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Megaplasmids study in polycyclic aromatic hydrocarbons degrading Sphingomonas Estudio de megaplásmidos en Sphingomonas degradadoras de hidrocarburos policíclicos aromáticos Starevich V.A., Madueño L., Salto I.P., Pistorio M., Morelli I.S. Bioremediation is a methodology that provides an economical, effective and permanent solution for the treatment of contaminated soils with polycyclic aromatic hydrocarbons (PAH). Inoculation with degrading microorganisms in contaminated environments (bioaugmentation) is one of the strategies used in last years. The genus Sphingomonas (sensu latu) has been intensively studied because of their large catabolic diversity, being able to degrade a wide range of xenobiotic compounds, different herbicides and pesticides. It was shown in the last years that many sphingomonads possess (often several) plasmids and especially large plasmids commonly designated as megaplasmids. Furthermore, there is increasing evidence for the existence of plasmids that only can be disseminated among sphingomonads and which undergo after conjugative transfer pronounced rearrangements. The aim of this work was to demonstrate the presence of megaplasmids in our collection of PAH-degrading Sphingomonas strains (1A, 22A, 22B, S. paucimobilis 20006FA, AM) isolated from soils of different regions with distinct pollution histories; to characterize and to find evidence of the presence of PAH-degrading genes located in megaplasmids. The presence of megaplasmids in PAH-degrading strains was demonstrated using lysis in situ technique. The same electrophoresis mobility bands in all megaplasmid preparations were observed, showing that the PAH-degrading strains would have at least one megaplasmid. In order to show differences in sequence and molecular weight, the restrictions pattern of the megaplasmids were obtained from purified plasmids by Kieser protocol. The same restrictions profiles were visualized for the strains tested with EcoRI, XbaI, HindIII, and molecular weight was calculated in 40-50 kpb. These results suggest that despite having been isolated from different soils, the studied PAH-degrading Sphingomonas strains could have closer related megaplasmids. The location of PAH-degrading genes in megaplasmids was evidenced by genus-specific primer sets targeted at the catechol 2,3-dioxygenase gene of proteobacterias. The expected molecular weight band was sequenced showing 100% identity and 97% of coverage with catechol 2,3 dioxygenase of PAH-degrading Sphingomonas PNB and Sphingobium BNQ31, confirming the probably location of the gene. Different mating assays between PAH-degrading Sphingomonas, E. coli S17 1 and E.coli DH5α was made in order to increase the clue of PAH-degrading location. No evidences of conjugation transfer between Sphingomonas to other proteobacteria were obtained. The conjugation failure between α and γ proteobacteria showed that megaplasmids would be unable to conjugate or replicate in different groups of proteobacteria. This result together with the high similarity found between megaplasmids from PAH-degrading Sphingomonas phenotypically diverse, suggest that the degrading property could be specifically transfers among this bacterial genus.