New linear plasmids in Micrococcus strains isolated from extreme environments

DIB JR; DÖBBER, JOHANNES; FARIAS MARIA EUGENIA; MEINHARDT F

Santander

Conferencia; International Plasmid Biology Conference; 2012

Laguna Diamante and Laguna Azul are pristine high-altitude lakes in the northwest of Argentina; they are extreme environments 4,400 meters above sea level with a high concentration of salts and heavy metals and exposed to high UV radiation. We have previously demonstrated the presence of large linear plasmids in actinobacteria from similar environments. The aim of this work is the molecular characterization of large plasmids from Micrococcus strains from above locations to identify genetic determinants presumably being beneficial for survival in these environments. Minimum inhibitory concentrations (MIC) for heavy metals and antibiotics were determined. Plasmids pJD12 (85 kb) and pLMA7 (80 kb) from two different Micrococcus spp. were isolated by pulse field gel electrophoresis (PFGE) and electroelution. Purified plasmids were tested for 5´ blocked ends. Sequencing of plasmids is currently being performed with the Sanger technology. Both hosting bacteria displayed a high degree of resistance to heavy metals, particularly to As and Pb. The order of toxicity of the metals was found to be Cd>Co>Zn>Ni>Pb>Cu>As and Cd>Co>Zn>Ni=Cu>Pb>As, respectively. One of the strains showed a high MIC for erythromycin and azithromycin, and was susceptible to other antibiotics as well. The other displayed resistance only for erythromycin. Different pulse time conditions during PFGE revealed constant migration distances of plasmid bands. Thus, the plasmids are linear rather than circular. Exonuclease III degraded both of the plasmids, whereas λ-exonuclease did not, proving that the 5´ ends are protected, presumably by covalently attached terminal proteins as for other linear plasmids. Preliminary sequence analyses suggest these plasmids to be novel elements. Whether these extrachromosomal genetic elements may contribute to the successful colonization, adaptation and persistence in oligotrophic extreme habitants as well as to the tolerance to heavy metals or toxic organic compounds is currently under investigation. Anyway, in light of the important role of Micrococcus in biodegradation of xenobiotics, bioremediation, production of biotechnologically important enzymes and bioactive compounds, pLMA7 and pJD12 will certainly provide new genetic tools. Julián R. Dib thanks the support from Alexander von Humboldt Foundation.