Identification of the Integration/Excision Module and Regulatory Elements Involved in the Mobility of IME8, an...

Lysinibacillus sphaericus, a bacterium successfully used in the control of mosquitoes, bears its insecticidal traits in GI8, a recently identified mosquitocidal genomic island. GI8 is renamed IME8 in the present work, as it displays a typical genetic organization of an Integrative and Mobilizable Element (IME) and its circularized form is not self-conjugative but mobilizable by the pBsph-like plasmid p1593. The IME8 integration module (int-operon) encodes two integrase-like proteins (Int1 and Int2) belonging to the family of tyrosine recombinases, and a hypothetical protein (Hp3). All three ORFs are necessary and function as an essential excision unit of IME8. The chimeric construct "attL-int1-int2-hp3-kan-attR" (hereafter named mini-IME8 cassette) displays integrating property. The integration is specific to an acnL-yolD(attB)-uvrX operon target region, which is not only distributed in various L. sphaericus isolates but is also present among other Lysinibacillus species. The regulation module, reg-operon, encodes an HTH-domain-carrying protein (Reg16) and a putative lytic polysaccharide monooxygenase (LPMO17). Knockout of the reg-operon remarkably increases IME8 excision and transcription levels of int1/int2/hp3 compared to the wild-type situation. However, expression of reg16 or the complete reg-operon both increase the int-operon promoter (P_int) activity in β-galactosidase activity assays, suggesting a complex regulation of the int-operon.