1.E.5 The PRD1 Phage P35 Holin (P35 Holin) Family
The prototype for this family is the lipid-containing PRD1 enterobacterial phage holin protein P35 (12.8 kDa) encoded by gene XXXV (orfT) (Rydman and Bamford, 2003). It is a component of a typical holin-endolysin system which functions to lyse the host bacterial cell. The autolysin is the gene XV product, P15, a soluble β1,4-N-acetylmuramidase that causes lysis following digestion of the Gram-negative bacterial cell wall. A defect in its structural gene can be corrected by expression of the lambda phage S gene encoding the lambda holin.
P35 holin has 3 TMSs with 5 positively charged residues between TMSs 1 and 2 and 4 at the C-terminus (Rydman and Bamford, 2003). It is therefore likely that the N-terminus is in the periplasm and the C-terminus is in the cytoplasm. Homologues of 109 aa, which also have 3 putative TMSs, are encoded in the genomes of Xylella fastidiosa strains (25% identity; 46% similarity; no gaps).
PRD1 infects gram-negative cells harboring a conjugative IncP plasmid. Here we studied the lytic functions of PRD1. Using infected cells and plasmid-borne lysis genes, we demonstrated that a two-component lysis system (holin-endolysin) operates to release progeny phage particles from the host cell. Monitoring of ion fluxes and the ATP content of the infected cells allowed us to build a model of the sequence of lysis-related physiological changes. A decrease in the intracellular level of ATP is the earliest indicator of cell lysis, followed by the leakage of K+ from the cytosol approximately 20 min prior to the decrease in culture turbidity. However, the K+ efflux does not immediately lead to the depolarization of the cytoplasmic membrane or leakage of the intracellular ATP. These effects are observed only approximately 5 to 10 min prior to cell lysis. Similar results were obtained using cells expressing the holin and endolysin genes from plasmids.
The reaction catalyzed by P35 holin is:
(autolysin)in → (autolysin)out