SinI and SinR function differently in biofilm formation, rhizosphere colonization, and biocontrol efficacy between Bacillus velezensis and B. subtilis.

Abstract

Numerous Bacillus species, in particular B. subtilis and B. velezensis, are usually used as effective biocontrol agents against plant diseases, leveraging their ability to form biofilms for robust colonization of the rhizosphere. In B. subtilis, SinI positively influences biofilm formation, rhizosphere colonization, and biocontrol efficacy, whereas SinR has a negative impact. To boost the biocontrol efficacy of B. velezensis R9 against tobacco bacterial wilt, we engineered the deletion of sinI and sinR genes in this strain, respectively. Contrary to expectations, deleting sinR impaired biofilm formation, rhizosphere colonization, plant resistance induction, and bacterial wilt control. Conversely, the R9ΔsinI strain showed notably enhanced biofilm, colonization, and biocontrol efficacy relative to both R9 and R9ΔsinR strains. Complementing R9ΔsinI with sinI and R9ΔsinR with sinR confirmed that SinI negatively and SinR positively regulate biofilm formation in R9, regardless of originating from B. velezensis or B. subtilis. By contrast, sinI knockout in B. subtilis M6 caused a marked decline in biofilm formation but could be partially reversed by complementary expression of sinI whether it was from B. subtilis or B. velezensis. Conversely, sinR knockout in M6 sharply decreased biofilm formation. In summary, SinI negatively and SinR positively regulate biofilm formation in B. velezensis, contrasting with their roles in B. subtilis. Consequently, deleting sinI, not sinR, in B. velezensis enhances biofilm formation, promoting root colonization, plant resistance, and disease control.

Importance: Bacillus species, exemplified by B. subtilis as a model organism for Gram-positive bacteria, have been extensively studied, particularly regarding biofilm formation. Biofilms represent a form of quorum sensing in microbial communities, and the biocontrol efficacy of Bacillus species in the rhizosphere, against plant pathogens, hinges on their biofilm-forming capabilities. In B. subtilis, the regulatory proteins SinI and SinR are known to have opposing functions in biofilm formation, with SinI facilitating and SinR inhibiting biofilm development. Drawing from this foundational knowledge, we endeavored to knock out the sinR gene in B. velezensis, a biocontrol bacterium, to enhance biofilm formation and, consequently, its colonization of the rhizosphere and biocontrol efficacy. Unexpectedly, the deletion of sinR reduced the bacterium's proficiency in biofilm formation and its ability to colonize the rhizosphere, resulting in a decrease in biocontrol effectiveness. On the contrary, the knockout of sinI promoted biofilm formation, bolstered the strain's colonization capacity in the rhizosphere, and thus fortified its biocontrol efficacy. These findings underscore that SinI and SinR exert divergent, even antithetical effects in Bacillus species. Insights gleaned from B. subtilis research cannot be extrapolated to encompass all Bacillus species, at least not B. velezensis, indicating the need for species-specific investigations.