Heterogeneous endolysins in Listeria monocytogenes bacteriophages: a new class of enzymes and evidence for conserved holin genes within the siphoviral lysis cassettes

Abstract

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.

Bactofection of mammalian cells by Listeria monocytogenes: improvement and mechanism of DNA delivery

“…Our new bactofection system consists of the L. monocytogenes EGD-e strain 21 with deletion in trpS and for additional attenuation in aroA, carrying a plasmid pSP118 with four characteristic features: (1) a reasonably small size (9.2 kb); (2) an origin of replication highly stable in L. monocytogenes; (3) the trpS gene with its own promoter allowing in vitro and in vivo growth of the thus stabilized DtrpS carrier strain at a similar rate as the wild-type strain; (4) the previously reported lysis cassette 13 which expresses the phage lysin 118 19 once the carrier bacteria enter the cytosol of the infected mammalian cells. Although this phage lysin remains in the cytoplasm of the producing carrier bacteria and does not lyse all intracellular bacteria, it proved to be sufficient for lysis of the intracellular bacteria, particularly in combination with the DaroA mutation.…”

“…Next, the trpS gene encoding tryptophanyltRNA synthetase was introduced into pUNK1, resulting in plasmid pSP0. We then inserted into pSP0 the previously described listerial autolysis cassette consisting of the lysis gene of phage A118 (ply118) 19 under the control of the actA promoter (P actA ) which is activated in the cytosol of infected mammalian host cells; 13 this plasmid is called pSP118. The two plasmids (pSP0, pSP118) are present in 95-100 copies per listerial cell as determined by real-time PCR (data not shown).…”

“…In most reported cases, disruption of the bacterial carriers with subsequent release of the plasmid DNA occurs either spontaneously 9 within the infected cell or is facilitated by treatment with antibiotics 14 or use of specific auxotrophic mutants [6][7][8]10,11 that have a higher tendency of disintegration in the host cell intracellular milieu. The suicide L. monocytogenes carrier system that we have recently developed for DNA delivery into mammalian cells takes advantage of a phage lysin 19 that is specifically expressed once the bacteria enter the host cell cytosol. 13 The highly improved system reported here is at least as efficient as all other previously described bacteriamediated DNA delivery systems [6][7][8][9][10][11][12][13][14] with respect to the efficacy of bactofection and by far superior with respect to stability and biosafety.…”

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

“…We began by amplifying ply118 from purified DNA of L. monocytogenes phage A118 (22) by using PCR and the primers ply118-5Ј-ex and ply118-3Ј-ex (listed in Table 2). An artificial ribosome binding site (boldface) and spacer sequence (5Ј-GGAGGAT TTAAAATG-3Ј) was added upstream of the ATG start codon (underlined) via the 5Ј primer.…”

“…We have previously isolated and characterized the Ply endolysins from L. monocytogenes bacteriophages (22). Ply118 represents a 30.8-kDa enzyme from bacteriophage A118 which cleaves between the L-alanine and D-glutamate residues of the Listeria peptidoglycan, whereas the ply511 gene product encodes an N-acetylmuramoyl-L-alanine amidase with a molecular mass of 36.5 kDa.…”