Colicin Immunity Proteins of Pathogenic Bacteria Detected by Antibiotic-Induced SOS Response, Plasmid Sequencing, MALDI-TOF-TOF Mass Spectrometry, and Top-Down Proteomic Analysis.

Rationale: Plasmids can play a major role in the survival of pathogenic bacteria. Plasmids are acquired through horizontal gene transfer resulting in their spread across various strains, species and genera of bacteria. Colicins are bacterial protein toxins expressed by plasmid genes and released against co-located bacterial competitors.

Methods: Three Shiga toxin-producing E. coli (STEC), whose genomes were sequenced previously, were analyzed using a combination of antibiotic induction, MALDI-TOF-TOF mass spectrometry, top-down proteomic analysis, and small plasmid sequencing. Protein biomarkers were identified using in-house software that matches protein mass and fragment ions of backbone cleavage by the aspartic acid effect. Predicted in silico protein structures assisted in the interpretation of protein ion fragmentation.

Results: In addition to proteomic identification of phage-encoded Shiga toxin, we were able to identify plasmid-encoded immunity proteins for colicin D and E3. The genes for these plasmid-encoded proteins were not found in the previous genomic sequencing. However, resequencing of these strains for small plasmids revealed the genes to be present on 7-8 kb sized plasmids. Upstream of the colicin/immunity genes was an inverted repeat of the SOS/LexA box that represses gene expression until antibiotic challenge.

Conclusions: Our top-down proteomic method demonstrates that it is possible to screen putative pathogenic bacteria (whose genomes have been sequenced in full, in part or not at all) for the presence of phage- and plasmid-encoded toxin and colicin genes under SOS control. Small plasmid sequencing confirmed the presence of colicin/immunity genes (and their regulatory control) suggested from induction and top-down proteomic analysis.