All plasmids, strains and primers used in this study are listed in Tables S1–S3 in the Supplemental Material. The GAS strains used in this study were MGAS5005, an M1-serotype strain isolated from the cerebral fluid of a patient with bacterial meningitis (Sumby et al., 2005) and the spyA isogenic mutant 5005ΔspyA (Hoff et al., 2011). Whole-genome sequencing was performed for 5005ΔspyA to confirm the deletion in spyA and the absence of additional mutations. The genome sequencing was conducted essentially as described in Price et al. (2013) using Illumina Genome Analyzer IIx. Illumina whole-genome sequence data sets were aligned against the chromosome of a published MGAS5005 reference genome (GenBank accession no. CP000017) (Sumby et al., 2005) using the short-read alignment component of the Burrows-Wheeler Aligner. Mutations were identified as described in Price et al. (2013).

GAS cultures were grown in Todd-Hewitt broth supplemented with 0.2% yeast extract (THY), or on THY agar plates. When indicated, strains were cultured in a chemically defined medium [CDM (Chang et al., 2011)] supplied with 1% glucose. GAS strains were incubated without aeration at 37°C. Escherichia coli strains were grown in Luria-Bertani (LB) medium or on LB agar plates at 37°C. When required, antibiotics were included at the following concentrations: ampicillin at 100 μg ml⁻¹ for E. coli; chloramphenicol at 10 μg ml⁻¹ for E. coli and 5 μg ml⁻¹ for GAS.

To analyze the effect of hemin on growth of GAS strains, frozen aliquots of GAS strains were prepared for experiments. To make frozen aliquots GAS strains cultured overnight in THY medium were diluted 1:100 into fresh THY medium and allowed to grow to mid-logarithmic phase (OD₆₀₀ = 0.7). Bacteria were collected by centrifugation, washed twice with PBS and re-suspended in PBS with 15% glycerol. The volume of PBS/glycerol was equal to a half volume of THY medium used for culturing. The aliquots were frozen and stored at −80°C. On experiment days, the aliquots were thawed, diluted 1:20 into THY medium. Bacteria were grown in 12-ml THY medium tightly sealed in 15-ml conical tubes without aeration at 37°C. Hemin chloride (Sigma) from stock solution prepared in DMSO was added to the growth media at a concentration of 2 μM.

Plasmid DNA was isolated from E. coli by commercial kits (Qiagen) according to the manufacturer's instructions and used to transform E. coli and GAS strains. Plasmids were transformed into GAS by electroporation as described previously (Hoff et al., 2011). Chromosomal DNA was purified from GAS as described in Caparon and Scott (1991). Constructs containing mutations were identified by sequence analysis. Primers for site-directed mutagenesis are listed in Table S3. All constructs were confirmed by sequencing analysis (Eurofins MWG Operon).

Total RNA was isolated from GAS grown to mid-exponential phase (OD₆₀₀ = 0.6) using the miRNeasy kit (Qiagen) according to manufacturer's instructions. Contaminating genomic DNA was removed from RNA samples with Turbo DNase (Applied Biosystems). cDNA for qRT-PCR was obtained as described in Falaleeva et al. (2014). The efficacy of the DNase step was controlled for by including a reverse transcriptase negative control. qRT-PCR was employed to examine relative mRNA abundance of spyA with the house keeping gene plr used as an internal standard for normalization. qRT-PCR reactions were performed using a Mx3005P qPCR system (Agilent Technologies) under standard reaction conditions. The reaction mixture contained Power SYBR green PCR Master Mix (Applied Biosystems), cDNA and primers spyArt-f and spyArt-r (Table S2). plr was analyzed in each cDNA reaction using the pair of primers plr-f and plr-r (Table S2). The expression levels of spyA in each condition tested were normalized to the levels of plr transcript. No-template and no RT controls were included for each primer set and template. Data is reported as the mean values of fold change in mRNA levels in the mutants relative to those of MGAS5005 WT strain. Each experiment was performed in triplicate, and mean values ± standard deviations (SDs) are shown.

The MBP-SpyB protein was expressed in E. coli Rosetta (DE3) harboring pmalEspyB plasmid. The frozen cells were resuspended in 20 mM Tris pH 7.5, 300 mM NaCl at 4°C. The suspension was passed twice through a microfluidizer cell disrupter and the lysate centrifuged for 45 min at 17,000 × g. The protein was purified by Ni-NTA affinity chromatography and dialyzed overnight at 4°C in 20 mM Tris pH 7.5, 300 mM NaCl in the presence of TEV protease. Cleavage with TEV protease leaves the first six residues of the TEV site (ENLYFQ) at the C-terminus of MBP-SpyB.

The cleaved protein solution was purified by Ni-NTA affinity chromatography. The removed His-tag binds to the resin while MBP-SpyB, without His-tag, immediately elutes from the column. The protein was concentrated to around 10 mg mL⁻¹, treated with 1 mM DTT and purified on the size-exclusion chromatography column Superose 6 10/300, pre-equilibrated with 20 mM HEPES pH 7.5, 100 mM NaCl, 1 mM DTT, and 2 mM maltose. The protein was present as a monomer and heme-bound dimer. The protein fractions corresponding to the monomeric form were pooled, concentrated and used for reconstitution with hemin.

MBP protein was expressed in E. coli Rosetta (DE3) harboring pKV1111 plasmid (Dunstan et al., 2013). The protein was purified by Ni-NTA affinity chromatography, followed by size-exclusion chromatography on a Superose 6 10/300 column, as for MBP-SpyB.

Hemin chloride was dissolved in 0.1 M NaOH at 10 mM concentration and diluted in protein buffer as desired. MBP-SpyB monomer was reconstituted with hemin in the presence of 1 mM DTT at room temperature at a protein:hemin ratio of 1:4 for 1 h. The sample was then loaded onto a Sephadex G-25 column and the holoprotein was eluted with 20 mM Tris-HCl and 100 mM NaCl, pH 7.5. The holoprotein was further purified on the Superose 6 size-exclusion chromatography column, as described above. The protein fractions corresponding to the holoprotein dimer form were pooled, concentrated and used for experiments.

TMBZ staining of native-PAGE to detect for the presence of bound hemin was carried out according to the protocol described in Thomas et al. (1976). Briefly, 6.3 mM TMBZ in methanol was freshly made and mixed with 0.25 mM sodium acetate pH 5.0 in a ratio of three parts to seven. The gel was incubated with the TMBZ solution in the dark for 1–2 h. The bands were developed with the addition of 30 mM H₂O₂ for 30 min. The gel was fixed with three washes of isopropanol and 0.25 mM sodium acetate pH 5.0 in a ratio of three parts to seven.

The SpyB peptide was chemically synthesized (GenScript, NJ). The MBP-SpyB, MBP-SpyB C7A/C13A, MBP-SpyB C7A/C13A/C30A/C35A monomers and MBP were purified as described above. Hemin binding was monitored by fluorescence quenching of the tryptophan residues of SpyB at 20°C using a PerkinElmer LS 55 Fluorescence spectrometer as described in Shepherd et al. (2007). Hemin (prepared as described above, in various dilutions) or protoporphyrin IX (prepared as a 10 mM stock in ddH₂O and diluted in protein buffer) were added to 50 μl of protein solution (5 μM, in 20 mM HEPES, 100 mM NaCl, and 1 mM DTT, pH 7.5) in 50 μl aliquots. The excitation wavelength was 280 nm and the emission wavelength was from 300 to 500 nm. The dissociation constant (K_D) was calculated using the change in fluorescence (ΔF) and concentration of porphyrin (hemin or protoporphyrin IX; [porphyrin]) using SigmaPlot v12.3 and the following equation: ΔF=ΔFmax×[porphyrin]KD+[porphyrin].

The Stern-Volmer constant (K_sv) was calculated to determine whether porphyrin binding was dynamic or static. K_sv was calculated using [porphyrin] and fluorescence intensities (F) according to the following equation: F0/F=1+Ksv[porporphyrin].

Absorbance spectrums were recorded between 300 and 700 nm for 50 μM hemin, and 50 μM hemin mixed with 2.5 μM MBP-SpyB or MBP, purified as described above. After mixing in the presence of 1 mM DTT, the spectrums were immediately recorded on a SpectraMax M5 (Molecular Devices).

Protein concentrations were determined using a modified BCA protein assay kit (Pierce, Rockford, IL) with bovine serum albumin (BSA) as a standard. The heme to protein ratio was measured by pyridine hemochrome assay according to the method (Berry and Trumpower, 1987). Briefly, 100 μl of protein sample in Tris-HCl buffer was mixed with 10 μl 1 N NaOH, 23 μl pyridine, and ~2 mg sodium dithionite. The optical absorption spectrum was immediately recorded between 280 and 700 nm using a SpectraMax M5 (Molecular Devices) spectrophotometer. The heme content was calculated using the extinction coefficient ε₄₁₈ = 191.5 mM⁻¹ cm⁻¹.

The rate of hemin dissociation from SpyB was determined using the procedure described in Ortiz de Orué Lucana et al. (2014). Briefly, 5 μM MBP-SpyB was incubated with 20 μM hemin in 20 mM Tris-HCl, pH 7.5, 1 mM DTT at 4°C for 1 h. Free hemin was removed and MBP-SpyB:hemin holoprotein was collected by size-exclusion chromatography under the same conditions described above. The holoprotein was diluted to 4 μM and incubated with 4 μM apo-myoglobin for 20 min at 25°C with absorbance readings at 408 nm every 5 s. GraphPad Prism v 4.0 software was used to calculate the dissociation rate constant (k_off) from the change in absorbance at 480 nm. Apo-myoglobin was generated from hemoglobin by methyl ethyl ketone extraction as described in Ascoli et al. (1980).

All samples were filtered through a 0.22-μm filter before analysis. Dynamic light scattering was conducted on a DynaPro Nanostar (Wyatt Technologies). MBP-SpyB was analyzed at a final concentration of 40 μM. Measurements were taken over a 54 s time period with 10 replicates at room temperature. The samples were illuminated with a HeNe laser (633 nm) and experiments were conducted at a scattering angle of 90°. The associated DYNAMICS analysis software was used to determine average radii.

Synthetic SpyB (30 μM) was mixed with hemin in different molar ratios. The samples were centrifuged at maximum speed for 5 min to collect the supernatant and pellet. Samples were dissolved in SDS-PAGE loading buffer and resolved on 16% Tris-Tricine gel.

MBP-SpyB holoprotein dimer was purified as described above and split into three. One sample was first reduced (10 mM DTT, 30 min, 56°C), then alkylated with 50 mM iodoacetamide (IAA) for 30 min in the dark at room temperature. The sample was digested overnight with trypsin at a 1:100 (w/w) enzyme to substrate ratio for 12 h at 37°C. After these treatments all free cysteine residues and residues that participate in disulfide bond formation should be found alkylated with IAA.

The second sample was alkylated only (50 mM IAA, for 30 min in the dark at room temperature), and then processed as described above. After these treatments, all free cysteine residues should be found alkylated with IAA.

For the assignment of disulfide bonds, the final holoprotein dimer sample was neither reduced nor alkylated. The sample was dialyzed and digested as described above. Tryptic fragments with disulfide bonds that disappeared after the reduction/alkylation treatment were identified by comparison of the two LC-MS/MS runs (with and without reduction/alkylation). LC-MS/MS analysis was performed using an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific) coupled with an Eksigent Nanoflex cHiPLC system (Eksigent) through a nanoelectrospray ionization source. The LC-MS/MS data were subjected to database searches for protein identification using Proteome Discoverer software V. 1.3 (Thermo Fisher Scientific) with a local MASCOT search engine. A custom database containing the protein of interest was used.

Growth phenotypes of MGAS5005 and the 5005ΔspyA and 5005ΔspyB mutants were assessed using the Biolog plates at Biolog's PM Services facility. A total of 20 96-well PM plates constituting eight metabolic panels (PM1–PM8); and 12 sensitivity panels (PM9–PM20) were used in this study. Two replicates were conducted for each strain. Standard PM testing protocols are described in http://www.biolog.com.

GAS strains were harvested from the exponential growth phase (OD₆₀₀ = 0.5) by centrifugation. Bacteria were washed with phosphate-buffered saline (PBS) and subjected to light (Gram stain) and fluorescent microscopy. For fluorescent microscopy, GAS strains were incubated with vancomycin-BODIPY for 30 min, washed and spread on glass slides, and imaged using a Nikon Eclipse E600 as described in Kant et al. (2015). For transmission and scanning electron microscopy the bacterial pellets were washed once with PBS and then fixed with freshly made 2.5% glutaraldehyde and 4% paraformaldehyde, in 0.1 M cacodylate buffer, pH 7.4. The fixed samples were processed at the Ohio State University Microscopy and Imaging Facility and imaged by field-emission gun-equipped scanning (Nova NanoSEM 400, FEI) or cryo-capable transmission electron microscopy (Tecnai G2 Spirit, FEI) as described in Agarwal et al. (2011) and Kant et al. (2015).

Aliquots of frozen bacteria were used to analyze GAS resistance to β-lactam antibiotics, and were prepared as described above. On experiment days, the aliquots were thawed, diluted 1:20 into CDM and grown to OD₆₀₀ = 0.5. Bacteria were collected by centrifugation and re-suspended in PBS (10⁻² dilution). For the antibiotics challenge, 10 μl of bacterial solution was spotted on THY plates unmodified or supplemented with 0.05–0.25 μM cefoperazone, 130–920 μM methicillin or 30–140 μM ampicillin. The experiment has been conducted at least three times. The minimum inhibitory concentration (MIC) of an antibiotic is defined as the lowest concentration of the compound that completely inhibits bacterial growth on plates.

Bacteria from 10 ml of exponential culture at an OD₆₀₀ = 0.5 were harvested by centrifugation. The pellet was washed in PBS and re-suspended in PBS with 0.3, 0.45, or 0.6 μM cefoperazone. A reference sample was re-suspended in PBS without the antibiotic. After 30 min of incubation at room temperature, the bacteria were pelleted, washed in PBS, and re-suspended in PBS with 15 μM Bocillin FL (Boc-FL). After 30 min incubation at room temperature, the bacteria were pelleted and washed in PBS. To digest the GAS cell wall, the pellet was re-suspended in PBS supplemented with PlyC lysin as described in Raz and Fischetti (2008) and incubated for 45 min at 37°C. The protoplasts were collected by centrifugation at 3,000 × g for 10 min. The cells were lysed by treatment with BugBuster Protein Extraction Reagent (Novagen) according to the manufacturer's protocol. The protein concentration was measured with a NanoDrop 1000 spectrophotometer. The protein concentration was adjusted to 1.5 mg ml⁻¹ and mixed with 6 × SDS-PAGE loading buffer. The samples were analyzed on 4–12% SDS-PAGE gel as described in Kocaoglu et al. (2015). Densitometry analysis was performed using ImageJ software.

Strains cultured overnight in THY medium were diluted 1:10 into fresh THY medium in a 96 well plate and allowed to grow for 2 h. Fresh plates containing 100 μL of THY and serial dilutions of PlyC (0–125 ng mL⁻¹) were inoculated with 100 μL of culture (giving a final maximum concentration of 62.5 ng mL⁻¹ PlyC). The cultures were incubated for 1 h at 37°C, with OD₆₀₀ measurements obtained at 0 and 1 h. The percentage change in OD₆₀₀ was calculated from three biological replicates.

Cells were collected from exponential growing cultures (OD₆₀₀ = 0.6), washed three times with BSA-saline solution (0.5% BSA, 0.15 M NaCl) and resuspended in BSA-saline solution prior to a 30 min blocking step at 37°C with mixing. Alexa Fluor 555-WGA was subsequently added to a final concentrations of 0, 12.5, 25, 50, 100 μg ml⁻¹. After 60 min of incubation at 37°C with mixing, the cells were centrifuged, washed twice, and resuspended in BSA-saline. Bound Alexa Fluor 555-WGA was quantified in a fluorimeter SpectraMax M5 (Molecular Devices) using an excitation of 544 nm and emission of 590 nm.

Bacteria from 15 ml of exponential culture at an OD₆₀₀ = 0.5 were harvested by centrifugation. The pellet was washed five times in water and subjected to analysis of total carbohydrate content by the phenol-sulfuric acid method (DuBois et al., 1956). The BCA protein assay was used to quantify total protein and normalize samples.

GAS cell wall was prepared from exponential phase cultures (OD₆₀₀ = 0.8) by the SDS-boiling procedure as described for Streptococcus pneumoniae (Bui et al., 2012). Purified cell wall samples were lyophilized and used for analysis of glycosyl composition.

Carbohydrate composition analysis was performed at the Complex Carbohydrate Research Center (Athens, GA).

Unless otherwise indicated, statistical analysis was carried out from at least three independent experiments. Quantitative data were analyzed using the paired Student's t-test. A P-value equal to or less that 0.05 is considered statistically significant.

In order to identify SpyB function we generated an in-frame deletion in spyB of 51 bp (deletion of codons 9–25) in the hyperinvasive M1T1 serotype strain, MGAS5005 (Sumby et al., 2005), creating the 5005ΔspyB mutant (Figure S1). As we previously reported (Korotkova et al., 2012) this deletion did not have an effect on SpyA translational fusion using a plasmid-based reporter system. Moreover 5005ΔspyB expressed equivalent levels of spyA (Figure S2A) confirming that the deletion has no a polar effect on gene expression levels. The SpyA deficient mutant, 5005ΔspyA, was previously constructed in the same strain background (Hoff et al., 2011) and utilized for our experiments. Whole-genome sequencing was performed for both mutants to confirm the deletion and the absence of additional mutations.

5005ΔspyB demonstrated impairment in growth in nutrient-rich THY medium (Figure 1). Gram staining of bacteria revealed that the exponentially grown spyB deletion mutant forms large cellular aggregates instead of typical chains of ovococci formed by the WT strain and 5005ΔspyA (Figure 2A). By complementing the 5005ΔspyB mutant in cis with spyB (5005ΔspyB spyB⁺), we restored the WT phenotype (Figure 2A). This observation indicated that the spyB mutant is defective in cell division or cell separation. Fluorescent microscopy of bacteria labeled with a fluorescent derivative of a cell wall and septa-binding antibiotic, vancomycin, indicated no obvious defects in cell division for 5005ΔspyB (Figure 2B). Although some instances of improper septa formation were visible by transmission and scanning electron microscopy with comparison to WT (Figures 2C,D). These data suggest that the increased aggregation is most likely due to defective cell separation.

To identify additional phenotypes of the mutants, we utilized Biolog phenotype MicroArrays that allow quantitative screening of growth and metabolic activities of the WT, 5005ΔspyB, and 5005ΔspyA mutant strains under various metabolic conditions. The latter included various carbon, nitrogen, phosphate, and sulfur sources; osmolytes; metabolic inhibitors; and toxic compounds including several types of antibiotics. The analysis revealed stronger growth of 5005ΔspyB in comparison to WT and 5005ΔspyA in the presence of β-lactam antibiotics of the cephalosporin class: cefoperazone, cefamandole nafate, cefsulodin, and cefoxitin (Figure S3). The cellular targets of cephalosporins are penicillin-binding proteins (PBPs) that catalyze the final steps of peptidoglycan (PG) biosynthesis. The antibiotics block the formation of peptide cross-links in the PG catalyzed by the transpeptidase domain of PBPs. To verify the increased cephalosporin resistance of 5005ΔspyB, strains were grown in nutrient-limited CDM to middle exponential phase and then plated on THY agar supplied with different concentrations of cefoperazone. We found that the minimum inhibitory concentration (MIC) of cefoperazone for the WT, 5005ΔspyA, and 5005ΔspyB spyB⁺ strains was 0.15 μM (Figure 3A). 5005ΔspyB showed decreased susceptibility to the antibiotic with a MIC of 0.2 μM. Although the MIC-values varied by less than two-fold, differences between the strains were highly reproducible (P = 0, t-test; n = 5).

Bacteria have several PBPs that display variabilities in binding profiles for different β-lactams resulting in selective antimicrobial effects (Hakenbeck et al., 2012). The β-lactams include antibiotics of the penicillin class such as penicillin G, methicillin, and ampicillin. To determine whether SpyB deficiency confers resistance to the penicillin class of β-lactams, we analyzed the sensitivity of the strains to methicillin and ampicillin. We found no significant differences in penicillin antibiotic susceptibility between strains.

In contrast to enterococci and staphylococci, GAS does not produce β-lactamases, which cleave the β-lactam group of antibiotics. In many instances the mechanisms of resistance to β-lactam antibiotics are due to amino acid substitutions in PBPs, leading to altered activity or stability of PBPs (Albarracin Orio et al., 2011). GAS genomes encode four HMW PBPs that are homologs of S. pneumoniae PBPs —three are class A PBPs (PBP1A, 1B, and 2A) and one is a class B PBP, PBP2X (Philippe et al., 2014; Table S4). Furthermore, HMW PBPs might be present in the cell membrane in several molecular forms due to the presence of alternate start codons that are preceded by Shine-Dalgarno sequences. Additionally, we identified three low-molecular-weight (LMW) class C PBPs in the GAS genomes.

To investigate a putative role of SpyB in the regulation of PBPs, we set out to characterize the effect of cefoperazone on the abundance of PBPs in MGAS5005 and 5005ΔspyB. PBPs can be visualized using fluorescent β-lactams that form a covalent complex with PBPs (Kocaoglu et al., 2012). Strains were grown in THY broth or CDM, and exponentially growing cultures were treated with different concentrations of cefoperazone followed by incubation with an excess of Bocillin FL (Boc-FL), fluorescent penicillin. Gel-based analysis of THY-grown GAS identified seven major protein bands with molecular weights (MW) ranging between 63 and 98 kDa, and one major band with a MW of ~40 kDa (Figure 4A). The MW of the major protein bands correlated with the sizes of HMW and LMW PBPs encoded by the GAS genome (Table S4). Additionally, Boc-FL-treated bacteria displayed multiple minor bands (Figure 4A). Growth in CDM reduced the number of visible PBPs (Figure 4A). Treatment with an increasing concentration of cefoperazone resulted in a decrease in these HMW bands in THY and CDM-grown bacteria. Band 3 was the first to disappear from the gel indicating that the corresponding PBP has the strongest affinity for cefoperazone. Densitometry analysis of the bands revealed no significant differences in Boc-FL labeling in the MGAS5005 in comparison to 5005ΔspyB (Figure 4B). Thus, this analysis suggested that SpyB does not affect the abundance of PBPs in the cell membrane.

GAS cell wall is composed of PG and the covalently attached rhamnose (Rha)-containing group A-specific carbohydrate (GAC; Mistou et al., 2016). Fluorescent microscopy of the PG-binding antibiotic vancomycin indicated that 5005ΔspyB was not defective in PG formation (Figure 2B). However, we found that the mutant has decreased sensitivity to bacteriophage-encoded PG hydrolase PlyC (Figure 3B), whereas the sensitivity to PG hydrolase mutanolysin was not affected (data not shown). PlyC, is known to lyse streptococcal species bearing a polyrhamnose epitope (Nelson et al., 2001), indicating that similar to other bacteriophage PG hydrolases, PlyC recognizes a carbohydrate epitope in the cell wall (Nelson et al., 2006). Structural studies of GAC have shown that the polysaccharide consists of alternating α(1,2)- and α(1,3)-linked Rha units and side units of β-N-acetylglucosamine (GlcNA) linked to the O-3 position of the polyrhamnose backbone (Coligan et al., 1978; Huang et al., 1986). Given the changes in sensitivity to PlyC of 5005ΔspyB, we evaluated the ability of bacteria to bind to Alexa Fluor 555-labeled WGA, a lectin that specifically binds GlcNA. Deletion of spyB led to increased binding of WGA compared with the WT strain (Figure 3C). This suggests that the mutant has increased amounts of GlcNA-containing saccharides on the cell surface.

In order to investigate whether GAC production is altered in the spyB deletion mutant, we analyzed the concentration of total carbohydrates in bacteria. The results of total carbohydrate analysis showed that there was no difference between MGAS5005 and 5005ΔspyB (Table S5).

To determine whether the monosaccharide composition of GAC is modified in 5005ΔspyB, we performed glycosyl composition analysis on cell wall material isolated from MGAS5005 and 5005ΔspyB. The analysis was carried out on two independent biological replicates. The Rha/GlcNa/N-acetylmuramic acid (MurNA) content of isolated cell wall fractions was determined by GC-MS analysis of the trimethylsilyl methyl-glycosides following methanolysis, mild hydrolysis, (0.8 N methanolic HCl, 80°C, 16 h) and re-N-acetylation of amino sugars (Chambers and Clamp, 1971). The second analysis (strong hydrolysis) included a strong acid pre-treatment (4 M HCl, 105°C, 3 h) to promote better hydrolysis of 2-amino sugars. The strong acid pretreatment improves recovery of monosaccharides linked to 2-amino sugars, but it is important to bear in mind that certain labile sugars, such as Rha, may be partially destroyed under these conditions. Mild cleavage conditions promoted the release of only 3–28% w/w carbohydrates from MGAS5005 cell wall (Table 1, Table S6 and Figure S4). The same hydrolytic conditions released 47–49% w/w carbohydrates from 5005ΔspyB cell wall (Table 1, Table S6 and Figure S4). The use of strong hydrolytic conditions promoted a significant increase in Rha, GlcNA and MurNA recoveries from MGAS5005 cell wall (Table 1, Table S6 and Figure S4), with yields of 64–92%. In contrast, strong hydrolysis was as affective as methanolysis in recovery of the carbohydrates from 5005ΔspyB cell wall (Table 1, Table S6 and Figure S4), with yields of 32–45%. This observation could indicate that the WT cell wall contains an increase in GlcNA side-chains, that are resistant to hydrolysis. The strong acid treatment would promote cleavage of the –GlcNA–Rha glycosidic linkage, yielding larger amounts of Rha and GlcNA in the WT strain compared with mild hydrolysis. Alternatively, there could be differences in de-acetylation of GlcNA because cleavage of the bond between de-acetylated GlcNA (GlcN) and Rha would require stronger acidic conditions than hydrolysis of the bond between neutral sugars GlcNA and Rha (Merkle and Poppe, 1994; Jennings et al., 2015). De-acetylation of GlcNA would be undetectable due to the N-acetylation step prior to GC-MS. Our binding experiments with GlcNA-specific lectin WGA supported the latter explanation. In conclusion our analyses indicated that there are differences in the cell wall composition of 5005ΔspyB compared with MGAS5005.

A major problem in studying SpyB function in vitro is the small size of this protein, making it challenging to produce recombinant protein for thorough biochemical analysis. To circumvent this problem, a fusion of maltose binding protein (MBP) to SpyB was constructed. We observed that MBP-SpyB was purified as a brown colored protein following its expression in Escherichia coli and purification by Ni-NTA affinity chromatography (Figures S5A,B). When expressed alone, purified MBP was not found in a colored form, implying that the chromophore associates with SpyB. We suggest that the MBP-SpyB is brown-colored because of the interaction between the protein and the chromophore, heme. To test this hypothesis we separated MBP-SpyB on a native PAGE gel followed by staining with tetramethylbenzidine, a chromogenic compound that changes color in the presence of heme-associated peroxidase activity. This activity was localized to MBP-SpyB, and it was absent from the MBP only control lane (Figure S5C).

The term heme refers to reduced, ferrous or Fe (II) iron protoporphyrin IX, whereas the term hemin refers to the oxidized, ferric or Fe(III) form of the molecule. E. coli displays a complete heme biosynthetic pathway, predominantly producing the ferrous form. We attempted to confirm that heme is associated with SpyB by analyzing hemin binding to synthetic SpyB peptide. The peptide was water-soluble, however the addition of hemin to SpyB significantly changed the protein solubility resulting in the precipitation of SpyB in aqueous solution (Figure 5A). Furthermore, analysis of the synthetic peptide mixed with hemin by size-exclusion chromatography was hampered by a strong non-specific interaction between SpyB and the resin, when we utilized a Superdex Peptide 10/300 GL (GE Healthcare Life Sciences) column. We were however, able to determine the affinity using tryptophan fluorescence quenching (Shepherd et al., 2007). As shown in Figure 5B (diamonds), hemin quenched tryptophan fluorescence of the synthetic SpyB in a dose-dependent manner. The resulting saturation curve allowed us to estimate a K_D-value of 2.6 × 10⁻⁶ ± 0.6 × 10⁻⁶ M. Next, we investigated whether other porphyrins, such as protoporphyrin IX (the iron-free precursor of heme) and vitamin B₁₂, bind SpyB. As shown in Figure 5B (squares), SpyB interacted with protoporphyrin IX with a K_D-value of 4.9 × 10⁻⁶ ± 0.8 × 10⁻⁶ M. In contrast, vitamin B₁₂ did not modulate the intrinsic fluorescence of SpyB, indicating that there is no interaction (data not shown).

Further investigations into hemin binding utilized the MBP-SpyB construct. Firstly, we obtained pure MBP-SpyB for use in recording the absorption spectra in the presence of hemin. Because SpyB is a cysteine-rich protein, dithiothreitol (DTT) was included in the buffers used for size-exclusion chromatography and for heme-binding assays. DTT helps in preventing formation of non-specific intramolecular disulfide linkages. We found that DTT-treated MBP-SpyB preparations obtained from recombinant E. coli were eluted in two peaks following size-exclusion chromatography, with the majority eluting in the lower molecular weight peak, which we predicted to be monomeric MBP-SpyB (Figure S6). Further analysis of the protein present in the small, high molecular weight peak identified MBP-SpyB associated with heme. Therefore, to demonstrate hemin binding by MBP-SpyB, we used the protein from the low molecular weight, heme-free peak. The absorption spectrum of hemin at pH 7.5 exhibited a broad peak between 355 and 400 nm (Figure 5C, solid line). When MPB-SpyB was added to the hemin solution, a double peak was observed with absorption maxima at 360 and 385 nm (dashed line). The addition of MBP shifted the peak but did not alter the shape of the hemin spectrum (Figure 5C, dotted line). The affinity of the hemin/MBP-SpyB interactions was determined by quenching of intrinsic tryptophan fluorescence (Shepherd et al., 2007). This method estimated that MBP-SpyB binds hemin with a K_D-value of 1.2 × 10⁻⁵ ± 0.4 × 10⁻⁵ M (Figure 5D, circles). The addition of hemin did not quench the fluorescence of MBP (Figure 5D, triangles), compared with the control N-acetyltryptophanamide (NATA) (crosses), which further demonstrates that MBP alone does not bind hemin. In addition, the Stern-Volmer plots for these data (Figure S7) showed weak dynamic quenching of MBP and NATA by hemin under our experimental conditions.

To gain insights into the mechanism of hemin binding to SpyB, we measured the dissociation rate constant using apomyoglobin as a hemin scavenger (Figure 5E). The hemin dissociation rate constant K_off for MBP-SpyB reconstituted with hemin was 1.8 × 10⁻³ ± 0.07 × 10⁻³ s⁻¹. The associate rate constant K_on = 1.5 × 10² ± 0.3 × 10² M⁻¹ s⁻¹ was calculated using the equilibrium constant K_D for MBP-SpyB. The data indicated that although hemin forms a stable complex with MBP-SpyB, it is not bound tightly. The K_D-value of hemin binding by MBP-SpyB is lower than the reported affinities of heme-binding proteins involved in heme scavenging from host (Ortiz de Orué Lucana et al., 2014). However, the K_D-value is within the range reported for some bacterial heme-sensing proteins, such as a regulator of porphyrin efflux system, PefR (Sachla et al., 2014), a repressor of photosystem genes, PpsR (Yin et al., 2012), light-regulated antirepressor, AppA (Yin et al., 2013), and a redox stress regulator, HbpS (Ortiz de Orué Lucana et al., 2014). Thus, these results suggest that SpyB plays a role in binding heme and possibly serves as an environmental sensor in GAS.

The significant change in synthetic SpyB peptide solubility and the chromatogram for MBP-SpyB suggested that hemin induces structural changes, likely dimerization, in SpyB. To investigate whether hemin binding affects the multimeric state of SpyB, we analyzed the purified heme-free MBP-SpyB (Figure 6A) mixed with hemin in a 1:4 ratio by size-exclusion chromatography. The size-exclusion chromatogram revealed two distinct peaks (Figure 6B). The absorbance at 385 nm, for detection of hemin, aligned only with the high molecular weight peak, and the protein that eluted during this peak had a brown color (Figure 6D). Dynamic light scattering analysis of the main fraction collected during elution of the high molecular weight peak confirmed that MBP-SpyB was in a dimeric state (Figure S8). To calculate the amount of hemin bound to MBP-SpyB dimer, we employed the pyridine hemochrome assay. We found that the ratio of MBP-SpyB:hemin was two per dimer (Figure S9). The calculated extinction coefficient of MBP-SpyB:hemin was 31405 M⁻¹ cm⁻¹.

Additionally, we analyzed whether protoporphyrin IX binding to SpyB induces protein dimerization. In contrast to hemin, reconstitution of MBP-SpyB with protoporphyrin IX did not significantly change the monomeric state of MBP-SpyB (Figure 6C), and the protoporphyrin IX largely dissociated from MBP-SpyB. Taken together, our data demonstrated that the association of iron-chelated porphyrin with SpyB induces MBP-SpyB dimerization.

SpyB contains four cysteines, which indicates their functional significance given the small size of the protein. Cysteine residues can form a covalent bond with the vinyl groups of heme (Bowman and Bren, 2008). They can also participate in non-covalent heme binding and/or disulfide bond formation. To examine the role of the cysteine residues in heme binding and dimerization we employed iodoacetamide (IAA) treatment followed by mass spectrometry (MS) analysis. IAA treatment causes the addition of a carbamidomethyl group to free cysteines. Using this method, we detected that treatment of MBP-SpyB dimer with DTT followed by incubation with IAA resulted in the modification of all four cysteines. In contrast, treatment of MBP-SpyB dimer with IAA alone generated a protein with the first and second cysteine residues modified by IAA. The MS analysis identified two intermolecular disulfide bonds involving the third and fourth cysteines (Figure 7A).

From this experiment, we concluded that SpyB cysteine residues do not participate in the covalent binding of heme. To test whether the first and second cysteines are involved in non-covalent binding of heme, we generated double (C7A/C13A) and quadruple (C7A/C13A/C30A/C35A) mutant proteins. We noticed that only the quadruple mutant protein was colorless following expression in E. coli and purification by Ni-NTA affinity chromatography and size-exclusion chromatography (Figure 6D). In addition, the quadruple mutant protein was also eluted from the size-exclusion column as a single peak corresponding to a monomeric state of SpyB (data not shown). The double mutant protein had a pale brown color after the two purification steps (Figure 6D) and eluted from the size-exclusion column as a dimer and monomer peak (data not shown). The mutant proteins were reconstituted with hemin as described in Materials and Methods Section and analyzed by size-exclusion chromatography. As shown in Figures 7B–D, hemin induced dimerization of both mutant variants. However, the removal of all the cysteines resulted in an elution profile displaying a major monomer peak and a significantly smaller dimer peak. Analysis of hemin content in the dimers of the mutant SpyB proteins by pyridine hemochrome assay displayed no changes in the MBP-SpyB/hemin ratio (Figure S9). Tryptophan fluorescence quenching determination of hemin binding affinities for the mutant variants revealed that the removal of cysteines led to a significant decrease in affinity for hemin (Table 2). The Stern-Volmer plots (Figure S7) further demonstrated the decrease in quenching capacity of hemin for the double and quadruple mutants compared with WT. Taken together, our data demonstrated that SpyB cysteine residues are involved in disulfide bond formation between SpyB subunits and they contribute to non-covalent heme association with the protein.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.