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Keywords:

  • Streptococcus gordonii;
  • phosphoglucosamine mutase;
  • bacterial cell morphology;
  • biofilm;
  • antibiotics;
  • bacterial growth

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Phosphoglucosamine mutase (EC 5.4.2.10) catalyzes the interconversion of glucosamine-6-phosphate into glucosamine-1-phosphate, an essential step in the biosynthetic pathway leading to the formation of peptidoglycan precursor uridine 5′-diphospho-N-acetylglucosamine. The gene (glmM) of Escherichia coli encoding the enzyme has been identified previously. We have now identified a glmM homolog in Streptococcus gordonii, an early colonizer on the human tooth and an important cause of infective endocarditis, and have confirmed that the gene encodes phosphoglucosamine mutase by assaying the enzymatic activity of the recombinant GlmM protein. Insertional glmM mutant of S. gordonii did not produce GlmM, and had a growth rate that was approximately half that of the wild type. Morphological analyses clearly indicated that the glmM mutation causes marked elongation of the streptococcal chains, enlargement of bacterial cells, and increased roughness of the bacterial cell surface. Furthermore, the glmM mutation reduces biofilm formation and increases sensitivity to penicillins relative to wild type. All of these phenotypic changes were also observed in a glmM deletion mutant, and were restored by the complementation with plasmid-borne glmM. These results suggest that, in S. gordonii, mutations in glmM appear to influence bacterial cell growth and morphology, biofilm formation, and sensitivity to penicillins.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Streptococcus gordonii and other closely related species comprise a numerically prominent group of oral bacteria that occur primarily on the human tooth surface as members of the biofilm community, commonly referred to as dental plaque (Gibbons & van Houte, 1980; Gibbons, 1984; Hsu et al., 1994). Streptococcus gordonii coaggregates with other oral bacteria including other streptococci (Whittaker et al., 1996), actinomycetes (Cisar et al., 1995; Palmer et al., 2003), and Porphyromonas gingivalis, one of the periodontal pathogens (Lamont et al., 1992; Park et al., 2005), contributing to colonization of these bacteria on the tooth surface. Although the cariogenicity of S. gordonii is less appreciated than that of Streptococcus mutans, its cariogenicity in specific pathogen-free rats has been reported (Tanzer et al., 2001). Besides the oral infectious diseases, S. gordonii is well known for its ability to colonize damaged heart valves and is among the bacteria most frequently identified as the primary etiological agents of infective endocarditis (Baddour et al., 1989; Baddour, 1994; Durack, 1995; Takahashi et al., 2006).

Over the last decade, several strategies to control biofilm growth on medical devices have been suggested, including the use of topical antimicrobial ointments, minimizing the duration of catheterization, using catheters with a surgically implanted cuff (Flowers et al., 1989), and coating the catheter lumen with antimicrobial agents (Soboh et al., 1995; Stickler et al., 1996; Darouiche et al., 1999; Johnson et al., 1999; Ahearn et al., 2000; Domenico et al., 2001). Enzymes involved in bacterial cell wall synthesis could be novel targets for the development of antibiofilm agents (Cerca et al., 2005; Sheldon, 2005). Antibiotics are also used to prevent infective endocardits following dental and surgical interventions (Gould et al., 2006). On the other hand, the susceptibility of α-hemolytic streptococci, including S. gordonii, to certain β-lactam antibiotics has been reported (Wilcox et al., 1993).

Uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) is an essential common precursor for the synthesis of bacterial cell-wall peptidoglycan (van Heijenoort, 1996) and outer membrane lipopolysaccharide (Raetz, 1986). The metabolic route leading to the formation of UDP-GlcNAc constitutes a potential site for regulating the flow of metabolites going through the peptidoglycan and lipopolysaccharide pathways. Phosphoglucosamine mutase (GlmM, EC 5.4.2.10) catalyzes the interconversion of glucosamine-6-phosphate into glucosamine-1-phosphate, the first step in the biosynthetic pathway leading to the formation of UDP-GlcNAc (Jolly et al., 1999). Significantly, a mutation in the glmM gene affects both peptidoglycan and lipopolysaccharide synthesis in Escherichia coli (Mengin-Lecreulx & van Heijenoort, 1996). Homologs of glmM in Staphylococcus aureus, Helicobacter pylori, and Pseudomonas aeruginosa have been identified (De Reuse et al., 1997; Jolly et al., 1997; Tavares et al., 2000), and in S. aureus the glmM homolog is associated with sensitivity to antibiotics (Jolly et al., 1997; Glanzmann et al., 1999).

In the present study, the glmM homolog in S. gordonii DL1 was identified as the gene encoding phosphoglucosamine mutase. In addition, we describe how the glmM gene impacts on bacterial growth, bacterial cell morphology, biofilm formation, and sensitivity to antibiotics.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Bacterial strains, plasmids, and growth conditions

All the bacterial strains and plasmids, as well as the recombinants that were constructed in this study, are listed in supplementary Table S1. Streptococcus gordonii DL1 was used as the wild-type strain (Hsu et al., 1994). Unless otherwise indicated, streptococci were cultured in brain–heart infusion (BHI) broth or BHI agar plates (Becton, Dickinson and Company, Sparks, MD) at 37 °C. Escherichia coli strains were maintained in Luria–Bertani (LB) broth or LB agar plates (Sambrook et al., 1989). Antibiotics were purchased from Sigma-Aldrich (St Louis, MO), and supplemented, when necessary, with the following antibiotics: for streptococci, spectinomycin dihydrochloride (200 μg mL−1), erythromycin (10 μg mL−1), or chloramphenicol (8 μg mL−1); and for E. coli, ampicillin sodium salt (50 μg mL−1), spectinomycin dihydrochloride (50 μg mL−1), erythromycin (200 μg mL−1), chloramphenicol (30 μg mL−1), or kanamycin sulfate (50 μg mL−1).

DNA manipulations

Chromosomal DNA was prepared from S. gordonii DL1 as described previously (Andersen et al., 1993). Streptococcal plasmids were prepared from transformants of S. gordonii using the method of Takamatsu et al. (2000). Restriction enzymes, T4 DNA ligase, calf-intestinal alkaline phosphatase, and Klenow fragment (large fragment of E. coli DNA polymerase I) were purchased from Takara Shuzo Co. Ltd (Kusatsu, Japan) and used according to the manufacturer's instructions. Transformation of E. coli and S. gordonii with plasmids was performed as described previously (Perry et al., 1983; Sambrook et al., 1989). The primers used in this study are listed in supplementary Table S2. PCR was carried out using KOD-Plus-DNA polymerase (Toyobo, Tokyo, Japan). Preparation of DNA probes, colony hybridization (Sambrook et al., 1989; Abe et al., 1990), and Southern hybridization (Southern, 1975) were carried out using a DIG DNA labeling and detection kit (Roche Diagnostics, Indianapolis, IN). Other methods were as described previously (Sambrook et al., 1989).

Database searching and sequence analysis

blastp (Altschul et al., 1990) searches (http://www.ncbi.nih.gov/BLAST/) were used for pair-wise deduced sequence comparisons. clustal x (http://www.embl.de/~chenna/clustal/darwin/index.html) (Thompson et al., 1994) was used for multiple alignments. DNA sequencing was performed using the BigDyeTerminator V3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) with a 3730xl DNA Analyzer (Applied Biosystems). The nucleotide sequence upstream of the glmM gene was analyzed for putative prokaryotic promoters using the Neural Network Promoter Prediction program (http://www.fruitfly.org/seq_tools/promoter.html).

Cloning of the S. gordonii DL1 glmM homolog

Chromosomal DNA from S. gordonii DL1 was digested with SacI and ligated using T4 DNA ligase into SacI-digested pZErO-2 (Invitrogen, Carlsbad, CA) after treatment with calf-intestinal alkaline phosphatase. The ligated plasmids were transformed into E. coli Top10 (Invitrogen) and the transformants were selected on LB agar plates containing 50 μg mL−1 kanamycin sulfate. Colonies were screened by colony hybridization probed with DNA fragment F001, the 0.4-kb PCR product homologous to glmM of E. coli (supplementary Table S2).

Preparation of His6-tagged GlmM enzyme

PCR product F201, containing glmM of S.gordonii DL1, was cloned into the expression vector pET200/D-TOPO (Invitrogen), generating plasmid pIRG201. DNA sequencing was performed to confirm that the sequence of the construct was correct. The plasmid was then transformed into E. coli BL21 Star (DE3) (Invitrogen). His6-tagged recombinant GlmM (i.e. His6-GlmM) enzyme was prepared from the recombinant as described previously (Jolly et al., 1997). Purified His6-GlmM was dialyzed against 20 mM potassium phosphate buffer (pH 7.4) containing 0.5 mM MgCl2 and 0.1%β-mercaptoethanol and stored with 40% (v/v) glycerol at −20 °C for later use. The purity of the prepared His6-GlmM was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (Laemmli, 1970) using 4–12% Bis-Tris Gel (NuPAGE; Invitrogen), followed by staining with Coomassie Brilliant Blue (Fairbanks et al., 1971). Protein concentrations were determined by the method of Bradford (Bradford, 1976), using bovine serum albumin as the standard.

Preparation of His6-tagged GlmU enzyme

His6-tagged GlmU (i.e. His6-GlmU) enzyme was prepared as described previously (Pompeo et al., 1998) from the recombinant E. coli BL21 Star (DE3) harboring pIRG202, in which glmU of E. coli W3110 (Jensen, 1993) was cloned. The purity of the prepared His6-GlmU was primarily confirmed by SDS-PAGE, followed by dye staining. The enzymatic activity of His6-GlmU was primarily confirmed by the assay described in the next section, except that glucosamine-1-phosphate was used as the substrate instead of glucosamine-6-phosphate (Mengin-Lecreulx & van Heijenoort, 1993).

Enzymatic assay

The coupled assay in which the glucosamine-1-phosphate synthesized from glucosamine-6-phosphate by the mutase was quantitatively converted to UDP-GlcNAc in the presence of purified bifunctional GlmU enzyme, possessing both glucosamine-1-phosphate acetyltransferase activity and GlcNAc-1-phosphate uridyltransferase activity (Mengin-Lecreulx & van Heijenoort, 1993, 1994), was performed as described previously (Mengin-Lecreulx & van Heijenoort, 1996) with some modifications. The chemicals used in this assay were purchased from Sigma-Aldrich. The assay mixture contained 50 mM Tris-HCl buffer (pH 8.0), 3 mM MgCl2, glucosamine-6-phosphate (0, 0.5 or 1 mM), 0.4 mM acetyl coenzyme A, 0.7 mM glucose-1,6-diphosphate, 2 mM uridine 5′-triphosphate (UTP), His6-GlmU (1 μg protein), and His6-GlmM (4 μg protein) in a final volume of 50 μL. The mixture was incubated at 37 °C for 30 min, and the reaction was terminated by heating at 100 °C for 5 min. The reaction products were isocratically separated by HPLC using a reverse-phase column (ODS-80Ts, Tosoh, Tokyo, Japan) with 20 mM triethylamine-acetic acid buffer (pH 4.0) at a flow rate of 1 mL min−1 (LC-9A pumps, Shimazdu Co. Ltd, Kyoto, Japan) (Rabina et al., 2001; Wopereis et al., 2006). The A260 nm of the eluate was monitored using an ultraviolet spectrophotometric detector (SPD-6A, Shimadzu). UDP-GlcNAc and UTP were eluted at 8.9 and 14 min, respectively, after the mixture was applied to the column (Fig. 1). The amount of UDP-GlcNAc produced was calculated by comparing the chromatogram with that of 1 mM UDP-GlcNAc as the standard.

image

Figure 1.  HPLC chromatogram for measurement of the amount of product in the coupled assay in which the glucosamine-1-phosphate synthesized from glucosamine-6-phosphate (GlcN-6-P) by phosphoglucosamine mutase (i.e. GlmM) was quantitatively converted into UDP-GlcNAc in the presence of purified bifunctional GlmU enzyme and its substrates, acetyl-coenzyme A and UTP. The standards contained 1 mM UDP-GlcNAc and 0.5 mM UTP. UDP-GlcNAc and UTP, which eluted at 8.9 and 14 min, respectively, are indicated by arrow heads.

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Insertional mutation, deletion and complementation of glmM in S. gordonii

An erythromycin-resistance gene (ermAM) was inserted into glmM of S. gordonii DL1 to obtain a glmM::erm mutant (EM231, supplementary Fig. S1). Initially, the ermAM fragment cleaved from pMDC10E (Shiroza et al., 1998) was inserted at the SspI site of the F001 fragment in plasmid pIRG001. The resulting plasmid, pIRE001, was linearized by digestion with HindIII and XbaI, and was transformed into S. gordonii DL1. The transformants thus produced were grown in the presence of erythromycin to select for recombination between the regions flanking ermAM and homologous regions in glmM. Insertion of ermAM into the expected locations in the transformant (i.e. EM231) was verified by Southern blotting of chromosomal DNA using the ermAM gene fragment as a probe and by PCR with primers flanking the predicted sites of ermAM insertion (primer pair for F001, Table S2).

The S. gordonii DL1 chromosomal DNA fragment containing glmM was replaced with a chloramphenicol-resistance gene (cat) to obtain ΔglmM mutant (CM201, supplementary Fig. S1) for use in genetic complementation studies. DNA fragments (c. 500 bp each) on either side of S. gordonii DL1 glmM, F201UP and F201DN (supplementary Table S2), were amplified by PCR, digested with the appropriate restriction endonucleases, and inserted either side of the cat gene into plasmid pR326 (Claverys et al., 1995). The resulting plasmid (pIRC201) was linearized by digestion with BglII and SphI, transformed into S. gordonii DL1, and a single transformant (CM201) was selected following growth on chloramphenicol. The replacement of glmM by cat was verified by Southern blotting of CM201 chromosomal DNA using the cat and glmM genes as probes, and by PCR with forward primer for F201UP and reverse primer for F201DN respectively, or a primer pair for cat.

Genetic complementation of S. gordonii CM201 with the glmM gene was performed by transformation with pAS201 (supplementary Fig. S1), a streptococcal plasmid containing the glmM gene and its putative promoter region. PCR-amplified F201C was inserted into pAS41S (supplementary Table S1), which was subsequently transformed into S. gordonii CM201. Spectinomycin-resistant (Spr) transformant was isolated as S. gordonii CM201(pAS201) (ΔglmM/glmM). Plasmids were isolated from the transformants and analyzed by digestion with appropriate restriction endonucleases to confirm the presence of the expected insert.

Immunological procedures

Rabbit antiserum against GlmM (anti-GlmM) was prepared by immunizing rabbits with His6-GlmM (0.3 mg protein injection−1) emulsified with an equal volume of Freund's complete adjuvant (Sigma-Aldrich) and administered subcutaneously at multiple sites. Anti-GlmM serum was obtained 1 week after three subsequent injections of the antigen in an incomplete adjuvant administered at biweekly intervals.

Sonic extracts of streptococci were prepared for Western blot analysis as described previously (Takahashi et al., 1997), separated by SDS-PAGE as described above, and electrophoretically transferred to nitrocellulose membranes (Towbin et al., 1979). The membranes were blocked with Tris-buffered saline (TBS) containing 1% bovine serum albumin, incubated with anti-GlmM (1 : 100 dilution), washed with TBS containing 0.05% Tween 20, and incubated with peroxidase-conjugated goat anti-rabbit IgG (1 : 1000 dilution; Bio-Rad, Hercules, CA). Bound antibody was detected using 4-chloro-1-naphthol and H2O2.

Bacterial growth

BHI broth (5 mL) in a glass tube was inoculated with an 18–24-h streptococcal culture (final A620 nm=0.01). Bacterial cell growth in a static culture at 37 °C was automatically recorded at A660 nm using the TVS062CA Bio-photorecorder (Advantec, Tokyo, Japan).

Biofilm assays

An assay measuring biofilm formation on a glass wall was performed as described previously (Olson et al., 1972) with some modifications. BHI broth supplemented with 5% sucrose (3 mL) in a glass tube was inoculated with an 18–24-h streptococcal culture (final A620 nm=0.01). The tube was incubated at 37 °C for 20 h while statically positioned at an angle of c. 15° from the horizontal to increase the surface area for biofilm formation. Bacteria nonadhered to the glass wall of the tube were taken off with the media into another glass tube. Adhered bacteria remaining on the glass wall were suspended in 3 mL of TBS (20 mM Tris-HCl [pH 7.8], 150 mM NaCl) by vigorous shaking. The A620 nm of each suspension was measured. Percentage adherence was defined as (A620 nm of adhered cell suspension)/[(A620 nm of adhered cell suspension)+(A620 nm of nonadhered cell suspension)] × 100.

Biofilm formation on plastic walls in culture with mixing was also assessed using the MBEC Physiology and Genetics assay of the Calgary Biofilm Device (Innovotech Inc., Edmonton, Canada) as described previously (Bernier & Sokol, 2005) with some modifications. The sucrose-supplemented BHI broth inoculated with an 18–24-h streptococcal culture (final A620 nm=0.01) was added to each well (150 μL well−1) of the 96-well tissue culture plate of the device, and the lid was vertically studded with the 96 polystyrene pegs corresponding to the 96 wells, primarily immersed in 10% horse serum in phosphate-buffered saline (pH 7.4) for 1 h, was placed. The plate was incubated at 37 °C for 20 h while being mixed using a gyrorotary shaker at c. 100 r.p.m. The pegs studded to the lid were immersed in 1% crystal violet in a 96-well microtiter plate (200 μL well−1) for 15 min, rinsed briefly twice with saline, and immersed in absolute ethanol in a 96-well microtiter plate (200 μL well−1) for 15 min to dissolve the dye. The A575 nm of the dye solution (Biofilm Abs) was measured. The culture medium containing planktonic cells was transferred to another 96-well microtiter plate, and the A575 nm of the suspension (Planktonic Abs) was measured. The biofilm formation index was defined as the ratio of Biofilm Abs/Planktonic Abs.

Morphological methods

Bacterial cells cultivated in BHI broth at 37 °C for 20 h were observed by Gram staining (Hucker's modification).

Bacterial cells were further observed by transmission electron microscopy (TEM). Bacterial pellets were fixed overnight with 2.5% glutaraldehyde and 0.05% ruthenium red in 0.1 M cacodylate buffer (pH 7.4) at 4 °C. Cells were washed in the same buffer and postfixed for 3 h in 1% osmium tetroxide containing 0.05% ruthenium red at 4 °C. Subsequently, the cells were rinsed with the same buffer again. Bacteria were suspended for 1 h at room temperature in 2% aqueous uranyl acetate and then washed in distilled water. Suspended cells were embedded in 1.5% agarose before dehydration with a graded ethanol series. The dehydrated blocks were embedded in Spurr resin. Ultrathin sections were cut with an ultramicrotome (Ultracut-N; Reichert-Nissei, Tokyo, Japan), mounted on copper grids, and stained with uranyl acetate and lead citrate. Images of sections were obtained using a transmission electron microscope (75 kV; H-7000; Hitachi, Tokyo, Japan).

Determination of minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs)

MICs and MBCs of various antibiotics were determined using the microdilution method as described elsewhere (Baker et al., 1981) with BHI broth as medium. In addition, MICs and minimum biofilm eradication concentration (MBECs) in biofilms of S. gordonii strains were determined using the MBEC High-throughput assay of the Calgary Biofilm Device (Innovotech) as described previously (Ceri et al., 1999; Kostenko et al., 2007), except that pegs of the device were coated with horse serum before the biofilm formation and the sucrose-supplemented BHI broth was used as medium. All antibiotics were purchased from Sigma-Aldrich.

Statistical analysis

Statistical differences in the means of obtained values were evaluated by an unpaired t-test. Differences were considered to be significant at P<0.05.

Nucleotide sequence accession number

The DDBJ/EMBL/GenBank accession number assigned to the glmM gene of S. gordonii DL1 is AB330077.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Cloning and DNA sequencing analysis of the S. gordonii DL1 glmM gene

A blast search for the glmM gene of E. coli against the S. gordonii NCTC7868 (Challis) genome database (the genome is currently being sequenced at the Institute for Genomic Research, http://www.tigr.org/) revealed a putative gene encoding a protein that was 43% identical to GlmM of E. coli (Mengin-Lecreulx & van Heijenoort, 1996). The glmM homolog of S. gordonii DL1 was cloned into E. coli plasmid pZErO-2 (i.e. pIRG101), and the DNA was sequenced. The glmM gene of S. gordonii consists of 1350 nucleotides and encodes a polypeptide (GlmM) of 450 amino acid residues with a molecular weight of 48 365. The gene is preceded by a putative prokaryotic promoter and a ribosome-binding site, and is followed by a stem-and-loop structure. The amino acid sequence of GlmM was compared with GenBank sequences using a blastp search; GlmM of S. gordonii DL1 was found to be completely identical to that of S. gordoniiNCTC7868, and 59%, 42%, and 39% identical to GlmM in S. aureus (Jolly et al., 1997), P. aeruginosa (Tavares et al., 2000), and H. pylori (De Reuse et al., 1997), respectively. Two serine residues that form the active site (Jolly et al., 1999) are conserved in GlmM in S. gordonii DL1 and all other bacteria (Fig. 2).

image

Figure 2.  Alignment of the predicted amino acid sequence of the active site of GlmM from Streptococcus gordonii DL1, Escherichia coli, Helicobacter Pylori, and Staphylococcus aureus. The conserved serine residues are indicated in bold and with arrows.

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Overexpression and enzymatic activity of His6-tagged recombinant GlmM of S. gordonii

His6-tagged recombinant protein encoded by glmM of S. gordonii DL1 (i.e. His6-GlmM) was overexpressed in E. coli BL21 Star (DE3). The His6-GlmM expressed was confirmed by SDS-PAGE to be a 50-kDa protein (Fig. 3a), which was consistent with the value calculated on the basis of the DNA sequence. The specific activity of purified His6-GlmM was 0.19 μmol min−1 mg−1 (Fig. 1), approximately twice that of the corresponding enzyme in S. aureus (Jolly et al., 1997), and one-tenth that in E. coli (Mengin-Lecreulx & van Heijenoort, 1996) and P. aeruginosa (Tavares et al., 2000). These results confirm that glmM in S. gordonii DL1 encodes phosphoglucosamine mutase.

image

Figure 3.  (a) Preparation of the recombinant phosphoglucosamine mutase (His6-GlmM). A sonic extract of Escherichia coli BL21 Star (DE3) harboring plasmid pIRG201 was clarified by ultracentrifugation (crude; 20 μg protein) and purified His6-GlmM (His6-GlmM, 2 μg protein) were analyzed by SDS-PAGE with molecular mass marker (marker) followed by staining with Coomassie Brilliant Blue. The positions of the molecular mass markers are indicated on the left. (b) Western blot of streptococcal sonic extracts (50 μg protein lane−1) and purified His6-GlmM (0.5 μg protein) with anti-GlmM (1 : 100 dilution). The positions of the molecular mass markers are indicated on the left. The positions of a nonspecific antigen (*), His6-GlmM and GlmM are indicated on the right.

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Insertional mutation, deletion and complementation of the glmM gene in S. gordonii

An insertional mutant (glmM::erm) was prepared for S. gordonii DL1 (EM231, supplementary Fig. S1) to assess the involvement of glmM in biofilm formation, sensitivity to antibiotics, and bacterial cell morphology in this organism. That the glmM::erm mutant, in which ermAM was inserted at a position corresponding to nucleotide 1123 of glmM, did not produce GlmM was confirmed by Western blotting of sonicated cell extracts (Fig. 3b). Purified His6-GlmM and sonicated extracts of wild-type DL1 cells served as positive controls in the experiment. GlmM production of ΔglmM mutant (CM201, supplementary Fig. S1) was also abolished, but the production was restored by plasmid-borne glmM complementation (ΔglmM/glmM) (Fig. 3b).

Mutation of the glmM gene reduces bacterial growth

Certain genes encoding enzymes involved in bacterial cell wall synthesis have been associated with bacterial growth and cell separation (Kajimura et al., 2005), (Komatsuzawa et al., 2004). These findings prompted us to examine the possible role of GlmM in bacterial growth. Compared with the wild-type strain, the growth rates of the glmM::erm mutant and the ΔglmM mutant were reduced, as assessed by cultivating the strains statically overnight in BHI broth and the growth was monitored on the basis of the turbidity of the broth culture (Fig. 4a). The growth rates of ΔglmM mutant were restored in the ΔglmM/glmM strain (Fig. 4a). Similar results were obtained when growth was monitored on the basis of CFU, bacterial total protein, and bacterial wet weight (data not shown). Interestingly, most bacterial cells of the glmM mutants sank, but did not adhere, to the bottom of the tube, whereas those of wild-type and ΔglmM/glmM strains grew dispersed in the broth (Fig. 4b).

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Figure 4.  Bacterial growth of Streptococcus gordonii in BHI broth. (a) Growth curve. (b) Overnight cultures in glass tubes.

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Mutation of the glmM gene reduces biofilm formation

It has been reported that a transposon-insertional glmM mutant of S. gordonii reduces biofilm formation in static culture (Loo et al., 2000). To further investigate the role of GlmM, biofilm formation by glmM mutants in static and mixed cultures was compared with that by the wild-type strain. As shown in Fig. 5, the glmM mutants resulted in a significant reduction in biofilm formation on the glass surface in static cultures and on the polystyrene surface of the Calgary Biofilm Device in mixed cultures, when the bacteria were grown in BHI broth containing 5% sucrose, the substrate of glucosyltransferase for production of glucan. These biofilm formations of the ΔglmM mutant were restored in the ΔglmM/glmM strain.

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Figure 5.  Biofilm formation by Streptococcus gordonii in BHI broth containing 5% sucrose. (a) Adhesion of streptococcal cells to the glass surface in static cultures. Mean and SD (n=5) of percent adherence are indicated. (b) Adhesion of streptococcal cells to the polystyrene surface in mixed cultures. Mean and SD (n=6) of biofilm formation index are indicated.

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The glmM gene has a role in determining bacterial cell morphology

Given that GlmM is involved in bacterial cell wall synthesis, it must also play an important role in bacterial cell morphology. We found that bacterial chains in a static culture of the glmM::erm mutant were markedly elongated relative to wild type (Fig. 6a). The chains appeared to be entangled with other chains. Mutation of glmM, however, seemed not to affect the Gram-staining character of the cells. The average number of bacterial cells per chain was 22 for the glmM::erm mutant, c. 10-fold the number in the wild-type culture (Fig. 6b). Similar elongation of bacterial chains was observed in the ΔglmM mutant. However, the length of the chains observed in ΔglmM/glmM was the same as that of the wild type (Fig. 6).

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Figure 6.  Morphological analysis of Gram-stained Streptococcus gordonii cells. (a) Light micrographs of Gram-stained streptococci. Scale bar, 10 μm. (b) Lengths of the streptococcal chain. Mean and SD (n=30) of bacterial cell number per streptococcal chain are indicated.

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Furthermore, TEM revealed variations in cell size and shape in the glmM::erm mutant culture relative to the wild type. As shown in Fig. 7, most mutant cells were larger, and with a rougher cell surface than the wild-type cells. In addition, variations in cell size and shape were found in the mutant cells.

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Figure 7.  Transmission electron micrographs of thin-sectioned Streptococcus gordonii cells. Scale bars in the upper panels (high magnification), and the lower panels (low magnification) represent 200 and 500 nm, respectively.

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Mutation of the glmM gene increases sensitivity to antibiotics

That the glmM mutation in S. aureus results in reduced methicillin resistance has been reported (Jolly et al., 1997; Glanzmann et al., 1999). To investigate whether glmM mutations in S. gordonii also affect sensitivity to antibiotics, we examined the MICs and MBCs of antibiotics including penicillins and inhibitors of protein synthesis with respect to their effect on S. gordonii cells. As shown in Table 1, the glmM mutants were c. 10-fold more sensitive to penicillins than the wild-type or the ΔglmM/glmM strain. However, all strains were similarly sensitive to inhibitors of protein synthesis. No clear differences of antimicrobial susceptibility were found between planktonic bacteria and in the biofilm of each strain.

Table 1.   MIC and MBC/MBEC of antibiotics in planktonic and in biofilm of Streptococcus gordonii strains
S. gordonii strainMIC (μg mL−1) in planktonic (upper) and in biofilm (lower)
PenicillinsInhibitors of protein synthesis
AmpicillinMethicillinPenicillin GSpectinomycinKanamycinChloramphenicol
Wild type0.250.0630.0078100253.1
0.500.0630.0078100253.1
glmM::erm0.0310.00780.0009810012.51.6
0.0630.00780.002010012.51.6
ΔglmM0.0630.0160.0020ND25ND
0.130.0310.0020 25 
ΔglmM/glmM0.250.0630.0078ND25ND
0.500.0630.0078 25 
S. gordonii strainMBC (μg mL−1) in planktonic (upper) and MBEC (μg mL−1) in biofilm (lower)
PenicillinsInhibitors of protein synthesis
AmpicillinMethicillinPenicillin GSpectinomycinKanamycinChloramphenicol
  1. ND, not done.

Wild type880.5100100100
881100100100
glmM::erm0.50.130.01620050100
0.50.130.01620050100
ΔglmM0.50.130.031ND50ND
0.50.130.031 50 
ΔglmM/glmM220.25ND100ND
220.25 100 

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

In the present study, the glmM gene of S. gordonii DL1 was cloned on the basis of amino acid sequence similarity to the homologous enzyme in other bacteria. We confirmed the enzymatic activity of the recombinant GlmM, showing that glmM encodes phosphoglucosamine mutase. In the enzyme assay, isocratic elution of UDP-GlcNAc and UTP by reverse-phase HPLC allowed us to assess the phosphoglucosamine mutase activity without using radiolabeled reagents. The activity of S. gordonii GlmM is quantitatively similar to that of the GlmM of S. aureus (Jolly et al., 1997), but is c. 10-fold less than that of GlmM from Gram-negative bacteria such as E. coli and P. aeruginosa (Mengin-Lecreulx & van Heijenoort, 1996).

After the glmM gene of E. coli JM83 was identified (Mengin-Lecreulx & van Heijenoort, 1996), homologs in S. aureus, H. pylori and P. aeruginosa were found subsequently (De Reuse et al., 1997; Jolly et al., 1997; Tavares et al., 2000). The present study is the first report in which a streptococcal glmM has been identified and characterized. Using a biofilm assay involving static cultures in 96-well microtiter plates, Loo et al. (2000) found that a glmM transposon mutant of S. gordonii loses the ability to form biofilms in the normal way. In the present study, we clearly demonstrated that the glmM gene is involved in biofilm formation in both static and mixed cultures of S. gordonii DL1 by comparison of wild-type and glmM mutant cells.

In S. aureus, certain factors associated with peptidoglycan metabolism influence resistance to methicillin and vancomycin (Berger-Bachi et al., 1989; Maidhof et al., 1991; Henze et al., 1993; Gustafson et al., 1994; Pinho et al., 2001; Komatsuzawa et al., 2004). Similarly, in the present study, we found that in S. gordonii DL1 mutation of glmM affects sensitivity to penicillins. However, no clear differences of antimicrobial susceptibility were found between planktonic bacteria and in the biofilm of each strain. These findings suggest that impaired peptidoglycan synthesis in the glmM mutant leads to an increase in sensitivity to cell wall inhibitors.

Our morphological analyses showed that the glmM mutation causes marked elongation of the streptococcal chains in S. gordonii (Fig. 6), suggesting that the mutation may affect bacterial cell division. The unusual long chain formation may explain why the mutant cells were found at the bottoms of the culture tubes (Fig. 4b). Further morphological analysis using electron microscopy revealed that the mutant cells were larger, with a rougher cell surface than the wild-type cells (Fig. 7), suggesting that unusual peptidoglycan synthesis occurs in the mutant. Furthermore, variations in cell size and shape were found in mutant cells. These morphological findings resemble those observed for S. aureus with a mutation in sle1, the gene encoding N-acetylmuramyl-l-amidase (Kajimura et al., 2005), suggesting that mutations in genes encoding factors involved in peptidoglycan metabolism influence cell shape, size, and division in Gram-positive bacteria.

The glmM mutation results in malfunction of glucosamine-1-phosphate synthesis. The glmM gene is essential for the growth of E. coli (Mengin-Lecreulx & van Heijenoort, 1996); however, in S. gordonii, although the growth of glmM mutants is reduced, cells are still viable, suggesting that an alternative pathway for UDP-GlcNAc production exists. A possible substitute of phosphoglucosamine mutase is phosphoglucomutase (EC 5.4.2.2), converting glucose-6-phosphate into glucose-1-phosphate. Even though the substrate specificity to hexosamines is probably very low, phosphoglucomutase may be able to catalyze the interconversion of glucosamine-6-phosphate to glucosamine-1-phosphate (Brown, 1953). Perhaps the morphological findings and penicillin susceptibility observed in glmM mutants may be because the supply of UDP-GlcNAc, one of the peptidoglycan precursors, without GlmM is insufficient to be equal to production of the other bacterial components.

Plasmid-borne glmM complemented a glmM chromosomal deletion mutant of S. gordonii, restoring GlmM production, morphological parameters, and biofilm formation to wild-type levels. This provides unequivocal evidence that phosphoglucosamine mutase plays an important role in normal growth of S. gordonii. In future studies, we will examine in detail the kinetics and the virulence of phosphoglucosamine mutase in S. gordonii. The results of such studies may provide important insights into the overall peptidoglycan synthetic pathway and preventing biofilm formation in medical and dental applications.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by Grants-in-Aid for Scientific Research No. 18592014 and No. 17591928 from the Japan Society for the Promotion of Science.

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  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1. Alignment of the physical map of the glmM gene on the chromosomal DNA of S. gordonii DL1, and the corresponding regions of the S. gordonii DL1 derivatives EM231 (glmM::erm) and CM201 (ΔglmM).

Table S1. Bacterial strains and plasmids used in this study.

Table S2. PCR-amplified DNA fragments.

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