Novel linear megaplasmid from Brevibacterium sp. isolated from extreme environment

DIB, JULIÁN RAFAEL; WAGENKNECHT, MARTIN; HILL, RUSSELL T.; FARÍAS, MARIA EUGENIA; MEINHARDT, FRIEDHELM

San Miguel de Tucuman

Congreso; XLV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular.; 2009

SAIB

Brevibacterium sp. Ap13, isolated from flamingo’s feces in Laguna Aparejos, a high-altitude lake located at approx. 4,200 m in the northwest of Argentina was previously found to be resistant to multiple antibiotics, and was therefore screened for plasmids that may be implicated in antibiotic resistance. Brevibacterium sp. Ap13 was found to contain two megaplasmids of ca., 87 and 436 kb, designated pAp13 and pAp13c, respectively. Using different pulsed-field gel electrophoresis (PFGE) running conditions, no changes in the electrophoretic mobility of pAp13 were observed, indicating that the plasmid is linear rather than circular. However, a considerably altered electrophoretic mobility became evident for pAp13c, suggesting that this molecule is circular. Only plasmid pAp13 was stably maintained and was extensively characterized. pAp13 was isolated by PFGE and electroelution, and subsequently subjected to exonuclease treatment. Exonuclease III, which hydrolyzes DNA in 3´-5´ direction, completely degraded the plasmid, whereas ë exonuclease, which acts in 5´-3´ direction, did not, indicating that the DNA 5´ ends are protected, presumably by covalently attached terminal proteins. In conclusion, we have detected a linear megaplasmid in a Brevibacterium sp., which to our knowledge is the first report of a plasmid greater than 70 kb found in Brevibacterium, and the first description of a linear replicon for that genus. In light of the important role of Brevibacterium in the dairy industry, pAp13 may provide a new tool for genetic manipulation of this commercially important genus.