Expression of the genes encoding important transport system proteins in silver resistant Escherichia coli mutants

Thesis Type: Postgraduate

Institution Of The Thesis: Middle East Technical University, Graduate School of Natural and Applied Sciences, Biotechnology, Turkey

Approval Date: 2019

Thesis Language: English


Principal Supervisor (For Co-Supervisor Theses): Ayşe Gül Gözen

Co-Supervisor: Çağdaş Devrim Son


Heavy metals, such as silver (Ag), copper (Cu) or mercury (Hg), have been used as antimicrobial agents for quite some time in human history. Silver has inhibitory or lethal effects on a broad range of bacteria and fungi. Furthermore, heavy metals are useful against viruses. The applications of silver dealing with bacteria comprise industrial, agricultural, medical and healthcare areas. They were used as disinfectants in hospitals as well as in the treatment of several infectious diseases such as tuberculosis, leprosy, gonorrhea, and syphilis. Silver is not an essential metal for bacterial metabolism and low concentrations of its ions are toxic. Increasing usage of silver has brought about a resistance problem, especially in Gram- negative bacteria. There are several reported cases in the literature indicating the acquisition of silver resistance in Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Silver ions must enter into the cells to show their antimicrobial effect. In order to cope with the silver stress, the Gram- negative bacteria take advantage of active efflux pumps and outer membrane- mediated decreased permeability. In this study, the relative gene expression levels of important transport system proteins, namely as CusB, CusF, and CusS, were determined in E.coli ATCC 8739 strain using RT-qPCR method. At the beginning of the study, UV-induced and spontaneous silver resistant E.coli mutants were generated. Both the UV-induced and spontaneous mutants were examined for three of the indicated proteins. Gene expression levels of each mutant were compared with the original E.coli ATCC 8739 strain and among themselves. The significance of the relative expression levels of the transport proteins was determined statistically by using one-way ANOVA with Tukey's multiple comparison post-test. The results indicated that cusB, cusF, and cusS genes’ expressions were significantly higher than the parent E.coli strain. It appeared that the cusCFBA efflux pump has crucial importance for the export of silver ions from the bacterial cells. The increase in gene expression levels in mutants was independent of the way that the mutants had been generated. In this regard, E.coli evidently relies heavily on efflux pumps to adapt themselves to the environment containing high concentrations of silver.