Listeria monocytogenes bacteriophages A118, A500 and A511 are members of three distinct phage groups with characteristic host ranges. Their endolysin (ply) genes were cloned and expressed in Escherichia coli as demonstrated by the conferred lytic phenotype when colonies of recombinant cells were overlaid with a lawn of Listeria cells. The nucleotide sequences of the cloned DNA fragments were determined and the individual enzymes (PLY118, 30.8 kDa; PLY500, 33.4 kDa; PLY511, 36.5 kDa) were shown to have varying degrees of homology within their N-terminal or C-terminal domains. Transcriptional analysis revealed them to be 'late' genes with transcription beginning 15-20 min post-infection. The enzymes were overexpressed and partially purified and their individual specificities examined. When applied exogenously, the lysins induced rapid lysis of Listeria strains from all species but generally did not affect other bacteria. Using hydrolysis of purified listerial cell walls, PLY511 was characterized as an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) and shows homology in its N-terminal domain to other enzymes of this type. In contrast, PLY118 and PLY500 were shown to represent a new class of cell wall lytic enzymes which cleave between the L-alanine and D-glutamate residues of listerial peptidoglycan; these were designated as L-alanoyl-D-glutamate peptidases. These two enzymes share homology in the N-terminal domain which we propose determines hydrolytic specificity. Highly conserved holin (hol) gene sequences are present upstream of ply118 and ply500. They encode proteins of structural similarity to the product of phage lambda gene S, and are predicted to be membrane proteins which form pores to allow access of the lysins to their peptidoglycan substrates. This arrangement of conserved holin genes with downstream lysin genes among the siphoviral lysis cassettes explains why the cytoplasmic endolysins alone are not lethal, since they require a specific transport function across the cell membrane.

Different approaches were used to examine the function of teichoic acids (TA) as phage receptors among selected Listeria strains, and to identify and characterize specific receptor structures of host cells belonging to different serovars. This included successive removal of cell wall constituents, preparation and purification of TA, and GLC analysis of TA components. Adsorption of Listeria monocytogenes bacteriophages could be inhibited by polyvalent antisera, specific lectins and addition of purified TA. The results confirmed the necessity of TA in general and of rhamnose and glucosamine in particular for adsorption of Listeria phage Al18, which is a temperate Siphovirus (morphotype Bl), attacking predominantly serovars 1/2. Host binding of siphoviral phage A500 (predominantly lysing serovars 4b), was also dependent on cell wall TA. A phage-resistant L. monocytogenes strain was shown to lack glucosamine in its TA. These results support the view that TA substituents may play an important role not only in antigenicity of Listeria cells, but also in specificity of host recognition by two temperate Listeria phages. In contrast, the broad-host-range virulent phage A51 1 (Myovirus, morphotype A l ) uses the listerial peptidoglycan as primary receptor. This corresponds well with the observation that A511 is capable of lysing the majority of L. monocytogenes strains. , 1982;Keogh & Pettingill, 1983; Sijtsma etal., 1988; Valyasevi etal., 1994;Yokokura, 1971). In particular, cell wall teichoic acids (TA) have been found to be important for phage adsorption in Stapblococcas spp. and Bacilhs spp. (Archibald, 1976;Chatterjee, 1969; Coyette & Ghuysen, 1968; Givan e t al., 1982 ; Glaser e t al., 1966 ;Schleifer & Steber, 1974;Young, 1967 D-galactose and N-acetylglucosamine (Fiedler et al., 1984 ;Fiedler & Ruhland, 1987; Kamisango etal., 1983;Knox & Wicken, 1973 ; Ullmann & Cameron, 1969 Busse, 1990;Loessner, 1991). Propagating strains are given in parentheses. Propagation of phages was done according to Loessner & Busse (1990). For adsorption assays, host strains were grown overnight in tryptose broth (Merck) at 30 "C. Cells were washed with SM buffer (Sambrook e t al., l989), resuspended in equal volumes, and titrated. One millilitre volumes of suspensions were used for adsorption assays. KeywordsPreparation of listerial cell walls. Broth cultures (late exponential phase) were inactivated by steaming (100 OC, 10 min), followed by concentration and washing by ultrafiltration (Sartocon module, cellulose acetate, pore size 0.2 pm, Sartorius). Finally, cells were harvested by centrifugation (10 000 g , 15 min), resuspended in SM buffer, and mechanically disrupted by double passage through a French pressure cell (SLM Aminco) at 40000 p.s.i. (276 MPa). Cell walls were prepared by methods similar to those described by Fiedler et al. (1984) and Valyasevi e t al. (1990). Crude cell walls were sedimented by differential centrifugation (1 5 000 g , 30 min), resuspended in SM buffer, and treated with DNase/RNase (3 h) and proteina...

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...