Bacillus subtilis builds structurally and functionally different spores in response to the temperature of growth

Abstract

SummaryBacterial spores are commonly isolated from a variety of different environments, including extreme habitats. Although it is well established that such ubiquitous distribution reflects the spore resistance properties, it is not clear whether the growing conditions affect the spore structure and function. We used Bacillus subtilis spores of similar age but produced at 25, 37, or 42°C to compare their surface structures and functional properties. Spores produced at the 25°C were more hydrophobic while those produced at 42°C contained more dipicolinic acid, and were more resistant to heat or lysozyme treatments. Electron microscopy analysis showed that while 25°C spores had a coat with a compact outer coat, not tightly attached to the inner coat, 42°C spores had a granular, not compact outer coat, reminiscent of the coat produced at 37°C by mutant spores lacking the protein CotG. Indeed, CotH and a series of CotH‐dependent coat proteins including CotG were more abundantly extracted from the coat of 25 or 37°C than 42°C spores. Our data indicated that CotH is a heat‐labile protein with a major regulatory role on coat formation when sporulation occurs at low temperatures, suggesting that B. subtilis builds structurally and functionally different spores in response to the external conditions.

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

“…Effects of the temperature on the recombinant display system CotB, CotC and CotG are abundant coat proteins widely used as carriers to display heterologous proteins on the spore surface [8]. All three proteins have been recently found differentially represented in spores produced at 25, 37 or 42°C, with CotB and CotG more abundant in spores prepared at 25°C and CotC more abundant in 42°C spores [22]. We used isogenic B. subtilis strains carrying DNA coding for the model antigen TTFC (tetC) fused to the gene coding for either CotB (cotB) [5] or CotC (cotC) [23] to evaluate the abundance of the fusion proteins at the different temperatures.…”

“…We used isogenic B. subtilis strains carrying DNA coding for the model antigen TTFC (tetC) fused to the gene coding for either CotB (cotB) [5] or CotC (cotC) [23] to evaluate the abundance of the fusion proteins at the different temperatures. Spores of strains RH103 (cotB::tetC) and RH114 (cotC::tetC) were produced at 25, 37 and 42°C and puri ed, as previously reported [22]. Surface proteins were extracted from RH103 and RH115 spores by the SDS-DTT or NaOH treatments, respectively and used for western blotting analysis with anti-CotB [5] or anti-CotC [23] antibodies.…”

“…Figures 1 and 2 indicated, respectively, the amounts of fusion proteins extracted and exposed on the spore surface but did not allow to exclude that other amounts of each fusion were actually present (but not extracted or not exposed) on spores produced at different temperatures. To address this issue, we used different isogenic strains of B. subtilis RH238, carrying the Green Fluorescent Protein (GFP) fused to CotC [23], and RH296, carrying the Red Fluorescent Protein (RFP) fused to CotG [22]. A uorescence microscopy analysis on spores prepared at 25, 37 or 42°C and the quanti cation of the uorescence signals performed by the ImageJ software, as previously reported [24], indicated that the CotG-based fusion was more abundant at 25°C, less abundant at 37°C and almost undetectable at 42°C while the CotC-based fusion showed an opposite pattern ( Figure 3).…”

“…with the non-recombinant display a modest effect is observed with small proteins (PPT and LYS) adsorbed more e ciently by 37 or 42°C spores than by 25°C spores; the localization of adsorbed RFP within the spore surface layers is modi ed by the temperature, indicating that spores produced at the low temperatures (CotB/CotG type coat) or at high temperature (CotC type coat) [22] have different adsorption properties.…”

“…Western blotting analysis of coat proteins. Puri ed spores of strains carrying the cotB::tetC (upper panel) or cotC::tetC (lower panel) were produced at the three temperatures and used to extract coat proteins by SDS-DTT (upper panel) or NaOH (lower panel) treatment as previously reported [22]. Proteins were reacted with anti-CotB (upper panel) or anti-CotC (lower panel) antibody.…”