Biofilm-associated toxin and extracellular protease cooperatively suppress competitors inBacillus subtilisbiofilms

In nature, most bacteria live in biofilms where they compete with their siblings and other species for space and nutrients. Some bacteria produce antibiotics in biofilms; however, since the diffusion of antibiotics is generally hindered in biofilms by extracellular polymeric substances, i.e., the biofilm matrix, their function remains unclear. TheBacillus subtilis yitPOMoperon is a paralog of thesdpABCoperon, which produces the secreted peptide toxin SDP. UnlikesdpABC,yitPOMis induced in biofilms by the DegS-DegU two-component regulatory system. HighyitPOMexpression leads to the production of a secreted toxin called YIT. Expression ofyitQ, which lies upstream ofyitPOM, confers resistance to the YIT toxin, suggesting that YitQ is an anti-toxin protein for the YIT toxin. The alternative sigma factor SigW also contributes to YIT toxin resistance. In a mutant lackingyitQandsigW, the YIT toxin specifically inhibits biofilm formation, and the neutral protease NprB is required for this inhibition. The requirement for NprB is eliminated by Δepsand ΔbslAmutations, either of which impairs production of biofilm matrix polymers. Overexpression of biofilm matrix polymers prevents the action of the SDP toxin but not the YIT toxin. These results indicate that, unlike the SDP toxin and conventional antibiotics, the YIT toxin can pass through layers of biofilm matrix polymers to attack cells within biofilms with assistance from NprB. When the wild-type strain and the YIT-sensitive mutant were grown together on a solid medium, the wild-type strain formed biofilms that excluded the YIT-sensitive mutant. This observation suggests that the YIT toxin protectsB. subtilisbiofilms against competitors. We propose that some bacteria have evolved specialized antibiotics that can function within biofilms.Author SummaryBiofilms are multicellular aggregates of bacteria that are formed on various living and non-living surfaces. Biofilms often cause serious problems, including food contamination and infectious diseases. Since bacteria in biofilms exhibit increased tolerance or resistance to antimicrobials, new agents and treatments for combating biofilm-related problems are required. In this study, we demonstrated thatB. subtilisproduces a secreted peptide antibiotic called the YIT toxin and its resistant protein in biofilms. A mutant lacking the resistance gene was defective in biofilm formation. This effect resulted from the ability of the YIT toxin to pass through the biofilm defense barrier and to attack biofilm cells. Thus, unlike conventional antibiotics, the YIT toxin can penetrate biofilms and suppress the growth of YIT toxin-sensitive cells within biofilms. Some bacteria produce antibiotics in biofilms, some of which can alter the bacterial composition in the biofilms. Taking these observations into consideration, our findings suggest that some bacteria produce special antibiotics that are effective against bacteria in biofilms, and these antibiotics might serve as anti-biofilm agents.