Two novel thermally resistant endolysins encoded by pseudo T-even bacteriophages RB43 and RB49

Abstract

Identification and cloning of genes as well as biochemical characterization of the gene products were carried out for two novel endolysins of pseudo T-even lytic bacteriophages RB43 and RB49, which represent different myovirus groups of the subfamily Tevenvirinae. Genes RB43ORF159c and RB49р102 were cloned in E. coli cells, and their products were purified to electrophoretic homogeneity with an up to 80 % yield of total activity. In respect to substrate specificity, both enzymes were found to be lytic l-alanoyl-d-glutamate peptidases belonging to the M15 family. The pH optimum functioning of both endolysins was within the range 7.0-9.0, whereas the optimal values of ionic strength were different for the two proteins (25 mM vs 100 mM for the RB43 and RB49 endolysins respectively). Both peptidases were thermally resistant, with the RB43 endolysin being more stable (it restored 81 % of enzyme activity and 96 % of secondary structure after a 10 min heating at 90 °C) than its RB49 counterpart (27 and 77% respectively). The possible origin of genes of lytic l-alanoyl-d-glutamate peptidases of myoviruses as a result of horizontal transfer in the variable parts of genomes between unrelated phages having a common host is discussed.

Chemical synthesis of peptidoglycan mimetic–disaccharide-tetrapeptide conjugate and its hydrolysis by bacteriophage T5, RB43 and RB49 L-alanyl-D-glutamate peptidases

“…Active homogeneous preparations of the recombinant enzymes EndoT5, EndoRB43 and EndoRB49 were obtained from producer strains obtained earlier, using the previously developed method ( Mikoulinskaia et al, 2018 ) and stored frozen at −20 °С.…”

“…In peptidoglycans of type A, which are characteristic of most Gram-positive and all Gram-negative bacteria, the l-Ala-d-Glu bond is always present. This bond is the target of endolysins of a number of bacteriophages, including coliphages T5, RB43, and RB49 ( Mikoulinskaia et al, 2018 ; Mikoulinskaia et al, 2013 ).…”

Lysis of cells of diverse bacteria by l,d ‐peptidases of Escherichia coli bacteriophages RB43, RB49 and T5

“…Homogeneous preparations of recombinant endolysins of bacteriophages RB43, RB49 and T5 were obtained in the laboratory from the cells of producer strains, as described earlier (Mikoulinskaia et al 2009, 2018…”

“…Homogeneous preparations of recombinant endolysins of bacteriophages RB43, RB49 and T5 were obtained in the laboratory from the cells of producer strains, as described earlier (Mikoulinskaia et al2009(Mikoulinskaia et al , 2018. Hen egg white lysozyme (HEWL) was purchased from Serva (Heidelberg, Germany).…”

“…Earlier, we described a group of single-domain endolysins isolated from three unrelated coliphages: T5 (Siphoviridae), RB43 and RB49 (pseudo T-even Myoviridae). These endolysins (EndoT5, endolysin of bacteriophage T5; EndoRB43, endolysin of bacteriophage RB43 and EndoRB49, endolysin of bacteriophage RB49) are small proteins (14-15 kDa), which have a relatively weak amino acid homology but belong to the same group of zinc-containing peptidases of the M15_3 family (Mikoulinskaia et al2009(Mikoulinskaia et al , 2018. EndoT5, EndoRB43 and EndoRB49 are globular proteins with high conformational stability and temperature resistance, which makes them attractive as potential pharmaceuticals.…”