An Investigation of Type I Toxin-Antitoxin Sytems from E.coli

Bacteria are evolving to be multidrug resistant, this is probably a combination effect of selection for previously existing resistance genes and more modern evolution through mutagenesis. Understanding the processes contributing to resistance development is important in an attempt to produce novel antibiotics. Escherichia coli is a well-studied organism but function of many small proteins are still unknown. Some of the small proteins are SOS regulated and are important in cellular processes such as regulation, signaling and antibacterial action. Toxin-antitoxin (TA) loci in bacteria consist of two genes, of which one of the genes encodes a small protein which is often highly toxic upon moderate overexpression. The toxin DinQ has shown to be highly lethal upon modest overexpression and DinQ has been suggested to be a candidate for anti-cell-envelope antibiotic against E.coli infections and possibly infections by other gram-negative bacteria. Overproduction of the toxin TisB has shown to form persister cells, which are dormant cells that are highly tolerant to antibiotics, and it is concerning that DNA-damaging antibiotics induce multidrug tolerant cells. Based on the recently characterized TA-system, dinQ-agrB, the function of the TA-systems tisB-istR, shoB-ohsC and ldrD-rdlD was investigated. The mutants tisB, istR, shoB, ohsC, ldrD, rdlD, dinQ and agrB were stressed in several ways and biophysical responses of the bacteria was observed by their ability to replicate and form colonies. Various genetic backgrounds were combined to define the epistasis groups of these systems. The results obtained show that ohsC mutant had dramatically reduced survivors by almost 9000-fold compared to wild type (MG1655) under 1 hour alkaline challenge. Under chronic oxidative stress, shoB and ohsC mutants showed opposite result when stressed with hydrogen peroxide and superoxide, indicating a specific role of ShoB under oxidative stress. Possibility of ShoB increasing oxidative stress through Fe2+ uptake proteins was investigated. Removing both shoB and iscA is indicating there is a strong genetic interaction between shoB and IscA as replication is strongly inhibited.