Positive control of the integrative and conjugative element Tn916 and its limited gene expression in transconjugants

Integrative and conjugative elements (ICEs) are widespread in bacteria and typically confer important phenotypes to their hosts. Following excision from the host chromosome, an ICE can transfer to a new cell through the element-encoded conjugation machinery (a type IV secretion system) to create a transconjugant. Tn916, a well-studied ICE that confers tetracycline resistance, is found in several Gram-positive pathogens and has been widely studied in Bacillus subtilis. Unlike most ICEs, Tn916 causes host-cell death following its activation (excision and gene expression). We found that orf7 and orf8 (renamed sig7 and sig8), two regulatory genes in Tn916, encode a two-subunit (split) sigma factor of RNA polymerase, Sig7-8, that activates transcription from Porf7 (now Psig7) and is required for excision of the element. Importantly, production of Sig7-8, either from Psig7 or another strong promoter, caused cell death. Using a Cre recombinase reporter to detect gene expression, we detected little or no transcription from Psig7 in transconjugants. This lack of transcription from Psig7 in transconjugants was essential for fitness of both the element and its host: forcing transcription from Psig7 in transconjugants caused either loss of Tn916 or death of nascent transconjugants. Our findings indicate that, unlike other well-studied ICEs (e.g., ICEBs1) that use repressors to control gene expression and are expressed in nascent transconjugants, most Tn916 genes remain off following transfer to a new host. This regulation prevents Tn916 from killing transconjugants and allows successful horizontal transfer and maintains the potential for future activation in the new host.