Three Bacillus cereus bacteriophage endolysins are unrelated but reveal high homology to cell wall hydrolases from different bacilli

Abstract

The ply genes encoding the endolysin proteins from Bacillus cereus phages Bastille, TP21, and 12826 were identified, cloned, and sequenced. The endolysins could be overproduced in Escherichia coli (up to 20% of total cellular protein), and the recombinant proteins were purified by a two-step chromatographical procedure. All three enzymes induced rapid and specific lysis of viable cells of several Bacillus species, with highest activity on B. cereus and B. thuringiensis. Ply12 and Ply21 were experimentally shown to be N-acetylmuramoyl-L-alanine amidases (EC 3.5.1.28). No apparent holin genes were found adjacent to the ply genes. However, Ply21 may be endowed with a signal peptide which could play a role in timing of cell lysis by the cytoplasmic phage endolysin. The individual lytic enzymes (PlyBa, 41.1 kDa; Ply21, 29.5 kDa, Ply12, 27.7 kDa) show remarkable heterogeneity, i.e., their amino acid sequences reveal only little homology. The N-terminal part of Ply21 was found to be almost identical to the catalytic domains of a Bacillus sp. cell wall hydrolase (CwlSP) and an autolysin of B. subtilis (CwlA). The C terminus of PlyBa contains a 77-amino-acid sequence repeat which is also homologous to the binding domain of CwlSP. Ply12 shows homology to the major autolysins from B. subtilis and E. coli. Comparison with database sequences indicated a modular organization of the phage lysis proteins where the enzymatic activity is located in the N-terminal region and the C-termini are responsible for specific recognition and binding of Bacillus peptidoglycan. We speculate that the close relationship of the phage enzymes and cell wall autolysins is based upon horizontal gene transfer among different Bacillus phages and their hosts.Numerous bacteriophages for the genus Bacillus have been isolated, and at present, they are grouped into 33 phage species. With one exception, all of them belong to the tailed phages (1). Lytic activity is not necessarily restricted to a single species, especially with respect to the phages used for typing strains of Bacillus cereus and Bacillus thuringiensis (2, 35). B. cereus is genotypically closely related to B. thuringiensis, and it has been suggested that they be grouped into a single species (4). In contrast to the well-studied phages infecting B. subtilis (29,40), no information is available on the molecular biology and relationships of viruses for the B. cereus-B. thuringiensis group.At the end of their multiplication cycle, most bacteriophages are released from the cells through the action of endogenous cell wall hydrolases, termed endolysins or phage lysins (for a review, see reference 38). Several genes encoding lysins from phages infecting both gram-negative and gram-positive hosts have been cloned and sequenced. In some cases, the catalytic mechanisms have been determined, which place the phage lysins into three distinct groups: amidases, muramidases (glycosidases and transglycosylases), and endopeptidases (38). Recently, a fourth type of enzyme was described (L-alanoyl-Dglutamate pe...

Structure and Lytic Activity of a Bacillus anthracis Prophage Endolysin

“…Ply21 is 61% identical with PlyL in its catalytic domain, whereas its C-terminal domain has no obvious homology. Ply21 has previously been shown to lyse B. cereus strains when added exogenously, whereas B. subtilis cells are resistant (16). We confirmed this specificity for the full-length endolysin, but found a dramatic increase in lytic activity toward B. subtilis by the free N-terminal domain.…”

Molecular Aspects and Comparative Genomics of Bacteriophage Endolysins

“…This would explain the variability of this module and the highly selective lytic activity of endolysins. Therefore, in some cases, CBDs can confer highly specific ligand recognition to endolysins, resulting in a very narrow substrate spectrum of the full-length endolysin, often limited to the host species or strain (76) or, to a lesser extent, genus specific (67,77). Well-studied examples of ligand recognition are the pneumococcal endolysins derived from phages Cp-1 (Cpl-1 endolysin), Dp-1 (PAL endolysin), and Ej-1 (Ej-1 endolysin).…”

Gene Cloning and Expression and Secretion of Listeria monocytogenes Bacteriophage-Lytic Enzymes in Lactococcus lactis

“…We have shown here that endolysins may also be exported with the aid of a signal peptide. However, with some putative exceptions (20), this situation has not yet been shown to naturally occur in phages, presumably due to the paramount importance of the independently expressed holins for lysis timing.…”