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1

Spore heat-activation requirements and germination responses correlate with sequences of germinant 1

receptors and with the presence of a specific spoVA2mob operon in food-borne strains of Bacillus subtilis 2

3

Antonina O. Krawczyk1,2, Anne de Jong1,2, Jimmy Omony1,2, Siger Holsappel1,2, Marjon H. J Wells-4

Bennik2,3, Oscar P. Kuipers1,2# and Robyn T. Eijlander1,2,3 5

6

Affiliations: 7

1. Laboratory of Molecular Genetics, University of Groningen, Nijenborgh 7, 9747 AG, Groningen, the 8

Netherlands 9

2. Top Institute Food and Nutrition (TIFN), Nieuwe kanaal 9A, 6709 PA, Wageningen, the Netherlands 10

3. NIZO Food Research B.V., Kernhemseweg 2, 6718 ZB, Ede, the Netherlands 11

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#Corresponding author: Oscar P. Kuipers, University of Groningen, Nijenborgh 7, 9747AG, Groningen, the 13

Netherlands; E-mail address: [email protected]; Phone: +31 50 3632093 14

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Running title: Spore heat-activation requirements and germination 16

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Abbreviations: Ca-DPA - calcium chelate of dipicolinic acid; GR(s), Ger - Germinant receptor(s); HA – 19

heat-activation, heat-activated; HR - heat resistance; IM - inner membrane; nH - non-heat-activated. 20

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Keywords: spore germination, Bacillus subtilis, spore heat-activation, spore heat resistance, germinant 22

receptors, spoVA, food spoilage 23

AEM Accepted Manuscript Posted Online 27 January 2017Appl. Environ. Microbiol. doi:10.1128/AEM.03122-16Copyright © 2017 American Society for Microbiology. All Rights Reserved.

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Abstract 24

Spore heat resistance, germination and outgrowth are problematic bacterial properties compromising 25

food safety and quality. Large inter-strain variation in these properties makes prediction and control of 26

spore behavior challenging. High-level heat resistance and slow germination of spores of some natural B. 27

subtilis isolates, encountered in foods, have been attributed to the occurrence of the spoVA2mob operon 28

carried on the Tn1546 transposon. In this study, we further investigate the correlation between the 29

presence of this operon in high-level heat resistant spores and their germination efficiencies before and 30

after exposure to various sub-lethal heat treatments (heat-activation, HA), which are known to 31

significantly improve spore responses to nutrient germinants. We show that high-level heat resistant 32

spores harboring spoVA2mob required higher HA temperatures for efficient germination than spores 33

lacking spoVA2mob. The optimal spore HA requirements additionally depended on the nutrients used to 34

trigger germination, L-alanine (L-Ala) or a mixture of L-asparagine, D-glucose, D-fructose and K+ (AGFK). 35

The distinct HA requirements of these two spore germination pathways are likely related to differences 36

in properties of specific germinant receptors. Moreover, spores that germinated inefficiently in AGFK 37

contained specific changes in sequences of the GerB and GerK germinant receptors, which are involved 38

in this germination response. In contrast, no relation was found between transcription levels of main 39

germination genes and spore germination phenotypes. The findings presented in this study have great 40

implications for practices in the food industry, where heat treatments are commonly used to inactivate 41

pathogenic and spoilage microbes, including bacterial spore-formers. 42

43

Importance 44

This study describes a strong variation in spore germination capacities and requirements for a 45

heat-activation treatment, i.e. an exposure to sub-lethal heat that increases spore responsiveness to 46

nutrient germination triggers, among 17 strains of B. subtilis including 9 isolates from spoiled food 47

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products. Spores of industrial food-borne isolates exhibited on average less efficient and slower 48

germination responses and required more severe heat-activation than spores from other sources. High 49

heat-activation requirements and inefficient, slow germination correlated with elevated resistance of 50

spores to heat and with specific genetic features, indicating a common genetic basis of these three 51

phenotypic traits. Clearly, inter-strain variation and numerous factors that shape spore germination 52

behavior, challenge standardization of methods to recover highly heat resistant spores from the 53

environment and have an impact on the efficacy of preservation techniques used by the food industry to 54

control spores. 55

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Introduction 56

Bacillus subtilis spores, which are widely present in nature, can easily contaminate food products 57

(1, 2). Because of their resistance to environmental stresses such as extreme temperatures, desiccation, 58

radiation and exposure to different chemicals, spores are able to survive preservation treatments that 59

are applied in the food industry (2–4). Surviving spores can germinate and resume vegetative growth in a 60

food product and subsequently cause spoilage (1, 5, 6). Spore germination is a necessary step in 61

resuming vegetative growth; thus, the probability that the spores germinate in food products needs to 62

be taken into account when assessing the risk of spoilage or outgrowth of pathogenic spore-formers. 63

Moreover, both inhibition of spore germination as well as induction of germination before inactivation 64

treatments (which renders the spore sensitive) are used in the food industry to improve food safety (7–65

12). However, none of these strategies are completely effective. 66

In nature, spores typically germinate in response to nutrients such as amino acids, sugars and/or 67

nucleosides. Nutrients need to permeate the spore coat through a process facilitated by the GerP 68

proteins (13–15), and the other outer layers of the spore to gain access to specific nutrient germinant 69

receptor complexes (Ger receptors, GRs) that are located in the spore inner membrane (IM) (16, 17). 70

Germinant receptors are predominantly built-up by three subunits: A, B and C, which are encoded in 71

tricistronic operons (18, 19). Subunit A contains five to six predicted transmembrane (TM) helices and a 72

hydrophilic domain at both N- and C-termini (18, 20). Subunit B, which might play a role in recognition of 73

germinants (21), appears to be an integral inner membrane protein with ten to twelve TM helices (18). 74

Subunit C is a predominantly hydrophilic lipoprotein. It is anchored to the outer surface of the inner 75

membrane by a diacylglycerol anchor that itself is attached to an N-terminal lipobox cysteine (22). Some 76

ger operons encode a fourth small subunit D that consists of two TM helices and likely modulates the 77

function of the respective GRs (23, 24). 78

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A number of germinant receptors and the nutrients to which they respond vary between 79

different Bacillus species and strains. The extensively studied laboratory strain, B. subtilis 168, contains 80

three functional germinant receptors: GerA, GerB and GerK (25). The GerA complex responds to L-81

alanine (L-Ala). The GerB and GerK receptors cooperatively initiate germination in response to a mixture 82

of L-asparagine, D-glucose, D-fructose and potassium ions (AGFK). In fact, GerB responds predominantly 83

to L-asparagine, whereas GerK responds to sugars; but neither of these receptors can trigger germination 84

alone (19, 26). Additionally, B. subtilis 168 contains two GR operons yndDEF and yfkQRTS that seem to be 85

inactive and poorly transcribed (25). Moreover, certain food-borne B. subtilis strains comprise the 86

incomplete and likely non-functional gerX germination operon that encodes putative GR subunits A and 87

C but lacks a gene for the B subunit (27, 28). 88

Germinant receptors form one or two germination clusters (germinosomes) in the spore IM, 89

together with another small lipoprotein, GerD, which is required for this process (29). Formation of the 90

germination clusters increases the local concentration of GRs and likely facilitates cooperativity and 91

synergism between them (29–31). Germination responses can be increased by applying a sublethal heat 92

treatment (so called heat-activation, HA) prior to exposure of spores to nutrient germinants (32). Heat-93

activation is thought to act either directly on GR subunits or indirectly by altering properties of IM, rather 94

than on the GerD protein or the germinosome formation (32). 95

After sensing germinants by the respective GRs, an unknown signal is transduced to downstream 96

effectors. This leads to the release of monovalent cations and of dipicolinic acid chelated by Ca2+ ions 97

(Ca-DPA) from the spore core (19, 26). Ca-DPA is transported across the IM by a channel formed by 98

products of the conserved heptacistronic spoVA operon, which in B. subtilis 168 consists of seven genes: 99

spoVAA-D, spoVAEb, spoVAEa and spoVAF (19, 26, 33). Release of Ca-DPA allows for partial rehydration 100

of the spore core and activation of the cortex lytic enzymes (CLEs), CwlJ and SleB (19, 26). Degradation of 101

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the protective peptidoglycan cortex leads to further water uptake and the completion of germination 102

(19, 26). 103

Germination responses can vary strongly between Bacillus species and strains (28, 34, 35). For 104

this reason, studies performed on the model strains, which are adapted to laboratory conditions, do not 105

accurately reflect spore germination properties of strains that contaminate food products. This variation 106

hinders the ability to predict the germination behavior of problematic spores and therefore complicates 107

a risk assessment and the development of efficient spore inactivation treatments. 108

Recently, variation in spore germination and spore heat resistance has been partly attributed to 109

inter-strain differences in the absence or presence (and copy number) of the heptacistronic spoVA2mob 110

operon that is usually carried on the Tn1546-like transposon element (which lost the ability of 111

transposition) (27, 28). Tn1546 additionally carries four other operons that alone do not have apparent 112

effects on spore heat resistance or germination (27, 28). Different B. subtilis strains have between 0 and 113

3 copies of spoVA2mob (two on the Tn1546-like elements integrated within the yitF gene and between the 114

yxjA and yxjB genes and one in a chromosomal location that could have not been determined in the non-115

closed genome sequences) (27). Strains carrying an increasing copy number of spoVA2mob produce spores 116

with elevated heat resistance and an affected germination phenotype in a nutrient-rich medium. The 117

exact functions of the encoded proteins on this operon remain unclear. Three of them—SpoVAC2mob, 118

SpoVAD2mob and SpoVAEb2mob—are homologous to the conserved SpoVAC, SpoVAD and SpoVAEb 119

proteins (27, 28) that are involved in Ca-DPA transport via the spore IM (26, 36, 37). Another four, out of 120

which two are predicted to be associated with the spore membrane, constitute putative proteins 121

containing domains of unknown functions (27, 28). The spoVA2mob products have been hypothesized to 122

affect spore germination (and HR) by altering properties of the spore IM in which the majority of the 123

spore germination apparatus is located (28). 124

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In this study, we investigated spore germination requirements and efficiencies for seventeen B. 125

subtilis strains with known genomic sequences (some of which constitute problematic food-spoilers) that 126

contain different copy numbers (0 to 3) of the spoVA2mob operon (4, 27) (Table 1). Eight strains (168, 127

B4055, B4056, B4057, B4058, B4060, B4061 and B4143) lack spoVA2mob in their genomes and produce 128

low heat resistant spores. Nine strains have either one (B4146), two (B4068, B4069, B4071, B4072, 129

B4073) or three (B4070, B4067, B4145) spoVA2mob copies and form spores with an increasing level of high 130

heat resistance (4, 27) (Table 1). The presence and sequences of specific germination genes were 131

analyzed to link differences in spore germination phenotype and spore germination requirements to 132

genetic properties of the strains. This study demonstrates that the presence of the spoVA2mob operon 133

correlates with increased spore HA requirements and, if spoVA2mob is present in multiple copies, with 134

inefficient germination in response to AGFK. Additionally, sequences of germinant receptor proteins 135

seemingly affect the final spore germination phenotypes and heat-activation requirements. 136

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Materials and methods 137

Strains and growth conditions 138

Strains used in this study are listed in Table 1. Spores were prepared during a seven-day 139

incubation at 37°C on Schaeffer-agar plates without antibiotics as described before (4, 27, 28). Harvested 140

spores were washed for 4 days with cold sterile Milli-Q water and stored at 4°C for at least 1 month prior 141

to the germination experiments to ensure full maturation of the spores (38). The spore crops consisted 142

of ≥95% dormant phase-bright spores as determined by phase-contrast microscopy. 143

144

Spore germination assays 145

Prior to the experiments, spores were washed with cold sterile MilliQ water. Before exposure to 146

nutrients, spore suspensions with an optical density of 10 at 600 nm (OD600) were subjected or not (non-147

heat-activated spores, nH) to a 30-minute heat treatment at 70, 80, 87, 95 and/or 100°C. Spores 148

produced by individual strains had substantially different levels of heat resistance as indicated by their 149

distinct decimal reduction times, D-values, at 100°C (for comparison purposes, D-values have been 150

extrapolated from higher temperatures to 100°C for high-level heat resistant spores) (4, 27)(Table 1). For 151

this reason, the choice of applied HA temperature was tailored for each strain, depending on the spore 152

heat resistances (Table 2, Table 3). Thus, low-level heat resistant spores (decimal reduction times, D-153

values, between 2.4±0.7 and 12.0±4.3 minutes at 100°C, Table 1) produced by strains 168, B4055-B4058, 154

B4060-B4061 and B4143 were heat-activated in most cases at 70°C and maximally at 87°C. Moderately 155

high heat resistant spores of strain B4146 (D-value of 83.0±32.3 minutes at 100°C, Table 1) and high-level 156

heat resistant spores of strains B4067-B4073 and B4145 (D-values between 310±137 and 4224±2470 157

minutes at 100°C, Table 1) were heat-activated in temperatures up to 100°C. After heating, spores were 158

cooled on ice and assessed by phase-contrast microscopy to ensure that the applied heat treatment did 159

not cause spore phase transformation in the absence of germination triggers. Spores were diluted to an 160

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OD600 of 1 in 10 mM L-alanine or 10 mM AGFK mixture (L-asparagine, D-glucose, D-fructose and KCl) in 25 161

mM Tris-HCl buffer (pH 7.4) with addition of 0.01% Tween20 to prevent spore clumping and absorption 162

to the plate wells (39). The 10 mM germinant concentrations were used as these are known to be 163

saturating for B. subtilis 168 spores (15, 40, 41). Germination was monitored at 37°C in a 96-well plate-164

reader (Tecan Infinite 200, Tecan) by measuring the decrease in OD600, which corresponds with the 165

change of dormant phase-bright spores to germinated phase-dark spores. Measurements were taken 166

every 2-3 minutes for 3 hours with shaking in-between. All spore germination experiments were 167

performed on two independently prepared spore crops. 168

169

Phase-contrast microscopy 170

Plate-reader samples were investigated by phase-contrast microscopy 3 hours after the addition 171

of nutrients as described before (42). Germination efficiency was calculated as a percentage of phase-172

dark (germinated) spores seen using phase contrast microscopy in a total of 100 to 700 spores per spore 173

crop and condition, using the Fiji software (43) (http://fiji.sc/Fiji), similarly to what has been described 174

before (42). 175

176

Genome mining 177

For all the analyzed B. subtilis strains, the phylogenetic tree was prepared (Figure 1) with the 178

PhyloPhlAn program based on the sequences of the selected 400 most conserved microbial proteins 179

(https://huttenhower.sph.harvard.edu/phylophlan) (44) and displayed with the use of the iTOL tool 180

(http://itol.embl.de) (45). For all the predicted protein sequences encoded in the genomes of the 181

seventeen B. subtilis strains, an orthology prediction was performed applying two programs: Ortho-MCL 182

(46) and ProteinOrtho (47) (data not shown), the latter with use of B. subtilis 168 as a reference. To find 183

potential functional equivalents for a selection of germinant receptor genes (Supp. Table S1), additional 184

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protein BLAST searches (48) were made using B. subtilis 168 sequences as queries to scan genomes of 185

the food-borne strains. For the selected GR proteins found in this manner, protein sequence alignments 186

were made using MUSCLE (49). Additionally, the genomic context of the selected genes was manually 187

inspected after visualization of the genomes with Clone Manager software (Clone Manager v8, Scientific 188

& Educational Software, Denver, CO, USA). In relevant cases, to verify operon structures, operon 189

predictions were performed with Glimmer (50, 51), and visualized using the draw context tool in the 190

Genome2D server (http://genome2d.molgenrug.nl) (52). The membrane topologies of the selected A, B 191

and D GR subunits were modeled using TOPCONS (http://topcons.cbr.su.se/) (53) and the secondary 192

structures of the C subunits were predicted with PredictProtein (http://www.predictprotein.org) (54), 193

except for the GerBC protein, the structure of which has been solved (22). 194

195

Transcriptome analysis by RNA-Seq 196

Sporulation of five B. subtilis strains: 168, B4143, B4146, B4072, 4067 was induced by the 197

resuspension method similarly as described by Nicolas et al. (55). Shortly, strains were grown with 198

shaking (200 rpm) at 37°C in the casein hydrolysate (CH) medium till an OD600 of 0.6. Subsequently, 199

whole bacterial cultures were spun down at 6000 rpm for 8 minutes and resuspended in the same 200

volume of the pre-warmed Sterlini-Mandelstam (SM) medium to induce sporulation. Samples for RNA 201

isolation and for microscopic analysis were collected at various time-points of the sporulation process. 202

For RNA extraction, 15 ml of the cultures was centrifuged (12000 rpm, 1 min) and the cell pellets were 203

frozen in liquid nitrogen and stored at -80°C. For the microscopic analysis, 300 µl of cultures were 204

precipitated by centrifugation at 10,000 rpm for 2 minutes, washed with PBS and fixed using 4% para-205

formaldehyde as described before (56). The microscopic samples were used to assess the stages of 206

sporulation at the selected time-points (data not shown). 207

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Total RNA was isolated from the samples of sporulating cells by phenol:chloroform extraction 208

and precipitation with ethanol and sodium acetate, as described before (57). To ensure homogenization 209

of both mother cell and forespore compartments, sporulating cells were exposed to five 45 second-long 210

bead-beating cycles, with at least 1 minute intervals of cooling on ice. The RNA samples were subjected 211

to the next generation directional sequencing on an Ion Proton™ Sequencer at the PrimBio Research 212

Institute (Exton, PA, USA), and T-REx (58) was used for the RNA-Seq transcriptome analysis. The 213

transcripts were either mapped on the reference genome of B. subtilis 168 or on the genomes of the 214

respective individual strains. Transcripts mapped on the genome of B. subtilis 168 were visualized in 215

JBrowse (59). In order to compare expression of selected germination genes between the strains or 216

between two different genes within one strain, average ratios of maximal gene transcription signals 217

(expressed as reads per kilobase of transcript per million mapped reads, RPKM) during sporulation were 218

calculated based on the results of two independent experiments. 219

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Results 220

Low-level heat resistant spores mostly do not require heat-activation for efficient nutrient-induced 221

germination 222

Under laboratory conditions, spores of B. subtilis are often heat-activated (HA) for 30 223

minutes at 70°C to increase their germination responses to nutrients (60–62). In this study, 224

germination (which under a phase-contrast microscope is reflected by the phase-bright to phase-225

dark transition of spores) in response to L-alanine and AGFK was assessed for HA and non-HA spores 226

of various B. subtilis strains that exhibit low to extremely high heat-resistance. When not heat-227

activated, almost all low heat-resistant spores (B4055, B4056, B4057, B4060, B4061, 168) responded 228

efficiently to L-alanine (mean ± absolute deviation of the mean between 84.8 ±14.1% and 98.4 229

±1.6% phase-dark spores within three hours), with an exception of B4058 and B4143 that showed no 230

to poor germination (2.3 ±1.6% and 20.6 ±6.3% phase-dark spores) (Table 2). Spores with high-level 231

heat resistance (B4067-B4073, B4145 and B4146), however, responded at best moderately (up to 232

38.0 ±0.6% phase-dark spores) without heat-activation (Table 2). When heat-activated at 70°C, the 233

germination percentage increased at least 2-fold for the poorly responding high-level heat resistant 234

spores of seven strains (namely, B4068-B4073 and B4146), whereas spores of strains B4058, B4067 235

and B4145 remained unresponsive to L-alanine (Table 2). 236

Using AGFK as a germination trigger, all but one low-heat resistant spore types germinated 237

almost entirely without the need for HA (Table 3). The exception (B4058) reached complete 238

germination after activation at 70°C in response to AGFK (but not to L-alanine with or without HA) 239

(Table 3). In contrast, almost all high heat-resistant spores germinated very poorly (0.8±0.2% - 240

10.6±4.9% phase-dark spores) in response to AGFK, even after HA at 70 ˚C. Only spores of strain 241

B4146, which exhibit moderately elevated heat resistance (4, 27), germinated moderately 242

(52.0±16.9% germinated spores) in these conditions (Table 3). 243

244

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Optimizing heat-activation conditions allows for efficient germination of high-level heat resistant 245

spores in L-alanine but not in AGFK 246

More severe heat-activation has been proposed to improve germination of highly resistant 247

spores of certain species and strains (63–65). Thus, to test whether optimizing HA conditions can 248

improve germination of the nine high heat resistant B. subtilis spore types (Table 1), we pre-249

incubated these spores at 80, 87, 95 and 100°C for 30 minutes. 250

In general, activation at temperatures exceeding 70°C increased the yields of germinated 251

high heat resistant spores in response to both germinants, with L-alanine-induced germination (Table 252

2) being optimally supported by somewhat lower HA temperatures (predominantly 80°C, with 87°C 253

giving similar results in a few cases) than required for AGFK-induced germination (mostly 87 - 95°C 254

and/or 100°C, Table 3). The only exception was L-alanine-induced germination of B4146 spores, for 255

which the best results (41.7±11.2% germination) were obtained after treatment at 70°C. In L-alanine, 256

the pre-treatment at 87°C gave slightly lower germination levels than at 80°C for the majority of the 257

high heat resistant spores. 95°C severely reduced L-alanine spore germination for all the strains 258

except for B4068 and B4073. Regardless of the activation temperature, L-alanine did not trigger 259

responses of B4067 and B4145 spores. 260

AGFK-induced germination of high heat resistant spores was rather poor (between 261

14.2±4.1% and 42.7±17.7%), even when the most optimal HA treatments at 87, 95 and/or 100°C 262

were applied (Table 3). The only exception were B4146 spores that germinated efficiently in AGFK 263

yielding 81.7±9.2% and 87.7±1.8% phase-dark spores after a pre-treatment at 87°C and 95°C, 264

respectively (Table 3). In some cases pre-heating at 100°C led to a phase change of up to 10.9±5.3% 265

spores in the absence of germinants (Table 3). 266

In comparison, heat-activation of low heat resistant spores of the reference strain B. subtilis 267

168 was not required for efficient germination in our experiments as similar results were obtained 268

for non-HA spores and for spores pre-heated at 70°C and 80°C (Table 2, Table 3). In contrast, pre-269

heating at 87°C caused a phase-bright to phase-dark transition of 18.4±1.5% of 168 spores in the 270

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absence of germinants (Table 2, Table 3), possibly due to spore damage and an increase in 271

permeability of outer spore layers leading to a rehydration of the core. 272

Besides the differences in germination efficiency (Table 2, Table 3), the high heat resistant 273

spores also germinated less rapidly than the low heat resistant spores in response to both L-alanine 274

and AGFK, even after optimal heat-activation (Figure 2). These differences in germination kinetics 275

observed between the various spore types are consistent with the previously described negative 276

effect of the spoVA2mob operon on the spore germination rate (28). 277

278

Genomic analysis reveals the presence of essential germination genes in all investigated strains 279

and uncovers differences in the structure of the gerB operon in two food-borne isolates 280

The results described above indicate a correlation between the presence of the spoVA2mob 281

operon, increased spore HA requirements and low germination efficiencies, especially in AGFK (Table 282

2, Table 3). Nevertheless, strong variations in a spore germination capacity and required HA were 283

seen between different strains that contain the same spoVA2mob copy numbers and between 284

different germination pathways (L-alanine and AGFK). Hence, factors other than spoVA2mob likely 285

contribute to the eventual germination behavior, to a greater extent in L-alanine and to a lesser 286

extent in AGFK. 287

To find genetic features other than components of the spoVA2mob operon that tune spore 288

germination properties, we investigated genomic sequences of the seventeen B. subtilis strains in 289

regard to genes that play key roles in nutrient-induced spore germination (19, 66, 67). In general, all 290

key germination genes are present in all B. subtilis strains investigated in this study, including those 291

encoding the three main germinant receptors, GerA, GerB, GerK (Supp. Table S1); six GerP A-F 292

proteins; the SpoVAA-AB-AC-AD-AEb-AEa-AF proteins; and the CwlJ and SleB cortex lytic enzymes 293

(data not shown). Of the two operons yndDEF and yfkQRST that encode putative and probably non-294

functional germinant receptors in B. subtilis 168 (25), yndDEF was present in all the strains except 295

B4057 (Supp. Table S1). In comparison, the intact yfkQRST operon occurs in seven (168, B4055, 296

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B4056, B4057, B4060, B4061 and B4143) out of the seventeen strains (Supp. Table S1), although its 297

residues could be found in the form of pseudogenes in genomes of the remaining strains (Supp. 298

Table S1). As reported recently, nine strains (B4067-B4073, B4145 and B4146) additionally contain 299

the gerXA and gerXC genes that encode the putative GR subunits A and C (Supp. Table S1) (27, 28). 300

However, the encoded GerXA and GerXC are very likely non-functional due to severe truncations 301

(especially at the N- and C-termini) that result in a decreased number of predicted TM helices in 302

GerXA and a loss of the N-terminal lipobox in GerXC in all strains except in B4146 (Supp. Figure S1). 303

Even though the three main GR operons (gerA, gerB and gerK) are present in all the 304

investigated strains, the food-borne isolates B4067 and B4145 contain strong deviations in the 305

structure of the gerB operon (Supp. Figure S2A). In the laboratory strain B. subtilis 168, this operon 306

encodes two integral membrane proteins GerBA and GerBB, and a lipoprotein, GerBC (Supp. Figure 307

S2B). In the strains B4067 and B4145, however, a codon stop at the nucleotide position 538-540 308

divides the gerBA gene into two predicted open reading frames (ORFs) gerBA-N and gerBA-C (Supp. 309

Figure S2A). Moreover, gerBB and gerBC are fused into one ORF (gerBB-BC) (Supp. Figure S2A). A 310

topology prediction suggests that the GerBB- and GerBC-parts of the encoded GerBB-BC fusion 311

protein might be arranged in the IM similarly (the N-terminus of the GerBB-part localizes inside the 312

spore core, followed by 11 TM helices and the majority of the GerBC-part localizes outside the spore 313

core) as the “regular” non-fused GerBB and GerBC proteins (Supp. Figure S2B). The encoded GerB 314

receptor of B4067 and B4145 might retain partial functionality, as spores produced by these two 315

strains were capable of moderate germination in AGFK (Table 3), which in B. subtilis 168 requires 316

both GerB and GerK (25). 317

318

Differences in germinant receptor protein sequences correlate with specific germination and heat 319

resistance phenotypes of the investigated B. subtilis strains 320

Germinant receptors are known to play a fundamental role in spore responsiveness 321

(specificity and affinity) to nutrient germinants (25, 68, 69). Moreover, besides the spore IM, GRs are 322

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the most probable spore component targeted when heat is applied to activate spore germination 323

(32). Thus, in addition to chromosomal presence of the spoVA2mob operon, properties of GR proteins 324

likely contribute to the diversity in germination efficiencies and HA requirements observed among 325

the tested spore types. To find features distinguishing GRs of the spores with strongly affected 326

germination phenotypes (very poor/no germination or high HA requirements) we analyzed multiple 327

amino acid sequence alignments of the GR subunits (Supp. data S2). 328

The analysis revealed an 82.5% - 100% amino acid sequence identity between the 329

corresponding GR proteins of individual strains, with the GerB receptor being the most variable 330

(Supp. data S1). Spores with the highest heat resistance level and high HA requirements for AGFK 331

germination (B4067-B4073, B4145) contained several distinctive amino acid residues found in various 332

regions of the GerBB, GerKA, GerKB and GerKC proteins (Table 4). The same amino acid substitutions 333

(Table 4) also concurred with the weak responsiveness of spores to AGFK as the high-level heat 334

resistant spores harbouring 2 or 3 spoVA2mob copies in their genome germinated rather poorly in this 335

cogerminant mixture (Table 3). Three of the distinct amino acids were additionally found in GerKB 336

(V147; I181) and GerKC (D398) of slightly high heat resistant B4146 spores (Table 4), which needed 337

moderate HA and responded strongly to AGFK (Table 2, Table 3). 338

No distinctive features typical for all high heat resistant spores were found in sequences of 339

the GerAA, GerAB, GerAC, GerBA and GerBC proteins (Table 4, Supp. data S2). However, a few 340

common amino acid substitutions were present in GerAA, GerAB and GerBC of B4068 and B4073 341

spores (Table 4) whose responses to L-alanine were not decreased by heating at 95°C (Table 2); L373I 342

in GerAA and S92L in GerAC (Table 4) are present in the protein regions that have previously been 343

shown to play a role in GR functionality (70, 71). For the spores that germinated weakly (B4143, 344

B4146, B4071 and B4070) or not at all (B4067, B4145 and B4058) in L-alanine, no common 345

alternations in the GerA subunits were observed (Table 4, Supp. data S2). Nevertheless, B4058, 346

B4143 as well as B4067 and B4145 contained various unique strain-specific amino acid residues in 347

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GerAA, GerAB and GerAC that distinguished them from the spores that responded efficiently to L-348

alanine (Table 4). 349

350

Transcription of germination genes in the selected food-borne strains does not correlate with the 351

germination phenotype of their spores 352

Besides genetic content and specific protein sequences, the expression levels of germination 353

genes during sporulation play an important role in the eventual germination behavior of the spores. 354

Thus, we analyzed transcription of germination genes during sporulation for B. subtilis 168, B4143, 355

B4146, B4072 and B4067 that each shows a distinct germination phenotype: i) 168 germinates well in 356

both L-alanine and AGFK; ii) B4143 only responds to AGFK; iii) B4146 germinates efficiently in AGFK 357

but only moderately in L-alanine; iv) B4072 germinates only in L-alanine; v) B4067 germinates rather 358

poorly in AGFK and not at all in L-alanine (Table 2, Table 3). 359

As can be seen in Supplementary Table S2 and Supplementary Figure S3, the three main 360

germination operons (gerA, gerB and gerK) as well as other important germination genes produced 361

transcripts with similar lengths and mostly comparable expression levels in all investigated strains. 362

Also, transcription of gerB in B4067 was comparable as in the other strains despite differences in the 363

genetic organization of this operon (Supp. Table S2, Supp. Figure S3). The putative GR operons, 364

yndDEF and yfkQRST (25) were also transcribed, with yndDEF exhibiting a somewhat higher 365

expression in B4143 and B4067 than in 168, B4146 and B4072 (Supp. Table S2, Supp. Figure S3). 366

Moreover, the part of the yfkQRST transcripts coding for the yfkT gene appeared to be unstable as 367

visualized in JBrowse (Supp. Figure S3).The potential germination genes located on the Tn1546 368

transposon present in the B4146, B4067 and B4072 strains were also expressed (Supp. Table S2). The 369

gerXA and gerXC genes, which encode likely non-functional GR subunits, were transcribed 1.8±0.1 to 370

8.0±0.3-fold stronger in B4146 and B4072 than the gerAA and gerAC genes of these strains, 371

respectively. The spoVAC2mob, spoVAD2mob and spoVAEB2mob genes of the spoVA2mob operon were 372

expressed similarly as the corresponding genes from the “regular” non-transposon spoVAA-AF 373

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operon (spoVAC, spoVAD and spoVAEB, respectively). Transcription of the first gene of the operon, 374

which encodes a putative protein of unknown function, was exceptionally high (57-99-fold higher 375

than transcription of the spoVAC gene in the respective strains), while the second and the last gene 376

of spoVA2mob, which also code for hypothetical proteins (27, 28), were expressed to a similar level as 377

spoVAC. The second copy of the spoVA2mob operon present in B4067 strain in an unknown genomic 378

context was transcribed around 2.3- to 8.5-fold weaker than the operon on the Tn1546-like 379

transposon element (Supp. Table S2). Again, expression of the first hypothetical gene was the 380

strongest. Whereas the last hypothetical gene, which encodes a putative membrane protein, has 381

been suggested to affect spore germination and heat resistance, the role of the second and the 382

(highly expressed) first gene of spoVA2mob remains unknown (27, 28). 383

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Discussion 384

This study shows that the spore germination capacities in response to nutrients and 385

accompanying heat-activation requirements are likely shaped by the presence (and copy number) of 386

the spoVA2mob operon in the genome and sequences of germinant receptor proteins. In contrast, the 387

transcription of germination genes during sporulation, which was similar in the five analyzed strains 388

(168, B4067, B4145, B4072, B4143, B4146) (Supp. Table S2, Supp. Figure S3), does not correlate with 389

spore germination phenotypes. 390

The spoVA2mob operon has been shown to i) increase spore heat resistance (27), (ii) prolong 391

the time required for spore germination and to (iii) decrease germination rates (28). Consistent with 392

our previous work in rich LB medium (28), this current study shows that the isolates containing the 393

spoVA2mob operon exhibited slower L-alanine- and AGFK-induced spore germination (Figure 2). Here, 394

we additionally demonstrate that these differences in germination kinetics between spores with and 395

without spoVA2mob persisted even after optimal heat-activation (HA) treatments (Figure 2). 396

Importantly, the present work extends former findings by revealing additional correlations between 397

the spoVA2mob presence and two other spore properties, namely higher spore HA requirements and 398

lower germination efficiencies (Table 2, Table 3). Furthermore, we show that two copies of the 399

spoVA2mob operon are transcribed, however to different levels (Supp. Table S2). 400

Overall, low-level heat resistant spores germinated well in response to L-alanine and AGFK, 401

with or without HA (at 70°C). High-level heat resistant spores generally germinated poorly in L-402

alanine without HA and in AGFK after relatively mild HA (70°C). Interestingly, an optimized HA 403

treatment strongly increased germination efficiency of high-level heat resistant B. subtilis spores that 404

contain the spoVA2mob operon only in response to L-alanine (usually after HA at 70-80°C) and for 405

strain B4146 (harboring only one copy of spoVA2mob) in response to AGFK (87-95°C) (Table 2, Table 3). 406

In contrast, AGFK-induced germination of spores with 2 or 3 spoVA2mob copies remained relatively 407

poor, even though HA at very high temperatures (95 - 100°C) improved yields of germinated spores 408

up to ~40% (Table 3). 409

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A few important implications arise from these results. First of all, gene products encoded by 410

the spoVA2mob operon (and possibly other operons on Tn1546) are likely directly or indirectly 411

responsible for inefficient germination of non-HA spores and for an increase in spore HA 412

requirements (Table 2, Table 3), with these effects being amplified when spoVA2mob is present in 413

multiple copies. This implies a direct genetic link between spore high heat resistance (27), slow 414

germination kinetics (28), low germination efficiencies and elevated requirements for HA. The exact 415

mechanism by which spoVA2mob affects these processes is not fully understood. Three out of seven 416

products of the spoVA2mob operon, namely SpoVAC2mob, SpoVAD2mob and SpoVAEb2mob, display 55%, 417

49%, and 59% amino acid sequence identity, respectively (27, 28), with the SpoVAC, SpoVAD and 418

SpoVAEb proteins encoded by the “regular” conserved heptacistronic spoVA (spoVAA-spoVAF) 419

operon (72, 73). The latter conserved SpoVA proteins are required for DPA uptake during sporulation 420

(36, 72) and DPA release during germination (26, 36, 37, 73). The products of spoVA2mob seem to 421

elevate spore heat resistance partly via an auxiliary role in DPA uptake as indicated by significantly 422

higher (~1.5-fold) DPA concentrations in spores of the B. subtilis 168 strain engineered to harbor the 423

Tn1546 transposon with the spoVA2mob operon (strain B4417) and of B. subtilis 168 amyE::spoVA2mob 424

than in the wild type 168 spores (27). However, this phenomenon does not correlate with improved 425

efflux of DPA upon germination. In fact, during the two-hour exposure to 10 mM AGFK, DPA was 426

released less efficiently from B4417 spores (31.6 ± 0.7% DPA released) when compared to 427

B4417ΔspoVA2mob (45.8 ± 3.1%) and 168 (67.1 ± 6.3%) spores, as monitored by fluorescence of 428

released DPA with terbium (Tb3+:DPA) (data not shown) measured in a fluorescence plate-reader, 429

similarly as described before (41). In contrast to the “regular” conserved spoVA operon, spoVA2mob 430

does not encode homologs of the SpoVAEa and SpoVAF proteins, which are reportedly important for 431

DPA release during GR-dependent spore germination, but not for DPA uptake during spore formation 432

(74). For this reason, the spoVA2mob products may not be able to support DPA transport during 433

germination. Instead, they might compete with the “regular” SpoVA channels or interfere with the 434

SpoVA proteins and/or GRs (37, 75), either directly or indirectly by altering the spore IM properties. 435

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Such interference could be caused not only by the three SpoVA homologs but also by the four 436

proteins of unknown function, as suggested by a somewhat improved spore germination after the 437

deletion of the last gene that encodes a putative membrane protein from B4417 strain (28). 438

Secondly, findings from this study indicate that various GR types in the high-level heat 439

resistant spores differ in their HA requirements and thermal stabilities. Regardless of the spoVA2mob 440

copy number, germination in L-alanine, which occurs via GerA (25), was optimally activated at lower 441

temperatures than germination in the AGFK mixture (Table 2, Table 3), which is mediated by a 442

cooperative action of GerB and GerK (25). Moreover, L-alanine germination was easily diminished by 443

too severe pre-heating (Table 2). Thus, consistently with a previous report on low heat resistant 444

spores of B. subtilis 168 (32), the GerA receptor of the high heat resistant spores also seems to 445

require the mildest HA treatment and appears the most thermolabile (Table 2). Differences in HA 446

requirements of individual GRs support the notion that HA directly affects the GR proteins (32). 447

However, the results from our current study make it plausible that HA acts in a dual manner: both 448

indirectly on the IM and directly on the GRs. Alternatively, HA could improve spore germination by 449

affecting the IM, but the exposure to (excessive) heat could simultaneously negatively influence GR 450

proteins. Therefore variation in the thermal stabilities of specific GRs could lead to differences in the 451

way HA affects individual nutrient-induced germination pathways. 452

Thirdly, our results suggest that in addition to spoVA2mob, the amino acid sequences of GR 453

proteins likely affect GR thermal stabilities and HA requirements. Spores of B4068 and B4073, which 454

germinate efficiently with L-alanine even after exposure to 95°C (Table 2), have several distinct 455

amino acid residues in the three GerA subunits (Table 4). Similarly, characteristic amino acids occur in 456

GerBB, GerBC and GerKA-KD (Table 4) of the food-borne strains that germinate moderately in AGFK 457

only after severe HA at 95 or 100°C (Table 3). Contradictory to the current work, our previous study 458

(28) has shown that HA at higher temperatures does not improve germination rates and efficiencies 459

of spores of the B. subtilis 168 strain with the spoVA2mob operon introduced on the Tn1546 460

transposon (B4417). This discrepancy could be explained by an assumption that GRs of B. subtilis 168 461

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are intrinsically less thermostable than GRs of high-level heat resistant spores; thus more severe HA 462

would simultaneously counteract the effect of spoVA2mob and reduce activity of B4417 GRs. This 463

explanation further supports the hypothesis that final spore HA requirements are concurrently 464

shaped by the spoVA2mob operon and by the (sequence-dependent) properties of the individual GRs. 465

Another observation from this work is that certain germination pathways, in particular 466

involving the GerA receptor and L-alanine, may be (at least temporarily) deactivated by pre-heating 467

at less severe conditions than are required for the inactivation of spores. High-level heat resistant 468

spores included in this study have decimal reduction times (D-values) at 100°C of 83.0±32.3 minutes 469

for B4146 spores and between 310±137 and 4224±2470 minutes for B4067-B4073 and B4145 spores 470

(Table 1) (4, 27) and their counts are reduced no more than 0.1 log (1.26-fold) after heating at 100°C 471

for 1 h (4). In contrast, germination responses in L-alanine of some high-level heat resistant spores 472

are substantially decreased after a 30-minute heat treatment at 80-95°C (Table 2). Most strikingly, L-473

alanine-induced germination of B4072 spores decreased ~6-fold (94.9±1.0% to 15.7±2.8%) after a 474

heat-treatment at 95°C when compared with heating at 80°C (Table 2). Although a certain low 475

degree of spore inactivation could potentially occur at the highest used activation treatments (95-476

100°C), the observed reduction in germination cannot be attributed solely to spore killing since the 477

D-value for B4072 spores at 100°C reaches 340±197 minutes (Table 1) (27). Taken together, the 478

observed decrease in spore germination in L-alanine after such heat treatments is more likely due to 479

specific inactivation of the GerA receptor complex than spore killing. Since the complete spore is not 480

inactivated, germination may still occur in response to other germination triggers. 481

The presence of multiple spoVA2mob copies in the genomes of eight B. subtilis strains (B4067-482

B4073 and B4145) correlates with modest (maximally 42.7±17.7%) spore germination in AGFK (Table 483

3) even after HA at 95-100°C. A causative relationship between these two factors is however unclear. 484

First of all, this was not observed for L-alanine-induced germination, which becomes efficient after 485

proper spore HA (Table 2) for spores of strains harboring spoVA2mob. Secondly, the same spores that 486

harbor 2 to 3 spoVA2mob copies (and germinate weakly in AGFK) share several changes in amino acid 487

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sequences of the GerBB, GerBC and GerKA-KD subunits that distinguish them from spores that 488

respond strongly to AGFK (and that contain 0 to 1 copies of spoVA2mob) (Table 4). Unfortunately, 489

despite multiple attempts we were unable to introduce the respective mutations in the relevant ger 490

genes in B. subtilis 168 in order to directly test their effect on spore germination and HA 491

requirements. Still, it cannot be excluded that some of these distinctive amino acid residues 492

contribute to the lower responsiveness of these spores to the AGFK mixture. Notably, the same 493

unique amino acids might simultaneously cause reduced functionality and higher thermostability of 494

these GRs, as a decreased conformational flexibility that is characteristic for thermostable proteins 495

has been suggested to compromise their activities (76, 77). Potentially stabilizing amino acid 496

changes, such as substitutions to L-alanine that tend to stabilize α-helices (78, 79) or to hydrophobic 497

amino acids that can increase a degree of protein packing (80), can be found in the GR subunits of 498

the investigated high heat resistant strains (Table 4). However, the prediction of mutations with 499

thermostabilizing effects is generally challenging (81, 82) and in case of GRs additionally hindered by 500

their membrane-associated localization and predominantly unknown protein structures and 501

mechanisms of action (26, 32). 502

In contrast to GerB and GerK, no common distinctive residues in the GerA subunits were 503

found for all five strains whose spores germinate barely (B4143, B4058, B4067 and B4145) or 504

moderately (B4146) in response to L-alanine (Table 4, Supp. data S2). A few identical amino acid 505

changes were found in GerAB and GerAC of closely related B4067 and B4145 strains (Figure 1) and 506

one common substitution (G135C) was present in GerAC of strains B4143 and B4146 (Table 4, Supp. 507

data S2). The three GerA subunits of strain B4058 contain multiple unique amino acid residues 508

distinguishing them from the GerA proteins of all other analyzed strains (Table 4, Supp. data S2). The 509

very poor or moderate responsiveness to L-alanine observed for B4143, B4067, B4145, B4058 and 510

B4146 spores may therefore be caused by various strain-specific changes in the GerA protein 511

sequences (Table 4, Supp. data S2). This constitutes an interesting subject for future investigation. 512

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In summary, we show that apart from inter-strain phenotypic variation in spore HR, 513

germination and HA requirements that correlates to spoVA2mob presence, the influence of other 514

intrinsic factors, in particular GR protein sequences, further complicates the prediction of spore 515

properties and behavior in response to heat and nutrients. These findings have major impacts on 516

practice in the food industry and challenge standardization of risk assessment and preservation 517

techniques. Firstly, in quality assessment, the recovery and enumeration of spores from food 518

products and food processing equipment involves specific heat treatment methods and commonly 519

applied plating techniques, which can result in an underestimation of viable spores due to poor, 520

heterogeneous and unpredictable spore germination properties. In addition to optimization of 521

germinants conditions (42), this study shows that the use of optimal HA treatments, especially for 522

high-level heat resistant spores, could help alleviate this problem. Furthermore, our findings suggest 523

that some of heat treatments that are used in food processing for spore inactivation can result in the 524

activation rather than inactivation of high-level heat resistant spores and thus increase unwanted 525

spore germination and outgrowth in food products, thereby decreasing food safety. Finally, as 526

processing conditions in food manufacturing likely select for spores with properties that are different 527

from those of the commonly-studied laboratory strains, our study underlines the importance of 528

extending scientific research from model laboratory organisms to industrially-relevant species and 529

strains. 530

531

Funding information 532

The authors have declared that no competing interests exist. The research was funded by TI Food 533

and Nutrition, a public-private partnership on pre-competitive research in food and nutrition. The 534

funding organization had no role in study design, data collection and analysis, decision to publish, or 535

preparation of the manuscript. 536

537

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Acknowledgements 538

The authors would like to thank Cyrus A. Mallon for proofreading the manuscript, Erwin M. 539

Berendsen for providing decimal reduction values of spores, described in (4, 27), and Gerwin 540

Kamstra for technical assistance. 541

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92. Caspers MPM, Schuren FHJ, van Zuijlen ACM, Brul S, Montijn RC, Abee T, Kort R. 2011. A 776 mixed-species microarray for identification of food spoilage bacilli. Food Microbiol 28:245–777 251. 778

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95. Oomes SJCM, Brul S. 2004. The effect of metal ions commonly present in food on gene 785 expression of sporulating Bacillus subtilis cells in relation to spore wet heat resistance. Innov 786 Food Sci Emerg Technol 5:307–316. 787

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Figure 1. The phylogenetic tree for the seventeen analyzed B. subtilis strains constructed on the 788

basis of sequences of the 400 most conserved microbial proteins using the PhyloPhlAn software (44). 789

790

Figure 2. Germination kinetics in L-alanine (A) and AGFK (B) of low (black and dark gray symbols) 791

and high (white and light gray symbols) heat resistant spores, subjected to strain- and nutrient-792

optimized heat-activation temperatures (shown in the legend, either one HA temperature for both 793

germinants or two different HA temperatures for L-alanine/AGFK). For clarity of the figure, absolute 794

deviations of the mean are only shown for the slopes of the most outer curves in each group. 795

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Ta le . Strai s used i this study. Spores of the se e tee B. subtilis strai s differ i their heat

resista e HR properties, hi h orrespo d to the op u er to of the spoVA o opero . D-

alues for spores of i di idual strai s at °C or e trapolated to °C for high-le el heat resista t

spores , hi h ha e ee assessed i the pre ious studies , , are e pressed as ea s a d a solute

de iatio s of the ea ased o data for t o i depe de tl prepared spore rops.

Strai HR spoVA o opies

Spore D-value at °C Strai des riptio Refere e

B lo . ± . La orator strai , deri ed fro ; alter ati e a es: JH , BGSC A ,

B lo . ± . La orator strai , alter ati e a es: PY , BGSC A ,

B lo . ± . La orator strai ; alter ati e a es: W , BGSC A , ,

B lo . ± . E iro e tal isolate Sahara Desert ; alter ati e a es: TU-B- T, BGSC A ,

B lo . ± . E iro e tal isolate; alter ati e a e: NCIB T, BGSC A T ,

B lo . ± . E iro e tal isolate Moja e Desert ; alter ati e a es: RO-NN- , BGSC A ,

B lo . ± . Food strai suri i

lo . ± . La orator strai ,

B high . ± . Food strai urr sau e ,

B high ± Food strai urr rea soup ; alter ati e a e: CC , ,

B high ± Food strai i di g flour i gredie t ; alter ati e a e: IIC , ,

B high ± Food strai urr rea soup ; alter ati e a e: CC , ,

B high ± Food strai red lasag a sau e ; alter ati e a e: RL , ,

B high ± Food strai urr soup ; alter ati e a e: MC , ,

B high ± Food strai pea ut hi ke soup ; alter ati e a e: A ,

B high ± Food strai pea ut hi ke soup ; alter ati e a e: A

, , , –

B high ± Food strai pasta ,

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Ta le . Ratios i per e tages of ger i ated phase-dark spores after e posure to L-ala i e, as

assessed ith phase- o trast i ros op . Mea s a d a solute de iatio s of the ea ere al ulated

fro at least t o e peri e ts perfor ed o t o i depe de tl prepared spore rops of ea h strai .

Spores ere or ere ot heat-a ti ated at differe t te peratures. Values i ra kets i di ate the results

for the egati e o trol to orre t for pote tial spore phase tra sitio i du ed se ere heat

treat e ts.

Heat-a ti atio te perature*

Strai HR

spoVA o opies

H °C °C °C °C °C

B JH lo . ± . . ± . NT NT NT NT

B PY lo . ± . . ± . NT NT NT NT

B W lo . ± . . ± . NT NT NT NT

B TU-B- lo . ± . . ± . . ± . . ± .

. ± . NT NT

B NCIB lo . ± . . ± . NT NT NT NT

B RO-NN- lo . ± . . ± . NT NT NT NT

B lo . ± . . ± .

NA . ± . NT NT NT NT

lo . ± . . ± . . ± . NA . ± . NT NT

B high . ± . . ± . . ± . . ± . . ± . . ± .

. ± .

. ± .

B high . ± . . ± . . ± . . ± . . ± . . ± .

. ± . . ± .

B high . ± . . ± . . ± . . ± . . ± . . ± .

. ± . . ± .

B high . ± . . ± . . ± . . ± . . ± . . ± .

. ± .

. ± .

B high . ± . . ± . . ± . . ± . . ± . . ± .

. ± . . ± .

B high . ± . . ± . . ± . . ± . . ± . . ± .

. ± . . ± .

B high . ± . . ± . . ± . . ± . . ± . . ± .

. ± . . ± .

B high . ± . . ± . . ± . . ± . . ± . . ± .

. ± . . ± .

B high . ± . . ± . . ± . . ± . . ± . . ± .

. ± . . ± .

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* Prior to i u atio ith ger i a ts at °C , spores ere either heat-a ti ated for i utes at te peratures

et ee °C a d °C or the heat-a ti atio treat e t as o itted H= o -heated .

A re iatio s: HR – heat resista e; NA – ot appli a le: the spe ifi heat-a ti atio HA treat e t alo e aused

the phase tra sitio phase- right to phase-dark , likel due to spore da age, of a sig ifi a t fra tio % i

ra kets of spores i the a se e of ger i atio triggers, there hi deri g a a urate a al sis of spore

ger i atio ; H – o -heated; NT – ot tested: the spe ifi HA treat e t as ot tested either due to earl

o plete ger i atio of spores a hie ed after HA at a other te perature, lo pro a ilit of i pro e e t of

spore ger i atio after the i di ated HA treat e t a d/or e pe ted spore killi g.

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Ta le . Ratios i per e tages of ger i ated phase-dark spores after e posure to AGFK, as assessed

ith phase- o trast i ros op . Mea s a d a solute de iatio s of the ea ere al ulated fro at

least t o e peri e ts perfor ed o t o i depe de tl prepared spore rops of ea h strai . Spores

ere or ere ot heat-a ti ated at differe t te peratures. Values i ra kets i di ate the results for the

egati e o trol to orre t for pote tial spore phase tra sitio i du ed se ere heat treat e ts.

Heat-a ti atio te perature*

Strai HR

spoVA o opies

H °C °C °C °C °C

B JH lo ± . . ± . NT NT NT NT

B PY lo . ± . ± . NT NT NT NT

B W lo . ± . . ± . NT NT NT NT

B TU-B- lo . ± . ± . NT NT NT NT

B NCIB lo ± . ± . NT NT NT NT

B RO-NN- lo ± . ± . NT NT NT NT

B lo . ± . . ± .

NA . ± . NT NT NT NT

lo . ± . ± . . ± . NA . ± . NT NT

B high NT . ± . . ± . . ± . . ± . . ± .

. ± . . ± .

B high NT . ± . . ± . . ± . . ± . . ± .

. ± . . ± .

B high NT . ± . . ± . . ± . . ± . . ± .

. ± . . ± .

B high NT . ± . . ± . . ± . . ± . . ± .

. ± .

. ± .

B high NT . ± . . ± . . ± . . ± . . ± .

. ± . . ± .

B high NT . ± . . ± . . ± . . ± . . ± .

. ± . . ± .

B high NT . ± . . ± . . ± . . ± . . ± .

. ± . . ± .

B high NT . ± . . ± . . ± . . ± . . ± .

. ± . . ± .

B high NT . ± . . ± . . ± . . ± . . ± .

. ± . . ± .

* Prior to i u atio ith ger i a ts at °C , spores ere either heat-a ti ated for i utes at te peratures

et ee °C a d °C or the heat-a ti atio treat e t as o itted H= o -heated .

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A re iatio s: HR – heat resista e; NA – ot appli a le: the spe ifi heat-a ti atio HA treat e t alo e aused

the phase tra sitio phase- right to phase-dark , likel due to spore da age, of a sig ifi a t fra tio % i

ra kets of spores i the a se e of ger i atio triggers, there hi deri g a a urate a al sis of spore

ger i atio ; H – o -heated; NT – ot tested: the spe ifi HA treat e t as ot tested either due to earl

o plete ger i atio of spores a hie ed after HA at a other te perature, lo pro a ilit of i pro e e t of

spore ger i atio after the i di ated HA treat e t a d/or e pe ted spore killi g.

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Ta le 4. Va iatio s i a i o a id se ue es i ge i a t e epto su u its that oi ide ith spe ifi

ge i atio a d heat-a ti atio phe otypes of the i estigated B. subtilis st ai s. The a i o a id

esidue positio s a d p otei egio s a e as i ed a o di g to p otei se ue es a d st u tu e

p edi tio s fo the espe ti e su u its of B. su tilis . “e ue e a iatio s p ese t i Ge KB o Ge KC

su u its of st ai s B -B a d B that a e also p ese t i B a e u de li ed.

Phe otype St ai P otei Se ue e a iatio P otei egio s

No/poo espo se to

L-Ala

B TU-B-

Ge AA T/Q A T- , Q- ; T K; D/A E D- ; A-; E K

glo ula N- pos - a d C-te i i pos -

Ge AB F/V I F- ; V- ; I L; I V; V I; T ; I V

TM ; TM ; TM ; TM ; TM ; L /

Ge AC K T DIII β B Ge AC G C also i Ge AC-B ; K N DII, DIII α

B , B

Ge AB T “; M I TM ; TM Ge AC A/“ Δ A- ; “- ; K Δ; “ N; T I DI; DI; DII α ; DIII α

°C HA & st o g

espo se to L-Ala

B , B

Ge AA G “; “ L; F Y; L I* TM ; TM ; TM ; TM Ge AB A/T “ A- ; T- L /

Ge AC “/A F “- ; A- ; “ L** DI β ; DII li ke

Poo espo se to

AGFK High HA te p.

B - B , B , B

Ge BB “ N; K T L / Ge BC E D DIII

Ge KA V A; Q E; I “; V F; I L; F Y; K/D Q K- , D- ; Q K

glo ula N-te i i; L / ; L / ; L / ; TM ; glo ula C-te i i pos -

Ge KB A V; V I; I L TM ; L / ; TM

Ge KC A/T V A- ; T- ; V A; A D; M I; T “; Y F; F L; N D

“ig al se ue e pos - ; DI; DI α ; DII β ; DII β ; DIII β ; DIII β

Ge KD M I TM “t o g

espo se to AGFK

despite high HR

B

Ge BC N K DIII α Ge KA V G; L M; P Q TM ; L / ; TM Ge KB D E OL Ge KC “ Y; T K; R W DI β ; DII; DII

E pla atio s: α – alpha heli ; β – eta st a d; Δ – deletio ; DI, DII, DIII – do ai I, II a d III, espe ti el ; L / – loop et ee t a s e a e heli es u e a d ; TM – t a s e a e heli ; T/Q A T- , Q- - T p ese t

i st ai s a d Q p ese t i st ai s at positio su stituted A i the st ai s that sho espe ti e phe ot pe et . * L F utatio i Ge AA auses st o g ge i atio defe t i L-ala i e . **“ esidue e hi its % o se atio a o g the se e Ge BC ho ologs f o B. su tilis, Ba illus a yloli uefa ie s, Ba illus pu ilis, Ba illus lausii a d Ba illus e eus ; Δ - i Ge BC auses loss of espo se to AGFK .

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AEM Accepted Manuscript Posted Online 27 January 2017 Appl ... · ñ óõ vµu }( P u]vv } v Z vµ...

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