SO-LAAO, a Novel l-Amino Acid Oxidase That Enables Streptococcus oligofermentans To Outcompete Streptococcus mutans by Generating H₂O₂ from Peptone

Bacterial strains and culture conditions.Bacterial strains and plasmids used in this study are listed in Table S1 in the supplemental material. All Streptococcus strains were routinely grown in brain heart infusion (BHI) medium (Difco, BD) or TPYG (0.5% tryptone-0.5% soy peptone-1% yeast extract-1% glucose) broth and incubated at 37°C as static culture unless indicated otherwise. For the biofilm assay, bacteria were grown in BHI or TPYG broth plus 0.5% sucrose and 1% glucose. For the antagonism assay on amino acids, bacteria were grown on an 0.8% modified chemically defined agar plate (33) containing the following components (per ml): 5 mg of glucose; 5 μg of l-glutamine; 100 μg of l-alanine, l-histidine, l-proline, l-cysteine, l-glycine and l-threonine; 200 μg of l-aspartic acid, l-isoleucine, l-phenylalanine, l-valine, l-methionine, l-serine, and l-leucine; 300 μg of l-glutamic acid; 400 μg of l-lysine; 100 μg of riboflavin, nicotinic acid, and pyridoxine; 50 μg of thiamine HCl and α-pantothenate; 10 μg of d-biotin, p-aminobenzoic acid, and folic acid; 208 mg of K₂HPO₄; 292 mg of KH₂PO₄; 120 mg of MgCl₂ [6H₂O]; 2 mg of MnCl₂ [4H₂O]; 11 mg of CaCl₂ [2H₂O]; 2 mg of FeSO₄ [7H₂O]; and 800 mg of agar.

H₂O₂ assay.Hydrogen peroxide in liquid culture was quantified using a modified method described previously (10, 32). Briefly, 1.3 ml of culture supernatant was added to 1.2 ml of solution containing 2.5 mM 4-amino-antipyrine (4-amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one; Sigma) and 0.17 M phenol. The reaction proceeded for 4 min at room temperature, and then horseradish peroxidase (Sigma) was added to a final concentration of 50 mU ml⁻¹ in 0.2 M potassium phosphate buffer (pH 7.2). After a 4-min incubation at room temperature, the A₅₁₀ was measured with a Beckman DU-800 spectrophotometer. A standard curve was generated with known concentrations of chemical H₂O₂.

In vitro interspecies interaction assays between oral streptococci.Overnight cultures of Streptococcus species were adjusted to the same optical density at 600 nm (OD₆₀₀) (∼1.0). All 10-μl aliquots of Streptococcus species were inoculated adjacently on 0.8% agar plates. The plates were incubated in a candle jar at 37°C for 24 h.

Amplification and cloning of the entire l-amino acid oxidase gene of S. oligofermentans (aao_So).The genomic DNA of S. oligofermentans was extracted and purified using the method of Marmur (22) with slight modification (8). Based on the known LAAOs, using flavin adenine dinucleotide (FAD) as a cofactor (37), the protein sequence of amino acid oxidase of Rhodococcus opacus was used as a probe to search the homologue against the complete genome of S. sanguinis SK36 because of its close relationship with S. oligofermentans and production of hydrogen peroxide with a similar amino acids profile (data not shown). The open reading frame (ORF) SSA-0323, annotated as “flavoprotein, putative” in the completed S. sanguinis SK36 genome, was selected as the candidate. To identify a conserved DNA sequence for primer design, SSA-0323 was used as a probe to search for homologous proteins in the Streptococcus pyogenes M1 GAS genome and an ORF, SPy_1866, was identified. By aligning the coding sequences of SPy_1866 (1,170 bp) and SSA-0323 (1,176 bp) using DNAman, a pair of degenerate primers, solaaoF (5′-GCNGGNATTCCNGGNAATGG-3′) (nucleotide positions 186 to 205 of SPy_1866 and SSA-0323) and solaaoR (5′-TCNANNCCNCCNTTGGTCAC-3′) (nucleotide positions 999 to 1018 of SPy_1866 and SSA-0323) were designed and synthesized by Sangon Company (Shanghai, China). A partial aao_So gene of S. oligofermentans was amplified by using the primer pair solaaoF/solaaoR and chromosomal DNA as a PCR template. The 25-μl PCR mixture contained 100 ng DNA, 0.4 μM (each) primer, 100 μM (each) deoxynucleoside triphosphate mix, 10× PCR buffer, and 0.1 U Taq DNA polymerase (Takara Company, Dalian, China). PCR was performed at 95°C for 5 min, followed by 30 cycles of 94°C for 30s, 55°C for 1 min, and 72°C for 1 min and then one cycle at 72°C for 10 min. The PCR product (832 bp) was cloned into pUCM-T (Shenergy Biocolor Company, Shanghai, China) and verified by DNA sequencing.

To obtain the entire aao_So gene, inverse PCR was employed to amplify the flanked sequences of the 832-bp fragment. Genomic DNA from S. oligofermentans was digested with EcoRI, BamHI, NheI, StuI, and XbaI at 37°C for 2 h. The digestion mixture was extracted by chloroform, and DNA was precipitated by ethanol. The digested DNA was self-circularized using T₄ DNA ligase (Shenergy Biocolor Company, Shanghai, China) at 14°C for 24 h. The resultant ligation mixture was used as the template, and primers solaao-inverseF (5′-CGGAGACGATTTCAGTATTGGTG-3′) and solaao-inverseR (5′-TTACCCAGCCCTTTCCCGAG-3′) were applied for the inverse PCR. The PCR was performed as described above except that LA Taq DNA polymerase (Takara Company, Dalian, China) was used and the extension time was 5 min. The 5-kb PCR product obtained from XbaI-digested DNA mixtures was purified, ligated to pCR2.1-Topo (Invitrogen), and sequenced.

Construction of the expression vector of the aao_So gene.The complete aao_So gene was amplified using a pair of 5′-modified primers (restriction sites are underlined, and modified sequences are in italics): solaao-expressF (with an NdeI restriction site) (5′-AACATATGATGAACCATTTCGACAC-3′) and solaao-expressR (with a BamHI restriction site) (5′-AAGGATCCTTAATCATAATGCAAACTTC-3′). Pfu DNA polymerase (Promega) was used in a PCR to amplify the 1,176-bp aao_So gene. This fragment was subsequently digested with NdeI-BamHI and then cloned into the NdeI-BamHI restriction sites of expression vector pET-15b (Novagen) to generate the pTH3 plasmid. The His₆-tag fusion sites and nucleotide sequences of double-stranded template DNA were confirmed by DNA sequencing using the ABI 3730xl sequencer.

Overexpression and purification of SO-LAAO protein.The pTH3 plasmid inserted with a PCR-amplified aao_So gene fragment was transformed into Escherichia coli BL21(DE3)pLysS (Novagen) cells and cultured in LB medium supplemented with 100 μg ml⁻¹ of ampicillin and 34 μg ml⁻¹ of chloramphenicol. Cells were grown at 37°C to an OD₆₀₀ of 0.4 to 0.6. Overproduction of the SO-LAAO protein was induced by addition of 5 mM isopropyl-β-d-thiogalactopyranoside and 1.2 μM FAD. The culture was allowed to grow for an additional 3 to 4 h before being harvested. Cells were collected by centrifugation at 8,200 × g for 10 min, resuspended in a 1/10 volume of binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 30 mM imidazole, pH 7.4), and then broken by sonication for 10 min. After the cell lysate was spun down at 18,449 × g for 15 min, the supernatant was filtered through a 0.22-μm polyvinylidene difluoride membrane (Millipore) and then applied to a Ni²⁺-charged chelating column (GE Healthcare) previously equilibrated with binding buffer. Proteins were eluted by elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4), and the fractions of elution were run on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel. The fractions with desired protein were pooled and dialyzed against 20 mM Tris-HCl buffer (pH 8.0).

Assay of LAAO activity of SO-LAAO.LAAO activity of SO-LAAO was determined by measuring hydrogen peroxide production with the enzyme-coupled assay described above. The assay mixtures contained 1.2 ml of 2% l-amino acid solution, 12.5 U of the horseradish peroxidase (250 U mg⁻¹; Sigma), 1.2 ml of solution containing 2.5 mM 4-aminoantipyrine and 0.17 M phenol, and 200 nM SO-LAAO in Tris-HCl buffer, pH 8.0, and the incubation was performed at 25°C.

Construction of aao_So insertional mutant of S. oligofermentans.A pair of primers, solaao-knockF (5′-GAAAATTGCAGAGCTGGG-3′) and solaao-knockR (5′-AAAGTCTGCCAAATCCT-3′), were designed and synthesized by Sangon Company (Shanghai, China). The 447-bp internal fragment of aao_So (located from 357 bp to 803 bp) was generated by PCR using the chromosomal DNA as a template and cloned into pCR2.1-Topo (Invitrogen). The fragment was subsequently excised using BamHI and XbaI (New England Biolabs), purified, and ligated to the compatible sites on the pFW5-luc (28) vector using T₄ DNA ligase (Shenergy Biocolor Company, Shanghai, China) to generate plasmid pTH4. The ligation mixture was then transformed into E. coli. Plasmid pTH4 was confirmed by restriction analysis and PCR and then transformed into S. oligofermentans using the method described previously (35). The transformants were then selected on BHI agar containing spectinomycin (800 μg ml⁻¹) and confirmed by PCR amplification of the spectinomycin-resistant gene aad9 and by Southern blotting with probes for aao_So and aad9.

Confocal laser scanning microscopy of mixed-species biofilms.Overnight cultures of S. oligofermentans::Φ(ldhp-gfp) (35) and S. mutans UA140::Φ(mutAp-mrfp) (15) were diluted (1:10) into BHI-SG (BHI supplemented with 0.5% sucrose and 1% glucose) or TPYG-SG (TPYG supplemented with 0.5% sucrose and 1% glucose) broth and then inoculated into the Lab-Tek II chamber slide system (Nalge Nunc International, Naperville, IL). After a 16-h incubation at 37°C in a candle jar, the culture supernatant was removed from the chambers. Mature biofilms were exposed to air for 10 min in the dark at room temperature and then washed with phosphate-buffered saline buffer. The biofilms were observed using a confocal laser scanning microscope (LEICA TCS SP2).

Luciferase measurement.An overnight bacterial culture was diluted (1:30) into fresh culture medium. The culture was sampled every 60 min from early log phase to early stationary phase to measure luciferase activity and OD₆₀₀. Twenty-five microliters of 1 mM d-luciferin (Sigma) solution (suspended in 1 mM citrate buffer, pH 6.0) was added to 100-μl samples, and luciferase assays were performed essentially as previously described (19) using a TD 20/20 luminometer (Turner system). The OD₆₀₀ was read with a 721 spectrophotometer (Shanghai Analytical Manufactory). All the measurements were done with duplicate samples, and all the readings were from three repeated experiments.

Nucleotide sequence accession number.The DNA sequence of the aao_So gene has been deposited in the GenBank database under accession number EU495328.

S. oligofermentans produces hydrogen peroxide from peptone and amino acids.It had been demonstrated that S. oligofermentans inhibits S. mutans in carbohydrate-rich medium by generating pronounced H₂O₂ from lactate with Lox. While the lox mutant completely lost the inhibition on TYG (36), partial inhibition still was retained on 0.5% soy peptone (Difco, BD)-supplemented TYG (TPYG) (Fig. 1A). This suggested that S. oligofermentans might have other H₂O₂ production pathways in addition to Lox. Since peptone was the only different component between TYG and TPYG, it was speculated that peptone was involved in H₂O₂ generation by S. oligofermentans in addition to lactate. To verify this, H₂O₂ production from peptone by S. oligofermentans was measured. By suspending the BHI overnight culture of S. oligofermentans in 0.5% peptone and exposing it to air for 20 min at 37°C, the H₂O₂ yield was determined to be 33.11 ± 0.22 μmol mg⁻¹ cell mass.

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FIG. 1.

Growth inhibition of Streptococcus mutans by Streptococcus oligofermentans. Each 10-μl overnight culture of S. oligofermentans wild type or the lox or aao_So mutant of S. oligofermentans and S. mutans at the same OD₆₀₀ was spotted adjacently on plates and incubated in a candle jar at 37°C for 24 h. Inhibition by the S. oligofermentans lox mutant on peptone-present TPYG medium (A), by the S. oligofermentans wild type (WT) (B), or by the S. oligofermentans aao_So mutant on an amino acid-containing chemically defined agar plate (C) is shown.

It is well known that l-amino acids are the constitutive components of peptone; therefore, 20 l-amino acids were tested for hydrogen peroxide production by S. oligofermentans. It was determined that H₂O₂ was produced mainly from seven l-amino acids (final concentration, 2%) (Table 1), including l-aspartic acid, l-tryptophan, l-lysine, l-isoleucine, l-arginine, l-asparagine, and l-glutamine. Therefore, a conclusion could be drawn that the H₂O₂ produced from peptone was derived from the seven l-amino acids.

Cloning of putative LAAO gene (aao_So) from S. oligofermentans.According to the previous biochemical studies (9), LAAO was the enzyme most likely to implement the reaction of oxidizing amino acids to form ketoacids, ammonia, and H₂O₂. To further verify the capability of S. oligofermentans to produce H₂O₂ from amino acids, a possible amino acid oxidase gene(s) in this oral streptococcus was searched for as described in Materials and Methods, and an open reading frame, named the aao_So gene, was identified. A BLASTP search at the NCBI website revealed the presence of the deduced amino acid sequence (SO-LAAO) of aao_So homologues in the genomes of five other streptococci (Fig. 2), at the identity levels of 96% with “hypothetical protein TIGR00275” of Streptococcus gordonii, 95% with “putative flavoprotein SSA-0323” of S. sanguinis, 85% with “hypothetical protein SP_0741” of Streptococcus pneumoniae, 79% with “hypothetical protein SPy_1866” of S. pyogenes, and 75% with “hypothetical protein SMU.392c” of S. mutans. This indicated that SO-LAAO homologous proteins might be widely present in streptococci. Partial sequence alignment (Fig. 2) of six streptococcal ORFs and four known LAAOs from bacteria, snakes, and fungi revealed that all the sequences contained a typical FAD binding site for the flavoprotein GxGxxG (37), whereas SO-LAAO had two amino acid difference (RxGKK) from the LAAOs' characteristic sequence motif (RxGGR) (37), so that SO-LAAO could represent a novel LAAO.

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FIG. 2.

Sequence alignment of SO-LAAO and homologous proteins of five streptococci and four known LAAOs. The highly conserved dinucleotide-binding central sequence GxGxxG and LAAO's characteristic sequence motif RxGGR are shown in bold characters. The amino acid differences in LAAO's characteristic sequence motif from those of the other four LAAOs in six streptococcal sequences are underlined. AY053450, LAAO of Rhodococcus opacus; AAY89681, LAAO of Notechis scutatus, YP_171306, LAAO of Synechococcus elongates; BAC55901, LAAO of Aspergillus oryzae; SO-LAAO, LAAO of S. oligofermentans; TIGR00275, conserved hypothetical protein of S. gordonii; SSA-0323, putative flavoprotein of S. sanguinis; SP_0741, hypothetical protein of S. pneumoniae; SPy_1866, hypothetical protein of S. pyogenes; SMU.392c, hypothetical protein of S. mutans.

Amino acid oxidase activity of aao_So gene product.To test the amino acid oxidase activity of the aao_So gene product, it was overexpressed in the E. coli BL21(DE3)pLysS strain. A His₆-tag sequence was fused to the N terminus of SO-LAAO to facilitate subsequent purification by immobilized metal ion affinity chromatography. Purified recombinant protein was shown as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a molecular mass of about 43 kDa (data not shown), slightly lower than the predicated molecular mass (44,973.42 Da) of the recombinant SO-LAAO protein. The purified protein was analyzed for amino acid oxidase activity using 20 l-amino acids as substrates as described in Materials and Methods. The reaction mixture without either purified enzyme, l-amino acid substrate, or horseradish peroxidase was included as a blank control. Enzyme assays showed that SO-LAAO catalyzed H₂O₂ production from l-aspartic acid, l-tryptophan, l-lysine, l-isoleucine, l-arginine, l-asparagine, and l-glutamine (each at a 2% concentration) (Table 1), while no measurable H₂O₂ was detected from the other tested l-amino acids and the derivates, such as N-acetyl-l-cysteine and cis-4-hydroxyl-l-proline.

aao_So mutant of S. oligofermentans abolishes H₂O₂ production from amino acids.Since the SO-LAAO protein exhibited LAAO activity, to test the in vivo role of SO-LAAO, an aao_So gene knockout mutant of S. oligofermentans was constructed by single-crossover homologous recombination with the suicide vector pFW5, fused with a luc reporter gene (pFW5-luc [28]). Hydrogen peroxide production from amino acids was then measured for the aao_So mutant. The result demonstrated that the aao_So mutant no longer produced H₂O₂ from the seven l-amino acids shown in Table 1, indicating that SO-LAAO did function in H₂O₂ production from amino acids in S. oligofermentans. Furthermore, the contribution of SO-LAAO in S. oligofermentans' competition with S. mutans was tested by using the aao_So mutant of S. oligofermentans in an interspecies antagonism experiment. To do this, the TPYG overnight cultures of S. oligofermentans or S. oligofermentans Δaao_So and S. mutans UA140 (29) with the same OD₆₀₀ were spotted side by side on an 0.8% chemically defined agar plate containing amino acids. After growth for 16 h in a candle jar, growth of S. mutans UA140 beside the S. oligofermentans wild-type strain was obviously inhibited (Fig. 1B); however, the inhibition zone beside S. oligofermentans Δaao_So was greatly reduced (Fig. 1C). This demonstrated that SO-LAAO contributed to the competitive potential of S. oligofermentans against S. mutans in growth on amino acid-rich plates.

Peptone up-regulates expression of aao_So.To get insight into the newly identified enzyme in in vivo H₂O₂ production, the expression profile of the aao_So gene was determined. Since the insertional mutation of the aao_So gene also resulted in an aao_So-luc fusion (see Materials and Methods), we used this strain to determine aao_So gene expression in cells grown in BHI (peptone absent) and TPYG (peptone-rich) broth by recording luciferase activity, expressed as relative light units per OD₆₀₀. The results indicated that the aao_So gene was constitutively expressed throughout growth in both media; however, the expression level in TPYG broth was nearly triple that in BHI broth.

SO-LAAO contributes to competitive edge of S. oligofermentans over S. mutans in mixed-species biofilm with peptone.It has been known that saliva is a great reservoir of proteinaceous materials, while the oral streptococci are dominant species in the dental biofilm. Therefore, it would be interesting to test the role of SO-LAAO in the competition of S. oligofermentans against S. mutans in the artificial two-species biofilm formed in a medium supplemented with peptone. A gfp reporter strain of S. oligofermentans (35) was mix incubated with an mrfp reporter strain of S. mutans, UA140 (15), in either BHI (peptone absent) or TPYG (peptone-rich) broth to form mixed-species biofilms. After 16 h of incubation, growth inhibition in both culture media was visualized under a confocal laser scanning microscope (Fig. 3A -1 and A-2). The images showed that S. mutans (red cells) was more suppressed in peptone-rich TPYG broth than in BHI. Similar results were obtained from quantitative determination of colonies. Though cell numbers of S. mutans in mixed-species biofilms with S. oligofermentans decreased dramatically in both media by comparison with its single-species one, the number was almost 10 times lower in TPYG than in BHI broth (Fig. 3B). In comparison with its single-species biofilm, the cell numbers of S. oligofermentans in the mixed-species biofilm did not decrease significantly under both culture conditions (data not shown). These results indicated that the amino acid-derived H₂O₂ could also contribute to the inhibition of S. mutans by S. oligofermentans.

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FIG. 3.

Interspecies competition between S. oligofermentans and S. mutans in the biofilm formed in BHI-SG and TPYG-SG. (A) Interspecies competition assay using confocal laser scanning microscopy. Overnight cultures of S. oligofermentans ldhp-gfp and S. mutans UA140 mutAp-mrfp were adjusted to the same OD₆₀₀ and then were inoculated (1:10) into chambers to form mixed-species biofilms. After 12 h of incubation, the biofilms were visualized under a confocal laser scanning microscope. The pictures were 1,000-fold magnified. A-1, mixed-species biofilm in BHI-SG broth; A-2, mixed-species biofilm in TPYG-SG broth. Green cells, S. oligofermentans ldhp-gfp; red cells, S. mutans UA140 mutAp-mrfp. (B) Interspecies competition assay by colony counting of S. mutans. Mixed-species biofilms of the S. oligofermentans wild type or aao_So mutant and S. mutans UA140 were formed by the same procedure described for panel A, and then colony numbers of S. mutans were counted after 16 h of incubation. 1, colony numbers in S. oligofermentans wild type-S. mutans mixed-species biofilm in BHI-SG broth; 2, colony numbers in S. oligofermentans wild type-S. mutans mixed-species biofilm in TPYG-SG broth; 3, colony numbers in aao_So mutant-S. mutans mixed-species biofilm in TPYG-SG broth; 4, colony numbers in S. mutans single-species biofilm. The results were expressed as means ± standard errors from three independent experiments.

The SO-LAAO contribution in the interspecies competition was also verified by comparing the survival rate of S. mutans in mixed-species biofilm with that of the wild-type strain and aao_So mutant strain of S. oligofermentans after 16 h of growth in TPYG broth. The single-species biofilms of S. oligofermentans Δaao_So and S. mutans were included as controls. As shown in Fig. 3B, about 10 times more cells of S. mutans survived in a biofilm mixed with the aao_So mutant than in that with the S. oligofermentans wild type. These results indicated that SO-LAAO conferred on S. oligofermentans an extra ability to compete with S. mutans, especially in peptone-abundant environments.