In situ monitoring of natural genetic transformation of Acinetobacter calcoaceticus BD413 in monoculture biofilms

Confocal laser scanning microscopy (CLSM) was used for the detection of nat ural genetic transformation in a defined pure culture Acinetobacter calcoac eticus BD413 biofilm, with the enhanced yellow fluorescent protein (EYFP) a s a nonselective genetic marker. The effect of free DNA concentration and o f exposure time to free DNA on transformation frequency was explored. Trans formation studies indicated that Acinetobacter calcoaceticus BD413 cells ex posed to as little as 0.1 ng free DNA/ml resulted in detectable transformat ion frequencies (1x10(-4) transformed cells/recipient cell). Within 2 hours about 20% of the biofilm cells expressed eyfp after exposure to 1 mu g fre e DNA/ml. Microscopic analysis revealed a unique distribution of transforma tion frequencies in the biofilm. The highest frequencies were found near th e substratum or near the biofilm/medium interface.