Expression of genes encoding fundamental copper homeostasis proteins in copper 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 are used as antimicrobial agents for thousands of years. Their areas of usage include water and food disinfection, preservation of food and beverages, prevention of dysentery, treatment of leprosy, tuberculosis, gonorrhea, syphilis, an alternative to laundry detergents and biocontrol agents for crops of fruits in developing countries. Copper is one of the widely used heavy metals for its antimicrobial property. On the other hand, it is an essential heavy metal for the most living systems. Over 30 proteins were shown to contain copper. Copper alternates between reduced Cu(I) and oxidized state Cu(II). Therefore, copper containing enzymes use this alternation in the redox reactions they catalyze. These proteins take part in important metabolic reactions such as respiration, electron transport, and protection against oxidative stress. However, under aerobic conditions when copper is in excess, redox cycling of copper results in the production of highly reactive hydroxyl radicals. These radicals damage proteins, DNA, and lipids. Consequently, copper homeostasis is strictly controlled in the bacterial cells. In this study, spontaneous and UV-induced copper mutants were generated from Escherichia coli ATCC 8739 parent strain. Then, among the known genes that contribute to copper homeostasis copA, cusS and cusF were selected, and their expression levels were measured. The expression levels of these genes in mutants were compared to the levels in parent strain in the presence or absence of copper in the medium. Expression differences of these genes between control strain (E.coli ATCC 8739) and mutant strains (spontaneous and UV-induced, obtained from E.coli ATCC 8739) were determined via RT-qPCR technique. The statistical significance of the results was established with one-way ANOVA with Tukey’s multiple comparison test. In the end, it has been shown that expressions of copA, cusS, and cusF genes increase significantly both in spontaneous and UV-induced mutants with respect to the parent strain. That indicates that copA, cusF and cusS genes are crucial for the copper- resistance. The analyses of the results indicated that there was no difference between the spontaneously obtained mutants and the UV-induced mutant in terms of copper homeostasis genes’ expressions. That implies that the gene expression levels were independent of the processes through which mutants were obtained. This study showed that the copper homeostasis genes’ expressions augmented 6 times for cusS, 21 times for copA, and to 24 times for cusF over the parent strain’s expressions. These increases in expressions obviously provide a mean for survival under copper stress.