A greenish yellow pigmented bacterial strain, designated GR5, was recently isolated from a freshwater culture pond for a soft-shell turtle. Phylogenetic analyses based on 16S rRNA gene sequences indicate that strain GR5 belongs to the genus Rheinheimera and its only closest neighbor is the type strain of Rheinheimera texasensis (98.2%). Based on the antibiogram assay, strain GR5 possesses a broad spectrum of antimicrobial activity including Gram-positive and Gram-negative bacteria, yeast, algae, and strain GR5 itself. Strain GR5 can synthesize a macromolecule with antimicrobial activity due to the generation of hydrogen peroxide and this antimicrobial effect can be inhibited by catalase. This antimicrobial activity is active only in complex culture media or chemically defined culture media containing L-lysine. This antimicrobial macromolecule in strain GR5 is shown to be a monomeric protein with a molecular mass of 71 kDa and isoelectric point of approximately 3.68. Liquid chromatography-tandem mass spectrometry analyses reveal close similarity of a 19-amino acid fragment derived from this protein to the antibacterial protein, AlpP from the marine bacterium Pseudoalteromonas tunicata D2, and to the antibacterial protein, marinocine, from the marine bacterium Marinomonas mediterranea. This study explores the nature of antimicrobial macromolecule such as L-lysine oxidase. This is the first report on a freshwater bacterium producing antimicrobial activity by generating hydrogen peroxide through its enzymatic activity of L-lysine oxidase. (C) 2010 Elsevier Inc. All rights reserved.