Transcription termination and antitermination are critical for the fitness and function of the integrative and conjugative element Tn916

Premature expression of genes in mobile genetic elements can be detrimental to their bacterial hosts. Tn916, the founding member of a large family of integrative and conjugative elements (ICEs; aka conjugative transposons), confers tetracycline-resistance and is found in several Gram-positive bacterial species. We identified a transcription terminator near one end of Tn916 that functions as an insulator that prevents expression of element genes when Tn916 is integrated downstream from an active host promoter. The terminator blocked expression of Tn916 genes needed for unwinding and rolling circle replication of the element DNA, and loss of the terminator caused a fitness defect for the host cells. Further, we identified an element-encoded antiterminator (named canT for conjugation-associated antitermination) that is essential for transcription of Tn916 genes after excision of the element from the host chromosome. We found that the antiterminator is orientation-specific, functions with heterologous promoters and terminators, is processive and is most likely a cis-acting RNA. Insulating gene expression in conjugative elements that are integrated in the chromosome is likely a key feature of the interplay between mobile genetic elements and their hosts and appears to be critical for the function and evolution of the large family of Tn916-like elements. AUTHOR SUMMARYHorizontal gene transfer allows bacteria to rapidly acquire new traits that can enhance their adaptability to different conditions. Integrative and conjugative elements (ICEs) are mobile genetic elements that reside integrated in a bacterial chromosome and can transfer to another cell via cell-to-cell contact through the element-encoded secretion system. ICEs often confer beneficial traits to their hosts, including antibiotic resistances, symbiotic/pathogenic determinants, metabolic capabilities, and anti-phage defense systems. Tn916, the first-described ICE, was identified based on its ability to transfer tetracycline resistance in the pathogen Enterococcus faecalis, and is found in several Gram-positive species. Once transferred into a new cell, Tn916 integrates into AT-rich sequences, sometimes downstream from a host promoter. We found that Tn916 has a transcription terminator near one end of the element that blocks transcription from an upstream host promoter, thereby protecting cells from detrimental effects of premature expression of element genes. Further, we found that Tn916 has a transcription antitermination system that is essential for expression of element genes after excision from the host chromosome. Our findings highlight the complex layers of transcriptional regulation that have evolved in ICEs, impacting host cell viability and the spread of the element.