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Journal of Bacteriology, July 2008, p. 4716-4721, Vol. 190, No. 13
0021-9193/08/$08.00+0     doi:10.1128/JB.00363-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China,¹ School of Dentistry, University of California, Los Angeles, California 90095,² Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California 90095,³ Oral Biology, College of Dentistry, and Microbiology and Immunology, College of Medicine, University of Oklahoma Health Sciences Center BRC366, Oklahoma City, Oklahoma 73104⁴

Received 13 March 2008/ Accepted 27 April 2008

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 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 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 aao_So 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 aao_So mutant, indicating the gene's importance in interspecies competition.

Published ahead of print on 9 May 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

^# Present address: College of Animal Science and Technology in Tarim University, Alar, Singkiang 843300, China.