Molecular genetics of ØX174 Lysis

Abstract

The 91 codon E gene of the small ssDNA phage OX 1 74, the only viral cistron required for lysis of the host, is entirely imbedded in an alternate reading frame of another essential gene. E has a hydrophobic N-terminus with one putative membrane-spanning domain, whereas the C-terminal 2/3 of the primary sequence is rich in basic and hydrophilic residues. E has been localized to the inner membrane but its mechanism of action is unknown. Phage-infected cells lyse because of a catastrophic defect in the developing septum. Recessive knockout mutations in single host locus slyD eliminate the ability of the E gene, expressed in its normal context of the phage or from a plasmid vector, to lyse the host. slyd encodes a 196 aa cytoplasmic protein of moderate abundance. The sequence of the N-terminal 150 residues of S]yD is homologous to FK506binding proteins (FKBP), an ubiquitous family of peptidyl prolyl cis-trans isomerases (PPlases), suggesting that S]yD is a member of the FKBP protein family. In this study, E mutants restoring the ability to lyse the host cells carrying slvd null alleles were isolated by different mutagenesis methods followed by direct selection of lysis proficient E plasmids in a slyd- host. The pos4b mutant, comprising two missense changes in E, has been repeatedly isolated and no other E mutant has been found, suggesting that the pos4b changes are the only changes sufficient to overcome the slyD-block. Host mutants eliminating the lysis ability of the E pos4b mutant were isolated, but did not map to a chromosomal locus, probably because multiple loci were involved. slyd is able to complement the growth defect caused by slyd] in E. coli C, and the H. influenza slyd gene is fully capable of complementing E. coli slyd in terms of lysis function. A group of slyd missense and deletion mutants that block the lytic function of the OX174 E gene but retain S]yD protein stability were isolated, characterized, and mapped to a structure modeled on a mammalian FKBP. These slyd mutants provide important tools for further genetic and biochemical studies on the unique molecular mechanism of lysis by OX174.