Rippa, Valentina (2009) ADAPTIVE RESPONSE OF ESCHERICHIA COLI TO ALKYLATING AGENTS: MOLECULAR ASPECTS AND BIOTECHNOLOGICAL APPLICATIONS IN THE BIOREMEDIATION FIELD. [Tesi di dottorato] (Inedito)

Anteprima

PDF thesis_Valentina_Rippa.pdf Download (4MB) | Anteprima

Abstract

The increasingly stringent environmental regulations on hazardous wastes has encouraged the search for innovative solutions for the remediation of contaminated wastes. In this field, bioremediation is seen as an attractive solution due to its reputation as a low cost, environmentally friendly and publicly acceptable treatment technology. The aim of this research project was to explore new potential candidates for the bio-treatment of wastes and environments contaminated by alkylating agents. The study has been specifically focused on AidB, an enigmatic component of the response to alkylation stress in bacterium Escherichia coli. First, AidB protein was functionally characterized: it was showed to bind with high affinity DNA regions containing an upstream element and to have transcriptional activity. At this regard, it was intriguing to speculate that AidB might stimulate the transcription of genes whose products are responsible for alkylation resistance. Successively, given that the knowledge of the domain architecture is necessary for understanding the multifunctional properties of a protein, structural and functional characterization of domains present in AidB was performed. Specifically, its N-terminal region was shown to be exhibit acyl-CoA dehydrogenase activity while the short C-terminal domain was shown to be responsible for the DNA binding activity and for regulatory function. The study was then aimed at investigate the mechanism by which AidB directly protects E. coli cells against alkylating compounds. It was demonstrated that this protein prevents alkylation damage and it does so by protecting DNA and, presumably, by inactivating alkylators before they are able to react with their target. Interestingly, a recent report on the three dimensional structure of AidB bound to double strand DNA supported this model, revealing that the protein is well equipped to sterically occlude DNA from attack by damaging agents. Importantly, the unique chemical environment of FAD active site provided a rationale for a possible role of AidB in deactivation of nitrosoguanidines such as N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and N-ethyl-N′-nitro-N-nitrosoguanidine (ENNG). Coupled with structural analysis, the results obtained in this work supported the hypothesis that AidB might act as a detoxification enzyme to destroy nitrosoguanidines: indeed, it was demonstrated that aidB mutant cells display decreased resistance to MNNG and ENNG and no change in sensitivity to other classes of alkylators; besides, AidB was showed to allow more efficient gene transcription in E. coli cells exposed to nitrosoguanidines rather than to other mutagens. Therefore, AidB represents a promising tool for the bio-treatment of sites contaminated by certain alkylating agents. On the basis of data described above, this experimental work was ultimately targeted at identify as well as at characterize E. coli AidB homologues in bacteria used for bioremediation applications. Specifically, the acyl-CoA dehydrogenase coded by the PP4780 gene from Pseudomonas putida KT2440 was the object of this investigation. The PP4780 gene was expressed in aidB and wild type E. coli strains and its involvement in the protection against alkylating agents was tested. Interestingly, the complementation of the aidB mutation by PP4780 restored the resistance phenotype to lethal and mutagenic effects of MNNG and ENNG; besides, recombinant cells that overexpress PP4780 were shown to possess increased resistance to nitrosoguanidines as compared with wild type and aidB-overexpressing cells. On the basis of these observations, the acyl-CoA dehydrogenase from P. putida has been demonstrated to be involved in the response to alkylation stress, presumably functioning as a detoxification enzyme. In conclusion, the data obtained strongly support the possibility of developing new successful strategies for the bioremediation of sites contaminated by alkylating compounds.

Downloads

Actions (login required)

Modifica documento