SO-LAAO, a novel L-amino acid oxidase that enables Streptococcus oligofermentans to outcompete Streptococcus mutans by generating H2O2 from peptone

Abstract

We previously demonstrated that Streptococcus oligofermentans suppressed the growth of Streptococcus mutans, the primary cariogenic pathogen, by producing hydrogen peroxide (H(2)O(2)) 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 H(2)O(2) 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 H(2)O(2) 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 aao(So), 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 H(2)O(2) 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 aao(So) mutant of S. oligofermentans did lose H(2)O(2) 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 aao(So) mutant, indicating the gene's importance in interspecies competition.

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.

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.

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.