SinR Controls Enterotoxin Expression in Bacillus thuringiensis Biofilms

Abstract

The entomopathogen Bacillus thuringiensis produces dense biofilms under various conditions. Here, we report that the transition phase regulators Spo0A, AbrB and SinR control biofilm formation and swimming motility in B. thuringiensis, just as they control biofilm formation and swarming motility in the closely related saprophyte species B. subtilis. However, microarray analysis indicated that in B. thuringiensis, in contrast to B. subtilis, SinR does not control an eps operon involved in exopolysaccharides production, but regulates genes involved in the biosynthesis of the lipopeptide kurstakin. This lipopeptide is required for biofilm formation and was previously shown to be important for survival in the host cadaver (necrotrophism). Microarray analysis also revealed that the SinR regulon contains genes coding for the Hbl enterotoxin. Transcriptional fusion assays, Western blots and hemolysis assays confirmed that SinR controls Hbl expression, together with PlcR, the main virulence regulator in B. thuringiensis. We show that Hbl is expressed in a sustained way in a small subpopulation of the biofilm, whereas almost all the planktonic population transiently expresses Hbl. The gene coding for SinI, an antagonist of SinR, is expressed in the same biofilm subpopulation as hbl, suggesting that hbl transcription heterogeneity is SinI-dependent. B. thuringiensis and B. cereus are enteric bacteria which possibly form biofilms lining the host intestinal epithelium. Toxins produced in biofilms could therefore be delivered directly to the target tissue.

Bacillus cereus phospholipases, enterotoxins, and other hemolysins

“…A recent microarray study on B. licheniformis biofilm revealed that SinR, a member of the transition state regulator family, controls HBL expression, together with PlcR [122]. HBL was expressed only in a small subpopulation of the biofilm, whereas almost all the planktonic population transiently expresses HBL.…”

Bacillus cereus growth and biofilm formation: the impact of substratum, iron sources, and transcriptional regulator Sigma 54

“…Although some overlap has been noticed, biofilm formation in B. cereus involves additional unique factors. Similar to B. subtilis, the SinR/SinI biofilm repressor and anti-repressor pair (Pflughoeft et al 2011, Fagerlund et al 2014) and the master regulator for sporulation, Spo0A, play a role in B. cereus biofilm formation (Hamon andLazazzera 2001, Gao et al 2015). Additional regulators which affect B. cereus biofilm formation indirectly or via a yet unknown pathway have been reported (reviewed in ).…”

“…Sporulation and biofilm formation mechanisms are extensively studied in B. subtilis, which shares many of the regulatory pathways with B. cereus group members (Fagerlund et al 2014. Whether the cells will sporulate or initiate biofilm formation depends on the phosphorylation degree of Spo0A, which is a regulator involved in both of these processes (Hamon and Lazazzera 2001).…”

“…Examples of direct regulators are Spo0A, AbrB and SinI/SinR (Fagerlund et al 2014. SinR and its antagonist SinI influence biofilm formation by regulating the production of EPS components and motility .…”

“…These include the biofilm repressor AbrB (Fagerlund et al 2014), SigH, virulence regulator PlcR (Hsueh et al 2006), necrotrophic regulator NprR (Dubois et al 2012) and CodY (Hsueh et al 2008, Lindbäck et al 2012. CodY is a transcriptional regulator which senses the nutrient status of the cell and mediates stationaryphase adaptations (Hsueh et al 2008, Frenzel et al 2012.…”