT (1 mM) and SUMO Ulp1 protease were added, the solution was incubated at room temperature for 2 hours and dialyzed into dialysis buffer overnight at 4°C. Following dialysis, SUMO-free ScoC was purified by loading the solution onto 3 mL of pre-equilibrated Ni-NTA resin by collecting the flow through. SUMO-free ScoC fractions were determined via SDS-PAGE, pooled, quantified by the Bradford assay, diluted with glycerol for a final concentration of 25%, and stored at -80°C. Expression vectors for PchR and AbrB were constructed similar to ScoC. Growth of BL21 E. coli harboring PchR was identical to ScoC. Growth of BL21 E. coli harboring AbrB had the following changes. First, 1 mL LB Kan was inoculated with a single colony of E. coli harboring the AbrB expression vector and grown for 6 hours at 37°C, shaking at 200 RPM. The 1 mL culture was diluted 1:10 in 9 mL LB Kan in a 125 mL flask and grown overnight at 37°C while shaking at 200 RPM. The next day, the culture was used to inoculate 400 mL of LB Kan at a starting OD 600 of 0.1 and grown at 30°C until the OD 600 reached between 0.6 and 0.7, at which point IPTG was added at a final concentration of 1 mM and the culture was moved to 16°C with shaking at 160 RPM for 16 hours. For purification, slight modifications to the lysis buffer and elution buffers were made. Frozen pellets of PchR-SUMO and AbrB-SUMO were resuspended in 40 mL lysis buffer (50 mM Tris pH 8, 500 mM NaCl, 10% glycerol, 20 mM imidazole, supplemented with 1 EDTA-free protease inhibitor tablet), lysed by sonication, and the lysate cleared by centrifugation. Clarified lysate was applied to 3 mL Ni-NTA resin columns, the columns were washed with 60 mL lysis buffer, and proteins were eluted by step elution using 5 mL each of increasing imidazole concentrations (elution buffer: 50 mM Tris pH 8, 500 mM NaCl, 10% glycerol, imidazole at 50, 100, 200, and 350 mM). Elution fractions were assayed for relative protein concentration by the Bradford assay (BioRad) and fractions containing protein were electrophoresed on SDS-PAGE to ensure proper expression and purification. Removal of the SUMO tag and purification of SUMO-free protein was carried out as described above. SUMO-free AbrB required an additional anion exchange purification step using a HiTrap qFF (Cytivia 17515601) anion exchange column attached to an AKTA FPLC. The column was equilibrated with 10% Q Buffer B (50 mM Tris, 5% glycerol, and 500 mM NaCl). Sumo-free AbrB was diluted to 50 mM NaCl in Q Buffer A and loaded into the column. Protein fractions (2 mL) were collected as the system increased the percentage of Q Buffer B while monitoring A260 readings. High A260 peaks were measured for AbrB on SDS-PAGE and correct fractions were pooled, dialyzed, concentrated by dialysis, mixed with glycerol at 25% final concentration, and stored at -80°C.

Electrophoretic mobility shift assays 5′ IRDye 700-labeled probes of the yvmC promoter (-244 to +9 relative to the ATG start codon) were generated by PCR using the primer pair oNLF433-oNLF387 using pNF035 as a template. PCR products were purified by gel extract and quantified by nanodrop. To generate the PyvmCΔ59 probe, two PCR reactions were carried out with primer pairs oLVG025A-oNLF467 and oNLF468-oNLF387. The two PCR products were gel extracted and fused together by SOE PCR (see Cloning). The fragment was then used as a template for PCR with primer pairs oNLF433-oNLF387, resulting in a 5′ IRDye 700 labeled DNA fragment lacking 59-bp. Binding reactions were assembled by first generating a binding solution: 1X binding buffer (5X binding buffer: 250 mM Tris pH 8, 5 mM EDTA, 150 mM KCl, 10 mM MgCl 2 , 12.5 mM DTT, 1.25% Tween 20, and 2.5 mg/mL BSA), 1X DNA probe (10X probe, 100 nM), 1 μL protein of interest (5X stock concentration), and water up to 5 μL. The binding reactions were incubated for 30 minutes at 37°C. When indicated, 1 μL of 1X heparin (6X heparin: 0.06 mg/mL) was added to each reaction after incubation and 3 μL were loaded into the wells of a 15-well 6% polyacrylamide gel and electrophoresed for 60 minutes at 150V at room temperature. After gel electrophoresis, the gels were left in the glass plates and imaged using an Odyseey xCl imager (1.5 mm offset height, 84 μm resolution). The resulting images were adjusted in Fiji [27] and cropped and annotated in Adobe Illustrator. EMSA experiments were carried out at least three times with separate aliquots for each protein.

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