Bacterial strains and plasmids used in this study are listed in Table 1 . S. mutans strains were maintained in brain heart infusion (BHI) medium. For biofilm formation, S. mutans was grown in modified biofilm medium (BM) with glucose (20 mM, BMG), sucrose (20 mM, BMS), or glucose (18 mM) plus sucrose (2 mM) as supplemental carbon and energy sources (BMGS) (Bitoun et al., 2012c). All solid media were prepared similarly with inclusion of Bacto agar (Difco Laboratories, Franklin Lakes, NJ) at the level of 1.5% (w/v). When needed, erythromycin (Erm, 10 µg/ml), kanamycin (Kan, 1 mg/ml), and/or spectinomycin (Spc, 1 mg/ml) were added. Unless otherwise stated, cells were grown at 37°C in an aerobic environment with 5% CO₂. All E. coli strains were grown in Luria Bertani medium at 37°C aerobically, with or without inclusion of kanamycin (40 µg/ml), ampicillin (100 µg/ml), spectinomycin (100 µg/ml), and/or erythromycin (300 µg/ml).

Standard recombinant DNA procedures were used (Lau et al., 2002; Bitoun et al., 2013). All restriction and modifying enzymes were purchased from Invitrogen (Carlsbad, CA) or New England Biolabs (Ipswich, MA) and used as recommended by the suppliers. All primers ( Supplementary Table 1 ) were synthesized by Integrated DNA Technologies, Inc. (Iowa City, IA). A perR deletional mutant was constructed using our well-established PCR-Ligation-Mutation protocol (Burne et al., 2011). Briefly, the 5’ and 3’ region of perR were amplified using high fidelity DNA polymerase Q5 (NEB Bioloabs) using gene-specific primers ( Supplementary Table 1 ), and following proper restriction enzyme digestions, the flanking fragments were ligated with a non-polar spectinomycin resistant marker. Then, the ligation mix was used to transform S. mutans with inclusion of the competence stimulating peptide, and mutants lacking perR was isolated from plates with spectinomycin. Isolated mutants were verified by PCR and Sanger sequencing with gene-specific primers ( Supplementary Table 1 ). S. mutans strains with deletion of dpr, sloR, and cysR were generated using similar strategies.

The transcription start sites were determined using the Template Switching RT Enzyme Mix (NEB #M0466) by following the 5’ RACE protocol (NEBLABS, Ipswich, MA). For total RNA, wild-type S. mutans UA159 cells were harvested at middle exponential phase (OD₆₀₀ ≈ 0.3) and immediately treated with RNAProtect^® (Qiagen, Inc). Total RNAs were extracted using the hot phenol method and purified using RNAeasy kit (Qiagen, Inc.), and DNase I treatment including an in-Column DNase I treatment was used to clean up the residual genomic DNA. 1 µg total RNA was used in the template switching reverse transcription, and the resulting cDNA with inclusion of a universal sequence attached to its 3’ were amplified by PCR using high fidelity DNA polymerase Q5, and the transcription initiation site was determined by Sanger sequencing.

To analyze the regulation of suf expression, a promoterless luciferase gene (luc) was used as a reporter as previously described (Kreth et al., 2005). Briefly, the cognate suf promoter region and its derivatives generated by PCR-based deletions and/or site-directed mutations were amplified using high fidelity DNA polymerase Q5 (NEB Biolabs), and following proper restriction digestions, cloned directly in front of the promoterless luc gene in integration vector pFW11-luc (Kreth et al., 2005), which also contains a Shine-Dalgarno sequence optimized for group A streptococci (Podbielski et al., 1999). Following confirmation of the cloned element by sequencing, the resulting constructs were introduced into S. mutans UA159 and its selected derivatives such as the perR deletion mutant, ΔperR, and the expression of suf were analyzed using luciferase assay by following the protocol of Podbielski (Kreth et al., 2005; Bitoun et al., 2012a, 2012). For controls, pFW11 with promoterless reporter and a luciferase reporter fused with the gyrase promoter (Pgyr) were used (Ellepola et al., 2021).

For expression and purification of recombinant PerR (rPerR), recombinant CysR (SMU.852), and recombinant Sigma factor (rSigA), the respective coding regions were amplified by PCR with gene-specific primers ( Supplementary Table 1 ) using high fidelity DNA polymerase Q5 (NEB Biolabs), and cloned in pQE30 (Qiagen, Inc.). Following Sanger sequencing to confirm accuracy of the cloned DNA, the resulting constructs were transformed into E. coli M15, and PerR, CysR and SigA expression were induced by addition of IPTG at 0.5 mM (final conc.), and the His-tagged rPerR, rCysR and rSigA were purified using nickel resins (Fisher Scientific) under native conditions similarly as described previously (Bitoun et al., 2012c). The purified proteins were then applied to Amicon Ultra 3 Centrifugal Filters (3 kDa MWCO, Millipore) for desalting and re-constituted in 20 mM Tris, pH 7.6 plus 20% glycerol. For PerR, 10 mM EDTA was also included in the elution buffer, desalting and exchange buffers (Lee and Helmann, 2006).

For in vitro transcription assays, crude native S. mutans RNA Polymerase (RNAP) was obtained based upon the methods described by Seepersaud et al. (Seepersaud et al., 2006) and Galvão et al. (Galvao et al., 2015). Briefly, S. mutans cells of mid-exponential phase were harvested by centrifugation, resuspended and incubated in protoplast preparation buffer at 37°C for 90 minutes. Protoplasts were then pelleted and resuspended in lysis buffer, lysed by glass bead beating and, the supernatants were applied to Affi-Gel heparin resin (Bio-Rad) and eluted with a NaCl gradient of 0.1 to 1 M. Fractions containing visible subunits were pooled and applied to Macro-Prep High-Q ion exchange resin (Bio-Rad) and eluted with a gradient of 0.1–0.8 M NaCl. The desired fractions were again pooled and dialyzed.

To assess if and how transcriptional factor PerR interacts with the sufCDSUB promoter, EMSA was employed similarly as described previously (Bitoun et al., 2012a). Briefly, the intact promoter region and its derivatives with deletions and/or site-directed mutations were amplified by PCR using Q5 DNA polymerase with the primers listed in Supplementary Table 1 . The PCR products, with biotinylation at the N-terminus or without (for cold-competition control), were incubated with increasing amounts of rPerR in the reaction buffer at room temperature (Bitoun et al., 2012a). The reaction mixtures were separated on a 5% polyacrylamide gel under native conditions, and DNA mobility shift was visualized via LightShift Chemiluminescent EMSA Kit by following procedures recommended by the manufacturer (Pierce, Rockford, IL) or by SYBR Gold staining (Invitrogen, CA) and a GelDoc Go Imaging system (BioRad).

In vitro transcription assay was performed by following the protocols of Dou et al (Dou et al., 2021) and Galvao et al (Galvao et al., 2015) using native S. mutans RNAP and rSigA. In some experiments, E. coli RNA polymerase holoenzyme (M0251S, NEB Biolabs) (2 units) was also used. Briefly, a DNA fragment carrying the sufCDSUB promoter plus 834 bp of the coding sequence was amplified by Q5 DNA polymerase and purified using Qiagen PCR purification kit (Qiagen, Inc.). For IVT, ~100 fM of the DNA template, 25 nM of S. mutans RNAP, and 25 nM of rSigA were incubated with and without rPerR or rCysR at different µM concentrations in buffer containing 20 mM Tris-HCl (pH 7.5), 50 mM KCl, 5 mM MgCl₂, 0.1 mM DTT, 0.01% Triton X-100, 5 mM of NTP mix at 37°C for 1 hour. The resulting transcripts were analyzed using 1.4% denaturing agarose gel and visualized by SYBR Gold staining (InVitrogen, CA) similarly as described above.

Proteomics analysis was carried out similarly as described previously (Wen et al., 2006; Yue and Guidry, 2019). Briefly, cultures were grown in regular BHI until mid-exponential phase (OD_600nm~0.4) and homologized in Tris-HCl buffer, 50 mM pH 7.4 containing 10% glycerol using a glass bead beater as described (Wen et al., 2006). Following protein concentration estimation by BCA assay (Pierce, ThermoFisher), 100 µg of each protein sample was digested with trypsin, and labeled using a Tandem Mass Tag (TMT) 10-plex Reagent set (Pierce, ThermoFisher). HPLC-MS/MS analysis was done in four technical replicates with a false discovery rate (FDR) of < 1% at the LSUHSC Proteomics Core by following established protocols.

At least three biological and two technical replicates each were used for all the experiments. Quantitative data were analyzed using paired Student t test. A P value of 0.05 or less is considered statistically significant.

First, rapid amplification of cDNA ends (5’RACE) was employed to determine the transcription initiation site of sufCDSUB, and it was done using total RNA isolated from mid-exponential phase cultures grown in regular BHI broth. The results showed that under these conditions, the transcription initiation site of the sufCDSUB operon was consistently mapped at nucleotide 22 from the translation initiation site ATG ( Figure 1A ). The promoter region was then subjected to virtual footprinting analysis (https://www.prodoric.de/vfp/), and multiple regions were identified with potential roles in sufCDSUB regulation, which include two regions, termed Fur-box1 and Fur-box2 with high similarity to the binding site of Fur (consensus: GGCAATAGTGATTACCGTC) and two regions with similarity to the PerR-binding site (consensus: TTATAATnATTATAA) ( Figure 1B ). To further examine the role of these respective regions in sufCDSUB expression, PCR-based mutations and deletions of the sufCDSUB promoter were made using primers listed in Supplementary Table 1 , and the resulting constructs were directly cloned in front of the promoterless luciferase reporter in pFW11 (Kreth et al., 2005; Bitoun et al., 2012a). As shown in Figure 1C , mutations of the PerR-box1 from TTGGACTTTTTTTGTT to aaGatCaTTTTTTGTT (low case indicated mutated nucleotides) in PerRm resulted in elevation of the reporter activity by 1.65-fold (P<0.05), compared to the intact promoter-reporter fusion Psuf, indicative of a role as a potential binding site of a transcriptional repressor. Site-directed mutation of Fur-box 2 (AGAAGAAAAAGTAATTTTT, Box2m), where nt GAAAAAGT were changed to ctttttgt, showed no changes in the luciferase activity compared to the intact promoter-reporter fusion. Interestingly, deletion of AGAAAAAG in Fur-box 2 (dBox2, shown in red-box) led to >4.23-fold reduction of the reporter activity, while no significant effect was observed with the deletion of Fur-box1 (dBox1) ( Figure 1C ). The results suggest that Fur-box2 is likely a binding site for a positive regulator.

S. mutans possesses a PerR (SMU.593) (Kajfasz et al., 2021), the sole Fur family of metal ion-dependent transcriptional regulator that modulates the expression of target genes by sensing and responding to oxidative stress (Bsat et al., 1998). To assess the role of PerR in sufCDSUB regulation, an allelic exchange mutant of perR was constructed with a major portion of the coding region replaced by a non-polar kanamycin resistance marker (Burne et al., 2011). As expected, deletion of perR led to de-repression of sufCDSUB expression, especially when grown in Fe-rich biofilm medium. Relative to the wild-type, the perR deletion mutant displayed an increase of >2-fold in luciferase reporter activity when the cultures were grown in regular BHI (P<0.05) ( Figure 2 ). Such differences were increased to as much as 10-fold compared to the wild-type, when the reporter strains were transferred from BHI to semi-defined biofilm medium. Consistent with our recent studies (Ellepola et al., 2021), luciferase reporter activity of the wild-type UA159 was increased by >5.3(±1.1)-fold when the BHI-grown overnight cultures were transferred to semi-defined biofilm medium with glucose (BMG), especially when FeCl₃ was omitted from the medium recipe (BMG-Fe) ( Figure 2 ). The highest reporter activity of the wild-type was measured when the cultures were grown in BMG medium with inclusion of 2,2’-pyridyl (ACROS Organics, #366-18-7), an iron chelating agent at 50 µM. However, no such effects were measured when overnight cultures were also grown in BMG or BMG-Fe. Like the wild-type, the luciferase reporter activity of the perR mutant was also increased when it was transferred from regular BHI to BMG. Compared to the wild-type, the reporter activity of the perR mutant was increased by >10-fold of the wild-type (P<0.001). However, unlike the wild-type, no significant differences in luciferase reporter activities were measured between the perR mutant when grown in BMG and those in BMG-Fe and BMG plus 2,2’-pyridyl at 50 µM ( Figure 2 ). These results further suggest PerR plays a major role as a repressor in sufCDSUB expression in response to Fe-availability in the culture medium, and consistent with other Fur regulators, it requires Fe as a co-factor.

To further examine if PerR interacts with the sufCDSUB promoter influencing its transcription, PerR was expressed in E. coli by isopropyl β-d-1-thiogalactopyranoside (IPTG) induction and purified using Ni²⁺ affinity columns, and EMSA assays were conducted using the purified rPerR. Results showed that inclusion of rPerR in the reaction mix resulted in mobility shift of the sufCDSUB promoter probe, and such activity was concentration dependent ( Figure 3 ). The mobility shift was negated or reduced significantly when rPerR protein was first incubated with non-biotinylated promoter oligo. Reduced mobility shift was also observed when whole cell lysates of the wild-type S. mutans UA159 were used in the reaction. Interestingly, no major differences were observed in mobility shift when rPerR was mixed with oligo PerRm, which has the PerR-box1 mutated from TTGGACTTTTTTTGTT to aaGatCaTTTTTTGTT using PCR-based site-directed mutagenesis (see Figure 1 ). These results further suggest that PerR interacts with the sufCDSUB promoter, although the exact nucleotides that interact with PerR repressor remain unclear. In an IVT assay using native S. mutans RNA polymerase (RNAP) and recombinant Sigma factor 70 (rRpoD) with the whole promoter plus 834 bp of the coding sequence of sufC, a transcript of expected size was detected. However, inclusion of rPerR in the reaction mix only slightly reduced the amount of the transcripts, but no such effects were observed when recombinant LuxS protein or none was added as controls.

A proteomics approach was also used to examine the protein profile of the perR mutant, and the results showed that when compared to the wild-type UA159, the expression of more than 35 proteins were altered as a result of perR deletion, including 17 up-regulated proteins and 18 down regulated ( Supplementary Table 2 ). Among the up-regulated proteins were those with roles in oxidative stress tolerance responses including Dpr, thiol peroxidase Tpx, and putative metal transporter SMU.635, consistent with the recent RNA-seq studies by Kajfasz et al (Kajfasz et al., 2021). Interestingly, several enzymes of sugar transport and metabolism pathways were also found to be altered significantly, including down-expression of β-exo-fructanase FruA, a well-known virulence attributor, and up-expression of sucrose- and mannose-specific transporters ( Supplementary Table 2 ). When whole cell lysates were separated by SDS-PAGE and probed in a Western blotting using polyclonal antibodies (a kind gift of Dr. RA Burne), the results further confirmed the reduction of FruA expression in the perR mutant as well as the sufCDSUB mutant ( Figure 4 ), compared to the parent strain, UA159. However, it awaits further investigation if the expression of these genes is directly regulated by PerR.

Our previous study showed that expression of the sufCDSUB operon is regulated in response to oxidative stressors (Ellepola et al., 2021). Relative to cultures grown in regular BHI, the luciferase reporter activity was increased by >2-fold in cultures growing in the presence of methyl viologen (at 2.5 mM) ( Figure 5 ), a molecule that induces superoxide production inside the cells. Like the wild-type, luciferase reporter activity of the perR mutant was also increased following exposure to methyl viologen at 2.5 mM, but unlike growing in regular BHI, the differences between the wild-type and the perR mutant under the condition with 2.5 mM methyl viologen were not statistically significant ( Figure 5 ). These results suggest that factors other than PerR are likely involved in the oxidative stress mediated regulation of sufCDSUB.

Dpr is known to as a major factor in resistance to reactive oxygen species. To test if Dpr mediated oxidative stress tolerance response influences sufCDSUB expression, an allelic exchange dpr mutant was constructed, and the impact of dpr deletion on sufCDSUB expression was analyzed using luciferase reporter fusion assays similarly as described above. Consistent with Ganguly et al (Ganguly et al., 2018), dpr deletion led to major reductions in growth rate and viability of the deficient mutant under aerobic conditions due to compromises in oxidative stress tolerance. Consistently, the luciferase reporter activity in the dpr mutant was increased, as compared to the wild-type, when the reporter strains were grown in an anaerobic box ( Figure 5 ). However, no such effects were observed when methyl viologen at 2.5 mM was included in the growth medium especially under aerobic conditions, which again indicates a compromised oxidative stress tolerance response of the dpr mutant. Similar trends were also observed with the gyrase A promoter fusion as a reference. These results further suggest that oxidative stressors strongly influence the expression of sufCDSUB, and that Dpr-deficiency induces oxidative stresses of the deficient mutant, indirectly affecting sufCDSUB expression.

S. mutans possesses two Spx homologues, SpxA1 and SpxA2, which serve as the activator of genes involved in oxidative stress response (Galvao et al., 2015; Kajfasz et al., 2015). By RNA-seq analysis, Kajfasz et al (Kajfasz et al., 2017) found that members of the sufCDSUB operon were up-regulated in the wild-type following exposure to sub-lethal amount of hydrogen peroxide, but they were significantly reduced in spxA1 mutant and more so in a spxA1/spxA2 double mutant, compared to the wild-type, UA159. To further investigate how the SpxA regulators influence the expression of the SUF machinery, the suf promoter-reporter fusion construct was transformed into the allelic exchange spxA1 and spxA2 mutants, and the impact of spxA deletion on sufCDSUB expression was analyzed using luciferase reporter assays with the gyrase gyrA promoter PgyrA-reporter fusion serving as a reference. As compared to the wild-type, the luciferase reporter activities under the direction of Psuf promoter were reduced by >2.8-fold (P<0.01) ( Figure 6 ) in the spxA2 mutant, which in part can also be attributed to the fact that the spxA2 mutants grew more slowly as compared to the wild-type, UA159 (Galvao et al., 2015). No significant differences were observed between the spxA1 mutant and the wild-type, UA159. The reference PgyrA-reporter fusion showed no significant change in expression under the conditions tested.

SloR is known as a metal-dependent repressor of sloABC when Fe is plentiful in the growth environment (Rolerson et al., 2006). SloR also facilitates its own expression as a positive autoregulator (Monette et al., 2018). To investigate potential roles of SloR in sufCDSUB expression, an allelic exchange sloR (SMU.186) mutant was constructed and further analyzed, along with a perR/sloR double mutant, using luciferase reporter assays as described above. Relative to the parent strain, UA159 and the perR mutant, the sloR mutant displayed some aberrant growth phenotypes including extended lag phase (>24 hours) and aggregations when growing in regular BMG medium. However, both the sloR single and sloR/perR double mutant grew similarly well as the wild-type in BMG medium with omission of iron in the recipe (BMG-Fe) or BMG with inclusion of chelating agent 2,2’-pyridyl at 50 µM. When analyzed with the full promoter-reporter fusion construct (Psuf), luciferase reporter activity in the sloR mutant was reduced by >2-fold (P<0.01) when the strains were grown in BMG medium, especially in BMG with exclusion of Fe (BMG-Fe), as compared to the parent strain under the same conditions ( Figure 7 ). Similar results were measured with the sloR/perR double mutant when grown in BMG, although no significant differences were measured when the double mutant was grown in BMG-Fe or BMG plus chelating agent ( Figure 7 ).

To examine if SloR directly interacts with the promoter, regulating sufCDSUB expression, recombinant SloR (rSloR) was used in an EMSA assay with rSloR-sloA promoter interaction as a positive control (Spatafora et al., 2015). As expected, the inclusion of rSloR led to mobility shift of sloA probe. However, no apparent effect was observed when rSloR was mixed with the sufCDSUB probe on its gel mobility under the conditions studied ( Supplementary Figure 1 ). Similar results were observed when S. mutans whole cell lysates were used in the EMSA assay. These results suggest that SloR is not directly involved in regulation of sufCDSUB expression, although it is also possible that other unknown factor(s) are involved in the SloR-mediated regulation, and in the absence of its co-factor, SloR will not bind to its promoter DNA in EMSA.

To test if cysteine also plays a direct role in SUF expression, the modified semi-defined biofilm medium with the omission of cysteine in the amino acid mix was used to grow the luciferase reporter strains, and the reporter activities were measured with and without addition of cysteine one hour prior to the luciferase assay. The results showed that reporter activity without cysteine was at its base line, while addition of cysteine at 4 mM was found to induce the reporter activity by 84.6 (± 7.4)-fold, compared to the ones receiving solvent control (P<0.001) ( Figure 8 ). Addition of serine at 4 mM also led to increases of reporter activity, but to a much less degree with an average of 3.63(± 0.43)-fold (P<0.05). SMU.852, also cysR (Sperandio et al., 2010), encodes a LysR-family transcriptional regulator, which we have recently identified in a study of the regulation of brpA expression. To analyze if CysR plays a role in sufCDSUB expression, an allelic exchange mutant, TW660, was constructed, and relative to its parent strain, UA159, the cysR mutant displayed a reduced growth rate and optical density overnight when grown in regular BHI broth ( Supplementary Figure 2 ). When transformed with the Psuf promoter luciferase reporter fusion, the reporter activity in the cysR mutant was shown to be reduced by >11.06-fold, compared to the parent strain (P<0.001) ( Figure 8 ). However, the reporter activity of the cysR mutant was still inducible with the addition of cysteine, when grown in the modified BM medium similarly as the wild-type, albeit to a reduced degree, which in part could be attributed to the reduced growth rate of the cysR mutant ( Supplementary Figure 2 ). These results suggest that expression of sufCDSUB requires cysteine as an inducer, and CysR is a positive regulator of sufCDSUB expression.

When analyzed, two regions with some similarities to the consensus of the LysR-type of regulators (https://regprecise.lbl.gov/sites.jsp?regulog_id=4369) were identified in the promoter region with one overlapping the -10 and the perR-box2 region and the other overlapping the Fur-Box1 site ( Supplementary Figure 3 ). To test if CysR interacts with the sufCDSUB promoter, CysR was expressed and purified using affinity chromatography, and as shown in Figure 9 , inclusion of the recombinant CysR protein (rCysR) was shown to cause mobility shift of the promoter probes, indicative of rCysR-promoter interaction. Consistent with the luciferase reporter fusion assays, rCysR was also shown in an IVT assay to enhance the transcription of sufC under the direction of sufCDSUB promoter ( Figure 9 ). These results further support the role of CysR as a positive regulator of sufCDSUB in S. mutans.