Monitoring of biofilm production in Xylella fastidiosa strain De Donno via biochemical signalling modulation

Diffusible lipid species are exploited by bacteria for regulating cell motility, cell-to-cell communication, activation of metabolism and proliferation. The most widely investigated lipid family is represented by diffusible signal factors (DSFs) responsible for quorum sensing. Xylella fastidiosa (Xf) uses the DSFs to coordinate genes involved in the expression of virulence and biofilm formation. These moieties are mainly cis-2-unsaturated fatty acids which directly enhance xylem infection and biofilm production, which are the two processes involved in the genesis of severe plant diseases such as the noticeable Olive Quick Decline Syndrome, associated with Xf subsp. pauca strain De Donno and affecting olives in the Apulia Region. Here we report the results of studies aiming to identify DSFs molecules of Xf De Donno and exploit strategies for modulating its biofilm formation.
We started inducing DSF expression in Escherichia coli using a plasmid vector recombinant for the rpfF gene of Xf De Donno and verifying the production of exogenous proteins and fatty acids. We set up extraction, mechanical treatments, and methyl ester derivatisation of the extracted crude oils from Xf and E. coli cultures. We compared the GC-MS profiles of fatty acids belonging to the metabolic activity of bacteria harbouring the bare and rpfF-recombinant plasmids. Completing previous studies, we speculated on the production of unsaturated fatty acids with a chain length of 12-18 carbon atoms, with α-unsaturated functions. Isolated and treated crude extracted oils obtained from the same bacterial sources, were tested in vitro to investigate their phenotypic effect on biofilm growth and the expression of key genes related to surface adhesion, biofilm formation and cell movement.
Furthermore, we set the synthesis of new, no commercially available, cis-2-unsaturated fatty acids with a chemical structure related to the DSFs family, in order to test the in vitro alteration of biofilm production in Xf De Donno. The exploited reaction was the stereoselective Still-Gennari olefination which leads to the synthesis of unsaturated fatty acids in cis (Z) conformation starting from commercial aldehydes. Results of these activities will be presented.