Flexibility of the Prograamme of Spore Coat Formation in Bacillus subtilis: Bypass of CotE Requirement by Over-Production of CotH

Abstract

Bacterial spores are surrounded by the coat, a multilayered shell that contributes in protecting the genome during stress conditions. In Bacillus subtilis, the model organism for spore formers, the coat is composed by about seventy different proteins, organized into four layers by the action of several regulatory proteins. A major component of this regulatory network, CotE, is needed to assemble the outer coat and develop spores fully resistant to lysozyme and able to germinate efficiently. Another regulator, CotH, is controlled by CotE and is present in low amounts both during sporulation and in mature spores. In spite of this CotH controls the assembly of at least nine outer coat proteins and cooperates with CotE in producing fully resistant and efficiently germinating spores. In order to improve our understanding of CotH role in spore formation, we over-produced CotH by placing its coding region under the control of a promoter stronger than its own promoter but with a similar timing of activity during sporulation. Over-production of CotH in an otherwise wild type strain did not cause any major effect, whereas in a cotE null background a partial recovery of the phenotypes associated to the cotE null mutation was observed. Western blot, fluorescence microscopy and Surface-Enhanced Raman Scattering spectroscopy data indicate that, in the absence of CotE, over-production of CotH allowed the formation of spores overall resembling wild type spores and carrying in their coat some CotE−/CotH-dependant proteins. Our results suggest that the B. subtilis spore differentiation programme is flexible, and that an increase in the amount of a regulatory protein can replace a missing partner and partially substitute its function in the assembly of the spore coat.

The spore surface of intestinal isolates ofBacillus subtilis

“…Western blot analyses were performed by standard procedures. Nitrocellulose membranes were used for electrotransfer and proteins and then hybrated with anti‐CotZ, anti‐CotB, anti‐CotG or anti‐CotC antibodies as described previously (Isticato et al ., ). A quantitative determination of the amount of LTB was obtained by dot blot experiments with specific anti‐LTB antibodies analyzing serial dilutions of purified LTB and the supernatant of binding assays (Isticato et al ., ).…”

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

“…Spore coat proteins were extracted from a suspension of spores by SDS-DTT or NaOH treatment [29]. The concentration of extracted proteins was determined by using Bio-Rad DC protein assay kit (Bio-Rad), and 20 μg of total spore coat proteins were fractionated on 12.5% SDS polyacrylamide gels and staining by Brilliant Blue Coomassie or electro-transferred to nitrocellulose lters (Bio-Rad) for western blot analysis following standard procedures.…”

Mucosal immunity induced by gliadin-presenting spores of Bacillus subtilis in HLA-DQ8-transgenic mice

“…2. Accordingly, we suggest that most gliadin escaped from antibody recognition, probably because it was incorporated inside structural niches originating from genetic [25,26] or physical modifications. Another important result was the high stability of gliadin-spore interactions.…”