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An open reading frame with homology to known endolysin genes was identified in the genome of Streptomyces sp. strain 212, which is a newly isolated soil bacterium. The heterologously expressed gene product of this endolysin-like gene, called Mitrecin A, demonstrated bacteriolytic activity against several Gram-negative bacteria. The genome of the bacterial strain was sequenced to draft quality using pyrosequencing followed by genome assembly and gene annotation. Within the sequence, a chromosomally located endolysin-like open reading frame was predicted. The gene product, designated Mitrecin A, was heterologously expressed and isolated from contaminating proteins as a fusion protein to a 6-histidine tag. Mitrecin A consists of 127 amino acids arranged in modular domains of activity. It has an estimated molecular weight of 14·3 kDa and retains sequence homology to the M15C peptidase subfamily of zinc metallocarboxypeptidases. The heat-labile purified recombinant protein has an overall positive charge, has optimal catalytic activities at 26°C in solution of pH 9 with 1% saline and has bacteriolytic activity against Gram-negative bacteria of the medically important genera Aeromonas, Escherichia, Salmonella, Shigella, Vibrio and Yersinia.Significance and Impact of the Study: The gene of a new protein antimicrobial, Mitrecin A, was discovered in the genome of a soil bacterium. The purified recombinant enzyme, resulting from heterologous over expression of the gene, was found to be tolerant of increased pH conditions and to have bacteriolytic activity against Gram-negative bacteria of the medically important genera Aeromonas, Escherichia, Salmonella, Shigella, Vibrio and Yersinia. Characterization of enzymes such as Mitrecin A from previously uncharacterized bacteria provides potential options for new biocontrol agents in medically and economically important applications like therapeutics, disinfectants, food preservatives, agricultural livestock antimicrobials, and inhibitors of biofilm production.