Broad host-range gene transfer: Conjugation in Tn916

The 18 kb conjugative transposon Tn916 is a member of a group of highly promiscuous elements found in both Gram positive and Gram negative bacteria. It confers tetracycline resistance, tetM, on its host, and contains regions responsible for recombination ( int and xis), regulation of expression ( orf7 and orf8), and conjugal transfer of the element (tra/tec region). Conjugal transfer of elements like Tn 916 requires an origin of transfer (oriT) and a relaxase enzyme that initiates transfer by cleaving oriT. Two proteins, Orf20 and Orf23, were identified as possible relaxases by BLASTP analysis. Attempts to purify His6-Orf20 failed to produce active soluble protein. Orf20 was subsequently purified as a chimeric protein fused to Maltose Binding Protein and assayed for its ability to cleave the Tn916 oriT. The chimeric protein possessed endonucleolytic activity, cleaving both strands of oriT at several distinct sites, favoring GT dinucleotides. Incubation of the oriT DNA with purified Tn916 integrase (Int - a tyrosine recombinase) and MBP-Orf20 resulted in strand- and sequence-specific cleavage of oriT at a TG↓GT motif in the transferred strand. This cleavage product could be labeled by [alpha32P] ddATP and terminal transferase, signifying the presence of a 3'-OH group as expected for a relaxase. Mutation of the cleavage site from TGGT to GTTG prevented cleavage. The role of a tyrosine recombinase as a specificity factor in a separate DNA cleavage reaction is completely unprecedented.; His6-Orf23 was successfully purified and subsequently used in DNA binding assays. Specific complex formation was not initially observed in gel shift experiments although use of shorter DNA fragments aided in complex resolution. Protection of oriT from DNase I by His6 -Orf23 binding was also not observed; however, denaturant-resistant aggregates formed at higher protein concentration that could be dissociated by proteinase K treatment. His6-Orf23 was able to form defined complexes with the unrelated sagA promoter DNA and again formed denaturant-resistant complexes in footprinting assays. His6-Orf23 was shown to prefer double-stranded oriT DNA in competition assays with sagA, and similarities between the two DNAs indicate a probable sequence specific component of the protein-DNA interaction. Differences between aggregate formation with two fragments of oriT possessing different levels of similarity with sagA support this conclusion. This protein likely interacts with DNA to change its secondary structure as evidenced by the formation of denaturant-resistant aggregates in the absence of proteinase K. These two proteins interact with their cognate oriT in a manner unlike proteins from other conjugal systems. This uniqueness of the proteins involved in conjugation of Tn916 has led to a proposed name change from tra (the designator applied to most proteins involved in conjugation) to tec (t&barbelow;ransposon-e&barbelow;ncoded c&barbelow;onjugation proteins).