Transposition of Tn916 in the four replicons of the Butyrivibrio proteoclasticus B316(T) genome

Abstract

The rumen bacterium Butyrivibrio proteoclasticus B316(T) has a 4.4-Mb genome composed of four replicons (approximately 3.55 Mb, 361, 302 and 186 kb). Mutagenesis of B316(T) was performed with the broad host-range conjugative transposon Tn916 to screen for functionally important characteristics. The insertion sites of 123 mutants containing a single copy of Tn916 were identified and corresponded to 53 different insertion points, of which 18 (34.0%), representing 39 mutants (31.7%), were in ORFs and 12 were where transposition occurred in both directions (top and bottom DNA strand). Up to eight mutants from several independent conjugation experiments were found to have the same integration site. Although transposition occurred in all four replicons, the number of specific insertion sites, transposition frequency and the average intertransposon distance between insertions varied between the four replicons. In silico analysis of the 53 insertion sites was used to model a target consensus sequence for Tn916 integration into B316(T) . A search of the B316(T) genome using the modelled target consensus sequence (up to two mismatches) identified 39 theoretical Tn916 insertion sites (19 coding, 20 noncoding), of which nine corresponded to Tn916 insertions identified in B316(T) mutants during our conjugation experiments.