Filling in the gaps for the master regulator of biofilm formation in Bacillus subtilis: A structural and biochemical look at SinR and SinI
Authors: Milton Morgan Eilise , Draughn G. Logan , Bobay Benjamin G , Stowe Sean D , Olson Andrew L , Feldmann Erik A , Thompson Richele J , Myers Katherine H , Santoro Michael T , Kearns Daniel B , Cavanagh John 
Published: June 2021

Bacillus subtilis is one of the most well studied bacterial species, serving as an archetype model organism which has been used in numerous genetic and biochemical studies. Unfortunately, B. subtilis also has a dark side. Strains have been identified in hospitals which are highly resistant to cleaning agents. Bacteria have developed numerous protection strategies to survive in harsh environments, but the most robust method is through the formation of biofilm. Pathogenic bacteria, such as Staphylococcus aureus, can hide in B. subtilis biofilms, taking shelter from extensive disinfecting techniques. B. subtilis is also able to transfer antibiotic resistance genes to serious human health threats such as Clostridium difficile. With these behaviors coming to light, new interest has emerged in what was once thought to be a harmless bacterium.

The transition into a biofilm state in B. subtilis is regulated by the transcriptional repressor SinR, through inactivation by its primary antagonist, SinI. Extensive work has been undertaken over the years to understand the interactions between SinR and SinI, but many gaps in knowledge remain. In this work, we present the first full‐length structural models of tetrameric SinR and dimeric SinI using a hybrid approach, combining solution NMR, chemical crosslinking, mass spectrometry, and molecular docking. Our molecular dynamics studies highlight the highly flexible nature of these proteins, which appears to play a critical role in the ability of SinR to recognize a wide range of DNA binding sites. We also probe the mechanism behind the SinR‐SinI interaction using a combination of biochemical and biophysical techniques. As a result of these findings, we conclude that SinI utilizes a residue replacement mechanism to block SinR multimerization, resulting in decreased repressor activity. Finally, building on decades of work, we provide the first biochemical evidence to support the ability of SinR to bend DNA by binding significantly separated binding sites. The bending of a DNA promoter site is likely the key mechanism for gene repression by SinR, and is facilitated by the formation of the flexible SinR tetramer. SinI disrupts the SinR tetramers, inhibiting SinR from effectively binding DNA. Since derepression of SinR by SinI induces a biofilm state, targeting the SinR‐SinI protein interaction could be a viable therapeutic intervention point. Inhibition of the SinR‐SinI interaction would impede biofilm formation, making B. subtilis more vulnerable and unable to harbor dangerous pathogens.

Support or Funding Information

This work was supported by NIH grant RO1 GM055769 and the V Foundation for Cancer Research.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.