Group I intron homing in Bacillus phages SPO1 and SP82: a gene conversion event initiated by a nicking homing endonuclease

Abstract

Many group I introns encode endonucleases that promote intron homing by initiating a double-stranded break-mediated homologous recombination event. In this work we describe intron homing in Bacillus subtilis phages SPO1 and SP82. The introns encode the DNA endonucleases I-HmuI and I-HmuII, respectively, which belong to the H-N-H endonuclease family and possess nicking activity in vitro. Coinfections of B. subtilis with intron-minus and intron-plus phages indicate that I-HmuI and I-HmuII are required for homing of the SPO1 and SP82 introns, respectively. The homing process is a gene conversion event that does not require the major B. subtilis recombination pathways, suggesting that the necessary functions are provided by phage-encoded factors. Our results provide the first examples of H-N-H endonuclease-mediated intron homing and the first demonstration of intron homing initiated by a nicking endonuclease.

FIG. 1.

Exclusion of intron-minus allele in progeny of SPO1 and SPO1ΔI coinfections. Plaques from progeny of mixed infections of SPO1ΔI and either wild-type SPO1 (A) or SPO1I-HmuI_oc (B) on B. subtilis CB312 (Sup⁻) or CB313 (Sup⁺) were replicated, lifted onto nylon membranes, and probed with end-labeled oligonucleotides specific for the intron-minus (SPpolIΔI) and intron-plus (K4O) DNA polymerase gene. Note that the oligonucleotide, although having two mismatches with the wild-type sequence, still exhibits specific hybridization to wild-type introns under these conditions.

FIG. 2.

Nucleotide sequence alignment around the I-HmuII cleavage site. The sequence of the top (coding) strand is shown, with the cleavage site of I-HmuII on the bottom strand indicated by an arrow. Differences from the SP82 sequence are highlighted. Oligonucleotide SP82c1 has eight mismatches to the SPO1 sequence, four on each side of the cleavage site, requiring bidirectional conversion to hybridize with SPO1 DNA.