Insertional inactivation of hblC encoding the L2 component of Bacillus cereus ATCC 14579 haemolysin BL strongly reduces enterotoxigenic activity, but not the haemolytic activity against human erythrocytes

Toril Lindbäck1, Ole Andreas Økstad1, Anne-Lise Rishovd1 and Anne-Brit Kolstø1

Biotechnology Centre of Oslo and School of Pharmacy, Department of Microbiology, University of Oslo, PO Box 1125 Blindern, N-0349 Oslo, Norway1

Author for correspondence: Anne-Brit Kolstø. Tel: +47 22958460. Fax: +47 22694130. e-mail: annebko{at}biotek.uio.no


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Haemolysin BL (HBL) is a Bacillus cereus toxin composed of a binding component, B, and two lytic components, L1 and L2. HBL is also the enterotoxin responsible for the diarrhoeal food poisoning syndrome caused by several strains of B. cereus. The three genes encoding the HBL components constitute an operon and are transcribed from a promoter 608 bp upstream of the hblC translational start site. The first gene of the hbl operon, hblC, in the B. cereus type strain, ATCC 14579, was inactivated in this study. Inactivation of hblC strongly reduced both the enterotoxigenic activity of B. cereus ATCC 14579 and the haemolytic activity against sheep erythrocytes, while maintaining full haemolytic activity against human erythrocytes.

Keywords: Bacillus cereus, enterotoxin, haemolysin, HBL

Abbreviations: HBL, haemolysin BL


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacillus cereus is a Gram-positive, spore-forming, motile, aerobic rod, commonly found in soil and water. B. cereus has also been isolated from a variety of foods, including rice, spices, meat, eggs and dairy products (Kramer & Gilbert, 1989 ), and from drugs including both topical and oral pharmaceutical products (Garcia-Arribas et al., 1988 ). B. cereus is the causative agent of two distinct food poisoning syndromes, the emetic and the diarrhoeal syndromes (Turnbull et al., 1979 ). Both forms of the illness are toxin-mediated. The emetic toxin, a cereulide, has been isolated and described by Agata et al. (1994 , 1995b ).

At least three diarrhoeal toxins have been isolated from B. cereus strains, the best characterized being the enterotoxic haemolysin BL (HBL) (Beecher & Macmillan, 1991 ). HBL is composed of three components, B, L1 and L2, forming a protein complex possessing haemolytic, cytotoxic, dermonecrotic and enterotoxigenic activities (Beecher & Wong, 1994a ; Beecher et al., 1995 ). In addition to its haemolytic and enterotoxic activities, B. cereus is considered one of the most destructive organisms to affect the eye (O’Day et al., 1981 ; Pflugfelder & Flynn, 1992 ). B. cereus ocular virulence is multifactorial and HBL is one of the major factors contributing to this virulence (Beecher et al., 1995 ).

The genes encoding the B, L1 and L2 components, hblA, hblD and hblC, respectively, have previously been cloned and sequenced from the B. cereus strain F837/76 (Heinrichs et al., 1993 ; Ryan et al., 1997 ). Beecher & Macmillan (1991) have shown that a combination of all three components, B+L1+L2, is required for complete lysis of sheep erythrocytes. Neither B+L1 nor any of the components alone was haemolytic. Using mAbs against the B component, it was shown by immunofluorescence staining of erythrocytes that protein B binds to the cell surface before the L components can act to cause cell lysis (Beecher & Macmillan, 1991 ). The potency of HBL has been compared to that of cholera toxin, and HBL has therefore been suggested to be the primary virulence factor in diarrhoea caused by B. cereus (Beecher et al., 1995 ).

Studies by Agata et al. (1995a ) and Lund & Granum (1996) have identified other proteins responsible for a diarrhoeal-type of B. cereus food poisoning. Agata et al. (1995a ) cloned a gene encoding a 41 kDa protein, bc-D-ENT, identified in 10 strains isolated from food or outbreaks of foodborne illness. The amino acid sequence of the protein showed no homology to the amino acid sequence of the HBL components, did not possess an N-terminal sequence characteristic of Bacillus signal peptides (Simonen & Palva, 1993 ), and has not yet been shown to be secreted by B. cereus. Lund & Granum (1996) have characterized a non-haemolytic enterotoxin (NHE) complex from B. cereus isolated after a foodborne outbreak. The enterotoxin consisted of three proteins of 39, 45 and 105 kDa, and the 39 kDa protein showed 70% identity in a stretch of 23 aa to the L1 component of HBL (Lund & Granum, 1996 , 1997 ). In addition, several haemolysins have been identified in B. cereus strains (Baida & Kuzmin, 1995 ; Honda et al., 1991 ). Thus the number and nature of enterotoxins and haemolysins in B. cereus is complex, and doubts have been raised whether the HBL complex expresses both haemolytic and enterotoxigenic activity.

To further investigate the role of HBL in the various aspects of B. cereus toxicity, we have constructed a gene inactivation mutant of the first gene in the hbl operon which in addition resulted in loss of transcription of the two downstream genes of the operon. The B. cereus type strain, ATCC 14579, was chosen for our studies, since this strain carries the hbl locus (Carlson et al., 1996 ) and has been used previously in gene inactivation experiments (Lindbäck & Kolstø, 1997 ; Økstad et al., 1997 ). The present study shows that disruption of the hblC gene causes a major reduction in the enterotoxigenic activity as well as in haemolytic activity against sheep erythrocytes, while no change was detected in haemolytic activity against human erythrocytes. The mutant described in this report will be employed in future work to further unravel the effects of HBL.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and growth conditions.
All bacteria were routinely grown in 100 ml LB broth (Sambrook et al., 1989 ) in 1000 ml flasks, or on LB agar at 37 °C. The B. cereus strains ATCC 14579 (type strain) and ATCC 10987 were obtained from the American Type Culture Collection (Manassas, VA, USA). B. cereus F837/76 was obtained from the Public Health Laboratory Service (London, UK). Escherichia coli strains DH5{alpha} and BK2118 (Evensen & Seeberg, 1982 ) were used as recombinant hosts. The DNA library was constructed in pUC19 and all subcloning was done in pUC18 or pUC19. Ampicillin was used at a concentration of 50 µg ml-1 and spectinomycin at a concentration of 100 µg ml-1, where appropriate. Bacterial culture supernatants used in immunoblotting and in the Vero cell toxicity test were obtained by centrifuging a culture of B. cereus grown to OD550 2·5 in Brain Heart Infusion broth (Difco) containing 0·5% glucose.

DNA manipulation and library preparation.
Preparation, cloning and analysis of DNA were performed according to standard protocols (Sambrook et al., 1989 ). E. coli BK2118 was used for electroporation according to Chuang et al. (1995) . Genomic DNA from B. cereus was isolated from 200 ml cultures grown to mid-exponential phase. The bacteria were washed in TES (30 mM Tris, pH 8·0; 5 mM EDTA; 50 mM NaCl) and resuspended in 8 ml TES. One millilitre of lysozyme (5 mg ml-1) and 0·4 ml RNase (2 mg ml-1) were added and the suspension was incubated for 30 min at 37 °C. Triton X-100 (1·4 ml, 8%) and 0·6 ml proteinase K (5 mg ml-1) were added and the bacteria were lysed for 1 h at 37 °C. PMSF (0·2 ml, 100 mM) was added before the DNA was purified by ultracentrifugation in a CsCl gradient (Sambrook et al., 1989 ).

A genomic library was constructed by digesting the purified B. cereus ATCC 14579 DNA with BglII followed by ligation into BamHI-digested pUC19. The library was screened using a probe covering the hblC and hblD genes from B. cereus F837/76. The probes were obtained as a gift from James D. Macmillan (Rutgers University, NJ, USA).

Amplification of hblA and hblB by PCR.
Subregions of the hblA and hblB genes were amplified by PCR using the primers P3 (5'-GGC ACA TGC TGT TAC TTG GG-3') and P4 (5'-ACC CAA AAT AGC ACC CTT CA-3') for hblA, and P5 (5'-TGA ATC AGC TTC GTC CAT CA-3') and P6 (5'-AAT GAA AGC CAA TCA GCC C-3') for hblB. PCR was run as recommended by the suppliers for 30 cycles in a 50 µl volume using an annealing temperature of 56 °C, 0·4 µM of each primer, 100 ng of the cloned Bc300 fragment (Fig. 1) as DNA template and 1 U Dynazyme (Finnzymes Oy). The forward primers P3 and P5 are located at positions 13771 and 16086, respectively, and the reverse primers P4 and P6 are located at positions 14185 and 16275, respectively, in the EMBL sequence AJ237785.



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Fig. 1. Restriction map of a genomic region from B. cereus ATCC 14579 covering the hbl operon. The two BglII fragments, Bc300 and Bc301, are indicated. The transcriptional start site, location of primers (P1, P2, P3, P4, P5, P6) and the PlcR box are indicated by arrows. The construction of pAT113-hbls used in conjugation experiments is shown.

 
RNA isolation and analysis.
RNA was isolated from 10 ml samples as described by Igo & Losick (1986) . Mapping of the 5' terminus of the hbl mRNA was performed on total RNA isolated from B. cereus ATCC 14579, using two different 5' end-labelled oligonucleotides, P1 (5'-TCC TTT CTG TCT GGT CAT ACC-3') and P2 (5'-TCA GCA AAC TCC TTA CTA GAC-3'), located 580 and 363 bp upstream of the putative translational start site of hblC, respectively. Annealing and extension were performed according to Luna et al. (1994) using 10 U AMV Reverse Transcriptase (Promega) in the extension reaction. Products were resolved in 6% polyacrylamide gels together with sequence reactions prepared according to the dideoxy chain-termination method using T7 DNA polymerase (Pharmacia). Northern blotting and hybridization were performed according to Narahara et al. (1992) .

SDS-PAGE and immunoblotting.
Supernatant (10 µl) from cultures harvested in early stationary phase was applied on 10% SDS-PAGE gels. SDS-PAGE and immunoblotting were performed according to standard protocols (Harlow & Lane, 1988 ). Polyclonal antibodies directed against the L1, L2 and B components of HBL were obtained as a gift from Douglas J. Beecher (Rutgers University, NJ, USA) and used at a dilution of 1:5000. Anti-rabbit horseradish-peroxidase-conjugated antibodies from donkey (Amersham) were used as secondary antibodies (1:5000) and ECL Western blotting detection reagent (Amersham) was used to develop the immunoblot.

Vero cell toxicity and haemolytic activity assays.
Vero cells were obtained from the Norwegian Radium Hospital and were grown in Dulbecco’s MEM medium supplemented with 5% foetal calf serum. Cells were seeded into 24-well plates the day before testing. Bacterial culture supernatants (100 µl) were added to triplicate wells containing a Vero cell monolayer. Cell cultures were incubated further at 37 °C under 5% CO2 for up to 1 h. Before and immediately after the addition of bacterial culture supernatants, and at 10, 20 and 60 min post addition, cell cultures were examined with a Nikon TMS microscope.

Blood agar plates were prepared with sheep, human, horse or chicken erythrocytes (10%), and bacteria or culture supernatant was spotted on the plates. The plates were incubated at 20, 30 or 37 °C overnight. After incubation the plates were checked for haemolytic zones.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning of the hbl operon from B. cereus ATCC 14579
Screening of 20000 colonies from a genomic library with the hblC/hblD probe from B. cereus F837/76 resulted in the identification of two DNA fragments, Bc300 and Bc301, of 9·9 kb and 11·3 kb, respectively. Sequencing and analysis of the complete Bc300 and Bc301 clones is reported elsewhere (Økstad et al., 1999 ). The DNA sequence of the hbl operon showed 97 % identity to the previously reported sequence from B. cereus F837/76 (Heinrichs et al., 1993 ; Ryan et al., 1997 ; Økstad et al., 1999 ).

Insertional inactivation of hblC
The most downstream HindIII fragment of Bc301 (Fig. 1), with one HindIII site located in the pUC19 polylinker, was cloned into the HindIII site of the conjugative suicide vector pAT113 (Trieu-Cuot et al., 1991 ) (Fig. 1). Subsequently a 1·2 kb EcoRI fragment covering the hblD gene (Fig. 1) was cloned into the EcoRI site and a spectinomycin-resistance cassette was cloned into the BamHI site of the pAT113 construct. This new construct, pAT113-hbls (Fig. 1), was used in transconjugal transfer from E. coli JM83(pRK24) (Thomas & Smith, 1987 ) to B. cereus ATCC 14579, and chromosomal integration was verified by hybridization. Three separate mating experiments between B. cereus ATCC 14579 and E. coli JM83(pRK24)(pAT113-hbls) produced exclusively single cross-over transconjugants. One double cross-over transconjugant was identified after growing a single cross-over transconjugant at 45  °C for five passages of 24 h each. Hybridization of chromosomal DNA isolated from the double cross-over transconjugant, assigned Bc{Delta}hblC, showed that the 0·9 kb BglII–EcoRI fragment located within hblC (Fig. 1) had been replaced by the spectinomycin-resistance cassette of 1·4 kb (Fig. 2).



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Fig. 2. Southern blot analysis of chromosomal DNA digested with HindIII. Lanes: 1, Bc{Delta}hblC; 2, single cross-over mutant used to create Bc{Delta}hblC; 3, wild-type B. cereus ATCC 14579. The membrane was probed with both the 0·9 kb BglII–EcoRI fragment replaced by the spectinomycin-resistance cassette in Bc{Delta}hblC, and the spectinomycin-resistance cassette of 1·4 kb.

 
Expression analysis of the hbl locus
Northern blots of RNA isolated from different B. cereus strains probed with the 0·9 kb BglII–EcoRI fragment from hblC (Fig. 1) revealed a 5·5 kb transcript in B. cereus ATCC 14579 and B. cereus F837/76, while no transcript was detected in RNA from B. cereus ATCC 10987 and Bc{Delta}hblC (Fig. 3). Using a part of the hblD gene and a PCR-amplified subregion of hblA (primers P3 and P4; Fig. 1) as probes, hybridization was observed to the 5·5 kb transcript in wild-type B. cereus ATCC 14579 while no hybridization was detected in Bc{Delta}hblC (data not shown). PCR was used to amplify a 3' region of hblB that does not show sequence similarity to hblA, using oligonucleotide primers P5 and P6 (Fig. 1). When this hblB-specific probe was used in Northern hybridization, no transcript was detected in RNA isolated from wild-type or mutant cells during stationary phase (data not shown). These data show that the disruption of the hblC gene abolished transcription of the downstream genes, hblD and hblA, and indicated that hblB is not transcribed at a level detectable by Northern blot analysis, even in wild-type B. cereus ATCC 14579.



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Fig. 3. Northern blot analysis of total RNA isolated from different B. cereus strains. Lanes: 1, 0·24–9·5 kb RNA ladder from Gibco-BRL; 2, B. cereus ATCC 10987; 3, B. cereus F837/76; 4, B. cereus ATCC 14579; 5, Bc{Delta}hblC. The membrane was probed with a 0·9 kb BglII–EcoRI fragment covering the hblC gene (Fig. 1).

 
Mapping of the 5' terminus of the hbl mRNA in B. cereus ATCC 14579 by primer extension analysis using oligonucleotide P2 (Fig. 1) revealed a product of 246 bp, thereby identifying a transcriptional start site at an adenine (Fig. 4) positioned 608 bp upstream of the putative translational start site. A putative -10 promoter sequence, TTTAAT, with T in position -10, was identified upstream of the putative transcriptional start site. Primer extension analysis using oligonucleotide P1 (Fig. 1) revealed no alternative transcriptional start site upstream of the one described above. The transcriptional start site is located 271 bp downstream of the putative binding site of the transcriptional regulator PlcR (Agaisse et al., 1999 ).



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Fig. 4. Mapping of the 5' terminus of the hbl mRNA of B. cereus ATCC 14579 by primer extension. The transcriptional start site is indicated by an arrow. PE, primer extension.

 
By using polyclonal antibodies directed against components B, L1 and L2, respectively, in immunoblotting, no trace of the L2 component, the product of the first gene of the operon (hblC), was found in the culture supernatant of Bc{Delta}hblC (Fig. 5). Faint bands observed when anti-B or anti-L1 sera were used may be due to non-specific binding of the polyclonal anti-B and anti-L1 antibodies.



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Fig. 5. Western blotting of B. cereus ATCC 14579 and Bc{Delta}hblC culture supernatant probed with antisera to the individual HBL components. Lanes: 1, 3 and 5, B. cereus ATCC 14579 culture supernatant; 2, 4 and 6, Bc{Delta}hblC culture supernatant. Lanes 1 and 2 are probed with antiserum against L2, lanes 3 and 4 are probed with antiserum against L1 and lanes 5 and 6 are probed with antisera against the B component of HBL. The size marker used was a prestained broad-range marker from Bio-Rad.

 
Enterotoxic and haemolytic activity
Vero cell monolayers were examined microscopically after addition of culture supernatants from B. cereus ATCC 14579, B. cereus ATCC 10987, B. cereus F837/76 and Bc{Delta}hblC. A rapid cytopathogenic effect was observed within a few minutes for B. cereus ATCC 14579, B. cereus ATCC 10987 and B. cereus F837/76, while a reduced effect was observed when the Bc{Delta}hblC supernatant was added to the cells (Fig. 6). As a control we also included a mutant strain inactivated by the spectinomycin-resistance cassette in the bctL gene encoding a putative transporter protein (Økstad et al., 1997 ). The effect of the Bc{Delta}bctL disruption mutant was similar to the effect of wild-type B. cereus ATCC 14579.



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Fig. 6. Effect of supernatant from B. cereus ATCC 14579 wild-type and Bc{Delta}hblC on a Vero cell monolayer. (a) Total destruction of the monolayer 20 min after addition of a 10xdilution of B. cereus ATCC 14579 culture supernatant. (b) Confluent monolayer 20 min after addition of a 10xdilution of Bc{Delta}hblC culture supernatant. (c) Confluent monolayer with added LB medium.

 
Wild-type B. cereus ATCC 14579 and Bc{Delta}hblC were grown on agar plates containing sheep, chicken, human or horse erythrocytes (10%). There was a pronounced reduction in the haemolytic activity of the mutant strain against sheep erythrocytes (Fig. 7), while no difference was seen against erythrocytes from human (Fig. 7) and other sources (data not shown). Interestingly, the strong haemolytic activity against sheep erythrocytes was observed when the cells were grown at 37 °C, but not at 30 °C, although a ‘paradoxic haemolytic zone’ described for HBL (Beecher & Wong, 1994b ) was sometimes observed also at 30 °C (Fig. 7).



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Fig. 7. Haemolytic pattern shows reduction of haemolytic activity of Bc{Delta}hblC compared to wild-type B. cereus ATCC 14579 against sheep erythrocytes at 37 °C, but not against human erythrocytes. At 30 °C, the haemolytic activity of the wild-type strain was less profound.

 
To analyse whether B. cereus ATCC 14579 harboured the gene(s) for the non-haemolytic enterotoxin NHE (Lund & Granum, 1996 ), a probe covering the promoter region and the 5' part of the first gene in the nhe operon (Agaisse et al., 1999 ) was used to hybridize a Southern blot of digested genomic DNA. nhe was present in both wild-type and mutant B. cereus ATCC 14579, and also in strain B. cereus ATCC 10987. The expression of the gene was analysed by Northern blot hybridization, and while the gene was highly transcribed in ATCC 10987, it was only very weakly transcribed in both wild-type B. cereus ATCC 14579 and Bc{Delta}hblC (data not shown).


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
HBL consists of three components which are all required to obtain full enterotoxigenic activity (Beecher & Macmillan, 1991 ). In this study we have inactivated the first gene of the hbl locus, hblC, encoding the L2 component of HBL from B. cereus ATCC 14579. This strain was chosen since it was previously used for inactivation of other genes (Lindbäck & Kolstø, 1997 ; Økstad et al., 1997 ). Other strains, like B. cereus F837/76 and B. cereus ATCC 10987, are not well suited for targeted gene disruption by the method used in this study. In addition, Southern blot analysis revealed no indication of more than one hbl locus in the ATCC 14579 strain.

Insertional inactivation of hblC, which is the first gene in the hbl operon, resulted in complete loss of transcription of the operon (Fig. 3). This effect rendered it difficult to correlate the phenotype with the inactivation of hblC. Immunoblot analysis (Fig. 5) showed that no L2 component was synthesized in the mutant strain, while very weak bands were detected with antisera against L1 and B components (Fig. 5). These weak bands were most likely due to cross-reaction with other unrelated proteins since no indication was found of separate transcription initiation from either of the two downstream genes of the operon by Northern blot analysis of the Bc{Delta}hblC strain (data not shown). However, it can not be excluded that small amounts of L1 and B proteins are present due to a low level of independent transcription of the corresponding genes not detected by the Northern blot experiments. Beecher & Macmillan (1991) showed that L1+B did not exhibit detectable haemolytic activity. We therefore presume that if any traces of B and L1 are present in the disruption mutant they would not contribute to cytotoxic and haemolytic activity of the mutant strain.

The use of McCoy cells and Vero cells to assay B. cereus enterotoxin activity is well-established (Jackson, 1993 ; Agata et al., 1995a ; Lund & Granum, 1997 ). Other toxins like phospholipases and haemolysins other than HBL do not induce the characteristic destruction of the McCoy cell monolayer (Jackson, 1993 ). When testing the bacterial culture supernatants on Vero cell monolayers, we observed that inactivation of L2 strongly reduced the rapid cytopathogenic activity observed in the wild-type strain of B. cereus ATCC 14579. However, we still observed a slower destruction of the cell monolayer when adding the culture supernatant from Bc{Delta}hblC.

The fact that Bc{Delta}hblC had a strongly reduced toxic effect towards Vero cells clearly indicated that HBL caused the main cytopathogenic activity of B. cereus ATCC 14579. The weak cytopathogenic effect observed from Bc{Delta}hblC was most likely due to small amounts of NHE, since low levels of nhe mRNA were observed in the wild-type and mutant strain of ATCC 14579. In accordance, strain ATCC 10987, which lacks the hbl operon, synthesized high levels of nhe mRNA and exhibited a strong cytopathogenic activity towards Vero cells. Both hbl and nhe are regulated by the PlcR regulator, present in B. cereus and Bacillus thuringiensis strains (Agaisse et al., 1999 ). Sequencing of the nhe promoter region from both ATCC 14579 and ATCC 10987 strains and further analysis of the nhe and the hbl upstream regions may provide better understanding of the complexity of regulation of these operons.

Analysis of the upstream region of the hbl operon of B. cereus F837/76 revealed the presence of the highly conserved palindromic region postulated to be the specific recognition target for transcriptional activation by the pleiotropic regulator PlcR (Agaisse et al., 1999 ). The transcriptional start site was located 271 bp downstream of the putative PlcR recognition target (Agaisse et al., 1999 ). The transcriptional start site of the B. cereus ATCC 14579 hbl operon was identified by primer extension analysis at a location 608 bp upstream of the putative translational start site of hblC (Fig. 4). This differed from the one in F837/76 reported by Agaisse et al. (1999) by 2 bp, probably due to variation between strains.

The hbl transcript from the B. cereus ATCC 14579 wild-type strain was about 5·5 kb, similar to that which has been reported for B. cereus F837/76 (Heinrichs et al., 1993 ; Ryan et al., 1997 ). The expected size of a transcript including the three genes hblC, hblD and hblA would be about 4·4 kb. A transcript of 5·5 kb transcribed from the start site as determined by primer extension in this report will be terminated within the hblB gene. No evidence was found supporting the presence of a second transcriptional start site upstream of hblC. This indicates that a putative stem–loop structure found downstream of hblA (Økstad et al., 1999 ) is not functional as a transcription terminator. Considering the primer extension analysis and the Northern blot analysis results, we conclude that transcription of the hbl operon most likely starts 608 bp upstream of the translational start site, 271 bp downstream of the PlcR regulatory sequence, and terminates within hblB. This conclusion is reinforced by the fact that no transcript was observed using a probe located at the 3' end of hblB in Northern blot analysis. Hybridization with this hblB-specific probe also indicated that hblB is not transcribed alone, although it cannot be excluded that at other time points or growth conditions, transcription of hblB may occur.

HBL is most active against sheep and calf erythrocytes (Beecher & Wong, 1994b ). Accordingly, inactivation of the hbl locus reduced the haemolytic activity of Bc{Delta}hblC compared to wild-type ATCC 14579. A smaller difference was seen at 30 °C, indicating a temperature dependence perhaps due to conformational changes of the HBL toxin. Since there was no reduction of the haemolysis when comparing mutant and wild-type ATCC 14579 strains on human blood agar plates, the HBL toxin does not contribute significantly to B. cereus haemolytic activity against human erythrocytes. The reason for the preferential lysis of sheep erythrocytes is not known, but the presence of a higher amount of sphingomyelin in the plasma membrane in the sheep erythrocytes could perhaps be of importance.

We conclude that the enterotoxic HBL is responsible for the major enterotoxigenic activity of B. cereus ATCC 14579. The haemolytic activity of B. cereus ATCC 14579 against sheep erythrocytes is partly caused by HBL, while HBL does not contribute significantly to the haemolytic activity against human, chicken or horse erythrocytes.


   ACKNOWLEDGEMENTS
 
We gratefully acknowledge James D. Macmillan for providing us with the probes used for screening of the genomic library, and Douglas J. Beecher for providing the polyclonal antibodies directed against the L1, L2 and B components of HBL. The authors are thankful to Ewa Jaroszewicz for skilful technical assistance.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 26 May 1999; revised 27 July 1999; accepted 5 August 1999.