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Cited by 3Bacterial Genetics and BiotechnologyBacteriophages and microbial interactionsAntimicrobial Peptides and Activities

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A Predicted Helix-Turn-Helix Core Is Critical for Bacteriophage Kil Peptide to Disrupt Escherichia coli Cell Division

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Naha, Arindam, Cameron, Todd A., Margolin, William·2025·Antibiotics
DOI 10.3390/antibiotics14010052PMC PMC11762379
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Abstract

Background/objectives: 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 λ inhibits Escherichia coli cell division by disrupting FtsZ ring assembly, and this inhibition requires the presence of the essential FtsZ membrane anchor protein ZipA. Methods: To investigate Kil’s molecular mechanism further, we employed deletions, truncations, and molecular modeling to identify the minimal residues necessary for its activity. Results: Modeling suggested that Kil’s 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 the 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 structure and function. Another Kil-like peptide from a closely related enterobacterial phage also disrupted FtsZ ring assembly and required ZipA for this activity. Consistent with its broader activity against FtsZ, λ Kil was able to efficiently inhibit cell division of a uropathogenic E. coli (UPEC) strain. Conclusions: Understanding the structure and function of Kil and similar peptides can potentially reveal additional ways to target FtsZ for antimicrobial therapies and elucidate how FtsZ functions in bacterial cell division.

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Figure 2 The ability of the λ Kil peptide to inhibit cell division requires an intact HTH core. (A) DIC (differential interference contrast) microscopic images of WT E. coli strain WM1074 expressing His-tagged FL Kil or its HTH deletion variants from pBAD24 under repressing (0.2% glucose, Glu) or inducing (0.2% arabinose, Ara) conditions. All derivatives are fused to an N-terminal His epitope. (A,B) Linear schematics and AlphaFold 3-modeled structures of the various derivatives are presented below and above each column of corresponding micrographs. Scale bars, 5 µm. (B) Fluorescence microscopic images of strain WM4952 expressing zapA-GFP plus either FL Kil or HTH deletion derivatives of Kil as described in part A. Arrowheads highlight intact Z-rings. (C) Immunoblot of His-FLAG-FL Kil (lanes 1 and 4) and His-FLAG-Kil variants with either helix 1 (lanes 2 and 5) or helix 2 (lanes 3 and 6) deleted, expressed from a phage T7 promoter in pET15b ± IPTG induction in strain WM5017 (ΔzipA ftsA*) to suppress Kil toxicity and probed with α-FLAG to detect expression of the tagged Kil derivatives. The predicted sizes of the His-FLAG-tagged FL, Δhelix 1, or Δhelix 2 Kil derivatives are 10.5, 8.8, and 8.2 kDa, respectively. A portion of the Ponceau S-stained blot is shown below the immunoblot as a loading control.
Figure 2 The ability of the λ Kil peptide to inhibit cell division requires an intact HTH core. (A) DIC (differential interference contrast) microscopic images of WT E. coli strain WM1074 expressing His-tagged FL Kil or its HTH deletion variants from pBAD24 under repressing (0.2% glucose, Glu) or inducing (0.2% arabinose, Ara) conditions. All derivatives are fused to an N-terminal His epitope. (A,B) Linear schematics and AlphaFold 3-modeled structures of the various derivatives are presented below and above each column of corresponding micrographs. Scale bars, 5 µm. (B) Fluorescence microscopic images of strain WM4952 expressing zapA-GFP plus either FL Kil or HTH deletion derivatives of Kil as described in part A. Arrowheads highlight intact Z-rings. (C) Immunoblot of His-FLAG-FL Kil (lanes 1 and 4) and His-FLAG-Kil variants with either helix 1 (lanes 2 and 5) or helix 2 (lanes 3 and 6) deleted, expressed from a phage T7 promoter in pET15b ± IPTG induction in strain WM5017 (ΔzipA ftsA*) to suppress Kil toxicity and probed with α-FLAG to detect expression of the tagged Kil derivatives. The predicted sizes of the His-FLAG-tagged FL, Δhelix 1, or Δhelix 2 Kil derivatives are 10.5, 8.8, and 8.2 kDa, respectively. A portion of the Ponceau S-stained blot is shown below the immunoblot as a loading control.CC BY · PubMed Central
Figure 3 Shorter λ Kil truncations differentially inhibit E. coli cytokinesis. (A) Additional truncation derivatives of His-tagged λ Kil are diagrammed, using the same color schemes as in Figure 1. (B) DIC microscopic images of WM1074 expressing His-tagged Kil truncations from pBAD24 under repressing (0.2% glucose, Glu) or inducing (0.2% arabinose, Ara) conditions. Scale bar, 5 µm. AlphaFold 3-modeled structures of the Kil derivatives are included. (C) Fluorescence microscopic images of strain XTL937 expressing ZipA-GFP at its native chromosomal locus along with the indicated His-tagged Kil derivatives in pBAD24 are shown. Arrowheads indicate intact Z-rings. Schematic diagrams of the Kil derivatives are presented at the bottom of each corresponding micrograph, along with AlphaFold 3-modeled structures. Scale bar, 5 µm. (D) Quantitation of ZipA-GFP rings from the strains shown in panel C induced with arabinose.
Figure 3 Shorter λ Kil truncations differentially inhibit E. coli cytokinesis. (A) Additional truncation derivatives of His-tagged λ Kil are diagrammed, using the same color schemes as in Figure 1. (B) DIC microscopic images of WM1074 expressing His-tagged Kil truncations from pBAD24 under repressing (0.2% glucose, Glu) or inducing (0.2% arabinose, Ara) conditions. Scale bar, 5 µm. AlphaFold 3-modeled structures of the Kil derivatives are included. (C) Fluorescence microscopic images of strain XTL937 expressing ZipA-GFP at its native chromosomal locus along with the indicated His-tagged Kil derivatives in pBAD24 are shown. Arrowheads indicate intact Z-rings. Schematic diagrams of the Kil derivatives are presented at the bottom of each corresponding micrograph, along with AlphaFold 3-modeled structures. Scale bar, 5 µm. (D) Quantitation of ZipA-GFP rings from the strains shown in panel C induced with arabinose.CC BY · PubMed Central
Figure 4 Comparing cell division inhibition activities of Kil from phage HK629 and λ. (A) Sequence alignment of Kil peptides produced by bacteriophages λ and HK629 using the Clustal Omega multiple sequence alignment tool. HK629 Kil has several potential initiator methionines; longer (Kil) and shorter (Kil’) versions are depicted. Similar amino acids are highlighted in cyan and non-conservative amino acid positions are highlighted in yellow. λ Kil HTH domains are depicted above the alignment. (B) Predicted structure of His-tagged HK629 Kil’ from AlphaFold 3, showing a HTH fold similar to λ Kil. (C) DIC microscopy of pBAD24-His-HK629-Kil’ expressed in WT strain WM1074. Scale bar, 5 µm. (D) DIC (top) or fluorescence (bottom) microscopy of pBAD24-His-HK629-Kil’ expressed in strain XTL937 with ZipA-GFP co-expressed from the native locus (left panels) or in strain WM4952 expressing chromosomal ZapA-GFP (right panels). Expression of His-HK629 Kil’ was either repressed (Glu) or induced (Ara), showing that HK629 Kil’ inhibits cell division and disrupts Z rings. Arrowheads highlight intact Z-rings. Scale bar, 5 µm.
Figure 4 Comparing cell division inhibition activities of Kil from phage HK629 and λ. (A) Sequence alignment of Kil peptides produced by bacteriophages λ and HK629 using the Clustal Omega multiple sequence alignment tool. HK629 Kil has several potential initiator methionines; longer (Kil) and shorter (Kil’) versions are depicted. Similar amino acids are highlighted in cyan and non-conservative amino acid positions are highlighted in yellow. λ Kil HTH domains are depicted above the alignment. (B) Predicted structure of His-tagged HK629 Kil’ from AlphaFold 3, showing a HTH fold similar to λ Kil. (C) DIC microscopy of pBAD24-His-HK629-Kil’ expressed in WT strain WM1074. Scale bar, 5 µm. (D) DIC (top) or fluorescence (bottom) microscopy of pBAD24-His-HK629-Kil’ expressed in strain XTL937 with ZipA-GFP co-expressed from the native locus (left panels) or in strain WM4952 expressing chromosomal ZapA-GFP (right panels). Expression of His-HK629 Kil’ was either repressed (Glu) or induced (Ara), showing that HK629 Kil’ inhibits cell division and disrupts Z rings. Arrowheads highlight intact Z-rings. Scale bar, 5 µm.CC BY · PubMed Central
Figure 5 HK629 Kil’ requires ZipA for its cell division inhibition activity. DIC microscopic images of E. coli WM1657 (ΔzipA ftsA*) or WM1659 (zipA+ ftsA*) expressing His-tagged FL λ Kil or HK629 Kil’ from pBAD24 under repressing (0.2% glucose, Glu) or inducing (0.2% arabinose, Ara) conditions. Cells harboring the FtsAR286W variant (FtsA*) that allows bypass of zipA (ΔzipA::aph) are resistant to the inhibitory effects of λ Kil or HK629 Kil’, but are not resistant when containing FtsAR286W in a zipA+ background. Ribbon diagrams of both Kil peptides are shown as modeled by AlphaFold 3. Scale bar, 5 µm.
Figure 5 HK629 Kil’ requires ZipA for its cell division inhibition activity. DIC microscopic images of E. coli WM1657 (ΔzipA ftsA*) or WM1659 (zipA+ ftsA*) expressing His-tagged FL λ Kil or HK629 Kil’ from pBAD24 under repressing (0.2% glucose, Glu) or inducing (0.2% arabinose, Ara) conditions. Cells harboring the FtsAR286W variant (FtsA*) that allows bypass of zipA (ΔzipA::aph) are resistant to the inhibitory effects of λ Kil or HK629 Kil’, but are not resistant when containing FtsAR286W in a zipA+ background. Ribbon diagrams of both Kil peptides are shown as modeled by AlphaFold 3. Scale bar, 5 µm.CC BY · PubMed Central
Figure 6 Kil peptides produced from phages HK629 or λ inhibit cell division of Uropathogenic E. coli (UPEC) strain UTI89. DIC microscopic images of E. coli cells expressing His-tagged FL λ Kil or HK629 Kil’ from pBAD24 under repressing (0.2% glucose, Glu) or inducing (0.2% arabinose, Ara) conditions. Ribbon diagrams of both Kil peptides are shown as modeled by AlphaFold 3. Scale bar, 5 µm.
Figure 6 Kil peptides produced from phages HK629 or λ inhibit cell division of Uropathogenic E. coli (UPEC) strain UTI89. DIC microscopic images of E. coli cells expressing His-tagged FL λ Kil or HK629 Kil’ from pBAD24 under repressing (0.2% glucose, Glu) or inducing (0.2% arabinose, Ara) conditions. Ribbon diagrams of both Kil peptides are shown as modeled by AlphaFold 3. Scale bar, 5 µm.CC BY · PubMed Central
Figure 1 Schematic representation of the λ Kil peptide and deletion derivatives. Green and blue bars indicate helix 1 and 2, respectively, with the turn highlighted in magenta and the 6xHis tag highlighted in grey. This color scheme is the same for Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6. The full-length (FL) Kil peptide with its intact HTH domains is depicted in the top row, followed by derivatives deleted for helix 1, the turn, or helix 2, shown below. Amino acid numbering of the native Kil peptide is shown above the FL sequence. Ribbon diagrams of each His-tagged Kil derivative are shown as high confidence predictions from AlphaFold 3 with corresponding colors representing the different domains. The predicted local difference test (plDDT) confidence values for each residue of the Kil peptide are shown below the FL Kil peptide schematic.
Figure 1 Schematic representation of the λ Kil peptide and deletion derivatives. Green and blue bars indicate helix 1 and 2, respectively, with the turn highlighted in magenta and the 6xHis tag highlighted in grey. This color scheme is the same for Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6. The full-length (FL) Kil peptide with its intact HTH domains is depicted in the top row, followed by derivatives deleted for helix 1, the turn, or helix 2, shown below. Amino acid numbering of the native Kil peptide is shown above the FL sequence. Ribbon diagrams of each His-tagged Kil derivative are shown as high confidence predictions from AlphaFold 3 with corresponding colors representing the different domains. The predicted local difference test (plDDT) confidence values for each residue of the Kil peptide are shown below the FL Kil peptide schematic.CC BY · PubMed Central

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Metadata

Authors
Naha, A., Cameron, T. A., & Margolin, W.
Affiliations
The University of Texas Health Science Center at Houston (US)
Corresponding author
Margolin, William
ORCID
Venue
Antibiotics
Publisher
MDPI AG
Published
2025-01-08
Volume
14
Issue
1
Pages
52
ISSN
2079-6382 (ISSN-L)
DOI
10.3390/antibiotics14010052
PMC
PMC11762379
OpenAlex
W4406173551
Cited by
3
References
37
Citations by year
2026: 1 · 2025: 2
FWCI
1.75
Type
journal article
Language
en
Open access
Licence
Locations
  • Antibiotics (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
he National Institutes of Health (NIH) (AI171856)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Immunology and Microbiology › Virology › Bacteriophages and microbial interactions
  • Life Sciences › Immunology and Microbiology › Microbiology › Antimicrobial Peptides and Activities
Keywords
FtsZ, bacterial cell division inhibition, divisome, ZipA, antimicrobial peptides, Escherichia coli, uropathogenic Escherichia coli

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