From DNA to lytic proteins: transcription and translation of the bacteriophage T5 holin/endolysin operon.

The analysis of transcriptional activity of the bacteriophage T5 hol/endo operon conducted in the paper revealed a strong constitutive promoter recognized by E. coli RNA polymerase and a transcription initiation point of the operon. It was also shown that the only translational start codon for holin was a non-canonical TTG.

Conservative Tryptophan Residue in the Vicinity of an Active Site of the M15 Family l,d-Peptidases: A Key Element in the Catalysis.

Bioinformatics analysis of the sequences of orthologous zinc-containing peptidases of the M15_C subfamily revealed the presence of a conserved tryptophan residue near the active site, which is not involved in the formation of the protein core. Site-directed mutagenesis of this Trp114/109 residue using two representatives of the family, l-alanoyl-d-glutamate peptidases of bacteriophages T5 (calcium-activated EndoT5) and RB49 (EndoRB49, without ion regulation) as examples, and further analysis of the H NMR spectra of the mutants showed that a decrease in the volume of the W → F → A residue leads to changes in the hydrophobic core and active center of the protein, and also decreases the affinity for regulatory Ca in the EndoT5 mutants. The inactive T5W114A mutant lacks the ability to bind the substrate.

A rapid and efficient technique for the isolation of Bacillus genomic DNA using a cocktail of peptidoglycan hydrolases of different type.

The paper suggests a rapid and efficient technique for isolation of genomic DNA from the bacteria of the genus Bacillus, which is based on the hydrolysis of cell wall peptidoglycan by a cocktail of peptidoglycan hydrolases of different type (L,D-peptidase and N-acetylmuramidase). The comparing of conventional techniques for the isolation of genomic DNA using: a microwave treatment; a treatment with ionic detergents (SDS, CTAB) or a chaotropic agent (GuSCN); and enzymatic hydrolysis (nonspecific, with proteinase K, or specific, with peptidoglycan hydrolases) conducted on Bacillus megaterium, B. subtilis, B.

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

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.

Comparative analysis of the active sites of orthologous endolysins of the Escherichia lytic bacteriophages T5, RB43, and RB49.

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.