The gene product was named PlyBt33. In this study, we analyzed Omipalisib nmr the functional domain composition of PlyBt33 using bioinformatics, and then demonstrated its biological activity after separately expressing the catalytic and cell wall binding domains in Escherichia coli. PlyBt33 showed a broad lytic spectrum against the tested Bacillus strains. Additionally, its cell wall binding domain exhibited low amino acid sequence similarity to previously reported domains. Results Identification and domain composition of endolysin from phage BtCS33 Position-specific iterated BLAST (PSI-BLAST) analysis of the phage BtCS33 genome identified orf18 as the gene encoding the endolysin PlyBt33.

Amino acid sequence alignment of PlyBt33 with several endolysins from Bacillus phages or prophages (ISRIB Figure 1a) revealed high similarity to PlyPH [9] and PlyBa04 [23] (about 67% and 71%, respectively), but low similarity to PlyG [18], PlyL [17], and Ply21 [27] (less than 15%). Figure 1 Amino acid sequence alignment TPCA-1 and structural composition of the studied Bacillus endolysins. (a) Alignment of the amino acid sequences of PlyBt33 with other bacteriophage endolysins. PlyPH, PlyBa04, and PlyL were the putative B. anthracis prophage endolysins [9, 16, 22]; PlyG was the endolysin from B. anthracis phage Gamma [17, 28]; Ply21 was the endolysin from B. cereus phage TP21[9, 29]. Residues critical for the cell wall binding activity

of PlyG to B. anthracis[30] and the corresponding residues in the other endolysins were boxed in red. (b) Schematic representation of PlyBt33 and other Bacillus. sp. endolysins. Amidase_2 and GH-25 represented the catalytic region of each endolysin; Amidase02_C and SH3_5 represented the cell wall binding region of each endolysin. The numbers above the rectangles corresponded to amino acid residue positions. Pfam and CDD analysis showed that PlyBt33 was composed

of two functional domains (Figure 1b), the N-terminal catalytic domain (amino acid residues 5–186) and the C-terminal cell wall binding domain (amino acid residues 224–269). Figure 1b showed the Pfam analysis of four endolysins from Bacillus phages, and indicated that the N-terminus PRKACG of PlyBt33 was a GH25 family hydrolase domain, while the C-terminus was an amidase02_C domain. PlyBt33 exhibited the same domain composition as PlyPH, but differed from PlyG and Ply21. According to homology-based endolysin classification [1], PlyBt33 is a putative member of the N-acetylmuramoyl-L-alanine amidases. Expression and purification of endolysin To determine the function of the entire PlyBt33 protein, the N-terminal region (PlyBt33-N, amino acids 1–186), and the C-terminus combined with the internal region (PlyBt33-IC, amino acids 187–272) (Figure 2a), we constructed three recombinant strains and induced protein expression with isopropyl-β-D-thio-galactoside (IPTG).