Factors influencing germination of Bacillus subtilis spores via activation of nutrient receptors by high pressure

Abstract

Different nutrient receptors varied in triggering germination of Bacillus subtilis spores with a pressure of 150 MPa, the GerA receptor being more responsive than the GerB receptor and even more responsive than the GerK receptor. This hierarchy in receptor responsiveness to pressure was the same as receptor responsiveness to a mixture of nutrients. The levels of nutrient receptors influenced rates of pressure germination, since the GerA receptor is more abundant than the GerB receptor and elevated levels of individual receptors increased spore germination by 150 MPa of pressure. However, GerB receptor variants with relaxed specificity for nutrient germinants responded as well as the GerA receptor to this pressure. Spores lacking dipicolinic acid did not germinate with this pressure, and pressure activation of the GerA receptor required covalent addition of diacylglycerol. However, pressure activation of the GerB and GerK receptors displayed only a partial (GerB) or no (GerK) diacylglycerylation requirement. These effects of receptor diacylglycerylation on pressure germination are similar to those on nutrient germination. Wild-type spores prepared at higher temperatures germinated more rapidly with a pressure of 150 MPa than spores prepared at lower temperatures; this was also true for spores with only one receptor, but receptor levels did not increase in spores made at higher temperatures. Changes in inner membrane unsaturated fatty acid levels, lethal treatment with oxidizing agents, or exposure to chemicals that inhibit nutrient germination had no major effect on spore germination by 150 MPa of pressure, except for strong inhibition by HgCl2.

FIG. 1.

Flow cytometry of dormant and germinated spores. Spores of PS533 (wild-type) either dormant (A), treated for 6 min with a pressure of 150 MPa (B), or killed 98% with H₂O₂ and treated for 7 min with a pressure of 150 MPa (C) were stained with Syto 16 and analyzed by flow cytometry as described in Materials and Methods. Arrows labeled days and g denote dormant and germinated spores, respectively. The fluorescence of dormant spores killed 98% with H₂O₂ was identical to that of dormant untreated spores, the fluorescence of untreated spores germinated for 30 min with l-alanine was identical to that of pressure-germinated spores, and the fluorescence of spores killed 97 and 98% with CuOOH and tBHP, respectively, and then treated for 7 min with a pressure of 150 MPa was the same as that of pressure-germinated H₂O₂-killed spores.

FIG. 2.

Pressure germination of spores with different levels of various nutrient receptors. Spores were treated with a pressure of 150 MPa, and germination was assessed by flow cytometry as described in Materials and Methods. The symbols for the spores of the various strains are as defined as follows. (A) ○, PS533 (wild type); •, FB87 (gerB gerK); ▴, PS3651 (gerA gerK); <▪>, PS3615 (gerA gerB); ▾, FB72 (gerA gerB gerK). (B) ○, PS533 (wild type); •, PS3476 (PsspD::gerA); ▴, PS3651 (gerA gerK); □, PS3655 (gerA gerK PsspD::gerB); ▾, PS3654 (gerA gerK PsspB::gerB). (C) ○, PS533 (wild type); •, FB22 (gerA gerBA*); □, PS3665 (gerA gerK gerBB*); ▾, PS3666 (gerA gerK PsspB-gerBB*); ▴, PS3667 (gerA gerK PsspD::gerBB*).

FIG. 3.

Effect of spore DPA content on pressure germination. Spores of strains PS533 (wild type) or FB122 (sleB spoVF) prepared with or without DPA were treated with 150 MPa of pressure, and germination was assessed by flow cytometry as described in Materials and Methods. Symbols: ○, PS533 spores; •, FB122 spores prepared without DPA containing <3% of wild-type spore DPA levels; ▴, FB122 spores prepared with DPA and containing 77% of wild-type spore DPA levels.

FIG. 4.

Effects of changes in unsaturated fatty acid composition and sporulation temperature on pressure germination. Spores of different strains were prepared at various temperatures and treated with 150 MPa of pressure, and germination was assessed by flow cytometry as described in Materials and Methods. (A) Spores prepared at 27°C (○, □, and ▾) and spores prepared at 40°C (•, ▪, and ▴). Circles, PS533 (wild type); squares, PS3624 (↑Des); triangles, PS3628 (Des). (B) PS533 spores prepared at 23°C (○), 30°C (•), 37°C (□), and 44°C (▪).

FIG. 5.

Pressure germination of spores killed by oxidizing agents. Spores of strain PS533 (wild type), either untreated or killed with H₂O₂ or tBHP, were exposed to a pressure of 150 MPa, and spore germination was assessed by flow cytometry as described in Materials and Methods. Symbols: ○, untreated spores; •, spores killed 96% with H₂O₂; ▴, spores killed 98% with tBHP. Spores killed 97% with CuOOH and exposed to a pressure of 150 MPa germinated at the same rate as the tBHP-treated spores.