Roles of the Crp/Fnr Family Regulator ArcR in the Hemolysis and Biofilm of Staphylococcus aureus

Staphylococcus aureus is an opportunistic human pathogen that is often involved in severe infections such as pneumonia and sepsis in which bacterial virulence factors play a key role. Infections caused by S. aureus are often difficult to eradicate, particularly when they are associated with biofilm. The physiological roles of the Crp/Fnr family regulator ArcR are elusive in S. aureus. In this study, it was found that the deletion of arcR increased the hemolytic ability and biofilm formation in S. aureus. Differential gene expression analysis by RNA-seq and real-time quantitative reverse transcription PCR showed that genes associated with hemolytic ability (hla and hlb) and biofilm formation (icaA, icaB, icaC and icaD) were significantly upregulated compared with those in the wild-type strain. The results revealed that ArcR regulated the expression of the hla and ica operon by binding to their promoter regions, respectively. This study provided new insights into the functional importance of ArcR in regulating the virulence and biofilm of S. aureus.


Introduction
Staphylococcus aureus is a globally prevalent pathogen that causes a variety of infections ranging from slight infections of the skin to serious cephalomeningitis [1][2][3]. S. aureus produces many virulence factors such as exotoxins, enzymes, and surface protein adhesins contributing to the initiation of adherence, the inhibition or avoidance of the host immune system, and the survival and expansion of S. aureus [4,5]. Among these virulence factors, αand β-hemolysins, as membrane-damaging toxins, are pore-forming toxin (PFT) factors which are encoded by hla and hlb. α-Hemolysin plays an important role in acute S. aureus infections, including skin and bloodstream infections. [6]. Multiple regulators participate in the regulation of hla in S. aureus. The two-component system (TCS) of SaeRS activated hla transcription via a well-defined SaeR binding site [7]. RNAIII performed as an antisense RNA to active hla mRNA translation [8]. Agr and SarA also affected the expression of hla in vitro and in vivo [9].
Biofilm formation increases the antibiotic resistance and host defense of bacteria contributing to persistent and chronic infections [10,11]. In S. aureus, polysaccharide intercellular adhesin (PIA) is vital in maintaining the structural integrity of biofilms [12][13][14][15]. PIA is produced by enzymes are that encoded by the icaADBC operon, which is responsible for the synthesis, modification, and export of PIA. The icaR gene located downstream of the icaADBC operon in S. aureus encodes a transcriptional repressor that regulates the expression of the ica operon [16,17]. In addition, studies have shown that LuxS/AI-2, SarA, MazEF, SrrAB, and YycFG participate in the regulation of the ica operon in S. aureus [1,[18][19][20][21][22][23].
Crp/Fnr family regulators are among the most widely studied transcriptional regulators, and they can respond to a variety of environmental stimuli by activating and The bacterial strains and plasmids are shown in Table 1. The primers used in this study were designed using Primer Premier 5 and are listed in Supplementary Table S1. S. aureus RN4220 was used to modify the plasmids before transformation into the other S. aureus strains. Luria-Bertani (LB) medium was used to culture Escherichia coli, and tryptic soy broth medium (TSB) containing 0.5% glucose was used to culture S. aureus. Antibiotics were used for constructed plasmid selection and maintenance at the following concentrations: 50 µg/mL kanamycin, 100 µg/mL ampicillin, 15 µg/mL chloramphenicol, and 5 µg/mL erythromycin. temp-sensitive plasmid, E. coli and S. aureus shutle vector, Apr, Cmr, for the construction of allelic-exchange mutants [35] pBTs-arcR pBTs derivative, for arcR deletion This study pBTs-icaA pBTs derivative, for icaA deletion This study

Construction of the S. aureus Mutant and Complementary Strains
The ∆arcR mutant and ∆arcR ∆icaA double mutant strains were obtained as described previously [38]. The ∆arcR ∆icaA double mutant strain was constructed on the background of the ∆arcR mutant. Briefly, the fragments of upstream and downstream were amplified from the NCTC8325 genome and then the fragments were ligated via overlap PCR. The fusion segments were cloned into the temperature-sensitive shuttle plasmid T1-pBTS. The constructed plasmid was transformed into RN4220 and subsequently transformed into the NCTC8325 wild type or ∆arcR mutant. The mutant strains were screened using a previously described method and verified via PCR and sequencing [39]. The shuttle vector pCN51 was used to construct complementary strains.

Detection of Biofilm Production
The tube method described previously was used to detect biofilm production under static conditions [40]. The bacteria were incubated in TSB for 14-16 h at 37 • C and 200 rpm. Cultures were diluted at 1:100 with fresh TSB and 200 µL of diluted suspension was added into each well of the 96-well plates. The plates were cultured at 37 • C for certain time courses. The wells were washed with water three times and dried at 37 • C. The wells were stained with 0.5% crystal violet at room temperature for 20 min and washed again with water. Then, the film around the wall and bottom of the wells was observed to confirm biofilm production. For the semi-quantitative assessment of biofilm formation, 100 µL per well of bleaching solution (methanol: glacial acetic acid: H 2 O (v/v/v) = 4:1:5) was added to each well with shaking for 1 h. Absorbance at 560 nm was then measured using an enzyme-linked immunosorbent assay reader.

Confocal Laser Scanning Microscopy (CLSM) for the Observation of Biofilm Production
Confocal laser scanning microscopy (CLSM) was introduced for observing biofilm production and form. To detect the function of ArcR in biofilm production, CLSM was used to analyze the biofilm formation in ∆arcR mutant strains. Overnight cultures were diluted with fresh TSB (2% glucose) at a ratio of 1:100. Then, 1 mL of diluted bacteria was added to 15 mm cell culture dishes and cultured at 37 • C for 24 and 48 h to form an artificial biofilm at the bottom. The biofilm at the bottom of the dish was washed with phosphatebuffered saline (PBS) buffer three times and then fixed with 2.5% glutaraldehyde at 4 • C for 1.5 h. After washing twice with PBS, 400 µL of 50 µg/mL FITC-conA solution was added to stain extracellular polysaccharides in the biofilm at 4 • C for 1 h. PBS was used to wash the biofilm and then the bacteria in the biofilm were stained with 10 µg/mL propidium iodide (PI) at 4 • C for 15 min. After washing with PBS, the dish was dried at room temperature. All confocal images were digitized using the CLSM system. Image J 1.53a was used for the analysis of integrated fluorescent intensity, biofilm production, bacterial density, and other parameters.

Determination of Hemolytic Activity
The WT, ∆arcR mutant, and arcR complementary strains were cultured in TSB at 37 • C. The optical density 600 (OD600) of overnight cultures was adjusted to 5 and 2 µL of the cultures was spotted onto sheep blood agar (SBA) plates for the hemolytic activity assay on solid medium. The SBA plates were left to dry and incubated at 37 • C, and hemolysis zones were measured after 24 h.
Hemolytic activity was quantitatively evaluated by incubating samples with 3% sheep red blood cells. The WT, ∆arcR mutant, and arcR complementary strains were grown in TSB and collected after being cultured overnight. Then, 100 µL of bacterial supernatants was mixed with 900 µL of PBS buffer containing 3% sheep red blood cells, and then incubated at 37 • C for 30 min. After centrifugation, the absorption at 543 nm of the supernatant was measured. The ddH 2 O containing 3% sheep red blood cells and the PBS containing 3% sheep red blood cells were used as the positive and negative control, respectively. The proportion of hemolytic activity was calculated relative to that of the positive control, which was regarded as having 100% hemolytic activity.
2.6. Total RNA Extraction, cDNA Generation, qRT-PCR, and RNA-seq Overnight cultures were added to fresh TSB at a 1:100 ratio and then grown to a certain cell density. Indicated cultures were collected by centrifugation. The precipitate was resuspended in 100 µL of 1 × RNase-free TAE and then incubated at 37 • C for 6-8 min. An RNAprep Pure Cell/Bacteria Kit (Tiangen, Beijing, China) was used for RNA isolation. The residual DNA was cleared using Recombinant DNase I at 37 • C for 60 min. For reverse transcription, 1 µg of RNA was used for cDNA synthesis. qRT-PCR was performed with cham Q universal SYBR qRT-PCR master mix (Vazyme, Nanjing, China). 16S rRNA was used as the reference target. Each reaction was performed in triplicate parallel experiments.
To perform RNA-seq, RNA was prepared as described above. The RNA quality was detected by agarose gel electrophoresis. The Bacterial rRNA Removal Kit (Vazyme, Nanjing, China) was used to eliminate the rRNA. The quality of RNA was evaluated using the Agilent 2100 BioAnalyzer (Agilent, Santa Clara, CA, USA) prior to library synthesis. The first strand of cDNA was synthesized using M-MuLV reverse transcriptase, DNA polymerase I, and dNTPs. The purified cDNA was end-repaired and sequencing joints were connected. Then, the beads of AMPure XP were used to filter the cDNA of approximately 370-420 bp. The beads were used to purify PCR products. After the library was constructed, it was initially quantified using Qubit 2.0 The insert size of the library was tested using the Agilent 2100 Bioanalyzer. qRT-PCR was used to accurately quantify the effective concentration of the library to ensure its quality. High-quality libraries were pooled for Illumina sequencing. NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, Houston, TX, USA) was used. DESeq2 was used to identify differentially expressed genes.

Construction of LacZ Reporter Vectors
To construct the reporter vectors pOShla for the detection of hla expression, a fragment containing the hla promoter region and a region of the first 78 bp of the coding sequence of the hla was amplified from the S. aureus genome with the primers pOS1-PhlaF/R. The PCR product was purified and then digested with EcoR1 and BamH1 at 37 • C for 1 h. The digested hla fragment was cloned into pOS1-lacZ to obtain reporter plasmid pOShla. The same method was used to construct the reporter plasmids pOShlb, pOSsbi, pOSvraX, and pOSsplA using the primers pOS1-PhlbF/R, pOS1-PsbiF/R, pOS1-PvraXF/R, and pOS1-PsplAF/R, respectively. These reporter plasmids were transformed into the NCTC8325 WT and ∆arcR mutant strains.

β-Galactosidase Activity Assay
The WT and ∆arcR mutant strains containing LacZ reporter plasmids were cultured in TSB at 37 • C overnight for the β-galactosidase assay. The cultures were diluted at a ratio of 1:100 in TSB with 15 µg/mL chloromycetin and grown for 4 and 8 h. Collected cultures were resuspended by 100 µL of ABT-LSA buffer (40 mM KH 2 PO 4 , 100 mM NaCl, 0.1% Triton X-100, 60 mM K 2 HPO 4 , 50 µg/mL lysostaphin) and then incubated at 37 • C for 30 min. Next, 100 µL of ABT buffer and 100 µL of 4 mg/mL ONPG (o-nitrophenyl-β-Dgalactopyranoside) were added to start the reaction. The samples were incubated at 37 • C until a yellow color appeared, and 1 mL of Na 2 CO 3 was immediately added to stop the reaction. The absorbance of samples was read at 420 nm and units were calculated using the following formula: units = (1000 × OD420)/(T × V × OD600), where T (measured in minutes) was the incubation time and V (in milliliters) was the volume of culture used in the assay.

PIA Detection
Overnight cultures were collected by centrifugation at 12,000× g and precipitates were stored in a −80 • C refrigerator. The precipitates were resuspended with 50 µL of EDTA (pH 8.0) and then incubated at 100 • C for 5 min. Subsequently, the samples were centrifugated at 12,000× g and 40 µL of the supernatant was collected for subsequent experiments. Then, 10 µL of 2 mg/mL proteinase K was added to the supernatants at 37 • C for 30 min. The extracted PIA was blotted onto a nitrocellulose filter membrane using a dot-blot apparatus as previously described [41]. Then, the membrane was dried at 65 • C and soaked in PBS containing 3% bovine serum albumin and 0.05% Tween-20 for 1 h. Next, the membrane was incubated in a solution containing 0.8 mg/mL wheat germ agglutinin conjugated with biotin (WGA-biotin) (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 1 h. After washing four times with PBS, the membrane was incubated in a solution with 0.1 µg/mL horseradish peroxidase-conjugated streptavidin (HRP-streptavidin) at room temperature for 15 min. After washing as described above, chemiluminescence detection was undertaken for PIA quantification.

ArcR Protein Expression and Purification
ArcR was expressed by the pET28a plasmid in BL21 and purified using the following procedures. The expression strains were grown in LB containing 50 µg/mL kanamycin at 37 • C for 3 h. Then, 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) was added to induce ArcR expression at 16 • C for 14-16 h. The cultures were then lysed in buffer (25 mM HEPES, 200 mM NaCl, 5 mM β-mercaptoethanol, 10% glycerol, pH 7.8). This fusion protein was purified though a nickel nitrogen triacetate agarose solution (Qiagen, Hilden, Germany). Lysis buffer containing 100, 200, or 400 imidazole was used to elute the bound protein.

Electrophoretic Mobility Shift Assay (EMSA)
The icaA and icaR promoter region DNA fragments were amplified from S. aureus NCTC8325 genomic DNA. The DNA fragments were incubated with purified ArcR protein at room temperature for 30 min in incubation buffer (25 mM HEPES, 300 mM NaCl, 1 mM dithiothreitol, 10% glycerol, pH 7.8). The incubation mixtures were added to 5 µL of loading buffer and electrophoresed on an 8% native polyacrylamide gel in 1 × Tris-borate-EDTA (TBE) buffer for 1-2 h. The shift bands were detected by staining with 0.5 µg/mL ethidium bromide (EB) and imaged using a gel imager (Bio-Rad, Hercules, CA, USA).

Deletion of arcR Increased the Hemolytic Activity in S. aureus
Qualitative and quantitative hemolytic methods were used to determine whether ArcR modulates the hemolytic activity of S. aureus NCTC8325. As shown in Figure 1A, the hemolytic zone of the ∆arcR mutant was larger than that of the WT after 24 h of incubation. The quantitative of hemolytic activity was performed with the sheep red blood cells, and then the proportion of hemolytic activity was calculated by measuring the optical density at 543 nm. The ∆arcR mutant displayed significantly greater hemolytic activity than that of the WT ( Figure 1B,C, Supplementary Table S2). Activity levels were restored in the arcR complementary strain. These results above demonstrated that ArcR participated in the regulation of hemolysis in S. aureus.

Deletion of arcR Increased Biofilm Formation in S. aureus
The effect of ArcR on biofilm formation in S. aureus NCTC8325 was evaluated by monitoring the WT, ∆arcR mutant, and arcR complementary strain using 96-well microtiter plate assays. As shown in Figure 2A, the arcR mutation resulted in an increased biofilm formation ability. This alteration could be complemented by the pCN51-arcR plasmid. The biofilms of the WT, ∆arcR mutant, and arcR complementary strains were stained with FITC-ConA and PI and were observed by CLSM. FITC-ConA stained the biofilm to green, and PI stained the bacterial cells to red. We observed an increase in adhered cells on the glass dishes in the ∆arcR mutant compared with that of the WT ( Figure 2B). In addition, the growth curves for the WT, ∆arcR mutant, and the arcR complementary strain grown in TSB were determined and the results showed no significant differences among these strains ( Figure 2C). These findings indicated that ArcR could repress biofilm formation in S. aureus.

Gene Expression Profiling of the ∆arcR Mutant
To understand the mechanism underlying the ArcR-mediated regulation of bacterial biofilm and hemolysis, the global gene expression profiles of the WT and ∆arcR mutant were determined via RNA-seq. Differentially expressed genes were identified by adjusted p < 0.05 and fold change log2 > 1. The results of these experiments revealed 224 of 2638 gene transcripts that were differentially expressed between the WT and ∆arcR mutant. A volcano plot of the RNA-seq results showed 69 downregulated and 155 upregulated genes in the ∆arcR mutant ( Figure 3A). The expression levels of ica operon genes icaA, icaB, icaC, and icaD increased by 7.4-, 11.0-, 5.1-, and 3.8-fold over levels in the WT. Moreover, the expression levels of the virulence genes hla, hlb, sbi, vraX, and splA increased by 11.0-, 5.4-, 4.3-, 3.9-, and 4.5-fold over levels in the WT, respectively ( Figure 3B). The downregulated and upregulated genes are listed in Supplementary Tables S3 and S4. Microorganisms 2023, 11, x FOR PEER REVIEW 9 of 17

Gene Expression Profiling of the ΔarcR Mutant
To understand the mechanism underlying the ArcR-mediated regulation of bacterial biofilm and hemolysis, the global gene expression profiles of the WT and ΔarcR mutant were determined via RNA-seq. Differentially expressed genes were identified by adjusted p < 0.05 and fold change log2 > 1. The results of these experiments revealed 224 of 2638 gene transcripts that were differentially expressed between the WT and ΔarcR mutant. A volcano plot of the RNA-seq results showed 69 downregulated and 155 upregulated genes in the ΔarcR mutant ( Figure 3A). The expression levels of ica operon genes icaA, icaB, icaC, and icaD increased by 7.4-, 11.0-, 5.1-, and 3.8-fold over levels in the WT. Moreover, the expression levels of the virulence genes hla, hlb, sbi, vraX, and splA increased by 11.0-, 5.4-, 4.3-, 3.9-, and 4.5-fold over levels in the WT, respectively ( Figure 3B). The downregulated and upregulated genes are listed in Supplementary Tables S3 and S4.

ArcR Regulates the Transcription of HEMOLYTIC Genes
The results presented above demonstrated that ArcR is involved in the regulation of hemolysis in S. aureus. To further determine whether the expression of hemolytic genes was altered in the ΔarcR mutant, qRT-PCR was performed. The qRT-PCR results showed that the expression levels of hla and hlb were significantly higher in the ΔarcR mutant strain at different growth phases than in the WT strain, and these expression changes could be fully restored by arcR complementation (Figure 4A-C). A β-galactosidase assay was performed to confirm the negative regulatory effect of arcR on the expression of hla and hlb. The reporter plasmids pOShla and pOShlb were constructed and transformed into the WT and ΔarcR mutant strains. The β-galactosidase activities in the ΔarcR mutant strain were significantly higher than those in the WT strain ( Figure 4D), consistent with the results of the qRT-PCR. These results suggested that the increase in hemolytic activity in the ΔarcR mutant strain was mainly due to the increased expression levels of hla and hlb.

ArcR Regulates the Transcription of HEMOLYTIC Genes
The results presented above demonstrated that ArcR is involved in the regulation of hemolysis in S. aureus. To further determine whether the expression of hemolytic genes was altered in the ∆arcR mutant, qRT-PCR was performed. The qRT-PCR results showed that the expression levels of hla and hlb were significantly higher in the ∆arcR mutant strain at different growth phases than in the WT strain, and these expression changes could be fully restored by arcR complementation (Figure 4A-C). A β-galactosidase assay was performed to confirm the negative regulatory effect of arcR on the expression of hla and hlb. The reporter plasmids pOShla and pOShlb were constructed and transformed into the WT and ∆arcR mutant strains. The β-galactosidase activities in the ∆arcR mutant strain were significantly higher than those in the WT strain ( Figure 4D), consistent with the results of the qRT-PCR. These results suggested that the increase in hemolytic activity in the ∆arcR mutant strain was mainly due to the increased expression levels of hla and hlb.

ArcR Influences Biofilm Formation through ica Operon
To verify the RNA-seq results, the transcript levels of genes of the ica operon were measured using a qRT-PCR with RNA extracted from the WT strain and its isogenic ΔarcR mutant in planktonic bacteria and biofilm. The transcript levels of icaA, icaB, icaC, and icaD were upregulated in the ΔarcR mutant not only in planktonic bacteria, but also in biofilm ( Figure 5A,B). To determine whether ArcR affects biofilm formation by regulating the expression of the ica operon, the icaA mutant was constructed from the ΔarcR mutant. Biofilm production was evaluated in the WT, ΔarcR mutant, and ΔarcR ΔicaA double mutant and was detected using 96-well microtiter plate assays. Biofilm production was lower in the ΔarcR ΔicaA double mutant than in the ΔarcR mutant ( Figure 5C). There were no significant differences in the growth rates between the WT, ΔarcR mutant, and ΔarcR ΔicaA double mutant ( Figure 5D).
The genes of the ica operon encode proteins involved in PIA synthesis. The production of PIA is important for biofilm formation in S. aureus. To investigate whether ArcR inhibits biofilm formation by regulating ica operon and subsequent PIA production, we detected the PIA production. As shown in Figure 5E, the PIA production in the ΔarcR

ArcR Influences Biofilm Formation through ica Operon
To verify the RNA-seq results, the transcript levels of genes of the ica operon were measured using a qRT-PCR with RNA extracted from the WT strain and its isogenic ∆arcR mutant in planktonic bacteria and biofilm. The transcript levels of icaA, icaB, icaC, and icaD were upregulated in the ∆arcR mutant not only in planktonic bacteria, but also in biofilm ( Figure 5A,B). To determine whether ArcR affects biofilm formation by regulating the expression of the ica operon, the icaA mutant was constructed from the ∆arcR mutant. Biofilm production was evaluated in the WT, ∆arcR mutant, and ∆arcR ∆icaA double mutant and was detected using 96-well microtiter plate assays. Biofilm production was lower in the ∆arcR ∆icaA double mutant than in the ∆arcR mutant ( Figure 5C). There were no significant differences in the growth rates between the WT, ∆arcR mutant, and ∆arcR ∆icaA double mutant ( Figure 5D). mutant was higher than that in the WT. Taken together, these results indicated that ArcR inhibited biofilm formation by regulating ica operon manner in S. aureus.  The genes of the ica operon encode proteins involved in PIA synthesis. The production of PIA is important for biofilm formation in S. aureus. To investigate whether ArcR inhibits biofilm formation by regulating ica operon and subsequent PIA production, we detected the PIA production. As shown in Figure 5E, the PIA production in the ∆arcR mutant was higher than that in the WT. Taken together, these results indicated that ArcR inhibited biofilm formation by regulating ica operon manner in S. aureus.

ArcR Could Bind to the Promoter Regions of the ica Operon and hla
As a member of the Crp/Fnr family of regulatory proteins, ArcR also contained two typical domains: the N terminus of ArcR contained a circular nucleotide binding domain and the C terminus contained a DNA-binding helix-turn-helix region. To explore whether ArcR could directly bind to the promoter regions of ica and hla, the binding affinities of ArcR to ica (233 bp) and hla (283 bp) were analyzed with EMSA. The locations of fragments of ica and hla are shown in Figure 6A,B. The results of EMSA are shown in Figure 6C,D, the shifted bands emerged and strengthened as the dose of ArcR increased. ArcR did not bind to 267-bp DNA of the tetA promoter region ( Figure 6E). These results suggested that ArcR could regulate the expression of icaA and hla by binding to their promoter regions.

ArcR Could Bind to the Promoter Regions of the ica Operon and hla
As a member of the Crp/Fnr family of regulatory proteins, ArcR also contained two typical domains: the N terminus of ArcR contained a circular nucleotide binding domain and the C terminus contained a DNA-binding helix-turn-helix region. To explore whether ArcR could directly bind to the promoter regions of ica and hla, the binding affinities of ArcR to ica (233 bp) and hla (283 bp) were analyzed with EMSA. The locations of fragments of ica and hla are shown in Figure 6A,B. The results of EMSA are shown in Figure 6C,D, the shifted bands emerged and strengthened as the dose of ArcR increased. ArcR did not bind to 267-bp DNA of the tetA promoter region ( Figure 6E). These results suggested that ArcR could regulate the expression of icaA and hla by binding to their promoter regions. Figure 6. ArcR regulated the expression of the ica operon and hla by directly binding to their promoter regions. The promoter sequence of the icaA and hla and the locations of the probes were marked by arrows (A,B). The interactions between ArcR and the DNA fragments of their promoter regions were detected with EMSA. ArcR was incubated with the ica promoter region (C), ArcR was incubated with the hla promoter region (D), and ArcR was incubated with the tetA promoter region (E) at 37 °C for 30 min. The mixtures were electrophoresed on 8% native polyacrylamide gels and the bands were imaged under UV light after EB staining.  A,B). The interactions between ArcR and the DNA fragments of their promoter regions were detected with EMSA. ArcR was incubated with the ica promoter region (C), ArcR was incubated with the hla promoter region (D), and ArcR was incubated with the tetA promoter region (E) at 37 • C for 30 min. The mixtures were electrophoresed on 8% native polyacrylamide gels and the bands were imaged under UV light after EB staining.

Discussion
Crp/Fnr family regulators are always found in bacteria, and their physiological roles, such as energy metabolism, biofilm formation, antibiotic resistance, and virulence, have been recognized. Previous studies showed that some Crp/Fnr family regulators positively regulated the bacterial virulence and biofilm formation in other bacteria [26][27][28][29][30][31]. In this study, ArcR negatively regulated the biofilm formation and hemolytic activity of S. aureus. These differences might be related to the fact that ArcR has low overall similarity with other Crp/Fnr family members, which resulted in a difference in their response to environmental stress. Certainly, ArcR is not an exception. Other Crp/Fnr family members can also repress the expression of functional genes such as Fnr, RedB, DnrD, and DnrF [25,42,43], as the NCTC8325 strain is known to be defective in sigma factor SigB at the stationary phase [44]. Meanwhile, to exclude strain-specific differences, the virulence-and biofilm-associated experiments were repeated with the arcR mutant under USA300 background. The deletion of arcR in USA300 increased bacterial virulence and biofilm, which was consistent with those in NCTC8325 (Supplementary Figures S1-S3).
Previous studies have shown that YycFG could directly bind to the promoter regions of icaA and positively regulate their expression [23,45]. The promoter region of yycFG contains a conserved Crp/Fnr family-like binding motif (-TGTGAttttttaCACA-). To determine whether ArcR regulates the biofilm formation of S. aureus via yycFG, the expression levels of yycF and yycG in the WT and ∆arcR mutant were evaluated using the results of qRT-PCR and RNA-seq assays. There was no significant difference between the ∆arcR mutant and the WT (Supplementary Figure S4). Taken together, the regulation of ArcR in biofilm formation was not dependent on yycFG in our study; however, it is possible that ArcR could regulate the expression of yycFG under certain conditions.
The RNA-seq results showed that the virulence-associated genes sbi, vraX, and splA were upregulated ( Figure 3B). The second immunoglobulin-binding protein (Sbi) is a multifunctional immune evasion factor that helps S. aureus to avoid innate immune defenses [46,47]. VraX is a protein secreted by S. aureus and could bind to C1q protein and inhibit the formation of the C1 complex. The deletion of vraX reduced the pathogenesis of S. aureus [48]. Serine protease-like protein A (SplA) is a member of Spl proteases and is unique to S. aureus [49]. SplA could cleave the mucin 16 glycoprotein on the CalU-3 lung cell line, which promotes the spread of S. aureus [50]. qRT-PCR and β-galactosidase activity assays further showed that ArcR regulated the expression of sbi, vraX, and splA (Supplementary Figure S5A-D). These results above suggested that ArcR may globally regulate the virulence of S. aureus, rather than affecting hemolysis alone. Therefore, other potential mechanisms by which ArcR regulates virulence and the effect of arcR deletion on S. aureus virulence in vivo need to be further explored.
It is generally believed that the success of S. aureus as a human pathogen is due to its ability to cause a wide range of infections, which requires its various virulence factors and sometimes biofilm formation. Studies on the regulators of virulence and biofilms have shown that the regulatory network of S. aureus is highly complex. Thus, further studies are needed to fully understand the molecular mechanisms underlying the regulation of virulence and biofilm formation in S. aureus. The regulation of ArcR in S. aureus is shown in Figure 7. ArcR binds to the promoter region of arcABDC operon and activates its transcription, facilitating the utilization of arginine under anaerobic conditions [32]. ArcR potentially binds to the yycFG promoter region and regulates its expression under certain conditions. Our previous study showed that ArcR contributed to tolerance to fluoroquinolone antibiotics by regulating katA in S. aureus. This study indicated that ArcR could bind to the promoter regions of ica operon and hla. Here, the physiological roles of ArcR in S. aureus NCTC8325 were investigated. These findings may not only provide novel insight into the regulatory networks involved in virulence and biofilm regulation networks, but also expand the physiological roles of ArcR in S. aureus and the function of the Crp/Fnr family in pathogenic bacteria.
Microorganisms 2023, 11, x FOR PEER REVIEW 14 of 17 Figure 7. The ArcR regulatory model in S. aureus. ArcR binds to the promoter region of the arcABDC operon and activates its transcription. ArcR may bind to the yycFG promoter region and regulate its expression under certain conditions. Our previous study showed that ArcR contributed to tolerance to fluoroquinolone antibiotics by regulating katA in S. aureus. Furthermore, ArcR could bind to the promoter regions of ica operon and hla by directly binding to their promoter regions. ArcR could also participate in the regulation of the virulence-associated genes sbi, vraX, and splA.

Supplementary Materials:
The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Table S1: Primers used in this study; Table S2: The quantitative hemolytic activity; Table S3: Genes downregulated by ArcR. (p < 0.05 and fold change log2 < −2); Table S4: Genes upregulated by ArcR. (p < 0.05 and fold change log2 >2); Figure S1: ArcR regulates the hemolytic activity of S. aureus USA300; Figure S2: ArcR negatively regulates the transcription of hemolytic genes in S. aureus USA300; Figure S3: Roles of ArcR in biofilm formation of S. aureus USA300; Figure  S4: The transcriptional levels of yycFG in S. aureus NCTC8325; Figure S5: ArcR negatively regulates the transcriptional of virulence genes in S. aureus NCTC8325.  . The ArcR regulatory model in S. aureus. ArcR binds to the promoter region of the arcABDC operon and activates its transcription. ArcR may bind to the yycFG promoter region and regulate its expression under certain conditions. Our previous study showed that ArcR contributed to tolerance to fluoroquinolone antibiotics by regulating katA in S. aureus. Furthermore, ArcR could bind to the promoter regions of ica operon and hla by directly binding to their promoter regions. ArcR could also participate in the regulation of the virulence-associated genes sbi, vraX, and splA.