Plasmid-encoded gene expression of pathogenic bacteria by antibiotic induction as detected by MALDI-TOF-TOF mass spectrometry and top-down proteomic analysis

Three previously genomically sequenced Shiga toxin-producing E. coli (STEC) strains of serotypes O43:H2, O103:H11, O111:H8 were analyzed by antibiotic induction, MALDI-TOF-TOF mass spectrometry and top-down proteomic analysis. In addition to detection and identification of phage-encoded Shiga toxin, we identified plasmid-encoded immunity proteins for colicin E8 and colicin D. Protein biomarkers were identified from b- and y-type fragment ions generated by the aspartic acid effect (AAE) that favors backbone cleavage on the C-terminal side of aspartic acid (D), glutamic acid (E) and asparagine (N) residues. Our in-house software was used for protein biomarker identification. Consistent with our previous report, we observed an apparent enhancement of the AAE when the residue on the C-terminal side of D-, E− or N-residues is a glycine (G). In silico predicted protein structures revealed that these DG, EG, NG sites of backbone breakage often correspond to flexible linker regions of the backbone. Finally, each pathogenic strain carried one or more large plasmids as well as a 6–8 kb plasmid that contained the colicin gene and its cognate immunity gene. Immediately upstream of the colicin gene was an SOS/LexA box to which the repressor protein (LexA) binds and blocks expression of genes downstream. Upon initiation of the SOS response by antibiotic induction, LexA undergoes self-cleavage and detachment allowing downstream gene expression. Thus, the presence of these small plasmids, their genes and the mechanism of expression is consistent with antibiotic induction and top-down proteomic analysis.