Investigating the effects of over expressed genes on boron tolerance in Escherichia coli and gene expression analyzes pertaining to boron

Boron (B) is a strategic and enigmatic element in many aspects. Boric acid and most boron compounds cause death or inhibit the growth of various bacterial cells. However, it is not well understood which biomolecules are targeted in the cell and response mechanisms are used against these boron compounds. Consequently, boron-related biomolecules, particularly genes, proteins and metabolites should be investigated using appropriate model organisms. Towards reaching these goals, the approaches of genomics, proteomics, metabolomics and gene expression analyzes were applied. In the first part of this thesis, genomic approach was used and a number of 4123 strains of the ASKA clone set belonging to Escherichia coli were subjected to genome-wide screening experiments in the presence of increasing boric acid concentrations. In order to induce the expression of the genes contained in the plasmids, 100 µM and 33 µM IPTG were used. Comparing to the control, the strains surviving at relatively high boric acid concentrations were selected. As a result of genome-wide screens using 100 µM IPTG, a number of 17 strains were found to grow in relatively higher levels of boric acid and the results were confirmed by various additional methods. The number of strains that were selected using 33 µM IPTG was 52, which were shown to grow in the presence of relatively higher boric acid concentrations owing to the over-expressed genes that they contain. The strains that have relatively higher boron tolerance possess the following cloned genes: aceK, rfaQ, yjcF, ymdE, bdm, yeeY, yddV, recQ, ygcU, ptsA, ydeE, lacI, lacA, cobT, gatD, paaE, ugpC, prfH, yaiB, nusB, cbpM, malI, ydhD, yeaC, yeeT, yfjL, hcaD, ,elaB, hypC, yfjU, yqeK, yhiJ, rep, thiC, yifB, aceA, sgbE, fre, ytfG, sgcQ, rpsJ, yhcE, yhdL, tnaC, ytfA, ulaB, yjgX, yjhX, sgcA, chpS, yceP, yjhD, yrbB, gatR, yoeF, arpB, hybF, ygeM, ymfP, yicR, hpt, frlC, menD, yadI, ydiA, yfcT, arsB, yeiQ ve yddK. In the second part of the study, "ASKA clone genomic library pool" containing 4123 gene clones was generated and selection was carried out by the enrichment method under the conditions of high boric acid levels. In addition, the "in vitro" plasmid library of the ASKA clone genomic library pool was obtained. After the plasmids were transformed into strain DH10b, the enrichment method was used again for selection and the tolerant colonies were chosen. The genes in the recombinant plasmids of the tolerant clones obtained from both studies were identified by the Sanger sequencing method. A number of 9 genes were found namely dtd, yedL, yjbO, elfC, yfjK, yfiC, sufD, yecF and idnR. In the third part, proteomic technology was applied. For 2-DE (two-dimensional gel electrophoresis), the strains AG1(pCA24N::aceK) and AG1(pCA24N::ptsA), which showed growth in the medium containing relatively high boric acid concentrations, were selected. Due to the gene(s) expressed in the plasmid(s), the clones could cope with boric acid stress and the protein profiles of them subjected to boric acid stress were compared. As a result of the study, 8 protein spots were observed to be up-regulated for the strain AG1(pCA24N::aceK) and for AG1(pCA24N::ptsA) strain, 7 up-regulated protein spots were selected. Identification of the proteins within the spots were determined by MALDI-TOF-TOF analysis. Proteomic analyzes of the strains AG1(pCA24N::aceK) and AG1(pCA24N::pstA) revealed PykF, Pgk, EftS, OmpA, CysK, KduD, AtpA, PrpD proteins and PykF, MalE, G3P1, AhpC, Alf, Mdh, KduI proteins, respectively. In the fourth part of the thesis, by evaluating the gene information obtained from previous studies, gene expression analyzes were conducted. Real-time PCR experiments were performed on selected genes and including the control, relative mRNA levels of 30 genes at different boric acid concentrations were revealed. Of the genes tested, in particular, the transcript levels of kduD, kduI, sodA, ompR, deoB, yoaC and icd genes were found to increase dramatically in response to boric acid stress. At last, metabolomics approach was used and NMR (nuclear magnetic resonance) analyzes were used to search for boron stress related metabolites. Metabolite extracts were obtained from AG1(pCA24N::aceK) and E. coli DH10b strains exposed to none as well as 3 different boric acid concentration and 1H-NMR experiments were performed. The results indicate that, for AG1(pCA24N::aceK), the levels of Adenine, Galactarate, Gluconate, Glutamate, Guanidoacetate, Guanosine, Inosine, Lactulose, Oxypurinol, Putrescine, Pyroglutamate, Threonine, Uridine metabolites increased, whereas Erythritol and Saccharopine levels decreased in the boric acid media. For E. coli(DH10b) however, the levels of Oxypurinol, Uracil, Xanthine, Glucarate, Putrescine, Thymine, Galactonate, Hypoxanthine, Maleate, Adenosine, Pantothenate, 1,6-Anhydro-β-D-glucose, Galactarate, Nicotinate, Nicotinurate, Pyridoxine, Thymidine, Uridine, Xanthosine metabolites increased and the levels of Inosine, Niacinamid, Saccharopine, Glycine ve Lactate decreased. In conclusion, this thesis study is a significant and comprehensive study that has provided valuable information by identifying genes, proteins and metabolites involved in boron (tolerance)-bacterial relationship using genomics, proteomics, metabolomics, molecular genetics and microbiological approaches. Keywords: Boron, Tolerance, E. coli, Genomics, Proteomics, Metabolomics