Colicin‐Bearing Plasmids Carried by Shiga Toxin‐Producing E. coli (STEC) Analyzed by Targeted Top‐Down MS Analysis

Rationale Colicins are protein toxins produced by bacteria that destroy the nucleic acids or outer membranes of their bacterial neighbors. Their genes are encoded in small extra‐chromosomal plasmids that play an important role in bacterial survival. Although colicins do not contribute to bacterial pathogen virulence, it is important to develop methods to identify molecular determinants that facilitate pathogen survival. Methods Shiga toxin‐producing Escherichia coli (STEC) were analyzed for the presence of colicin‐bearing plasmids using antibiotic induction, MALDI‐TOF‐TOF, Orbitrap mass spectrometry, and targeted top‐down analysis using in‐house software. Small plasmid sequencing was used to confirm plasmid‐encoded genes as well as their upstream regulation. AlphaFold3 was used to rationalize expected (as well as anomalous) fragment ions detected by MS/MS post‐source decay (PSD). Results Colicin immunity (Imm) proteins were detected and identified by targeted top‐down mass spectrometry in two STEC strains (serotypes O26:H11 and O104:H7) that carried one or more colicin plasmids. ImBac, ImmD, and a 7.8 kDa hypothetical protein (whose gene resides on a plasmid that encodes a pore‐forming colicin) were detected and identified. Whole genome sequencing (by other groups) and our small plasmid sequencing confirmed the colicin/immunity genes as well as their upstream regulatory control. Conclusions MALDI‐TOF‐TOF‐MS/MS‐PSD is an effective platform for rapid detection and identification of inducible antibacterial protein toxins. We also note that the previously reported glycine‐enhanced aspartic acid effect (AAE) appears to occur most often at unstructured/linker regions of the polypeptide backbone.