PMC

Western blots of TP901-1erm and mutant derivatives using anti-N-terminal Tal₂₀₀₉ antibodies. Lanes 1 and 6, molecular weight ladder; lanes 2–5, TP901-1erm, TP901-1_Gly>Arg, TP901-1_H892A, and TP901-1_Gly603*, respectively.

Reverse phase HPLC separation of muropeptides obtained by digestion of L. lactis PG by mutanolysin (A) and mutanolysin + TalM23-LysM (B). Peak numbers refer to structures identified by MALDI-TOF (supplemental Table 2).

Comparative analysis of the TMP, Dit, and Tal proteins from P335 species phages. The locations of predicted peptidoglycan hydrolase domains are highlighted by colored boxes, as indicated in the legend. The percentage amino acid identity between protein sequences was estimated by BLAST2p and is indicated in the shaded gray boxes.

Analysis of peptidoglycan hydrolase activity associated with M23 peptidase of Tal protein constructs. A, diagrammatic representation of Tal₂₀₀₉ tail fiber and recombinant proteins Tal₂₀₀₉-5ΔN and TalM23-LysM. B, hydrolysis of L. lactis UC509.9 cell wall. The negative control (water) is marked as black diamonds, mutanolysin-only is marked with dark gray squares, mutanolysin and Tal₂₀₀₉-5ΔN is marked with white triangles, and mutanolysin and TalM23-LysM are marked with light gray circles. Error bars indicate S.D., whereas asterisks are located above statistically significant values (p < 0.001). C, diagram displaying Tal_901-1 tail fiber and recombinant proteins Tal_901-1-5ΔN and Tal_901-1-5ΔN_H892A. The histidine or alanine amino acid at position 892 in the M23 peptidase catalytic site of constructs Tal_901-1-5ΔN and Tal_901-1-5ΔN_H892A, respectively, is highlighted. D, hydrolysis of L. lactis 3107 cell wall. The negative control (water) is marked as black diamonds, mutanolysin-only is marked with dark gray squares, mutanolysin and Tal_901-1-5ΔN_H892A is marked with white triangles, and mutanolysin and Tal_901-1-5ΔN is marked with light gray circles. Error bars indicate S.D., whereas asterisks are located above statistically significant values (p < 0.001).

Bar graphs showing phage assays of TP901-1erm and mutant derivatives. TP901-1erm results are in solid gray, TP901-1erm_Gly>Arg are white with gray dots, TP901-1erm_H892A are gray and white checkers, and TP901-1erm_Gly603* are gray with white dots. Error bars indicate S.D., whereas asterisks indicate mutants that are statistical significance (p < 0.05). A, plaque assays of TP901-1erm and mutant derivatives. B, adsorption assays of TP901-1erm and mutant derivatives, measuring the percentage of bacterial bound phage in 10 min. C, ECOI formation by TP901-1erm and mutant derivatives. Each ECOI was calculated as the number of COIs formed on stationary phase cells relative to the number of COIs formed on exponentially growing cells. D, competition assays between TP901-1erm_Gly>Arg and TP901-1erm_Gly603*, comparing their ability to form COIs within 5 min. TP901-1erm_Gly>Arg are white with gray dots, and TP901-1erm_Gly603* are gray with white dots. Mutant phage TP901-1erm_Gly>Arg permanently retains the C terminus of its tail fiber protein, which possesses the 0, whereas mutant TP901-1erm_Gly603* incorporates only a truncated derivative of Tal_901-1.