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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Publications at this Location » Publication #405829

Research Project: Elucidating the Factors that Determine the Ecology of Human Pathogens in Foods

Location: Produce Safety and Microbiology Research

Title: Top-down proteomic identification of plasmid-encoded immunity proteins from pathogenic bacteria using MALDI-TOF-TOF tandem mass spectrometry

item Fagerquist, Clifton - Keith
item Shi, Yanlin
item PARK, JIHYUN - Orise Fellow

Submitted to: Symposium Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 8/15/2023
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Pathogenic bacteria often carry plasmids (small circular pieces of DNA separate from the host genome) that encode genes that confer survival benefits to a microorganism. Plasmids can encode genes for antibiotic resistance, virulence factors, stress response, etc. Although there is a metabolic cost to retain and maintain these plasmids, the benefits to the host outweigh the metabolic burden. Plasmids can also be transferred to other bacteria by horizontal gene transfer which can spread antibiotic resistance and virulence factors. Characterizing the protein products of these plasmid genes is critical to understanding their role in bacterial survival and virulence. Using plasmid induction techniques, DNA sequencing, Alphafold2 protein structures, MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis, we have identified and characterized proteins (and their post-translational modifications) of immunity proteins whose genes are encoded by plasmids carried by a Shiga toxin-producing Escherichia coli (STEC) isolated from an agricultural region in Arizona (USA). Colicin E8 is a DNAse protein whose gene (and its immunity cognate) is encoded by the plasmid ColE8. Colicin E8 attaches to and invades neighboring bacteria destroying their DNA and thus conferring a competitive survival advantage to the host that produced it. Its immunity cognate protein (ImmE8) inhibits the action of colicin E8 until such time as both are deployed from the host against neighboring bacteria. Interestingly, ImmE8 was detected with and without an attached acyl group (+308 Da) at its only cysteine residue. Such a PTM has not been previously reported for immunity proteins and suggests that acyl attachment via a thioester bond (based on reduction experiments) may temporarily anchor this protein to the host inner membrane until it is needed to inhibit colicin E8 before it is transported from the bacterial host.