The Temperature of Growth and Sporulation Modulates the Efficiency of Spore-Display in Bacillus Subtilis

Abstract

Background: Bacterial spores displaying heterologous antigens or enzymes have long been proposed as mucosal vaccines, functionalized probiotics or biocatalysts. Two main strategies have been developed to display heterologous molecules on the surface of Bacillus subtilis spores: i) a recombinant approach, based on the construction of a gene fusion between a gene coding for a coat protein (carrier) and DNA coding for the protein to be displayed, and ii) a non-recombinant approach, based on the spontaneous and stable adsorption of heterologous molecules on the spore surface. Both systems have advantages and drawbacks and the selection of one or the other depends on the protein to be displayed and on the final use of the activated spore. It has been recently shown that B. subtillis builds structurally and functionally different spores when grown at different temperatures, based on that B. subtilis spores prepared at 25, 37 or 42°C were compared for their efficiency in displaying various model proteins by either the recombinant or the non-recombinant approach.Results: Immune- and fluorescence-based assays were used to analyze the display of several model proteins on spores prepared at 25, 37 or 42°C. Recombinant spores displayed different amounts of the same fusion protein in response to the temperature of spore production. In spores simultaneously displaying two fusion proteins, each of them was differentially displayed at the various temperatures. The display by the non-recombinant approach was only modestly affected by the temperature of spore production, with spores prepared at 37 or 42°C slightly more efficient than 25°C spores in adsorbing at least some of the model proteins tested. Conclusion: Our results indicate that the temperature of spore production allows to control the display of heterologous proteins on spores and, therefore, that the spore-display strategy can be optimized for the specific final use of the activated spores by selecting the display approach, the carrier protein and the temperature of spore production.

CotG controls spore surface formation in response to the temperature of growth in Bacillus subtilis

“…Chromosomal DNA‐mediated transformation of competent cells of the B . subtilis strain RH406 ( amyE :: cotG :: rfp cotC :: gfp ) (Petrillo et al ., 2020) with chromosomal DNA of B . subtilis strain AZ603 ( ΔcotG ΔcotH :: neo ) (Saggese et al ., 2014) was used to yield strain AZ720 ( ΔcotG ΔcotH :: neo amyE :: cotG :: rfp cotC :: gfp) .…”

“…Chromosomal DNA-mediated transformation of competent cells of the B. subtilis strain AZ603 (ΔcotG ΔcotH:: neo) (Saggese et al, 2014) with chromosomal DNA of B. subtilis strain RH296 (amyE::cotG::rfp) (Petrillo et al, 2020) was used to yield strain RH298 (ΔcotG ΔcotH::neo amyE::cotG::rfp).…”

Sporulation efficiency and spore quality in a human intestinal isolate of Bacillus cereus

“…λ max values for the absorption and fluorescence emissions of the complex with DNA are 488 and 518 nm, respectively. Then, spores were analysed in a flow cytometer (BD Accuri C6; BD Biosciences, San Jose, CA) and detected in FL1 by using the standard C6 filter configuration (FL1 =530/30 BP) separating germination-unspecific and germination-specific fluorescence (threshold 10 4 ) (Petrillo et al 2020).…”

Regulation of pro‐σK activation: a key checkpoint in Bacillus subtilis sporulation

“…Additionally, recent reports also showed that B . subtilis builds spores with different structures when grown at different temperatures (Isticato et al ., 2020; Petrillo et al ., 2020). Bacterial spores are resistant to physical and chemical insults, making them a major burden for the public health and food industry.…”

“…de Andrade Cavalcante et al ( 2019)) proposed that much of this diversity was driven by adaptation to varying environments and their niche-specific stresses. Additionally, recent reports also showed that B. subtilis builds spores with different structures when grown at different temperatures (Isticato et al, 2020;Petrillo et al, 2020). Bacterial spores are resistant to physical and chemical insults, making them a major burden for the public health and food industry.…”