Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
    • JB Special Collection
    • JB Classic Spotlights
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JB
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Journal of Bacteriology
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
    • JB Special Collection
    • JB Classic Spotlights
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JB
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
MICROBIAL COMMUNITIES AND INTERACTIONS

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

Huichun Tong, Wei Chen, Wenyuan Shi, Fengxia Qi, Xiuzhu Dong
Huichun Tong
1State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Wei Chen
1State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Wenyuan Shi
2School of Dentistry, University of California, Los Angeles, California 90095
3Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California 90095
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Fengxia Qi
4Oral Biology, College of Dentistry, and Microbiology and Immunology, College of Medicine, University of Oklahoma Health Sciences Center BRC366, Oklahoma City, Oklahoma 73104
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Xiuzhu Dong
1State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: dongxz@sun.im.ac.cn
DOI: 10.1128/JB.00363-08
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

We previously demonstrated that Streptococcus oligofermentans suppressed the growth of Streptococcus mutans, the primary cariogenic pathogen, by producing hydrogen peroxide (H2O2) through lactate oxidase activity. In this study, we found that the lox mutant of S. oligofermentans regained the inhibition while growing on peptone-rich plates. Further studies demonstrated that the H2O2 produced on peptone by S. oligofermentans was mainly derived from seven l-amino acids, i.e., l-aspartic acid, l-tryptophan, l-lysine, l-isoleucine, l-arginine, l-asparagine, and l-glutamine, indicating the possible existence of l-amino acid oxidase (LAAO) that can produce H2O2 from l-amino acids. Through searching the S. oligofermentans genome for open reading frames with a conserved flavin adenine dinucleotide binding motif that exists in the known LAAOs, including those of snake venom, fungi, and bacteria, a putative LAAO gene, assigned as aaoS o , was cloned and overexpressed in Escherichia coli. The purified protein, SO-LAAO, showed a molecular mass of 43 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and catalyzed H2O2 formation from the seven l-amino acids determined above, thus confirming its LAAO activity. The SO-LAAO identified in S. oligofermentans differed evidently from the known LAAOs in both substrate profile and sequence, suggesting that it could represent a novel LAAO. An aaoS o mutant of S. oligofermentans did lose H2O2 formation from the seven l-amino acids, further verifying its function as an LAAO. Furthermore, the inhibition by S. oligofermentans of S. mutans in a peptone-rich mixed-species biofilm was greatly reduced for the aaoS o mutant, indicating the gene's importance in interspecies competition.

The oral cavity harbors more than 500 microbial species (26), which maintain a dynamic balance through interacting with each other. This natural microflora contributes to the host defenses by excluding exogenous microorganisms. However, this stability can be perturbed by significant changes in the oral environment. Changes in diet, saliva flow, and general health, etc., can lead to overgrowth of some opportunistic pathogens, which cause such diseases as dental caries and bacterial endocarditis (23). Therefore, understanding the interactions among different oral microbial species is an important aspect of microbial ecology and essential for studies of oral microbial pathogenesis.

Streptococcus mutans is a primary pathogen causing dental caries (tooth decay) (18). The virulence attributes of S. mutans include biofilm formation (14, 24), lactic acid production and tolerance (18), and bacteriocin production (29, 40). It has also been reported that some other oral streptococcal species, such as Streptococcus sanguinis and Streptococcus oligofermentans, a recently identified oral streptococcal species (34), can suppress the growth of S. mutans by producing hydrogen peroxide (H2O2) (16, 31, 36). These in vitro interactions were also substantiated by epidemiological studies, in which a reverse relationship between the level of S. mutans and that of S. sanguinis or S. oligofermentans has been demonstrated (3, 5; unpublished data for S. oligofermentans). This interspecies competition and inhibition probably help maintain the microbial homeostasis in the oral cavity.

S. oligofermentans has been isolated from dental plaques of caries-free human subjects in Beijing, China (34), and belongs to the “mitis” group of oral streptococci, which are believed to be the “pioneer” colonizers during the initial stages of dental biofilm formation. In a previous study, we demonstrated that S. oligofermentans inhibited the growth of S. mutans by producing abundant H2O2: more specifically, by converting the large amount of lactic acid produced by S. mutans through its enzymatic activity of lactate oxidase (Lox). The lox mutant of S. oligofermentans indeed lost its inhibitory activity toward S. mutans on peptone-deprived tryptone-yeast extract-glucose (TYG) plates (36); however, it was found that the lox mutant regained its inhibitory activity while growing on peptone-rich plates in this study. This suggests that S. oligofermentans may have other H2O2 production pathways independent of Lox. In this study, a novel l-amino acid oxidase (LAAO) (SO-LAAO) was identified and was demonstrated to catalyze H2O2 production from peptone in S. oligofermentans. Thus, a novel H2O2 production pathway was determined for this oral streptococcus.

MATERIALS AND METHODS

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 A 510 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 (aaoSo ).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 aaoS o 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 aaoSo 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 aaoS o gene.The complete aaoS o 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′-AA CATATGATGAACCATTTCGACAC-3′) and solaao-expressR (with a BamHI restriction site) (5′-AA GGATCCTTAATCATAATGCAAACTTC-3′). Pfu DNA polymerase (Promega) was used in a PCR to amplify the 1,176-bp aaoS o 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 aaoS o 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 aaoS o 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 aaoS o (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 aaoS o 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 aaoS o gene has been deposited in the GenBank database under accession number EU495328.

RESULTS

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.

FIG. 1.
  • Open in new tab
  • Download powerpoint
FIG. 1.

Growth inhibition of Streptococcus mutans by Streptococcus oligofermentans. Each 10-μl overnight culture of S. oligofermentans wild type or the lox or aaoS o 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 aaoS o 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.

View this table:
  • View inline
  • View popup
TABLE 1.

Hydrogen peroxide production by Streptococcus oligofermentans and heterogeneous expressed SO-LAAOa

Cloning of putative LAAO gene (aaoS o ) 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 aaoS o gene, was identified. A BLASTP search at the NCBI website revealed the presence of the deduced amino acid sequence (SO-LAAO) of aaoS o 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.

FIG. 2.
  • Open in new tab
  • Download powerpoint
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 aaoS o gene product.To test the amino acid oxidase activity of the aaoS o 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.

aaoS o 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 aaoS o 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 aaoS o mutant. The result demonstrated that the aaoS o 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 aaoS o mutant of S. oligofermentans in an interspecies antagonism experiment. To do this, the TPYG overnight cultures of S. oligofermentans or S. oligofermentans ΔaaoS o 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 ΔaaoS o 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 aaoS o .To get insight into the newly identified enzyme in in vivo H2O2 production, the expression profile of the aaoS o gene was determined. Since the insertional mutation of the aaoS o gene also resulted in an aaoS o -luc fusion (see Materials and Methods), we used this strain to determine aaoS o 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 aaoS o 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.

FIG. 3.
  • Open in new tab
  • Download powerpoint
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 aaoS o 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 aaoS o 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 aaoS o mutant strain of S. oligofermentans after 16 h of growth in TPYG broth. The single-species biofilms of S. oligofermentans ΔaaoS o 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 aaoS o 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.

DISCUSSION

Hydrogen peroxide is widely used by lactic acid bacteria as a biological weapon to compete with other bacteria inhabiting the same ecological niche (16, 27), as with lactobacilli, which can prevent pathogens causing bacterial vaginosis from colonizing the vagina by forming H2O2 (11). H2O2 could be produced via NADH oxidase, pyruvate oxidase, or Lox (4, 20, 32). However, it was found in our study that S. oligofermentans, an oral streptococcus, produces large amounts of H2O2 not only from lactate but also from peptone (Fig. 1A) and from seven l-amino acids (Table 1). By an integrated approach using physiology, biochemistry, and genetics, an ORF protein assigned as SO-LAAO, which is the homologue of flavoprotein SSA-0323 in S. sanguinis, was verified to possess LAAO activity and to be responsible for H2O2 production from l-amino acids in S. oligofermentans.

LAAOs, which catalyze the oxidative deamination of amino acids to yield ammonia, hydrogen peroxide, and ketoacids with oxygen consumed (9), have been widely detected in snake and insect venoms (1, 30) and in some fungi, algae, and bacteria (17, 25, 38). According to the substrate spectra, LAAOs can be divided into two categories, one with a broad spectrum of substrates, like the LAAO of Rhodococcus opacus DSM 43250, which catalyzes not only almost all the 20 l-amino acids but also some derivatives (9), and another with a restricted substrate spectrum, like lysine oxidase of Marinomonas mediterranea, which uses lysine exclusively (21). However, SO-LAAO of S. oligofermentans in this study can represent a novel amino acid oxidase by showing detectable activity against only seven l-amino acids (Table 1).

So far, all the described LAAOs, except the lysine oxidase of Marinomonas mediterranea (21), are flavoproteins possessing a FAD binding domain (37). It is reported that LAAOs possess another characteristic sequence motif, RxGGR, and this motif is also present in several families of flavoproteins, including achacin and aplysianin A, monoamine oxidase, corticosteroid-binding proteins, and tryptophan 2-monooxygenases (37). Great differences have been shown between the deduced SO-LAAO sequence and those of the known LAAOs, at identities from 12% to 16% (data not shown), and the characteristic amino acid differences in the RxGGR motif of SO-LAAO, as well as its highly homologous ORFs (75 to 96% identities), present in five other streptococci (Fig. 2), have been shown, so that SO-LAAO can represent a novel type of LAAO commonly existing in oral streptococci like S. sanguinis and S. gordonii. Although a SO-LAAO homologue (SMU.392c) also exists in the genome of S. mutans, no detectable hydrogen peroxide production from peptone has been measured for this streptococcus (data not shown). This implies that either SMU.392c does not function as an amino acid oxidase, possibly due to the relatively lower sequence identities (75%) to SO-LAAO, or a NADH-dependent peroxidase (AhpC) present in S. mutans (12) can scavenge the endogenous H2O2.

Although the intact cells of S. oligofermentans and SO-LAAO showed the same substrate profile, a large difference (3- to 10-fold) in hydrogen peroxide yield was detected for four l-amino acids, namely l-aspartic acid, l-lysine, l-arginine, and l-glutamine (Table 1). This can possibly be attributed to the fact that charged l-amino acids need a transporting apparatus, such as amino acid permease (13), to get into the cell, while neutral l-amino acids (l-tryptophan, l-isoleucine, and l-asparagine) might permeate cells freely.

In comparison with that mediated by Lox, SO-LAAO catalyzes a relative lower level of H2O2 production, and Lox-mediated inhibition on S. mutans (difference between the colony numbers of lanes 1 and 4 in Fig. 3B) is about 10 times higher than that mediated by SO-LAAO (difference between the colony numbers of lanes 1 and 2 of Fig. 3B) in S. oligofermentans. Therefore, lox can play the predominant role in H2O2 formation by S. oligofermentans when lactate is abundant. However, oral saliva is also in rich in polypeptides (39), and oral streptococci take up salivary oligopeptides and then degrade them into free amino acids by intracellular aminopeptidase (2, 6, 7). Aminopeptidase activity has been detected in S. oligofermentans (data not shown), implying that it might follow a path similar to that of other oral streptococci to obtain amino acids from the environment. Thus, SO-LAAO can give oral streptococci a competitive advantage in dental plaque biofilm. In addition, we also found that the aaoS o mutant of S. oligofermentans not only lost peptone derivate H2O2 production but also reduced the growth to some extent (data not shown), implying that this protein can be involved in other biological process, probably amino acid metabolism.

ACKNOWLEDGMENTS

This study was supported by a China NSFC grant, 30428025, to W. Shi and X. Dong and by the High Technology Research and Development Program of China (863 program), grant 2007AA10Z353.

FOOTNOTES

    • Received 13 March 2008.
    • Accepted 27 April 2008.
  • Copyright © 2008 American Society for Microbiology

REFERENCES

  1. 1.↵
    Ahn, M. Y., K. S. Ryu, Y. W. Lee, and Y. Kim. 2000. Cytotoxicity and l-amino acid oxidase activity of crude insect drugs. Arch. Pharm. Res. 23 : 477-481.
    OpenUrlCrossRefPubMed
  2. 2.↵
    Andersson, C., M. Sund, and L. Linder. 1984. Peptide utilization by oral streptococci. Infect. Immun. 43 : 555-560.
    OpenUrlAbstract/FREE Full Text
  3. 3.↵
    Becker, M. R., B. J. Paster, E. J. Leys, M. L. Moeschberger, S. G. Kenyon, J. L. Galvin, S. K. Boches, F. E. Dewhirst, and A. L. Griffen. 2002. Molecular analysis of bacterial species associated with childhood caries. J. Clin. Microbiol. 40 : 1001-1009.
    OpenUrlAbstract/FREE Full Text
  4. 4.↵
    Carlsson, J., Y. Iwami, and T. Yamada. 1983. Hydrogen peroxide excretion by oral streptococci and effect of lactoperoxidase-thiocyanate-hydrogen peroxide. Infect. Immun. 40 : 70-80.
    OpenUrlAbstract/FREE Full Text
  5. 5.↵
    Caufield, P. W., A. P. Dasanayake, Y. Li, Y. Pan, J. Hsu, and J. M. Hardin. 2000. Natural history of Streptococcus sanguinis in the oral cavity of infants: evidence for a discrete window of infectivity. Infect. Immun. 68 : 4018-4023.
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    Cowman, R. A., and S. S. Baron. 1990. Influence of hydrophobicity on oligopeptide utilization by oral streptococci. J. Dent. Res. 69 : 1847-1851.
    OpenUrlCrossRefPubMed
  7. 7.↵
    Cowman, R. A., and S. S. Baron. 1993. Comparison of aminopeptidase activities in four strains of mutans group oral streptococci. Infect. Immun. 61 : 182-186.
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    Dong, X. Z., Y. H. Xin, W. Y. Jian, X. L. Liu, and D. W. Ling. 2000. Bifidobacterium thermacidophilum sp. nov., isolated from an anaerobic digester. Int. J. Syst. Evol. Microbiol. 50 : 119-125.
    OpenUrlCrossRefPubMedWeb of Science
  9. 9.↵
    Geueke, B., and W. Hummel. 2002. A new bacterial L-amino acid oxidase with a broad substrate specificity: purification and characterization. Enzyme Microb. Technol. 31 : 77-87.
    OpenUrlCrossRef
  10. 10.↵
    Gopalan, K. V., and D. K. Srivastava. 1997. Inhibition of acyl-CoA oxidase by phenol and its implication in measurement of the enzyme activity via the peroxidase-coupled assay system. Anal. Biochem. 250 : 44-50.
    OpenUrlCrossRefPubMed
  11. 11.↵
    Hawes, S. E., S. L. Hillier, J. Benedetti, C. E. Stevens, L. A. Koutsky, P. Wolner-Hanssen, and K. K. Holmes. 1996. Hydrogen peroxide-producing lactobacilli and acquisition of vaginal infections. J. Infect. Dis. 174 : 1058-1063.
    OpenUrlCrossRefPubMedWeb of Science
  12. 12.↵
    Higuchi, M., Y. Yamamoto, L. B. Poole, M. Shimada, Y. Sato, N. Takahashi, and Y. Kamio. 1999. Functions of two types of NADH oxidases in energy metabolism and oxidative stress of Streptococcus mutans. J. Bacteriol. 181 : 5940-5947.
    OpenUrlAbstract/FREE Full Text
  13. 13.↵
    Hosie, A. H., and P. S. Poole. 2001. Bacterial ABC transporters of amino acids. Res. Microbiol. 152 : 259-270.
    OpenUrlCrossRefPubMed
  14. 14.↵
    Kreth, J., E. Hagerman, K. Tam, J. Merritt, D. T. Wong, B. M. Wu, N. V. Myung, W. Shi, and F. Qi. 2004. Quantitative analyses of Streptococcus mutans biofilms with quartz crystal microbalance, microjet impingement and confocal microscopy. Biofilms 1 : 277-284.
    OpenUrlCrossRefPubMed
  15. 15.↵
    Kreth, J., J. Merritt, C. Bordador, W. Shi, and F. Qi. 2004. Transcriptional analysis of mutacin I (mutA) gene expression in planktonic and biofilm cells of Streptococcus mutans using fluorescent protein and glucuronidase reporters. Oral Microbiol. Immunol. 19 : 252-256.
    OpenUrlCrossRefPubMed
  16. 16.↵
    Kreth, J., J. Merritt, W. Shi, and F. Qi. 2005. Competition and coexistence between Streptococcus mutans and Streptococcus sanguinis in the dental biofilm. J. Bacteriol. 187 : 7193-7203.
    OpenUrlAbstract/FREE Full Text
  17. 17.↵
    Le, K. H., and V. R. Villanueva. 1978. Purification and characterization of epsilon-N-trimethyllysine L-amino acid oxidase from Neurospora crassa. Biochim. Biophys. Acta 524 : 288-296.
    OpenUrlPubMed
  18. 18.↵
    Loesche, W. J. 1986. Role of Streptococcus mutans in human dental decay. Microbiol. Rev. 50 : 353-380.
    OpenUrlFREE Full Text
  19. 19.↵
    Loimaranta, V., J. Tenovuo, L. Koivisto, and M. Karp. 1998. Generation of bioluminescent Streptococcus mutans and its usage in rapid analysis of the efficacy of the antimicrobial compounds. Antimicrob. Agents Chemother. 42 : 1906-1910.
    OpenUrlAbstract/FREE Full Text
  20. 20.↵
    Lorquet, F., P. Goffin, L. Muscariello, J. B. Baudry, V. Ladero, M. Sacco, M. Kleerebezem, and P. Hols. 2004. Characterization and functional analysis of the poxB gene, which encodes pyruvate oxidase in Lactobacillus plantarum. J. Bacteriol. 186 : 3749-3759.
    OpenUrlAbstract/FREE Full Text
  21. 21.↵
    Lucas-Elio, P., D. Gomez, F. Solano, and A. Sanchez-Amat. 2006. The antimicrobial activity of Marinocine, synthesized by Marinomonas mediterranea, is due to hydrogen peroxide generated by its lysine oxidase activity. J. Bacteriol. 188 : 2493-2501.
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from microorganism. J. Mol. Biol. 3 : 208-218.
    OpenUrlCrossRefPubMedWeb of Science
  23. 23.↵
    Marsh, P. D., and R. S. Percival. 2006. The oral microflora—friend or foe? Can we decide? Int. Dent. J. 56 : 233-239.
    OpenUrlPubMed
  24. 24.↵
    Motegi, M., Y. Takaqi, H. Yonezawa, N. Hanada, J. Terajima, H. Watanabe, and H. Senpuku. 2006. Assessment of genes associated with Streptococcus mutans biofilm morphology. Appl. Environ. Microbiol. 72 : 6277-6287.
    OpenUrlAbstract/FREE Full Text
  25. 25.↵
    Nishizawa, T., C. C. Aldrich, and D. H. Sherman. 2005. Molecular analysis of the Rebeccamycin l-amino acid oxidase from Lechevalieria aerocolonigenes ATCC 39243. J. Bacteriol. 187 : 2084-2092.
    OpenUrlAbstract/FREE Full Text
  26. 26.↵
    Paster, B. J., S. K. Boches, J. L. Galvin, R. E. Ericson, C. N. Lau, V. A. Levanos, A. Sahasrabudhe, and F. E. Dewhirst. 2001. Bacterial diversity in human subgingival plaque. J. Bacteriol. 183 : 3770-3783.
    OpenUrlAbstract/FREE Full Text
  27. 27.↵
    Pericone, C. D., K. Overweg, P. W. Hermans, and J. N. Weiser. 2000. Inhibitory and bactericidal effects of hydrogen peroxide production by Streptococcus pneumoniae on other inhabitants of the upper respiratory tract. Infect. Immun. 68 : 3990-3997.
    OpenUrlAbstract/FREE Full Text
  28. 28.↵
    Podbielski, A., M. Woischnik, B. A. Leonard, and K. H. Schmidt. 1999. Characterization of nra, a global negative regulator gene in group A streptococci. Mol. Microbiol. 31 : 1051-1064.
    OpenUrlCrossRefPubMedWeb of Science
  29. 29.↵
    Qi, F., P. Chen, and P. W. Caufield. 2001. The group I strain of Streptococcus mutans, UA140, produces both the lantibiotic mutacin I and a nonlantibiotic bacteriocin, mutacin IV. Appl. Environ. Microbiol. 67 : 15-21.
    OpenUrlAbstract/FREE Full Text
  30. 30.↵
    Raibekas, A. A., and V. Massey. 1996. Glycerol-induced development of catalytically active conformation of Crotalus adamanteus L-amino acid oxidase in vitro. Proc. Natl. Acad. Sci. USA 93 : 7546-7551.
    OpenUrlAbstract/FREE Full Text
  31. 31.↵
    Ryan, C. S., and I. Kleinberg. 1995. Bacteria in human mouths involved in the production and utilization of hydrogen peroxide. Arch. Oral Biol. 40 : 753-763.
    OpenUrlCrossRefPubMedWeb of Science
  32. 32.↵
    Seki, M., K. Iida, M. Saito, H. Nakayama, and S. Yoshida. 2004. Hydrogen peroxide production in Streptococcus pyogenes: involvement of lactate oxidase and coupling with aerobic utilization of lactate. J. Bacteriol. 186 : 2046-2051.
    OpenUrlAbstract/FREE Full Text
  33. 33.↵
    Terleckyj, B., N. P. Willett, and G. D. Shockman. 1975. Growth of several cariogenic strains of oral streptococci in a chemically defined medium. Infect. Immun. 11 : 649-655.
    OpenUrlAbstract/FREE Full Text
  34. 34.↵
    Tong, H., X. Gao, and X. Dong. 2003. Streptococcus oligofermentans sp. nov., a novel oral isolate from caries-free humans. Int. J. Syst. Evol. Microbiol. 53 : 1101-1104.
    OpenUrlCrossRefPubMedWeb of Science
  35. 35.↵
    Tong, H., B. Zhu, W. Chen, F. Qi, W. Shi, and X. Dong. 2006. Establishing a genetic system for ecological studies of Streptococcus oligofermentans. FEMS Microbiol. Lett. 264 : 213-219.
    OpenUrlCrossRefPubMed
  36. 36.↵
    Tong, H., W. Chen, J. Merritt, F. Qi, W. Shi, and X. Dong. 2007. Streptococcus oligofermentans inhibits Streptococcus mutans through conversion of lactic acid into inhibitory H2O2: a possible counteroffensive strategy for inter-species competition. Mol. Microbiol. 63 : 872-880.
    OpenUrlCrossRefPubMed
  37. 37.↵
    Vallon, O. 2000. New sequence motifs in flavoprotein: evidence for common ancestry and tools to predict structure. Proteins 38 : 95-114.
    OpenUrlCrossRefPubMedWeb of Science
  38. 38.↵
    Vallon, O., L. Bulte, R. Kuras, J. Olive, and F. A. Wollman. 1993. Extensive accumulation of an extracellular L-amino-acid oxidase during gametogenesis of Chlamydomonas reinhardtii. Eur. J. Biochem. 215 : 351-360.
    OpenUrlCrossRefPubMedWeb of Science
  39. 39.↵
    Van der Hoeven, J. S., M. H. de Jong, A. H. Rogers, and P. J. M. Camp. 1984. A conceptual model for the coexistence of Streptococcus spp. and Actinomyces spp. in dental plaque. J. Dent. Res. 63 : 389-392.
    OpenUrlCrossRefPubMed
  40. 40.↵
    Yonezawa, H., and H. K. Kuramitsu. 2005. Genetic analysis of a unique bacteriocin, Smb, produced by Streptococcus mutans GS5. Antimicrob. Agents Chemother. 49 : 541-548.
    OpenUrlAbstract/FREE Full Text
View Abstract
PreviousNext
Back to top
Download PDF
Citation Tools
SO-LAAO, a Novel l-Amino Acid Oxidase That Enables Streptococcus oligofermentans To Outcompete Streptococcus mutans by Generating H2O2 from Peptone
Huichun Tong, Wei Chen, Wenyuan Shi, Fengxia Qi, Xiuzhu Dong
Journal of Bacteriology Jun 2008, 190 (13) 4716-4721; DOI: 10.1128/JB.00363-08

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Journal of Bacteriology article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
SO-LAAO, a Novel l-Amino Acid Oxidase That Enables Streptococcus oligofermentans To Outcompete Streptococcus mutans by Generating H2O2 from Peptone
(Your Name) has forwarded a page to you from Journal of Bacteriology
(Your Name) thought you would be interested in this article in Journal of Bacteriology.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
SO-LAAO, a Novel l-Amino Acid Oxidase That Enables Streptococcus oligofermentans To Outcompete Streptococcus mutans by Generating H2O2 from Peptone
Huichun Tong, Wei Chen, Wenyuan Shi, Fengxia Qi, Xiuzhu Dong
Journal of Bacteriology Jun 2008, 190 (13) 4716-4721; DOI: 10.1128/JB.00363-08
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

hydrogen peroxide
L-Amino Acid Oxidase
Peptones
streptococcus
Streptococcus mutans

Related Articles

Cited By...

About

  • About JB
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #Jbacteriology

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0021-9193; Online ISSN: 1098-5530