Mutations in the Lactococcus lactis Ll.LtrB group II intron that retain mobility in vivo

BACKGROUND: Group II introns are mobile genetic elements that form conserved secondary and tertiary structures. In order to determine which of the conserved structural elements are required for mobility, a series of domain and sub-domain deletions were made in the Lactococcus lactis group II intron (Ll.LtrB) and tested for mobility in a genetic assay. Point mutations in domains V and VI were also tested. RESULTS: The largest deletion that could be made without severely compromising mobility was 158 nucleotides in DIVb(1–2). This mutant had a mobility frequency comparable to the wild-type Ll.LtrB intron (ΔORF construct). Hence, all subsequent mutations were done in this mutant background. Deletion of DIIb reduced mobility to approximately 18% of wild-type, while another deletion in domain II (nts 404–459) was mobile to a minor extent. Only two deletions in DI and none in DIII were tolerated. Some mobility was also observed for a DIVa deletion mutant. Of the three point mutants at position G3 in DV, only G3A retained mobility. In DVI, deletion of the branch-point nucleotide abolished mobility, but the presence of any nucleotide at the branch-point position restored mobility to some extent. CONCLUSIONS: The smallest intron capable of efficient retrohoming was 725 nucleotides, comprising the DIVb(1–2) and DII(ii)a,b deletions. The tertiary elements found to be nonessential for mobility were alpha, kappa and eta. In DV, only the G3A mutant was mobile. A branch-point residue is required for intron mobility.