Characterization of a conjugative transposon integrase, IntDOT

Area of Science:

• Molecular Biology
• Genetics
• Microbial Genetics

Background:

• The Bacteroides conjugative transposon CTnDOT utilizes an integrase, IntDOT, for its transposition.
• Sequence analysis suggests IntDOT belongs to the tyrosine recombinase family due to conserved catalytic residues.
• However, IntDOT's catalytic mechanism may differ from well-studied tyrosine recombinases like phage lambda integrase.

Purpose of the Study:

• To investigate the role of conserved residues in IntDOT's catalytic activity.
• To confirm IntDOT's classification within the tyrosine recombinase family.
• To elucidate the precise cleavage sites within the CTnDOT attachment (attDOT) sites.

Main Methods:

• Site-directed mutagenesis was used to alter conserved residues in IntDOT.
• In vivo integration assays were performed to assess the activity of mutant IntDOT proteins.
• In vitro cleavage and ligation assays were conducted using mutant IntDOT proteins and attDOT DNA.

Main Results:

• Mutagenesis of conserved residues significantly reduced or abolished IntDOT's in vivo integration activity.
• In vitro cleavage and ligation activities generally correlated with in vivo results.
• The H345A mutant showed wild-type in vivo integration but reduced in vitro activity, suggesting a distinct catalytic mechanism.
• Cleavage assays revealed that one cleavage site is located 2 bp from the CTnDOT-coupling sequence junction, within a conserved homologous region.

Conclusions:

• IntDOT is confirmed as a member of the tyrosine recombinase family.
• The catalytic core of IntDOT exhibits a unique organization compared to other tyrosine recombinases.
• The attDOT site cleavage mechanism involves a site within a conserved homologous region, not solely the non-homologous coupling sequences.