All strains and plasmids used in this study are listed in Table 1. Primers used for the amplification of DNA fragments are listed in Table S1. As reference strains, B. subtilis 168 and B. cereus ATCC 14579 were used. Foreign DNA was introduced into B. cereus via electroporation (Masson et al., 1989), either by using the multicopy plasmid pNW33n or by single crossover DNA integration via non-replicative pMAD and PSG1151 derivatives.

For the construction of the pNWVT vector, the spoVT gene (BC0059) including its own promoter was amplified from B. cereus ATCC 14579 chromosomal DNA using primers AKupVT-F and TIFN16. The resulting product was cut with EcoRI and KpnI (Fermentas, FastDigest) and ligated into the corresponding sites of pNW33n (Genbank Accession number, AY237122), which resulted in pNWVT. The pVTBsu2 vector was created by replacing spoVT_BCE with spoVT_BSU in pNWVT, which was amplified using primers BsuVTF2 and BsuVTR. The primers were specifically designed to ensure that spoVT_BSU expression would be driven by the B. cereus spoVT promoter already present in pNWVT. The spoVT_BCE gene was cut out of pNWVT using BclI and HindIII and replaced with spoVT_BSU containing compatible sticky ends. Correct construction was verified using restriction analysis and sequencing.

For the construction of the B. cereus spoVT disruption mutant, we constructed the pDCVT vector by amplifying an upstream spoVT flanking region from B. cereus ATCC 14579 gDNA using primers dCVT-F1 and dCVT-R1. A downstream flanking region was amplified using primers dCVT-F2 and dCVT-R2. Both flanking regions were fused to HindIII compatible ends of a spectinomycin resistance cassette originating from pDG1726. The fused fragments were ligated into the NcoI and EcoRI sites of pMAD to create pDCVT. The pDCVT was introduced into B. cereus ATCC 14579 to disrupt the spoVT gene according to the method described by Arnaud et al. (2004). Disruption of spoVT was verified using PCR analysis and sequencing of the integration site to ensure there were no second-site mutations.

For the construction of a chromosomally integrated P_spoVA-gfp fusion in B. cereus, a 1.5 kb fragment of the upstream region of the B. cereus ATCC 14579 spoVAA gene (BC4070) was amplified using primers TIFN41 and TIFN42A. The TIFN42A primer was designed as such that the original RBS plus the first two codons of the spoVAA gene were included in the amplified fragment. This was then cleaved with EcoRI and KpnI and introduced into the corresponding sites of pSG1151. The resulting pSGCVA vector was introduced into B. cereus ATCC 14579 and B. cereus ATCC 14579 ΔspoVT via electroporation and checked for single crossover integration on the original locus using PCR analysis and sequencing.

A strain bearing an in-frame deletion of the ORF encoded at locus BC1117 was created using a marker-less gene replacement method (Janes and Stibitz, 2006; Lindbäck et al., 2012). Essentially, a modified pMAD-derived vector containing ~500 bp of the respective up- and downstream regions of DNA flanking the BC1117 ORF was introduced by electroporation to B. cereus ATCC 14579 cells. Plasmid pBKJ233, which encodes I-SceI enzyme, was introduced subsequently to cells identified by blue-white (X-gal) screening and PCR as having inserted the pMAD-derived plasmid at locus BC1117. Clones that had undergone a second recombination event, deleting the BC1117 ORF and leaving behind only the start and stop codons, were identified by screening for erythromycin sensitivity. Finally, the ΔBC1117 strain was validated for the correct construction by PCR.

All B. cereus strains were cultured at 30°C with aeration at 220 rpm. Media was supplemented with chloramphenicol (4 μg/ml), erythromycin (2 μg/ml), or spectinomycin (300 μg/ml) when appropriate. For the preparation of cells for time-lapse microscopy, cells were prepared as previously described (Eijlander and Kuipers, 2013). For all other sporulation experiments and for the preparation of sporulating cells for RNA isolation, cells were cultured in maltose sporulation medium (MSM) (van der Voort et al., 2010). Spore crops were prepared on MSM agar plates for 7 days at 30°C. Spores were collected from the plates and washed (6000 rpm, 15 min at 4°C) twice a day for 14 days in 10 mM phosphate buffer (pH 6.2) with decreasing concentrations of Tween20 to prevent clumping of the spores (0.1, 0.075, 0.05, 0.04, 0.03, 0.02, and 0.01%). Phase contrast microscopy analysis of the spore crops showed >98% free-lying spores. Spores were stored at 4°C in 10 mM phosphate buffer with 0.01% added Tween20 and were washed twice a week to prevent spontaneous germination.

The vectors pNWVT (containing spoVT_BCE expressed by its own promoter) and pVTBsu2 (containing spoVT_BSU expressed by the P_spoVTBCE promoter) were introduced in the B. cereus ATCC 14579 ΔspoVT strain by electroporation as described above. Overnight cultures were diluted 1:100 in MSM medium and incubated at 30°C while shaking for 6 h. Exponentially growing cells were again diluted 1:100 in fresh MSM medium and allowed to grow in the same conditions for 65 h. Samples for phase contrast microscopy analysis were taken after 17, 22, 44, and 65 h. Efficiency in complementation of sporulation was determined using the Cell Counter plugin in ImageJ¹.

B. cereus spores were washed twice before the experiment and resuspended in ice-cold sterile demineralized water. Spores were heat-activated at 70°C for 15 min and immediately placed on ice for 2 min to cool down. The washing step was repeated after which spores were resuspended in ice-cold sterile germination buffer (10 mM Tris-HCl 7.4 + 10 mM NaCl) to a final OD₆₀₀ of 10. Spores were diluted 10 times in germination buffer containing nutrients (10 mM alanine or 1 mM inosine) or BHI medium supplemented with chloramphenicol (5 μg/ml) to prevent outgrowth. Changes in optical density were monitored every 2 min for 4 h at 30°C in a TECAN plate reader and the obtained data plotted in Excel.

All microscopy imaging was performed using the IX71 Microscope (Olympus) with CoolSNAP HQ2 camera (Princeton Instruments) and DeltaVision softWoRx 3.6.0 (Applied Precision) software. For B. subtilis the 100x phase contrast objective was used, for B. cereus the 60x phase contrast objective. For the visualization of green fluorescence from GFP or FM46 dye the GFP filterset was used (Chroma, excitation at 470/40 nm, emission at 525/50 nm). Images were taken using 32% APLLC White LED light and 0.05 s exposure for bright field pictures and 10% Xenon light with 0.5 s exposure for fluorescence detection. Pictures were analyzed using ImageJ software.

To monitor gene expression in single cells during sporulation, the time-lapse microscopy technique was applied for the promoter-gfp fusion strains of B. cereus as previously described (Eijlander and Kuipers, 2013). For quantification of the GFP fluorescence signal distribution, the fluorescence intensity was measured for every cell in the microcolony for every time frame using the ImageJ ROI tool. Fluorescence intensities per cell per frame were exported to Excel and normalized by subtracting background fluorescence levels of the microscopy slide (agarose) and autofluorescence of the cells or spores (highest fluorescent value measured for wt strains). Mean fluorescence values were calculated from ten frames per cell and binned according to set fluorescence value categories. This data was represented in fluorescence distribution plots in Excel.

Cells were cultured in sporulation media to induce the sporulation event. Individual stages in sporulation were determined via fluorescence microscopy on agarose patches with added FM® 1–43 membrane stain (Invitrogen, Ex 479 nm, Em 598 nm) at an end concentration of 1 μg/μl.

Cell samples (5 ml, including biological and technical replicates) were taken every hour for 6 h from transition point onwards (T0, reached after 7 h of growth in MSM). Cells were collected by centrifugation in a pre-cooled centrifuge (4°C, 4000 rpm, 3 min) and immediately frozen in liquid nitrogen. For RNA extraction, cells were thawed on ice in 500 μl TRI-reagent (Life Technologies, Carlsbad, CA USA) and glass beads (<100 μm, Sigma-Aldrich), resuspended and immediately disrupted by 4 rounds of bead beating [45 s at maximal settings (3450 rpm)] in a Mini-Beadbeater-16 (BioSpec products, Bartlesville, OK USA). Direct-zol RNA MiniPrep (Zymo Research, Irvine, CA USA) was used according to manufacturer's instruction for on column RNA purification. Residual chromosomal DNA was removed using the Ambion DNA-free™ kit (Life Technologies®, Thermo Fisher Scientific, USA). The RNA concentration was measured on a NanoDrop ND-1000 spectrophotometer. RNA quality was determined using an Agilent BioAnalyzer RNA 6000 nanokit. The concentration of RNA isolated from wt sporulation cells was a little lower than for ΔspoVT sporulating cells (3000 ng/μl compared to 3800 ng/μl) but still comparable. Quality of all isolations was within the specified levels for all samples (an OD_260/280 ratio between 1.8 and 2.0 and an OD_260/230 ratio of >1.7).

RNA samples (> 1 μg) were sent to PrimBio Research Institute (Exton, PA, USA) where rRNA depletion (using the Ambion MICROBExpress™ Kit) and a library prep (using the Ion Total RNA-Seq Kit v2) were performed. Samples were multiplexed in sets of nine and loaded on an Ion Proton™ chip. In total 80 M reads with an average length of 120 bases were derived per chip. The reads were mapped against the reference genomes for B. cereus ATCC 14579 using Bowtie 2 (Langmead and Salzberg, 2012) with optimized parameters setting for Ion Proton data: “-D 20 -R 3 -N 1 -L 20 -i S, 1, 0.50 –local”. Gene expression profiling was done using SAMtools (Li et al., 2009) and Cufflinks (Trapnell et al., 2012). Subsequent statistical analysis was performed using a modified EdgeR routine on the MolGen webserver².

Spearman Rank correlation analysis was performed to show clustering of biological replicates within one strain and between strains. Correctly clustered data sets were further filtered based on intra-variation < inter-variation. Excel conditional formatting was applied to visualize the degree of intra- and inter-variation for each differentially expressed gene. The biological ratio in gene expression was calculated using log-transformed data in the following formula: Biological ratio=log2(wt1 + wt2)-log2(dVT1 + dVT2) where wt1 and wt2 are replicate rpkm values for the wt strain and dVT1 and dVT2 are replicate rpkm values for the ΔspoVT strain. The fold change in gene expression between the two strains was furthermore determined by raising the number 2 to the power of the absolute value for the ratio (2^ABS(ratio)).

Orthologs of B. subtilis sporulation genes (SporeWeb³, Eijlander et al., 2014) in B. cereus ATCC 14579 were determined using OrthoMCL (Chen et al., 2006) and (predicted) gene function using the KEGG database⁴ or NCBI Blast⁵.

All present knowledge on the SpoVT regulator protein so far originates from studies in B. subtilis. Cells of B. subtilis that lack SpoVT are still able to produce spores, albeit with a defective spore coat structure, an increased initial germination rate in response to nutrients and a severe defect in further germination and outgrowth (Bagyan et al., 1996; Ramirez-Peralta et al., 2012). Despite a strong conservation of SpoVT in endospore-forming bacteria, the effect of removing SpoVT from B. cereus cells (SpoVT_BCE) proves more severe. In this organism, a similar spoVT disruption mutant is unable to complete the sporulation process and produces very few, if any, free-lying spores with a severely defective morphology (Figure 2 ΔspoVT). Re-introduction of spoVT_BCE only partly restored this defect (14% of the total number of cells restored sporulation, Figure 2 ΔspoVT+). The partial complementation result could be due to differences in plasmid copy number between individual spores. Interestingly, similar complementation experiments with the spoVT wt allele of B. subtilis (spoVT_BSU driven by the PspoVT_BCE promoter) produced comparable results (complementation restored sporulation in 12% of the total number of cells, Figure 2, ΔspoVT +Bsu). This indicates that the working mechanism of SpoVT_BSU and SpoVT_BCE is comparable, which is expected given the high sequence conservation between both proteins. The observed difference in phenotypic effect after removal of SpoVT in both organisms must thus have a basis in the promoters of these regulatory proteins interact with to modulate gene expression.

Time-lapse fluorescence microscopy is a powerful technique to visualize dynamics in gene expression in time in individual cells and was recently optimized for application with B. cereus cells (Eijlander and Kuipers, 2013). In this study, we applied time-lapse fluorescence microscopy to follow the events of forespore-specific gene expression in time in a spoVT deletion background of B. cereus. Unfortunately, multiple attempts to construct a variety of sporulation-specific promoter-gfp fusions (e.g., P_gerI, P_cwlJ, P_gerR, P_cotD, P_sspA) stably integrated into the B. cereus genome failed, except for one (i.e., P_spoVA-gfp). This limited our ability to determine true effects of SpoVT removal on the expression levels of late sporulation genes, but did enable us to provide insights in the sporulation line of events in the absence of SpoVT.

Time-lapse image analysis of strain B. cereus ΔspoVT P_spoVA−BCE-gfp (Movie S2) clearly shows that during the earlier stages of sporulation prespores are formed like in the wild type background (Movie S1), but that progression of sporulation is stalled at a later stage. First, the formed pre-spores lyse, thereby spreading the produced GFP throughout the mother cell (Figure 3B and Movie S2). Finally, the mother cell also lyses (Figure 3A).

A quantitative analysis of the obtained images was performed to substantiate the difference in spoVA gene expression levels in a spoVT background. Before pre-spore lysis, the intensity of fluorescence originating from the P_spoVA-gfp fusion was significantly higher in the absence of SpoVT_BCE than in wild type cells (Figure 3C and Movies S1, S2). In addition, the distribution of the signal strength was more wide-spread amongst individual cells. It must be noted, however, that it is possible that this observed effect is not the result of increased (heterogeneity in) promoter activity in the absence of SpoVT, but rather an artifact in the detection of the GFP signal—the spoVT spores are severely weakened shortly after the completion of engulfment and, furthermore, it is known that SpoVT enhances rather than represses spoVA expression in B. subtilis (Wang et al., 2006; Ramirez-Peralta et al., 2012).

To further investigate the role of SpoVT in gene expression during sporulation of B. cereus, we isolated RNA from sporulating cells at three sequential time points (3, 4, and 5 h after the transition point to stationary growth was reached; Figure S1). Differentially expressed genes in the spoVT deletion strain were compared to the wild type situation using a transcriptomics approach. Details on RNA isolation, preparation, sequencing, downstream normalization, and statistical analysis of the resulting data are described in the Materials and Methods Section. Resulting normalized expression values [reads per kilobase per million (rpkm)] are provided in Table S2.

Sporulation is a very heterogeneous process, which complicates the synchronization of gene expression in individual cells. This generates a large variety in the absolute gene expression values generated by RNA sequencing. Spearman Rank correlation of the available data showed that two biological replicates of the same strain (wt or ΔspoVT) clustered together only for the samples taken at T5. Therefore, further data analysis was performed for that time point only.

The log2-transformed data was used to calculate the fold change in gene expression in the spoVT mutant strain compared to the wt strain. A total list of significantly up- and down-regulated genes is provided in Table S3.

Of the differentially expressed genes (with a cut-off of ≥2-fold difference), a total number of 52 genes were down-regulated and 87 genes were up-regulated (Table 2). Both groups contain genes involved in sporulation, germination, regulation, metabolism, and transport and both groups contain a large number of genes encoding hypothetical or unknown proteins. What stands out, is that genes involved in protein synthesis (ribosomal protein subunits) were specifically upregulated in a spoVT mutant. In addition, a large number of genes involved in metabolism was also upregulated.

Concerning sporulation and/or germination-specific genes, almost no B. cereus orthologs of known SpoVT-regulated genes (as determined by studies in B. subtilis; Bagyan et al., 1996; Wang et al., 2006) are present in this list. Only spoIIIG (BC3903) and tepA (BC3795) were, similarly to their counterparts in B. subtilis, repressed by SpoVT in B. cereus (Table S3). Amongst significantly down-regulated sporulation genes (>2-fold difference in gene expression) were some genes encoding spore coat proteins, one germination receptor subunit (GerIA) and a few sporulation proteins of unknown function. Upregulated sporulation genes, on the other hand, were those encoding CwlJ1, SpoIIQ, SpmA, CotD, YaaH, SpoIVA, SpoIIIAH, CdaS, SpoIIIAC, genes encoding sporulation-specific regulators RsfA, SigK, GerE, SigG, and AbrB and a few sporulation proteins of unknown function.

Interestingly, quite a few hypothetical or genes of unknown function were extremely, or at least considerably, downregulated in the spoVT deletion strain (Table 3). Although no putative function could be deducted from homology searches using their protein sequences, most of them seem to be specific for B. cereus group organisms. One such example is the gene encoded at locus BC1117, expression of which was down-regulated approximately 3000-fold in the spoVT mutant strain. BC1117 is predicted to encode a small (50 residue, ~6 KDa) lysine-rich polypeptide. A preliminary examination of the role of BC1117 in B. cereus ATCC 14579 was conducted by constructing a strain with a marker-less deletion in this locus. However, in contrast to the ΔspoVT strain, the ΔBC1117 strain was observed to grow and sporulate normally, releasing mature spores after approximately 24 h of culture (Figure S2).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.