SinR Controls Enterotoxin Expression in Bacillus thuringiensis Biofilms

et al. (2014) SinR Controls Enterotoxin Expression in Bacillus thuringiensis Biofilms. PLoS ONE 9(1): e87532. doi:10.1371/journal.pone.0087532 SinR Controls Enterotoxin Expression in Bacillus thuringiensis Biofilms Annette Fagerlund 0 Thomas Dubois 0 Ole-Andreas kstad 0 Emilie Verplaetse 0 Nathalie Gilois 0 Ime` ne Bennaceur 0 Ste phane Perchat 0 Myriam Gominet 0 Ste phane Aymerich 0 Anne-Brit Kolst 0 Didier Lereclus 0 Michel Gohar 0 Theresa M. Koehler, The University of Texas-Houston Medical School, United States of America 0 1 Laboratory for Microbial Dynamics (LaMDa) and Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo , Oslo, Norway, 2 Micalis, INRA (UMR1319), Domaine de Vilvert, Jouy-en-Josas, France, 3 Micalis, AgroParistech (UMR1319), Domaine de Vilvert, Jouy-en-Josas , France , 4 Institut Pasteur , CNRS URA 2172 , Unite de Biologie des Bacte ries Pathoge`nes a` Gram positif , Paris , France 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. - Funding: TD was funded by the Direction Generale a` LArmement, France and IB was funded by the Region Ile de France, France. AF, OA and ABK were funded by a project grant from the Norwegian Research Council through the FUGE II Programme (Channel 3 grant ; project nr. 183421). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Bacillus subtilis and pathogenic bacteria of the Bacillus cereus group (B. cereus, B. thuringiensis and B. anthracis) are all Gram-positive, flagellated, sporulating, and aerobic bacteria clustering closely in the phylogenetic tree of the Bacillus genus ([1]; http://www. patricbrc.org/portal/portal/patric/Phylogeny?cType = taxon&cId = 1386). They share a large number of transcriptional factors, including the sporulation regulator Spo0A, the stress response sigma factor sB, and the phase-transition regulators SinI, SinR, CodY and AbrB [2]. However, there are also important differences in the regulatory pathways between B. subtilis and B. cereus sensu lato. For example, the stress regulator sB is not activated in the same way in these species [3]; the two-component system DegU/DegS and the motility regulator SigD are absent from B. cereus sensu lato [2]; the virulence regulator PlcR, which promotes the transcription of numerous genes for extracellular enzymes and toxins and plays an important role in B. cereus and B. thuringiensis physiology [46], is absent from B. subtilis. These differences may well be the consequences of adaptation of these species to different ecosystems. B. subtilis is a saprophyte living on soil organic matter, whereas, B. thuringiensis is an entomopathogenic bacterium, genetically closely related to the human opportunistic pathogen B. cereus [7,8], and to the human pathogen B. anthracis [9]. Both B. subtilis and B. thuringiensis, or B. cereus, can form biofilms at air-liquid interfaces. Biofilms are widely found structures in which microorganisms are protected against various stresses, allowing them to persist in adverse environmental conditions. The regulatory pathways in B. subtilis leading either to biofilm formation or to sporulation share the same initial steps. The transcriptional regulator Spo0A controls entry into sporulation [10], and is required for biofilm formation [11]. Spo0A represses abrB transcription [12] and promotes the transcription of sinI [13], the product of which is the SinR antagonist SinI. Both AbrB and SinR repress the two polycistronic operons tapA-sipW-tasA and epsA-O [14,15]. The 15-gene epsA-O operon is involved in the biosynthesis of the exopolysaccharide component of the biofilm matrix [16] and the three-gene tapA-sipW-tasA operon is involved in the production of the protein component of the biofilm matrix [17,18]. An inhibitor of flagellar motility is encoded by the epsE gene which is part of the epsA-O operon [19]. Therefore, deletion of sinR from B. subtilis results in an overproduction of biofilm and in impaired motility, whereas deletion of sinI results in the reverse phenotype. A paralogue of SinR, SlrR, is also involved in the control of biofilm formation and motility through its interaction with SinR [20,21]. How biofilm formation is regulated in B. thuringiensis or in B. cereus is still unknown. In B. anthracis, SinR strongly represses the sipW-tasA operon [22], but the effect of sinR deletion on biofilm formation has not been studied. The quorum sensing molecule AI2 is produced by B. cereus and inhibits biofilm formation when added exogenously [23], and the transcriptional regulators PlcR and CodY affect biofilm formation in the B. cereus reference strain ATCC 14579 [2426]. PlcR is the main virulence regulator in B. cereus [6] and CodY, which represses the biosynthesis of branched amino-acids, might also be involved in the pathogenicity of B. cereus [2629]. These findings suggest a connection between biofilm formation and virulence in this species. Here we report an investigation of the roles of Spo0A, AbrB and SinI/SinR in biofilm formation in the B. thuringiensis strain 407, which produces dense pellicles at the air-liquid interface. We found that SinI/SinR had a large effect on biofilm formation. We therefore analyzed the B. thuringiensis sinR regulon, which was found to include the sipW-tasA operon (...truncated)