Frequent integration of short homologous DNA tracks during Acinetobacter baylyi transformation and influence of transcription and RecJ and SbcCD DNases

The minimal length of integrated homologous donor DNA tracks in Acinetobacter baylyi transformation and factors influencing the location and length of tracks were determined. Donor DNA contained the nptII gene region (kanamycin resistance, KmR). This region carried nine approximately evenly spaced silent nucleotide sequence tags and was embedded in heterologous DNA. Recipient cells carried the normal nptII gene with a central 10 bp deletion (kanamycin-sensitive). The Km(R) transformants obtained had donor DNA tracks integrated covering on average only 4.6 (2-7) of the nine tags, corresponding to about 60 % of the 959 nt homologous donor DNA segment. The track positions were biased towards the 3' end of nptII. While the replication direction of recipient DNA did not affect track positions, inhibited transcription (by rifampicin) shifted the beginning of tracks towards the nptII promoter. Absence of the RecJ DNase decreased the length of tracks. Absence of SbcCD DNase increased the integration frequency of the 5' part of nptII, which can form hairpin structures of 43-75 nt, suggesting that SbcCD DNase interferes with hairpins in transforming DNA. In homology-facilitated illegitimate recombination events during transformation (in ...Continue Reading