Synthesis, function and regulation of the lambda holin

Abstract

Two X lysis genes S and R are absolutely required for lysis of E. coli cell. The R gene encodes cytoplasmic endolysin, a transglycosylase, that degrades the peptidoglycan in the periplasm. The R protein has no signal sequence and requires inner membrane lesions, or "holes", for access to the peiiplasm. The S gene encodes the protein, holin, required for hole formation. Hole formation is scheduled at a precise time, leading to cell lysis. Another lysis gene, Rz is dispensable for lysis at low concentration of divalent cations. The scheduling of lysis ("lysis clock") is strictly attributed to the action of S holin and is independent of the intracellular level of endolysin. The S gene has been identified as an open reading frame of 107 codons with the unusual feature of two AUG start codons, specifying residues Met l and Met3. Genetic and biochemical analysis has shown that both start codons are functional: the protein initiated at Met3 is the lethal lysis effector, designated S 105; the protein initiated at Met 1, S 107 is apparently a lysis inhibitor. In these studies, utilizing an antibody raised against the Cterminal peptide of S protein, in vivo evidence is presented that the "lysis clock" is determined by (1) the ratio of S 105 to S 107, (2) the total expression of S gene, and (3) the intrinsic structure of S. The ratio of the two S proteins is primarily determined by an RNA secondary structure, the s& stem-loop and the strength of the ribosome binding site. The 190-nt RNA sequence upstream of the sdi stem-loop is essential for efficient S translation. However, the intrinsic structure of the S protein determines the biggest component of lysis timing. The selective advantage of the scheduled lysis time controlled by the "dual start" motif and the upstream sequence of the sdi stem-loop are discussed. The quaternary structure and membrane topology of S protein are analyzed by using specific antibodies. It is found that biochemically useful quantities of S protein can be produced using a T7 transcriptional and translational expression system. S protein standards has been prepared and used to measure the quantity of the S protein at the onset of cell lysis in various conditions. The kinetics of S protein synthesis in vivo is also detrrnined. A model for the "clock" which determines lysis timing is discussed in the light of these findings.