Novel Anti-Repression Mechanism of H-NS Proteins by Phage’s “Early Proteins”

Abstract

Bacteria and their associated bacteriophages are in a continuous battle, co-evolving defence, and offence mechanisms. Bacterial xenogeneic silencers, H-NS family proteins, play important roles in bacterial phage defence and evolution by silencing incoming genes and genes acquired through horizontal gene transfer. It is anticipated that the repression of these genes is directly dependent on the DNA binding modes of H-NS proteins. H-NS proteins bind along DNA, forming lateral protein filaments, in which the H-NS protomers adopt a “half-open” conformational state. Under specific environmental conditions and without dissociation from DNA, these proteins adopt an “open” conformational state which can bridge two DNA duplexes. This switching is a direct effect of environmental conditions on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of H-NS proteins. The Pseudomonas lytic phage LUZ24 encodes the gp4 protein which binds the H-NS family protein MvaT of Pseudomonas aeruginosa. Binding of gp4 was suggested to inhibit the silencing of phage LUZ24 DNA by MvaT. However, the mechanism by which gp4 modulates MvaT action remains elusive. in this study, we show that gp4 interferes with the formation and stability of the bridged MvaT-DNA complex. Structural investigations by NMR and paramagnetic NMR spectroscopies revealed that gp4 acts as an “electrostatic zipper” between the N-terminal and DNA binding domains of MvaT protomers and stabilizes their “half-open” conformation. Based on these observations we propose that binding of Mip promotes the half-open - bridging incompetent - state of MvaT, resulting in relief of MvaT-mediated gene silencing, which has a negative impact on P. aeruginosa homeostasis and fitness. Our survey uncovers a novel anti-repression mechanism of H-NS proteins by phage “early proteins” and opens a new avenue in emulating this antimicrobial mechanism by small chemical compounds to fight Pseudomonas multidrug-resistance.