Sporulation conditions influence the surface and adhesion properties of Bacillus subtilis spores

Abstract

Spore-forming bacteria of the Bacillus subtilis group are responsible for recurrent contamination of processing lines in the food industry which can lead to food spoilage. The persistence of B. subtilis would be due to the high resistance of spores to extreme environmental condition and their propensity to contaminate surfaces. While it is well known that sporulation conditions modulate spore resistance properties, little is known about their effect on surface and adhesion properties. Here, we studied the impact of 13 sporulation conditions on the surface and adhesion properties of B. subtilis 168 spores. We showed that Ca²⁺ or Mg²⁺ depletion, lower oxygen availability, acidic pH as well as oxidative stresses during sporulation lead to the release of more hydrophobic and adherent spores. The consequences of these sporulation conditions on crust composition in carbohydrates and proteins were also evaluated. The crust glycans of spores produced in a sporulation medium depleted in Ca²⁺ or Mg²⁺ or oxygen-limited conditions were impaired and contained lower amounts of rhamnose and legionaminic acid. In addition, we showed that lower oxygen availability or addition of hydrogen peroxide during sporulation decreases the relative amount of two crust proteins (CgeA and CotY) and the changes observed in these conditions could be due to transcriptional repression of genes involved in crust synthesis in late stationary phase. The fact that sporulation conditions affect the ease with which spores can contaminate surfaces could explain the frequent and recurrent presence of B. subtilis spores in food processing lines.

Keywords: Bacillus subtilis; adhesion; crust; glycans; hydrophobicity; spores; sporulation.

Figure 1

Influence of sporulation conditions on spore adhesion to polypropylene. Adhesion (%) was determined after 10 successive adhesion steps of the spores to polypropylene tubes. The whole adhesion kinetics are presented in Supplementary Figure 3. Error bars represent the SDs of the means. ns, not significant. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001 for each condition versus Spo8 by Mann–Whitney.

Figure 2

Observation of spores by phase contrast microscopy after India ink staining. FP: Spores after a French press treatment (negative control). A white halo around the spores (white arrow) indicates the presence of crust glycans.

Figure 3

Influence of sporulation conditions on the monosaccharide composition of the crust. The relative amounts of Rha, Qui, and Leg in the crust were evaluated by RP-HPLC-FL. The experiments were performed on the crust of spores released from B. subtilis 168 cells produced in Spo8 and in the sporulation conditions shown to affect spore surface properties: -CaCl₂ (A), -MgSO₄ (B), -O₂ (C), -O₂ + CO₂ (D), H₂O₂ (E), peracetic acid (F), and pH 5 (G). The results were standardized by the OD_600nm of the spore preparations. Error bars represent the SDs of the means. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 for each condition versus Spo8 by Mann–Whitney.

Figure 4

Influence of sporulation conditions on the relative amount of CgeA and CotY in the crust. Fluorescence of spores of the B. subtilis 168 amyE::cgeA-GFP and B. subtilis 168 amyE::cotY-GFP strains was measured by flow cytometry after sporulation of both strains in Spo8 and in the sporulation conditions shown to affect spore surface properties: -CaCl₂ (A), -MgSO₄ (B), -O₂ (C), -O₂ + CO₂ (D), H₂O₂ (E), peracetic acid (F) and pH 5 (G). The fluorescence of spores is proportional to the amount of CgeA-GFP and CotY-GFP fusion proteins on the spore surface. Autofluorescence of the B. subtilis 168 strain is represented in red in all the panels (negative control). The data are presented as overlaid histograms that represent the distribution of fluorescence per cell of the same number of events.

Figure 5

Influence of sporulation conditions on the transcription of genes involved in crust synthesis. (A) Schematic representation of the genetic organization of the cge, cot, and sps genes involved in crust synthesis. Broken arrows, arrows and, stem-loop, respectively, represent the σ^K dependent promoters, direct or indirect activation of transcription by a transcriptional regulator and transcriptional terminators defined previously (Zhang et al., 1994; Roels and Losick, 1995; Eichenberger et al., 2004; Kuwana et al., 2005; Nicolas et al., 2012; Cangiano et al., 2014; Arrieta-Ortiz et al., 2015). Primers used for qRT-PCR are indicated by half arrows. Relative transcript levels of the cotY, cgeA, spsI, and spsM genes and SPβ prophage excision were evaluated by qRT-PCR in the late stationary phase (t8). SPβ prophage excision was assessed using primers flanking the prophage (SPβ-F/SPβ-R). Relative transcript levels were calculated as the ratio of the mRNA level (arbitrary units) of each gene in the sporulation conditions shown to affect spore surface properties: -CaCl₂ (B), -MgSO₄ (C), pH 5 (D), -O₂ (E), -O₂ + CO₂ (F) and H₂O₂ (G), compared to that obtained in the Spo8 condition. The fold change (FC) was calculated by the ΔΔCT method using the polC gene for normalization. Error bars represent the median with the interquartile range. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 for each condition versus Spo8 by t-test.