The effects of heat activation on Bacillus spore germination, with nutrients or under high pressure, with or without various germination proteins

Abstract

Nutrient germination of spores of Bacillus species occurs through germinant receptors (GRs) in spores' inner membrane (IM) in a process stimulated by sublethal heat activation. Bacillus subtilis spores maximum germination rates via different GRs required different 75 °C heat activation times: 15 min for l-valine germination via the GerA GR and 4 h for germination with the L-asparagine-glucose-fructose-K(+) mixture via the GerB and GerK GRs, with GerK requiring the most heat activation. In some cases, optimal heat activation decreased nutrient concentrations for half-maximal germination rates. Germination of spores via various GRs by high pressure (HP) of 150 MPa exhibited heat activation requirements similar to those of nutrient germination, and the loss of the GerD protein, required for optimal GR function, did not eliminate heat activation requirements for maximal germination rates. These results are consistent with heat activation acting primarily on GRs. However, (i) heat activation had no effects on GR or GerD protein conformation, as probed by biotinylation by an external reagent; (ii) spores prepared at low and high temperatures that affect spores' IM properties exhibited large differences in heat activation requirements for nutrient germination; and (iii) spore germination by 550 MPa of HP was also affected by heat activation, but the effects were relatively GR independent. The last results are consistent with heat activation affecting spores' IM and only indirectly affecting GRs. The 150- and 550-MPa HP germinations of Bacillus amyloliquefaciens spores, a potential surrogate for Clostridium botulinum spores in HP treatments of foods, were also stimulated by heat activation.

FIG 1

Effects of heat activation on germination of spores via various GRs. Spores of strains PS533 (wild type) (A and B) or FB10 (gerB*) (C) were germinated with 10 mM l-valine (A), 10 mM (each) AGFK (B), or 10 mM l-asparagine (C) after various heat activation times as described in Materials and Methods. Spore germination was monitored by Tb-DPA fluorescence, with values given either in relative fluorescence units (RFU) or as percent spore germination as described in Materials and Methods. Values shown are the averages of results from measurements on duplicate germinations done simultaneously, and the individual measurements differed by ≤6% from average values. The symbols representing the heat activation times are as follows: ○, 0 min; ●, 5 min; △, 15 min; ▲, 30 min; □, 1 h; ■, 2.5 h; and ◊, 4 h. A 6-h heat activation did not increase AGFK germination further (data not shown). For the samples analyzed in panels A and B, the maximum percentages of spore germination at 100 min were 92 and 88%, respectively.

FIG 2

Effects of heat activation times on rates and levels of spore germination. Spores of strain PS533 (wild type) or FB10 (gerB*) were prepared at 37°C and heat activated at 75°C for various times; spores were germinated in duplicate with either 10 mM l-valine, 10 mM (each) AGFK, or 10 mM l-asparagine as described in Materials and Methods. Spore germination was measured and germination rates (A) and percentages of spore germination (B) after 100 min were determined as described in Materials and Methods. Values shown are averages of duplicate determinations in two experiments with the same spore preparations and are ≤±12%. The symbols used are follows: ○, PS533 spores, l-valine germination; ●, PS533 spores, AGFK germination; and △, FB10 spores, l-asparagine germination.

FIG 3

Effects of heat activation on germination of spores made at various temperatures. Spores of strain PS533 (wild type) were prepared at 23°C (○), 30°C (●), 37°C (△), or 43°C (▲) and heat activated at 75°C for various times, and spores were germinated in duplicate with either 10 mM l-valine (A) or 10 mM (each) AGFK (B) as described in Materials and Methods. Spore germination was measured and germination rates were determined as described in Materials and Methods. Values shown are averages of duplicate determinations in two experiments with the same spore preparations and were ≤±19%.

FIG 4

Effects of heat activation on germination of spores with overexpressed GerA. PS3476 (PsspD::gerA) spores were heat activated for various times and germinated in duplicate with either 10 mM l-valine (○) or 10 mM AGFK (●), spore germination was measured, and germination rates were determined as described in Materials and Methods. Values shown are averages of duplicate determinations in two experiments with the same spore preparation and were ≤±22%, with the largest variations in AGFK germinations at short heat activation times.

FIG 5

Effects of heat activation on the synergy between GerA and GerB plus GerK and GerA and GerB* in spore germination. Spores of strain PS533 (wild type) (A) or FB10 (gerB*) (B) were either left unheated (○), heat activated for 30 min (●), or heat activated for 4 h (△) (A) or 2 h (B). These spores were germinated in duplicate with various concentrations of l-valine or l-asparagine (plus 10 mM [each] GFK) (A) or l-valine or l-asparagine (B) as described in Materials and Methods. The extents of spore germination at various times were determined as described in Materials and Methods and added together to give the predicted extents of spore germination, p, if there was no synergy. The spores were also germinated in duplicate with various concentrations of both l-valine and l-asparagine (plus 10 mM [each] GFK) (A) or both l-valine and l-asparagine (B), and the actual extents of spore germination with the germinant mixtures, a, were also determined. The degree of synergy (D_s) in germination at various concentrations of l-valine and l-asparagine was calculated as described previously (44) as D_s = a/p, and D_s values of >1 indicate synergy. In panel A the concentrations of l-valine and l-asparagine were equal, and in panel B the l-valine concentrations were 5-fold higher than the l-asparagine concentrations. The germination times selected for calculation of D_s values were the same for all data points for a particular germinant mixture, and these germination times gave the highest D_s values throughout the germinant concentration range. D_s values shown are averages from a and p values determined from duplicate measurements of extents of spore germination in two experiments with the same spore preparations and differed by ≤±32%.

FIG 6

Effects of heat activation on the germination of gerD spores made at various temperatures. Spores of strain FB62 (gerD) made at 23°C (○), 30°C (●), 37°C (△), and 43°C (▲) were heat activated for various times and germinated in duplicate with either 10 mM l-valine (A) or 10 mM AGFK (B) as described in Materials and Methods. Rates of spore germination were also determined as described in Materials and Methods. Values shown are averages of duplicate determinations in two experiments with the same spore preparations and were ≤±22%.

FIG 7

HP germination (150 MPa) of spores of various B. subtilis strains with and without heat activation. Spores of B. subtilis strains PS533 (wild type) (A), FB20 (ΔgerA) (B), FB61 (ΔgerA ΔgerB) (C), and PS3651 (ΔgerA ΔgerK) (D), without heat activation (○) or heat activated at 75°C for 30 min (●) or 4 h (△), were germinated in one experiment for various times with an HP of 150 MPa at 37°C; the extents of spore germination were measured as described in Materials and Methods.

FIG 8

HP germination (550 MPa) of spores of various B. subtilis strains with and without heat activation. Spores of B. subtilis PS533 (wild type) (A), B. subtilis FB20 (ΔgerA) (B), B. subtilis FB61 (ΔgerA ΔgerB) (C), and B. subtilis PS3651 (ΔgerA ΔgerK) (D), without heat activation (○) or heat activated at 75°C for 30 min (●) or 4 h (△), were germinated in one experiment for various times with an HP of 550 MPa at 50°C; the extents of spore germination were measured as described in Materials and Methods.

FIG 9

HP germination of B. amyloliquefaciens spores with and without heat activation. Spores of B. amyloliquefaciens prepared at 37°C and without heat activation (○) or heat activated at 70°C for 30 min (△) or 4 h (□) were germinated in one experiment for various times with an HP of 150 MPa (A) or 550 MPa (B); the extents of spore germination were measured as described in Materials and Methods. Similar results were obtained with two independent spore preparations, and HP germination of spores prepared at 30°C was affected similarly by heat activation (data not shown).