Genetic dissection of T4 lysis.
t is the holin gene for coliphage T4, encoding a 218-amino-acid (aa) protein essential for the inner membrane hole formation that initiates lysis and terminates the phage infection cycle. T is predicted to be an integral membrane protein that adopts an N(in)-C(out) topology with a single transmembrane domain (TMD). This holin topology is different from those of the well-studied holins S105 (3 TMDs; N(out)-C(in)) of the coliphage lambda and S68 (2 TMDs; N(in)-C(in)) of the lambdoid phage 21. Here, we used random mutagenesis to construct a library of lysis-defective alleles of t to discern residues and domains important for holin function and for the inhibition of lysis by the T4 antiholin, RI. The results show that mutations in all 3 topological domains (N-terminal cytoplasmic, TMD, and C-terminal periplasmic) can abrogate holin function. Additionally, several lysis-defective alleles in the C-terminal domain are no longer competent in binding RI. Taken together, these results shed light on the roles of the previously uncharacterized N-terminal and C-terminal domains in lysis and its real-time regulation.
Department of Biology, Texas A&M University, College Station, Texas, USA Center for Phage Technology, Texas A&M University, College Station, Texas, USA.
Samir H
SH
Moussa
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA.
Jessica L
JL
Lawler
Center for Phage Technology, Texas A&M University, College Station, Texas, USA Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA ryland@tamu.edu.
Ry
R
Young
D000595:Amino Acid Sequence; D001433:Bacteriolysis / Q000502:physiology*; D017122:Bacteriophage T4 / Q000235:genetics* / Q000502:physiology*; D004926:Escherichia coli / Q000821:virology*; D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D014764:Viral Proteins; C120332:holin t protein, Enterobacteria phage T4