The strategy of bacterial lysis by bacteriophages of the Caudovirales taxon consists in the destruction of the host cell wall “from within” with the help of a group of lytic proteins, represented at least by the holin-endolysin dyad. Holins have membrane activity, and the main function in lysis is performed by endolysins, enzymes that degrade cell wall peptidoglycan. Endolysins alone are capable of causing osmotic lysis of bacteria when applied “from without”. The study of them as antibacterial agents - "enzybiotics", whose mechanism of action is different from antibiotics - is very important given the wide spread of resistant bacterial pathogens. Globular L,D-peptidases are a large family of single-domain endolysins, the first representative of which was isolated by the authors of the project in 2009. Unlike modular phage endolysins of Gram-positive bacteria, which consist of separate catalytic and substrate-binding domains, globular endolysins combine the functions of binding and catalysis in one domain. How catalysis is carried out in this case has not yet been studied, primarily due to the limited structural data and the complexity of the substrate structure. Establishment of exact amino acid residues of the consensus sequence responsible for substrate binding and catalysis, and the molecular mechanism of hydrolysis by globular L,D-peptidases is one of the aims of the project. Potentially, the family of L,D-peptidases has more than a hundred members only in lytic phages, not counting the representatives found in prophages and bacterial metagenomes. However, only 5 globular L,D-peptidases have been characterized so far, of which 3 in our laboratory are endolysins of T5 siphovirus, RB43 and RB49 myoviruses. A new type of calcium-binding loop was found in T5 peptidase; this variant of ionic regulation is still unique among endolysins. Meanwhile, there is reason to believe that periplasmic activation by calcium ions is a common pathway for the regulation of lysis in a number of lytic bacteriophages that infect Gram-negative hosts. Verification of this hypothesis requires new experimental data on the ionic activation of L,D-peptidases. Search, characterization and structural study of new ion-regulated endolysins is the second aim of the project. The genes of lytic proteins in phages are usually organized into "cassettes" and are under the control of common regulatory elements. Often, they have an early promoter, i.e., transcription begins at the initial stage of the phage life cycle. However, the products of lytic genes are toxic to the host cell, and careful control of the timing of lysis is necessary in order for the phage progeny to be fully formed and numerous. So far, the responsibility for the timing of lysis has been attributed almost exclusively to holins, since some holins have been shown to be translationally regulated by dual start. However, many holins do not have a dual start, remaining toxic membrane proteins. There are practically no data on other transcriptional and translational mechanisms of phagolysis regulation. The search for natural regulatory sequences, analysis of transcriptional activity, evaluation of translation efficiency, and identification of new factors that affect the level of expression of various lytic operon genes will help to understand how the phage-host interaction during phagolysis is coordinated at the molecular level, and this is the third aim of the project. It should be emphasized that the target of L,D-peptidases, the L-Ala- D-Glu bond, is present in the peptidoglycan of the vast majority of bacterial species, both Gram-negative and Gram-positive. Thus, these enzymes potentially have a wide spectrum of antibacterial activity. Already characterized members of the family have high specific activity and thermal resistance, which expands the possibilities for their therapeutic (in biomedicine) and non-therapeutic (in biotechnology) applications.