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7 January 2015

A Conserved Domain Is Crucial for Acceptor Substrate Binding in a Family of Glucosyltransferases


Serine-rich repeat glycoproteins (SRRPs) are highly conserved in streptococci and staphylococci. Glycosylation of SRRPs is important for bacterial adhesion and pathogenesis. Streptococcus agalactiae is the leading cause of bacterial sepsis and meningitis among newborns. Srr2, an SRRP from S. agalactiae strain COH1, has been implicated in bacterial virulence. Four genes (gtfA, gtfB, gtfC, and gtfD) located downstream of srr2 share significant homology with genes involved in glycosylation of other SRRPs. We have shown previously that gtfA and gtfB encode two glycosyltransferases, GtfA and GtfB, that catalyze the transfer of GlcNAc residues to the Srr2 polypeptide. However, the function of other glycosyltransferases in glycosylation of Srr2 is unknown. In this study, we determined that GtfC catalyzed the direct transfer of glucosyl residues to Srr2-GlcNAc. The GtfC crystal structure was solved at 2.7 Å by molecular replacement. Structural analysis revealed a loop region at the N terminus as a putative acceptor substrate binding domain. Deletion of this domain rendered GtfC unable to bind to its substrate Srr2-GlcNAc, concurrently abolished the glycosyltransferase activity of GtfC, and also altered glycosylation of Srr2. Furthermore, deletion of the corresponding regions from GtfC homologs also abolished their substrate binding and enzymatic activity, indicating that this region is functionally conserved. In summary, we have determined that GtfC is important for the glycosylation of Srr2 and identified a conserved loop region that is crucial for acceptor substrate binding from GtfC homologs in streptococci. These findings shed new mechanistic insight into this family of glycosyltransferases.


Streptococcus agalactiae, also known as group B Streptococcus (GBS), has gained worldwide attention over the past few decades because of its pathogenicity in newborns and pregnant women (1, 2). GBS is the leading cause of sepsis and meningitis in newborns due to its ability to adhere to the mother's vaginal tract (3). It also has caused an increasing infection rate in adults, including immunocompromised patients, the elderly, and diabetics (4). In all cases, the bacteria must adhere to the host cell surface first before the virulence is induced and pathogenesis ensues. Two serine-rich repeat glycoproteins (Srr1 and Srr2) identified in various S. agalactiae strains (5, 6) are recognized to mediate bacterial attachment to the host cell surface (3, 4, 7, 8).
Serine-rich repeat glycoproteins (SRRPs) belong to a growing family of adhesins in Gram-positive bacteria, and many of them contribute to bacterial pathogenesis (9). Besides Srr1 and Srr2, the SRRP family also includes PsrP of Streptococcus pneumoniae (10), Fap1 of Streptococcus parasanguinis (11), GspB and Hsa of Streptococcus gordonii (12, 13), and SraP of Staphylococcus aureus (14). A unique feature shared among these SRRPs is that all SRRPs are glycosylated and that glycosylation plays a central role in the biogenesis and pathogenesis of SRRPs (9, 15, 16). Therefore, it is important to understand the glycosylation mechanism of the SRRPs. Genes involved in the glycosylation process have been studied for various SRRPs over the past few years (12, 1630). However, little is known about the genes involved in the glycosylation of Srr2. Srr2 is a surface protein found in hypervirulent serotype III GBS strains (6), while another SRRP, Srr1, is present in strains that are commonly associated with neonatal infection such as the Ia, Ib, V, and certain III serotype groups. Like other SRRP genes, the gene encoding Srr1 or Srr2 is located within a conserved gene cluster that contains two regions: a core region, which is highly conserved in every SRRP locus (9, 16, 31), and a variable region (16). The core region contains two essential glycosyltransferases, GtfA and GtfB, and several accessory secretory components, SecA2, SecY2, Asp1, Asp2, and Asp3. The variable region contains a number of putative glycosyltransferases, which are species and strain dependent. For instance, the organization of the glycosyltransferases is different between S. agalactiae strains that contain the serine-rich proteins Srr1 and those that contain Srr2 (6, 16). The number of glycosyltransferases in this locus differs from the number in the PsrP locus from various S. pneumoniae strains (16).
In the variable region, a putative glycosyltransferase, gtfC, from the hypervirulent GBS strain COH1 is located at the 3′ end of the srr2 locus and lies downstream of gtfA and gtfB (16) (see Fig. S1 in the supplemental material). GtfC is a homolog of a glucosyltransferase (Gtf3) from S. parasanguinis, which belongs to a new subfamily of glycosyltransferases (28). Despite the fact that the catalytic domain of Gtf3 has been well characterized (28), there is no study determining the acceptor substrate binding site of Gtf3 or Gtf3-like glycosyltransferases. Functional acceptor substrate binding sites are hallmarks of all glycosyltransferases and crucial for enzyme specificity and activity; however, they do not share sequence homology among different families of glycosyltransferases, and thus, it is difficult to infer the binding sites through the primary sequence alignment. In this study, we determined the three-dimensional X-ray crystal structure of GtfC and demonstrated its glucosyltransferase feature. Biochemical studies have also revealed that GtfC catalyzes the second step of the Srr2 glycosylation by transferring glucosyl residues to GlcNAc-modified Srr2. Structural analysis revealed that a flexible loop region is required for the substrate binding. Structure-based mutagenesis and functional studies have determined that the key loop region is required for binding to the acceptor substrate. Furthermore, this loop region is functionally conserved in this family of glycosyltransferases.


Bacterial strains and plasmids.

The bacterial strains and plasmids used in this study are listed in Table 1. Antibiotics were used at the following concentrations: 10 μg/ml erythromycin (Erm) and 1,500 μg/ml kanamycin (Kan) in Todd-Hewitt (TH) broth or agar plates for S. agalactiae; 300 μg/ml erythromycin, 50 μg/ml kanamycin, and 250 μg/ml chloramphenicol (Cm) in Luria-Bertani (LB) broth or agar plates for Escherichia coli. Isolation of plasmid DNAs was carried out with a QIAprep Miniprep kit (Qiagen). The primers used in this study are listed in Table 2. PCR was carried out with Taq DNA polymerase (Promega) or KOD DNA polymerase (Novagen). PCR products were purified with a QIAquick PCR purification kit (Qiagen). DNA digestion, ligation, and transformation were performed using standard methods. Competent cells for S. agalactiae electroporation were prepared as described previously (32).
TABLE 1 Strains and plasmids used in this study
Strain or plasmidRelevant propertiesSource or reference
    E. coli strain Top10Host for propagation of the recombinant plasmidsInvitrogen
    E. coli strain BL21-Gold(DE3)pET system host strainStratagene
    S. agalactiae COH1Wild type43
    S. agalactiae COH1 srr2 deletion mutantWild type; srr2::aphA3; KanrThis study
    S. agalactiae COH1 gtfC deletion mutantWild type; gtfC knockout; gtfC::aphA3This study
    Strains expressing protein:  
        GST-Srr2-GlcNAcpGEX-Srr2 and pVPT-GtfAB(COH1) transformed into Top10This study
        GST-Srr2pGEX-Srr2 transformed into Top10This study
        His-GtfCpET-SUMO-GtfC transformed into BL21This study
        His-GtfCΔ106-111aapET-SUMO-GtfCΔ106-111 transformed into BL21This study
        GST-GtfCpGEX-GtfC transformed into Top10This study
        GST-GtfCΔ106-111aapGEX-GtfCΔ106-111 transformed into Top10This study
        GST-rFap1-GlcNAcpGEX-rFap1 and pVPT-GtfAB transformed into Top1025
        His-Gtf3pET-SUMO-Gtf3 transformed into BL2128
        His-Gtf3Δ106-111aapET-SUMO-Gtf3Δ106-111 transformed into BL21This study
        His-Psrp-GlcNAcpGEX-GtfAB-His-Psrp transformed into Top1044
        GST-GtfC(TIGR4)pGEX-Gtf3(TIGR4) transformed into Top1028
        GST-GtfC(TIGR4)Δ106-111aapGEX-Gtf3(TIGR4)Δ106-111transformed into Top10This study
    pGEX-6p-1GST fusion protein expression vector; AmpraAmersham
    pET-SUMOHis fusion protein expression vector; Kanr28
    pGEX-GtfCgtfC cloned in pGEX-6p-1; AmprThis study
    pGEX-GtfCΔ106-111aagtfC with 106-111aa deletion cloned in pET28-SUMOThis study
    pGEX-GtfC(TIGR4)gtfC from TIGR4 cloned in pGEX-6p-128
    pGEX- GtfC(TIGR4)Δ106-111aa106-111aa deletion of pGEX-GtfC(TIGR4)This study
    pET-SUMO-GtfCgtfC cloned in pET28-SUMOThis study
    pET-SUMO-GtfCΔ106-111aa106-111aa deletion of pET-SUMO-GtfCThis study
    pET-SUMO-Gtf3Δ106-111aa106-111aa deletion of pET-SUMO-Gtf3This study
    pVPT-GtfCgtfC cloned in pVPT-GFP; ErmrThis study
    pVPT-GtfCΔ106-111aa106-111aa deletion of pVPT-GtfC; ErmrThis study
    pVPT-GFPE. coli-streptococci shuttle vector; Ermr45
    pJL1055E. coli-streptococci shuttle vector; Cmr32
    pJL1055-ΔgtfC-aphA3pJL1055 vector containing gtfC with aphA3 insertionThis study
Amp, ampicillin.
TABLE 2 Primers used in this study
Underlining in the sequence refers to the underlined restriction enzyme site in the first column.

In vitro glycosylation assay.

To prepare substrates and enzymes for in vitro glycosyltransferase assays, an Srr2 DNA fragment (bp 1 to 3252) was amplified from genomic DNA of S. agalactiae using primer pair Srr2-1F-BamHI and Srr2-3252R-XhoI. The amplified fragment was digested by BamHI and XhoI and then ligated into pGEX-6P-1 to construct pGEX-Srr2. A plasmid harboring two glycosyltransferases, GtfA and GtfB, and a pGEX-Srr2 plasmid were cotransformed into E. coli Top10 cells to yield a strain carrying Srr2/GtfAB. This strain was used to generate recombinant GlcNAc-modified Srr2 substrate. To produce the enzyme, the strain harboring pGEX-GtfC was used to purify GtfC. The purification was completed using glutathione-Sepharose 4B beads (Amersham Biosciences) according to the manufacturer's instructions.
To examine the importance of the potential acceptor substrate binding domain, a truncated GtfC with deletion of amino acids 106 to 111 (Δ106-111aa) was created by inverse PCR. In brief, the plasmid pGEX-6p-1-GtfC was used as a template. The primers used for the mutagenesis are listed in Table 2. Mutant alleles were confirmed by sequencing. The resulting plasmids were transformed into E. coli Top10 for protein expression.
To examine the importance and conservation of this domain, the same deletion (Δ106-111aa) allele was amplified by inverse PCR usingpGEX-6p-1-GtfC/Gtf3 or pET-SUMO-GtfC/Gtf3 forGtfC homologs from S. parasanguinis FW213 and S. pneumoniae TIGR4 as described above.
In vitro glycosyltransferase assays were performed as previously described (25). Briefly, the substrate and enzyme bound to glutathione-Sepharose beads were washed five times with glycosylation buffer (50 mM HEPES, pH 7.0, 10 mM MnCl2, 0.01% bovine serum albumin) and then incubated with 0.2 μCi of UDP-[3H] glucose (28 Ci/mmol; American Radiolabeled Chemicals, Inc.) at 37°C. The beads in the glycosylation assays were washed three times with NETN buffer (20 mM Tris-HCl [pH 7.2], 150 mM NaCl, 1 mM EDTA, 0.2% NP-40) and then transferred to scintillation vials (read by liquid scintillation counter) to measure the radioactivity transferred to the substrates from the radiolabeled activated sugars. Heat-inactive enzymes were used as a negative control. The assays were performed in triplicate in three independent experiments.

Protein expression, purification, and crystallization.

The full-length gene gtfC was amplified from genomic DNA of S. agalactiae using primer set GtfC-NotI-F and GtfC-XhoI-R (Table 1). The PCR product was purified and cloned into pET-SUMO. The resulting plasmid pET-SUMO-GtfC was transformed in E. coli BL21 Gold (DE3) cells. The recombinant strain was expressed and purified as described previously (27). GtfC was crystallized at 20°C by using the hanging-drop vapor diffusion method. Each drop contains 1 μl of protein solution (30 mg/ml protein and 10 mM UDP-glucose) with 1 μl reservoir (14% polyethylene glycol 3350, 0.3 M ammonium sulfate, 0.1 M Tris [pH 8.5], 15% glycerol). The crystals were cryoprotected by the addition of 25% glycerol and flash-frozen in liquid nitrogen.

Data collection and structure determination and refinement.

A single crystal was flash-vitrified in liquid nitrogen using 10% glycerol as a cryoprotectant. Diffraction data were collected on SER-CAT beamline 22-ID at the Argonne National Laboratory (APS). The data were collected using a MAR 300 charge-coupled-device (CCD) detector. During data collection, the crystal-to-detector distance was kept at 200 mm. A total of 360 images covering a Phi range of 360° were collected and processed using HKL2000 (33). X-ray diffraction data and refinement statistics are listed in Table 3. The structure of GtfC was determined by molecular replacement in the Phenix (34) using Gtf3 (27) (PDB entry 3QKW) as the template. Model building and structure refinement were carried out using Autobuild and Refinement in Phenix. The structure was validated in Procheck (35).
TABLE 3 Data collection and refinement statistics for GtfC
ParameterResult for GtfCa
Resolution range (Å)29.91–2.70 (2.85–2.7)
Space groupP212121
Unit cell (Å)a = 77.204, b = 99.27, c = 188.214
Unit cell (°)α = β = γ = 90
No. of unique reflections84,174 (7,971)
Multiplicity5.6 (3.5)
Completeness (%)98.03 (94.00)
Mean I/sigma(I)16.241 (2.227)
Wilson B-factor33.91
R-merge0.090 (0.479)
    R-work0.1867 (0.4354)
    R-free0.2774 (0.4114)
No. of nonhydrogen atoms10,892
    No. of macromolecules10,419
    No. of ligands100
    No. of water molecules373
Protein residues1296
    RMS (bonds)0.009
    RMS (angles)1.34
Clash score16.97
Values in parentheses are for the highest-resolution shell.

In vivo genetic complementation.

A gtfC mutant was generated by allele replacement mutagenesis with a kanamycin resistance cassette, aphA-3 (30). Briefly, the gtfC gene was amplified from genomic DNA of S. agalactiae COH1 and cloned to an E. coli-Streptococcus shuttle/suicide vector, pJL1055 (32), to yield plasmid pJL1055-GtfC. A 100-bp gtfC internal fragment from pJL1055-gtfC was deleted by inverse PCR using primers gtfC-in-F and gtfC-in-R. The inverse PCR product was ligated with aphA3 from pALH124 to generate pJL1055-ΔgtfC-aphA3.The resulting plasmid pJL1055-ΔgtfC-aphA3 was transformed to S. agalactiae COH1 competent cells by electroporation (32). The transformants were selected on TH agar plates containing chloramphenicol and kanamycin at 28°C first. Then, single colonies were incubated in TH broth containing chloramphenicol at 28°C until the optical density at 470 nm (OD470) reached 0.6. The bacterial culture was then transferred to 37°C for a further 3-h incubation. Finally, the gtfC insertion mutant was obtained by the selection of kanamycin-resistant colonies.
To make gtfC complementation, the full length of gtfC gene was cloned to pVPT-gfp vector and then transformed into the gtfC knockout strain via electroporation. The transformants were selected on TH agar plates containing kanamycin and erythromycin. All gene constructs and mutants were verified by PCR and DNA sequencing.

GST pulldown assays.

To examine in vitro protein interactions, glutathione S-transferase (GST) pulldown assays were used to determine the acceptor substrate binding site of GtfC and its homologs. Three groups of recombinant proteins, i.e., (i) GST-Srr2-GlcNAc, His-GtfC, and His-GtfCΔ106-111aa, (ii) His-Psrp-GlcNAc, GST-GtfC (TIGR4), and GST-GtfC (TIGR4)Δ106-111aa, and (iii) GST-rFap1-GlcNAc, His-Gtf3 and His-Gtf3Δ106-111aa, were prepared as follows. The GST fusion proteins were bound to glutathione-Sepharose 4B beads (Amersham). Five micrograms of the glutathione-Sepharose-GST fusion proteins was mixed with 5 μl of the His-tagged proteins in the presence of pulldown binding buffer (20 mM Tris-HCl [pH 7.2], 100 mM NaCl, 1 mM EDTA, 0.2% NP-40) and incubated overnight on a rotary shaker at 4°C. The beads were washed three times with 600 μl NETN buffer (20 mM Tris-HCl [pH 7.2], 150 mM NaCl, 1 mM EDTA, 0.2% NP-40), and the proteins were eluted with SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer, boiled for 10 min, and subjected to Western blotting with anti-His or anti-GST antibody.

Statistical analysis.

Results are presented as the means ± standard deviations (SD). Differences between groups were determined with the use of Student t tests or 1-way analysis of variance (ANOVA) where appropriate. Significance was defined at P values of <0.05.


GtfC mediates the second step of the Srr2 glycosylation.

GtfC shares 57.7% protein sequence identity to Gtf3 from S. parasanguinis (25, 28). Gtf3 is a glycosyltransferase that mediates the second step of Fap1 glycosylation by transferring glucose residues to Fap1-GlcNAc (25). To determine whether GtfC is responsible for the second step of Srr2 glycosylation, a well-established in vitro glycosylation assay (25) was performed using 3H-labeled UDP-glucose as a sugar donor. The recombinant GtfC and GlcNAc-modified Srr2 were purified and used as an enzyme and a substrate, respectively. As shown in Fig. 1, the active GtfC enzyme can transfer 3H-labeled glucose from UDP-glucose to Srr2-GlcNAc. Heat inactivation of GtfC abolished its enzymatic activity. Use of unmodified Srr2 as a substrate failed to support the transfer. These results demonstrated that GtfC directly catalyzed the transfer of glucose to GlcNAc-modified Srr2 but not to the unmodified Srr2 protein, suggesting that GtfC catalyzes the second step of the Srr2 glycosylation. To further determine the specificity of the sugar donors, we also performed UDP-Glo glycosyltransferase assays (36) using UDP-GlcNAc or UDP-galactose as an alternate sugar donor (see Fig. S2 in the supplemental material). Only UDP-Glc promoted the transfer of the Glc to the Srr2-GlcNAc by GtfC, further demonstrating that GtfC is specific for glucosyl residues.
FIG 1 GtfC is a glucosyltransferase that directs the second step of the Srr2 glycosylation. An in vitro glycosyltransferase assay (25) was used to determine whether GtfC is a glucosyltransferase. 3H-labeled UDP-glucose was transferred to Srr2-GlcNAc but not unmodified Srr2. CPM, counts per minute; HI, heat inactivated. Significant differences are indicated by asterisks (**, P < 0.01; ***, P < 0.001).

GtfC possesses a typical bacterial glycosyltransferase structural fold.

The structure of GtfC was determined at 2.7-Å resolution in a space group of P212121with four monomers per asymmetric unit (Table 3). Like its homolog Gtf3, GtfC exists as a tetramer with two very similar dimers (28). Four monomers of GtfC form tight binding interfaces with each other (Table 4; Fig. 2A).
TABLE 4 Interface statistics for GtfC
ChainNo. of interface residuesInterface area (Å2)No. of salt bridgesNo. of hydrogen bondsNo. of nonbonded contacts
C:D23:241,062:1,052 17136
A:C7:6285:292  20
B:D8:6304:312  32
A:D20:201,180:1,185 6120
B:C20:211,192:1,218 5110
FIG 2 Overall structure of GtfC. (A) Structural overview of the GtfC tetramer. The four molecules are colored as follows; molecule A, green; molecule B, cyan; molecule C, purple; molecule D, yellow. AD and BC interfaces share similar features through the long helix that links the N and C termini in each monomer. (B) Structural overview of a GtfC monomer. A single GtfC molecule structure is shown in cartoon. The loop region is shown in red, and the UDP ligand is shown in ball-and-stick motifs.
Each monomer contains a Rossmann fold on both the N terminus and the C terminus, which is a typical feature for the GT-B type glycosyltransferase. The N-terminal domain (residues 1 to 150) and the C-terminal domain (residues 170 to 330) are linked by a long loop (residues 151 to 169) (Fig. 2B). Each monomer binds a UDP molecule. Weak or no electron density was observed for a loop region (Met-106 to Phe-111) in some monomers of GtfC as illustrated in Fig. 2B. Degree of flexibility (openness) and residue orientation (hydrophobicity) are two main characteristic features for a potential substrate binding site (3739). Surface-exposed Phe-111 might provide the potential interaction site, and the flexibility of the loop may allow it to adapt the right orientation to bind to its substrate. Therefore, we hypothesize that this flexible loop region, which is also seen in the structure of its homolog Gtf3 (see Fig. S3 in the supplemental material), is important for the acceptor substrate binding.

The loop region in GtfC is critical for its substrate binding and its enzymatic activity.

To determine if the loop region (Met-106 to Phe-111) is involved in the acceptor substrate binding, we performed site-directed mutagenesis in this region and then evaluated its binding to the acceptor substrate, GlcNAc-modified Srr2, using a GST pulldown assay. The GST pulldown assay was performed using purified recombinant proteins GST-Srr2-GlcNAc, His-GtfC, and His-GtfCΔ106-111aa. As shown in Fig. 3A, His-GtfC was pulled down from GST-Srr2-GlcNAc but deletion of the loop region 106-111aa failed to support the binding, indicating that the loop region is crucial for acceptor substrate binding. Of note, the deletion of the loop region does not affect the protein stability of GtfC (see Fig. S4 in the supplemental material). To examine whether the substrate binding is essential for GtfC enzymatic activity, a GtfC variant lacking the loop region (aa 106 to 111) was examined for in vitro enzymatic activity. GtfC without the putative acceptor substrate binding region failed to transfer glucosyl residues to the Srr2-GlcNAc substrate in vitro (Fig. 3B), suggesting the importance of this domain in GtfC enzymatic activity.
FIG 3 The loop region (aa 106 to 111) of GtfC is important for its binding to acceptor substrate and its enzymatic activity. (A) Deletion of the GtfC loop region abolished its binding to its acceptor substrate, Srr2-GlcNAc. Equal amounts of purified His-GtfC and His-GtfCΔ106-111 were incubated with GST-Srr2-GlcNAc, which was bound to glutathione-Sepharose 4B beads. The pulldown protein complexes were analyzed by Western blotting using anti-His monoclonal antibody. Input represents the His-tagged proteins. (B) Deletion of the GtfC loop region abolished its enzymatic activity. The GtfC variants were used to determine enzymatic activity using an in vitro glycosyltransferase assay. Significant differences are indicated by asterisks (***, P < 0.001).

GtfC and its acceptor substrate binding are important for the maturation of Srr2.

To determine the role of GtfC in the Srr2 maturation, we constructed a GtfC insertional mutant and examined the production of Srr2 using polyclonal anti-Srr2 antibody. As shown in Fig. 4, Srr2 from the GtfC mutant migrated faster than the mature Srr2 from the wild-type strain (lanes 1 and 3), indicating that some modification on Srr2 is missing in the GtfC mutant. The migration of Srr2 was restored when Gtf3 was complemented (lane 4), further suggesting that GtfC plays an important role in the maturation of Srr2. The complementation allele that had the potential substrate binding domain deleted failed to restore the production of mature Srr2 (lane 5), suggesting that the acceptor substrate binding is critical for the Srr2 maturation.
FIG 4 GtfC and its acceptor substrate binding are important for the maturation of Srr2. Wild-type GtfC and GtfCΔ106-111aa cloned in E. coli-streptococcal shuttle vector pVPT-GFP were used to transform the gtfC mutant to construct the gtfC mutant variants. Whole-cell lysates prepared from the same number of bacterial cells of wild-type bacteria (WT), srr2 mutant (srr2-), gtfc mutant (gtfC-), and the gtfC mutant transformed with the wild-type gtfC gene (gtfC-/pVPT-gtfC) and with the substrate binding-deficient mutant (gtfC-/pVPT-gtfCΔ106-111aa) were subjected to Western blot analysis using Srr2-specific antibody. A homolog of FimA, Lmb (46), determined by anti-FimA antibody was used as a loading control.

The loop region crucial for acceptor substrate binding is conserved in streptococci.

GtfC homologs belong to a new family of glycosyltransferases and are highly conserved in streptococci that possess SRRPs (25, 28). To determine whether the acceptor substrate binding site is conserved as well, deletion of the loop region (aa 106 to 111) in Gtf3 from S. parasanguinis FW213 and GtfC from S. pneumoniae TIGR4 was carried out and the binding ability of each deletion was evaluated by GST pulldown assay. As shown in Fig. 5A, wild-type Gtf3 or GtfC was able to bind to its own substrate but the loop region deletion mutant abolished the binding ability, suggesting that the loop region represents a conserved acceptor substrate binding among these streptococci. To further determine the importance of the conserved substrate binding site of these GtfC homologs, we performed in vitro glycosyltransferase assays. The variants lacking the loop region (aa 106 to 111) were constructed and examined for in vitro enzymatic activity. Gtf3 from S. parasanguinis FW213 or GtfC from S. pneumoniae TIGR4 without the putative substrate binding domain failed to support the transfer of glucosyl residues to its substrate in vitro (Fig. 5B), furthering supporting that the acceptor substrate binding is critical for the enzymatic activity of this family of glycosyltransferases.
FIG 5 The loop region crucial for acceptor substrate binding is functionally conserved in streptococci. (A) Deletion of the Gtf3 (FW213) and GtfC (TIGR4) loop regions abolished binding to their acceptor substrates rFap1-GlcNAc and Psrp-GlcNAc, respectively. Equal amounts of purified His-Gtf3 and His-Gtf3Δ106-111 were incubated with GST-Fap1-GlcNAc, which was bound to glutathione-Sepharose 4B beads. Equal amounts of GST-GtfC and GtfCΔ106-117aa were incubated with His-Psrp-GlcNAc. The pulldown protein complexes were analyzed by Western blotting using anti-His monoclonal antibody. Inputs represent the His-tagged Gtf3 variants or GST-tagged GtfC variants. (B) Deletion of the Gtf3 (FW213) and GtfC (TIGR4) loop regions abolished their enzymatic activity. Gtf3 (FW213) or GtfC (TIGR4) variants were used to determine enzymatic activity using an in vitro glycosyltransferase assay. Significant differences are indicated by asterisks (***, P < 0.001).


The serine-rich repeat glycoproteins (SRRPs), a family of adhesins produced by Gram-positive bacteria, have emerged as important virulence factors (9). Srr2, one of the SRRPs found in serotype III GBS strains, plays a key role in binding fibrinogen and promotes GBS attachment to endothelial cells and bacterial virulence (8). Glycosylation of SRRPs is crucial for bacterial pathogenesis (15). Thus, characterization of the individual enzymes involved in the glycosylation of Srr2 is important for our understanding of the biosynthetic pathway of this family of glycoproteins and for developing effective vaccines targeting GBS infection.
Four genes, gtfA, gtfB, gtfC, and gtfD, downstream of srr2 share significant homology with genes involved in glycosylation of SRRPs. In previous studies, we have demonstrated that gtfA and gtfB encode two proteins, GtfA and GtfB, which function in concert to transfer the first sugar residue, GlcNAc, to the Srr2 polypeptide backbone. But the role of GtfC in the glycosylation of Srr2 in GBS is unknown. In the current study, we employed biochemical and structural biology approaches and obtained evidence that supports the role of GtfC in the glycosylation of Srr2. GtfC directs the second step of Srr2 glycosylation by transferring glucose to GlcNAc-modified Srr2; this datum is consistent with our early finding that GtfC was able to transfer glucose to GlcNAc-modified Fap1 (Srr2-like protein) (27). These results indicate that GtfC is a glucosyltransferase.
Furthermore, structural analysis of GtfC has revealed that GtfC has a typical bacterial glycosyltransferase GT-B structural fold. The structure is very similar to that of Gtf3 from S. parasanguinis (root mean square deviation [RMSD], 0.48). Both GtfC and Gtf3 structures have a UDP molecule in each monomer and share key residues in the UDP binding pocket (see Fig. S3 in the supplemental material). And they also have one flexible region/loop close to the sugar-nucleotide binding site, which is common in many glycosyltransferases. However, the exact locations and primary amino acid sequences of this type of loop differ significantly among different families of glycosyltransferases (39, 40), which makes it difficult to predict the substrate binding region in the absence of structural information. Due to its openness and hydrophobicity, the flexible loop is predicted to be an acceptor substrate binding site, and it is believed that the closure of the flexible loop leads to the complete formation of the acceptor recognition site (3739, 41). The flexible loop has been demonstrated to play an important role in substrate binding in both GT-A family and bacterial sialyltransferases (40). However, few studies have investigated the loop region thoroughly in the GT-B family because the loop region is flexible and often does not present clear electron density in the absence of acceptor substrates. In this study, through structure-directed mutagenesis and GST pulldown studies, we demonstrated the flexible loop region (Met-106 to Phe-111) in GtfC is crucial for acceptor substrate binding. The deletion of this region affects the maturation of Srr2. Furthermore, the same loop region also plays a critical role in acceptor substrate binding in this family of glycosyltransferases from other Gram-positive bacteria such as Gtf3 in S. parasanguinis and GtfC in S. pneumoniae, suggesting the functional conservation and importance of this loop region. There are some amino acid differences across this region (see Fig. S5 in the supplemental material), which could be responsible for the binding specificity of the acceptor substrate. Indeed, GtfC from S. pneumoniae TIGR4 failed to transfer glucose to GlcNAc-modified Fap1 but was able to transfer glucose to its native substrate, GlcNAc-modified rPsrP (28). This is also consistent with the notion that the N-terminal domains of GT-B family members are more variable sequence-wise than the C-terminal domains, because the loop regions in the N-terminal domain have evolved to accommodate different acceptors (42).
Taken together, we found that GtfC is a glucosyltransferase that is important for glycosylation of Srr2. The loop region at the N terminus is critical for acceptor substrate binding, and this putative substrate binding site is also conserved in this new subfamily of glycosyltransferases from other streptococci. These findings provide valuable insights into the study of glycosylation mechanisms of SRRPs in GBS.


We thank Jeanine Brady for providing us with anti-Srr2 antibody and Craig Ruben for the S. agalactiae COH1 strain.
This study was supported by NIH/NIDCR F33DE022215 and R01DE017954 (H. Wu).

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Published In

cover image Journal of Bacteriology
Journal of Bacteriology
Volume 197Number 31 February 2015
Pages: 510 - 517
Editor: V. J. DiRita
PubMed: 25404702


Received: 1 September 2014
Accepted: 12 November 2014
Published online: 7 January 2015


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Fan Zhu
Department of Pediatric Dentistry, Schools of Dentistry and Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA
Department of Microbiology, Schools of Dentistry and Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA
Hua Zhang
Department of Pediatric Dentistry, Schools of Dentistry and Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA
Hui Wu
Department of Pediatric Dentistry, Schools of Dentistry and Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA
Department of Microbiology, Schools of Dentistry and Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA


V. J. DiRita


Address correspondence to Hui Wu, [email protected].

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