Lsr2-like nucleoid-associated proteins play an important role as xenogeneic silencers (XS) of horizontally acquired genomic regions in actinobacteria. In this study, we systematically analyzed the in vivo constraints underlying silencing and counter-silencing of the Lsr2-like protein CgpS in Corynebacterium glutamicum. Genome-wide analysis revealed binding of CgpS to regions featuring a distinct drop in GC profile close to the transcription start site (TSS) but also identified an overrepresented motif with multiple A/T steps at the nucleation site of the nucleoprotein complex. Binding of specific transcription factors (TFs) may oppose XS activity, leading to counter-silencing. Following a synthetic counter-silencing approach, target gene activation was realized by inserting operator sites of an effector-responsive TF within various CgpS target promoters, resulting in increased promoter activity upon TF binding. Analysis of reporter constructs revealed maximal counter-silencing when the TF operator site was inserted at the position of maximal CgpS coverage. This principle was implemented in a synthetic toggle switch, which features a robust and reversible response to effector availability, highlighting the potential for biotechnological applications. Together, our results provide comprehensive insights into how Lsr2 silencing and counter-silencing shape evolutionary network expansion in this medically and biotechnologically relevant bacterial phylum. IMPORTANCE In actinobacteria, Lsr2-like nucleoid-associated proteins function as xenogeneic silencers (XS) of horizontally acquired genomic regions, including viral elements, virulence gene clusters in Mycobacterium tuberculosis, and genes involved in cryptic specialized metabolism in Streptomyces species. Consequently, a detailed mechanistic understanding of Lsr2 binding in vivo is relevant as a potential drug target and for the identification of novel bioactive compounds. Here, we followed an in vivo approach to investigate the rules underlying xenogeneic silencing and countersilencing of the Lsr2-like XS CgpS from Corynebacterium glutamicum. Our results demonstrated that CgpS distinguishes between self and foreign by recognizing a distinct drop in GC profile in combination with a short, sequence-specific motif at the nucleation site. Following a synthetic counter-silencer approach, we studied the potential and constraints of transcription factors to counteract CgpS silencing, thereby facilitating the integration of new genetic traits into host regulatory networks.

Quorum sensing (QS) is widely accepted as a procedure that bacteria use to converse. However, prevailing thinking places acyl homoserine lactones (AHLs) at the forefront of this communication pathway in Gram‐negative bacteria. With the advent of high‐throughput genomics and the subsequent influx of bacterial genomes, bioinformatics analysis has determined that the genes encoding AHL biosynthesis, originally discovered to be indispensable for QS (LuxI‐like proteins and homologues), are often absent in QS‐capable bacteria. Instead, the sensing protein (LuxR‐like proteins) is present with an apparent inability to produce any outgoing AHL signal. Recently, several signals for these LuxR solos have been identified. Herein, advances in the field of QS are discussed, with a particular focus on recent research in the field of bacterial cell–cell communication.

Bacteriophages represent the "dark matter" of the biological world (Hatfull, 2015;Ofir & Sorek, 2018;Rohwer & Youle, 2011). With the recent massive expansion of the genomic sequence space, the number of functionally unknown open reading frames (ORFs) in phage genomes is continuously increasing (Yin & Fischer, 2008). By targeting diverse cellular processes and regulatory hubs in their host cell, bacteriophages represent a rich source for the identification of novel antibacterial proteins as well as for the establishment of highly efficient molecular tools (

Background The insect pathogenic bacterium Photorhabdus luminescens exists in two phenotypically different forms, designated as primary (1°) and secondary (2°) cells. Upon yet unknown environmental stimuli up to 50% of the 1° cells convert to 2° cells. Among others, one important difference between the phenotypic forms is that 2° cells are unable to live in symbiosis with their partner nematodes, and therefore are not able to re-associate with them. As 100% switching of 1° to 2° cells of the population would lead to a break-down of the bacteria’s life cycle the switching process must be tightly controlled. However, the regulation mechanism of phenotypic switching is still puzzling. Results Here we describe two novel XRE family transcriptional regulators, XreR1 and XreR2, that play a major role in the phenotypic switching process of P. luminescens. Deletion of xreR1 in 1° or xreR2 in 2° cells as well as insertion of extra copies of xreR1 into 2° or xreR2 into 1° cells, respectively, induced the opposite phenotype in either 1° or 2° cells. Furthermore, both regulators specifically bind to different promoter regions putatively fulfilling a positive autoregulation. We found initial evidence that XreR1 and XreR2 constitute an epigenetic switch, whereby XreR1 represses xreR2 expression and XreR2 self-reinforces its own gene by binding to XreR1. Conclusion Regulation of gene expression by the two novel XRE-type regulators XreR1 and XreR2 as well as their interplay represents a major regulatory process in phenotypic switching of P. luminescens. A fine-tuning balance between both regulators might therefore define the fate of single cells to convert from the 1° to the 2° phenotype.

Bacteriophages represent a powerful source for the identification of novel antimicrobial proteins. In this study, a screening of small cytoplasmic proteins encoded by the CGP3 prophage of Corynebacterium glutamicum, resulted in the identification of the novel gyrase-inhibiting protein Cg1978 (Gip), which shows a direct interaction with the gyrase subunit A (GyrA). In vitro supercoiling assays further suggest a stabilization of the cleavage complex by Gip. Overproduction of Gip in C. glutamicum resulted in a severe growth defect as well as an induction of the SOS response. The cells adapted to gip overexpression by increasing expression levels of gyrAB and by reducing topA expression reflecting the homeostatic control of DNA topology. Interestingly, Gip features a similar activity profile towards gyrases of Escherichia coli, Mycobacterium tuberculosis and C. glutamicum. Therefore, the detailed elucidation of the mechanism of Gip action may provide novel directions for the design of drugs targeting DNA gyrase.