Control of a gene transfer agent cluster in Caulobacter crescentus by transcriptional activation and anti-termination

Gene transfer agents (GTA) are domesticated prophages that cannot self-multiply and be infectious but might have been co-opted to perform biological functions for the host bacteria. Recently, Caulobacter crescentus, a bacterium best known as a model organism to study bacterial cell biology and cell cycle regulation1, has been demonstrated to produce bona fide GTA particles (CcGTA). Two direct activators of the CcGTA biosynthetic gene cluster, GafY and GafZ, have been identified, however, it is unknown how GafYZ controls transcription mechanistically or how they coordinate gene expression of the CcGTA gene cluster with other accessory genes elsewhere on the genome for an ultimate CcGTA synthesis. Here, we show that the CcGTA gene cluster is transcriptionally co-activated by GafY and an integration host factor (IHF), and by a GafZ-mediated transcription anti-termination. We present evidence that GafZ is a novel transcription anti-terminator that likely forms an anti-termination complex with RNA polymerase, NusA, NusG, and NusE to bypass transcription terminators within the 14kb CcGTA cluster. Overall, we reveal a two-tier regulation (by transcription activation and anti-termination) that coordinates the synthesis of GTA particles in C. crescentus. Overall design: Chromatin-immunoprecipitation with deep sequencing experiments (ChIP-seq) were performed on stationary phase Caulobacter crescentus (see the supplementary Materials and Methods for the exact treatment that were applied to each strain).