title: Protein determinants of phage T4 lysis inhibition.
abstract: Genetic studies have established that lysis inhibition in bacteriophage T4 infections occurs when the RI antiholin inhibits the lethal hole-forming function of the T holin. The T-holin is composed of a single N-terminal transmembrane domain and a ~20 kDa periplasmic domain. It accumulates harmlessly throughout the bacteriophage infection cycle until suddenly causing permeabilization of the inner membrane, thereby initiating lysis. The RI antiholin has a SAR domain that directs its secretion to the periplasm, where it can either be inactivated and degraded or be activated as a specific inhibitor of T. Previously, it was shown that the interaction of the soluble domains of these two proteins within the periplasm was necessary for lysis inhibition. We have purified and characterized the periplasmic domains of both T and RI. Both proteins were purified in a modified host that allows disulfide bond formation in the cytoplasm, due to the functional requirement of conserved disulfide bonds. Analytical centrifugation and circular dichroism spectroscopy showed that RI was monomeric and exhibited ~80% alpha-helical content. In contrast, T exhibited a propensity to oligomerize and precipitate at high concentrations. Incubation of RI with T inhibits this aggregation and results in a complex of equimolar T and RI content. Although gel filtration analysis indicated a complex mass of 45 kDa, intermediate between the predicted 30 kDa heterodimer and 60 kDa heterotetramer, sedimentation velocity analysis indicated that the predominant species is the former. These results suggest that RI binding to T is necessary and sufficient for lysis inhibition.
authors.affiliation: Center for Phage Technology, Texas A&M University, College Station, Texas 77843-2128, USA.
authors.forename: Samir H
authors.initials: SH
authors.lastname: Moussa
authors.forename: Vladimir
authors.initials: V
authors.lastname: Kuznetsov
authors.forename: Tram Anh T
authors.initials: TA
authors.lastname: Tran
authors.forename: James C
authors.initials: JC
authors.lastname: Sacchettini
authors.forename: Ry
authors.initials: R
authors.lastname: Young
mesh_terms: D000595:Amino Acid Sequence; D001433:Bacteriolysis; D017122:Bacteriophage T4 / Q000737:chemistry* / Q000472:pathogenicity / Q000502:physiology; D002463:Cell Membrane Permeability; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000821:virology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019897:Periplasm / Q000737:chemistry; D033903:Periplasmic Proteins / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D012995:Solubility; D014461:Ultracentrifugation; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D004220:Disulfides; D033903:Periplasmic Proteins; D014764:Viral Proteins; C120332:holin t protein, Enterobacteria phage T4; D003545:Cysteine