Received 25 September 2009/Accepted 14 December 2009

0099-2240/10/$12.00

doi:10.1128/AEM.02317-09

Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Identification of the Main Promoter Directing Cereulide Biosynthesis

in Emetic

Bacillus cereus

and Its Application for Real-Time

Monitoring of

ces

Gene Expression in Foods

䌤

†

Monica K. Dommel,

1

# Elrike Frenzel,

1

# Bernd Strasser,

1

Claudia Blo

¨chinger,

1

Siegfried Scherer,

1,2

and Monika Ehling-Schulz

3

*

Microbiology Unit, Center for Nutrition and Food Research ZIEL, Technische Universita

¨t Mu

¨nchen, 85350 Freising, Germany

1

_;

Microbial Ecology Group, Department of Biosciences, Technische Universita

¨t Mu

¨nchen, 85350 Freising, Germany

2

_{; and}

Food Microbiology Unit, Clinic for Ruminants, Department for Farm Animals and

Veterinary Public Health, University of Veterinary Medicine Vienna,

1210 Vienna, Austria

3

Received 25 September 2009/Accepted 14 December 2009

Cereulide, the emetic

Bacillus cereus

toxin, is synthesized by cereulide synthetase via a nonribosomal peptide

synthetase (NRPS) mechanism. Previous studies focused on the identification, structural organization, and

biochemical characterization of the

ces

gene locus encoding cereulide synthetase; however, detailed information

about the transcriptional organization of the

ces

genes was lacking. The present study shows that the

ces-PTABCD

genes are transcribed as a 23-kb polycistronic transcript, while

cesH

, encoding a putative hydrolase,

is transcribed from its own promoter. Transcription initiation was mapped by primer extension and rapid

amplification of cDNA ends (RACE). Deletion analysis of promoter elements revealed a main promoter located

upstream of the

cesP

coding sequence, encoding a 4

ⴕ

-phosphopantetheinyl transferase. This promoter drives

transcription of

cesPTABCD

. In addition, intracistronic promoter regions in proximity to the translational

start sites of

cesB

and

cesT

were identified but were only weakly active under the chosen assay conditions. The

identified main promoter was amplified from the emetic reference strain

B. cereus

F4810/72 and fused to

luciferase genes in order to study promoter activity in complex environments and to establish a biomonitoring

system to assess cereulide production in different types of foods.

ces

promoter activity was strongly influenced

by the food matrix and varied by 5 orders of magnitude. The amount of cereulide toxin extracted from spiked

foods correlated well with the bioluminescence data, thus illustrating the potential of the established reporter

system for monitoring of

ces

gene expression in complex matrices.

Bacillus cereus

is the causative agent of two types of food

poisoning: diarrhea and emesis. The toxicoinfection referred

to as diarrheal disease occurs after consumption of

B. cereus

spores or vegetative cells, most likely due to the action of

heat-labile enterotoxins, which are produced by cells

multiply-ing in the small intestine (4, 22, 30, 39). Contrarily, the emetic

type of food-borne illness is caused by intoxication with the

heat-stable peptide cereulide, which is preformed in foods and

elicits vomiting a few hours after ingestion (18, 51, 58).

Regulation of enterotoxin expression of

B. cereus

has been

studied in some detail (e.g., references 16, 29, and 46; for a

review, see reference 58), revealing a major role of the

pleio-tropic transcription regulator PlcR in activation of enterotoxin

genes and other virulence factors (1, 29, 45). Cereulide

syn-thesis in emetic

B. cereus

, in contrast to synthesis of

B. cereus

enterotoxins, is not controlled by PlcR but by the Spo0A

phos-phorelay. The global transition state factor AbrB was identified

as one factor repressing cereulide production in early

expo-nential phase (38). Although

B. cereus

enterotoxins and the

emetic toxin cereulide seem to belong to completely different

regulatory networks, production of both types of toxins

de-pends substantially on nutritional and environmental factors

(3, 46, 55), and it is expected that great effort is required to

unravel in detail the intrinsic and extrinsic factors and

mech-anisms controlling toxin expression in

B. cereus

.

Cereulide, which is produced by specific subgroups of

B.

cereus

(20, 62) and a few

Bacillus weihenstephanensis

isolates

(60), is a small, acid- and proteolytically stable cyclic

dodeca-depsipeptide that is structurally related to the potassium

iono-phore valinomycin (2). It is toxic to mitochondria and has been

implicated in severe forms of food poisoning resulting in acute

liver failures (14, 41, 48). The peptide toxin [

D

-

O

-Leu-

D

-Ala-L

-

O

-Val-

L

-Val]

₃

is synthesized enzymatically by a

nonriboso-mal peptide synthetase (NRPS) (21).

NRPSs are responsible for catalyzing the synthesis of a broad

range of bioactive low-molecular-weight peptides, with chain

lengths of 2 to 48 residues (42). Several

Bacillus

species produce

siderophores and peptide antibiotics via NRPSs, such as the

broadly distributed bacillibactin-related siderophores or

surfac-tins or the species-specific antibiotics tyrocidine and gramicidin S

in

Bacillus brevis

(for a review, see references 13, 24, and 57);

however, few transcriptional analyses of the corresponding

bio-synthetic enzyme complexes have been performed.

* Corresponding author. Mailing address: Food Microbiology Unit,

Clinic for Ruminants, Department for Farm Animals and Veterinary

Public Health, University of Veterinary Medicine Vienna, 1210

Vi-enna, Austria. Phone: 43-1-25077-5223. Fax: 43-1-25077-5291. E-mail:

[email protected].

# These authors contributed equally to this work.

† Supplemental material for this article may be found at http://aem

.asm.org/.

䌤

_{Published ahead of print on 28 December 2009.}

1232

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The complete genetic locus encoding cereulide

biosyn-thetase (

ces

) in the emetic

B. cereus

reference strain F4810/72

is a 24-kb gene cluster located on a megaplasmid (pBCE) that

shows high homology to the

B. anthracis

toxin plasmid pXO1

(17). Besides the typical genes, such as a gene encoding a

4

⬘

-phosphopantetheinyl (4

⬘

-PP) transferase (

cesP

) essential for

priming the NRPS, a gene encoding a putative type II

thioes-terase (

cesT

) which removes misprimed monomers, and the

structural genes responsible for the assembly of the peptide

product (

cesA

and

cesB

), it includes a coding sequence (CDS)

encoding a putative hydrolase (

cesH

) in the 5

⬘

region and a

putative ABC transporter (

cesC

and

cesD

) in the downstream

part (17). The cereulide NRPS is unique in that the substrates

for the

cesA1

(

D

-

O

-Leu) and

cesB1

(

L

-

O

-Val) modules are

␣

-keto acids which are then chirally reduced (40).

Although the genetic locus responsible for cereulide

produc-tion has been identified and partially characterized,

informa-tion about the transcripinforma-tional organizainforma-tion of the

ces

operon is

still lacking. Such information is crucial for the understanding

of cereulide toxin biosynthesis and might also contribute to the

fundamental knowledge of nonribosomal biosynthetic

path-ways. A complete transcriptional analysis of the

ces

gene

clus-ter was carried out by using reverse transcription-PCR

(RT-PCR), rapid amplification of cDNA ends (RACE), and

promoter deletion analysis to identify the promoters and

char-acterize the

ces

transcripts. Our studies revealed a central

promoter that drives the polycistronic transcript of

cesPTABCD

.

This promoter sequence was used to establish a bioluminescence

reporter system for noninvasive real-time monitoring of cereulide

synthetase promoter activity in different environments. This is a

first step toward deciphering conditions at a transcriptional level

that are favorable or unfavorable for emetic toxin production in

food. Such data may contribute to reducing the risk and incidence

of food-borne disease.

MATERIALS AND METHODS

Bacterial strains and culture conditions.The emetic reference strainBacillus cereusF4810/72 (AH187) (61) was routinely cultivated on standard plate count agar (5 g peptone, 2.5 g yeast extract, 1 g glucose per liter) at 30°C. For experiments with food,B. cereusF4810/72 transformed with pMDX[P1/luxABCDE] was cultured in

lysogeny broth (LB) (5) for 16 h at 30°C with 150-rpm rotary shaking.Escherichia coliwas routinely grown at 37°C in LB. When appropriate, chloramphenicol (5 ␮g ml⫺1_{) or ampicillin (100␮g ml}⫺1_{) was added.}

Sequence analysis.Putative transcriptional start sites and transcription factor binding sites were searched using promoter prediction at http://www.fruitfly.org /seq_tools/promoter.html (53) and the DBTBS search tool at http://dbtbs.hgc.jp/ (32). Termination structures were analyzed using the Heidelberg Unix Sequence Analysis Resource (7) at http://genius.embnet.dkfz-heidelberg.de and Mfold (64) at http://www.bioinfo.rpi.edu/applications/mfold/. Frameshift slippery sites were analyzed using FSFinder at http://wilab.inha.ac.kr/FSFinder/ (44).

PCR.PCR was used to amplify putative promoter regions both for cloning into vectors to produce sequencing ladders for primer extension and for cloning into fusion vectors to test promoter activity. The 50-␮l PCR mixture (10 ng DNA, 0.5 ␮M each primer, 1.5 mM MgCl2, 0.4 mM each deoxynucleoside triphosphate

[dNTP], 1.25 U ThermoStartTaqpolymerase [all from ABgene]) was activated (95°C for 15 min), followed by 30 amplification cycles (95°C for 30 s, 60°C for 45 s, and 72°C for 1 min) and an elongation step (72°C for 5 min).

Nucleic acid isolation.Total DNA was isolated as described previously (19), and plasmid DNA was prepared using standard procedures. RNA samples for RT-PCR, primer extension, and 5⬘RACE were taken during mid-exponential-phase growth: cells were collected (10,000⫻gat 4°C for 2 min), frozen in liquid nitrogen, and stored at⫺80°C. Total RNA was isolated by an adapted plant tissue protocol (37) after cell disruption by using 0.1-mm zirconia-silica beads (Carl Roth GmbH & Co. KG) and a RiboLyser (Hybaid). Contaminating DNA

was removed with 10 U RQ1 DNase (Promega). Subsequently, RNA was iso-lated by chloroform extraction and ethanol precipitation, resuspended in diethyl pyrocarbonate (DEPC)-treated double-distilled water (ddH2O), and stored at

⫺80°C. RNA purity and quantity were determined by measurement of absor-bance at 260 nm and 280 nm and by agarose gel electrophoresis. Efficacy of DNA removal was confirmed using RNA as the PCR template with 16S primers 16SA1 (5⬘-GGAGGAAGGTGGGGATGACG-3⬘) and 16SA2 (5⬘-ATGGTGTGACG GGCGGTGTG-3⬘) (43).

RT and RT-PCR.RT-PCR was used to investigate thecestranscript. cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen). In 10␮l, 100 ng total RNA, 2 pmol gene-specific reverse primer (see Table S1 in the supplemental material), and 1␮l 10 mM each dNTP were incubated at 75°C for 2 min, 70°C for 1 min, 65°C for 1 min, 60°C for 1 min, and 55°C for 1 min. At 55°C, 10␮l of RT mix (4␮l 5⫻RT buffer, 1␮l 0.1 M dithiothreitol [DTT], 100 U SuperScript III, 20 U RNaseOUT [all from Invitrogen]) was added, and the reaction mix was incubated at 55°C for 1 h, before inactivation at 70°C for 15 min. For negative controls, reverse transcriptase was omitted. Subsequent PCRs using a 2-␮l RT reaction volume (template) with 50 pmol each primer (Table S1) in 50 ␮l were carried out as described above. Fragments of expected lengths greater than 2 kb were amplified using the Expand high-fidelity PCR system (Roche Applied Science) by following the manufacturer’s instructions. All RT-PCR experiments were repeated with RNA samples from several independent cul-tures.

Transcript stability.To investigate the stability of thecesAtranscript, cultures were grown to mid-exponential phase and samples were taken at time zero (before rifampin addition) and in 10-min increments (up to 60 min) after the addition of 200␮g ml⫺1

rifampin. RNA was isolated, and cDNA synthesized as described above was used as the template for real-time PCR. Real-time quan-titative PCR (qPCR) was performed using a SmartCycler (Cepheid). Reaction volumes contained 1␮l cDNA (equivalent to 10 ng total RNA) and 80 nM each primer in qPCR master mix with SYBR green I (ABgene) in a total volume of 25␮l. Activation (15 min at 95°C) was followed by 40 amplification cycles with a temperature ramp rate of 2.5°C s⫺1_{(95°C for 30 s; melting temperature [T}

m] for 30 s with optics on; 72°C for 45 s) and a melt curve analysis from theTmto 95°C at 0.2°C s⫺1_{with optics on. (TheT}

mforcesAis 53°C, and that for the 16S rRNA gene is 63°C.) Relative expression was determined using primers forces

(cesA_for [5⬘-GATTACGTTCGATTATTTGAAG-3⬘] and cesA_rev [5⬘-CGTA GTGGCAATTTCGCAT-3⬘]) and normalized to 16S rRNA genes by using primers previously reported (43). Relative expression was calculated by the REST (relative expression software tool) method of analysis (47), using time point zero as the calibrator.

RACE.RACE was performed to find transcriptional start sites. The 5⬘RACE System for Rapid Amplification of cDNA Ends, version 2.0 (Invitrogen), was used with the indicated primers (see Table S2 in the supplemental material); cDNA was created using SuperScript III as described above. RACE products were visualized by agarose gel electrophoresis, TOPO TA cloned (Invitrogen), and sequenced to determine the transcription start site. All RACE experiments were repeated several times with RNA samples from independent cultures.

PE.Transcriptional start sites were confirmed using primer extension (PE). cDNA was synthesized by using 10 pmol IRD800-labeled gene-specific reverse primer (Table S2) and 10␮g RNA in a 7-␮l reaction volume and incubating at 75°C for 2 min, 70°C for 1 min, 65°C for 1 min, 60°C for 1 min, and 55°C for 1 min. Thirteen microliters of RT mix (4␮l 5⫻RT buffer, 1␮l 0.1 M DTT, 2␮l 10 mM dNTPs, 200 U SuperScript III, 20 U RNaseOUT [all from Invitrogen]) was added, and the reaction volume was incubated at 55°C for 1 h before inactivation (70°C for 15 min). For analysis, 9␮l Sequitherm Excel II stop/ loading buffer (Epicentre) was added, incubated at 92°C for 3 min, and loaded onto an 8% urea polyacrylamide gel in a LiCor 4200 sequencer (LiCor Bio-sciences). As markers and ladders on the gels, 1-kb regions containing putative promoters were PCR amplified, TOPO TA cloned into pCR2.1 (Invitrogen), and sequenced using IRD800-labeled primers with the SequiTherm Excel II DNA sequencing kit (Epicentre).

Construction of GFP transcriptional fusions andin vitrofluorescence mea-surements.Vector pAD123 with a promoterless red-shifted green fluorescent protein (GFP) gene (gfpmut3a) (11) was used for promoter studies. pAD43-25 with a constitutive promoter controllinggfpmut3aserved as a positive control (15). In brief, putativecespromoter regions and sequentially deleted putative promoters were PCR amplified and directionally cloned in the multiple-cloning site of pAD123, giving rise to the pMKD plasmids shown in Table 1.E. coli

TOP10 (Invitrogen) was used for cloning steps, and correct ligation was con-firmed by colony PCR and restriction analysis. The plasmids were passaged through the methylase-deficientE. colistrain INV110 (Invitrogen) and electro-porated intoB. cereusF4810/72 as described elsewhere (21).B. cereusmain

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cultures bearing the recombinantgfpmut3apromoter probe vectors were inocu-lated with 100␮l of a 100-fold-diluted preculture and then cultured at 30°C and 150 rpm for 24 h. Cells were harvested by centrifugation (8,000⫻gfor 2 min at 4°C), washed once with phosphate-buffered saline, and collected by centrifuga-tion. Fluorescence was measured as described previously (8). Fluorescence val-ues (relative fluorescence units [RFU]) were normalized to the culture optical density at 600 nm (OD600) at the time of sampling. Background fluorescence

from strains transformed with promoterless pAD123 was recorded.

Assembly of thecespromoter-luciferase reporter system.The promoterless luciferaseluxABCDEvector pXen1 (27), optimized to study gene regulation in Gram-positive bacteria, was used as a basis for constructing a bioreporter system for monitoringcestoxin gene transcription. A 238-bp region of the main cereu-lide synthetase promoter was amplified by PCR from genomic DNA ofBacillus cereusF4810/72 by using the primers Pshort_for (5⬘-TAGAGAATTCCTGTTA

GCCAATATAACGAGTC-3⬘) and Pshort_rev (5⬘-TTCCGGATCCTTCATTG AAAAATCCCTCT-3⬘) (EcoRI and BamHI restriction sites are underlined). This region corresponds to nucleotides (nt) 36891 to 37128 on the plasmid pCER270 (also designated pBCE) (GenBank accession number DQ889676 [52]). The fragment was cloned into the EcoRI and BamHI sites of the plasmid pXen1, creating pMDX[P1/luxABCDE], and further amplified inE. coliTOP10

(Invitrogen). The construct was then passaged through nonmethylatingE. coli

INV110 (Invitrogen) prior to transfer intoB. cereusF4810/72 by electroporation, giving rise to F4810/72(pMDX[P1/luxABCDE]). To exclude cryptic promoter

activity,B. cereusF4810/72 with a promoterless luciferase construct was used as a negative control. Furthermore, the luciferase reporter plasmid was used to transform two other emetic strains by electroporation: one clinical isolate and one food isolate from a recent emetic outbreak caused by a rice dish. All constructs were verified by restriction analysis and double-strand sequencing.

Inoculation of growth media.Brain heart infusion (BHI; Merck), Trypticase soy agar (TSA; Merck), fortified nutrient agar (FNA; Oxoid), Columbia blood agar (Oxoid), and BCMBacillus cereusgroup plating medium (Biosynth) were obtained from diverse distributors as prepared media, while the standard plate count (PC) medium, sodium lactate medium (27 ml of a 50% sodium lactate solution, 10 g yeast extract, 10 g peptone, 5 g KH2PO4per liter), and

mannitol egg yolk polymyxin agar (MYP) (1 g beef extract, 10 g peptone, 10 g mannitol, 10 g sodium chloride, 25 mg phenol red, 100 ml egg yolk solution, 100,000 IU polymyxin B supplement per liter) were prepared using standard chemicals and ingredients. All media were solidified with 15 g agar per liter. The media were inoculated with two 25-␮l drops of overnight cultures ofB. cereusF4810/72(pMDX[P1/luxABCDE]) and the negative control strain,B. cereus(pXen1[luxABCDE]), and incubated at 24°C for 24 h.

Inoculation of food samples.Various foods were obtained from local con-sumer markets and prepared according to the manufacturer’s instructions if necessary. Under sterile conditions, 30-g portions were filled in petri dishes and were spot inoculated with four 25-␮l drops ofB. cereusF4810/72 orB. cereus

pMDX[P1/luxABCDE] overnight cultures. Plates were sealed to prevent

mois-ture evaporation and were incubated for 24 h at 24°C. The initial cell inoculum per gram of food was determined by plating appropriate dilutions of the over-night culture on LB.

Growth ofBacillus cereusin food.Spiked food samples (30 g) were commi-nuted for 1 min with 270 ml of a 0.025% Tween 80 solution (pH 7.0) using a stomacher. The homogenates were serially 10-fold diluted in LB, and 100␮l was spread in duplicate onto LB plates. For growth experiments with bioluminescent

B. cereus, LB agar with 5␮g ml⫺1

chloramphenicol was used. Colonies showing the typical morphology of emeticBacillus cereuswere counted after 16 to 20 h of incubation at 30°C. The increase of viable cell counts was calculated as logarith-mical growth units per gram of food.

Luciferase (lux) assay. Luciferase activity of F4810/72 transformed with pMDX[P1/luxABCDE] was visualized on growth media and on food with a

photon-counting intensified-charge-coupled-device (ICCD) camera (model 2400-32; Hamamatsu Photonics). Images were acquired for 2 s with a binning factor of 1 (without filter; relative aperture, 1), and the bioluminescence intensity was superimposed as false-color renderings. For quantification of the biolumi-nescence signals, region of interest (ROI) analysis was performed according to the manufacturer’s instructions by using Living Image 2.10 software (Caliper Life Sciences) along with the IGOR Pro 4.01 software (WaveMetrics). In brief, the circle ROI tool was used to manually define the entire petri dish area as the ROI (dimensions were kept constant throughout all experiments), and the biolumi-nescence intensities was recorded as the total photon counts for all pixels inside the ROI (total counts). An empty area in the image was included as the average background ROI to correct for autofluorescence and was used for background correction of the signals.

Determination of cereulide contents in spiked food samples.Whole portions of inoculated food were extracted with 20 ml of 96% ethanol for 24 h at room temperature on a rotary shaker. Extracts were centrifuged twice (9,000⫻gfor 20 min at 24°C). To estimate the toxin recovery rate, controls were spiked with valinomycin (Fluka) to a final concentration of 25␮g g⫺1_{, left for 1 h at room}

temperature, and extracted as described above. The HEp-2 cell-based bioassay was carried out as described previously (38). Additionally, extracts of control food samples were measured in order to exclude an interfering toxic background effect of the different ethanolic extracts on the HEp-2 cells. For calculation of the recovery rate of the toxins from food, samples were spiked with valinomycin and extracted as described above.

RESULTS

Polycistronic transcript of

ces

genes.

A complete

transcrip-tional analysis using RT-PCR on the

ces

gene cluster showed

that the

cesPTABCD

gene cluster forms a polycistronic operon.

As RT-PCR primers were designed such that the amplicons

overlap, the RT-PCR analysis covered the entire

ces

gene

cluster (Fig. 1A). A transcript was observed between the genes

encoding the modules incorporating

D

-

O

-Leu and

D

-Ala (

cesA

)

and

L

-

O

-Val and

L

-Val (

cesB

), and another transcript was

ob-served in the noncoding regions between the CDSs for

cesP

,

cesT

,

cesA

,

cesB

,

cesC

, and

cesD

(Fig. 1B) but not between

cesH

and

cesP

. PCR amplicons from the cDNA and DNA used as

the positive control were the same size, whereas control

reac-tions using RNA produced no amplicon. No transcript was

detected by using a forward primer in the

cesD

coding region

with a reverse primer located after a predicted hairpin

down-stream of

cesD

(Fig. 1B, lane 10), indicating that this inverted

repeat represents the terminator of

ces

gene transcription.

Thus,

cesPTABCD

are transcribed as a single 23-kb transcript,

while

cesH

is transcribed as monocistronic transcript from its

own promoter. As bacteria regulate virulence factor expression

not only at the level of transcription initiation but also by

mRNA turnover rates, the decay rate of the mRNA of the

main

ces

transcript was assessed to determine its stability. The

ces

transcript was relatively short-lived. Eighty percent of

cesA

mRNAs were degraded within 10 min (data not shown).

Transcription initiation sites for the

ces

gene cluster

mapped by RACE.

Transcription initiation sites were mapped

using 5

⬘

RACE (Fig. 2) and were confirmed by primer

exten-sion (data not shown). Analysis of RNA from liquid cultures

grown to mid-log phase by using RACE revealed a

transcrip-tion start site for

cesH

76 bp upstream of the translational start

site (corresponding to position 3266 in the

ces

sequence

TABLE 1. Promoter fusion vectors

Vector Promoter Promoter regiona

Mean promoter activity⫾SD (RFU/OD600)

pAD123

b

_None

₇₄

_⫾

₃₈

pAD43-25

b

_upp

₉₇₆

_⫾

₄₃₄

pMKD

关

P

1

/

gfpmut3a

兴

P

1

4909–5146

10,342

⫾

765

pMKD

关

P

2

/

gfpmut3a

兴

P

2

4798–4930

74

⫾

11

pMKD

关

P

1

-P

2

/

gfpmut3a

兴

P

1

and P

2

4798–5146

8,499

⫾

248

pMKD

关

P

0

/

gfpmut3a

兴

P

0

(3

⬘

region

of P

1

)

5060–5146

147

⫾

68

pMKD

关

P

H

/

gfpmut3a

兴

P

H

3173–3346

526

⫾

38

pMKD

关

P

T

/

gfpmut3a

兴

P

T

6081–6170

65

⫾

29

pMKD

关

P

B2

/

gfpmut3a

兴

P

B

16733–17612

232

⫾

88

a_{nt positions in thecessequence (GenBank DQ360825).} b_{From reference 15.}

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[GenBank DQ360825 {17}]) and two transcriptional start sites

for

cesP

, located 100 bp upstream (P

₁

) and 256 bp (P

₂

)

up-stream of the translational start (Fig. 2). In addition,

intracis-tronic promoters that might enhance transcription or be active

under specific conditions were detected 56 bp upstream of

cesT

and 80 bp upstream of

cesB

(corresponding to position 6110

[

cesT

] and position 17248 [

cesB

] in the

ces

sequence [GenBank

DQ360825]). In contrast, no intracistronic promoters were

de-tected for

cesA

or

cesC

(neither by RACE nor by PE).

Transcriptional promoter fusions.

To investigate the active

promoter via deletion analysis, the putative promoter regions

were PCR amplified and cloned into pAD123 (15) upstream of

the GFP gene

gfpmut3a

(11). An overview of the

transcrip-tional fusions and their promoter activities is provided in Table

1. The fusion of a DNA fragment designated P

₁

upstream of

the

cesP

gene produced the strongest fluorescence signals,

which were about 5- to 10-fold higher than the fluorescence

signals from the constitutive promoter

upp

, used as the positive

control. P

₂

alone was not active, and P

₀

, a sequence

down-stream of P

1

, did not show activity, either. The

cesH

,

cesT

, and

cesB

promoters were only weakly active under the growth

con-ditions tested.

cesH

promoter activity was in the same range as

FIG. 1. Transcriptional analysis of the

ces

gene cluster. (A) Bars indicate overlapping primer pairs used for transcriptional analysis of the

ces

operon (listed in Table S1 in the supplemental material). Bent arrows indicate promoters as determined by primer extension. (B) RT-PCR showed

the presence of consecutive transcripts between

cesP

and

cesD

(lane 3 to 9) but no transcripts between

cesH

and

cesP

(lane 2). There were no

transcripts from a forward primer in

cesD

and reverse primer downstream of

cesD

after the predicted hairpin termination structure (lane 10).

Negative controls (RNA; see lane H-) and positive controls (DNA; see lane H

⫹

). M, marker ladder mixture (Fermentas).

FIG. 2. 5

⬘

RACE mapping of

ces

promoter sites. RACE products detected for the

ces

genes indicate intracistronic promoters present (

cesT

and

cesB

). Central promoters upstream of

cesP

(P

1

and P

2

), indicated by an asterisk, and the

cesP

promoter region are depicted. Transcription (

⫹

1)

sites and translation start sites are boxed. A putative ribosome binding site (RBS) and putative

⫺

10 and

⫺

35

␴

A

_{recognition sites (deduced from}

the consensus sites for

B. subtilis

) are underlined. Putative transcription starts determined by RACE were confirmed by primer extension. No

intracistronic promoter was detected for

cesA

(by RACE or by PE). M, marker ladder mixture (Fermentas).

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the positive control, while the intracistronic

cesB

promoter

fragment showed about 50% of the fluorescence signal

ob-served for the positive control. The

cesT

promoter fusion

yielded a fluorescence signal of about 20% of the signal from

the positive control. Therefore, P

1

appears to be the main

promoter active under these culture conditions.

Real-time monitoring of cereulide synthetase promoter

ac-tivity by using a bioluminescent

B. cereus

reporter strain.

To

monitor the activity of the main P

1

promoter of the

ces

gene

cluster in different environments, a 238-bp region of the

cereu-lide synthetase promoter P

1

was fused to a luciferase cassette

on the pXen1 plasmid, creating pMDX[P

₁

/

luxABCDE

] (see

Materials and Methods for details). F4810/72 transformed with

a promoterless luciferase construct was used as the negative

control. After spot inoculation of media of diverse nutrient

compositions, the reporter strain could be easily detected with

the ICCD camera system, whereas luminescence was not

emit-ted by the negative control strain (see Fig. S1 in supplemental

material). The intensity of the luminescence signals was

strongly dependent on the growth substrates available,

indicat-ing that transcription of the cereulide synthetase genes was

promoted by media containing carbohydrates (MYP, PC

me-dium, and BCM agar), while the P

₁

promoter activity was lower

on media such as Columbia agar, BHI, and TSA, which contain

larger amounts of rich proteinaceous ingredients (e.g., sheep

blood, brain heart infusion, and peptones [

ⱖ

20 g liter

⫺1

_]).

Media which promoted P

1

activity also contained yeast or meat

extracts (MYP, PC medium, LB, BCM, and FNA) and/or

larger amounts of sodium chloride (MYP and LB).

Categorization of foods by cereulide production potential.

To evaluate the suitability of the luciferase reporter strain for

analyzing the risk of cereulide formation in foods, a selection

of food products with different nutrient compositions was

in-oculated with four 25-

␮

l drops of a bioluminescent reporter

strain culture and incubated at 24°C, mimicking temperature

abuse. To further assess the influence of the luciferase reporter

plasmid on cell multiplication, viable cells for both wild-type

B.

cereus

F4810/72 and the bioluminescent derivative strain were

enumerated after growth in different model food systems by

conventional plate counting. Almost identical cell numbers

were observed for the two strains after incubation for 24 h at

24°C (Table 2). To determine the activity of the P

₁

promoter in

the artificially inoculated foods, software-assisted

quantifica-tions of the bioluminescence signals were performed using a

Hamamatsu image collector and the Living Image software

along with IGOR Pro 4.01 software (for details, see Materials

and Methods). According to the transcription efficiency of the

ces

gene cluster, foods were categorized into three main classes

depending on their potential to support cereulide production:

high-risk, risk, and low-risk foods (Fig. 3A and Table 2). To

correlate the P

₁

-driven transcript synthesis with the amount of

cereulide produced by wild-type

B. cereus

F4810/72, the toxin

quantity was determined with the HEp-2 assay. As illustrated

in Table 2, cereulide amounts correspond to the

luminescence-defined risk categories.

To further evaluate the functionality of the luciferase-P

1

construct in different genetic backgrounds, two other emetic

B.

cereus

strains were grown on rice, and luminescence was

re-corded with the ICCD camera. As shown in Fig. 3B, P

₁

was

also active in the clinical isolate as well as in a recently isolated

emetic food-borne outbreak strain.

DISCUSSION

Central promoter drives polycistronic transcription of the

Bacillus cereus

cereulide toxin genes.

The transcriptional

anal-ysis of the

ces

gene locus presented here shows that

cesH

is

transcribed from its own promoter, while the

cesPTABCD

genes are cotranscribed as a single large 23-kb polycistronic

transcript (Fig. 1). A hairpin structure, predicted by Mfold and

confirmed by real-time RT-PCR analysis (Fig. 1), directly

downstream of

cesD

represents the terminator of the

ces

operon. Other large NRPS gene clusters are also expressed as

polycistronic transcripts (see, e.g., references 12, 36, and 63),

thus favoring coordinated expression of related genes. Due to

the size of the NRP synthesis machinery and the wide range of

unusual substrates used by NRPS, timing of transcription of

NRPS genes is of special importance. Currently, over 350

non-proteinogenic amino acids are known, and most of them are

activated and incorporated into microbial NRP metabolites

(9). It would be energetically wasteful if the biosynthetic

machin-eries required for NRP production were not tightly regulated and

TABLE 2. Correlation of cereulide synthetase promoter activity, cell counts, and cereulide production of emetic

B. cereus

F4810/72 in different foods after 24 h at 24°C

a

Food pH

MeanB. cereuscell count⫾SD (log CFU g⫺1

) _P

1activity

(total counts in ROI)b

Mean cereulide concn⫾SD

(␮g g⫺1₎

Categoryc F4810/72 _(pMDX关PF4810/72

1/luxABCDE兴)

Béarnaise sauce

5.8

7.4

⫾

0.1

7.3

⫾

0.2

8.2E

⫹

06

8.0

⫾

1.6

HR

Liver sausage

6.2

7.9

⫾

0.0

7.9

⫾

0.2

3.1E

⫹

05

2.4

⫾

1.7

HR

Cooked rice

7.0

7.6

⫾

0.1

7.5

⫾

0.2

1.9E

⫹

06

1.8

⫾

0.6

HR

Camembert cheese

7.9

6.7

⫾

0.1

6.9

⫾

0.4

6.7E

⫹

04

0.6

⫾

0.4

R

Quark dessert with vanilla

flavor

5.1

6.0

⫾

0.0

6.0

⫾

0.3

2.3E

⫹

04

1.0

⫾

0.4

R

Pastry snack with sweetened

milk filling

5.9

6.2

⫾

0.4

5.9

⫾

0.3

8.3E

⫹

02

ND

LR

Cre

`me fraiche

4.5

4.5

⫾

0.1

4.3

⫾

0.3

7.1E

⫹

02

ND

LR

a

Tests were performed in triplicate. ND, not detectable. b

Average of three independent measurements. c

Potential to support cereulide synthesis. HR, high-risk food; R, risk food; LR, low-risk food.

on January 9, 2021 by guest

http://aem.asm.org/

temporally coordinated. Surprisingly, the main

ces

promoter is

located upstream of

cesP

and not

cesA

(Fig. 1 and 2), although

4

⬘

-PP transferase domains are highly conserved and not

neces-sarily specific to their respective NPRS. In gramicidin S, which is

produced by

Bacillus brevis

, the 4

⬘

-PP transferase gene (

gsp

) is

located upstream of the structural

grsTAB

genes and transcribed

from its own promoter P

_gsp

(36) yet can be used to complement

in

trans

the deleted 4

⬘

-PP gene (

sfp

) required for surfactin

bio-synthesis (6). In the case of cereulide, it appears that the 4

⬘

-PP

transferase encoded by

cesP

forms an integral part of the

poly-cistronic

ces

gene cluster and the

cesP

promoter is essential for

transcription of the

ces

gene cluster. Several

⫹

1 frameshifts occur

in the

ces

operon, and although

⫹

1 frameshift mutations are less

common than

⫺

1 frameshift mutations, they have been described

for both prokaryotes and eukaryotes (23).

The function of internal promoters is unknown.

In addition

to the central

cesP

promoter, intracistronic promoters for

cesT

and

cesB

, but not for

cesA

, were found. However, as shown by

GFP promoter fusions, the internal promoters were only weakly

active (Table 1). The occurrence of unusually long NRPS gene

transcripts with multiple promoters and long leader sequences

has been previously described for the

mcy

gene cluster

responsi-ble for microcystin biosynthesis in

Microcystis aeruginosa

(34);

however, regulation of the intracistronic promoters and their

im-pact on transcription are still cryptic. Intracistronic promoters,

such as those found for

cesT

and

cesB

, might ensure adequate

expression of distal genes and/or might be activated under

par-ticular growth conditions.

Bioluminescent reporter system for real-time monitoring of

ces

gene transcription.

In this study, the

in situ

application of

FIG. 3. Real-time monitoring of cereulide synthetase promoter activity in food. (A) The bioluminescent reporter strain

B. cereus

F4810/

72(pMDX[P

1

/

luxABCDE

]) was inoculated into various foods. After incubation for 24 h at 24°C, the luciferase gene expression controlled by the

main cereulide synthetase promoter P

1

was visualized with a photon-counting ICCD camera. According to the intensity of luminescence signals,

foods were designated as low-risk, risk, and high-risk products depending on their potential to support cereulide synthesis (Table 2). (B) P

1

promoter activity in different emetic strains on cooked rice. Depicted are the emetic reference strain F4810/72, one clinical isolate, and one isolate

from a recent food-borne emetic outbreak transformed with the pMDX[P

1

/

luxABCDE

] luciferase reporter plasmid.

on January 9, 2021 by guest

http://aem.asm.org/

the

B. cereus

F4810/72(pMDX[P

₁

/

luxABCDE

]) reporter

strain allowed a direct monitoring of

ces

promoter activity in

complex environments. The results from various growth

me-dia and foods illustrated that the influence of environmental

parameters is reflected by gradual promoter responses,

which were displayed at the various intensities of

biolumi-nescence signals (Table 2 and Fig. 3A; see also Fig. S1 in the

supplemental material). The activity of the P

₁

promoter was

strongly dependent on the nutrients provided. Thus, the

lux

reporter system described here visualizes the findings of

previous studies reporting substantial variances in cereulide

production on different growth media for a certain strain

(50, 59). Media routinely used for

B. cereus

detection and

experimental procedures (MYP, PC medium, LB, and

BCM) led to higher P

₁

activities than protein-enriched

me-dia such as Columbia blood, TSA, and BHI agar (see Fig. S1

in the supplemental material). However, blood, TSA, and

BHI agar are still frequently used to study cereulide

pro-duction by emetic

B. cereus

, though it was reported earlier

that less toxin was detected than with, e.g., skim milk- or

rice-based media (26, 59). Correspondingly, high promoter

activity was detected in the carbohydrate-rich cooked rice,

while only intermediate promoter activity was observed in

the proteinaceous Camembert cheese (Fig. 3 and Table 2),

although final cell numbers of the

lux

reporter strain in the two

model food systems did not significantly differ. These findings

correlate with reports of emetic food poisoning cases that

implicate farinaceous rather than proteinaceous foods (3, 14,

18, 41) and underscore the risk of emetic intoxications, for

instance, in mass catering quantities of rice- or pasta-based

dishes that have been improperly cooled.

B. cereus

F4810/72 was originally isolated from rice

in-volved in an emetic food poisoning case in the United

King-dom in 1972 (61). As decades of laboratory culturing might

have modified the original features of this strain, two other

recently obtained emetic isolates were transformed with the

luciferase reporter vector and included in this study (Fig.

3B). The responses of the main cereulide promoters in the

background of a clinical isolate and of an emetic outbreak

isolate did not differ discernibly on cooked rice, indicating

that F4810/72 is an appropriate tool for real-time

monitor-ing of

ces

gene expression in different environments.

Categorization of foods by potential for cereulide

produc-tion.

A selection of retail food products was inoculated with

a bioluminescent emetic

B. cereus

reporter strain and

incu-bated at 24°C, the temperature reported to be optimal for

cereulide production (31). Under these conditions, the

foods could be divided into three main categories by their

potential to support toxin synthesis (Fig. 3A and Table 2).

As shown by cereulide quantification, the amount of toxin

produced correlated to the signal intensity of the

ces

pro-moter-driven

lux

gene expression (Table 2). Several rice

dishes were implicated in food poisoning cases or previously

reported to support toxin synthesis to a great extent (e.g.,

see references 25, 28, and 35). Consistently, cooked rice was

classified as a high-risk food in our system. The system

revealed other products besides this classical

outbreak-re-lated food to be potentially at risk, such as a sweetened

dairy-based dessert and Camembert cheese. Interestingly,

cereulide was detected in another Camembert cheese

sam-ple investigated previously (49). The proposed risk group

classification is further supported by the fact that around 8

to 10

␮

g cereulide per kg of body weight must be consumed

by healthy adults to provoke a clinical manifestation of the

emetic syndrome (33, 56). Thus, foods containing

⬎

1

␮

g g

⫺1

cereulide would be hazardous to a child with a body weight

of 10 kg if about 100 g were consumed. Indeed, foods related

to emetic poisoning contained about 1 to 3

␮

g g

⫺1

_cereulide

(3, 33). But the risk-group foods should also be considered

critical, as other studies revealed that food poisoning cases

implicated meals which contained as little as 0.01

␮

g g

⫺1

cereulide (3).

A comprehensive evaluation concerning cereulide

pro-duction in different groups of food is still missing, most

probably due to the time-consuming and laborious methods

required for toxin quantification in these complex matrices.

The extraction of the highly lipophilic cereulide molecule,

especially from fat-rich foods, is often prone to error (for

example, the toxin recovery rate from different food

matri-ces varies from 22% to 100% [M. Ehling-Schulz and E.

Frenzel, unpublished data]), and matrix-specific extraction

protocols are often required (55). The assay presented here

offers the advantage of a high-throughput system for a basic

categorization of foods and food ingredients by the risk of

cereulide production. Thus, the real-time monitoring of

ces

gene expression might have the potential as an industrial

application to support hazard identification in terms of

haz-ard analysis and critical control point concepts and offers

the possibility of basic identification of a potential source of

risk to the consumer.

This study focuses on the classification of foods by their

potential to support cereulide toxin synthesis. However, it

should also be taken into consideration that

B. cereus

is able

to produce additional food spoilage and virulence factors,

including phospholipases, proteases, hemolysins, and

ente-rotoxins, which are unambiguously of medical and

econom-ical impact (10, 35, 54).

lux

gene reporter systems, similar to

the one described in this work, might also be useful for

monitoring enterotoxin expression and could add important

information to the hazard identification of different food

systems.

ACKNOWLEDGMENTS

We thank Daniel Zeigler of the Bacillus Genetic Stock Center at the

Ohio State University and Jo Handelsman of the Department of Plant

Pathology at the University of Wisconsin—Madison for the kind gifts

of pAD123 and pAD43-25, respectively. We thank Kevin Francis

(Xenogen Corporation) for the gift of pXen1 and Romy Renner and

Laura Tschernek for excellent technical assistance.

This work was supported in part by the Forschungskreis der Erna

¨hr-ungsindustrie e.V. (FEI), by the Arbeitskreis fu

¨r Industrielle

For-schung (AiF), by the Ministry of Economics and Technology (BMWi;

project no. 15186N), and in part by the Bavarian Ministry for

Agricul-ture and Forestry (project no. M2-7606.2-526).

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