The Kil Peptide of Bacteriophage λ Blocks Escherichia coli Cytokinesis via ZipA-Dependent Inhibition of FtsZ Assembly

Abstract

Assembly of the essential, tubulin-like FtsZ protein into a ring-shaped structure at the nascent division site determines the timing and position of cytokinesis in most bacteria and serves as a scaffold for recruitment of the cell division machinery. Here we report that expression of bacteriophage λ kil, either from a resident phage or from a plasmid, induces filamentation of Escherichia coli cells by rapid inhibition of FtsZ ring formation. Mutant alleles of ftsZ resistant to the Kil protein map to the FtsZ polymer subunit interface, stabilize FtsZ ring assembly, and confer increased resistance to endogenous FtsZ inhibitors, consistent with Kil inhibiting FtsZ assembly. Cells with the normally essential cell division gene zipA deleted (in a modified background) display normal FtsZ rings after kil expression, suggesting that ZipA is required for Kil-mediated inhibition of FtsZ rings in vivo. In support of this model, point mutations in the C-terminal FtsZ-interaction domain of ZipA abrogate Kil activity without discernibly altering FtsZ-ZipA interactions. An affinity-tagged-Kil derivative interacts with both FtsZ and ZipA, and inhibits sedimentation of FtsZ filament bundles in vitro. Together, these data inspire a model in which Kil interacts with FtsZ and ZipA in the cell to prevent FtsZ assembly into a coherent, division-competent ring structure. Phage growth assays show that kil+ phage lyse ∼30% later than kil mutant phage, suggesting that Kil delays lysis, perhaps via its interaction with FtsZ and ZipA.

Evidence That Bacteriophage λ Kil Peptide Inhibits Bacterial Cell Division by Disrupting FtsZ Protofilaments and Sequestering Protein Subunits

“…Construction of DPH677 Strain-To overproduce an N-terminally hexahistidine-tagged Kil peptide (His 6 -Kil), the kil gene from CC4506 genomic DNA was amplified using primers DPH170 and DPH211 (31) and cloned into pET28a (Stratagene) between the NdeI and HindIII sites, creating pDPH100. The his 6 -kil-containing sequence was then cleaved from pDH100 using XbaI and XhoI and cloned into pET15b to produce pDH148.…”

“…The his 6 -kil-containing sequence was then cleaved from pDH100 using XbaI and XhoI and cloned into pET15b to produce pDH148. pDH148 was then introduced into E. coli strain DPH673 (31), to create the BL21(DE3) expression strain DPH677. To reduce the toxicity of expressed kil, this strain also carries plasmid pBS58, which expresses extra ftsA and ftsZ, as well as a deletion of the zipA gene and an ftsA* mutation to bypass zipA.…”

“…These viruses constitute a potential tool to fight against pathogenic bacteria, but to date the molecular details of the action of their inhibitory factors on division ring assembly remain largely unknown. It has been recently reported that the kil gene from E. coli bacteriophage encodes a 47-amino acid protein that antagonizes FtsZ polymerization in vivo and in vitro, blocking cell division at the latest stages of the viral lytic phase (31). According to this study, the inhibitor requires the presence of ZipA for its activity in vivo, although this requirement is overcome when Kil is overproduced.…”

“…According to this study, the inhibitor requires the presence of ZipA for its activity in vivo, although this requirement is overcome when Kil is overproduced. In vitro, Kil interacts with FtsZ in pulldown experiments and precludes the sedimentation of FtsZ polymer bundles elicited by GTP plus Ca 2ϩ or truncated ZipA (31). When present, this inhibitor produces a slight increase in the GTPase activity of FtsZ, under conditions promoting bundle formation through lateral interaction of protofilaments (31).…”

“…In vitro, Kil interacts with FtsZ in pulldown experiments and precludes the sedimentation of FtsZ polymer bundles elicited by GTP plus Ca 2ϩ or truncated ZipA (31). When present, this inhibitor produces a slight increase in the GTPase activity of FtsZ, under conditions promoting bundle formation through lateral interaction of protofilaments (31). Although these results clearly identify Kil as an FtsZ polymerization inhibitor, new research is still required to explore the molecular mechanisms by which this protein counteracts FtsZ assembly and particularly to address the influence of this new antagonist on the one subunit thick protofilaments that constitute the basic unit of the Z-ring.…”