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

摘要

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 KmR 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 which a homologous DNA segment serves as a recombinational anchor to facilitate illegitimate recombination in neighbouring heterologous DNA), on average only about half of the approximately 800?nt long tagged nptII anchor sequences were integrated. From donor DNA with an approximately 5000?nt long homologous segment having the nptII gene in the middle, most transformants (74?%) had only a part of the donor nptII integrated, showing that short track integration occurs frequently also from large homologous DNA. It is discussed how short track integration steps can also accomplish incorporation of large DNA molecules.