Cameron, T. A. | bioRxiv Science

bioRxiv2024

Bacteriophage Kil peptide folds into a predicted helix-turn-helix structure to disrupt Escherichia coli cell division

FtsZ, a eukaryotic tubulin homolog and an essential component of the bacterial divisome, is the target of numerous antimicrobial compounds as well as proteins and peptides, most of which inhibit FtsZ polymerization dynamics. We previously showed that the Kil peptide from bacteriophage lambda; inhibits Escherichia coli cell division by disrupting FtsZ ring assembly, and this inhibition requires the presence of the essential FtsZ membrane anchor protein ZipA. To investigate the Kil molecular mechanism further, we employed truncation mutants and molecular modeling to identify the minimal residues necessary for its activity. Modeling suggests that the Kil core segment folds into a helix-turn-helix (HTH) structure. Deleting either the C-terminal 11 residues or the N-terminal 5 residues of Kil still allowed inhibition of E. coli cell division, but removing both termini nearly abolished this activity, indicating that a minimal region within the Kil HTH core is essential for its function. Another Kil-like peptide from a closely related enterobacterial phage also disrupts FtsZ ring assembly and requires ZipA for this activity. Consistent with its broader activity against FtsZ, lambda Kil was able to efficiently inhibit cell division of a uropathogenic E. coli (UPEC) strain. Understanding the function of Kil and similar peptides can potentially reveal how FtsZ functions in bacterial cell division and additional ways to target FtsZ for antimicrobial therapies.

molecular biology↗

bioRxiv2022

A fluorescent reporter for FtsA is functional as the sole FtsA in Escherichia coli and has hypermorphic properties

FtsA, a homolog of actin, is essential for cell division of Escherichia coli and is widely conserved among many bacteria. FtsA helps to tether polymers of the bacterial tubulin homolog FtsZ to the cytoplasmic membrane as part of the cytokinetic Z ring. GFP fusions to FtsA have illuminated FtsAs localization in live E. coli, but these fusions have not been fully functional and required the presence of the native FtsA. Here, we characterize "sandwich" fusions of E. coli FtsA to either mCherry or msfGFP that are fully functional for cell division and exhibit fluorescent rings at midcell that persist throughout constriction until cell separation. FtsA within the Z ring moved circumferentially like FtsZ, and FtsA outside the rings formed highly dynamic patches at the membrane. Notably, both FtsA-mCherry and FtsA-msfGFP acted as mild hypermorphs, as they were not toxic when overproduced, bypassed the essential cell division protein ZipA, and suppressed several thermosensitive fts alleles, although not as effectively as the prototypical hypermorph FtsA*. Overall, our results indicate that fluorescent FtsA sandwich fusions can be used as the sole FtsA in E. coli and thus should shed new light on FtsA dynamics during the cell division cycle in this model system. ImportanceFtsA is a key conserved cell division protein, and E. coli is the most well studied model system for bacterial cell division. One obstacle to full understanding of this process is the lack of a fully functional fluorescent reporter for FtsA in vivo. Here, we describe a fluorescent fusion to E. coli FtsA that divides cells efficiently in the absence of the native FtsA and can be used to monitor FtsA dynamics during cell division.

microbiology↗