In spite of the high degree of amino acid sequence similarity between the newly discovered phage endolysin SAL-1 and the phage endolysin LysK, SAL-1 has an approximately 2-fold-lower MIC against several Staphylococcus aureus strains and higher bacterial cell-wall-hydrolyzing activity than LysK. The amino acid residue change contributing the most to this enhanced enzymatic activity is a change from glutamic acid to glutamine at the 114th residue.Staphylococcus aureus is a highly virulent human pathogen, and S. aureus infections are a significant cause of morbidity and mortality, particularly in settings such as hospitals, nursing homes, and infirmaries (24). In addition to the more severe consequences of contact with S. aureus, this pathogen is also responsible for many cases of food poisoning (14). Many recent isolates of S. aureus show innate resistance to currently available antibiotics (18), leading to challenges in managing S. aureus infections.Since the discovery of bacteriophages, their destructive effect on their host organisms has been exploited as a way of treating infectious bacteria (10). In addition, phage endolysins, also termed lysins, have been proposed as potent antibacterial agents (15,17). Phage endolysins derived from bacteriophages are bacteriophage-encoded peptidoglycan hydrolases that have evolved to rapidly break down the bacterial cell wall, thereby allowing the release of phage progeny (30). Many phage endolysins have shown promise in preclinical trials involving animal models of human diseases (3,5,7,16,20,27), and they thus constitute a promising route for the discovery and development of novel antibacterial therapeutic agents.The phage endolysin LysK from staphylococcal phage K (22) is a valuable endolysin due to its broad-spectrum activity against the staphylococcal genus (23). A large number of articles have been published about this phage endolysin (2, 6, 11) due to its potency and promise as an antibacterial agent.The present study was prompted by the serendipitous observation that phage endolysin SAL-1, derived from the bacteriophage SAP-1 recently isolated in our laboratory, had a significantly lower MIC than phage endolysin LysK in spite of the high level of amino acid sequence similarity between the two proteins. Based on sequencing studies, we determined that phage endolysin SAL-1 only differs from LysK at three residues: isoleucine instead of valine at the 26th residue, glutamine instead of glutamic acid at the 114th residue, and histidine instead of glutamine at the 486th residue.This article describes a comparative study of the newly discovered phage endolysin SAL-1 and the well-studied phage endolysin LysK.An S. aureus bacteriophage was isolated from an environmental sample by a conventional method (29) and designated bacteriophage SAP-1. The gene encoding the endolysin of bacteriophage SAP-1 was identified by comparison with reported sequences and subcloned into the pBAD-TOPO vector (Invitrogen, Carlsbad, CA) by conventional PCR cloning methods. Escherichia coli TOP10 (I...