Estudio de megaplásmidos en Sphingomonas degradadoras de hidrocarburos policíclicos aromáticos

STAREVICH, V. A; MADUEÑO L.; SALTO, I. P; PISTORIO, M; MORELLI I. S.

Mar del Plata

Congreso; X Congreso de Microbiología General (SAMIGE); 2014

Sociedad Argentina de Microbiología General (SAMIGE)

Bioremediation is a methodology that provides an economical and effective 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. 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, 20006FA and 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 in the megaplasmids of a gene implicated in PAH-degradation pathway was evidenced from purified plasmids, by PCR using 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 were made to determine if a complete degradation pathway are located in the plasmid. For this purpose, mobilization functions were incorporated in megaplasmid by transposition mutagenesis, then the megaplasmid was forced to move using a triparental mating assay. Mobilization of the megaplasmid was not achieved. This result could be due to a system failure in mating strategy or absence in replication capacity of the megaplasmid in gammaproteobacteria. The conjugation failure, together with the high similarity found between megaplasmids from PAH-degrading Sphingomonas phenotypically diverse, suggest that the degrading property could be specifically transferred among this bacterial genus.