Differential gene expression for investigation of the effect of germinants and heat activation to induce germination in Bacillus cereus spores

Food Research International 119 (2019) 462–468

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Differential gene expression for investigation of the effect of germinants and heat activation to induce germination in Bacillus cereus spores Aswathi Sonia, Indrawati Oeya,b, Patrick Silcocka, Elizabeth Perminac, Phil J. Bremera,d,

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a

Department of Food Science, University of Otago, PO Box 56, Dunedin 9054, New Zealand Riddet Institute, Palmerston North, New Zealand c Otago Genomics & Bioinformatics Facility, University of Otago, New Zealand d New Zealand Food Safety Science Research Centre, New Zealand b

A R T I C LE I N FO

A B S T R A C T

Keywords: Bacillus spores Heat activation L-alanine Ger-A, ABC transporters

Differential gene expression was used to explore the mechanisms underpinning the differences in the impact of heat activation (70 °C for 30 min) on the germination of Bacillus cereus spores in the presence and absence of a germinant (L-alanine). The number of germinated cells, after heat activation plus L-alanine (3.5 ± 0.02 log CFU/ml) in the spore only initial population was found to be higher than that in only heat activated spores (2.01 ± 0.02 log CFU/ml). The concentration of DPA released by heat activated spores in the presence of Lalanine was 68.3 ± 0.1 and 112.1 ± 0.02 μg/ml after 30 and 60 min, compared to 96.5 and 166.2 ± 0.01 μg/ ml after 30 and 90 min, respectively released by spores subjected only to heat activation. Gene (BC0784) encoding for the spore germination protein, gerA operon was up-regulated with a log2-transformed fold change value of 1.2 due to heat activation in the presence of L-alanine. The GerA operon located in the inner membrane is known to be involved in the uptake of L-alanine by B. cereus and has been reported to be involved in L-alanine mediated germination. In addition the up-regulation of genes involved in the uptake of L-alanine is proposed to provide the answer to the synergistic effect of heat and L-alanine in inducing germination in B. cereus spores. In short, heat activation increases the ability of L-alanine to penetrate into the spore's inner membrane, where it can be recognized by the receptors for initiation of the germination pathway. In the current study, the majority of the ribosomal proteins were down-regulated (when spores were heat treated in presence of germinants) this process also appeared to slow down protein synthesis by restricting the protein translation machinery. Differential gene expression revealed the genes responsible for the pathways related to transport and recognition of L-alanine into the spore that could have led to the accelerated germination process along with partial shutting down of protein synthesis pathway and ABC transporters. Knowledge of gene regulation in spores during heat activation will help in the development of approaches to prevent spore germination, which could provide an additional safeguard against bacterial growth and toxin production in improperly cooled heat treated foods.

1. Introduction Bacillus cereus spores pose a challenge to the food industry as they are resistant to heat treatments (except retorting) and have been detected in a wide range of food products including cooked rice, lowmoisture food like spices and milk powders as well as pasteurized chilled food products (Choma et al., 2000; Dierick et al., 2005; Dong, 2013; Luby, Jones, Dowda, Kramer, & Horan, 1993; Naranjo et al., 2011; Svensson et al., 2006). Under favourable conditions, B. cereus spores can germinate into vegetative cells, which are able to produce two types of toxins. An emetic toxin is produced in the food where more than approximately 103 cells are present and diarrheal toxins (Nhe, Hbl

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and CytK) are produced in the human intestine on ingestion of > 103 cells (Agata, Ohta, & Yokoyama, 2002; Granum & Lund, 1997; Turnbull, Kramer, Jørgensen, Gilbert, & Melling, 1979). The germination of spores into outgrown vegetative cells is of considerable interest to food microbiologists as it is not only a checkpoint to prevent toxin production but also a method to reduce the dormancy and resistance of the spores. The factors leading to spore germination vary widely but specific components, called germinants, including L-alanine, glucose, or inosine can bind to GerA and GerB receptors in the inner membrane of the spore coat and induce germination (Collado, Fernandez, Rodrigo, & Martinez, 2006; Setlow, 2003). Binding of the germinants to Ger proteins (Ger L, Ger N, and Ger T) in B. cereus initiates a cascade of

Corresponding author at: Department of Food Science, University of Otago, Dunedin 9054, New Zealand. E-mail address: [email protected] (P.J. Bremer).

https://doi.org/10.1016/j.foodres.2018.12.041 Received 11 August 2018; Received in revised form 10 December 2018; Accepted 22 December 2018 Available online 24 December 2018 0963-9969/ © 2018 Elsevier Ltd. All rights reserved.

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2. Materials and methods

reactions leading to the expression of proteins required to transform the dormant spores to viable vegetative cells (Senior & Moir, 2008). Though it is expected that the spores recognize germinants in the medium and germinate, the process is not very consistent among different strains of the same group of spores (Behravan, Chirakkal, Masson, & Moir, 2000; Setlow, 2003). The effect of germinants is very specific to species or strain, for example, L-alanine is recognized by B. subtilis 168 AJ12866 and B. cereus with GerA receptors (Paidhungat, Setlow, Driks, & Setlow, 2000; Setlow, 2003). B. subtilis spores have been reported to respond to L-alanine and a combination of asparagine, glucose, fructose, and potassium ions (AGFK) via the receptor GerK (Zhang, Griffiths, Cowan, Setlow, & Yu, 2013) whereas, B. cereus responds to potassium ions or L-alanine using GerA receptors (Barlass, Houston, Clements, & Moir, 2002). B. megaterium has been reported to respond to glucose via the receptors Ger UA, UC and WB, however this has not been reported for B. cereus (Christie, Götzke, & Lowe, 2010). These differences in receptors suggest that the germinants might also trigger a different set of pathways depending on the strain and the extent of germination induced. Once the receptors recognize the germinants, the subsequent steps involve (but are not limited to) ion exchange, followed by the intake of water by the spore, and the activation of lytic enzymes (Dion & Mandelstam, 1980). Since food is a complex medium, the presence or deliberate addition of some compounds can also inhibit the germination of spores. Sorbic acid has been reported to completely block B. cereus spore germination in nutrient rich brain heart infusion (BHI) broth (van Melis, Almeida, Kort, Groot, & Abee, 2012). For some bacterial strains, heat activation can induce germination or accelerate the effect of germinants on germination (Fernández, Ocio, Fernández, & Martı́nez, 2001; Luu et al., 2015). Heat activation of dormant bacterial spores can be defined as a short treatment at a sublethal temperature that potentiates and synchronizes spore germination (Ghosh, Zhang, Li, & Setlow, 2009). The time and temperatures used for heat activation vary between 15 and 30 min at 70–80 °C for different strains, with a single method to cover all species or strains not being defined. For example, B. cereus T has been reported to be activated at 65 °C for 30 min (Ghosh & Setlow, 2009), B. cereus INRA AVZ421 spores at 80 °C for 10 min (Fernández et al., 2001), B. subtilis PS832 spores at 70 °C for 30 min (Ghosh et al., 2009), and B. subtilis CMCC 604 spores at 65 °C for 10 min (Leuschner & Lillford, 1999). The mechanism of heat activation has been explained using the glass transition theory by Sapru and Labuza (1993), wherein the dormant spore protoplasm is compared to a glassy state configuration of vital macromolecules and supramolecular assemblies in the spore protoplast that change extremely slowly when heated. The germinant receptors (GR) in B. subtilis spores are believed to be located together in a small cluster in the inner membrane (IM) termed the germinosome (Griffiths, Zhang, Cowan, Yu, & Setlow, 2011). Heat activation has been postulated to affect germination either directly by altering the GR structure and affinity or indirectly by affecting the IM; however, the exact mechanisms involved are unclear. It has previously been reported that L-alanine was not able to induce significant spore germination (Soni, Oey, Silcock, & Bremer, 2018) unless it was used in conjunction with heat activation. In the current study, the mechanism by which heat activation in the presence of germinant (L-alanine) increases germination by B. cereus spores was investigated using microbial plate counts, the estimation of DPA and by monitoring differential gene expression. Understanding the role that germinants play along with heat activation is of importance for two reasons. Firstly, as food microbiologists commonly use heat activation to synchronize germination in Bacillus spores, a better understanding of the mechanism in the presence of germinants in the medium will help to standardize the procedure. Secondly, a better understanding of the underlying mechanisms associated with heat activated germination will help in the development of approaches to prevent spore germination, which could provide an additional safeguard against bacterial growth and toxin production in improperly cooled heat-treated foods.

2.1. Preparation of spore suspension A stock culture of B. cereus NZAS01 spores was obtained from the Department of Food Science, University of Otago culture collection. To prepare the spore suspensions, an inoculum from the stock culture at −80 °C was streaked on to a plate count agar (PCA) plate, which was incubated at 30 °C for 48 h. A single colony was subsequently inoculated into a 100 ml of tryptic soy broth (TSB) in a conical flask (250 ml) with a cotton plug and incubated at 25 °C for 72 h in an orbital shaker incubator at 75 rpm. The resulting culture was held at 80 °C for 15 min, then transferred to ice slurry for 10 min, followed by centrifugation (15,300 g for 8 min at 4 °C) prior to purification by washing three times in autoclaved distilled water. The crude spore suspension in distilled water and stored at −18 °C until used. 2.2. Experimental set up The effect of the presence of L-alanine (0.9 mg/ml) on heat activation in inducing germination was monitored by comparing spores that were heat activated in the presence or absence of L-alanine. The process of heat activation was carried out by incubating B. cereus spores (108 CFU /ml) in phosphate buffer (pH 7.2, 50 mM) in a water bath at 70 °C for 30 min, followed by their immediate transfer to an ice slurry for 15 min. To assess the effect of germinant in combination with heat, L-alanine (0.9 mg /ml) was added to B. cereus spores (108 CFU /ml) in phosphate buffer (pH 7.2, 50 mM) along with immediate heat activation (70 °C for 30 min). Once cooled, the suspension was held at 30 °C for 90 min and total microbial number (TMN) and spore number (SN) were estimated and DPA was quantified after every 30 min as described in Section 2.3. 2.3. Monitoring germination Germination was monitored by estimating the number of vegetative cells present in the initial spore only population during the set time points. The total microbial number (TMN) in the suspension was determined by plating the solution onto PCA plates (in triplicates). The spore number (SN) in the suspension was determined by heating the suspension at 80 °C for 15 min and immediately transferring it to ice slurry before diluting and plating onto PCA as previously reported (Soni et al., 2018). The release of DPA during the germination of B. cereus spores into the suspension/media was estimated using the method described by Scott and Ellar (1978). The supernatant from samples (1 ml) at all time points was collected by centrifugation (15,300 g for 5 min at 4 °C). The absorption maxima of the supernatant were found to be 270 nm using wave scan (200 nm–800 nm range). The absorption maxima (270 nm) were in agreement to that of Ca-DPA as well as DPA secreted by B. cereus during germination (Bailey, Karp, & Sacks, 1965; Scott & Ellar, 1978). A standard curve of DPA (Sigma Aldrich) was used to determine the unknown concentrations. 2.4. RNA extraction and analysis To assess the effect of heat activation and germinants on the gene expression, RNA was extracted immediately post-treatment, from the spores held in an ice slurry. RNA was extracted as per the manufacturer's instructions using the Ribopure RNA extraction kit that includes glass beads for rupturing the spores for better extraction (Thermofisher Scientific). The purity of the RNA isolated was measured spectrophotometrically (NanoDrop, Wilmington, DE) by monitoring the ratio of absorbance at 260 nm and 280 nm using the undiluted RNA preparation and RNase-free H2O as a blank. The 260:280 ratio of 1.6–2.0 indicated good quality of RNA (Moeller et al., 2006). To measure the 463

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30 min was 96.5 μg/mL, which increased to 166.2 ± 0.01 μg/ml by 90 min. For heat activation combined with L-alanine, the DPA concentration in the suspension was 68.3 ± 0.1 μg/ml within 30 min, which increased to 112.1 ± 0.02 μg/ml after 60 mins (Fig. 1). Heat activation of spores at sublethal temperatures (below the inactivation threshold) for a short period of time results in an endothermic transition that is postulated to be related to the breakage of hydrogen and disulphide bonds leading to partial denaturation of proteins, resulting in significant changes in the spore's inner and outer membranes (Zhang, Setlow, & Li, 2009). As a result of these changes, it may be easier for the germinants to penetrate the outer membrane of the spores and interact with specific receptor proteins located on the inner membrane, which in turn leads to series of events including the release of monovalent cations and DPA and spore hydration, which is followed by a loss of heat resistance. This sequence of events has previously been reported to be the first stage towards the germination of spores into vegetative cells (Moir, 2006). The results from the current study are in agreement with the earlier findings as germination was found to be enhanced if heat activation was combined with L-alanine (Fig. 1). It has previously been reported that L-alanine alone was not able to induce significant spore germination (Soni et al., 2018) unless it was used in conjunction with heat activation. To explore the mechanism behind the combined effect of heat activation and exposure to L-alanine, the B. cereus spores subjected to either heat activation alone or heat activation combined with L-alanine were analysed for differential gene expression.

intactness of the RNA extracted, an Agilent 2100 bioanalyzer was used to obtain the RNA Integrity Number (RIN). RIN is an algorithm used by the Agilent 2100 Bioanalyzer to assess the degradation of RNA using the RNA 6000 Nano lab chip kit. To achieve the desired threshold of RIN 7, zymoclean columns (Zymoclean™ Gel RNA Recovery Kit) were used to further purify the RNA samples following the manufacturer's instructions. Initially, MiSeq followed by HiSeq RNA sequencing was used for the genetic sequencing of the libraries. Reads were mapped to the B. cereus NCBI reference sequence (NC_004722.1) using Burrows-Wheeler Alignment (BWA) software and the reads were checked for quality using a FastQC software package (Li & Durbin, 2009). Read counts from six samples (triplicates of test and control) were obtained using the BED tools package (Quinlan & Hall, 2010), multicov functionality and an annotation file for B. cereus ATCC 14579 (NC_004722.1), obtained from Ensembl Bacteria (http://bacteria.ensembl.org/info/website/ftp/index. html). Raw read counts were processed using R package DESeq2 (Love, Huber, & Anders, 2014) to estimate the most significant differentially expressed genes. The analysis was done twice: once to determine a full list of transcripts present, and once to determine the top 1000 transcripts with the highest log2-transformed fold change value between the cold-stored and control samples. The functional annotation tool and pathway analysis using DAVID Bioinformatics Resources 6.8, NIAID/ NIH (https://david.ncifcrf.gov/summary.jsp) were used for enrichment analysis.

3. Results and discussion 3.1. Differential gene expression in heat activation vs heat activation and Lalanine

To understand the impact of heat activation on germination in the presence of L-alanine, B. cereus spores were subjected to heat activation in the presence or absence of added L-alanine (0.9 mg/ml) in phosphate buffer and germination was monitored every 30 mins. Germination as evaluated using the number of vegetative cells present in the initial spore only population at the end of 90 mins was higher (3.5 ± 0.02 log CFU/ml) for heat activation combined with L-alanine compared to heat activation alone (2.01 ± 0.02 log CFU/ml) (Fig. 1). After heat activation alone, the concentration of DPA in the spore suspension after

A total of 145 genes were found to be differentially expressed (sorted by a log2-transformed fold change value) as a result of heat activation in the presence of L-alanine (test) compared to heat activation alone (control). Genes that showed an FDR value ≤0.05 were subsequently selected for further analysis. The most consistently differentially expressed genes (p < 0.01) were classified into different functional categories (Fig. 2). Out of the 145 differentially expressed genes, 75 were up-regulated and 70 were down-regulated (Figs. 3 and 4). On the basis of molecular function and pathways involved, the genes that were up-regulated in response to added germinant (L-alanine) and heat activation were further classified into 7 pathways (Table 1). These pathways suggest that there was some general onset of metabolic regulation like amino acid biosynthesis and glucose breakdown, processes which are indicative of germinating spores. Most of the genes up-regulated (Table 1) were associated with the onset of metabolic activities as a result of germination. For example, the gene BC1569 coding for xanthine phosphoribosyl transferase (xpt) is involved in the uptake of xanthine in the salvage pathway, which is itself part of purine metabolism (Christiansen, Schou, Nygaard, &

Fig. 1. TMN (solid lines) and SN (dotted lines) in response to heat activation of B. cereus spores in the absence (a) or presence (b) of L-alanine added in the phosphate buffer (pH 7.4, 50 mM). DPA release (black bars) in response to heat activation of B. cereus spores in the absence (a) or presence (b) of L-alanine added in the phosphate buffer (pH 7.4, 50 mM). Data is shown as means ± S.D. (n = 9) and letters (a–c) represent values that differ significantly (p < 0.05) in each series.

Fig. 2. Functional classification of differentially expressed genes based on molecular function in response heat activation (70 °C, 30 min) alone as compared to heat activation with added germinant (L-alanine) in phosphate buffer (pH 7.4, 50 mM), where black solid bars represent the up-regulated and grey solid bars represent the down-regulated genes. 464

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Fig. 3. Volcano plot representation of the differentially expressed genes in a pair-wise comparison of heat-treated spores with and without L-alanine. The genes within the circle are up-regulated and those in the square are down-regulated.

in permeability of the spore membranes (inner and outer) in response to the heat activation and the presence of L-alanine in the suspension. Gene (BC0784) encoding for the spore germination protein, gerA operon was up-regulated with a log2-transformed fold change value of 1.2 due to heat activation in the presence of L-alanine. The gerA family, like most of its homologues, encodes the gerA, gerB and gerK operons in B. subtilis, and the gerI, gerQ and gerL operons in B. cereus and has been reported to be involved in L-alanine mediated germination (Barlass et al., 2002). The up-regulation of genes involved in either the uptake of L-alanine or the onset of germination provides the answer to the synergistic effect of heat and alanine in inducing germination in B. cereus spores. In short, heat activation is postulated to increase the ability of Lalanine to penetrate to the spore's inner membrane, where it can be recognized by the receptors for initiation of the germination pathway. This also explains why L-alanine was earlier reported to be ineffective in inducing germination in B. cereus spores (same strain) in the absence of heat activation (Soni et al., 2018).

Saxild, 1997). Apart from the genes involved in the regular metabolism of growing cells, the gene BC_5051 coding for a sodium/proton-dependent alanine carrier protein was up-regulated with a log2-transformed fold change value of 1.2 when heat activation was combined with L-alanine. This protein is a cellular component reported to be involved in the transport of L-alanine into the Bacillus stearothermophilus spores (Heyne, de Vrij, Crielaard, & Konings, 1991). The mechanism has not been reported for B. cereus; however, the current study indicates a strong possibility of this being a mechanism which enhances the availability of L-alanine to the receptor. In the current study, gene (BC_0211) encoding for oligopeptide-binding protein (oppA) was found to be up-regulated after heat activation when L-alanine was added with a log2-transformed fold change value of 1.8. Though not reported for B. cereus, the opuA system is encoded by an operon (opuA) comprising three structural genes: opuAA, opuAB, and opuAC in B. subtilis and the products of these are involved in protein-dependent transport systems, which show homology to glycine betaine uptake (Kempf & Bremer, 1995). Upregulation of opu A has been involved in the transport of glycine betaine and is a functional osmoprotectant for the spores; however, this has not been reported to be involved in the uptake of Lalanine. The gene (BC_5286) coding for a transcriptional regulator with an ABC transporter ATP-binding domain and the lytTR DNA-binding domain was up-regulated with a log2-transformed fold change value of 1.8. Though, the lytTR domain is found in several bacterial cytoplasmic proteins that regulate the production of important virulence factors, like extracellular polysaccharides, toxins and bacteriocin it has also been involved in regulation of cell autolysis in B. subtilis (Nikolskaya & Galperin, 2002). Though BC_5286 has not been reported in L-alanine mediated germination, the up-regulation due to heat activation indicates a possibility of its involvement in the onset of germination via Lalanine. The up-regulation of gene (BC_1955) encoding for the multidrug resistance ABC transporter ATP-binding and permease protein with a log2-transformed fold change value of 1.2 indicated an increase

3.2. Down-regulation of genes Out of the 70 genes which were found to be down-regulated, some of these belonged to specific pathways (Fig. 2). The down-regulation of these genes could be a result of the negative stringent response by germinating cells. Although the spores are germinating and outgrowing, the demand for energy and metabolic intermediates increases in the germinating cells and there can be a negative stringent response as a result of starvation if nutrients have not been added to the medium. This explains the down-regulation of genes belonging to the pathways of ATP synthesis, amino acid metabolism, purine biosynthesis and Krebs cycle (Table 2). Similar results have been earlier reported for B. subtilis cells undergoing glucose starvation, where the genes encoding for enzymes in the synthesis of amino acids, cofactors, or nucleotides were downregulated (Maaβ et al., 2014). It has been reported that the 465

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Fig. 4. Expression profiles of B. cereus spores when heat-treated without (C 1, 2, 3) or with germinant (L-alanine) (T1, 2, 3) in phosphate buffer (pH 7.4, 50 mM). Table 1 Classification of up-regulated B. cereus genes (up-regulated) on heat activation with L-alanine as compared to that without L-alanine. Gene name and gene id

Pathway

log2Fold Change

FDR value

Xanthine phosphoribosyl transferase(xpt) (BC1569) 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (gpmA) (BC2435) Cystathionine beta-lyase (BC_4254) N-acetylglucosamine-6-phosphate deacetylase (BC_4055) 3-methyl-2-oxobutanoate hydroxyl methyl transferase (pan B) (BC1540) Aspartate 1-decarboxylase (pan D) (BC1542) Aragonite dehydrogenase (BC_2939)

Adenine – hypoxanthine salvage pathway Glycolysis Methionine biosynthesis N-acetylglucoseamine metabolism Pantothenate biosynthesis Pantothenate biosynthesis Tyrosine biosynthesis

1.034 0.595 0.965 0.494 0.582 0.672 0.537

0.002 0.005 0.000 0.027 0.001 0.007 0.000

Note: FDR figures were obtained on the subsampled data (top 1000 top differentially expressed, sorted by log2transformed fold change). Table 2 Classification of down-regulated B. cereus genes on heat activation with L-alanine as compared to that without L-alanine. Gene name and gene id

Pathway

log2-transformed fold change value

FDR

ATP synthase subunit alpha (atpA) (BC5308) 4-aminobutyrate aminotransferase (BC_0355) Carbamoyl-phosphate synthase large chain (carB) (BC3886) Gluconokinase (BC_3369) Adenylate kinase (adk) (BC0152) 2-oxoglutarate dehydrogenase E1 component(odhA) (BC1252) Threonine synthase (BC1965)

ATP synthesis Amino butyrate degradation Arginine biosynthesis Ascorbate degradation De novo purine biosynthesis Krebs cycle Threonine biosynthesis and Vitamin B6 metabolism

−0.238 −1.606 −0.473 −0.868 −0.630 −0.442 −0.827

0.01 0.00 0.04 0.03 0.04 0.02 0.00

Note: FDR figures were obtained on the subsampled data (top 1000 top differentially expressed, sorted by log-transformed fold change).

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Table 3 Down-regulation of ribosomal protein genes in B. cereus spores in response when heat activation alone was compared with heat activation with L-alanine. Protein encoded and gene name

log2-transformed fold change value

FDR value

50S 50S 30S 30S 50S 50S 50S 30S 50S 30S

−0.56751 −0.33055 −0.28067 −0.34926 −0.38772 −0.43735 −0.26735 −0.42413 −0.45681 −0.34032

0.010 0.022 0.040 0.003 2.84E-17 0.003 0.042 0.001 0.002 0.036

ribosomal ribosomal ribosomal ribosomal ribosomal ribosomal ribosomal ribosomal ribosomal ribosomal

protein protein protein protein protein protein protein protein protein protein

L14 (rplN) (BC0141) L2 (rplB) (BC0134) S11(rpsK) (BC0157) S5 (rpsE) (BC0148) L5 (rplE)(BC0143) L16 (rplP) (BC0138) L15 (rplO) (BC0150) S19 (rpsS) (BC0135) L6 (rplF) (BC0146) S3 (rpsC) (BC0137)

Note: FDR figures were obtained on the subsampled data (top 1000 top differentially expressed, sorted by log-transformed fold change).

safeguard against bacterial growth and toxin production in improperly cooled, heattreated food.

lipid composition of various GRs differ and can influence their response to heat activation using the proteins embedded in the inner membrane which are attributed to the permeability of germinants (Luu et al., 2015). Interestingly, the gene (BC_1203) encoding for the protein FtsW which is involved in the transport of the peptidoglycan precursor lipid II and is also essential for cell division (Gamba, Hamoen, & Daniel, 2016; Mohammadi et al., 2011) was found to be down-regulated by a log2-transformed fold change value of −1.1. The reason behind this remains unclear because the process of germination ultimately proceeds to cell division (Stringer, Webb, George, Pin, & Peck, 2005). Once the spores are heat activated, a dormant spore undergoes rehydration and loss of heat and chemical resistance. This may or may not lead to the initiation of metabolism and macromolecular synthesis before the new vegetative cells start to grow. Once germinated, cell division seems likely to take place, but the down-regulation of ftsW seems to be contradicting this theory. Recent studies on B. subtilis spores suggest that the degradation of the majority of the 23S and 16S rRNAs occurs at 75 and 80 °C suggesting that protein synthesis is not essential for Bacillus spore germination (Korza, Setlow, Rao, Li, & Setlow, 2016). In the current study, the majority of the ribosomal proteins were down-regulated (when spores were heat treated in presence of germinants) indicating that nutrient-induced germination, when combined with heat activation, also shuts down protein synthesis by restricting the protein translation machinery (Table 3). Active 30S and 50S ribosomal subunits combine during protein synthesis to form a complete 70S intact ribosome, which is necessary for protein synthesis and have the specific functions characteristic of each ribosomal subunit; formyl-[3H] methionyl tRNA binding by 30S and peptidyl transferase activity by 50S (Guha & Szulmajster, 1975). Differential gene expression revealed genes responsible for the pathways related to the recognition and transport of L-alanine into the spore, which, along with shutting down of some protein synthesis pathway and ABC transporters, are likely to be responsible for the accelerated germination process However, in order to apply these findings to real food systems, studies using complex food models are required.

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