AbstractEfficient horizontal gene transfer of the conjugative plasmid pCF10 from Enterococcus faecalis depends on the sex pheromone cCF10, which induces the expression of the Type 4 Secretion System (T4SS) genes controlled by the PQ promoter. The pheromone responsive PQ promoter is strictly regulated to prevent overproduction of the prgQ operon, which contains the T4SS, and to limit the cell toxicity caused by overproduction of PrgB, a T4SS adhesin involved in cellular aggregation. PrgU plays an important role in regulating this toxicity by decreasing PrgB production. PrgU has an RNA-binding fold, prompting us to test whether PrgU exerts its regulatory control through binding of prgQ transcripts. With a combination of lacZ reporter fusion, northern blot, and RNAseq analyses, we provide evidence that PrgU binds a specific RNA sequence within the intergenic region (IGR), ca 400 bp downstream of the PQ promoter. PrgU-IGR binding reduces levels of downstream transcripts, with the strongest decrease seen for prgB messages. Consistent with these findings, we determined that pCF10-carrying cells expressing prgU decreased transcript levels more rapidly than isogenic cells deleted of prgU. Finally, purified PrgU bound RNA in vitro, but without sequence specificity, suggesting that PrgU requires a specific RNA structure or one or more host factors to bind its RNA target in vivo. Together, our results support a working model where PrgU binding to the IGR serves to recruit RNase(s) for targeted degradation of downstream transcripts.ImportanceBacteria utilize Type 4 Secretion Systems (T4SS) to efficiently transfer DNA from donor to recipient cells, thereby spreading genes encoding for antibiotic resistance as well as various virulence factors. The conjugative plasmid pCF10 from Enterococcus faecalis, originally isolated from clinical isolates, serves as a model system for these processes in Gram-positive bacteria. It is very important to strictly regulate the expression of the T4SS proteins for the bacteria, as some of these proteins are highly toxic to the cell. Here, we identify the mechanism by which PrgU performs its delicate fine tuning of the expression levels. As prgU genes are present in various conjugative plasmids and transposons, this provides an important new insight into the bacterial repertoire of regulation mechanisms of these clinically important systems.
Structural mechanism for the recognition and ubiquitination of a single nucleosome residue by Rad6–Bre1
