Silencing of cryptic prophages inCorynebacterium glutamicum

Abstract

DNA of viral origin represents a ubiquitous element of bacterial genomes. Its integration into host regulatory circuits is a pivotal driver of microbial evolution but requires the stringent regulation of phage gene activity. In this study, we describe the nucleoid-associated protein CgpS, which represents an essential protein functioning as a xenogeneic silencer in the Gram-positive Corynebacterium glutamicum. CgpS is encoded by the cryptic prophage CGP3 of the C. glutamicum strain ATCC 13032 and was first identified by DNA affinity chromatography using an early phage promoter of CGP3. Genome-wide profiling of CgpS binding using chromatin affinity purification and sequencing (ChAP-Seq) revealed its association with AT-rich DNA elements, including the entire CGP3 prophage region (187 kbp), as well as several other elements acquired by horizontal gene transfer. Countersilencing of CgpS resulted in a significantly increased induction frequency of the CGP3 prophage. In contrast, a strain lacking the CGP3 prophage was not affected and displayed stable growth. In a bioinformatics approach, cgpS orthologs were identified primarily in actinobacterial genomes as well as several phage and prophage genomes. Sequence analysis of 618 orthologous proteins revealed a strong conservation of the secondary structure, supporting an ancient function of these xenogeneic silencers in phage-host interaction.

Deciphering the rules underlying xenogeneic silencing and counter-silencing of Lsr2-like proteins

“…Recent genome-wide profiling studies revealed that the Lsr2-like xenogeneic silencer CgpS preferentially binds to AT-rich DNA sequences in the genome of C. glutamicum ATCC 13032 (4). To determine the parameters affecting CgpS binding and silencing in vivo, we systematically analyzed the peak sequences obtained from CgpS ChAP-seq analysis (4) and subsequently confirmed our conclusion by testing the silencing of synthetic promoter variants. Remarkably, an overlay of the GC-profiles of all 35 CgpS target promoters located within the prophage element CGP3 revealed a high degree of similarity with a distinct drop in GC-content matching the position of maximal CgpS coverage (Figure 1A).…”

“… A) Overlay and calculated mean (orange curve) of GC-profiles of CgpS target promoters located within the CGP3 prophage (n=35) (4). Profiles were calculated by a rolling mean with a window size of 50 bp and a step size of 10 bp.…”

“…Although XS were convergently evolved and show only low sequence similarity, the domain properties of their N-terminal oligomerization domains and their C-terminal DNA-binding domains are similar (19, 20, 22, 23). Their binding mechanisms are diverse but they all preferentially bind to horizontally acquired DNA, which has typically a higher AT-content than the genome of the recipient cell (4, 24). The broad distribution of XS among prokaryotes emphasizes the strong need to discriminate between “self” and “non-self” across phylogenetic clades (13).…”

“…The essentiality of Lsr2 for this human pathogen makes this XS protein a highly promising drug candidate (37). In Corynebacterium glutamicum the Lsr2-like XS protein CgpS was also shown to play an essential role as a silencer of cryptic prophage elements, whose entrance into the lytic cycle would otherwise cause cell death (4, 38). In contrast to mycobacteria and corynebacterial species, Streptomyces typically encode two Lsr2-like proteins.…”

“…In this study, we set out to systematically assess the rules underlying silencing and counter-silencing of Lsr2-like XS by using the Lsr2-like protein CgpS of Corynebacterium glutamicum as a model (4). To the best of our knowledge, it is the first detailed analysis of the counter-silencing mechanism of an Lsr2-like XS protein.…”