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.
Fang, Qiu (1996). Molecular genetics of ØX174 Lysis. Master's thesis, Texas A&M University. Available electronically from
https : / /hdl .handle .net /1969 .1 /ETD -TAMU -1996 -THESIS -F344.