How bacterial xenogeneic silencer rok distinguishes foreign from self DNA in its resident genome

Abstract

Bacterial xenogeneic silencers play important roles in bacterial evolution by recognizing and inhibiting expression from foreign genes acquired through horizontal gene transfer, thereby buffering against potential fitness consequences of their misregulated expression. Here, the detailed DNA binding properties of Rok, a xenogeneic silencer in Bacillus subtilis, was studied using protein binding microarray, and the solution structure of its C-terminal DNA binding domain was determined in complex with DNA. The C-terminal domain of Rok adopts a typical winged helix fold, with a novel DNA recognition mechanism different from other winged helix proteins or xenogeneic silencers. Rok binds the DNA minor groove by forming hydrogen bonds to bases through N154, T156 at the N-terminal of α3 helix and R174 of wing W1, assisted by four lysine residues interacting electrostatically with DNA backbone phosphate groups. These structural features endow Rok with preference towards DNA sequences harboring AACTA, TACTA, and flexible multiple TpA steps, while rigid A-tracts are disfavored. Correspondingly, the Bacillus genomes containing Rok are rich in A-tracts and show a dramatic underrepresentation of AACTA and TACTA, which are significantly enriched in Rok binding regions. These observations suggest that the xenogeneic silencing protein and its resident genome may have evolved cooperatively.

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

“…The binding to AT-rich DNA is a common feature of XS proteins (3) and was shown to represent an important fitness trait to avoid spurious transcription and the sequestering of the RNA polymerase (8). In vitro protein binding microarray experiments revealed a clear preference of the xenogeneic silencers H-NS, MvaT and Rok for DNA stretches containing flexible TpA steps, while no positive effect of TpA steps was observed for Lsr2 from Mycobacterium tuberculosis (20, 47). Here, our ‘design-test-build’ approach, where different synthetic promoter variants were tested in vivo with regard to CgpS-mediated silencing, however, revealed a certain degree of sequence specificity of CgpS towards a binding motif containing A/T steps.…”

“…It is important to note, that we almost exclusively relied on in vivo approaches including ChAP-seq analysis and reporter assays to define the parameters affecting silencing and counter-silencing at the systems level. Although in vitro analysis of protein-DNA fragments (often linear DNA) is frequently applied and has provided valuable insights into the binding behavior of XS proteins (20, 47, 49), the conditions do not reflect physiologically relevant situations (DNA topology, protein-protein interaction and interference) and consequently the results have to be interpreted with caution.…”

“…Known XS proteins belong to one of four currently described classes: H-NS-like proteins of proteobacteria like Escherichia coli , Yersinia and Salmonella (14–16), MvaT/U-like proteins found in gamma-proteobacteria of the Pseudomonodales order (17), Lsr2-like XS of Actinomycetes (18, 19) and Rok present in different bacilli, including Bacillus subtilis (20, 21). 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).…”

Nucleoid openness profiling links bacterial genome structure to phenotype

“…Instead, a wide variety of poorly conserved nucleoid-associated proteins (NAPs) control the dynamic organization of the nucleoid and directly affect how genetic information is accessed, interpreted, and implemented 1,2,3,4 . Among the most widely studied NAPs is H-NS in E. coli 5 and its functional analog, Rok in B. subtilis 6 , both known to have high affinity towards AT-rich regions 7,8 .…”

Deciphering the Rules Underlying Xenogeneic Silencing and Counter-Silencing of Lsr2-like Proteins Using CgpS of Corynebacterium glutamicum as a Model

“…Known XS proteins belong to one of four currently described classes: H-NS-like proteins of proteobacteria, like Escherichia coli, Yersinia, and Salmonella (14)(15)(16), MvaT/U-like proteins found in gammaproteobacteria of the Pseudomonodales order (17), Lsr2-like XS of Actinomycetes (18,19), and Rok, present in different bacilli, including Bacillus subtilis (20,21). Although XS were convergently evolved and show only low sequence similarity within the different classes, 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 typically has a higher AT content than the genome of the recipient cell (4,24).…”