The plasmid pTUC200, encoding Microcin J25, have unusual properties

CECILIA VALLEJOS, MÓNICA DELGADO, PAULA VINCENT, RAÚL SALOMÓN

Pinamar, Buenos Aires

Congreso; XLI Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología molecular; 2005

SAIB

Plasmid pTUC200 is a microcin J25 (MccJ25)-producing pBR322 derivative carrying a fragment from the wild-type MccJ25 plasmid, pTUC100. This fragment includes a copy of the insertion sequence IS1294 and the replicon of pTUC100. Plasmid pTUC200 has two interesting properties. First, it suffers spontaneous deletions at a low proportion. Second, it was stably maintained in a TolC– E. coli strain. Note that other microcin-producing plasmids are lethal for TolC– cells, since they cannot export the microcin produced. Regarding the deletion phenomenon, although it occurred spontaneously, its frequency was greatly increased in Tn5-carrying pTUC200, or when the transposon was present in trans. The nucleotide sequences of deletion junctions were determined for 4 deletants. All of them had lost a fragment containing the pTUC100 replicon. Deletions seem not to be the result of homologous recombination. One of the deletions end points is invariable and corresponds to the right end of IS1294. This suggests that the deletions are a result of IS1294 intramolecular transposition. We have as yet no explanation for the increase in deletion frequency in the presence of Tn5. As for the stability of pTUC200 in TolC– strains, genetic experiments showed that this is due to the prevalence of the pTUC100 low-copy-number replicon over that of the vector pBR322. This is unusual, since when two different replicons are fused it is the high-copy-number one which prevails. The resulting low expression of MccJ25 would maintain the intracellular antibiotic concentration  below a toxic level.