The Role of an Activating Peptide in Protease-mediated Suicide of Escherichia coli K12

Abstract

Activation of latent proteinases ensures that the timing of proteolysis is regulated precisely, a process that generally involves proteolytic excision of a pro-region or a tightly bound inhibitor. Here we define the activation mechanism for Lit, a dormant suicide proteinase in Escherichia coli K-12. Previous work has shown that Gol, a short sequence within the major capsid protein gp23, activates Lit during the latter stages of T4 phage infection. This results in cell death and exclusion of the phage from the culture. The Lit site specifically cleaves the host translation factor EF-Tu (elongation factor Tu) after it has formed a weak complex with Gol, which can be supplied as a 29-residue peptide. Gol is absolutely required for Lit activation. but its role in proteolysis is unknown. Using a purified three-component system and kinetic analysis, we demonstrate that under physiological conditions Lit hydrolyzes its substrate very slowly (k cat of ϳ1 s ؊1 ). Given the abundance of EF-Tu in the cell, this finding is consistent with a cell-killing mechanism in which a few cleaved EF-Tu proteins are able block translating ribosomes from functioning. We also demonstrate that less than half of the 29 Gol residues are needed for Lit activation and that the role of the peptide is not to provide catalytic groups but to influence catalysis indirectly through stabilization of the ternary Lit⅐Gol⅐EF-Tu complex. Hence, phage-elicited suicide of E. coli K-12 by Lit is a variant form of "cofactor-induced activation," a mechanism of protease activation that has only been documented previously in pathogen subversion of mammalian hemostasis cascades.

Post-transcriptional control by bacteriophage T4: mRNA decay and inhibition of translation initiation

“…Scanning mutagenesis showed 13 residues in a 20 amino acid core region of Gol to be most important for its activity [124]. Binding of Gol to EF-Tu is required to promote Lit reactivity.…”

“…Binding of Gol peptide is preferential for the open EF-Tu:GDP complex, and binding itself inhibits the EF-Tu GTPase of domain I. When Gol is bound to EF-Tu, it appears that EF-Tu domain I is more accessible to Lit, leading to "substrate-assisted" or "cofactor-induced" activation of cleavage by the protease [124,125]. Lit is a zinc metallo-protease with the active site motif HEXXH of this protease class, but Gol does not contribute directly to active site residues [124-126].…”

“…When Gol is bound to EF-Tu, it appears that EF-Tu domain I is more accessible to Lit, leading to "substrate-assisted" or "cofactor-induced" activation of cleavage by the protease [124,125]. Lit is a zinc metallo-protease with the active site motif HEXXH of this protease class, but Gol does not contribute directly to active site residues [124-126]. Kleanthous and colleagues [124] have noted that the gp23 Gol region is the most conserved region, in the overall conserved gp23 major head protein of sequenced T4-related phages.…”

SP10 Infectivity Is Aborted after Bacteriophage SP10 Infection Induces nonA Transcription on the Prophage SPβ Region of the Bacillus subtilis Genome

“…The phage T4 Gol and Stp proteins regulate the activity of E. coli Lit protease and PrrC anticodon nuclease, respectively, that abort infection (14). Gol promotes the Lit-mediated hydrolysis of elongation factor (EF)-Tu by binding to EF-Tu domains II and III (45). The activity of PrrC is neutralized by EcoprrI; Stp alters interaction between PrrC and EcoprrI, and activated PrrC is released and aborts infection (14).…”

“…B. subtilis was cultured in 300-ml conical flasks containing 60 ml of LB medium at 37°C to an OD 600 of 0.5 and then harvested before infection (0 min) and at 15,30,45, and 60 min after infection with SP10 at an MOI 10. The cells harvested from 1.0-ml cultures were washed once with an equal volume of 10 mM Tris-HCl buffer (pH 7.4), pelleted, frozen, and stored at Ϫ80°C.…”