Food Microbiology 55 (2016) 73e85

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Food Microbiology

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Transcriptome analysis of thuringiensis spore life, germination and cell outgrowth in a vegetable-based food model

Daniela Bassi a, Francesca Colla a, 1, Simona Gazzola a, Edoardo Puglisi a, * Massimo Delledonne b, Pier Sandro Cocconcelli a, a Istituto di Microbiologia e Centro Ricerche Biotecnologiche, Universita Cattolica del Sacro Cuore, via Emilia Parmense 84, 29122 Piacenza, Italy e via Milano 24, 26100 Cremona, Italy b Dipartimento di Biotecnologie, Universita degli Studi di Verona, Strada le Grazie 15, 37134 Verona, Italy article info abstract

Article history: Toxigenic species belonging to sensu lato, including , cause foodborne Received 23 April 2015 outbreaks thanks to their capacity to survive as spores and to grow in food matrixes. The goal of this Received in revised form work was to assess by means of a genome-wide transcriptional assay, in the food isolate B. thuringiensis 3 November 2015 UC10070, the gene expression behind the process of spore germination and consequent outgrowth in a Accepted 10 November 2015 vegetable-based food model. Scanning electron microscopy and Energy Dispersive X-ray microanalysis Available online 12 November 2015 were applied to select the key steps of B. thuringiensis UC10070 cell cycle to be analyzed with DNA- microarrays. At only 40 min from heat activation, germination started rapidly and in less than two Keywords: Bacillus cereus sensu lato hours spores transformed in active growing cells. A total of 1646 genes were found to be differentially Food model expressed and modulated during the entire B. cereus life cycle in the food model, with most of the Microarray significant genes belonging to transport, transcriptional regulation and protein synthesis, cell wall and Spores motility and DNA repair groups. Gene expression studies revealed that toxin-coding genes nheC, cytK and Gene expression hblC were found to be expressed in vegetative cells growing in the food model. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction et al., 2013) and Bacillus cytotoxicus, which is a thermotolerant species associated with food poisonings (Guinebretiere et al., 2013). The genus Bacillus comprises Gram-positive species that can be All these species are associated by their genome structure, but they found ubiquitously in the environment and can survive and persist often demonstrate specific traits that are due to large plasmid de- to starvation and adverse growth conditions thanks to their ability terminants (Jensen et al., 2003). Cry genes located on a large to form resistant and metabolically inert spores (Schmidt et al., plasmid in B. thuringiensis (Schnepf et al., 1998), cereulide gene on a 2011). Six different Bacillus species, namely Bacillus anthracis, Ba- mega virulence plasmid in B. cereus (Hoton et al., 2005) and capsule cillus cereus, Bacillus mycoides, Bacillus pseudomycoides, Bacillus genes on pXO1 and pXO2 plasmids in B. anthracis (Ehling-Schulz thuringiensis and Bacillus weihenstephanensis, although exhibiting et al., 2006) are only some examples. Moreover, the toxicological different biological characteristics, such as pathogenicity and toxin potential, the occurrence of B. cereus-related food-borne outbreaks production, have been classified on the basis of 16S sequencing (Bargabus et al., 2002), the possible food contamination of (Daffonchio et al., 2003), multilocus sequence typing (MLST) (Priest B. thuringiensis as a result of its wide biopesticide use (Hendriksen et al., 2004) and comparative genome sequencing (Rasko et al., and Hansen, 2006), and the high level of genomic similarities be- 2005) as a single taxonomical unit, the Bacillus cereus sensu lato tween the species forming the group (Schmidt et al., 2011), indicate (Helgason et al., 2000; Schmidt et al., 2011). Moreover, the B. cereus that the B. cereus sensu lato should be studied as a whole. sensu lato was recently extended to comprise two further species: Bacillus spores are indeed often present in food matrices, where Bacillus toyonensis, a strain employed in animal nutrition (Jimenez they can resist preservation treatments and germinate in the end- product if nutrients and proper conditions to return to vegetative life are available (Dworkin and Shah, 2010). Their presence is * Corresponding author. indeed responsible for serious food-borne illnesses (EFSA, 2015) E-mail address: [email protected] (P.S. Cocconcelli). and significant numbers of food spoilage cases (Drean et al., 2015; 1 Current address. Colla S.p.a., Via Sant'Anna 10, 29010 Cadeo, PC, Italy. http://dx.doi.org/10.1016/j.fm.2015.11.006 0740-0020/© 2015 Elsevier Ltd. All rights reserved. 74 D. Bassi et al. / Food Microbiology 55 (2016) 73e85

Markland et al., 2013). Factors triggering germination in food can be Spores were produced from cells cultured in BP medium (Ba- represented by amino acids, sugars and other molecules or condi- cillus Genetic Stock Center, Ohio State University, Columbus, OH, tions (pH, sub-lethal thermal treatments, aw, storage conditions) USA). BP plates were inoculated with 500 mlofB. thuringiensis that let the spore exit from its dormant state (Moir and Smith, 1990; UC10070 overnight cultures, and incubated for 4 days at 37 C. Moir et al., 2002), the precondition for the subsequent growth and Spores were harvested and purified by extensive washing with toxins production. distilled water at 4 C(Nicholson and Setlow, 1990). The spore The spore inert state has been demonstrated with the evidence crops, checked by phase-contrast microscopy, were free (<1%) of of a jelly core and a partial dehydration that forms an immobile vegetative cells and germinating spores. Spore suspensions were matrix (Cowan et al., 2003; Driks, 2002; Nakashio and Gerhardt, maintained at 4 C, and immediately used for the subsequent 1985), but it's still disputed if the spore maintains some minimal analyses. metabolic activity. What has been already demonstrated is that spores of different Bacillus and Clostridium species contain RNA 2.2. Toxin assays transcripts (Bassi et al., 2013; Bettegowda et al., 2006; Dembek et al., 2013; Keijser et al., 2007) and that the amount of RNA The presence of enterotoxin genes and their expression in B. seems to be affected by the spore incubation temperature and thuringiensis UC10070 were assessed by means of PCR and RT-PCR spore age (Segev et al., 2012). The function of these transcripts is analyses. Primer sets and conditions listed in Table 1 were used to still largely unknown: they could represent preserved RNA mole- detect nheBC, hblCD, cytK, cry and ces genes. cules ready for a new cycle, or degraded molecules useful to be the The ability of the strain to produce the diarrhoeal enterotoxin starting point for a de novo synthesis. The RNA state appears to HBL (haemolytic fraction L2) was also tested by the reverse passive affect the exit from dormancy together with the effects of envi- latex agglutination test using BCET-RPLA toxin detection kit ronmental signals (Segev et al., 2012). The Bacillus spores behaviour (Oxoid). After 1% inoculum of the strain in BHI broth, and incuba- in a food matrix in terms of RNA state and gene expression during tion at 37 C for 18 h on shaking (250 cycles/min), the culture was germination is also still unknown. Factors triggering germination centrifuged; supernatants were filter-sterilized and stored and outgrowth have been largely studied in Bacillus subtilis (Keijser at 20 C until the assay performance according to the manufac- et al., 2007) and B. cereus (Abee et al., 2011), while germination turers' instructions. studies of B. cereus in foodstuffs have been recently performed in milk (Bartoszewicz et al., 2013), cream, bechamel sauce and mixed 2.3. Food model and germination assays vegetable soup (Samapundo et al., 2014) and as a probabilistic model of predictive microbiology in refrigerated processed foods of Three commercial thermally treated vegetable creams based extended durability (Daelman et al., 2013). The germination pro- respectively on pepper, artichoke and spinach, and an experimental cess has been studied also in terms of gene expression by DNA- pasteurized soup, made with courgettes, potatoes and milk (CPM), microarrays particularly in B. cereus for screening purposes were tested in triplicates. CPM food model was prepared by mixing (Sergeev et al., 2006), and to assess the microorganism' response to homogeneously fresh courgettes and potatoes, previously washed, salt stress (Den Besten et al., 2009), preservatives such as sorbic trimmed and peeled, to UHT milk with a ratio of 3:1:1. The mixture acid (Van Melis et al., 2011), disinfectants (Ceragioli et al., 2010) and obtained was sterilized at 121 C for 15 min. pH and aw values of modified atmosphere (Passalacqua et al., 2009). DNA-microarrays each soup were determined before proceeding with the experi- were also applied to assess the germination of spores in B. subtilis ments. The samples were inoculated with 105 CFU/ml and incu- (Berka et al., 2002; Keijser et al., 2007), Clostridium novyi bated at 20 C and at 4 C for seven days. Growth dynamics for (Bettegowda et al., 2006), Clostridium sporogenes (Bassi et al., 2013) B. thuringiensis UC10070 were analyzed by plate counts. and Clostridium difficile (Dembek et al., 2013). Germination assays were carried out at room temperature by The aim of this work was to simulate a B. cereus contamination inoculating 100 mlofB. thuringiensis UC10070 spore suspension in an experimental vegetable food model and to study the gene (107 CFU/ml) in selected food products followed by anaerobic expression profile during the initial colonization of food as spore packaging with Anaerocult A packs (Merck, Darmstadt, Germany) entities and during germination and outgrowth phases. To do this, and by a heat treatment for 15 min at 80 C mimicking processing we investigated the phenotypic behaviour of a strain isolated from conditions. spoilt food through OD measurements, scanning electron micro- The germination process in the CPM food model was monitored scopy (SEM) and Energy Dispersive X-ray (EDAX) microanalysis by optical and scanning electron microscopy (SEM) together with that allowed us to determine the critical steps representing the Energy Dispersive X-ray (EDAX) analyses as described below. Mi- entire time-course of Bacillus life cycle in a foodstuff. An array- croscopy data were confirmed by CFU counts plating before and based transcriptome analysis was then applied to study genes after pasteurization for 20 min at 80 C respectively for total cells differentially expressed in these steps, particularly during the (vegetative and spores) and spore counts. germination process. For expression studies, the time points T0 (dormant spores), 40 min (GSP, germinating spores), 2 h (C2h, early-log phase) and 2. Materials and methods 12 h (C12h, mid-log phase) after thermal treatment, were selected as the most representative of the B. thuringiensis UC10070 life cycle 2.1. Bacterial strain isolation, growth conditions and spore under the tested conditions. Samples used for microscopy and production molecular analyses were taken from the inoculated CPM food model at the above time points and immediately stored at 80 C The strain UC10070 was isolated from a biofilm on a spoilt for RNA extraction. All studies were carried out in three indepen- vegetable-based puree using a B. cereus selective agar medium dent experiments. (Oxoid, Milan Italy). It was assigned to the Bacillus cereus sensu lato by means of 16S rRNA gene analysis, and specifically to 2.4. Microscopy examinations B. thuringiensis on the basis of cry toxins identification. The strain was cultured in Brain Heart Infusion (BHI) broth at 37 C in aerobic The transition from spores to vegetative cells was monitored by conditions on continuous shaking. optical phase contrast microscopy, SEM and EDAX microanalyses. D. Bassi et al. / Food Microbiology 55 (2016) 73e85 75

Table 1 Primer set for detection of enterotoxin genes in B. thuringiensis UC10070 isolated strain. For HBL and NHE complex, PCR analysis were performed to amplify at least two genes in the three component operons: hblC and hblD genes for the haemolytic toxin, nheB and nheC genes for the non-haemolytic toxin.

Target genes Primer name Sequence (50e30) Reference

cryIV DiplA CAAGCCGCAAATCTTGTGGA (Carozzi et al., 1991) Dip1B ATGGCTTGTTTCGCTACATC cry1Ia1 X62821 Fw GCTGTCTACCATGATTCGCTTG (Song et al., 2003) X62821 Rv CAGTGCAGTAACCTTCTCTTGCA hblC L2A Fw AATGGTCATCGGAACTCTAT (Hansen and Hendriksen, 2001) L2B Rv CTCGCTGTTCTGCTGTTAAT hblD HBLD-N AATCAAGAGCTGGTCACGAAT (Hansen and Hendriksen, 2001) HBLD-C CACCAATTGACCATGCTAAT nheB nheB 1500 S Fw CTATCAGCACTTATGGCAG (Hansen and Hendriksen, 2001) nheB 2269 A Rv ACTCCTAGCGGTGTTCC nheC nheC 2820 S Fw CGGTAGTGATTGCTGGG (Hansen and Hendriksen, 2001) nheC 3401 A Rv CAGCATTCGTACTTGCCAA cytK cytKf GATAATATGACAATGTCTTTAAA (Swiecicka and Mahillon, 2006) cytKr GGAGAGAAACCGCTATTTGT ces CesF1 GGTGACACATTATCATATAAGGTG (Ehling-Schulz et al., 2005) CesR2 GTAAGCGAACCTGTCTGTAACAACA rrn P1 GCGGCGTGCCTAATACATGC (Klijn et al., 1991) P4 ATCTACGCATTTCACCGCTAC

Optical microscopy was made at regular time points (every 10 min) temperature for each primer set was determined prior to the observing at least 15 optical fields for the presence of phase-bright analysis of experimental samples. Reverse transcription was per- spores, dark spores and vegetative cells. Samples for SEM obser- formed using 200 ng DNA-free RNA, random primers, and the vations and EDAX were prepared according to the carbon coating Transcriptor First Strand cDNA Synthesis kit (Roche, Indianapolis, method previously described (Bassi et al., 2009). SEM and EDAX IN, USA), following the supplier's recommendations. A sample analyses were carried out on three replicates for each analyzed volume of 20 ml was used for all quantification assays, which con- condition with a Philips XL30 E-SEM microscope. tained a 1 final concentration of SYBR green PCR master mix, 0.5 mM gene specific primers, and 1 or 2 ml of template. Samples were heated at 95 C for 10 min before cycling for 45 cycles of 95 C 2.5. RNA isolation for 10 s, 56 Cor57C for 20 s, and 72 C for 25 s. In each step the temperature transition rate was 20 C/s. A melting curve plotted at RNA was isolated from CPM frozen samples collected at the the end of each run verified the specificity of the amplification above-defined time points and immediately frozen at 80 C (three product. For each independent experiment, three technical repli- biological replicates for each analyzed stage). RNA was isolated cates were run in real-time PCR analysis. Prior to quantitative an- from 1 ml of soup for each phase, homogenized with physiological alyses, standard curves were constructed for housekeeping and solution, centrifuged at 10,000 g for 10 min and pellets processed target genes using cDNA from log B. thuringiensis UC10070 cells using RNeasy mini kit (Qiagen, Ilden, Germany) according to the grown in BHI medium: serial decimal dilutions of the cDNA with manufacturer's instructions. Samples lysis was carried out in a nuclease-free water were used to generate standard curves for FastPrep instrument (MP Biomedicals, Irvine, CA, USA) with target genes, as well as with P1eP4 oligonucleotides, which different settings for spores (SP), germinating spores (GSP) and covered 3e5 orders of magnitude in the range of the samples in vegetative cells (C2h and C12h). SP and GSP were processed four order to calculate the specificefficiency (E) using LightCycler times for 50 s each in the FastPrep machine at setting 6.5 m/s using Software 3.5. Relative expression levels between samples were 0.1 mm zirconia/silica beads (Biospec Products Inc., Bartlesville, then calculated as fold changes, where each PCR cycle represents a USA); C2h and C12h cells were bead-beated one time for 50 s at two-fold change (Livak and Schmittgen, 2001). 6.5 m/s. Samples were kept on ice during the intermediate pro- cessing steps. RNA samples were finally resuspended in 40 mlof RNase-free water and rapidly frozen at 80 C. RNA quantities were 2.7. RNA amplification and hybridization determined with the Qubit Quantitation Platform (Invitrogen, Paisley, UK), while the quality was assessed with RNA 6000 Nano The complete genome sequence of B. thuringiensis sv konkukian Lab-Chip (Agilent Technologies, Santa Clara, CA, USA) using a 2100 str. 97e27 (NCBI Reference Sequence: NC_005957.1) was chosen for Bioanalyser (Agilent Technologies, Santa Clara, CA, USA). its high homology with B. thuringiensis UC10070, to design probes corresponding to 5197 genes spotted in duplicates onto Elec- 2.6. Relative quantification of enterotoxin gene expression in CPM traSenseH 12K microarrays chip (CombiMatrix Irvine, CA, USA). model Probes were designed using the Combimatrix Automated Probe- Design Suite (CombiMatrix, Irvine, CA, USA). Thirteen negative The mRNA level changes of enterotoxin genes hblC, nheC, and control probes designed on three Arabidopsis genes were synthe- cytK in the different tested stages were analyzed in real-time RT- sized in 30 replicates randomly distributed on the chip as previ- PCR. For relative quantification, the 16S rRNA gene was analyzed as ously described (Bassi et al., 2013). Nucleotides quantity and quality ® housekeeping gene by amplification with the P1eP4 primers pair. were determined by NanoDrop ND-1000 (ThermoFisher, Wal- The oligonucleotide primer sets used for reference and target genes tham, MA, USA) and analyzed on a RNA 6000 Nano LabChip (Agi- amplification are shown in Table 1. Primer sets for enterotoxin lent Technologies, Santa Clara, CA, USA) using a 2100 bioanalyzer genes were designed against the complete nucleotide sequence, as (Agilent Technologies, Santa Clara, CA, USA). deposited on GenBank, using LightCycler Probe Design Software 2.0 RNA amplification, labelling, fragmentation and hybridization (Roche, Indianapolis, IN, USA). The optimum annealing were performed using the MessageAmp™ II- Kit for 76 D. Bassi et al. / Food Microbiology 55 (2016) 73e85 prokaryotic RNA (Ambion, ThermoFisher, Waltham, MA, USA), a culture by immunological assay and gave positive results. Albeit linear in vitro transcription based RNA amplification system (Van some limitations of the BCET-RLPA kit employed were recently Gelder et al., 1990) as previously described (Bassi et al., 2013). outlined (Kaminska et al., 2014), the immunological and PCR results Slides previously coated with an imaging solution were scanned here obtained are in accordance, and confirm the enterotoxins with a ScanArray 4000 XL (PerkineElmer, USA) and raw data were production of the strain UC10070. The PCR approach, amplifying extracted with ScanArray Express 4.0 and Microarray Imager soft- the ces gene, demonstrated the absence of the non-ribosomal ware (CombiMatrix, Irvine, CA, USA). Experiments were carried out peptide synthetase gene responsible for the cereulide production. in three biological replicates per stage. Each gene was present at least in duplicate on the slide, so each biological replicate was hy- 3.2. Food model development to study B. thuringiensis UC10070 bridized in duplicate on the same microarray slide. germination kinetics and growth

2.8. Microarray data analysis A number of preliminary experiments were carried out in order to: (i) identify a food substrate which supported the growth and The fluorescence signal for Cy5-dye channel and background germination of B. thuringiensis UC10070; (ii) mimic the handling subtractions were determined with Microarray Imager software conditions of a ready to eat food, (iii) follow the germination ki- (CombiMatrix, Irvine, CA, USA). The fluorescence signal of each spot netics in the food models and (iv) select the key steps to reproduce was calculated as the difference between the mean of pixel in- and study gene expression during germination, outgrowth and cell tensities and the mean of background fluorescence signals, defined cycle of inoculated B. cereus sensu lato in a food model. Three by surrounding pixel intensity. Signals were log2 transformed and commercial vegetable soups based on pepper, artichoke and differentially expressed genes in the different conditions tested spinach, and one experimental pasteurized soup (CPM), were were identified with a one-way ANOVA test, with P value < 0.05 tested. A thermal treatment for 15 min at 80 C, and incubation in and for induction or repression ratio equal or higher than 1-fold. A anaerobic conditions, were used to reproduce, in our experimental preliminary quality control of the data was also carried out with food models, the steps of an industrial process. Chemical analysis of principal component analysis (PCA). Filtered data were then the primary factors affecting microbial growth, pH and water ac- analyzed using Microarray Expression Viewer software (Saeed tivity (aw), allowed a first selection of the best model to use for et al., 2006); in order to identify groups of genes with similar analysis. Pepper cream presented an optimum value of aw (0.97), as transcription profiles, the significantly regulated genes were sub- the CMP model, but the pH (4.63) seemed to be too restrictive to divided into clusters with different expression patterns by using K- support the growth of B. thuringiensis. The cream of artichoke and means clustering (Soukas et al., 2000). Significance Analysis of spinach showed pH values of respectively 5.63 and 5.94 suitable for Microarrays (SAM) with a one-class design (Tusher et al., 2001)was the development of the bacterium, but an aw of 0.94 that probably applied to calculate mRNA abundance in dormant spores. limited spores germination and the growth of vegetative cells. The For a functional interpretation of the transcriptional activity, the analysis of growth kinetics carried out in three replicates indicated B. thuringiensis sequence sv. konkukian str. 97-27 genome annota- that the CPM soup substrate (pH 5.95 and aw 0.97) better supported tions (http://img.jgi.doe.gov/) was used. Six groups of functionally the multiplication of vegetative cells at both 20 C(Fig. 2A) and 4 C related genes were identified and ordered by HCL hierarchical (Fig. 2B), the germination of spores and their subsequent clustering using Microarray Expression Viewer software (Saeed outgrowth: this substrate was therefore selected as the most et al., 2006). appropriate artificial food model and the germination kinetics were analyzed in this medium. 2.9. Microarray data accession number The selection of the crucial steps for B. thuringiensis UC10070 spores germination and outgrowth in CPM soup stored at room The data discussed in this publication have been deposited in temperature was performed on the basis of optical microscopy NCBI's Gene Expression Omnibus (Edgar et al., 2002) and are together with scanning electron microscopy (SEM) and EDAX mi- accessible through GEO Series accession number GSE65259 (http:// croanalyses results (Bassi et al., 2009). At room temperature, after www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc¼GSE65259). 20 min from thermal treatment, the optical microscopic analysis of food samples, based on the observation of at least 15 fields, showed 3. Results and discussion that the transition of Bacillus spores from phase-bright to phase- dark was commenced. This step coincides with the release of cal- 3.1. Characterization of the food isolate Bacillus strain UC10070 cium dipicolinate from spores, the entrance of water into the core environment and consequent increase in volume (Paidhungat et al., Bacillus strain UC10070 isolated from a biofilm on a spoilt 2000; Setlow, 2003). After 40 min about the 90% of spores showed vegetable based puree from organic farming was used for this the conversion to the dark germinating phase. This observation study. It was first of all characterized by means of 16S rRNA gene were confirmed by SEM analysis which allowed the identification sequencing and assigned accordingly to B. cereus sensu lato. Scan- of morphological changes of B. thuringiensis spores and cells. ning electron microscopy (SEM) analysis revealed the presence of Dormant spores (Fig. 1 A and B) in CPM food model, after toxin crystals (Fig. 1A) and PCR reaction confirmed the positivity of pasteurization, underwent morphological changes in their overall the strain to cry4 and cry4a1 genes, therefore indicating that the structure after 20 min (Fig. 1C) with an increase cell dimension strain belongs to B. thuringiensis. To clarify the presence and the (average spore length 1.4 ± 0.09 vs 1.2 ± 0.10) and a more elongated expression of genes coding for B. cereus toxins, PCR and reverse structure. To further investigate the germination process, X-ray transcriptase PCR were performed on DNA and RNA extracted from microanalysis (EDAX) (Hayat, 1980; Bassi et al., 2009)was vegetative cells using primers pairs (Table 1) targeted to the hbl and employed together with the high resolution carbon-coating SEM nhe operons. These experiments demonstrated that UC10070 strain technique. This approach allowed to identify calcium at the cellular harbours and expresses the genes coding for the two multipartite level and to monitor calcium-dipicolinate release (Ca-DPA) from enterotoxins Hbl and Nhe and for cytotoxin K. The production of the spores, an event that is considered a key step in the germination L2 component of Hbl enterotoxin, involved in the diarrhoeal syn- process (Santo and Doi, 1974; Vepachedu and Setlow, 2007). The drome, was also assessed in the supernatant of UC10070 broth release of calcium dipicolinate started already 20 min after D. Bassi et al. / Food Microbiology 55 (2016) 73e85 77

Fig. 1. Scanning electron microscopy (SEM) images using the gold coating technique; pictures show representative fields selected among all the performed observations: A, an enterotoxin crystal protein of B. thuringiensis UC10070; B, dormant spores of B. thuringiensis UC10070 in CPM food model at the beginning of the experiment (average length 1.2 ± 0.10); C, spores after 20 min (average length 1.4 ± 0.09); D, germinating spores at 40 min after heat treatment; E, vegetative cells at 2 h; F, log phase cells at 12 h. Arrows indicate: CP, crystal protein; DS, dormant spore; GS, germinating spore; VC, vegetative cell. pasteurization with approximately the 30% of the spores having from sporulating cells was not considered in the course of the study þ lost the Ca2 content (Fig. 3). The germination process was due to the lack of reliable methods to differentiate mRNA of the completed (>90% of the spores) 40 min after the thermal treatment, mother cell from transcripts in the prespore. For each sampling, þ when the spores had completely lost Ca2 and adopted an elon- cells were rapidly harvested and frozen in order to stabilize the gated shape (Fig. 1D). At 120 min only vegetative cells that initiated nucleic acids in the different steps of the biological cycle. RNA was the duplication, as demonstrated by the septa formation, were isolated efficiently from dormant spores and more easily from detected (Fig. 1E). At 12 h the log phase vegetative cells did not germinating spores and vegetative cells adopting a combination of enter yet in the sporulation process (Fig. 1F). a bead-beating mechanical method using zirconia beads, with a These microscopic examinations allowed to identify the critical column RNA purification in order to preserve its integrity (Bassi time-points that were used in the concomitant transcriptomic an- et al., 2013). Gene expression was found to be highly dynamic alyses of B. thuringiensis UC10070: the spore dormant state (S), the during the B. thuringiensis UC10070 life cycle and was found to onset of germination 40 min after heat activation (GSP), the involve a large number of genes, as demonstrated by the Principal beginning of the outgrowth process at 2 h after heat activation Component Analysis (data not shown). A total of 1646 genes were (C2h), and vegetative cells at 12 h after heat treatment (C12h). found to be differentially expressed and modulated in the four distinct analyzed conditions (Fig. 5). The large amount of genes differentially expressed was consistent with the relevant metabolic 3.3. Microarray analysis of B. thuringiensis UC10070 gene and morphologic changes undergoing along the Bacillus life cycle. expression during germination and growth cycle in an artificially contaminated vegetable soup model 3.4. Transcripts in spores A custom array analysis was performed on total RNA extractions at the above-described conditions and time-points S, GSP, C2h and The general assumptions that spores do not contain RNA tran- C12h to determine genes differentially expressed in B. thuringiensis scripts have been now rejected; all the transcriptional studies UC10070 grown in CPM food model. Even though we used a published in the last years showed that mRNA molecules are pre- vegetable food model to perform our experiments, the monitored sent in these dormant entities and the hypothesis of a functional time-points for the germination assay were consistent with the role is proposed but not yet confirmed (Alsaker and Papoutsakis, ones observed in B. subtilis in germination medium (Keijser et al., 2005; Fawcett et al., 2000; Liu et al., 2004; Segev et al., 2013; 2007) and in B. cereus with L-Alanine (Wang et al., 2011). RNA Setlow, 2003; Wang et al., 2006). In addition to the fact that 78 D. Bassi et al. / Food Microbiology 55 (2016) 73e85

Fig. 2. Kinetics of growth of B. thuringiensis UC10070 observed in four different vegetables mix at room temperature of 20 C (A) and refrigerator temperature of 4 C (B). Numbers are expressed as the mean values of triplicate experiments, with error bars; A artichoke, - pepper, : spinach, C CPM model.

Fig. 3. SEM (left) and X-ray microanalysis (right) maps of the same picture at incubation time of 20 min. The spores A, B and C showed no calcium inside, as represented by the absence of a fluorescent spot. These spores have already triggered the germination process. spore mRNA composition is clearly dissimilar from that of vegeta- 40 min this processed rRNA species has already disappeared, a sign tive cells, differences have been found also in ribosomal RNA spe- of an advanced germination phase evolving to outgrowth. The cies. As observed in B. subtilis (Keijser et al., 2007), C. novyi specific role of this shorter rRNA is not known, but its presence in (Bettegowda et al., 2006), C. sporogenes (Bassi et al., 2013) and all the analyzed species of Bacillus and Clostridium, and its disap- C. difficile (Dembek et al., 2013), B. thuringiensis UC10070 presented pearance after the first steps of germination suggests a possible evidence of a truncated 23S rRNA in spores that cannot be detected regulatory function associated with the spore cycle. According to in the vegetative cell forms (Fig. 4). In fact, in germinating spores at Segev et al. (2012), rRNA degradation seems to be related to the D. Bassi et al. / Food Microbiology 55 (2016) 73e85 79

variety of solutes, including drugs, across the membrane (Han et al., 2006). The prevalence in spores of these drug transporter tran- S1 S1 S2 S3 GSP1 C2H1 C2H2 C2H3 C12H1 C12H2 C12H3 GSP2 GSP2 GSP3 scripts may be correlated to previous spore maturation or either to the transport of metabolites necessary for the germination process. An mRNA encoding for a transposase IS660 was also abundant in spores. Insertion sequences may play an important role in internal 23S tr 23S genetic rearrangements in the genome, likely to support the 16S mechanisms of adaptation to extreme environments, such as those with extreme values of pH, temperature, pressure, or salinity (Takami et al., 2001). In this case, its function was difficult to explain 5S in spores since it could be related to a former insertional event. A 50% of transcripts found in spores coded for hypothetical proteins or proteins with unknown function as seen in previous Fig. 4. RNA integrity measured with Agilent 2100 Bioanalyser (Agilent Technologies). Two major bands (16S and 23S rRNA subunits) were present in all samples in the four studies (Bettegowda et al., 2006; Dembek et al., 2013) and arising analyzed stages. An extra band corresponding to a truncated 23S rRNA (tr 23S) the need to deeply investigate on their role in cell cycle of spore appeared only in all the three replicates of dormant spores (samples 1, 2, 3), while was formers. absent in GSP at 40 min after heat treatment (samples 4, 5, 6), after 2 h (sample 7, 8, 9) and after 12 h (samples 10, 11, 12). 3.5. Functional analysis and hierarchical clustering during germination dormant state to minimize normal activity and enter dormancy, and the small rRNA fragments could be a result of this growth ar- The presence of 407 (120 in cluster 2 and 287 in cluster 3) up- rest. All this process can be considered an adaptation to a precise regulated genes in GSP vs SP reflected the transfer of the environment where the spore finds itself and has to prepare all the dormant spore to an active state (Fig. 5). This initiates a cascade of “ready-to-use kit” of molecular tools to germinate (Segev et al., processes that gradually degrade the protective structures of the 2012). spore and resume cellular processes and its metabolism, ultimately In contrast to other reported studies on spore transcripts, in leading to the vegetative cell. In accordance with previously pub- B. thuringiensis UC10070 a high number of transcripts (824) was lished data on the temporal gene expression during Bacillus spp. found in the dormant spores used to inoculate the food model and spores outgrowth (Keijser et al., 2007), important house-keeping collected immediately after. This was a relatively abundant number genes, encoding proteins such as translation initiation factors, ri- if compared to what observed in previous studies in B. cereus and in bosomal proteins, and elongation factors, were found to be tran- fi B. subtilis, where respectively 46 (Van Melis et al., 2011), 23 (Keijser scribed in the rst 40 min after heat activation of dormant spores, et al., 2007) and 369 transcripts (Segev et al., 2012) were detected. together with ample modulation of their expression levels during Segev et al. (2012) studied the rRNA dynamics in ageing spores and outgrowth and vegetative cycle. observed that in B. subtilis, the number of mRNAs in spore is In order to have a functional interpretation of the transcriptional inversely related to the spore age and that the RNA degradation is activity during the germination process and outgrowth, groups of accelerated in spores held at high temperatures. functionally related genes were identified using B. thuringiensis 97- In our experiments, the spores were prepared, cooled to 4 C 27 genome annotations and ordered by hierarchical clustering. and directly used for transcription studies. This might explain the Functional categories were then analyzed in relation to their higher number of transcripts detected in UC10070 and its rapid expression patterns (Fig. 6) and the most representative genes for entrance in germination up to favourable environmental condi- each categories have been evaluated (Fig. 7). tions. The mRNAs found in the spore transcriptome coded for genes belonging to DNA metabolism, transport, transcription, translation 3.5.1. Transport fi and other metabolic pathways (sporulation, cell wall metabolism, One of the major signi cantly over-represented group of func- energy production), consistently with what already described in tional genes identified encoded for proteins involved in transport of B. subtilis and its relatives, C. novyi and C. sporogenes (Bassi et al., various molecules (116 differently regulated genes). ABC trans- 2013; Bettegowda et al., 2006; Keijser et al., 2007). porters specific for ions, sugars, and other organic compounds like Among the total 824 transcripts, we analyzed the most abun- drug/metabolites, are considered essential to restart the normal dant genes identified in spores of B. thuringiensis UC10070. The sspI vegetative behaviour after dormancy (Dembek et al., 2013); these mRNA for small-acid-soluble protein A, a protein responsible for transcripts were mostly found to be present in spores and they DNA protection from chemical and physical insults during the were differentially regulated during germination (Fig. 7). Among passage to spore, and the transcript for YhcN/YlaJ, a forespore these, 32 were up-regulated at 40 min and, particularly, the ones specific sporulation lipoprotein were found at this stage, being both coding for spermidine/putrescine ABC transporters, ferrous iron probably a rest of the gene expression during the sporulation stage. transport proteins, zinc transporters, cobalt transport proteins, Another transcript present in spores was a thioredoxin family glycerol-3-phosphate ABC transporters. The involvement of ions fi protein; since in B. subtilis thiol-disulfide oxidoreductases seemed during the germination process has been studied since the very rst to have a role in cortex synthesis and consequent spore maturation works by Levinson and Hyatt (1963). It could be explained as a (Erlendsson et al., 2004), we speculate also in this case an entrap- support to germinants action reinforcing their effect; moreover, it ping of transcripts during sporulation phases. Interestingly, other has been supposed that the suspension of the ion channels action is genes whose transcripts were quite abundant in spores included a critical for spore germination (Mitchell et al., 1986). The importance possible PAP2 BcrC_like subfamily protein, a bacitracin transport of metal ion homeostasis and particularly of iron was evidenced by permease involved in bacitracin resistance and a gene coding for a the fact that eight genes for iron-regulated transport proteins were fi Major Facilitator Superfamily transporter; the first is an ABC found to be signi cantly expressed during germination. transporter which pumps out bacitracin from the cell wall (Cao and Transcripts for glycine-betaine transport protein OpuAB, a Helmann, 2002) and the second a drug anti-porter that pumps a widely diffuse bacterial system for cell protection against high so- lute concentration, were found in spores and remained present in 80 D. Bassi et al. / Food Microbiology 55 (2016) 73e85

Fig. 5. Cluster graphics of the 4 studied stages. Using K-means clustering, the filtered genes were grouped into 5 different clusters of expression; the bars indicate the centroid value, which is the mean value in every group (in order: S, GSP, 2 h, 12 h). The Y-axis shows the normalized intensities (1, 0, 1).

GSP, suggesting that osmotic defence is important in the earliest were identified as common families to , Lactobacillales and stages of outgrowth, in agreement with previous studies on Clostridiales (Moreno-Campuzano et al., 2006) in addition to GntR, germination process (Keijser et al., 2007). A large number of ArsR, DeoR, Fur families of transcriptional regulators. These pro- transporter genes encoding putative multidrug transporters, such teins are responsible for rapid adaptation of bacteria to changing as SMR and Bcr/CflA family, which were up-regulated during the environmental conditions, including resistance to different anti- first 40 min of outgrowth, may also provide the germinated spore microbial compounds and oxidative stress agents, controlling the with a transient resistance against antimicrobial compounds. expression of drug efflux pumps. Members of GntR family proteins, also found in dormant spores, respond to environmental changes 3.5.2. Transcriptional regulation and protein synthesis affecting the carbohydrate metabolism of the cell and may provide Many genes involved in transcriptional regulation for rapid re- bacteria with the ability to grow in the presence of several carbon covery of cell functions were found to be already present among the sources and to rapidly adapt their gene expression to changing spore transcript pool. The end of dormancy is a process that needs nutrient conditions (Reizer et al., 2006). Transcripts coding for the the progressive re-starting of transcription and translation pro- fumarate and nitrate reduction regulator CRP-Fnr, member of the cesses. During the first 40 min, several ribosomal proteins were up- cyclic AMP receptor proteins, were present both in S and GSP; they regulated: among these, we found twenty-seven 50S ribosomal are known to play an important role in modulating the expression proteins and nineteen 30S subunit proteins (Fig. 7). This evidence of many metabolic genes in several facultative or strictly anaerobic can be interpreted on the basis of the data obtained by Segev et al. bacteria; their functions also include the control of virulence factors (2012); according to this work, to achieve quiescence, spores of (Baltes et al., 2005; Bartolini et al., 2006). In addition, transcripts for B. subtilis degrade their RNA to reduce their cellular activity. At the RNA polymerase s-factor, ECF type, that controls genes involved in onset of germination, transcription could restart within the spore cell envelope functions (protein transport and secretion processes), and rRNA resynthesized upon germination maintaining the same in response to extra-cytoplasmic stress in B. subtilis and Bacillus high level also during vegetative life. In addition, genes for RNA licheniformis (Wecke et al., 2006), and RNA polymerase sK factor, polymerase subunits a, b and d were up-regulated during germi- responsible for the expression of sporulation specific genes in the nation together with genes for transcriptional regulators. A large mother cell, were found in dormant spores. number of genes coding for proteins involved in the control of During outgrowth and cell vegetative growth (C2h and C12h), different biological processes were found. Particularly, 22 genes genes involved in DNA replication, transcription (DNA polymerase encoding for transcriptional regulators were up-regulated during III, DNA-directed RNA polymerase delta subunit) and RNA trans- germination; examples were members of TetR, MarR, MerR lation, like transcription elongation antitermination factor proteins (especially found in C12h) transcription factors families (TFs), that NusB and NusG, whose function was described in B. subtilis D. Bassi et al. / Food Microbiology 55 (2016) 73e85 81

Fig. 6. Gene expression profiles in the four steps of B. thuringiensis UC10070 life cycle in different functional categories. A) All genes; B) Transport; C) Ribosomal proteins; D) Transcription; E) Germination; F) Membrane biosynthesis and motility; G) DNA repair.

(Yakhnin et al., 2008), were over-represented. During the late the members of the B. cereus sensu lato, except for B. anthracis stages of outgrowth, cells appeared to prepare for septation, as where the PlcR regulator is present but inactive (due to a non-sense indicated by the overexpression of septation ring formation regu- mutation). lator ezrA (Errington, 2001), giving evidence that cells are in an active growing phase. Transcripts for the transcriptional regulator 3.5.3. Cell wall biosynthesis and motility PlcR (Phospholipase C Regulator) were found to be up-regulated Expression pattern analysis of genes coding for proteins 12 h after germination. This transcriptional regulator takes part in involved in cell wall biosynthesis, revealed in dormant spores the the control of most known virulence factors in B. cereus (entero- presence of mRNAs for transpeptidase enzymes (D-alanyl-D-alanine toxin, haemolysins, phospholipases and proteases) (Gohar et al., carboxypeptidase family proteins) that cross-link the peptido- 2008), acting as a quorum-sensing system whose molecular glycan chains to form rigid cell walls. During germination, the new mechanism has been explained by Grenha et al. (2013).Tobe emerging cell has to synthesize new cell wall: accordingly, D-Ala D- activated, PlcR needs its cognate PapR peptide, whose transcript Ala carrier protein ligase, UDP-N-acetylmuramate-L-alanine ligase, was also found to be overrepresented in vegetative cells. PlcR is the cell wall hydrolase possible N-acetylmuramoyl-L-alanine amidase, first example described of a pleiotropic regulator required for the a glycosyltransferase, murein hydrolase export regulator, were up- control of extracellular virulence factor expression in pathogenic regulated at 40 min in B. thuringiensis UC10070 (Fig. 7). Bacillus spp. (Agaisse et al., 1999) and seemed to be present in all Motility is known to be absent in dormant spores, while is a 82 D. Bassi et al. / Food Microbiology 55 (2016) 73e85

Fig. 7. Functional categories and expression patterns of the most representative genes modulated during germination and outgrowth phases.

prerequisite in the vegetative life; in B. thuringiensis UC10070 a allowing an efficient reactivation of transcription, likely after the single transcript for the flagellar motor switch protein was present partial degradation of the SASPs that coat and protect DNA during in the spore phase, while the majority of transcripts for flagellar spore dormant state by complete saturation and tight binding, biosynthesis-assembly proteins and chemotaxis were overex- changing the structure to a more stable conformation (Setlow, pressed at C2h and C12h. 1995).

3.5.4. DNA repair 3.6. Expression of enterotoxin genes in the food model A number of general DNA repair genes were expressed actively during vegetative life (C12h). At the same time some transcripts Although limited evidence on B. cereus toxins expression in food coding for proteins involved in more specific repair activity could is available (Delbrassinne et al., 2011; Rajkovic et al., 2013), en- be found in dormant spores. DNA mismatch repair protein and DNA terotoxins production has been demonstrated to be highly dynamic repair protein RadC, that was supposed to be involved in the pro- depending on pH, glucose availability and oxygen. Previous studies tection of spores against harsh environmental conditions and reported that the highest toxin levels can be achieved during the damaging following X- and UV-irradiation (Felzenszwalb et al., late log-early stationary phase for cultures grown in broth and that 1986) were an example (Fig. 7). DNA of dormant spores is no significant increase in toxin production occurs during the late believed to be in a supercoiled state, providing protection against stationary phase (Mckillip, 2000); more recently, Madeira et al. damage. The helicase activity of Holliday junction RuvB, and (2015), studying the exoproteome of B. cereus found that the ma- methionine gamma-lyase, observed during early stages of germi- jority of toxin-related proteins were produced during the expo- nation, might be necessary to relax rapidly the supercoiled DNA nential growth phase. In fact, when B. cereus reaches high cellular D. Bassi et al. / Food Microbiology 55 (2016) 73e85 83

Table 2 Lombardia founding scheme “GENOBACT” project n. Relative quantification of the expression of virulence genes by means of qRT-PCR. G41J10000400002. Data (average ± standard deviation) are expressed as fold changes in mRNA levels. GSP, germinating spores; C2h, vegetative cells two hours after the heat treatment; C12h, vegetative cells at mid-log phase. References GSP C2h C12h F value Abee, T., Groot, M.N., Tempelaars, M., Zwietering, M., Moezelaar, R., Van Der hblC 0.69 ± 0.18 b 2.70 ± 0.62 a 1.65 ± 0.24 b 19.251** Voort, M., 2011. Germination and outgrowth of spores of Bacillus cereus group ± ± ± nheC 1.05 0.24 b 2.17 0.25 a 2.01 0.46 a 9.48* members: diversity and role of germinant receptors. Food Microbiol. 28, ± ± ± ns cytK 0.69 0.39 a 0.93 0.37 a 1.42 0.48 a 2.30 199e208. * <0.05, ** < 0.01, *** <0.0001, ns ¼ not significant. Agaisse, H., Gominet, M., Økstad, O.A., Kolstø, A.B., Lereclus, D., 1999. PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis. Mol. Microbiol. 32, 1043e1053. Alsaker, K.V., Papoutsakis, E.T., 2005. Transcriptional program of early sporulation density, different toxic compounds are produced: the two enter- and stationary-phase events in Clostridium acetobutylicum. J. Bacteriol. 187, otoxic complexes, haemolysin BL (HBL) and non-haemolytic 7103e7118. Baltes, N., Mohamed, N., Jacobsen, I.D., Maas, A., Buettner, F.F.R., Gerlach, G.F., 2005. enterotoxin (NHE), several phospholipases-C, a collagenase and Deletion of the anaerobic regulator HlyX causes reduced colonization and several haemolysins/cytolysins (HlyI, HlyII, HlyIII and CytK) persistence of Actinobacillus pleuropneumoniae in the porcine respiratory tract. (Ramarao and Sanchis, 2013). Coherently with these observations, Infect. Immun. 73, 4614e4619. the transcriptome analysis of B. thuringiensis UC10070 demon- Bargabus, R., Zidack, N., Sherwood, J., Jacobsen, B., 2002. Characterisation of sys- temic resistance in sugar beet elicited by a non-pathogenic, phyllosphere- strated no signal during germination for probes related to genes colonizing Bacillus mycoides, biological control agent. Physiol. Mol. Plant P 61, coding for Hbl and Nhe enterotoxic complexes, Haemolysin II, 289e298. CryIA, CytK and haemolysin type III. In contrast, at 12 h after heat Bartolini, E., Frigimelica, E., Giovinazzi, S., Galli, G., Shaik, Y., Genco, C., Welsch, J.A., Granoff, D.M., Grandi, G., Grifantini, R., 2006. Role of FNR and FNR-regulated, activation, genes coding for delta-endotoxin CryIA, cytotoxin K, sugar fermentation genes in Neisseria meningitidis infection. Mol. Microbiol. 60, haemolysin type III, and possible phospholipases, were signifi- 963e972. cantly expressed. The analysis of microarray data showed that the Bartoszewicz, M., Kroten, M.A., Swiecicka, I., 2013. Germination and proliferation of emetic Bacillus cereus sensu lato strains in milk. Folia Microbiol. 58, 529e535. expression of both transcripts of nhe and hble operons were Bassi, D., Cappa, F., Cocconcelli, P.S., 2009. A combination of a SEM technique and X- significantly increased (P < 0.05) from GSP to C2h, but the fold ray microanalysis for studying the spore germination process of Clostridium increase was lower than the defined threshold. To better study the tyrobutyricum. Res. Microbiol. 160, 322e329. Bassi, D., Cappa, F., Cocconcelli, P.S., 2013. Array-based transcriptional analysis of enterotoxin expression, the total RNA from the same samples was Clostridium sporogenes UC9000 during germination, cell outgrowth and vege- analyzed by quantitative RT-PCR, evaluating hblC, nheC and cytK tative life. Food Microbiol. 33, 11e23. mRNA levels, during the B. thuringiensis UC10070 life cycle Beecher, D.J., Wong, A.C., 1994. Improved purification and characterization of he- molysin BL, a hemolytic dermonecrotic vascular permeability factor from Ba- (Table 2). The transcript of hbl and nhe operons were found to in- cillus cereus. Infect. Immun. 62, 980e986. crease in the passage from GSP to C2h, with values respectively Berka, R.M., Hahn, J., Albano, M., Draskovic, I., Persuh, M., Cui, X., Sloma, A., threefold and twofold greater than in germinating spore (GSP). The Widner, W., Dubnau, D., 2002. Microarray analysis of the Bacillus subtilis K- expressions were maintained in the log phase (C12h) cells. Simi- state: genome-wide expression changes dependent on ComK. Mol. 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The toxin was detected in low concentration two insecticidal activity of Bacillus thuringiensis strains by polymerase chain reac- hours after spore activation and its amount increased over time tion product profiles. Appl. Environ. Microbiol. 57, 3057e3061. reaching the higher value at 12 h. This is in agreement with studies Ceragioli, M., Mols, M., Moezelaar, R., Ghelardi, E., Senesi, S., Abee, T., 2010. e Comparative transcriptomic and phenotypic analysis of the responses of Ba- on Hbl characterization, where a growth period of 5 6 h was used cillus cereus to various disinfectant treatments. Appl. Environ. Microbiol. 76, for in vitro routine production of enterotoxins (Beecher and Wong, 3352e3360. 1994; Dietrich et al., 1999). These data indicate that B. thuringiensis Cowan, A.E., Koppel, D.E., Setlow, B., Setlow, P., 2003. 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