Category:Team Phage Queens

In Figure 4, an assay of lytic enzyme activity of PlyL and its mutant, Ply21 was conducted on the cell wall of five bacterial hosts. The mutant Ply21 is described as the full-length protein of PlyL with the removal of the C-terminal domain involved in cell wall binding, leaving behind only the presence of the N-terminal catalytic domain. In Panel B, the lysing abilities of Ply21 are shown, and the removal of the C-terminal cell wall binding domain did not have a significant effect on the lysing of the bacterial hosts. However, the N-terminal catalytic domain displayed a higher percent absorbance over time as compared to Panel A, (which displayed the lysing abilities of the full length protein). Furthermore, B. subtilis (yellow-orange line) displayed the highest lysing ability than all of the bacterial hosts, with the absorbance rate being higher in the graph of Panel B than Panel A. This concludes that the mutant PlyL 21, also known as the N-terminal catalytic domain of the full length PlyL protein with the C-terminal binding domain removed, is most efficient in cytolysis and that the removal of the C-terminal cell wall binding domain does not inhibit nor improve the lysing ability of PlyL on the cell wall of bacterial hosts.

Zinc binding and catalysis is highly conserved between Bacillus phages’ endolysins. This is indicated by the blue and red triangles located under the specific residues in the Figure 1. There is a high sequence alignment between PlyG and PlyLBa02 as shown by Figure 1 which is a sequence alignment diagram from ClustalX. PlyLBA02 and PlyG have a 93% identity in the enzymatic domain and a 60% identity in the C-terminal domain. Because of this similarity and the conserved residues, we can come to conclusion the PlyG is also involved in N-acetylmuramoyl-L-alanine amidase activity.

In Figure 2, the structures and desirable behaviors were compared between PlyL and XlyA, concluding them to be homologous amidase lysins. Specifically in Panel A of Figure 2, the lysing abilities of the strains were compared on their effect on host cell B. Subtilis. The full-length protein of XlyA was similar to its lysing abilities of the catalytic domain of PlyL, which is responsible for producing amidase, an enzyme that cleaves peptidoglycan in the cell walls of the bacterial host cells.

Figure 1 shows four different classes of endo-N-acetylmuramidases which all use lysosome structures and this is presented in the cellosyl. This shows that lysozyme activity is being used.

Figure 5 shows that catalytic activity of N-terminal domain binding to the C-terminal domain in which it shows the C-terminal domain to catalyze the cell wall component. This proves that the N-acetylmuramoyl-L-alanine amidase activity is being used.

Figure 2 shows the results of high performance liquid chromatography of a reaction between AmpD and compound 4. It shows that compound 4 was diminished and that two new compounds labeled 19 and 3 were created. The structure of compounds 19 and 3 were determined through mass spectrometry and indicates that AmpD breaks the amide bond in compound 4. Table one shows the same results for AmpD and compounds 4-9 further justifying the role of AmpD in N-acetylmuramoyl-l-alanine amidase activity.

Zinc binding and catalysis is highly conserved between Bacillus phages. There is a high sequence alignment between PlyLBa02 and PlyG as shown by Figure 1 which is a sequence alignment diagram from ClustalX. PlyG and PlyLBA02 have a 93% identity in the enzymatic domain and a 60% identity in the C-terminal domain. Because of this similarity, we can come to conclusion the PlylBa02 is also involved in N-acetylmuramoyl-L-alanine amidase activity.