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 . Genes RB43ORF159c and RB49р102 were cloned in 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.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1099/jgv.0.001014 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Chemical synthesis of peptidoglycan mimetic-disaccharide-tetrapeptide conjugate and its hydrolysis by bacteriophage T5, RB43 and RB49 L-alanyl-D-glutamate peptidases.
PeerJ
May 2021
Institute for Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.
Background: Endolysins of a number of bacteriophages, including coliphages T5, RB43, and RB49, target the peptidoglycans of the bacterial cell wall. The backbone of these bacterial peptidoglycans consist of alternating N-acetylglucosamine and N-acetylmuramic acid residues that is further "reinforced" by the peptide subunits. Because of the mesh-like structure and insolubility of peptidoglycans, the processes of the peptidoglycan binding and hydrolysis by enzymes cannot be studied by spectral methods.
View Article and Find Full Text PDFComparative analysis of the active sites of orthologous endolysins of the Escherichia lytic bacteriophages T5, RB43, and RB49.
Int J Biol Macromol
January 2021
Laboratory of New Methods in Biology, Institute for Biological Instrumentation of the Russian Academy of Sciences, Federal Research Center "Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences", Pushchino, Moscow Region 142290, Russia; Department of Molecular Medicine and USF Health Byrd Alzheimer's Research Institute, Morsani College of Medicine, University of South Florida, Tampa, FL, USA. Electronic address:
The methods of solution NMR, circular dichroism (CD), and differential scanning calorimetry (DSC) were used to study two zinc-containing L-alanyl-D-glutamate peptidases - endolysins of the pseudo T-even myoviruses RB43 and RB49 (EndoRB43 and EndoRB49, respectively), which are orthologous to the EndoT5, which is a zinc-containing L-alanyl-D-glutamate peptidase of the T5 siphovirus. The spatial conservation of the Zn-binding sites for the enzymes EndoT5, EndoRB43, and EndoRB49 was established, and the key role of Zn ions in the stabilization of the spatial structures of these three peptidases was confirmed. We are showing here that the binding of the Zn ion in the active center of EndoRB49 peptidase causes conformational rearrangements similar to those observed in the EndoT5 peptidase upon binding of Zn and Ca ions and lead to the formation of a catalytically active form of the enzyme.
View Article and Find Full Text PDFAims: The objective of this work was to study the antibacterial specificity and antibacterial effect of endolysins isolated from colibacteriophages RB43, RB49 and T5-as manifested on the exponential and stationary cell cultures of diverse bacteria depending on the growth stage, structure of peptidoglycan (PG) and antibiotic resistance.
Methods And Results: Enzyme activity was assayed by the spectrophotometric method. Antimicrobial activity was estimated by the number of colony forming units (CFUs), with the results represented as logarithmic units.
Two novel thermally resistant endolysins encoded by pseudo T-even bacteriophages RB43 and RB49.
J Gen Virol
March 2018
Skryabin's Institute of Biochemistry and Physiology of Micro-organisms RAS, Pushchino, Moscow region 142290, Russia.
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 . Genes RB43ORF159c and RB49р102 were cloned in 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.
View Article and Find Full Text PDFAn ORFan no more: the bacteriophage T4 39.2 gene product, NwgI, modulates GroEL chaperone function.
Genetics
March 2012
Department of Biochemistry, University of Utah, Salt Lake City, Utah 84112-5650, USA.
Bacteriophages are the most abundant biological entities in our biosphere, characterized by their hyperplasticity, mosaic composition, and the many unknown functions (ORFans) encoded by their immense genetic repertoire. These genes are potentially maintained by the bacteriophage to allow efficient propagation on hosts encountered in nature. To test this hypothesis, we devised a selection to identify bacteriophage-encoded gene(s) that modulate the host Escherichia coli GroEL/GroES chaperone machine, which is essential for the folding of certain host and bacteriophage proteins.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!