Investiagation of genes conferring resistance to the antibacterial effect of propolis in Escherichia coli using aska clone collection and functional genomic approach

The genomic approach and genome-wide plasmid libraries have been utilized in recent years to relatively rapidly screen thousands of genes in order to find and validate antimicrobial targets. As known, the need for scientific search for new drug candidate molecules still continues as a result of emerging resistance of pathogenic bacteria to current antibiotics. In addition to the discovery and approval of synthetic and natural compounds that can kill or inhibit the growth of harmful bacteria or other disease-causing cells, the cellular targets and mechanisms of action of these compounds need to be investigated. Escherichia coli ASKA genome-wide plasmid library collection (ASKA clone collection) consists of 4123 strains containing 4123 different genes cloned into recombinant plasmids, and this collection has been used in the literature to determine cellular targets of some antimicrobial compounds as well as genes that may be involved in the cellular response. The ASKA clone collection is a unique and powerful experimental resource for genomic studies. In this thesis, the antibacterial targets of the water extract of the natural product propolis, which is also abundant in our region, and the candidate genes that may play a role in the cellular response were investigated. Propolis is a natural resinous mixture of plant-derived compounds produced by bees. Antimicrobial and anticancer effects of propolis extracts are known. However, it is not known which genes or related products and processes propolis targets in the cell. At the same time, data on which genes are used when the cell is exposed to propolis are insufficient. In this thesis, the "ASKA clone pool" containing 4123 cloned genes was used in selection studies where increasing concentrations of propolis water extract was used. Cloned genes in the recombinant plasmids (pCA24N::genX) are under transcriptional control by the T5 promoter and lac operator, and levels of gene expression can be controlled using varying concentrations of IPTG. A number of 4123 strains in the ASKA clone pool were grown and inoculated on a gradient petri having LB medium with increasing concentrations of propolis water extract. As the concentration of propolis was increased, all bacterial strains died at certain concentrations, but some strains survived and grew. A number 28 strains with relatively high tolerance levels against propolis were obtained. The genes present in the recombinant plasmids in these strains were evaluated as genes providing direct or indirect resistance to the antibacterial effect of propolis and validation studies were carried out. In the following studies, 7 colonies were selected that showed a relatively better growth under higher concentrations compared to the control strain. Plasmid isolation was performed from the strains and restriction enzyme digestion was applied to the recombinant plasmids. The insert sizes of the plasmids within the tolerant colonies were determined. Sanger sequence analysis was performed to determine the sequences of target genes, and the information of aaeR, cmtB, prpE genes was obtained as a result of bioinformatics analyzes. Biofilm formations of the selected strains based on the gene information were evaluated. In order to determine the effect of propolis on target gene expression in wild-type bacterial strains with these genes, Real-Time PCR analyzes were carried out and mRNA levels related to propolis stress were examined. This thesis has been an original and relatively comprehensive study that used some of the experimental approaches from the disciplines of functional genomics, molecular microbiology and molecular genetics and produced novel scientific results to the field. Keywords: Propolis, ASKA, Escherichia coli, target gene, antibacterial