Mobile genetic elements carrying stress response systems, antibiotic resistance determinants, and catabolic pathways

In the present thesis, I studied the activation of an SOS-like response in Streptococcus pneumoniae encoded by the Streptococcus pyogenes prophage φ1207.3. This system leads to the temporary activation of an hypermutable phenotype, which resulted in increased survival and increased mutation rate upon exposure to mitomycin C or UV-C light. Then, a different type of stress response, the Envelope Stress Response (ESR), was exploited as a strategy for sensitization of Escherichia coli to several antibiotics, by disbalancing five pathways, namely σE, Cpx, Rcs, Bae, and Psp. Disbalancing the Psp pathway increased E. coli susceptibility to some beta-lactam antibiotics. Prophage φ1207.3, carrying a two-genes macrolide efflux system, was originally described as an Integrative and Conjugative Element (ICE). In this thesis, φ1207.3 was transferred to the standard pneumococcal laboratory strain Rx1, for which the whole genome sequence was obtained. It was demonstrated that φ1207.3 is a functional phage of the Siphoviridae family, able to form mature phage particles. It was shown that φ1207.3 does not enter the lytic cycle, even upon induction with mitomycin C. Since φ1207.3 transfers through a mechanism requiring cell-to-cell contact resembling conjugation, the cellular localization of φ1207.3 was investigated. It was demonstrated that the number of φ1207.3 phage particles on the bacterial cells exceeds the number of phages in the culture supernatant by 3 orders of magnitude. φ1207.3 transfer to a variety of streptococcal species was obtained by setting up a mating protocol for the transfer of large mobile genetic elements. Tn5253 is a composite ICE of Streptococcus pneumoniae carrying two elements: i) the ICE Tn5251, carrying the tet(M) tetracycline resistance gene, and ii) the Ωcat(pC194) not-conjugative element, harbouring the cat chloramphenicol resistance gene and able of intracellular transposition. The Tn5253 chromosomal integration site (attB) was investigated in S. pneumoniae with different backgrounds and in other streptococcal and enterococcal species. Finally, during the sequencing of two Mycobacterium chimaera strains, it was reported the presence of an ICE carrying putative genes involved in the catabolic degradation of polycyclic aromatic hydrocarbons, important environmental pollutants.