Expression of the 2Duf protein in wild-type Bacillus subtilis spores stabilizes inner membrane proteins and increases spore resistance to wet heat and hydrogen peroxide

Abstract

Aims: This work aimed to characterize spore inner membrane (IM) properties and the mechanism of spore killing by wet heat and H2O2 with spores overexpressing the 2Duf protein, which is naturally encoded from a transposon found only in some Bacillus strains with much higher spore resistance than wild-type spores.

Methods and results: Killing of Bacillus subtilis spores by wet heat or hydrogen peroxide (H2O2) was slower when 2Duf was present, and Ca-dipicolinic acid release was slower than killing. Viabilities on rich plates of wet heat- or H2O2 -treated spores +/- 2Duf were lower when NaCl was added, but higher with glucose. Addition of glucose but not Casamino acids addition increased treated spores' viability on minimal medium plates. Spores with 2Duf required higher heat activation for germination, and their germination was more wet-heat resistant than that of wild-type spores, processes that involve IM proteins. IM permeability and lipid mobility were lower in spores with 2Duf, although IM phospholipid composition was similar in spores +/- 2Duf.

Conclusions: These results and previous work suggests that wet heat and H2O2 kill spores by damaging an IM enzyme or enzymes involved in oxidative phosphorylation.

Keywords: Bacillus; antimicrobials; bacterial spores; disinfection; metabolism.

Figure 1.

(A–C) Viability and DPA remaining during heat or H₂O₂ treatment of spores with and without 2Duf. Spores of strains (A) PS4461, (B) PS4462, and (C) PS4461 and PS4462 at ∼10⁸ ml⁻¹ in water were incubated at (A and B) 90°C (PS4461) or 98°C (PS4462) or (C) in H₂O₂ at 23°C. Aliquots were serially diluted in cold water without (A and B) or with catalase (C), 10 μl aliquots spotted in duplicate on LB medium agar plates, plates were incubated and colonies were counted, as described in Material and methods section. Aliquots (0.5 ml) were taken at various times, centrifuged, and DPA quantitated in the supernatant fluid and pellet as described in Material and methods section. All values are +/- ≤12%. The symbols in (A and B) are: ○, DPA remaining; ●, viability, and in (C) are ○, ●–viability of PS4461 and PS4462 spores, respectively, and △—DPA remaining in PS4462 spores; slightly less DPA was released from PS4461 spores (data not shown).

Figure 2.

(A and B) Spore viability on minimal medium plates with various additions after treatment with (A) 93°C or (B) H₂O₂. PS533 spores (wt) were treated, cooled on ice or H₂O₂ eliminated, and spore viability determined on Spizizen’s minimal medium plates with various additions as described in Material and methods section. The symbols for additions to recovery plates are: 0.1% Casamino acids–△; 0.1% Casamino acids and 30 mmol l⁻¹ glucose–○; and 30 mmol l⁻¹ glucose plus 100 μmol l⁻¹ l-alanine–●. All values are +/- ≤ 16%.

Figure 3.

(A–C) Effects of heat activation time on AGFK germination of (A) PS4461 and (B) PS4462 spores. Spores at ∼3 × 10⁸ spores ml⁻¹ were incubated at 80°C for various times, and aliquots saved on ice. The spores were germinated at 37°C with AGFK as described in Material and methods section, and CaDPA release monitored fluorometrically in (A and B). Note that amounts of fluorescence at 0 min increased with increasing heating times, more for PS4461 spores than PS4462 spores, presumably due to CaDPA release by the 80°C treatment. (C) Slopes of the initial increases of CaDPA fluorescence for germination, defined as germination rate, after different heat activation times were measured and plotted vs heat activation times. Symbols in A and B denote heat activation times in min as: ○–0; ●–15; △–30; ▲–60; □–90; ■-120; ◊–150; ♦–180; ×- 210; *- 240. In (C) the symbols for the rates of germination of the spores are: ○—PS4461; and ●—PS4462.

Figure 4.

(A–D) Loss of ability to germinate following heat treatment of spores with and without 2Duf. (A) Spores of strains PS4461 (○) and PS4462 (●) at ∼8 × 10⁸ ml⁻¹ in water were incubated at 93°C (PS4461) or 98°C (PS4462). At various times, two 250 μl aliquots were diluted 4-fold in cold water, centrifuged, the pellets suspended in 500 μl of LB liquid medium with 10 mmol l⁻¹ l-valine, incubated at 37°C, and after 90 min, completion of spore germination was measured by phase contrast microscopy of ∼100 spores. (B) Spores of PS4461 (○) and PS4462 (●) at ∼3 × 10⁸ ml⁻¹ in water were heated at 93°C, aliquots cooled, germinated for 90 min in 40 mmol l⁻¹ CaDPA at 23°C and germination assessed by phase contrast microscopy of ∼85 individual spores. In (C) PS4461 and (D) PS4462 spores at ∼3 × 10⁸ ml⁻¹ in water were incubated at 85°C, aliquots were cooled, and their l-valine germination assessed by measuring CaDPA release in duplicate as described in Material and methods section. The symbols for heating times in min are: ○–0; ●–30; △–60; ▲–90; □–120; ■–150; and ◊−210; note there is no 30 min heating time in (D).

Figure 5.

(A and B) ¹⁴C-Methylamine uptake by (A) intact and (B) chemically decoated spores. Methylamine uptake was measured as described in Material and methods. All values are averages of duplicate determinations and have been corrected so that uptake into identical numbers of spores is compared. Symbols for spores are: PS4461–▲; and PS4462–△. Values in B are averages of results in two experiments with deviations of ≤15%.